Enhancement of intracellular glutathione protects endothelial cells against oxidant damage

We studied the role of glutathione in the endothelial cell defense against H2O2 damage. Treatment of endothelial cells with buthionine sulfoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, depleted the cells of GSH, while L-2-oxothiazolidine-4-carboxylate, an effective intracellular cysteine delivery agent, markedly enhanced endothelial cell GSH concentration. Depletion of intracellular GSH sensitized the endothelial cells to injury by H2O2 either preformed or generated by the glucose-glucose oxidase system. In contrast, an increase of intracellular GSH protected the cells from H2O2 damage. There was an inverse, linear relationship between the intracellular GSH concentrations and killing of endothelial cells by H2O2. Our results suggest that enhancement of endothelial cell GSH may be an alternative approach toward the prevention of oxidant-induced endothelial damage such as adult respiratory distress syndrome.     

Effect of steroid on hyperoxia-induced ICAM-1 expression in pulmonary endothelial cells

Intercellular adhesion molecule-1 (ICAM-1) of the vascular endothelium plays a key role in the development of pulmonary oxygen toxicity. We studied the effect of steroid on hyperoxia-induced ICAM-1 expression using cultured endothelial cells in vitro. Human pulmonary artery endothelial cells (HPAECs) were cultured to confluence, and then the monolayers were exposed to either control (21% O(2)-5% CO(2)) or hyperoxic (90% O(2)-5% CO(2)) conditions with and without a synthetic glucocorticoid, methylprednisolone (MP). MP reduced hyperoxia-induced ICAM-1 and ICAM-1 mRNA expression in a dose-dependent manner. Neutrophil adhesion to hyperoxia-exposed endothelial cells was also inhibited by MP treatment. In addition, MP attenuated hyperoxia-induced H(2)O(2) production in HPAECs as assessed by flow cytometry. An electrophoretic mobility shift assay demonstrated that hyperoxia activated nuclear factor-kappaB (NF-kappaB) but not activator protein-1 (AP-1) and that MP attenuated hyperoxia-induced NF-kappaB activation dose dependently. With Western immunoblot analysis, IkappaB-alpha expression was decreased by hyperoxia and increased by MP treatment. These results suggest that MP downregulates hyperoxia-induced ICAM-1 expression by inhibiting NF-kappaB activation via increased IkappaB-alpha expression.     

Tyrosine phosphorylation of cortactin is required for H2O2-mediated injury of human endothelial cells

Injury of endothelial cells induced by reactive oxygen species plays an important role in the development of early stages of vascular diseases such as hypertension and atherosclerosis. Exposure of human umbilical vein endothelial cells to hydrogen peroxide (H(2)O(2)), a common form of reaction oxygen species, triggers a series of intracellular events, including actin cytoskeletal reorganization, cytoplasm shrinkage, membrane blebbing and protein-tyrosine phosphorylation. The effect of H(2)O(2) on endothelial cells is dramatically enhanced when a survival pathway involving extracellular signal-regulated kinase is blocked by PD098059. In contrast, the injury of endothelial cells mediated by H(2)O(2) is inhibited by PP2, a selective specific inhibitor for protein-tyrosine kinase Src. Cortactin, a filamentous actin (F-actin)-associated protein, becomes phosphorylated at tyrosine residues upon stimulation by H(2)O(2) in a manner dependent on the activity of Src. The level of tyrosine phosphorylation of cortactin is correlated with the formation of membrane blebs. Overexpression of wild-type cortactin tagged with green fluorescent protein in endothelial cells via a retroviral vector substantiates the H(2)O(2)-induced morphological changes, whereas overexpression of a green fluorescent protein-cortactin mutant deficient in tyrosine phosphorylation renders endothelial cells resistant to H(2)O(2). The functional role of cortactin in H(2)O(2)-mediated shape changes was also evaluated in NIH 3T3 cells. Stable 3T3 transfectants expressing wild-type cortactin in the presence of either H(2)O(2)/PD098059 or H(2)O(2) alone at 200 microm exhibited a dramatic shape change characterized by rounding up or aggregation. However, the similar changes were not detected with cells overexpressing a cortactin mutant deficient in tyrosine phosphorylation. These data demonstrate an important role of the Src/cortactin-dependent actin reorganization in the injury of endothelial cells mediated by reactive oxygen species.     

