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PubMed1984

Influence of 1,25-dihydroxyvitamin D3 on cultured osteogenic sarcoma cells: correlation with the 1,25-dihydroxyvitamin D3 receptor.

We have characterized the effects of the steroid hormone 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on a series of rat osteogenic sarcoma cell lines of increasing osteoblastic-like nature (ROS 24/1, ROS 2/3, and ROS 17/2.8). When these cells were grown in monolayer culture in the presence of 10 nM 1,25-(OH)2D3, there was a dramatic and selective inhibition of proliferation in the ROS 17/2.8 line. Similar concentrations of other vitamin D metabolites did not elicit this effect. Furthermore, the aggregated cuboidal ROS 17/2.8 cells showed a marked change after 6 days of treatment with 10 nM 1,25-(OH)2D3 to an apparently less transformed spindle-like morphology. In contrast, ROS 2/3 displayed only a slight morphological alteration, and ROS 24/1 was unchanged by treatment with 1,25-(OH)2D3. Anchorage-independent growth studies performed in soft agar indicated that 1,25-(OH)2D3 inhibited colony formation to the greatest degree in ROS 17/2.8, with a lesser effect in ROS 2/3. Based upon analyses by sucrose gradient centrifugation, DNA cellulose chromatography, and saturation of specific binding, the level of the 1,25-(OH)2D3 receptor was quantitated in these cells. ROS 17/2.8 cells possess 18,000 copies of the receptor per cell, while ROS 2/3 contains only 500 binding sites per cell, and no detectable high-affinity 1,25-(OH)2D3 receptor is found in ROS 24/1. The receptor in ROS cells is indistinguishable from other mammalian 1,25-(OH)2D3 receptors in that it is a DNA-binding protein that sediments on sucrose gradients at 3.3S, and specifically binds the hormone with high affinity (Kd = 2 to 3 X 10(-11) M). Since the biological responses of these three cell lines to 1,25-(OH)2D3 exhibit a strong correlation with the respective number of receptor molecules per cell, we propose that the actions of this hormone are mediated by the specific 1,25-(OH)2D3 receptor.

Animals↗

PubMed1983

Defective ingestion of rod outer segments by cultured dystrophic rat pigment epithelial cells.

A new procedure for assaying the phagocytosis of rod outer segments (ROS) by cultured rat pigment epithelial (PE) cells has been developed. Using an ROS antiserum and a double immunofluorescent labeling procedure, ROS attached to the external surfaces of these cells can be distinguished from those that have already been ingested. We have used this procedure to study the phagocytosis of ROS by PE cells isolated from normal rats and rats with inherited retinal dystrophy (RCS rats). With this approach we have been able to show that the attachment of ROS to the external surfaces of dystrophic PE cells does take place to a normal extent. However, only a small number of these bound ROS are subsequently ingested, demonstrating that the ingestion phase of phagocytosis is defective. After a 4-hr incubation during which ROS are continuously present, normal rat PE cells ingest about 80% of the ROS that have bound to the cell surfaces. In contrast, after this time period, less than 20% of the ROS bound to the dystrophic PE cells have been ingested. These results, as well as the results of pulse-chase experiments in which ROS are rinsed away after two hours and the incubation continued without further addition of ROS, have demonstrated that normal PE cells rapidly ingest most of the bound ROS, whereas the dystrophic PE cells show no such rapid ingestion. Both cell types, however, are able to slowly ingest additional bound ROS with time.

Animals↗

PubMed1985

Thermotropic behavior of retinal rod membranes and dispersions of extracted phospholipids.

