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PubMed2005

Characterization of the differential expression of uncoupling protein 2 and ROS production in differentiated mouse macrophage-cells (Mm1) and the progenitor cells (M1).

The expression status of mitochondrial uncoupling protein 2 (UCP2) was investigated in undifferentiated mouse myeloid leukemia (M1) and its differentiated macrophage-like cells (Mm1). Mm1 cells have a high ability of phagocytosis along with significantly high levels of reactive oxygen species (ROS) production, UCP2 protein and manganese superoxide dismutase (Mn-SOD), in contrast to undifferentiated leukemia cells (M1). Mm1 cells expressed 10-fold more UCP2 protein compared with undifferentiated M1 cells, although the UCP2 mRNA levels in both cell types were similar. The higher expression of UCP2 in the Mm1 cells suggests a regulatory role of UCP2 in the ROS production. Furthermore, the transfection of UCP2-GFP-expression vector in Mm1 cells dissipated the mitochondrial membrane potential and reduced ROS production, which was shown by their direct visualization using MitoTracker Red CM-H2Xros. The macrophage gp91phox protein, a membrane catalytic component of the NADPH oxidase complex, was at a similar level in both of UCP2-GFP expressed and non-expressed Mm1 cells. These results suggest that the UCP2 protein of the undifferentiated cell is regulated at a quite low level and the higher UCP2 protein of the differentiated macrophages involves with the regulation of ROS production.

Animals↗

PubMed1991

The phagocytosis of ROS by RPE cells is not inhibited by mannose-containing ligands.

We have examined the ability of mannose and the mannose-rich ligands, mannan and mannosylated BSA, to inhibit the phagocytosis of rod outer segments (ROS) by cultured rat retinal pigment epithelial (RPE) cells. Mannose, at concentrations up to 0.25 M, had no effect on either the binding or the ingestion of ROS. At concentrations above 0.25 M, the cells were rounded and showed detachment from the substrate, and phagocytosis was markedly inhibited. Neither mannan (2 mg ml-1), nor mannosylated BSA(0.8 mg ml-1), affected the phagocytosis of ROS. These results suggest that the phagocytosis of ROS is probably not mediated by a mannose receptor on the surface of the RPE cells.

Animals↗

PubMed1996

Regulation of NGF, BDNF and LNGFR gene expression in ROS 17/2.8 cells.

The secosteroid hormone 1.25-dihydroxyvitamin D3 (1,25(OH)2D3) has been recently shown to enhance the synthesis of NGF to mouse L929 fibroblasts. In view of the critical role of 1,25(OH)2D3 on bone metabolism, it has been investigated if ROS 17/2.8 osteoblastic cells were able to express the nerve growth factor (NGF) gene and if this process was responsive to 1,25(OH)2D3. Results indicate that these cells respond in a dose-dependent manner to the presence of 1,25(OH)2D3 by an increase in NGF mRNA levels. However, the phorbol ester PMA, previously reported to augment the synthesis of NGF via the recruitment of AP-1 complexes, depressed the expression of the NGF gene in ROS cells. In contrast, the mRNA levels of an NGF-related trophic factor, brain-derived neurotrophic factor (BDNF), was increased by PMA but not following 1,25(OH)2D3 treatment. Binding of 125I-NGF to ROS cells displayed the properties of a low affinity NGF receptor (dissociation constant Kd approximately 10(-9) M). In agreement with this result, the mRNA encoding the low affinity NGF receptor (LNGFR) was detected in ROS 17/2.8 cells, unlike trkA transcripts which encode the high affinity receptor. These data suggest that neurotrophins and their low affinity receptor could play an unsuspected role in bone tissue.

Animals↗

PubMed2006

Effects of proteolysis and reduction on phosphatase and ROS-generating activity of human tartrate-resistant acid phosphatase.

