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PubMed1992

Release of reactive oxygen by hepatocytes on reoxygenation: three phases and role of mitochondria.

Reoxygenation of isolated hepatocytes in primary culture resulted in a three-phase response in the release of reactive oxygen species (ROS) as determined by peroxidase-dependent luminol chemiluminescence. Release of ROS within the first and second phase correlated well with the extent of reoxygenation injury, both being most significant after approximately 4 h of hypoxic incubation. During the third phase, some of the ROS were released by already nonviable cells. Both antimycin A and rotenone significantly increased release of ROS, indicating severe alterations of the mitochondrial respiratory chain caused by hypoxia and suggesting that the altered mitochondrial respiratory chain represents an important source for the release of ROS on reoxygenation. Generation of ROS rose sharply when the O2 content was increased from 0 to 2%, whereas a further increase in the O2 content, of up to 95%, resulted in only small but steady increases in the formation of ROS. The latter suggests that, in addition to enzymatic sources such as the mitochondrial respiratory chain, nonenzymatic reactions may also contribute to the formation of ROS on reoxygenation.

Animals↗

PubMed2003

Microvascular oxygenation, oxidative stress, NO suppression and superoxide dismutase during postischemic reperfusion.

Increased formation of reactive oxygen species (ROS) on reperfusion after ischemia underlies ischemia-reperfusion (I/R) damage. We measured, in real time, oxygen tension in both microvessels and tissue and oxidant stress during postischemic reperfusion in the hamster cheek pouch microcirculation. We measured Po2 by using phosphorescence quenching microscopy and ROS production in the systemic blood. We evaluated the effects of a nitric oxide synthase inhibitor (NG-monomethyl-L-arginine, L-NMMA) and SOD on the oxidative stress during reperfusion. Microvascular injury was assessed by measuring diameter change, the perfused capillary length (PCL), and leukocyte adhesion. During early reperfusion, arteriolar Po2 was significantly lower than baseline, whereas capillary Po2 varied between 7 and 0 mmHg. Arterial blood flow did not regain baseline values, whereas Po2 returned to baseline in arterioles and tissue after 30 min of reperfusion. During 5 and 15 min of reperfusion, ROS increased by 72 and 89% versus baseline, respectively, and declined to baseline after 30 min of reperfusion. Pretreatment with SOD maintained ROS at normal levels, increased arteriolar diameter, blood flow, and PCL, and decreased leukocyte adhesion (P < 0.05). L-NMMA decreased ROS only within 5 min of reperfusion, which increased significantly by 72% later during reperfusion. L-NMMA worsened leukocyte adhesion (P < 0.05). In conclusion, our results show that the early reperfusion is characterized by low Po2 linked to increased production of ROS. At early reperfusion both SOD and L-NMMA decreased ROS production, whereas only SOD reduced it during later reperfusion. We suggest that low-flow hypoxia profoundly affects vascular endothelial damage during reperfusion through changes in ROS and nitric oxide production.

Animals↗

PubMed2004

Acetylcholine and bradykinin trigger preconditioning in the heart through a pathway that includes Akt and NOS.

In the rabbit heart, bradykinin and ACh trigger preconditioning by a mechanism involving ATP-sensitive potassium channel-dependent production of reactive oxygen species (ROS). Recent evidence indicates that the pathway by which bradykinin causes ROS generation includes nitric oxide synthase (NOS) and protein kinase G (PKG). On the other hand, Akt was shown to be essential for ACh to generate ROS. This study determines whether these two G-coupled receptor agonists indeed have similar signaling targets, i.e., whether Akt is involved in bradykinin's pathway and whether NOS is involved in ACh's pathway. Isolated adult rabbit cardiomyocytes were incubated for 15 min in reduced MitoTracker red, which becomes fluorescent only after exposure to ROS. Bradykinin (400 nM) and ACh (250 microM) caused a 51.4 +/- 14.8% and 39.8 +/- 11.7% increase, respectively, in ROS production (P <0.005). Coincubation of either agonist with Akt inhibitor (20 microM) or infection of cells with an adenovirus containing dominant negative Akt abolished this increase. The NO donor S-nitroso-N-acetyl penicillamine (SNAP, 1 microM) also increased the ROS signal by 40.8 +/- 15.7%, but this increase was unaffected by Akt inhibitor (39.0 +/- 6.4%), implying that Akt is upstream of NOS. ACh-induced ROS production could be abolished by either of the NOS inhibitors Nomega-monomethyl-L-arginine monoacetate (100 microM) and L-N5-(1-iminoethyl)ornithine hydrochloride (L-NIO, 5 microM). L-NIO also blocked the anti-infarct effect of ACh (550 microM) in isolated rabbit hearts exposed to 30 min of regional ischemia. We conclude that both bradykinin and ACh trigger ROS generation by sequentially activating Akt and NOS.