High sensitivity, differential scanning calorimetry studies of bovine retinal rod outer segment (ROS) disk membranes and aqueous dispersions of the extracted ROS phospholipids have been performed. ROS disk membranes were found to exhibit a broad peak of excess heat capacity with a maximum at less than about 3 degrees C, ascribable to a gel-to-liquid crystalline phase transition of a fraction of the phospholipids. A similar thermotropic transition was observed for aqueous dispersions of the total extracted and purified ROS phospholipids. Comparison of the results obtained for the dispersion of total ROS phospholipids to those of the purified head group fractions suggests that the thermotropic behavior reflects a gel-to-liquid crystalline transition, leading to lateral phase separation, involving those phosphatidylcholine (PC) molecules containing saturated fatty acyl chains, possibly together with the highest melting ROS phosphatidylethanolamine (PE) and phosphatidylserine (PS) components. The interpretation of the thermal behavior of the ROS disk membranes depends on whether the transition is assumed to derive from the ROS PC and/or PE/PS fractions, and whether the transbilayer arrangement of the ROS phospholipids is assumed to be symmetric or asymmetric. The calorimetric data can be simply explained in terms of an asymmetric distribution of the major ROS disk membrane phospholipids (G.P. Miljanich et al., J. Membrane Biol. 60:249-255, 1981). In this case, the transition would arise from the PE/PS fractions in the outer ROS disk membrane monolayer, and the anticipated transition from the PC in the inner monolayer would be broadened due to interaction with cholesterol. For the ROS membranes at higher temperatures, two additional, irreversible transitions are observed at 57 and 72 degrees C, corresponding to the thermal denaturation of opsin and rhodopsin, respectively.

Animals↗

PubMed1996

The promotion of iron-induced generation of reactive oxygen species in nerve tissue by aluminum.

Aluminum is suspected to play a role in several neurological disorders. Reactive oxygen species (ROS) lead to oxidative stress, which is thought to be a possible mechanism for neurological damage. Interactions between aluminum and iron, a known promoter of prooxidant events, were studied in cerebral tissues using a fluorescent probe to measure rates of generation of ROS. Al2(SO4)3 alone failed to stimulate ROS production over a wide range of concentrations (50-1000 microM). The aluminum-deferrioxamine chelate in the absence of iron could also not potentiate ROS formation. However, Al2(SO4)3 potentiated FeSO4-induced ROS, with a maximal effect at 10 microM Fe and 500 microM Al. Kaolin, a hydrated aluminum silicate, did not potentiate iron-induced ROS formation. Ferritin had a minor stimulatory effect on ROS generation, but this was not potentiated by the concurrent presence of Al2(SO4)3. Transferrin had no effect on basal rates of ROS generation, but when Al2(SO4)3 was also present, ROS production was enhanced. It is concluded that: 1. There is a potentiation of iron-induced ROS by aluminum salts; 2. Free or complexed aluminum alone is not a key producer of ROS; and 3. High rates of ROS production are unlikely to be owing to the displacement by aluminum iron from its biologically sequestered locations.

Alum Compounds↗

PubMed1990

Phosphoinositide metabolism in frog rod outer segments.

Previous studies have shown that vertebrate rod outer segments (ROS) have a light activated phospholipase C which hydrolyzes phosphatidylinositol-4,5-bisphosphonate (PIP2). Three different experimental approaches have been used to test the hypothesis that the phosphatidylinositol (PI) biosynthetic cycle is present in ROS and that PIP2 can be regenerated from DG independent of rod inner segments. In the first study, enzyme activities of the PI cycle were assayed simultaneously in the presence of CTP, myo-inositol and [gamma-32P]ATP using endogenous lipids as substrates. Under these conditions, broken (leaky) ROS prepared by continuous sucrose gradient centrifugation showed PI, PIP and DG kinase activities similar to those found in intact ROS and non-ROS membranes, whereas PI synthetase activity was much lower in the leaky ROS than in the other two fractions. The relative distribution of PI synthetase specific activity in the three membrane preparations was similar to that of the microsomal enzyme marker cytochrome c reductase. ROS prepared by discontinuous sucrose gradient centrifugation showed only 2-3% of whole homogenate PI synthetase or phosphatidyl: cytidyl transferase activities, and the distribution of activities was the same as for microsomal and mitochondrial marker enzymes. In the second study, whole retinas were incubated with myo-[2-3H]inositol or [2-3H]glycerol in vitro, and the time course of incorporation of radioactivity into PI and other phospholipids was determined for ROS and three other retinal fractions. Over a 10-hr period, the rate of incorporation of myo-[2-3H]inositol or [2-3H]glycerol into PI in ROS was lowest among the various retinal fractions. In the third study, chemical analysis of the molecular species composition of PI, DG and phosphatidic acid (PA) from ROS shows that PA is substantially different from PI and DG, the latter two being quite similar. These results are consistent with a precursor-product relationship between PI and DG, but not with the conversion of DG to PA or of PA to PI. Taken together, these three studies indicate that ROS do not have PI synthetase or phosphatidyl: cytidyl transferase activities, but do have DG, PI and PIP kinase activities. Thus, the PI in ROS lost through rapid turnover must be replaced with molecules derived from de novo synthesis in the inner segment of the photoreceptor cell.