Resveratrol protects ROS-induced cell death by activating AMPK in H9c2 cardiac muscle cells

Resveratrol, one of polyphenols derived from red wine, has been shown to protect against cell death, possibly through the association with several signaling pathways. Currently numerous studies indicate that cardiovascular diseases are linked to the release of intracellular reactive oxygen species (ROS) often generated in states such as ischemia/reperfusion injury. In the present study, we investigated whether resveratrol has the capability to control intracellular survival signaling cascades involving AMP-activated kinase (AMPK) in the inhibitory process of cardiac injury. We hypothesized that resveratrol may exert a protective effect on damage to heart muscle through modulating of the AMPK signaling pathway. We mimicked ischemic conditions by inducing cell death with H(2)O(2) in H9c2 muscle cells. In this experiment, resveratrol induced strong activation of AMPK and inhibited the occurrence of cell death caused by treatment with H(2)O(2). Under the same conditions, inhibition of AMPK using dominant negative AMPK constructs dramatically abolished the effect of resveratrol on cell survival in H(2)O(2)-treated cardiac muscle cells. These results indicate that resveratrol-induced cell survival is mediated by AMPK in H9c2 cells and may exert a novel therapeutic effect on oxidative stress induced in cardiac disorders.     

Human serum catalase decreases endothelial cell injury from hydrogen peroxide.

Serum from normal human subjects contained variable amounts of catalase activity, which was inhibitable by heat, azide, trichloroacetic acid (TCA), or aminotriazole treatment. Serum also decreased hydrogen peroxide (H2O2) concentrations in vitro and H2O2-mediated injury to cultured endothelial cells. By comparison, heat-, azide-, TCA-, or aminotriazole-treated serum neither decreased H2O2 concentrations in vitro nor reduced H2O2-mediated damage to endothelial cells. We conclude that serum catalase activity can alter H2O2-dependent reactions. We speculate that variations in serum catalase activity may alter individual susceptibility to oxidant-mediated vascular disease or be a factor when added to test systems in vitro.     

Protection of human endothelial cells from oxidant injury by adenovirus-mediated transfer of the human catalase cDNA.

In a variety of disorders, endothelial cells are exposed to high levels of oxidants, generated within the cells and/or consequent to local inflammation. In the context of the sensitivity of endothelial cells to oxidant stress, particularly related to H2O2, we have designed a replication deficient recombinant adenovirus containing the human catalase cDNA (AdCL) to transfer the catalase cDNA to the endothelial cells, in order to augment intracellular anti-H2O2 protection. Human umbilical vein endothelial cells that were not infected or infected with control adenovirus maintained low levels of catalase mRNA. Endothelial cells infected with AdCL expressed AdCL-driven exogenous catalase mRNA, as early as 24 hr and at least for 7 days. Catalase protein levels were increased significantly over controls in cells infected with AdCL, as were catalase activity levels, with catalase activity correlated closely with levels of catalase protein. Importantly, when the endothelial cells were exposed to 500 microM H2O2, all the AdCL infected endothelial cells survived, compared to only 37% of the control cells. Thus, a recombinant adenovirus containing the human catalase cDNA is able to infect human endothelial cells in vitro and express high levels of functional intracellular catalase, protecting the cells against H2O2-mediated oxidant stress. These observations support the feasibility of the transfer of catalase cDNA to human endothelium to protect against oxidant injury.     

Zinc resistance impairs sensitivity to oxidative stress in HeLa cells: protection through metallothioneins expression.