Acetylcholine↗

PubMed2004

Mitochondrial-derived free radicals mediate asbestos-induced alveolar epithelial cell apoptosis.

Asbestos causes pulmonary toxicity by mechanisms that in part involve reactive oxygen species (ROS). However, the precise source of ROS is unclear. We showed that asbestos induces alveolar epithelial cell (AEC) apoptosis by a mitochondrial-regulated death pathway. To determine whether mitochondrial-derived ROS are necessary for causing asbestos-induced AEC apoptosis, we utilized A549-rho(omicron) cells that lack mitochondrial DNA and a functional electron transport. As expected, antimycin, which induces an oxidative stress by blocking mitochondrial electron transport at complex III, increased dichlorofluoroscein (DCF) fluorescence in A549 cells but not in A549-rho(omicron) cells. Compared with A549 cells, rho(omicron) cells have less asbestos-induced ROS production, as assessed by DCF fluorescence, and reductions in total glutathione levels as well as less caspase-9 activation and apoptosis, as assessed by TdT-mediated dUTP nick end labeling staining and DNA fragmentation. A mitochondrial anion channel inhibitor that prevents ROS release from the mitochondria to the cytoplasm also blocked asbestos-induced A549 cell caspase-9 activation and apoptosis. Finally, a role for nonmitochondrial-derived ROS with exposure to high levels of asbestos (50 microg/cm(2)) was suggested by our findings that an iron chelator (phytic acid or deferoxamine) or a free radical scavenger (sodium benzoate) provided additional protection against asbestos-induced caspase-9 activation and DNA fragmentation in rho(omicron) cells. We conclude that asbestos fibers affect mitochondrial DNA and functional electron transport, resulting in mitochondrial-derived ROS production that in turn mediates AEC apoptosis. Nonmitochondrial-associated ROS may also contribute to AEC apoptosis, particularly with high levels of asbestos exposure.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2004

Important role for Rac1 in regulating reactive oxygen species generation and pulmonary arterial smooth muscle cell growth.

Vascular NADPH oxidases have been shown to be a major source of reactive oxygen species (ROS). Recent studies have also implicated ROS in the proliferation of vascular smooth muscle cells. However, the components required for activation of the NADPH oxidase complex have not been clearly elucidated. Here we demonstrate that ROS generation in ovine pulmonary arterial smooth muscle cells (PASMCs) requires the activation of Rac1, implicating this protein as an important subunit of the NADPH oxidase complex. Our results, using a geranylgeranyl transferase inhibitor (GGTI-287), demonstrated a dose-dependent inhibition of Rac1 activity and ROS production. This was associated with an inhibition of PASMC proliferation with an arrest at G(2)/M. The inhibition of Rac1 by GGTI-287 led us to more specifically target Rac1 to investigate its role in the generation of ROS and cellular proliferation. To accomplish this, we utilized a dominant negative Rac1 (N17Rac1) and a constitutively active Rac1 (V12Rac1). These two forms of Rac1 were transiently expressed in PASMCs using adenovirus-mediated gene transfer. N17Rac1 expression resulted in decreased cellular Rac1 activity, whereas V12Rac1 infection showed increased activity. Compared with controls, the V12Rac1-expressing cells had higher levels of ROS production and increased proliferation, whereas the N17Rac1-expressing cells had decreased ROS generation and proliferation and cell cycle arrest at G(2)/M. However, the inhibition of cell growth produced by N17Rac1 overexpression could be overcome if cells were co-incubated with the Cu,Zn superoxide dismutase inhibitor DETC. These results indicate the importance of Rac1 in ROS generation and proliferation of vascular smooth muscle cells.

