Inhibition of mitochondrial phosphate carrier prevents high phosphate-induced superoxide generation and vascular calcification.

Vascular calcification is a serious complication of hyperphosphatemia that causes cardiovascular morbidity and mortality. Previous studies have reported that plasmalemmal phosphate (Pi) transporters, such as PiT-1/2, mediate depolarization, Ca2+ influx, oxidative stress, and calcific changes in vascular smooth muscle cells (VSMCs). However, the pathogenic mechanism of mitochondrial Pi uptake in vascular calcification associated with hyperphosphatemia has not been elucidated. We demonstrated that the phosphate carrier (PiC) is the dominant mitochondrial Pi transporter responsible for high Pi-induced superoxide generation, osteogenic gene upregulation, and calcific changes in primary VSMCs isolated from rat aortas. Notably, acute incubation with high Pi markedly increased the protein abundance of PiC via ERK1/2- and mTOR-dependent translational upregulation. Genetic suppression of PiC prevented Pi-induced ERK1/2 activation, superoxide production, osteogenic differentiation, and vascular calcification of VSMCs in vitro and aortic rings ex vivo. Pharmacological inhibition of mitochondrial Pi transport using butyl malonate (BMA) or mersalyl abolished all pathologic changes involved in high Pi-induced vascular calcification. BMA or mersalyl also effectively prevented osteogenic gene upregulation and calcification of aortas from 5/6 subtotal nephrectomized mice fed a high-Pi diet. Our results suggest that mitochondrial Pi uptake via PiC is a critical molecular mechanism mediating mitochondrial superoxide generation and pathogenic calcific changes, which could be a novel therapeutic target for treating vascular calcification associated with hyperphosphatemia.

Permeability transition pore-mediated mitochondrial superoxide flashes mediate an early inhibitory effect of amyloid beta1-42 on neural progenitor cell proliferation.

Cellular damage by reactive oxygen species and altered neurogenesis are implicated in the etiology of AD and the pathogenic actions of amyloid ß-peptide (Aß); the underlying mechanisms and the early oxidative intracellular events triggered by Aß are not established. In the present study, we found that mouse embryonic cortical neural progenitor cells exhibit intermittent spontaneous mitochondrial superoxide (SO) flashes that require transient opening of mitochondrial permeability transition pores (mPTPs). The incidence of mitochondria SO flash activity in neural progenitor cells (NPCs) increased during the first 6-24 hours of exposure to aggregating amyloid ß-peptide (Aß1-42), indicating an increase in transient mPTP opening. Subsequently, the SO flash frequency progressively decreased and ceased between 48 and 72 hours of exposure to Aß1-42, during which time global cellular reactive oxygen species increased, mitochondrial membrane potential decreased, cytochrome C was released from mitochondria and the cells degenerated. Inhibition of mPTPs and selective reduction in mitochondrial SO flashes significantly ameliorated the negative effects of Aß1-42 on NPC proliferation and survival. Our findings suggest that mPTP-mediated bursts of mitochondrial SO production is a relatively early and pivotal event in the adverse effects of Aß1-42 on NPCs. If Aß inhibits NPC proliferation in the brains of AD patients by a similar mechanism, then interventions that inhibit mPTP-mediated superoxide flashes would be expected to protect NPCs against the adverse effects of Aß.

Characterization of coal fly ash nanoparticles and induced oxidative DNA damage in human peripheral blood mononuclear cells.

The nano-sized particles present in coal fly ash (CFA) were characterized through the X-ray diffraction (XRD), transmission and scanning electron microscopy (TEM, SEM), atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) analyses. The XRD data revealed the average crystallite size of the CFA nanoparticles (CFA-NPs) as 14 nm. TEM and SEM imaging demonstrated predominantly spherical and some polymorphic structures in the size range of 11 to 25 nm. The amount of heavy metal associated with CFA particles (µg/g) were determined as Fe (34160.0±1.38), Ni (150.8±0.78), Cu (99.3±0.56) and Cr (64.0±0.86). However, the bioavailability of heavy metals in terms of percent release was in the order as Cr>Ni>Cu>Fe in CFA-dimethyl sulfoxide (DMSO) extract. The comet and cytokinesis blocked micronucleus (CBMN) assays revealed substantial genomic DNA damage in peripheral blood mononuclear (PBMN) cells treated with CFA-NPs in Aq and DMSO extracts. About 1.8 and 3.6 strand breaks per unit of DNA were estimated through alkaline unwinding assay at 1:100 DNA nucleotide/CFA ppm ratios with the Aq and DMSO extracts, respectively. The DNA and mitochondrial damage was invariably greater with CFA-DMSO extract vis-à-vis -Aq extract. Generation of superoxide anions (O(2)•(-)) and intracellular reactive oxygen species (ROS) through metal redox-cycling, alteration in mitochondrial potential and 8-oxodG production elucidated CFA-NPs induced oxidative stress as a plausible mechanism for CFA-induced genotoxicity.

