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PubMed2005

Reactive oxygen species and related haem pathway components as possible epigenetic modifiers in neurobehavioural pathology.

The neuroendocrine response to stress utilizes several bio-communicative pathways which also play a role in neurodevelopmental plasticity. The mechanism of action of steroidal compounds includes DNA alteration by reactive oxygen species (ROS) arising through redox cycling of reactive hormone derivatives. ROS and reactive nitrogen species play a significant role in signaling networks affecting gene transcriptional regulation during normal as well as stress-induced responses. ROS-associated synaptic and regulatory region plasticity may have been important for normal brain evolution, but probably simultaneously lowered the threshold for inducing neuropathology. A shift from 'plasticity' to 'instability' is likely to be associated with the emergence of complex effects depending on the timing, duration and intensity of the ROS insult, and is suggested to include heritable epigenetic chromatin/regulatory region remodeling differentially influencing expression levels of significant neuropsychiatric genes and their variant alleles. Neurobehavioural disorder clinical manifestations have been linked with ROS effects. The concepts discussed here relate to ROS-associated instability of DNA regulatory region sequences and a proposal that it may play an important modifying role in brain and neuro-behaviourally related gene expression. Genes encoding key steps in mitochondrial, haem, iron and bilirubin ROS metabolic pathways have been used as examples to illustrate how ROS-modified regulatory networks could possibly alter the context within which (even ostensibly unrelated) neuropsychiatric gene candidates may sometimes be recruited. Furthermore, reactions of certain radicals release sufficient energy to generate UV-photons. DNA conformational changes accompanied by changes in photon emission suggest that functional neuroimaging findings probably reflect interaction on the level of ROS/biophoton/genome regulatory region domains rather than the signatures of individual neurobehavioural disorder candidate genes.

Bilirubin↗

PubMed2006

Rosiglitazone improves insulin sensitivity in nonobese subjects with impaired glucose tolerance: the role of adiponectin and C-reactive protein.

To evaluate the effects of rosiglitazone (ROS) on serum adiponectin and C-reactive protein (CRP) in nonobese subjects with impaired glucose tolerance (IGT), we enrolled 21 patients with body mass index < or =24 kg/m(2) to receive ROS 4 mg daily for 12 weeks. Fifteen age-, sex-, and body mass index-matched healthy subjects were recruited as controls. A 75-g oral glucose tolerance test (OGTT), hemoglobin A(1c), fasting glucose, insulin, C-peptide, lipid profiles, adiponectin, and CRP levels were determined before initiation and at the end of the 12-week ROS treatment. Insulin resistance and beta-cell function were calculated using the homeostasis model assessment method (HOMA-IR and HOMA-beta, respectively). Compared with healthy controls, the ROS-treated subjects had significantly higher glycemic indices, HOMA-IR, CRP, and glucose and insulin concentrations in response to OGTT, and lower HOMA-beta level. After 12 weeks of ROS therapy, the results showed statistically significant changes from baseline in 2-hour plasma glucose during OGTT (9.4 +/- 0.3 vs 8.3 +/- 0.4 mmol/L, P < .05), HOMA-IR (2.6 +/- 0.2 vs 1.9 +/- 0.3, P < .05), HOMA-beta (63.4 +/- 12.5 vs 90.1 +/- 13.0, P < .05), and glucose and insulin concentrations during OGTT in nonobese subjects with IGT. In addition, elevation of serum adiponectin and decrease in CRP levels were significantly found after ROS treatment. Of 21 patients treated with ROS, 5 subjects were converted to normal (converter), 1 progressed to diabetes, and 15 remained in IGT status (nonconverter). There was a significant amelioration in HOMA-IR (-2.10 +/- 1.03 vs -0.07 +/- 0.33, P < .05) without significant changes in adiponectin and CRP levels in converter compared with nonconverter. We conclude that ROS effectively enhanced insulin sensitivity and beta-cell function to improve adiponectin and CRP levels in nonobese patients with IGT. The amelioration of insulin resistance may be a major determinant to predict the conversion of IGT independent of the changes in adiponectin and CRP.