Animals↗

PubMed1986

Reactive oxygen species: production and role in the kidney.

Reactive oxygen species (ROS) are formed by incomplete reduction of molecular oxygen. They include superoxide anion (O2-.), hydrogen peroxide (H2O2), hydroxyl radical (OH.), and singlet oxygen (1O2). ROS may induce different types of cell injury, particularly lipid peroxidation and membrane damage. ROS have been shown to play an essential role in the mechanisms of experimental models of several renal diseases: ischemic acute renal failure, renal graft rejection, acute glomerulonephritis, and toxic renal diseases. They are produced by the renal cells and also by the inflammatory bone marrow-derived cells invading the renal tissue. ROS, regardless of their origin, may degrade the glomerular basement membrane and alter the glomerular and tubular cell functions. Particularly, they produce an increase in cyclic AMP synthesis and prostaglandin production in the glomeruli. Recent studies have shown that the glomerular mesangial cells themselves generated ROS on stimulation by phagocytosis of foreign particles or exposure to the complement membrane attack complex or platelet-activating factor. Production of ROS is in narrow relationship with the metabolism of arachidonic acid. Conversion of this fatty acid via the lipoxygenase pathway is associated with an increase of ROS, whereas its transformation into prostaglandins via the cyclooxygenase pathway results in the opposite effect. Production of ROS in activated mesangial cells can be inhibited by glucocorticoids via a receptor-mediated mechanism. The fact that some of these characteristics are different in leukocytes suggests the possibility in the future of the more specific pharmacological control of the inflammatory process in the glomerular mesangium.

Acute Disease↗

PubMed1999

Extracellular calcium modulates generation of reactive oxygen species by the contracting diaphragm.

Recent studies have indicated that free radicals may play an important role in the development of muscle dysfunction in many pathophysiological conditions. Because the degree of muscle dysfunction observed in some of these conditions appears to be both free radical dependent and modulated by extracellular calcium concentrations, we thought that there may be a link between these two phenomena; i.e., the propensity of a muscle to generate free radicals may be dependent on extracellular calcium concentrations. For this reason, we compared formation of reactive oxygen species (ROS; i.e., free radicals) by electrically stimulated rat diaphragms (trains of 20-Hz stimuli for 10 min, train rate 0.25 trains/s) incubated in organ baths filled with physiological solutions containing low (1 mM), normal (2.5 mM), or high (5 mM) calcium levels. Generation of ROS was assessed by measuring the conversion of hydroethidine to ethidium. We found ROS generation with contraction varied with the extracellular calcium level, with low ROS production (3.18 +/- 0.40 ng ethidium/mg tissue) for low-calcium studies and with much higher ROS generation for normal-calcium (18. 90 +/- 2.70 ng/mg) or high-calcium (19.30 +/- 4.50 ng/mg) studies (P < 0.001). Control, noncontracting diaphragms (in 2.5 mM calcium) had little ROS production (3.40 +/- 0.80 ng/mg; P < 0.001). To further investigate this issue, we added nimodipine (20 microM), an L-type calcium channel blocker, to contracting diaphragms (2.5 mM calcium bath) and found that nimodipine also suppressed ROS formation (2.56 +/- 0.85 ng ethidium/mg tissue). These data indicate that ROS generation by the contracting diaphragm is strongly influenced by extracellular calcium concentrations and may be dependent on calcium transport through L-type calcium channels.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗

PubMed2006

MYC can induce DNA breaks in vivo and in vitro independent of reactive oxygen species.

