Generation of active oxygen species by iron nitrilotriacetate (Fe-NTA)

Ferric nitrilotriacetate (Fe3+-NTA) solution showed maximum absorbance at pH 7.5. The iron was in ferric high-spin state and coordinated octahedrally with a relatively symmetric structure and also probably pentagonally. A spin trapping technique employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) yielded a DMPO spin adduct of unknown radical with three doublets (DMPO-Z) and a simple nitroxide radical (Y-NO.) in serum from rats injected intraperitoneally with Fe3+-NTA. When the Fe3+-NTA solution was diluted 500-fold with 50 mM NTA solution, DMPO-Z, Y-NO. and an additional signal, DMPO-OH were observed. The DMPO-Z signal was suppressed by a decrease in oxygen tension, alpha-tocopherol and 3-tert-butyl-4-hydroxy-anisole (BHA). The DMPO-OH signal was suppressed in the presence of ethanol and catalase. Fe2+-NTA solution hardly produced DMPO spin adducts. The Fe3+-NTA solution produced a strong DMPO-OH signal in the presence of H2O2. Rose Bengal solution, a singlet oxygen generating system, produced the same DMPO adducts. Fe3+-NTA reacted with oxygen in solution. The oxygen was activated and might be similar to singlet molecular oxygen. In the presence of H2O2, the Fe3+-NTA solution generated a hydroxyl radical. Fe3+-NTA itself generated free radicals, but Fe2+-NTA did not.     

Inactivation of primary antioxidant enzymes in mouse keratinocytes by photodynamically generated singlet oxygen

Cellular antioxidant enzymes protect against damage caused by exposure to endogenous or exogenous prooxidants. Singlet oxygen ((1)O(2)) is a reactive form of oxygen that can be produced in vivo either in normal and pathophysiologic conditions or by photosensitizing chemicals, as during photodynamic treatment. We hypothesized that photodynamically generated (1)O(2) would decrease the enzymatic activities of cellular antioxidants. To test this hypothesis, we treated cultured mouse epidermal keratinocytes with the photosensitizer Photofrin plus visible light to produce (1)O(2), and then measured CuZnSOD, MnSOD, and catalase activities with both ingel and spectrophotometric enzyme activity assays. Our results demonstrated that the enzymatic activities of cellular CuZnSOD, MnSOD, and catalase were significantly decreased after keratinocytes were treated with Photofrin plus visible light. By contrast, the enzymatic activities of cellular CuZnSOD, MnSOD, and catalase were unaffected in control cells treated with Photofrin only or visible light only. Despite the decreased levels of enzymatic activities, the protein levels of all three primary antioxidant enzymes remained constant after photodynamic treatment, as determined by Western blotting. L-Histidine, a (1)O(2) quencher, protected against the inactivation of cellular CuZnSOD, MnSOD, and catalase enzymes induced by photodynamically generated (1)O(2). The conclusion from these experiments is that the primary cellular antioxidant enzymes CuZnSOD, MnSOD, and catalase can be inactivated by photodynamically generated (1)O(2) in nucleated mammalian cells. These findings may be useful in the future development of antineoplastic adjuvant therapies that use photodynamic generation of (1)O(2) to inactivate antioxidant defenses with a goal of sensitizing tumor cells to prooxidant-generating drugs.     

The mechanism of nitric oxide and/or superoxide cytotoxicity in endothelial cells.

We examined the mechanism of nitric oxide (NO) and/or superoxide (O2-)-induced cytotoxicity and the importance of thiols in endothelial cells by treating the cells with superoxide dismutase (SOD), catalase (CAT) and hemoglobin (Hb). Pyrogallol, a O2 generator and precursor of hydrogen peroxide (H2O2), had potent cytotoxic effects on the endothelial cells, but this effect was completely abolished by SOD/CAT. Hb, a NO scavenger, protected the endothelial cells from sodium nitroprusside-induced cytotoxicity. The cytotoxic effect of 3-morpholinosydnonimine (SIN-1), which is thought to form peroxynitrite (ONOO-) as a simultaneous O2- and NO generator, was completely blocked by SOD/CAT or Hb. On the other hand, pretreatment of endothelial cells with diethylmaleate, a glutathione depleter, aggravated the cytotoxicity induced by SIN-1, which was prevented by addition of exogenous glutathione and/or SOD/CAT. These data suggest that the cytotoxicity induced by NO, O2- and ONOO- can be blocked by glutathione, and that this is an important cellular protective mechanism against these reactive oxygen species.     