To analyze the effects of high concentrations of zinc ions on oxidative stress protection, we developed an original model of zinc-resistant HeLa cells (HZR), by using a 200 microM zinc sulfate-supplemented medium. Resistant cells specifically accumulate high zinc levels in intracellular vesicles. These resistant cells also exhibit high expression of metallothioneins (MT), mainly located in the cytoplasm. Exposure of HZR to Zn-depleted medium for 3 or 7 d decreases the intracellular zinc content, but only slightly reduces MT levels of resistant cells. No changes of the intracellular redox status were detected, but zinc resistance enhanced H2O2-mediated cytotoxicity. Conversely, zinc-depleted resistant cells were protected against H2O2-induced cell death. Basal- and oxidant-induced DNA damage was increased in zinc resistant cells. Moreover, measurement of DNA damage on zinc-depleted resistant cells suggests that cytoplasmic metal-free MT ensures an efficient protection against oxidative DNA damage, while Zn-MT does not. This newly developed Zn-resistant HeLa model demonstrates that high intracellular concentrations of zinc enhance oxidative DNA damage and subsequent cell death. Effective protection against oxidative damage is provided by metallothionein under nonsaturating zinc conditions. Thus, induction of MT by zinc may mediate the main cellular protective effect of zinc against oxidative injury.     

Nitric oxide attenuates H(2)O(2)-induced endothelial barrier dysfunction: mechanisms of protection

Nitric oxide (.NO) attenuates hydrogen peroxide (H(2)O(2))-mediated injury in porcine pulmonary artery endothelial cells (PAECs) and modulates intracellular levels of cGMP and cAMP. We hypothesized that.NO attenuates H(2)O(2)-induced PAEC monolayer barrier dysfunction through cyclic nucleotide-dependent signaling mechanisms. To examine this hypothesis, cultured PAEC monolayers were treated with H(2)O(2), and barrier function was measured as transmonolayer albumin clearance. H(2)O(2) caused significant PAEC barrier dysfunction that was attenuated by intracellular as well as extracellular.NO generation.NO increased PAEC cGMP and cAMP levels, but treatment with inhibitors of soluble guanylate cyclase or protein kinase G did not abrogate.NO-mediated barrier protection. In contrast, H(2)O(2) decreased protein kinase A activity, and inhibiting protein kinase A abrogated the protective effect of.NO. H(2)O(2)-induced barrier dysfunction was not associated with decreased levels of cGMP or cAMP. 3-Isobutyl-1-methylxanthine and the cGMP analog 8-bromo-cGMP had little effect on H(2)O(2)-mediated endothelial barrier dysfunction, whereas 8-bromo-cAMP plus 3-isobutyl-1-methylxanthine was protective. These results indicate that.NO modulates vascular endothelial barrier function through cAMP-dependent signaling mechanisms.     

重组nkx2.5腺病毒抑制过氧化氢诱导的H9c2细胞凋亡

为研究心脏发育关键基因nkx2.5的功能及应用价值,构建Ad-Nkx2.5重组腺病毒,并检测nkx2.5过表达拮抗氧化应激损伤的效应及机制。采用AdEasy腺病毒表达系统构建Ad-Nkx2.5重组腺病毒,建立H2O2诱导H9c2心肌细胞凋亡模型,分别用Ad-Nkx2.5重组病毒或对照病毒感染细胞,采用Hoechst33342染色观察细胞形态变化、MTT法检测细胞存活率,免疫印迹检测caspase-3活化、细胞色素C的胞浆含量。并通过Real-timePCR检测凋亡相关基因bcl-2和bax表达。结果发现,nkx2.5过表达促进H9c2细胞存活,抑制H2O2诱导的caspase-3活化及线粒体细胞色素C的释放。Nkx2.5过表达上调bcl-2表达,显著下调H2O2诱导的bax表达。并发现H2O2对Nkx2.5核定位无明显影响。结果显示重组腺病毒介导的Nkx2.5过表达可通过调控凋亡相关基因表达,抑制线粒体凋亡途径,保护心肌细胞抗氧化损伤。     

Toxicity by pyruvate in HepG2 cells depleted of glutathione: role of mitochondria.