MYC overexpression is thought to initiate tumorigenesis by inducing cellular proliferation and growth and to be restrained from causing tumorigenesis by inducing cell cycle arrest, cellular senescence, and/or apoptosis. Here we show that MYC can induce DNA breaks both in vitro and in vivo independent of increased production of reactive oxygen species (ROS). We provide an insight into the specific circumstances under which MYC generates ROS in vitro and propose a possible mechanism. We found that MYC induces DNA double-strand breaks (DSBs) independent of ROS production in murine lymphocytes in vivo as well as in normal human foreskin fibroblasts (NHFs) in vitro in normal (10%) serum, as measured by gammaH2AX staining. However, NHFs cultured in vitro in low serum (0.05%) and/or ambient oxygen saturation resulted in ROS-associated oxidative damage and DNA single-strand breaks (SSBs), as measured by Ape-1 staining. In NHFs cultured in low versus normal serum, MYC induced increased expression of CYP2C9, a gene product well known to be associated with ROS production. Specific inhibition of CYP2C9 by small interfering RNA was shown to partially inhibit MYC-induced ROS production. Hence, MYC overexpression can induce ROS and SSBs under some conditions, but generally induces widespread DSBs in vivo and in vitro independent of ROS production.

Animals↗

PubMed2004

Emodin enhances arsenic trioxide-induced apoptosis via generation of reactive oxygen species and inhibition of survival signaling.

Although arsenic trioxide (As(2)O(3)) induces apoptosis in a relatively wide spectrum of tumors, the sensitivity of different cell types to this treatment varies to a great extent. Because reactive oxygen species (ROS) are critically involved in As(2)O(3)-induced apoptosis, we attempted to explore the possibility that elevating the cellular ROS level might be an approach to facilitate As(2)O(3)-induced apoptosis. Emodin, a natural anthraquinone derivative, was selected because its semiquinone structure is likely to increase the generation of intracellular ROS. Its independent and synergistic effects with As(2)O(3) in cytotoxicity were studied, and the plausible signaling mechanism was investigated in HeLa cells. Cell Proliferation Assay and flow cytometry were used to assess cell viability and apoptosis. Electrophoretic mobility shift assay, luciferase reporter assay, and Western blotting were performed to analyze signaling alteration. The results demonstrated that coadministration of emodin, at low doses of 0.5-10 micro M, with As(2)O(3) enhanced As(2)O(3)-rendered cytotoxicity on tumor cells, whereas these treatments caused no detectable proproliferative or proapoptotic effects on nontumor cells. ROS generation was increased, and activation of nuclear factor kappaB and activator protein 1 was suppressed by coadministration. All enhancements by emodin could be abolished by the antioxidant N-acetyl-L-cysteine. Therefore, we concluded that emodin sensitized HeLa cells to As(2)O(3) via generation of ROS and ROS-mediated inhibition on two major prosurvival transcription factors, nuclear factor kappaB and activator protein 1. This result allows us to propose a novel strategy in chemotherapy that uses mild ROS generators to facilitate apoptosis-inducing drugs whose efficacy depends on ROS.

Apoptosis↗

PubMed2005

Chlamydia pneumoniae induces nitric oxide synthase and lipoxygenase-dependent production of reactive oxygen species in platelets. Effects on oxidation of low density lipoproteins.

There is increasing evidence that Chlamydia pneumoniae is linked to atherosclerosis and thrombosis. In this regard, we have recently shown that C. pneumoniae stimulates platelet aggregation and secretion, which may play an important role in the progress of atherosclerosis and in thrombotic vascular occlusion. The aims of the present study were to investigate the effects of C. pneumoniae on platelet-mediated formation of reactive oxygen species (ROS) and oxidation of low-density lipoprotein (LDL) in vitro. ROS production was registered as changes in 2',7'-dichlorofluorescin- fluorescence in platelets with flow cytometry. LDL-oxidation was determined by measuring thiobarbituric acid reactive substances (TBARs). We found that C. pneumoniae stimulated platelet production of ROS. Polymyxin B treatment of C. pneumoniae, but not elevated temperature, abolished the stimulatory effects on platelet ROS-production, which suggests that chlamydial lipopolysaccharide has an important role. Inhibition of nitric oxide synthase with nitro-L-arginine, lipoxygenase with 5,8,11-eicosatriynoic acid and protein kinase C with GF 109203X significantly lowered the production of radicals. In contrast, inhibition of NADPH-oxidase with di-phenyleneiodonium (DPI) did not affect the C. pneumoniae induced ROS-production. These findings suggest that the activities of nitric oxide synthase and lipoxygenase are the sources for ROS and that the generation is dependent of the activity of protein kinase C. The C. pneumoniae-induced ROS-production in platelets was associated with an extensive oxidation of LDL, which was significantly higher compared to the effect obtained by separate exposure of LDL to C. pneumoniae or platelets. In conclusion, C. pneumoniae interaction with platelets leading to aggregation, ROS-production and oxidative damage on LDL, may play a crucial role in the development of atherosclerotic cardiovascular disease.