Expression of reactive oxygen-species related enzymes in endothelial cells stimulated with glycated lipoproteins

Glycated lipoproteins, which elevate the blood in diabetic patients, cause direct attenuation of endothelial function. Increased glycation of apolipoproteins may play a trigger role in the accelerated development of atherosclerosis in the patient with diabetes. Here we assessed whether glycated lipoproteins affect on the endothelial function with particular emphasis on the role of reactive oxygen species in vitro. Incubation of human aortic endothelial cells(HAEC) with glycated LDL had little influence on the expression of antioxidant enzymes such as nitric oxide synthase(NOS), Cu2+Zn(2+)-superoxide dismutase (Cu2+Zn(2+)-SOD), catalase, and p22 phox in the cells. In contrast, exposure of glycated HDL induced a marked decrease of Cu2+Zn(2+)-SOD, catalase, and endothelial NOS as well as a slight increase of p22 phox in HAEC in term of both protein and mRNA expression, suggesting that increased formation of reactive oxygen species such as O2- and OH radical participate in the deterioration for the function of vascular endothelial cells in diabetic patients.     

Superoxide dismutase and pulmonary oxygen toxicity: lessons from transgenic and knockout mice (Review)

Superoxide (O2-) has been implicated in the pathogenesis of pulmonary O2 toxicity. The studies using transgenic and knockout mice of each of the three isoforms of superoxide dismutase (SOD) e.g. , CuZnSOD, MnSOD and extracellular SOD (EC-SOD), have demonstrated that O2- produced in the mitochondria from its electron transport system and extracellular O2- generated by infiltrating neutrophils, and possibly its derivatives e.g., hydroxyl radical and peroxynitrite, are important mediators of hyperoxia-induced pulmonary injury, while cytoplasmic O2- plays a limited, if any, role in the pathogenesis of pulmonary O2 toxicity. Distal airway epithelial cells including type II alveolar and non-ciliated bronchiolar epithelial cells, are important targets for O2 radicals under the hyperoxic condition. The accessibility of these distal airway epithelial cells to in vivo gene transfer through the tracheal route of administration, suggests the potential for in vivo transfer of MnSOD and EC-SOD genes as a future approach in the prevention of pulmonary O2 toxicity.     

Respiratory activity of isolated rat liver mitochondria following in vitro exposure to oxygen species: a threshold study

Respiratory activity of isolated rat liver mitochondria was assayed following in vitro exposure to oxygen radicals. Our results show that mitochondrial respiration is more sensitive to O2.(-) than to H2O2. However, ferrous ions drastically enhance the toxicity of the enzymatic system generating H2O2 because of the production of the hydroxyl radicals. A protection against those oxygen species could be given by SOD in the xanthine/xanthine oxidase system and by catalase with the glucose/glucose oxidase system. The most damaging system was the combination of Fe2+ with H2O2. In this case, OH. is formed in a Fenton-like reaction. The fact that the OH. is the most damaging molecule accounts for the finding that catalase and desferrioxamine were efficient protectors in this system. Threshold levels of O2.(-) and H2O2 able to inhibit the mitochondrial respiration have been estimated. It is concluded that under normal respiration such thresholds are not reached in vivo and that the impairment of the mitochondrial respiratory activity does not seem to originate only from the natural free radical production in those organelles. However, if the production of free radicals is such to exceed the defense capability, like under oxidative stress, then the critical threshold can be surpassed and the respiration impaired leading to irreversible damages.     

Response of human endothelial cell antioxidant enzymes to hyperoxia.