Genomic instability induced by mutant succinate dehydrogenase subunit D (SDHD) is mediated by O2(-•) and H2O2.

SDHD mutations are associated with human cancers but the mechanisms that may contribute to transformation are unknown. The hypothesis that mutations in SDHD increase levels of superoxide leading to genomic instability was tested using site-directed mutagenesis to generate a truncated SDHD cDNA that was expressed in Chinese hamster fibroblasts. Stable expression of mutant SDHD resulted in 2-fold increases in steady-state levels of superoxide that were accompanied by a significantly increased mutation rate as well as a 70-fold increase in mutation frequency at the hprt locus. Overexpression of MnSOD or treatment with polyethylene glycol conjugated (PEG)-catalase suppressed mutation frequency in SDHD mutant cells by 50% (P<0.05). Simultaneous treatment with PEG-catalase and PEG-SOD suppressed mutation frequency in SDHD mutant cells by 90% (P<0.0005). Finally, 95% depletion of glutathione using l-buthionine-[S,R]-sulfoximine (BSO) in SDHD mutant cells caused a 4-fold increase in mutation frequency (P<0.05). These results demonstrate that mutations in SDHD cause increased steady-state levels of superoxide which significantly contributed to increases in mutation rates and frequency mediated by superoxide and hydrogen peroxide. These results support the hypothesis that mutations in SDHD may contribute to carcinogenesis by increasing genomic instability mediated by increased steady-state levels of reactive oxygen species.

Deficiency of CuZn superoxide dismutase promotes inflammation and alters medial structure following vascular injury.

AIM: The anti-oxidant enzyme copper/zinc superoxide dismutase (CuZnSOD) metabolizes superoxide anion (O(2)(-)) in vascular cells. However, the role of CuZnSOD in vascular injury remains poorly understood. METHODS: Using CuZnSOD-deficient (CuZnSOD(-/-)) mice and wild-type (WT) mice, we investigated morphometric changes and the role of O(2)(-) in vascular remodeling after femoral artery injury induced by an external vascular cuff model. RESULTS: Three days post-injury, inflammatory cell infiltration increased significantly. Moreover, the percent positive area of tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in media were higher in CuZnSOD(-/-) mice than in WT mice (TNF-α: 34.8±8.4% versus 18.8±5.6%, p < 0.05, ICAM-1: 29.6±6.5% versus 11.0±2.8%, p < 0.05, VCAM-1: 23.5±7.5% versus 3.7±1.1%, p < 0.05). mRNA expression of iNOS was markedly increased in CuZnSOD(-/-) mice with cuff injury. Dihydroethidine staining revealed increased levels of vascular O(2)(-) in media from CuZnSOD(-/-) mice. Although neointimal formation remained unchanged, 14 days postinjury, we observed degeneration of the media, and the media/vessel wall ratio increased in CuZnSOD(-/-) mice (40.4±2.1% versus 26.8±1.4%, p < 0.05). Furthermore, SMemb/MHC-B-stained lesions increased markedly in CuZnSOD(-/-) mice. CONCLUSIONS: CuZnSOD-deficiency promoted inflammation, expressed adhesion molecules, and altered the structure of the media post-injury. Our results suggest that O(2)(-) participates importantly in the progression of early stage vascular inflammation, resulting in vascular remodeling in media but not neointimal formation, post-injury.

An in vitro examination of the antioxidant and anti-inflammatory properties of buckwheat honey.