Adiponectin↗

PubMed2005

Modulatory effect of glutathione status and antioxidants on methylmercury-induced free radical formation in primary cultures of cerebral astrocytes.

Excessive free radical formation has been implicated as one of the causative factors in neurotoxic damage associated with variety of metals, including methylmercury (MeHg). Although the mechanism(s) associated with MeHg-dependent neurotoxicity remains far from clear, overwhelming data give credence to a mediatory role for astrocytes, a major cell type that preferentially accumulates MeHg. To extend our recent findings of MeHg-induced increase in ROS formation (G. Shanker, J.L. Aschner, T. Syversen et al., Free radical formation in cerebral cortical astrocytes in culture induced by methylmercury, Mol. Brain Res. 128 (2004) 48-57), the present studies were designed to assess the effect of modulating intracellular glutathione (GSH) content, on ROS generation, in the absence and presence of MeHg. Intracellular GSH was reduced by treatment with 100 microM buthionine-L-sulfoxane (BSO) for 24 h, and increased by treatment with 1 mM l-2-oxothiazolidine-4-carboxylic acid (OTC) for 24 h. Additionally, the effects of the selective antioxidants, catalase (1000 U/ml for 1 h), an H2O2 scavenger, and n-propyl gallate (100 microM for 1 h), a superoxide radical (*O2-) and possibly hydroxyl radical (*OH) scavenger on MeHg-induced ROS formation were examined. After these treatments, astrocytes were exposed to +/-10 microM MeHg for 30 min, following which the fluorescent probes, CM-H2DCFA and CM-H2XRos were added; 20 min later, laser scanning confocal microscopy (LSCM) images were obtained. Exposure of astrocytes for 24 h to 100 microM BSO, a GSH synthesis inhibitor, led to a significant increase in mitochondrial ROS (i.e., *O2-, *NO, and ONOO-) formation, as assessed with CM-H2XRos mitotracker red dye. Similarly, BSO increased ROS formation in various intracellular organelles, as assessed with CM-H2DCFDA. BSO in combination with MeHg increased fluorescence levels in astrocytes to levels above those noted with BSO or MeHg alone, but this effect was statistically indistinguishable from either of these groups (BSO or MeHg). Pretreatment of astrocytes for 24 h with 1 mM OTC abolished the MeHg-induced increase in ROS. Results similar to those obtained with OTC were observed with the free radical scavenger, n-propyl gallate (n-PG). The latter had no significant effects on astrocytic fluorescence when administered alone. This *O2- and possibly *OH radical scavenger significantly attenuated MeHg-induced ROS formation. Catalase, an H2O2 scavenger, was less effective in reducing MeHg-induced ROS formation. Taken together, these studies point to the important protective effect of adequate intracellular GSH content as well as antioxidants against MeHg-triggered oxidative stress in primary astrocyte cultures.

Animals↗

PubMed2004

H2S cytotoxicity mechanism involves reactive oxygen species formation and mitochondrial depolarisation.

A number of scavengers of reactive oxygen species (ROS) were found to be protective against cell death induced by hydrogen sulfide (H2S) in isolated hepatocytes. The H2O2 scavengers alpha-ketoglutarate and pyruvate, which also act as energy substrate metabolites, were more protective against H2S toxicity than lactate which is only an energy substrate metabolite. All of these results suggest that H2S toxicity is dependent on ROS production. We measured ROS formation directly in hepatocytes using the fluorogenic dichlorofluorescin method. H2S-induced ROS formation was dose dependent and pyruvate inhibited this ROS production. Non-toxic concentrations of H2S enhanced the cytotoxicity of H2O2 generated by glucose/glucose oxidase, which was inhibited by CYP450 inibitors. Furthermore, hepatocyte ROS formation induced by H2S was decreased by CYP450 inhibitors cimetidine and benzylimidazole. These results suggest that CYP450-dependant metabolism of H2S is responsible for inducing ROS production. H2S-induced cytotoxicity was preceded by mitochondrial depolarization as measured by rhodamine 123 fluorescence. Mitochondrial depolarization induced by H2S was prevented by zinc, methionine and pyruvate all of which decreased H2S-induced cell death. Treatment of H2S poisoning may benefit from interventions aimed at minimizing ROS-induced damage and reducing mitochondrial damage.