To explore the level of regulation of the expression of the major antioxidant enzymes in response to hyperoxia, we exposed human umbilical vein endothelial cells to 95% O2 for 3 and 5 days and measured (1) the steady-state mRNA levels, (2) the activities, and (3) the immunoreactive content of CuZn and Mn superoxide dismutases (SOD), catalase (CAT), and glutathione peroxidase (GP). We found that a 3-day exposure to 95% O2 caused (1) an increase in CuZnSOD mRNA (by 41%), CAT mRNA (by 26%), and GP mRNA (by 173%); (2) an increase in CuZnSOD activity (by 30%), a decrease in CAT activity (by 37%), and an increase in GP activity (by 60%); and (3) an increase in CuZnSOD immunodetectable protein (by 26%) and a loss in CAT immunoreactive protein (by 27%). After a 5-day exposure to 95% O2, there was (1) a 93% increase in CuZnSOD mRNA, a 71% increase in CAT mRNA, and a 127% increase in GP mRNA; (2) a 56% increase in CuZnSOD activity, a 70% decrease in CAT activity, and an 89% increase in GP activity; and (3) a 35% increase in CuZnSOD immunoreactive protein and a 55% loss in CAT immunoreactive protein. There was no change in the steady-state MnSOD mRNA level after 3 days in 95% O2, but a 100% increase was observed on day 5 of oxygen exposure. MnSOD activity was unchanged in cells exposed to hyperoxia for 3 and 5 days. These data suggest that, in human umbilical vein endothelial cells, the regulation of antioxidant enzymes expression in response to O2 is complex and exerted at different levels.     

Endothelial cell killing by neutrophils. Synergistic interaction of oxygen products and proteases.

Killing of rat pulmonary artery endothelial cells by activated polymorphonuclear leukocytes (PMNs), as measured at 4 hours, is catalase sensitive, iron dependent, and unaffected by addition of protease inhibitors. If the time course for exposure of endothelial cells to activated PMNs is extended to 18 hours, progressive injury occurs. Endothelial cell injury resulting at 18 hours is partially inhibited by catalase and partially inhibited by soybean trypsin inhibitor. Together, these two inhibitors function synergistically to protect the cells from injury. Exposure of endothelial cells to reagent H2O2 and purified proteolytic enzymes (trypsin, chymotrypsin, elastase, and cathepsin G) mimics the effects of activated PMNs: H2O2 alone is cytotoxic with maximal killing achieved by 4 hours; proteolytic enzymes produce cytotoxicity only at high concentrations and only after prolonged incubation (longer than 8 hours); and, in combination, H2O2 and proteolytic enzymes act synergistically. These data provide compelling evidence that PMN-mediated injury of endothelial cells involves interaction between oxygen products and proteases.     

The effect of non-transferrin-bound iron on murine T lymphocyte subsets: analysis by clonal techniques.

A number of different immunological properties have been attributed to iron (Fe3+ and Fe2+) and iron-binding proteins. However, in many previous studies, high concentrations of iron were used and cell-cell interactions were not excluded as a possible cause of the observed immunomodulatory effects. In this study, clonal techniques have been used to examine the effect of non-transferrin-bound iron (Fe3+) on the T lymphocyte subsets required for the generation of cytotoxic T lymphocytes (CTL). Concentrations of non-transferrin-bound Fe3+ of 10 microM or greater were shown to inhibit the generation of C57, BALB/c and CBA allo-specific CTL in bulk culture. Limit-dilution analysis revealed that: (i) Fe3+ reduced the cloning efficiency of CTL-precursors (CTL-P) by up to 96% without affecting the rate of clone growth; (ii) Fe3+ did not affect the cloning efficiency of allo-stimulated Ly-2-ve T cell precursors but reduced the rate of clone growth of these cells; (iii) Fe3+ enhanced, by more than 13-fold, the function of clones of Concanavalin A (Con A)-induced suppressor T lymphocytes (STL) which suppressed in vitro the development of CTL from their precursor cells. The data provide further evidence that low concentrations of non-transferrin-bound Fe3+, of the same order as those reported to be present in the serum of patients with iron-overload, have significant immunoregulatory properties.     

高糖对脑微血管内皮细胞抗氧化酶系统的影响

探讨高糖刺激时脑微血管内皮细胞锰超氧化物歧化酶及其他抗氧化酶的基因表达改变。通过体外培养小鼠脑微血管内皮细胞系bEnd.3;H2DCF荧光法检测高糖刺激下脑微血管内皮细胞短期内氧自由基（ROS）生成,RT-PCR检测高糖刺激下内皮细胞铜锌超氧化物歧化酶（SOD1）、锰超氧化物岐化酶（MnSOD）、脂质过氧化物酶（GPX）及触酶（CAT）的表达,并通过werstern blot法检测内皮细胞内MnSOD蛋白水平。结果表明：高糖刺激下脑微血管内皮细胞短期内氧自由基（ROS）产生明显增多并持续增高;高糖刺激10小时抗氧化酶MnSOD,GPX,CAT的mRNA水平无明显变化,SOD1 mRNA水平略有增高;SOD1、MnSOD的mRNA水平在高糖刺激24小时后有明显增加,但是MnSOD蛋白水平在高糖刺激48小时内并无明显增加;抗氧化剂白藜芦醇或表没食子儿茶素没食子酸酯（EGCG）不能上调MnSOD蛋白水平。以上数据表明,高糖刺激能诱导细胞内多种抗氧化酶包括MnSOD基因表达上调,但也能抑制MnSOD蛋白水平的增加。     