Animals↗

PubMed2006

Arsenite and monomethylarsonous acid generate oxidative stress response in human bladder cell culture.

Arsenicals have commonly been seen to induce reactive oxygen species (ROS) which can lead to DNA damage and oxidative stress. At low levels, arsenicals still induce the formation of ROS, leading to DNA damage and protein alterations. UROtsa cells, an immortalized human urothelial cell line, were used to study the effects of arsenicals on the human bladder, a site of arsenical bioconcentration and carcinogenesis. Biotransformation of As(III) by UROtsa cells has been shown to produce methylated species, namely monomethylarsonous acid [MMA(III)], which has been shown to be 20 times more cytotoxic. Confocal fluorescence images of UROtsa cells treated with arsenicals and the ROS sensing probe, DCFDA, showed an increase of intracellular ROS within five min after 1 microM and 10 microM As(III) treatments. In contrast, 50 and 500 nM MMA(III) required pretreatment for 30 min before inducing ROS. The increase in ROS was ameliorated by preincubation with either SOD or catalase. An interesting aspect of these ROS detection studies is the noticeable difference between concentrations of As(III) and MMA(III) used, further supporting the increased cytotoxicity of MMA(III), as well as the increased amount of time required for MMA(III) to cause oxidative stress. These arsenical-induced ROS produced oxidative DNA damage as evidenced by an increase in 8-hydroxyl-2'-deoxyguanosine (8-oxo-dG) with either 50 nM or 5 microM MMA(III) exposure. These findings provide support that MMA(III) cause a genotoxic response upon generation of ROS. Both As(III) and MMA(III) were also able to induce Hsp70 and MT protein levels above control, showing that the cells recognize the ROS and respond. As(III) rapidly induces the formation of ROS, possibly through it oxidation to As(V) and further metabolism to MMA(III)/(V). These studies provide evidence for a different mechanism of MMA(III) toxicity, one that MMA(III) first interacts with cellular components before an ROS response is generated, taking longer to produce the effect, but with more substantial harm to the cell.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2005

Effect of roscovitine on nuclear maturation, MPF and MAP kinase activity and embryo development of prepubertal goat oocytes.

The low number of embryos obtained from IVM-IVF-IVC of prepubertal goat oocytes could be due to an incomplete cytoplasmic maturation. Roscovitine (ROS) inhibits MPF and MAP kinase activity and maintains the oocyte at Germinal Vesicle (GV) stage. The aim of this study was to determine if meiotic activity is arrested in prepubertal goat oocytes cultured with 0, 12.5, 25, 50 and 100 microM of ROS for 24 h. A group of oocytes from adult goats was cultured with 25 microM of ROS to compare the effect of ROS on prepubertal and adult goat oocytes. A sample of oocytes was stained to evaluate the nuclear stage at oocyte collection time and after ROS incubation. IVM-oocytes not exposed to ROS formed the control group. Prepubertal goat IVM-oocytes were inseminated and cultured for 8 days. The percentage of oocytes at GV stage, after exposition to ROS was significantly higher in adult goat oocytes (64.5%) than in prepubertal goat oocytes. No differences were found among 25, 50 and 100 microM ROS concentrations (29, 23 and 26%, oocytes at GV stage, respectively). After 8 days of culture, no differences in total embryos were observed between control oocytes and oocytes treated with 12.5 and 25 microM (45.2, 36.1 and 39.4%, respectively), however the percentage of blastocysts was higher in the control group. Western blot for the MAPK and p34(cdc2) showed that both enzymes were active in prepubertal goat oocytes after 24h of ROS exposition. In conclusion, a low percentage of prepubertal goat oocytes reached GV stage after ROS incubation; possibly because most of them had reinitiated the meiosis inside the follicle. ROS did not affect fertilization or total embryos but ROS showed a negative effect on blastocyst development.