OBJECTIVE: Hydroxyl radical and hypochlorite anion formed at the wound site from superoxide anion produced by activated polymorphonuclear neutrophils (PMNs) are considered important factors in impaired wound healing. Superoxide anion may also react with nitric oxide produced by macrophages to form peroxynitrite, a third strong oxidant that damages surrounding tissue. In order to select honey for use in wound-healing products, different samples were compared for their capacity to reduce levels of reactive oxygen species (ROS) in vitro. METHOD: Honey samples were tested in assays for inhibition of ROS production by activated human PMNs, antioxidant activity (scavenging of superoxide anion in a cell-free system) and inhibition of human complement (reducing levels of ROS by limiting formation of complement factors that attract and stimulate PMNs). For buckwheat honey (NewYork, US), moisture and free acid content were determined by refractive index measurement and potentiometric titration respectively. Honey constituents other than sugars were investigated by thin layer chromatography, using natural product reagent to detect phenolic compounds. Constituents with antioxidant properties were detected by spraying the chromatogram with DPPH. RESULTS: Although most honey samples were shown to be active, significant differences were observed, with the highly active honey exceeding the activities of samples with minor effects by factors of 4 to 30. Most pronounced activities were found for American buckwheat honey from the state of NewYork. Phenolic constituents of buckwheat honey were shown to have antioxidant activity. CONCLUSION: As buckwheat honey was most effective in reducing ROS levels, it was selected for use in wound-healing products. The major antioxidant properties in buckwheat honey derive from its phenolic constituents, which are present in relatively large amounts. Its phenolic compounds may also exert antibacterial activity, whereas its low pH and high free acid content may assist wound healing.

Pulmonary inflammation by ambient air particles is mediated by superoxide anion.

Lung inflammation is a key response to increased levels of particulate air pollution (PM); however, the cellular mechanisms leading to this response remain poorly understood. We have previously shown that oxidants are critical mediators of the inflammatory response elicited by inhalation of ambient air particles. Here we tested the possible role of a specific oxidant, superoxide anion, by using the membrane-permeable analog of superoxide dismutase, Mn(III) tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP). Adult Sprague-Dawley rats were instilled with either urban air particles (UAP) or saline. MnTBAP-treated rats received 10 mg/kg (ip) MnTBAP 2 h prior to exposure to UAP. Recruitment of inflammatory cells into bronchoalveolar lavage was evaluated 4 h after instillation. Rats exposed to UAP showed significant increases in the total cell number (8.9 +/- 0.6 x 10(6); sham: 5.1 +/- 0.6 x 10(6), p < .02), the numbers of polymorphonuclear leukocytes (26 +/- 4%; sham: 6 +/- 1%, p < .0001), protein levels (1.2 +/- 0.5 mg/ml, sham: 0.4 +/- 0.1 mg/ml, p < .001), and a trend of increase in myeloperoxidase levels (5 +/- 1; sham: 2 +/- 1 mU/ml) in bronchoalveolar lavage (BAL). Pretreatment with MnTBAP at a dose that prevented UAP-induced increases in oxidants effectively prevented increase in BAL cells (2.7 +/- 0.6 x 10(6), p < .0001 vs. UAP), PMN influx into the lungs (4 +/- 3%, p < .0001 vs. UAP), and increase in myeloperoxidase (2 +/- 1 mU/ml) and protein levels in BAL (0.1 +/- 0.1 mg/ml). These data indicate that superoxide anion is a critical mediator of the inflammatory response elicited by PM deposition in the lung.

Estrés oxidativo en el recién nacido y su influencia en la fisiología vascular pulmonar / Oxidative stress in the newborn and its influence on pulmonary vascular physiology