Oxygen radicals potentiate the genetic toxicity of tobacco-specific nitrosamines

Tobacco-specific nitrosamines (TSNAs), nitrosonornicotine (NNN) and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are metabolites of nicotine and the major carcinogens in cigarette smoke. To evaluate the effect of oxygen radicals on TSNA-induced genetic damage, MRC-5 fetal human lung cells were exposed to NNN and NNK (5 mM) and DNA single-strand breaks measured. Both NNN and NNK produced a dose-dependent increase in strand breaks up to 10 mM which was cytotoxic. In combination with enzymatically-generated oxygen radicals, strand breakage increased by approximately 50% for both NNN and NNK. Oxygen radical scavengers (superoxide dismutase, catalase, mannitol) significantly reduced the DNA damage caused by both the TSNAs and TSNAs plus oxygen radicals, suggesting that the genotoxicity is radical-mediated. Because both superoxide dismutase and catalase were protective, the hydroxyl radical may be playing an important role in the mediation of the DNA damage observed.     

An oxygen release system to augment cardiac progenitor cell survival and differentiation under hypoxic condition

Stem cell therapy has the potential to regenerate heart tissue damaged by myocardial infarction (MI), but it experiences extremely low efficacy. One of the major causes is the inferior cell survival under hypoxic condition of the infarcted hearts. We examined whether an oxygen-releasing system capable of sustainedly supplying oxygen to stem cells would augment cell survival and cardiac differentiation under hypoxic condition mimicking that of the infarcted hearts. The oxygen-releasing system consisted of hydrogen peroxide (H(2)O(2))-releasing microspheres, catalase and an injectable, thermosensitive hydrogel. The microspheres were based on poly(lactide-co-glycolide) (PLGA) and a complex of H(2)O(2) and poly(2-vinlypyrridione) (PVP). The oxygen was generated after the released H(2)O(2) was decomposed by catalase. The hydrogel was designed to improve the retention of microspheres and stem cells in the beating heart tissue during myocardial injection. The oxygen-releasing system was capable of sustainedly releasing oxygen for at least two weeks. The release kinetics was dependent on the ratio of H(2)O(2)/VP. The hydrogel was based on N-isopropylacrylamide (NIPAAm), acrylic acid (AAc), and a macromer hydroxyethyl methacrylate-oligo(hydroxybutyrate) (HEMA-oHB). The hydrogel had a stiffness matching that of the heart tissue and was able to stimulate the cardiosphere-derived cells (CDCs) to differentiate into cardiomyocytes. Under hypoxic condition mimicking that of the infarcted hearts (1% O(2)), CDCs encapsulated in the hydrogel experienced massive cell death. Introduction of oxygen release in the hydrogel significantly augmented cell survival; no cell death was found after seven days of culture, and cells even grew after seven days. Under hypoxic condition, cardiac differentiation of CDCs was completely silenced in the hydrogel, as confirmed at both mRNA and protein levels. However, introduction of oxygen release restored the differentiation. These results demonstrate that the developed oxygen-releasing system has great potential to improve the efficacy of cardiac stem cell therapy.     

低氧对人脐带间充质干细胞生物学特性及增殖的影响简

目的探讨低氧培养条件对人脐带间充质干细胞（hUC-MSCs）的生长和增殖的影响。方法采用人脐带酶消化法分离培养hUC-MSCs,分别将细胞置人体积分数20％、10％、3％0,条件下培养,通过细胞成脂、成骨分化诱导及流式细胞仪检测其表面标志物.鉴定hUC-MSCs：绘制细胞生长曲线,用流式细胞仪检测细胞周期及凋亡情况。结果低氧条件培养hUC-MSCs具有成脂、成骨等多项分化的潜能：流式细胞仪检测呈CD105、CD73、CD90、CD44、HLA-ABC高表达,CDl06、CD29、CD45、CD34、HLA-DR低表达;体积分数3％O2培养组与正常体积分数20％O2培养组及体积分数10％0,培养组相比（各组倍增时间分别为17.2h、20.8h、18.9h）,细胞增殖速度加快（P〈0.05）;G0/G1期细胞减少（各组G0／G1期细胞数分别为77.11％±3.89％、83.92％±5.59％、80.19％±5.16％）,S期细胞增多（S期细胞数分别为15.73％±2.56％、10.91％±1.86％、13.31％-4-2.31％）,增殖指数升高（P〈0.05）;细胞凋亡率降低（流式细胞仪检测各组凋亡率分别为13.41％±1.39％、20.83％±1.81％、19.11％±2.44％）（P〈0.05）。结论体积分数3％O：持续培养可促进hUC-MSCs的增殖.减少细胞凋亡。     