Animals↗

PubMed1999

Reactive oxygen species modulate endothelin-I-induced c-fos gene expression in cardiomyocytes.

OBJECTIVES: Recent evidence indicates that reactive oxygen species (ROS) may act as second messengers in receptor-mediated signaling pathways. The possible role of ROS during Et-1 stimulation in cardiomyocytes was therefore investigated. METHODS: Intracellular ROS levels were measured with fluorescence probe 2',7'-dichlorofluorescin diacetate by confocal microscopy in cultured neonatal rat cardiomyocytes. The ROS-inducible c-fos expression was analyzed by Northern blotting and promoter activity. RESULTS: Et-1 applied to cardiomyocytes dose-dependently increased intracellular ROS levels. The increase of ROS levels was attenuated by pretreating cardiomyocytes with Et-A receptor antagonist-BQ485, but not with Et-B receptor antagonist. Cardiomyocytes pretreated with catalase or an antioxidant N-acetylcysteine (NAC) reduced Et-1-induced ROS levels. Et-1 or H2O2 treatment of cardiomyocytes rapidly induced the expression of an immediate early gene c-fos. Et-1-treated cardiomyocytes enhanced the c-fos gene expression as revealed by functional analysis using a reporter gene construct containing c-fos promoter region (-2.25 kb) and reporter gene chloramphenicol acetyltransferase. The induction of mRNA levels and the promoter activities of c-fos gene by Et-1 or H2O2 were abolished by pretreating cardiomyocytes with catalase or NAC. Cells transiently transfected with the dominant positive mutant of p21ras (RasL61) led to a significant increase in intracellular ROS. Concomitantly, the mRNA levels and the promoter activities of c-fos were also induced. In contrast, cells transfected with the dominant negative mutant of Ras (RasN17) inhibited Et-1-induced ROS. Consistently, the increase of c-fos mRNA levels and promoter activities by Et-1 were also inhibited. CONCLUSIONS: These findings clearly indicate that Et-1 treatment to cardiomyocytes can induce ROS via Ras pathway and the increased ROS are involved in the increase of c-fos expression. Our studies thus emphasize the importance of ROS as second messengers in Et-1-induced responses on cardiomyocytes.

Animals↗

PubMed2000

Reactive oxygen species and vascular cell apoptosis in response to angiotensin II and pro-atherosclerotic factors.

Reactive oxygen species (ROS) are known to induce apoptotic cell death in various cell types. In the vessel wall, ROS can be formed by macrophages within the atherosclerotic plaque or can act on the endothelium after adhesion of monocytes or leucocytes. Moreover, ROS are endogenously synthesized by endothelial and vascular smooth muscle cells by NAD(P)H oxidase. Enhanced ROS production is a very early hallmark in the atherogenic process, suggesting a link between ROS and apoptosis. In endothelial cells, the endogenous generation of ROS is induced by different pro-inflammatory and pro-atherosclerotic factors such as Ang II, oxLDL or TNFalpha, which all promote the execution of programmed cell death. ROS synthesis is thereby causally involved in apoptosis induction, because antioxidants prevent endothelial cell death. The pro-apoptotic effects of endogenous ROS in endothelial cells mechanistically seems to involve the disturbance of mitochondrial membrane permeability followed by cytochrome c release, which finally activates the executioner caspases. In contrast to the pro-apoptotic capacity of ROS in endothelial cells, in vascular smooth muscle cells emerging evidence suggests that endogenous ROS synthesis promotes cell proliferation and hypertrophy and does not affect cell survival. However, high concentrations of exogenous ROS can also stimulate smooth muscle cell apoptosis as shown for other cell types probably via activation of p53. Taken together, the double-edged effects of endogenously derived ROS in endothelial cells versus VSMC may provide a mechanistic clue to the anti-atherosclerotic effects of antioxidants shown in experimental studies, given that the promotion of endothelial survival in combination with inhibition of VSMC proliferation blocks two very important steps in the pathogenesis of atherosclerosis.