Osteoclasts and macrophages express high amounts of tartrate-resistant acid phosphatase (TRACP), an enzyme with unknown biological function. TRACP contains a disulfide bond, a protease-sensitive loop peptide, and a redox-active iron that can catalyze formation of reactive oxygen species (ROS). We studied the effects of proteolytic cleavage by trypsin, reduction of the disulfide bond by beta-mercaptoethanol, and reduction of the redox-active iron by ascorbate on the phosphatase and ROS-generating activity of baculovirus-generated recombinant human TRACP. Ascorbate alone and trypsin in combination with beta-mercaptoethanol increased k(cat)/K(m) of the phosphatase activity seven- to ninefold. The pH-optimum was changed from 5.4-5.6 to 6.2-6.4 by ascorbate and trypsin cleavage. Trypsin cleavage increased k(cat)/K(m) of the ROS-generating activity 2.5-fold without affecting the pH-optimum (7.0). These results suggest that the protease-sensitive loop peptide, redox-active iron, and disulfide bond are important regulatory sites in TRACP, and that the phosphatase and ROS-generating activity are performed with different reaction mechanisms.

Acid Phosphatase↗

PubMed2005

High activity of mitochondrial glycerophosphate dehydrogenase and glycerophosphate-dependent ROS production in prostate cancer cell lines.

Most malignant cells are highly glycolytic and produce high levels of reactive oxygen species (ROS) compared to normal cells. Mitochondrial glycerophosphate dehydrogenase (mGPDH) participates in the reoxidation of cytosolic NADH by delivering reducing equivalents from this molecule into the electron transport chain, thus sustaining glycolysis. Here, we investigate the role of mGPDH in maintaining an increased rate of glycolysis and evaluate glycerophosphate-dependent ROS production in prostate cancer cell lines (LNCaP, DU145, PC3, and CL1). Immunoblot, polarographic, and spectrophotometric analyses revealed that mGPDH abundance and activity was significantly elevated in prostate cancer cell lines when compared to the normal prostate epithelial cell line PNT1A. Furthermore, both the glycolytic capacity and glycerophosphate-dependent ROS production was increased 1.68- to 4.44-fold and 5- to 7-fold, respectively, in prostate cancer cell lines when compared to PNT1A cells. Overall, these data demonstrate that mGPDH is involved in maintaining a high rate of glycolysis and is an important site of electron leakage leading to ROS production in prostate cancer cells.

Cell Line, Tumor↗

PubMed2007

Mtf-1 lymphoma-susceptibility locus affects retention of large thymocytes with high ROS levels in mice after gamma-irradiation.

Mouse strains exhibit different susceptibilities to gamma-ray-induced thymic lymphomas. Our previous study identified Mtf-1 (metal responsive transcription factor-1) as a candidate susceptibility gene, which is involved in the radiation-induced signaling pathway that regulates the cellular reactive oxygen species (ROS). To reveal the mechanism for the increased susceptibility conferred by Mtf-1 locus, we examined early effects of gamma-ray on ROS levels in vivo and its difference between Mtf-1 susceptible and resistant congenic mice. Here, we show the detection of clonally growing thymocytes at 4 weeks after irradiation, indicating the start of clonal expansion at a very early stage. We also show that large thymocytes with higher ROS levels and a proliferation capacity were more numerous in the Mtf-1 susceptible mice than the resistant mice when examined at 7 days after irradiation, although such tendency was not found in mice lacking one allele of Bcl11b tumor suppressor gene. This high retention of the large thymocytes, at a high risk for ROS-induced mutation, is a compensatory proliferation and regeneration response to depletion of the thymocytes after irradiation and the response is likely to augment the development of prelymphoma cells leading to thymic lymphomas.

Animals↗

PubMed2006

Platelet regulation by NO/cGMP signaling and NAD(P)H oxidase-generated ROS.