Overexpression of manganese superoxide dismutase protects lung epithelial cells against oxidant injury

To determine whether overexpression of antioxidant enzymes in lung epithelial cells prevents damage from oxidant injury, stable cell lines were generated with complementary DNAs encoding manganese superoxide dismutase (MnSOD) and/or catalase (CAT). Cell lines overexpressing MnSOD, CAT, or MnSOD + CAT were assessed for tolerance to hyperoxia or paraquat. After exposure to 95% O(2) for 10 d, 44 to 57% of cells overexpressing both MnSOD and CAT and 37 to 47% of cells overexpressing MnSOD alone were viable compared with 7 to 12% of empty vector or parental cells (P < 0.05). To assess if viable cells were capable of cell division after hyperoxic exposures (up to 5 d), a clonogenicity assay was performed. The clonogenic potential of cells overexpressing MnSOD + CAT and MnSOD alone were significantly better than those expressing CAT alone or empty vector controls. In addition, 54 to 72% of cells overexpressing both MnSOD and CAT survived in 1 mM paraquat compared with 58 to 73% with MnSOD alone and 27% with control cells. Overexpression of CAT alone did not improve survival in hyperoxia or paraquat. The combination of MnSOD + CAT did not provide additional protection from paraquat. Data demonstrate that overexpression of MnSOD protects cells from oxidant injury and CAT offers additional protection from hyperoxic injury when co-expressed with MnSOD.     

Degranulation effect of ferric nitrilotriacetate (Fe3+-NTA) on the pancreatic islet beta-cells: its acute toxic effect on glucose metabolism

A single injection of ferric nitrilotriacetate (Fe3+-NTA) caused a transitory increase in plasma immunoreactive insulin (IRI) and plasma immunoreactive glucagon (IRG) in rats. They reached maximum levels at 2 days after injection and returned to the normal range at 10 days. At 2 days after Fe3+-NTA injection, blood glucose level was normal but the glucose tolerance test (GTT) was impaired. There was a further increase in plasma IRI level and IRG level was suppressed after glucose loading. At 10 days after Fe3+-NTA injection, glucose tolerance was normal and IRI also returned to the normal range. No degenerative changes were found on H.E.-stained rat pancreatic tissue sections after Fe3+-NTA injection. Histochemical staining, however, showed a reduction in beta-granules and heavy metals (Timm's granules) from islet cells in the central area of the rat pancreatic islet 1 to 3 days after injection of Fe3+-NTA. The fading remained in some islets even at 10 days after injection, but by then the beta-granule distribution was restored in most islet cells. The results indicate a single Fe3+-NTA injection induced transitory instability of the pancreatic islet beta-cell granules and the glucose intolerance with a hyperresponse of IRI.     

Reversible modulation of cell cycle kinetics in NIH/3T3 mouse fibroblasts by inducible overexpression of mitochondrial manganese superoxide dismutase

To study the mechanism(s) by which manganese-containing superoxide dismutase (MnSOD) mediates cellular growth inhibition, an inducible retroviral vector system regulated by the lac repressor was used to overexpress MnSOD protein in NIH/3T3 cells. Increased MnSOD activity led to decreased cell growth due to prolonged cell cycle transition times in G(1) and S phases without significant changes in G(2)/M phase. Changes in cell cycle transition time were reversible and tightly correlated with MnSOD levels. A transient increase of reactive oxygen species and concomitant decrease in mitochondrial membrane potential were documented following MnSOD induction. N-Acetyl-L-cysteine prevented growth inhibition by MnSOD. Our data suggest that MnSOD may serve a physiological function of regulating cell cycle progression through its prooxidant activity of generating hydrogen peroxide, resulting in coordination of mitochondrial redox state and cellular proliferation.