Angiotensin II↗

PubMed2001

Reactive oxygen species involved in trichosanthin-induced apoptosis of human choriocarcinoma cells.

The type-I ribosome-inactivating protein trichosanthin (TCS) has a broad spectrum of biological and pharmacological activities, including abortifacient, anti-tumour and anti-HIV activities. We have found for the first time that TCS stimulated the production of reactive oxygen species (ROS) in JAR cells (a human choriocarcinoma cell line) in a time- and concentration-dependent manner by using the fluorescent probe 2',7'-dichlorofluorescein diacetate with confocal laser scanning microscopy. ESR spectral studies and the inhibition of ROS formation by the superoxide radical anion (O(2)(-.)) scavenger superoxide dismutase, the H(2)O(2) scavenger catalase and the hydroxyl radical (OH(.)) scavenger mannitol suggested the involvement of O(2)(-.), H(2)O(2) and OH(.). TCS-induced ROS formation was shown to be dependent on the presence of both extracellular and intracellular Ca(2+); moreover, ROS production paralleled the intracellular Ca(2+) elevation induced by TCS, suggesting that ROS production might be a consequence of Ca(2+) signalling. TCS-induced activation of caspase-3 was initiated within 2 h; however, TCS-induced production of ROS was initiated within 5 min, suggesting that the production of ROS preceded the activation of caspase-3. Simultaneous observation of the nuclear morphological changes via two-photon laser scanning microscopy and ROS production via confocal laser scanning microscopy revealed that ROS is involved in the apoptosis of JAR cells. The involvement of ROS was also confirmed by the inhibition of TCS-induced cell death by the antioxidant Trolox and the ROS scavengers catalase and mannitol. Diethylenetriaminepenta-acetic acid, an inhibitor of metal-facilitated OH(.) formation, markedly inhibited TCS-induced cell death, suggesting that TCS induced OH(.) formation via the Fenton reaction. The finding that ROS is involved in the TCS-induced apoptosis of JAR cells might provide new insight into the anti-tumour and anti-HIV mechanism of TCS.

Antineoplastic Agents, Phytogenic↗

PubMed1999

Reactive oxygen species influence the acrosome reaction but not acrosin activity in human spermatozoa.

It is now widely accepted that the higher levels of reactive oxygen species (ROS) produced by damaged or deficient spermatozoa are associated with a loss of motility and a decreased capacity for sperm-oocyte fusion. Furthermore, earlier studies show, under physiological conditions, that some ROS may be involved in capacitation and hyperactivation of human spermatozoa. We measured ROS levels, acrosome reaction (AR) and acrosin activity (AA) in semen samples from suspected subfertile men to reveal the influence of ROS on AR and AA of human spermatozoa. Semen samples were obtained from 60 patients. Samples with > or = 1 x 10(6) leukocytes/mL were excluded from the study. ROS production was determined using a chemiluminescence technique. AR was determined using a triple stain technique. The percentage of acrosome-reacted spermatozoa after low temperature induction of the AR (test value), and the inducibility of AR (= the difference between the test value and the control), were calculated. The AA was analysed by determining the proteolytic potential of spermatozoa on gelatin plates. The mean halo diameter and percentage of halo formation in each sample were measured as AA parameters. Scatter plots of ROS levels and AR parameters showed that the percentage of acrosome reacted spermatozoa and AR inducibility were better in samples with low rather than high ROS levels. On the other hand, there were no apparent similarities between ROS and the AA parameters. Therefore, the percentage of acrosome-reacted spermatozoa and AR inducibility were significantly higher in the low than in the high ROS group (p = 0.028, p = 0.0001, respectively). In addition, there was no significant difference in AA parameters between groups. These findings suggest that lower ROS in semen may have a role in AR but excessive ROS may exert a negative influence on AR, while ROS in semen has no relationship to AA.