Air Pollutants↗

PubMed2004

Differential susceptibility of osteosarcoma cells and primary osteoblasts to cell detachment caused by snake venom metalloproteinase protein.

The interaction of extracellular matrix with cells plays a key role in the regulation of cell adhesion, migration, proliferation as well as differentiation. Transformed cells express a different profile of adhesion molecules, which may mediate metastasis under specific matrix microenvironment. We here found that ROS 17/2.8 osteosarcoma cells and osteoblasts have different expression of alpha5 integrin, executing different fibronectin fibrillogenesis. As compared with ROS 17/2.8 cells, osteoblasts have higher expression of fibronectin, collagen, alpha5, beta1, alpha2 integrins and focal adhesion kinase as examined by immunostaining and flow cytometry. Crovidisin, a PIII snake venom metalloproteinase (SVMP) purified from venom of Crotalus viridis, exhibits collagen-binding activity and matrix metalloproteinase activity. Crovidisin selectively caused the detachment of ROS 17/2.8 osteosarcoma cells but not of primary cultured osteoblasts. On the other hand, triflavin, an RGD-dependent disintegrin purified from venom of Trimeresurus flavoviridis, did not cause the detachment of both osteoblasts and ROS 17/2.8 cells. Although ROS 17/2.8 cells detached from substratum after crovidisin treatment for 24 h, the loss of mitochondrial membrane potential was not observed unless a prolonged treatment for longer than 36 h. These results suggest that cultured primary rat osteoblasts and ROS 17/2.8 osteosarcoma cells possess different expression of integrins and matrix environment, and ROS 17/2.8 is much more susceptible to be detached by crovidisin. The matrix degradation by crovidisin may be responsible for the preferential detachment of ROS 17/2.8 osteosarcoma cells.

Animals↗

PubMed2006

Optimization of conditions for in vitro production of radical oxygen species and expression of tissue factor by canine mononuclear cells and granulocytes for use in high-throughput assays.