El radical libre es cualquier especial química que contenga uno más electrones no apareados, y es habitual que las reacciones en las que interviene sean procesos en cadena. Los lípidos, los ácido nucleicos, los hidratos de carbono y las proteínas son susceptibles al ataque de los radicales libre, que sufren un daño oxidativo in vivo, lo que degenera en fenómenos de necrosis o de apoptosis celular. En contraposición a tales sistemas de oxidación, las células cuentan con enzimas y moléculas antioxidantes. Este delicado equilibrio existente en el organismo en condiciones de normoxia entre los mecanismos oxidantes y antioxidantes se define como estrés oxidativo. El desequilibrio de estos sistemas, en particular el estrés oxidativo derivado de un déficit en la maduración de los sistemas antioxidantes en el neonato, participa directa o indirectamente en la génesis de la patología clínica, contribuyendo tanto a la patogenia como al pronóstico. Además, hay condiciones inherentes al recién nacido que incrementan su susceptibilidad al daño pulmonar, convirtiendo al pulmón en un órgano crítico en este proceso de adaptación a la vida extrauterina, sobre todo teniendo en cuenta que la maduración pulmonar continúa durante la etapa postnatal. En cualquier caso, se sabe que los recién nacidos tienen una mayor resistencia a la toxicidad por el oxígeno, posiblemente relacionada con su capacidad para aumentar la concentración de las enzimas oxidantes. Por otro lo anterior, se concluye que el conocimiento de estas vías de señalización puede contribuir en gran manera al desarrollo de futuros tratamientos, aunque en la actualidad la terapéutica con antioxidantes se está estudiando en modelos experimentales, aún no es una práctica habitual en niños recién nacidos

A free radical is any chemical species that has one or more unpaired electrons. These species usually take part in chain reactions. Lipids, nucleis acids, carbohydrates and proteins are susceptible to free radical attack, which produces in vivo oxidative damage that degenerates into cellular necrosis or apoptosis. To combat these oxidation systems, the cells are equipped with enzymes and antioxidant molecules. The delicate balance between oxidant and antioxidant mechanisms, which exists in the organism under conditions of normoxia, is defined as oxidative stress. An imbalance in these systems, in particular, oxidative stress derived from a lack of maturity of the antioxidant systems in the newborn infant, participates directly or indirectly in the clinical onset of disease, playing a role in both the pathogenesis and the prognosis. Moreover, there are conditions inherent in the newborn infant that increase his or her susceptibility to pulmonary damage, making the lung a critical organ in the process of adaptation of the infant to extrauterine life, especially since the lung continues to mature after birth. In any case, newborn infants are known to be more resistant to oxygen toxicity, possibly due, at least in part, to their ability to increase antioxidant enzyme levels. Thus, the authors conclude that the knowledge of these signalling pathways may contribute substantially to the development of future treatments. Although, at the present time, antioxidant therapy is being studied in experimental models, it is not yet a routine practice in newborn infants

Effect of oxidative stress on glial cell volume.

Cytotoxic brain edema is a major contributor of tissue damage following cerebral ischemia and traumatic brain injury. The pathophysiology of cytotoxic edema formation is still not well understood. Although it is widely believed that oxidative stress causes cytotoxic brain edema, experimental proof is lacking. The aim of the present study was therefore to examine the effect of oxidative stress on cell volume of glial cells. C6 glial cells were exposed to hydrogen peroxide and the superoxide forming complex hypoxanthine/xanthine oxidase (HX/XO). Exposure to hydrogen peroxide (0.5-5 mM) resulted in initial cell shrinkage by 5.7 +/- 1.5% (mean +/- SEM; p < 0.05) and was followed by a dose-dependent recovery to baseline. Exposure to superoxide anions generated by HX/XO provoked a delayed, but sustained decrease of cell volume by 11.8 +/- 0.9% (p < 0.05). Cell volume showed no tendency to recover upon sustained exposure to superoxide. Neither hydrogen peroxide nor HX/XO exposure was associated with a decrease of cell viability. Thereby, the present study demonstrates that oxidative stress by hydrogen peroxide and superoxide anions does not induce cytotoxic cell swelling and suggests that free radicals are not directly involved in the formation of cytotoxic brain edema.

Increased reactive oxygen species contributes to kidney injury in mineralocorticoid hypertensive rats.