Acrosin↗

PubMed2001

The benefits and hazards of antioxidants: controlling apoptosis and other protective mechanisms in cancer patients and the human population.

Cellular oxidants, called reactive oxygen species (ROS), are constantly produced in animal and human cells. Excessive ROS can induce oxidative damage in cell constituents and promote a number of degenerative diseases and aging. Cellular antioxidants protect against the damaging effects of ROS. However, in moderate concentrations, ROS are necessary for a number of protective reactions. Thus, ROS are essential mediators of antimicrobial phagocytosis, detoxification reactions carried out by the cytochrome P-450 complex, and apoptosis which eliminates cancerous and other life-threatening cells. Excessive antioxidants could dangerously interfere with these protective functions, while temporary depletion of antioxidants can enhance anti-cancer effects of apoptosis. Experimental data are presented supporting these notions. The human population is heterogeneous regarding ROS levels. Intake of exogenous antioxidants (vitamins E, C, beta-carotene and others) could protect against cancer and other degenerative diseases in people with innate or acquired high levels of ROS. However, abundant antioxidants might suppress these protective functions, particularly in people with a low innate baseline level of ROS. Screening human populations for ROS levels could help identify groups with a high level of ROS that are at a risk of developing cancer and other degenerative diseases. It also could identify groups with a low level of ROS that are at a risk of down-regulating ROS-dependent anti-cancer and other protective reactions. Screening populations could provide a scientifically grounded application of antioxidant supplements, which could significantly contribute to the nation's health.

Antineoplastic Agents↗

PubMed2002

Increased free radical production in hypertension due to increased expression of the NADPH oxidase subunit p22(phox) in lymphoblast cell lines.

OBJECTIVES: To confirm increased production of reactive oxygen species (ROS) in hypertension, to demonstrate the source of ROS and to analyse NADPH oxidase subcomponent expression in hypertension. DESIGN: A lymphoblast model was used, as this has previously been used in the study of hypertension and of NADPH oxidase. Chemiluminescence (CL) was chosen to assay ROS production, as it is simple and sensitive. METHODS: Lymphocytes from 12 hypertensive patients (HT), and 12 age- and sex-matched normotensive (NT) subjects, were immortalized. Luminol, isoluminol and Cypridina luciferin analogue (CLA) CL were used to assay ROS production. NADPH oxidase subunits were measured by Western blot analysis. RESULTS: Stimulation with 50 micromol/l arachidonic acid (AA) resulted in increased ROS production in HT cell lines with luminol, CLA and isoluminol CL. Stimulation with 500 nmol/l 12-O-tetradecanoylphorbol-13-acetate (TPA) produced a detectable increase in HT ROS production with luminol and with CLA, whereas there was no significant difference with isoluminol. The ROS production was abolished by diphenyleneiodonium chloride (DPI) but not by rotenone, indicating that a non-mitochondrial flavoprotein such as NADPH oxidase is the source of ROS. Analysis of NADPH oxidase subcomponents revealed an increase in p22(phox) in HT subjects. CONCLUSIONS: We have shown there is increased ROS production in lymphoblasts derived from hypertensive subjects, probably originating from NADPH oxidase. As the ROS production persists in transformed cells, this suggests a genetic predisposition to increased ROS production. Increased expression of p22(phox) in HT lymphoblasts may account for some of the increased ROS.

Arachidonic Acid↗

PubMed1997

Two Distinct Sources of Elicited Reactive Oxygen Species in Tobacco Epidermal Cells.