Platelets play a crucial role in the physiology of primary hemostasis and pathophysiological processes such as arterial thrombosis. Accumulating evidence suggests a key regulatory role of both NO and reactive oxygen species (ROS) in platelets. While the inhibitory role of NO/cGMP signaling in both murine and human platelets is well established, recent data suggest that intracellular ROS generation is involved in platelet activation. Thrombin-induced intracellular ROS production was inhibited by NAD(P)H oxidase inhibitors (DPI and apocynin), cyclooxygenase inhibitor (acetylsalicylic acid), and superoxide scavengers (tiron and MnTMPyP). Furthermore, thrombin (Trap6)-induced platelet aggregation and thrombus formation on collagen under high shear was inhibited by NAD(P)H oxidase inhibitors (DPI and apocynin), whereas secretion and platelet shape change were not affected. Inhibition of alphaIIbbeta3 activation by NAD(P)H oxidase inhibitors and superoxide scavengers was independent of NO/cGMP signaling demonstrating a direct role of platelet NAD(P)H oxidase-generated ROS for integrin alphaIIbbeta3 activation.

Animals↗

PubMed2005

Stimulation of glutathione depletion, ROS production and cell cycle arrest of dental pulp cells and gingival epithelial cells by HEMA.

2-Hydroxy-ethyl methacrylate (HEMA) is the major component released from resin-modified glass ionomer cements and dental adhesives. Human tissues mainly affected by HEMA are oral epithelium and dental pulp. We treated human gingival epithelial S-G cells and pulp fibroblasts (HPF) with various concentrations of HEMA, to evaluate its effects on cell growth, cell cycle progression, intracellular glutathione (GSH) level and reactive oxygen species (ROS) production. HEMA-induced growth inhibition in HPF and S-G cells in a dose-dependent manner, which may be partially explained by induction of cell cycle perturbation. G(2)/M phase arrest was noted after exposure of HPF to 5 and 10mm of HEMA, concomitant with glutathione depletion and ROS production. S-phase arrest occurred in S-G cells when treated with 2.5 and 5mm, while at 10mm a sub-G(0)/G(1) peak was noted, indicating the potential induction of apoptosis. GSH depletion was marked in S-G cells only at concentrations of 5 and 10mm, but excessive ROS production was noted at concentration of 1mm and rose with dose increase between 1 and 5mm, then lessened at 10mm. This suggested that the increase of ROS in S-G cells was not mainly caused by GSH depletion. These results helped to define the mechanism of the cytotoxicity caused by HEMA.

Apoptosis↗

PubMed2005

Characterisation of cytosolic FK506 binding protein 12 and its role in modulating expression of Cbfa1 and osterix in ROS 17/2.8 cells.

FK506 is a commonly used immunosuppressant that mediates its action by exclusively interacting with the cytosolic immunophilin, FK506 binding protein 12 (FKBP12). Although FK506-induced acute osteoporosis is now well recognised, its precise mode of action in osteoblasts remains unclear. Therefore, in the present study we characterised FKBP12 in osteoblasts and investigated the role of FK506 in modulating osteoblast-specific transcription factors, core-binding factor alpha1 (Cbfa1) and osterix gene expression in ROS 17/2.8 cells. RT-PCR, immunolocalisation and Western blotting studies were employed to identify and characterise FKBP12 in rat primary osteoblasts and osteoblast-like osteosarcoma ROS 17/2.8 cells. Western blotting extracts of these cells revealed the 12 kDa and hitherto unreported 10 kDa FKBP isoform that were immunolocalised predominantly to the cytosol. The transient exposure of ROS 17/2.8 cells to H2O2 (100 microM) was found to elevate FKBP12 mRNA after 10 min and protein expression after 24 h. Both PTH (10(-9) M) and 1,25 (OH)2D3 (Vitamin D3) (10(-7) M) suppressed FKBP12 protein expression. FK506 in the therapeutic range (25 nmol/L) suppressed expression of Cbfa1 and osterix mRNA. The inhibition of Cbfa1 isoforms II/III expression was evident at 30 min and the extent of inhibition was sustained at 6 h. Osterix inhibition was also seen after 30 min, however, it became maximal after 6 h. The dose-dependant inhibition of osterix in these cells, carried out using 1.25, 12.5 and 125 nmol/L of FK506 was maximal at 1.25 nmol/L. Cbfa1 isoforms II/III were also maximally inhibited at 1.25 nmol/L; interestingly, the inhibition became less marked at higher concentrations of FK506. Similar dose of FK506 was found to inhibit ROS 17/2.8 cell proliferation; the inhibitory effect however was greater in insulin-stimulated cells. The results of this study suggest that immunosuppressant-induced osteoporosis, which is known to involve accelerated bone resorption by increase in osteoclastogenesis, may in fact also be accentuated by the inhibition of osteoblast differentiation and function.