Atherosclerosis↗

PubMed2006

Nox2, Ca2+, and protein kinase C play a role in angiotensin II-induced free radical production in nucleus tractus solitarius.

The dorsomedial portion of the nucleus tractus solitarius (dmNTS) is the site of termination of baroreceptor and cardiorespiratory vagal afferents and plays a critical role in cardiovascular regulation. Angiotensin II (Ang II) is a powerful signaling molecule in dmNTS neurons and exerts some of its biological effects by modulating Ca(2+) currents via reactive oxygen species (ROS) derived from reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. We investigated whether a Nox2-containing NADPH oxidase is the source of the Ang II-induced ROS production and whether the signaling mechanisms of its activation require intracellular Ca(2+) or protein kinase C (PKC). Second-order dmNTS neurons were anterogradely labeled with 4-(4-[didecylamino]styryl)-N-methylpyridinium iodide transported from the vagus and isolated from the brain stem. ROS production was assessed in 4-(4-[didecylamino]styryl)-N-methylpyridinium iodide-positive dmNTS neurons using the fluorescent dye 6-carboxy-2',7'-dichlorodihydro-fluorescein di(acetoxymethyl ester). Ang II (3 to 2000 nmol/L) increased ROS production in dmNTS neurons (EC(50)=38.3 nmol/L). The effect was abolished by the ROS scavenger Mn (III) porphyrin 5,10,20-tetrakis (benzoic acid) porphyrin manganese (III), the Ang II type 1 receptor antagonist losartan, or the NADPH oxidase inhibitors apocynin or gp91ds-tat. Ang II failed to increase ROS production or to potentiate L-type Ca(2+) currents in dmNTS neurons of mice lacking Nox2. The PKC inhibitor GF109203X or depletion of intracellular Ca(2+) attenuated Ang II-elicited ROS production. We conclude that the powerful effects of Ang II on Ca(2+) currents in dmNTS neurons are mediated by PKC activation leading to ROS production via Nox2. Thus, a Nox2-containing NADPH oxidase is the critical link between Ang II and the enhancement of Ca(2+) currents that underlie the actions of Ang II on central autonomic regulation.

Angiotensin II↗

PubMed1998

Cyclic strain-induced reactive oxygen species involved in ICAM-1 gene induction in endothelial cells.

Vascular endothelial cells (ECs) are constantly subjected to pressure-induced strain. We have previously demonstrated that strain can induce intercellular adhesion molecule-1 (ICAM-1) expression in ECs. The molecular mechanisms of gene induction by strain, however, remain unclear. Recent evidence suggests that intracellular reactive oxygen species (ROS) may act as second messengers. The potential role of ROS in strain-induced ICAM-1 expression was examined. ECs grown on a flexible membrane base were deformed with various sinusoidal negative pressures to produce an average strain of 12%. Cyclic strain induced an increase in intracellular ROS measured by fluorescent intensity of dichlorofluorescein formed after peroxidation. Maximal levels of ROS were seen after 30 minutes. Levels subsequently decreased but remained elevated compared with unstrained groups. Concomitantly, a sustained increase of H2O2 decomposition activity was observed in strained ECs. Both ROS and H2O2 decomposition activity returned to basal levels after removal of the strain. ECs treated with an antioxidant (N-acetylcysteine or catalase) inhibited strain-induced ROS generation and ICAM-1 mRNA levels followed by decreased ICAM-1 expression on EC surfaces. This inhibition may account for the reduced monocytic cell adhesion in antioxidant-treated ECs but not in strained controls. Our findings indicate that cyclic strain-induced monocyte adhesion to ECs is mediated, at least in part, by an increase of ICAM-1 gene expression via the elevation of ROS levels in strained ECs. Our results support the importance of intracellular ROS in the modulation of hemodynamic force-induced endothelial responses.