Hypertension is associated with increased reactive oxygen species (ROS). Renal ROS production and their effects on renal function have never been investigated in mineralocorticoid hypertensive rats. In this study we hypothesized that increased ROS production in kidneys from deoxycorticosterone (DOCA)-salt rats contributes to adverse renal morphological changes and impaired renal function in DOCA-salt hypertensive rats. We also determined whether ROS-induced renal injury was dependent on blood pressure. DOCA-salt hypertensive rats exhibited a marked increase in blood pressure, renal ROS production, glomerular and tubular lesions, and microalbuminuria compared to sham rats. Treatment of DOCA-salt hypertensive rats with apocynin for 28 days resulted in attenuation of systolic blood pressure and improvement of renal morphology. Renal superoxide level in DOCA-salt rats was 215% of sham-operated rats and it was significantly decreased to 140% with apocynin treatment. Urinary protein level was decreased from 27 +/- 3 mg/day in DOCA-salt hypertensive rats to 9 +/- 2 mg/day. 28 days of Vitamin E treatment also reduced renal injury in regard to urinary protein level and renal morphology but had no effect on blood pressure in DOCA-salt rats. Increased urinary 8-isoprostane, a marker for oxidative stress, in DOCA-salt hypertensive rats (55 +/- 8 ng/day) was diminished by vitamin E treatment (24 +/- 6 ng/day). These data suggest that renal injury characteristic of mineralocorticoid hypertension is associated with oxidative stress and is partly independent of blood pressure.

Interplay between superoxide and nitric oxide in aging and diseases.

Free radicals, superoxide and nitric oxide, are important signaling molecules, which mediate numerous physiological functions (phagocytosis, vasorelaxation, etc.). However, regulation errors may lead to free-radical-mediated damaging processes in cells and tissues. In this work, the effects of an interplay between superoxide and NO, which may be responsible for the development of aging and diseases, are considered. We are suggesting that the superoxide-mediated proton leak leading to the inhibition of oxidative phosphorylation and the competition between NO and O2*- in their reactions with cytochrome oxidase can be a cause of mitochondrial aging.

A newly identified role for superoxide in inflammatory pain.

Novel classes of pain-relieving molecules are needed to fill the void between nonsteroidal anti-inflammatory agents and narcotics. Our studies have identified superoxide as a novel mediator of hyperalgesia (clinically defined as an augmented sensitivity to painful stimuli) and have exposed potential pathways through which this radical modulates the hyperalgesic response. The role of superoxide in pain was elucidated using a superoxide dismutase mimetic, M40403 [a manganese(II) complex with a bis(cyclo-hexylpyridine-substituted) macrocyclic ligand]. Intraplantar injection of carrageenan in rats led to time-dependent development of peripheral inflammation [measured parameters of inflammation included paw edema, cytokine release in the paw exudates, nitrotyrosine formation (a marker of peroxynitrite formation and oxidative stress), and poly-ADP-ribose-polymerase activation (the nuclear enzyme activated by superoxide/peroxynitrite)] and hyperalgesia. M40403 blocked all measured parameters of inflammation and hyperalgesia. Furthermore, when given therapeutically (2 h after the induction of hyperalgesia) either by intravenous or intrathecal administration, M40403 but not its inactive congener M40404 inhibited hyperalgesia with a rapid onset of action. Our results also show that, at the level of the spinal cord and time of peak hyperalgesia, endogenous manganese superoxide dismutase was nitrated and subsequently deactivated, losing its capacity to remove superoxide. The antihyperalgesic effects of M40403 were not reversed by naloxone excluding the potential involvement of an opiate pathway. Collectively, these studies have unraveled a critical role for superoxide in the nociceptive signaling cascade both peripherally and centrally. The discovery of this pathway opens a new therapeutic strategy for the development of novel nonnarcotic antihyperalgesic agents.

Long-chain polyunsaturated fatty acids interact with nitric oxide, superoxide anion, and transforming growth factor-beta to prevent human essential hypertension.