Several studies have shown that pyruvate can scavenge H(2)O(2) and protect from H(2)O(2)-mediated cell injury. Mitochondria are critical participants in the control of apoptotic and necrotic cell death. Mitochondrial GSH plays an important role in the maintenance of cell functions and viability by metabolism of oxygen free radicals generated by the respiratory chain. Since loss of GSH, especially mitochondrial GSH, is associated with increased production of reactive oxygen species and cell toxicity, the ability of pyruvate to protect against these actions was evaluated. Adding pyruvate to HepG2 cells depleted of GSH by treatment with l-buthionine sulfoximine (BSO) surprisingly caused loss of viability after 24 and 48 h of incubation. Anoxia, treatment with antioxidants, and infection with cytosolic catalase, and interestingly, catalase expressed in the mitochondrial compartment were able to rescue the HepG2 cells from this pyruvate plus BSO injury, suggesting a key role for H(2)O(2), and lipid peroxides as mediators in the cytotoxicity. This toxicity and cell death observed was linked to damage to the mitochondria as evidenced by the increased lipid peroxidation in total homogenate and mitochondrial fraction, loss of mitochondrial membrane potential, and a decrease in protein-sulfhydryl groups. The type of cell death observed under these conditions was a mixture of apoptosis and necrosis. These results suggest that the protective ability of pyruvate against oxidant damage requires a functional GSH pool, especially in the mitochondrial compartment, and that in the absence of GSH, pyruvate increases cell injury by damaging the mitochondria, presumably as a consequence of enhanced electron flow and reactive oxygen production by the respiratory chain.     

Effect of oxygen and endotoxin on lactate dehydrogenase release, 5-hydroxytryptamine uptake, and antioxidant enzyme activities in endothelial cells

We compared the effects of 95% O2 (hyperoxia) alone, endotoxin (20 ng/ml) alone, and 95% O2 plus endotoxin on the release of lactate dehydrogenase (LDH), uptake of 5-hydroxytryptamine (5-HT), and antioxidant enzyme activities in porcine pulmonary arterial and aortic endothelial cells in monolayer culture. Hyperoxia increased LDH release and decreased 5-HT in both endothelial cell types. Hyperoxia also caused a decrease in catalase (CAT) activity and an increase in total superoxide dismutase (SOD) and glutathione reductase (GSH-Red) activities in both cell types. Endotoxin alone had no effect on LDH release, 5-HT uptake, or antioxidant enzyme activities. However, endotoxin prevented the hyperoxic increase in LDH release and the hyperoxic decrease in 5-HT uptake. Endotoxin plus 95% O2 had no consistent effect on the antioxidant enzyme profile in pulmonary artery or aortic endothelial cells. These results indicate that (1) hyperoxia injures both pulmonary artery and aortic endothelial cells in culture and causes changes in the antioxidant enzyme profile that are similar in the two cell types; (2) hyperoxia-induced decreases in CAT activity and increases in SOD activity may be responsible for increased sensitivity of endothelial cells to O2 toxicity; and (3) endotoxin protects against hyperoxic injury to endothelial cells in vitro, but increases in antioxidant enzyme activities are not the mechanism for this protection.     

The endothelium-derived hyperpolarizing factor, H2O2, promotes metal-ion efflux in aortic endothelial cells: elemental mapping by a hard X-ray microprobe

Hydrogen peroxide (H(2)O(2)) is a physiologic oxidant implicated in vascular cell signaling, although little is known about the biochemical consequences of its reaction with endothelial cells. Submicrometer-resolution hard X-ray elemental mapping of cultured porcine aortic endothelial cells (PAEC) has provided data on the global changes for intracellular elemental density within PAEC and indicates an efflux of metal ions and phosphorus from the cytoplasm after H(2)O(2) treatment. The synchrotron-radiation-induced X-ray emission experiments (SRIXE) show that H(2)O(2)-treated cells are irregularly shaped and exhibit blebbing indicative of increased permeability due to the damaged membrane. The SRIXE results suggest that H(2)O(2)-induced damage is largely restricted to the cell membrane as judged by the changes to membrane and cytoplasmic components rather than the cell nucleus. The SRIXE data also provide a mechanism for cell detoxification as the metal-ion efflux resulting from the initial H(2)O(2)-mediated changes to cell membrane potentially limits intracellular metal-mediated redox processes through Fenton-like chemistry. They may also explain the increased levels of these ions in atherosclerotic plaques, regardless of whether they are involved in plaque formation. Finally, the SRIXE data support the notion that cultured endothelial cells exposed to H(2)O(2) respond with enhanced cellular metal-ion efflux into the extracellular space.     