Alkaline Phosphatase↗

PubMed2005

Mitochondrial complex III is required for hypoxia-induced ROS production and cellular oxygen sensing.

Multicellular organisms initiate adaptive responses when oxygen (O(2)) availability decreases, but the underlying mechanism of O(2) sensing remains elusive. We find that functionality of complex III of the mitochondrial electron transport chain (ETC) is required for the hypoxic stabilization of HIF-1 alpha and HIF-2 alpha and that an increase in reactive oxygen species (ROS) links this complex to HIF-alpha stabilization. Using RNAi to suppress expression of the Rieske iron-sulfur protein of complex III, hypoxia-induced HIF-1 alpha stabilization is attenuated, and ROS production, measured using a novel ROS-sensitive FRET probe, is decreased. These results demonstrate that mitochondria function as O(2) sensors and signal hypoxic HIF-1 alpha and HIF-2 alpha stabilization by releasing ROS to the cytosol.

Basic Helix-Loop-Helix Proteins↗

PubMed2003

Application of in vivo ESR spectroscopy to measurement of cerebrovascular ROS generation in stroke.

This study used an in vivo ESR spectroscopy/spin probe technique to measure directly the generation of reactive oxygen species (ROS) in the brain after cerebral ischemia-reperfusion. Transient middle cerebral artery occlusion (MCAO) was induced in rats by inserting a nylon thread into the internal carotid artery for 1 h. The in vivo generation of ROS and its location in the brain were analyzed from the enhanced ESR signal decay data of three intra-arterially injected spin probes with different membrane permeabilities. The ESR signal decay of the probe with intermediate permeability was significantly enhanced 30 min after reperfusion following MCAO, whereas no enhancement was observed with the other probes or in the control group. The enhanced in vivo signal decay was significantly suppressed by superoxide dismutase (SOD). Brain damage was barely discernible until 3 h of reperfusion, and was clearly suppressed with the probe of intermediate permeability. The antioxidant MCI-186 completely suppressed the enhanced in vivo signal decay after transient MCAO. These results clearly demonstrate that ROS are generated at the interface of the cerebrovascular cell membrane when reperfusion follows MCAO in rats, and that the ROS generated during the initial stages of transient MCAO cause brain injury.

Animals↗

PubMed2005

Formation of M1G-dR from endogenous and exogenous ROS-inducing chemicals.

The present study provides fundamental information regarding the production of M1G-dR by ROS. To investigate the production of M1G-dR from deoxyribose damage as caused by ROS, calf thymus DNA (CT-DNA) was incubated with NAD(P)H, CuCl2, and various concentrations of hydrogen peroxide (H2O2). The incubation of CT-DNA with H2O2 resulted in concentration-dependent increases in the number of M1G-dR adducts. In subsequent experiments, 1,4-tetrachlorobenzoquinone or catechol estrogens were evaluated for their effects on M1G-dR formation. In addition, the role of lipid peroxidation in the formation of M1G-dR was verified using an in vitro lipid peroxidation model which consisted of methyl esters of either fish oil or purified fatty acids found in cellular membranes. This experiment confirmed that M1G-dR is a major DNA adduct produced by lipid peroxidation. Furthermore, the number of double bonds in polyunsaturated fatty acids was found to be the key factor in the formation of M1G-dR. The findings obtained from this study provide important information regarding the molecular pathways for M1G-dR formation by ROS, which is an essential element in understanding and evaluating the genotoxicity of a variety of ROS-inducing chemicals.

DNA↗

PubMed2005

Aging sensitizes toward ROS formation and lipid peroxidation in PS1M146L transgenic mice.