Patients with uncontrolled essential hypertension have elevated concentrations of superoxide anion (O(2)(-*)), hydrogen peroxide (H(2)O(2)), lipid peroxides, endothelin, and transforming growth factor-beta (TGF-beta) with a simultaneous decrease in endothelial nitric oxide (eNO), superoxide dismutase (SOD), vitamin E, and long-chain polyunsaturated fatty acids (LCPUFAs). Physiological concentrations of angiotensin II activate NAD(P)H oxidase and trigger free radical generation (especially that of O(2)(-*)). Normally, angiotensin II-induced oxidative stress is abrogated by adequate production and release of eNO, which quenches O(2)(-*) to restore normotension. Angiotensin II also stimulates the production of endothelin and TGF-beta. TGF-beta enhances NO generation, which in turn suppresses TGF-beta production. Thus, NO has a regulatory role on TGF-beta production and is also a physiological antagonist of endothelin. Antihypertensive drugs suppress the production of O(2)(-*) and TGF-beta and enhance eNO synthesis to bring about their beneficial actions. LCPUFAs suppress angiotensin-converting enzyme (ACE) activity, reduce angiotensin II formation, enhance eNO generation, and suppress TGF-beta expression. Perinatal supplementation of LCPUFAs decreases insulin resistance and prevents the development of hypertension in adult life, whereas deficiency of LCPUFAs in the perinatal period results in raised blood pressure later in life. Patients with essential hypertension have low concentrations of various LCPUFAs in their plasma phospholipid fraction. Based on this, it is proposed that LCPUFAs serve as endogenous regulators of ACE activity, O(2)(-*), eNO generation, and TGF-beta expression. Further, LCPUFAs have actions similar to statins, inhibit (especially omega-3 fatty acids) cyclooxygenase activity and suppress the synthesis of proinflammatory cytokines, and activate the parasympathetic nervous system, all actions that reduce the risk of major vascular events. Hence, it is proposed that availability of adequate amounts of LCPUFAs during the critical periods of growth prevents the development of hypertension in adulthood.

Oxidative damage to a supercoiled DNA by water soluble peroxyl radicals characterized with DNA repair enzymes.

Earlier work (Paul, T., et al. (2000) Biochemistry 39, 4129-4135) has demonstrated that the water soluble positively charged peroxyl radical, (H(2)N)(2)(+)CC(CH(3))(2)OO(*) ((+)AOO(*)), caused direct strand scission of the Escherichia coli plasmid supercoiled DNA, pBR 322, with ca. 50% scission occurring at a (+)AOO(*)/base pair (bp) ratio of 0.2. There was no measurable direct scission with a negatively charged peroxyl ((-)BOO(*)) at (-)BOO(*)/bp = 24, nor with a neutral peroxyl (COO(*)) at COO(*)/bp = 5. Base modification (BM) of the same DNA by the same peroxyls has now been investigated using four base excision repair (BER) glycosylases. At (+)AOO(*)/bp = 0.04, there is 10% direct strand scission, and the Fpg protein recognized an additional 25% BM, while endonuclease (Endo) IV recognized an additional 20% BM and the other two BER enzymes did not give statistically significant BMs. None of the BER enzymes showed BMs in the DNA treated with -BOO(*). However, Fpg and Endo IV showed that at COO(*)/bp = 3.4 there was a BM comparable to that observed at (+)AOO(*)/bp = 0.04. Thus, COO(*) radicals are only ca. 1.2% as reactive toward the DNA's bases as (+)AOO(*). These results underline the importance of Coulombic forces in DNA reactions. It is also proposed that (+)AOO(*) has a higher intrinsic reactivity in H-atom abstractions and electron transfer processes than (-)BOO(*) or COO(*) radicals.

Pathophysiological mechanisms of diabetic angiopathy.

The sequelae of chronic hyperglycemia in diabetes of all phenotypes are divided into microvascular and macrovascular complications. Microvascular disease causes blindness, renal failure, and neuropathy, and diabetes-accelerated macrovascular disease causes excessive risk for myocardial infarction, stroke, and lower limb amputation. The link between chronic hyperglycemia and vascular damage has been established by four independent biochemical abnormalities: increased polyol pathway flux, increased formation of advanced glycation end-products (AGEs), activation of protein kinase C (PKC), and increased hexosamine pathway flux. These seemingly unrelated pathways have an underlying common denominator: overproduction of superoxide by the mitochondrial electron transport chain. Mitochondrial reactive oxygen species (ROS) partially inhibit the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, which diverts increased substrate flux from glycolysis to pathways of glucose overutilization. Preliminary experimental evidence in vivo suggests that this new paradigm provides a novel basis for research and drug development.

Nitric oxide and superoxide in inflammation and immune regulation.