Reactive oxygen species (ROS) play a prominent role in early and later stages of the plant pathogenesis response, putatively acting as both cellular signaling molecules and direct antipathogen agents. A single-cell assay, based on the fluorescent probe dichlorofluorescein, was used to scrutinize the generation and movement of ROS in tobacco epidermal tissue. ROS, generated within cells, quickly moved apoplastically as H2O2 into neighboring cells. Two classes of rapidly elicited intracellular ROS, originating from distinct sources, were distinguished. Cryptogein, the fungal elicitor from Phytophthora cryptogea, induced ROS from a flavin-containing oxidase source. ROS accumulation could be inhibited by a number of pharmacological agents, suggesting induction through an active signal transduction pathway. The insensitivity of the increase in ROS to the external addition of enzymes that dissipate ROS suggests that this oxidative increase is primarily intracellular. In contrast, amines and polyamines, compounds that form during wounding and pathogenesis, induced ROS at an apoplastic site from peroxidase- or amine oxidase-type enzyme(s). Salicylic acid, a putative inhibitor of cellular catalases and peroxidases, did not induce cellular ROS, as measured by dichlorofluorescein fluorescence. The physiological relevance of ROS-generated signals was indicated by the rapid alteration of the epidermal cell glutathione pool and the cellular redox state. In addition, induction of ROS by all elicitors was correlated with subsequent cell death.

Journal Article↗

PubMed2005

In high glucose protein kinase C-zeta activation is required for mesangial cell generation of reactive oxygen species.

BACKGROUND: We postulated that in mesangial cells exposed to high glucose, protein kinase C-zeta (PKC-zeta) is necessary for the generation of reactive oxygen species (ROS) by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and that the requirement of PKC-zeta for filamentous (F)-actin disassembly may involve ROS. To identify signaling mechanisms relevant to PKC-zeta activation and ROS generation, including phosphoinositide 3 kinase (PI3 kinase), we examined mesangial cell stimulation with platelet-derived growth factor (PDGF). METHODS: In primary rat mesangial cells cultured in 5.6 mmol/L or 30 mmol/L d-glucose, PKC-zeta expression was identified with immunoblotting and activity was analyzed in cell membrane immunoprecipitates and by confocal immunofluorescence imaging. ROS generation was measured by dichlorofluorescein fluorescence using confocal microscopy and was inhibited by transfection of antisense against NADPH subunits p22(phox) or p47(phox) or with Tempol. F-actin disassembly was observed by dual-channel confocal fluorescence imaging. PI3 kinase activity was detected by immunoblotting of phosphorylated Akt. RESULTS: In high glucose, generation of NADPH oxidase-dependent ROS was dependent on PKC-zeta. Conversely, sustained PKC-zeta activity was dependent on ROS generation, suggesting a positive feedback. PKC-zeta-dependent F-actin disassembly in high glucose required ROS generation. PDGF stimulated NADPH oxidase generation of ROS through a PKC-zeta mechanism that was independent of Akt phosphorylation and remained unchanged in high glucose. CONCLUSION: In high glucose, mesangial cell PKC-zeta is required for ROS generation from NADPH oxidase similar to PDGF stimulation of PKC-zeta-dependent ROS generation through a pathway independent of PI3 kinase. F-actin disassembly in high glucose also requires ROS. A positive feedback loop occurs between ROS and the activation of PKC-zeta in high glucose.

Actins↗

PubMed2002

Reactive oxygen species in cell responses to toxic agents.

This review first summarizes experimental data on biological effects of different concentrations of ROS in mammalian cells and on their potential role in modifying cell responses to toxic agents. It then attempts to link the role of steadily produced metabolic ROS at various concentrations in mammalian cells to that of environmentally derived ROS bursts from exposure to ionizing radiation. The ROS from both sources are known to both cause biological damage and change cellular signaling, depending on their concentration at a given time. At low concentrations signaling effects of ROS appear to protect cellular survival and dominate over damage, and the reverse occurs at high ROS concentrations. Background radiation generates suprabasal ROS bursts along charged particle tracks several times a year in each nanogram of tissue, i.e., average mass of a mammalian cell. For instance, a burst of about 200 ROS occurs within less than a microsecond from low-LET irradiation such as X-rays along the track of a Compton electron (about 6 keV, ranging about 1 microm). One such track per nanogram tissue gives about 1 mGy to this mass. The number of instantaneous ROS per burst along the track of a 4-meV alpha-particle in 1 ng tissue reaches some 70000. The sizes, types and sites of these bursts, and the time intervals between them directly in and around cells appear essential for understanding low-dose and low dose-rate effects on top of effects from endogenous ROS. At background and low-dose radiation exposure, a major role of ROS bursts along particle tracks focuses on ROS-induced apoptosis of damage-carrying cells, and also on prevention and removal of DNA damage from endogenous sources by way of temporarily protective, i.e., adaptive, cellular responses. A conclusion is to consider low-dose radiation exposure as a provider of physiological mechanisms for tissue homoeostasis.