The purpose of this study was to optimize conditions for high throughput measurement of radical oxygen species (ROS) production and expression of tissue factor, also termed procoagulant activity, by canine leukocytes. Granulocytes and mononuclear cells were separated by density gradient centrifugation from peripheral blood collected on several occasions from three healthy large breed dogs. To determine optimal conditions for ROS production, granulocytes were incubated for 1 or 3h in PBG (PBS containing 0.5% BSA and 5mM glucose) or RPMI containing 10% fetal bovine serum (FBS); lipopolysaccharide (LPS), zymosan, peptidoglycan (PGN) and phorbol myristate acetate (PMA) were used as stimuli. ROS was assessed by conversion of the nonfluorescent dye dihydrorhodamine 123 to fluorescent rhodamine 123 by radical species released into the media. To identify optimal conditions for expression of tissue factor, mononuclear cells were incubated for 5h in RPMI containing different concentrations of heat-inactivated FBS (HI-FBS), and LPS, zymosan, PGN or PMA as stimuli. Expression of tissue factor was determined using a one-stage recalcification assay performed in an automated nephelometric coagulation analyzer. Neither LPS nor zymosan increased ROS production by granulocytes incubated in PBG media. In contrast, granulocytes incubated in RPMI had dose-dependent increases in ROS production in response to zymosan and PGN. ROS production was significantly increased by incubation with concentrations of LPS of 0.01microg/ml or greater, and by zymosan concentrations of 0.1microg/ml or greater. ROS production in response to incubation with PMA was significantly increased starting at 10(-7)M, and was significantly greater for cells incubated in RPMI than cells incubated in PBG. LPS-, zymosan- and PGN-stimulated procoagulant activity increased in a dose-dependent manner, whereas PMA-stimulated procoagulant activity peaked at 10(-7)M. Increasing concentrations of HI-FBS significantly increased LPS-, zymosan- and PGN-induced procoagulant activity of mononuclear cells. Results obtained in this study indicate production of ROS by canine granulocytes is optimal when these cells are incubated for 3h in RPMI with LPS (0.1microg/ml), zymosan (10 microg/ml), PGN (10 microg/ml), and PMA (10(-7)M). Furthermore, canine mononuclear cells express procoagulant activity in response to LPS, zymosan, PGN, and PMA, and responses to LPS, zymosan and PGN are enhanced by the addition of HI-FBS. These findings suggest that HI-FBS retains important serum proteins that facilitate interactions between each of these bacterial or yeast derived products and the mononuclear cells. Consequently, future studies regarding the regulation of procoagulant activity by canine mononuclear cells should be performed in the presence of HI-FBS. Both assays utilized in this study allow high throughput of samples, and therefore are appropriate choices for rapid screening of conditions and/or therapeutic interventions affecting the canine inflammatory system.

Animals↗

PubMed2002

Effect of light and protein phosphorylation on photoreceptor rod outer segment acyltransferase activity.

Rod outer segments (ROS) exhibit high acyltransferase (AT) activity, the preferred substrate of which being lysophosphatidylcholine. To study factors possibly regulating ROS AT activity purified ROS membranes were assayed under conditions under which protein kinase C (PKC), cAMP-dependent protein kinase (PKA), and phosphatases were stimulated or inhibited. PKC activation produced a significant increase in the acylation of phosphatidylethanolamine (PE) and phosphatidylinositol (PI) with oleate, it inhibited phosphatidylcholine (PC) acylation, and phosphatidylserine (PS) and phosphatidic acid (PA) acylation remained unchanged. ROS PKA activation resulted in increased oleate incorporation into PS and PI while the acylation of PC, PE, and PA remained unchanged. Inhibition of ROS PKC or PKA produced, as a general trait, inverse effects with respect to those observed under kinase-stimulatory conditions. ROS phosphatase 2A was inhibited by using okadaic acid, and the changes observed in AT activity are described. These findings suggest that changes in ROS protein phosphorylation produce specific changes in AT activity depending on the phospholipid substrate. The effect of light on AT activity in ROS membranes was also studied and it is reported that acylation in these membranes remains unchanged independent of the illumination condition used.

Acyltransferases↗

PubMed2002

Reactive oxygen species production by the mitochondrial respiratory chain in isolated rat hepatocytes and liver mitochondria: studies using myxothiazol.

Increased production of reactive oxygen species (ROS) by the mitochondrion has been implicated in the pathogenesis of numerous liver diseases. However, the exact sites of ROS production within liver mitochondria and the electron transport chain are still uncertain. To determine the sites of ROS generation in liver mitochondria we evaluated the ability of a variety of mitochondrial respiratory inhibitors to alter the steady state levels of ROS generated within the intact hepatocyte and in isolated mitochondria. Treatment with myxothiazol alone at concentrations that significantly inhibit respiration dramatically increased the steady-state levels of ROS in hepatocytes. Similar results were also observed in isolated mitochondria oxidizing succinate. Coincubation with antimycin or rotenone had no effect on myxothiazol-induced ROS levels. Myxothiazol stimulation of ROS was mitochondrial in origin as demonstrated by the colocalization of MitoTracker Red and dichlorofluorescein staining using confocal microscopy. Furthermore, diphenyliodonium, an inhibitor that blocks electron flow through the flavin mononucleotide of mitochondrial complex I and other flavoenzymes, significantly attenuated the myxothiazol-induced increase in hepatocyte ROS levels. Together, these data suggest that in addition to the ubiquinone-cytochrome bc(1) complex of complex III, several of the flavin-containing enzymes or iron-sulfur centers within the mitochondrial electron transport chain should also be considered sites of superoxide generation in liver mitochondria.