Nitric oxide (NO) and reactive oxygen species exert multiple modulating effects on inflammation and play a key role in the regulation of immune responses. They affect virtually every step of the development of inflammation. Low concentrations of nitric oxide produced by constitutive and neuronal nitric oxide synthases inhibit adhesion molecule expression, cytokine and chemokine synthesis and leukocyte adhesion and transmigration. Large amounts of NO, generated primarily by iNOS can be toxic and pro-inflammatory. Actions of nitric oxide are however not dependent primarily on the enzymatic source, but rather on the cellular context, NO concentration (dependent on the distance from NO source) and initial priming of immune cells. These observations may explain difficulties in determining the exact role of NO in Th1 and Th2 lymphocyte balance in normal immune responses and in allergic disease. Similarly superoxide anion produced by NAD(P)H oxidases present in all cell types participating in inflammation (leukocytes, endothelial and other vascular cells etc) may lead to toxic effects, when produced at high levels during oxidative burst, but may also modulate inflammation in a far more discrete way, when continuously produced at low levels by NOXs (non-phagocytic oxidases). The effects of both nitric oxide and superoxide in immune regulation are exerted through multiple mechanisms, which include interaction with cell signalling systems like cGMP, cAMP, G-protein, JAK/STAT or MAPK dependent signal transduction pathways. They may also lead to modification of transcription factors activity and in this way modulate the expression of multiple other mediators of inflammation. Moreover genetic polymorphisms exist within genes encoding enzymes producing both NO and superoxide. The potential role of these polymorphisms in inflammation and susceptibility to infection is discussed. Along with studies showing increasing role of NO and free radicals in mediating inflammatory responses drugs which interfere with these systems are being introduced in the treatment of inflammation. These include statins, angiotensin receptor blockers, NAD(P)H oxidase inhibitors, NO-aspirin and others. In conclusion in this mini-review we discuss the mechanisms of nitric oxide and superoxide dependent modulation of inflammatory reactions in experimental animals and humans. We also discuss potential roles of nitric oxide as a mediator of allergic inflammation.

Induction of apoptosis by superoxide anion and the protective effects of selenium and Vitamin E.

OBJECTIVE: The purpose of this study is to investigate the effect of superoxide anion on the apoptosis of cultured fibroblasts and the protective role of selenium and Vitamin E. METHODS: Cultured fibroblasts (NIH3T3), with or without selenium or vitamin E in the medium, were treated by superoxide anion produced by xanthine/xanthine oxidase reaction system and changes in cell structure and DNA were observed microscopically and electrophoretically. RESULTS: Apoptosis was observed when superoxide anion at a concentration of 5 nmol/L or 10 nmol/L had acted on the fibroblasts for 5-10 h. Selenium and Vitamin E in the medium inhibited the apoptosis significantly when their concentrations reached 1.15 mol/L and 2.3 mol/L respectively. CONCLUSION: Selenium and vitamin E have protective effect against the apoptosis induced by superoxide anion. The effect of selenium is more remarkable than that of vitamin E.

Inhibitory effects of methanol extract of Cyperus rotundus rhizomes on nitric oxide and superoxide productions by murine macrophage cell line, RAW 264.7 cells.

The rhizomes of Cyperus rotundus (C. rotundus) have been used in oriental traditional medicines for the treatment of stomach and bowel disorders, and inflammatory diseases. Nitric oxide (NO) and superoxide (O2-) are important mediators in the pathogenesis of inflammatory diseases. This study was undertaken to address whether the metanol (MeOH) extract of rhizomes of C. rotundus could modulate NO and O2- productions by murine macrophage cell line, RAW 264.7 cells. The MeOH extract of rhizomes of C. rotundus showed the inhibition of NO production in a dose-dependent manner by RAW 264.7 cells stimulated with interferon-gamma plus lipopolysaccharide. The inhibition of NO production by the extract was due to the suppression of iNOS protein, as well as iNOS mRNA expression, determined by Western and Northern blotting analyses, respectively. In addition, the MeOH extract suppressed the production of O2- by phorbol ester-stimulated RAW 264.7 cells in dose- and time-dependent manners. Collectively, these results suggest that the MeOH extract of rhizomes of C. rotundus could be developed as anti-inflammatory candidate for the treatment of inflammatory diseases mediated by overproduction of NO and O2-.

Is insulin an antiinflammatory molecule?

I suggest that insulin suppresses the secretion and antagonizes the harmful effects of tumor necrosis factor-alpha, macrophage migration-inhibitory factor, and superoxide anion. Therefore, the glucose-insulin-potassium regimen might be beneficial in acute myocardial infarction and useful in the management of patients with septicemia, septic shock, and other inflammatory diseases in which tumor necrosis factor-alpha and macrophage migration-inhibitory factor have important roles.