Mutations in the presenilins (PS) account for the majority of familial Alzheimer disease (FAD) cases. To test the hypothesis that oxidative stress can underlie the deleterious effects of presenilin mutations, we analyzed lipid peroxidation products (4-hydroxynonenal (HNE) and malondialdehyde) and antioxidant defenses in brain tissue and levels of reactive oxygen species (ROS) in splenic lymphocytes from transgenic mice bearing human PS1 with the M146L mutation (PS1M146L) compared to those from mice transgenic for wild-type human PS1 (PS1wt) and nontransgenic littermate control mice. In brain tissue, HNE levels were increased only in aged (19-22 months) PS1M146L transgenic animals compared to PS1wt mice and not in young (3-4 months) or middle-aged mice (13-15 months). Similarly, in splenic lymphocytes expressing the transgenic PS1 proteins, mitochondrial and cytosolic ROS levels were elevated to 142.1 and 120.5% relative to controls only in cells from aged PS1M146L animals. Additionally, brain tissue HNE levels were positively correlated with mitochondrial ROS levels in splenic lymphocytes, indicating that oxidative stress can be detected in different tissues of PS1 transgenic mice. Antioxidant defenses (activities of antioxidant enzymes Cu/Zn-SOD, GPx, or GR) or susceptibility to in vitro oxidative stimulation was unaltered. In summary, these results demonstrate that the PS1M146L mutation increases mitochondrial ROS formation and oxidative damage in aged mice. Hence, oxidative stress caused by the combined effects of aging and PS1 mutations may be causative for triggering neurodegenerative events in FAD patients.

Aging↗

PubMed2006

ROS-triggered caspase 2 activation and feedback amplification loop in beta-carotene-induced apoptosis.

Reactive oxygen species (ROS) and caspases 8, 9, and 3 are reported to be crucial players in apoptosis induced by various stimuli. Recently, caspase 2 has been implicated in stress-induced apoptosis but the exact mechanism remains unclear. In this study, we report that ROS generation led to activation of caspase 2 during beta-carotene-induced apoptosis in the human leukemic T cell line Molt 4. The apoptosis progressed by simultaneous activation of caspases 8 and 9, and a cross talk between these initiator caspases was mediated by the proapoptotic protein Bid. Inhibition of caspases 2, 8, 9, and 3 independently suppressed the caspase cascade. The kinetics and function of caspase 2 were similar to those of caspase 3, suggesting its role as an effector caspase. Interestingly, beta-carotene-induced apoptosis was caspase 2 dependent but caspase 3 independent. The study also revealed cleavage of the antiapoptotic protein BclXL as an important event during apoptosis, which was regulated by ROS. The mechanistic studies identify a functional link between ROS and the caspase cascade involving caspase 2 and cleavage of BclXL. The interdependence of caspases 8, 9, 2, and 3 in the cascade provides evidence for the presence of an extensive feedback amplification loop in beta-carotene-induced apoptosis in Molt 4 cells.

Apoptosis↗

PubMed2004

Involvement of reactive oxygen species in the bactericidal activity of activated carbon fibre supporting silver; Bactericidal activity of ACF(Ag) mediated by ROS.

An activated carbon fibre supporting silver (ACF(Ag)) was tested for its antibacterial capacity against Escherichia coli (E. coli). Water that has passed through ACF(Ag) demonstrated strong bactericidal ability. This activity decreased over the time suggesting that generated bactericidal species were short lifespan. Since formation of reactive oxygen species (ROS) might be catalysed by silver impregnated and/or ACF itself, implication of ROS and silver was evaluated by the use of ROS scavengers and a silver ions neutralizing agent. The role of ROS in the E. coli mortality was confirmed by the use of a molecular approach which revealed a strong expression of oxidative stress genes.

Escherichia coli↗

PubMed2006

Advanced glycation endproduct induces ROS accumulation, apoptosis, MAP kinase activation and nuclear O-GlcNAcylation in human cardiac myocytes.