Animals↗

PubMed2001

The production of reactive oxygen species by irradiated camphorquinone-related photosensitizers and their effect on cytotoxicity.

Camphorquinone (CQ) is widely used as an initiator in modern light-cured resin systems but there are few reports about its effects on living cells. To clarify the mechanism of photosensitizer-induced cytotoxicity, the production of initiator radicals and subsequent reactive oxygen species (ROS) by CQ, benzil (BZ), benzophenone (BP), 9-fluorenone (9-F) in the presence of the reducing agent (2-dimethylaminoethyl methacrylate or N,N-dimethyl-p-toluidine, DMT) with visible-light irradiation was examined in a cell or cell-free system. Initiator radical production was estimated by the reduction rate of 1,1-diphenyl-2-picrylhydrazyl and by the conversion of poly-triethyleneglycol dimethacrylate; the results indicated that CQ/DMT had the highest activity among them. The cytotoxic effects of the photosensitizers on both human submandibular gland (HSG) adenocarcinoma cell line and primary human gingival fibroblast (HGF) showed that the 50% toxic concentration (TC(50)) declined in the order: CQ>BP>9-F>BZ. ROS produced in HSG or HGF cells by elicited, irradiated photosensitizers were evaluated in two different assays, one using adherent cell analysis and sorting cytometry against adherent cells and the other, flow cytometry against floating cells, with fluorescent probes. ROS production was dose- and time- dependent, and declined in the order: BZ>9-F>BP>CQ. Cytotoxic activity was correlated with the amount of ROS. Cytotoxicity and ROS generation in HGF cells was significantly lower than in HSG cells. ROS induced by aliphatic ketones (CQ) were efficiently scavenged by hydroquinone and vitamin E, whereas those by aromatic ketones (9-F) were diminished by mannitol and catalase, suggesting that OH radicals were involved in ROS derived from 9-F. A possible link between the cytotoxic activity and ROS is suggested.

Antioxidants↗

PubMed2003

Mitochondrial state 3 to 4 respiration transition during Fas-mediated apoptosis controls cellular redox balance and rate of cell death.

The role of reactive oxygen species (ROS) production in death receptor-mediated apoptosis is ill defined. We show that ROS levels play a novel role in moderating the rate of cell death in Fas-dependent apoptosis. Treatment of Jurkat T cells with oligomycin (ATP-synthase inhibitor) or FCCP (mitochondrial uncoupler) and Fas activating antibody (CH11), facilitated rapid cell death. ATP levels, DEVDase activity and cytochrome c release were not account for the synergistic killing effect. However, a decrease in cellular ROS production was associated with CH11 treatment and combinations of CH11 with oligomycin or FCCP further inhibited cellular ROS levels. Thus, decreased ROS production is correlated with accelerated cell death. A transition from state 3 to state 4 mitochondrial respiration following apoptotic stimuli accounted for an attenuated membrane potential and as a results mitochondria-derived ROS production capacity diminished. Similar observations were demonstrated in isolated rat liver mitochondria. Transfection with mitochondrial targeted catalase inhibited mitochondrial ROS production and potentiated cell death. These data show that ROS production is important in receptor-mediated apoptosis and may play a pivotal role in cell survival.

Apoptosis↗

PubMed2003

Alterations in mitochondria membrane potential and oxidative stress in infertile men: a prospective observational study.