Antioxidants↗

PubMed2004

Phosphoinositide-dependent kinase 1 and p21-activated protein kinase mediate reactive oxygen species-dependent regulation of platelet-derived growth factor-induced smooth muscle cell migration.

Smooth muscle cell migration in response to platelet-derived growth factor (PDGF) is a key event in several vascular pathologies, including atherosclerosis and restenosis. PDGF increases intracellular levels of reactive oxygen species (ROS) in vascular smooth muscle cells (VSMCs), but the ROS sensitivity of migration and of the signaling pathways leading to migration are largely unknown. In VSMCs, PDGF dose-dependently increased migration compared with nonstimulated cells, with a maximum increase at 10 ng/mL. Pretreatment with the antioxidant N-acetyl-cysteine, the flavin-containing enzyme inhibitor diphenylene iodonium, or the glutathione peroxidase mimetic ebselen significantly attenuated migration (PDGF alone, 5.0+/-1.1-fold; NAC, 1.8+/-0.2-fold; diphenylene iodonium, 1.4+/-0.3-fold migration; and ebselen, 2.0+/-0.5-fold migration), as did overexpression of catalase. Pretreatment of VSMCs with the Src inhibitor PP1 or dominant-negative Rac adenovirus significantly inhibited migration, but only Src activation was attenuated by ROS inhibitors. Phosphorylation of the Src- and Rac-effector p21-activated protein kinase (PAK) 1 on Thr423 (the phosphoinositide-dependent kinase-1 [PDK1] site) was attenuated by ROS inhibition, and infection of VSMCs with dominant-negative PAK1 adenovirus attenuated migration. Moreover, kinase-inactive K111N-PDK1 inhibited PAK1 phosphorylation on Thr423, and both K111N-PDK1 and Y9F-PDK1 significantly inhibited VSMC migration. PDK1 tyrosine phosphorylation was also ROS dependent. These data indicate that PDGF-induced VSMC migration is ROS dependent and identify the Src/PDK1/PAK1 signaling pathway as an important ROS-sensitive mediator of migration. Such information is critical to understanding the role of ROS in vascular diseases in which migration of VSMCs is an important component.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed1996

Creatine kinase is the main target of reactive oxygen species in cardiac myofibrils.

Reactive oxygen species (ROS) have been reported to alter cardiac myofibrillar function as well as myofibrillar enzymes such as myosin ATPase and creatine kinase (CK). To understand their precise mode and site of action in myofibrils, the effects of the xanthine/xanthine oxidase (X/XO) system or of hydrogen peroxide (H2O2) have been studied in the presence and in the absence of phosphocreatine (PCr) in Triton X-100-treated cardiac fibers. We found that xanthine oxidase (XO), with or without xanthine, induced a decrease in maximal Ca(2+)-activated tension. We attributed this effect to the high contaminating proteolytic activity in commercial XO preparations, since it could be prevented a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), and it could be mimicked by trypsin. In further experiments, XO was pre-treated with 1 mmo1/L PMSF. Superoxide anion production by the X/XO system, characterized by electron paramagnetic resonance spin-trapping technique, was not altered by PMSF. A slight increase in maximal force was then observed either with X/XO (100 mumol/L per 30 mIU/mL) or H2O2. pMgATP-rigor tension relationships have been established in the presence and in the absence of PCr to separate the effects of ROS on myosin ATPase and myofibrillar-bound CK. In the absence of PCr, pMgATP50, the pMgATP necessary to induce half-maximal rigor tension, was reduced from 5.03 +/- 0.17 (n = 21) to 4.22 +/- 0.22 (n = 4) after 25 minutes of incubation in the presence one of 30 mIU/mL. XO and 100 mumol/L xanthine or to 4.04 +/- 0.1 (n = 11) after incubation in the presence of 2.5 mmol/L H2O2. The ROS effects were partially prevented or antagonized by 1 mmol/L dithiothreitol. No effect was observed on pMgATP50 when PCr was absent. pCa-tension relationships have been evaluated to assess the effects of ROS on active tension development. Incubations with H2O2 induced on increase in Ca2+ sensitivity and resting tension when MgATP was provided through myofibrillar CK (PCr and MgADP as substrates) but not when MgATP was added directly. These results suggest that myofibrillar CK was inhibited by ROS. Active stiffness and the time constant of tension changes after quick stretches applied to the fibers were dose-dependently increased by H2O2 only in the presence of PCr. In addition, myofibrillar CK but not myosin ATPase enzymatic activity was depressed after incubation with either ROS. These results suggest that ROS mainly alters CK in myofibrils, probably by the oxidation of its essential sulfhydryl groups. Such CK inactivation results in a decrease in the intramyofibrillar ATP-to-ADP ratio. The effects of ROS on cytosolic and bound CKs may take part in the overall process of myocardial stunning after cardiac ischemia and reperfusion.