Accumulation of advanced glycation endproduct (AGE) has been implicated in the pathogenesis of diabetic complications. However, the precise role and mechanism behind AGE-associated diabetic heart injury are not fully clear. This study was designed to evaluate the effect of AGE on accumulation of reactive oxygen species (ROS), apoptosis, mitogen-activated protein kinase (MAPK) activation and nuclear O-GlcNAcylation in fetal human cardiac myocytes. Myocytes were maintained for 24-72 h in a defined culture medium containing high glucose, the AGE carbon precursor methylglyoxal (MG), and MG-AGE derived from MG and bovine serum albumin (BSA). Generation of ROS was detected by 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate. Apoptosis was evaluated by caspase-3 activity and quantitative DNA fragmentation. Both high glucose (25.5 mM) and MG (200 microM) significantly enhanced ROS and AGE formation with greater effects elicited by MG. Both high glucose and MG-AGE significantly facilitated apoptosis with a more predominant effect from MG-AGE. In addition, phosphorylation of MAPK cascade [extracellular signal-regulated kinase-1/2 (ERK1/2) and p38] and nuclear O-GlcNAcylation were enhanced in MG-AGE-treated myocytes, similar to those elicited by high glucose. MG-AGE-induced phosphorylation of ERK1/2 and p38 was nullified by neutralizing AGE with specific anti-AGE antibody but not nonspecific antiserum. Collectively, these results indicated that AGE or its precursor MG may trigger ROS generation, apoptosis, MAPK activation and nuclear O-GlcNAcylation in human cardiac myocytes, in a manner reminiscent of high extracellular glucose.

Acylation↗

PubMed2005

3-Hydroxy-3-methylglutaryl coenzyme A reductase deregulation and age-related hypercholesterolemia: a new role for ROS.

The microsomal enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR) is the rate-limiting enzyme in cholesterol synthesis and is highly regulated by a variety of factors. We have recently reported increased reductase activity during ageing, attributed to a higher activation state and impaired degradation of the hepatic enzyme. One of the widely recognized causes of age-related metabolic modifications is the large increase of reactive oxygen species (ROS). Therefore, the effect of ROS increase on the activity and the regulation of the HMGCoAR has been investigated in two different experimental models of ROS enriched tissue: liver from rats fed on diets deprived of either Vitamin E (Vit. E) or polyunsaturated fatty acids (Pufa). The results show that in these models, compared to that of old rats, full activation the HMGCoAR was detected while a different degradation rate is observed with the respect to old rats. Thus, our data show full correlation between ROS production and increased HMGCoAR activity. The possible therapeutic implications of these results are discussed.

Aging↗

PubMed2005

Oxygen-induced reperfusion-injury is caused by ROS: Amelioration is possible by recombinant-DNA antioxidant enzymes and mimics in selected tissues.

Billions of years of photolytic cleavage of the water molecule has led to a build-up at sea-level of a "toxic" oxygen concentration in the atmosphere (to 20%). Unfortunately, this dioxygen is likely to be converted in the mitochondria (cell organelles) during cellular respiration to generate reactive oxygen species (ROS); including free-radicals such as superoxide anion (.O2') and hydroxyl radical (.OH) (peroxide O2") a ROS is not a free radical because its electrons are paired). Development of recombinant-DNA improved isoenzyme forms (or mimics) of antioxidant enzymes such as superoxide dismutases is predicted in this hypothesis to be utilised to ameliorate reperfusion-injury (and other oxygen-induced molecular pathology). Introduction into the human genome of the genes for expression of antioxidant enzymes, to order in particular tissues, is imminent. Furthermore, O2 itself will be recognised universally as a harmful gas that can subject the cell to oxidative stress; because it produces ROS, such as the superoxide anion when it acts as the terminal electron acceptor in cellular respiration in the production of water from hydrogen ions in mitochondria. In conclusion, therefore, oxygen-induced injury in humans can no longer be accepted during medical techniques such as reperfusion procedures, because this is associated with reperfusion-injury that can be the cause of several serious medical conditions arising from biomolecular pathology. Some of this is caused by isoforms of cytochromes P450 (CYP; EC 1.14.14.1) such as 3A4, 2D6 and 2C19. These can generate ROS in the liver at low substrate concentrations by futile recycling of oxygen.

Animals↗