OBJECTIVE: To evaluate the mitochondrial membrane potential (MMP) of spermatozoa and its correlation with semen parameters and production of reactive oxygen species (ROS) in infertile men and healthy donors. DESIGN: Controlled prospective study. SETTING: Male infertility clinic, Glickman Urological Institute, The Cleveland Clinic Foundation, Cleveland, Ohio. PATIENT(S): Nineteen infertile men and 7 healthy volunteers. INTERVENTION(S): Standard semen analysis, assessment of MMP and ROS production in spermatozoa. The MMP was assessed by flow cytometry using the probe carbocyanine DiOC(6)(3) and ROS was measured with chemiluminescence assay using luminol. MAIN OUTCOME MEASURE(S): The results of MMP are reported as the median interquartile range (IQR) number of cells counted in different areas of fluorescence. Results of ROS measurement are expressed as x10(6) counted photons per minute per 20 million sperm (cpm). RESULT(S): The patients with abnormal semen parameters had a significantly lower MMP [1337.7 (1066.38, 1879.2)], and higher ROS [1.12 (0.26, 3.86)] than the donors [MMP: 2482.9 (2162.5, 3520.6)] and [ROS: 0.10 (0.01, 0.14)]. The MMP was positively correlated with sperm concentration (r = 0.62) and negatively correlated with the ROS produced (r = -0.45). CONCLUSION(S): Measuring MMP in spermatozoa provides useful information about a man's fertility potential. Increased ROS production by spermatozoa is associated with a decreased MMP.

Carbocyanines↗

PubMed1999

Effect of seminal oxidative stress on fertility after vasectomy reversal.

OBJECTIVE: To evaluate seminal oxidative stress in men after vasectomy reversal and to determine whether seminal oxidative stress could predict fertility after vasectomy reversal. DESIGN: Measurement of seminal reactive oxygen species (ROS) and total antioxidant capacity (TAC) in normal donors, men who were fertile after vasectomy reversal, and men who were infertile after vasectomy reversal. SETTING: A male infertility clinic of a tertiary care center. PATIENT(S): Thirty men who underwent vasectomy reversal and 17 normal donors. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Semen characteristics, seminal ROS, and TAC were measured with chemiluminescence assays in samples from donors and reversal patients. RESULT(S): Mean adjusted seminal ROS (log [ROS+1]) was higher in infertile reversal patients (2.38+/-0.25) than in normal donors (1.30+/-0.14). Seminal ROS was also higher in all (fertile and infertile reversal combined) reversal patients than in donors. Total antioxidant capacity did not differ between groups. The ROS-TAC score, a composite index of seminal oxidative stress, was a significant predictor of fertility. A ROS-TAC score of 45 or greater had a positive predictive value of 73% in predicting fertility. CONCLUSION(S): Seminal oxidative stress is associated with vasectomy reversal. The ROS-TAC score is a possible predictor of infertility after vasectomy reversal.

Adult↗

PubMed2001

17Beta-estradiol inhibits oxidized low density lipoprotein-induced generation of reactive oxygen species in endothelial cells.

Increase of intracellular reactive oxygen species (ROS) has been proposed to cause endothelial injury, and oxidized LDL (oxLDL) actions are associated with an early increase of ROS. Estrogen protects vascular cells partly via its antioxidant effects and by preventing lipid peroxidation. However, whether it can inhibit oxLDL-induced stimulation of ROS generation in endothelial cells is unknown. We utilized the fluorescent dye (DCFH-DA) to measure ROS generation and compared the stimulant effect of tert-butylhydroperoxide (TBH) and oxLDL in human umbilical vein endothelial cells (HUVECs). We found that TBH, H2O2, and oxLDL rapidly stimulated ROS generation, and in a dose-dependent manner with TBH. A concentration of estrogen effective in preventing lipid peroxidation was employed either by pretreatment of cells 18 h prior to or by direct co-incubation (30 min) with HUVEC and oxLDL. Estrogen (54 microM) pretreatment significantly suppressed both TBH- and oxLDL- induced stimulation of ROS generation. Both 1 and 54 microM concentration of estrogen could directly inhibit oxLDL-induced ROS production in HUVECs. Thus, either 18 h pretreatment or 30 min co-incubation with estrogen reduced stimulated ROS generation, suggesting that both cellular and direct actions of estrogen may be involved.