Animals↗

PubMed2006

The role of the NADPH oxidase complex, p38 MAPK, and Akt in regulating human monocyte/macrophage survival.

M-CSF induces PI 3-kinase activation, resulting in reactive oxygen species (ROS) production. Previously, we reported that ROS mediate macrophage colony-stimulating factor (M-CSF)-induced extracellular regulated kinase (Erk) activation and monocyte survival. In this work, we hypothesized that M-CSF-stimulated ROS products modulated Akt1 and p38 activation. Furthermore, we sought to clarify the source of these ROS and the role of ROS and Akt in monocyte/macrophage survival. Macrophages from p47(phox-/-) mice, lacking a key component of the NADPH oxidase complex required for ROS generation, had reduced cell survival and Akt1 and p38 mitogen-activated protein kinase (MAPK) phosphorylation compared with wild-type macrophages in response to M-CSF stimulation, but had no difference in M-CSF-stimulated Erk. To understand how ROS affected monocyte survival and signaling, we observed that NAC and DPI decreased cell survival and Akt1 and p38 MAPK phosphorylation. Using bone marrow-derived macrophages from mice expressing constitutively activated Akt1 (Myr-Akt1) or transfecting Myr-Akt1 constructs into human peripheral monocytes, we concluded that Akt is a positive regulator of monocyte survival. Moreover, the p38 MAPK inhibitor, SB203580, inhibited p38 activity and M-CSF-induced monocyte survival. These findings demonstrate that ROS generated from the NADPH oxidase complex contribute to monocyte/macrophage survival induced by M-CSF via regulation of Akt and p38 MAPK.

Animals↗

PubMed2003

Optic neuropathy induced by reductions in mitochondrial superoxide dismutase.

PURPOSE: Reactive oxygen species (ROS) are suspected to play a pivotal role in the pathogenesis of Leber hereditary optic neuropathy (LHON), caused by mutated complex I subunit genes. It seems surprising that optic neuropathy has not been described in animals with a knockout of genes encoding critical anti-ROS defenses. If ROS have a role in the optic nerve injury of LHON, then increasing mitochondrial levels of ROS should induce optic neuropathy. METHODS: To develop an animal model system for study of oxidative injury to the optic nerve, mitochondrial defenses were decreased against ROS by designing hammerhead ribozymes to degrade SOD2 mRNA. Several potential ribozymes were analyzed in vitro. The one with the best kinetic characteristics was cloned into a recombinant adeno-associated virus (rAAV) vector for delivery and testing in cells and animals. The effects of the AAV-expressing ribozyme on murine cell growth, SOD2 mRNA and protein, cellular ROS levels, and apoptosis were evaluated by RNase protection assay, immunoblot analysis, and ROS- and apoptosis-activated fluorescent probes. The rAAV-ribozyme was then injected into the eyes of DBA/1J mice, and the effect on the optic nerve was evaluated by ocular histopathologic examination. RESULTS: The AAV-expressing ribozyme decreased SOD2 mRNA and protein levels by as much as 85%, increased cellular superoxide, reduced mitochondrial membrane potential, and culminated in the death of infected cell lines by apoptosis without significantly altering complex I and III activity, somewhat spared in the most common LHON mutation (G11778A), although adenosine triphosphate (ATP) synthesis is markedly reduced. When inoculated into the eyes of mice, the AAV-expressing ribozyme led to loss of axons and myelin in the optic nerve and ganglion cells in the retina, the hallmarks of optic nerves examined at autopsy of patients with LHON. CONCLUSIONS: The striking similarity of the optic neuropathy to the histopathology of LHON is powerful evidence supporting ROS as a key factor in the pathogenesis of LHON.