Antioxidants↗

PubMedDate not supplied

Effects of time and sperm concentration on reactive oxygen species formation in human semen.

Although generally accepted standards exist for routine semen analysis, recent methods of assessing reactive oxygen species (ROS) in human semen lack a standardized protocol. The purpose of this study was to investigate (1) the relationship between ROS level and the time interval between semen collection and analysis, and (2) the effect of sperm concentration on the level of ROS formation. Semen specimens from men (n = 40) consulting for infertility treatment were divided in two groups: in 20, routine semen analysis was performed and ROS formation evaluated at 1, 3, 5, and 24 h after semen collection; in the other 20, ROS formation was evaluated at four sperm concentrations (60, 30, 15, and 7.5 x 10(6)/mL). White blood cell (WBC) concentration was assessed before ROS measurement using a myeloperoxidase staining technique (Endtz test). ROS level was measured by a chemiluminescence method. ROS formation decreased significantly over time. The mean ROS level 343.4 (1 h), 133.5 x 10(4) cpm (3 h, p = .004), 66.0 x 10(4) cpm (5 h, p < or = .001), and 22.2 x 10(4) cpm (24 h, p < or = .001), respectively. In the first group of 20 specimens, 14 were positive for ROS formation at 1 h after collection, and 4 of these were positive for the Endtz test (> 1 x 10(6) WBC/mL). The number of ROS-positive specimens after 3, 5, and 24 h was eight, six, and two, respectively. In the second group, eight patients were positive for ROS formation at 1 h after collection.(ABSTRACT TRUNCATED AT 250 WORDS)

Humans↗

PubMed2006

Impaired generation of reactive oxygen species during differentiation of dendritic cells (DCs) by Mycobacterium tuberculosis secretory antigen (MTSA) and subsequent activation of MTSA-DCs by mycobacteria results in increased intracellular survival.

We investigated the role of reactive oxygen species (ROS) in dendritic cell (DC) differentiation by 10-kDa Mycobacterium tuberculosis secretory Ag (MTSA) and survival of mycobacteria therein. Compared with GM-CSF, MTSA induced lower ROS production during DC differentiation from precursors. This result correlated with higher superoxide dismutase 1 expression in MTSA stimulated precursors as compared with GM-CSF stimulation. Furthermore, a negative regulation of protein kinase C (PKC) activation by ROS was observed during DC differentiation. ROS inhibited the rapid and increased phosphorylation of PKCalpha observed during DC differentiation by MTSA. In contrast, ROS inhibition increased the weak and delayed PKCalpha phosphorylation by GM-CSF. Similar to DC differentiation, upon activation with either M. tuberculosis cell extract (CE) or live Mycobacterium bovis bacillus Calmette-Guérin (BCG), DCs differentiated with MTSA (MTSA-DCs) generated lower ROS levels when compared with DCs differentiated with GM-CSF (GM-CSF-DCs). Likewise, a negative regulation of PKCalpha phosphorylation by ROS was once again observed in DCs activated with either M. tuberculosis CE or live M. bovis BCG. However, a reciprocal positive regulation between ROS and calcium was observed. Compared with MTSA-DCs, stimulation of GM-CSF-DCs with M. tuberculosis CE induced a 2-fold higher ROS-dependent calcium influx. However, pretreatment of MTSA-DCs with H(2)O(2) increased calcium mobilization. Finally, lower ROS levels in MTSA-DCs correlated with increased intracellular survival of M. bovis BCG when compared with survival in GM-CSF-DCs. Although inhibiting ROS in GM-CSF-DCs increased M. bovis BCG survival, H(2)O(2) treatment of MTSA-DCs decreased survival of M. bovis BCG. Overall our results suggest that DCs differentiated with Ags such as MTSA may provide a niche for survival and/or growth of mycobacteria following sequestration of ROS.

Animals↗