Antioxidants↗

PubMed1997

Cholinergic-induced production of reactive oxygen species in human neuroblastoma cells.

Stimulation of human SH-SY5Y neuroblastoma cells by a muscarinic receptor agonist, carbachol (CCh; 1 mM), elevated levels of free intracellular calcium and subsequently increased the production of reactive oxygen species (ROS). Quinuclidinylbenzilate (QNB) binding increased at 1 h after CCh, but returned back to the control level at 3 h. Production of ROS increased, however, during the 3 h time period. CCh also increased the translocation of protein kinase C (PKC) to the membrane. ROS production was completely blocked by atropine and a PKC inhibitor, Ro 31-8220. These results show that increased ROS production was a result of muscarinic receptor stimulation, and that PKC had an active role in this cellular stimulation. ROS production upon cellular stimulation by CCh was completely inhibited also by superoxide dismutase, and partially by catalase, indicating that the formation of superoxide anion dominated in cholinergic-induced generation of ROS in human neuroblastoma cells. These results also show that muscarinic stimulation causes sustained ROS production in human neuroblastoma cells. The slow increase in ROS production by CCh suggest a stepwise cascade of events leading to oxidative stress with a triggering role of cholinergic muscarinic receptors in this process.

Biological Transport↗

PubMed2002

Effects of glucocorticoids on generation of reactive oxygen species in platelets.

Since prednisolone and dexamethasone are known as potent anti-inflammatory agents, the effects of prednisolone and dexamethasone on production of intracellular reactive oxygen species (ROS) were investigated in human platelets. Platelet ROS were measured using the intracellular fluorescent dye dichlorofluorescein diacetate after activation of protein kinase C by phorbol-12-myristate-13-acetate (PMA) or 1-oleoyl-2-acetyl-sn-glycerol (OAG). NAD(P)H oxidase activity was measured photometrically. PMA and OAG significantly increased ROS in platelets (P<0.001). Prednisolone or dexamethasone concentration-dependently reduced the PMA-induced ROS production. The PMA-induced ROS increase was significantly reduced in the presence of 10 micromol/l prednisolone to 9+/-1% (n=31; P<0.001) or in the presence of 10 micromol/l dexamethasone to 9+/-1% (n=24; P<0.001). The inhibitory effect of prednisolone or dexamethasone could also be observed in the presence of the glucocorticoid receptor inhibitor, mifepristone (RU486). Administration of testosterone or aldosterone did not significantly reduce PMA-induced ROS increase. Prednisolone had no effect on platelet NAD(P)H oxidase activity. The inhibition of oxidative phosphorylation by sodium azide reduced platelets ROS to 8+/-1% (n=35). It is concluded that glucocorticoids, prednisolone and dexamethasone, directly inhibit production of intracellular ROS. This effect may contribute to the anti-inflammatory actions of these agents.

Blood Platelets↗

PubMed2002

Role of oxidants in male infertility: rationale, significance, and treatment.

Interest in the physiologic and pathologic effects of ROS on male fertility is growing. The controlled generation of very low amounts of ROS seems to regulate the acquisition of sperm-fertilizing ability. High levels of ROS endanger sperm function and viability. Oxidative stress can arise as a consequence of excessive production of ROS or impaired antioxidant defense mechanisms in semen. Oxidative stress precipitates a range of pathologies that are thought to affect the male reproductive system. ROS-mediated peroxidative damage to the sperm plasma membrane may account for the defective sperm functions observed in a high proportion of infertility patients. High levels of ROS in semen have been correlated with reduced sperm motility and damage to sperm nuclear DNA. Determining the levels and the sources of excessive ROS production in human semen and precise evaluation of the scavenger system may be useful tools to develop therapeutic strategies for male infertility. Therapeutic interventions directed toward the isolation of mature spermatozoa by in vitro separation techniques may benefit infertile men in whom oxidative stress has a significant role. Nevertheless, this approach may be limited by the fact that the prolonged centrifugation involved in these procedures may enhance ROS production by spermatozoa. Clinical trials using antioxidants in vivo and in vitro have resulted in a major debate, and further research is required before one can be optimistic about a role for antioxidants in the treatment of infertile men. A double-blind, placebo-controlled study underway at the authors' center is evaluating the effects of treating infertile men with a combination of vitamins E and C after adjusting for all causes of controversy.