3T3 Cells↗

PubMed1996

BCL-2 expression or antioxidants prevent hyperglycemia-induced formation of intracellular advanced glycation endproducts in bovine endothelial cells.

Hyperglycemia rapidly induces an increase in intracellular advanced glycation end products (AGEs) in bovine endothelial cells, causing an alteration in bFGF activity (Giardino, I., D. Edelstein, and M. Brownlee. 1994. J. Clin. Invest. 94:110-117). Because sugar or sugar-adduct autoxidation is critical for AGE formation in vitro, we evaluated the role of reactive oxygen species (ROS) in intracellular AGE formation, using bovine aortic endothelial cells. 30 mM glucose increased intracellular ROS formation by 250% and lipid peroxidation by 330%, while not affecting ROS in the media. In cells depleted of glutathione, intracellular AGE accumulation increased linearly with ROS generation as measured by immunoblotting and the fluorescent probe DCFH (AGE 0.258-3.531 AU* mm/5x10(4) cells, DCF 57-149 mean AU, r = .998, P < .002). Deferoxamine, alpha-tocopherol, and dimethylsulfoxide each inhibited hyperglycemia-induced formation of both ROS and AGE. To differentiate an effect of ROS generation on AGE formation from an effect of more distal oxidative processes, GM7373 endothelial cell lines were generated that stably expressed the peroxidation-suppressing proto-oncogene bcl-2. bcl-2 had no effect on hyperglycemia-induced intracellular ROS formation. In contrast, bcl-2 expression decreased both lipid peroxidation (100% at 3 h and 29% at 168 h) and AGE formation (55% at 168 h). These data show that a ROS-dependent process plays a central role in the generation of intracellular AGEs, and that inhibition of oxidant pathways prevents intracellular AGE formation.

Animals↗

PubMed1996

Benzene metabolites enhance reactive oxygen species generation in HL60 human leukemia cells.

Benzene is myelotoxic and leukemogenic in humans. The mechanisms leading to these effects, however have not been fully elucidated. One of the underlying mechanisms is believed to be the oxidative damage caused by its metabolites. A comparative study was undertaken to examine the relationships between reactive oxygen species (ROS) production, lipid peroxidation and subsequent cytotoxicity induced by five major benzene metabolites. The generation of ROS by benzene metabolites was demonstrated by the significant and dose-dependent increase of intracellular ROS formation in HL60 human promyelocytic leukemia cells in vitro. 1,4-Benzoquinone (BQ) was found to be the most potent metabolite in induction of ROS formation, followed by 1,2,4-benzenetriol (BT) and to a lesser extent, phenol (PH) and trans, trans-muconaldehyde (MD). No significant effect was observed when the cells were treated with trans, trans-muconic acid (MA). The enhancement of ROS production by BQ was effectively inhibited by the addition of catalase, deferoxamine (DFO) and dimethyl sulfoxide (DMSO), but unchanged by superoxide dismutase (SOD), suggest that hydrogen peroxide (H2O2) and hydroxyl radicals (OH) are the two major forms of ROS involved. The results also demonstrate that the ability of benzene metabolites in enhancing ROS generation is closely correlated to their capacity in causing lipid peroxidation and subsequent cytotoxicity. These findings together with earlier parallel observations on DNA damage suggest that ROS play an important role in the mechanism of carcinogenesis induced by benzene metabolites.

Aldehydes↗