Humans↗

PubMed1999

Ceramide prevents motoneuronal cell death through inhibition of oxidative signal.

We previously reported that cell death of rat spinal motoneurons, induced by trophic factor-deprivation, was attenuated by the application of exogenous cell-permeable ceramide (C6-Cer), or bacterial sphingomyelinase (SMase). Recently, motoneuronal cell death was demonstrated to be mediated by the generation of reactive oxygen species (ROS), including superoxide and peroxinitrite. In this study, to investigate the protective mechanism of ceramide (Cer), we examined the effects of Cer and sphingolipid metabolites against ROS generation and oxidative injury in enriched motoneuron cultures. Staining with C-DCDHF-DA, a fluorescent probe for detection of ROS, demonstrated that application of C6-Cer (2.5 mM) or bacterial SMase inhibited the increase of ROS generation. C6-dihydro-Cer, a biologically inactive analogue of C6-Cer, sphingosine, and sphingosine-1-phosphate did not affect ROS generation. This specificity corresponded to the results of cell survival assays. In addition, C6-Cer was shown to specifically inhibit ROS-induced reactions, such as tyrosine nitration and lipid peroxidation, in studies using antibodies against peroxinitrite and 4-hydroxinonenal, respectively. A potent neurotrophin for motoneurons, GDNF, had inhibitory effects against ROS generation and ROS-induced reactions. C6-Cer was also effective in the prevention of cytotoxicity induced by 1-buthionine-sulfoximine, an inhibitor of glutathione synthesis. These observations suggest that Cer plays a protective role in spinal motoneurons through inhibition of oxidative signals.

Aldehydes↗

PubMed1997

BDNF protection of auditory neurons from cisplatin involves changes in intracellular levels of both reactive oxygen species and glutathione.

Previous studies have shown that brain derived neurotrophic factor (BDNF) can protect auditory neurons from cisplatin toxicity in vitro. To explore the mechanism of BDNF mediated neuronal protection sequential confocal microscopic sampling of auditory neurons measured intracellular levels of reactive oxygen species (ROS) in response to withdrawal of BDNF supplementation, cisplatin exposure, and BDNF protection from cisplatin damage in normal and oxidative stress states. Additionally, we examined intraneuronal levels of the free radical scavenger glutathione (GSH) in response to withdrawal of BDNF. Withdrawal of BDNF resulted in increased production of ROS and decreased survival of auditory neurons. Levels of GSH within neurons increased after BDNF withdrawal, and this increase was shown to lag behind the production of ROS. Auditory neurons in cultures supplemented with BDNF and exposed to cisplatin showed significantly lower levels of ROS and increased survival compared to neurons in unprotected, cisplatin exposed cultures. Neurons treated with buthionine sulfoximine (an inhibitor of GSH synthesis), supplemented with BDNF, and exposed to cisplatin showed significantly higher intracellular levels of ROS compared to the neurons in BDNF supplemented cultures exposed to cisplatin. These results suggest that intracellular levels of ROS play an important role in cisplatin induced cell death of auditory neurons and that production of ROS can be ameliorated through supplementation with BDNF. GSH appears to mediate BDNF protection of these neurons from cisplatin induced ROS and subsequent damage.

Animals↗