MAPK pathway mediates the protective effects of onychin on oxidative stress-induced apoptosis in ECV304 endothelial cells

Our recent studies have shown that onychin could protect rabbit aortic rings from lysophosphatidylcholine-induced injury by preserving endothelium-dependent relaxation and alleviating acute endothelial damage mediated by oxidative stress. However, the effect of onychin on apoptosis of endothelial cells induced by oxidative stress was not evaluated. In the present study, we investigated the effect of onychin on Hydrogen Peroxide (H2O2) induced apoptosis of ECV304 endothelial cells. Cultured human umbilical vein endothelial cell line (ECV304) was pretreated with vehicle (DMSO), genistein, or different concentrations of onychin (0.1, 0.3, 1, 3, and 10 micromol/L) for 30 minutes and then exposed to 1 mmol/L H2O2 for 24 hours. Cell apoptosis was determined by TUNEL and flow cytometric analysis. Meanwhile, Western-blot was used to measure the expression of phospho-ERK1/2, phospho-p38 and caspase-3. Our data showed that onychin treatment exhibited a protective effect on ECV304 endothelial cells from H2O2-induced apoptosis in a concentration-dependent manner. Moreover, onychin attenuated H2O2-induced phosphorylation of p38MAPK and increased H2O2-induced phosphorylation of ERK1/2. Furthermore, onychin decreased the activation of caspase-3. The opposing effects of onychin on phosphorylation levels of p38MAPK and ERK1/2, and its caspase-3 inhibition might play a role in the beneficial effect of onychin on endothelial injury.     

Transferrin: a potential source of iron for oxygen free radical-mediated endothelial cell injury.

The ability of transferrin to potentiate oxygen free radical-mediated endothelial cell injury was assessed. 51Cr-labeled endothelial cells derived from rat pulmonary arteries (RPAECs) were incubated with hydrogen peroxide (H2O2) in the presence and absence of holosaturated human transferrin, and the effect of transferrin on H2O2-mediated endothelial cell toxicity was determined. Addition of holosaturated transferrin potentiated H2O2-mediated RPAEC cytotoxicity at concentrations of H2O2 greater than 10 microM, suggesting that transferrin may provide a source of iron for free radical-mediated endothelial cell injury. Free radical-mediated injury is dependent on non-protein-bound iron. The ability of RPAECs to facilitate the release of iron from transferrin was assessed. We determined that RPAECs facilitate the release of transferrin-derived iron by reduction of transferrin-bound ferric iron (Fe3+) to ferrous iron (Fe2+). The reduction and release of transferrin-derived Fe2+ were inhibited by apotransferrin and chloroquine, indicating a dependence on receptor-specific binding of transferrin to the RPAEC cell surface, with subsequent endocytosis, acidification, and reduction of transferrin-bound Fe3+ to Fe2+. The release of transferrin-derived Fe2+ was potentiated by diethyldithiocarbamate, an inhibitor of intracellular superoxide dismutase (SOD). In contrast, exogenous SOD did not alter iron release, suggesting that intracellular superoxide anion (O2-) may play an important role in mediating the reduction and release of transferrin-derived iron. Results of this study suggest that transferrin may provide a source of iron for oxygen free radical-mediated endothelial cell injury and identify a novel mechanism by which endothelial cells may mediate the reduction and release of transferrin-derived iron.     

Dimethylthiourea prevents hydrogen peroxide and neutrophil mediated damage to lung endothelial cells in vitro and disappears in the process

Dimethylthiourea (DMTU) progressively disappeared following reaction with increasing amounts of hydrogen peroxide (H2O2) in vitro. DMTU disappearance following reaction with H2O2 was inhibited by addition of catalase, but not aminotriazole-inactivated catalase (AMT-catalase), superoxide dismutase (SOD), mannitol, benzoate or dimethyl sulfoxide (DMSO) in vitro. By comparison, DMTU disappearance did not occur following addition of histamine, oleic acid, elastase, trypsin or leukotrienes in vitro. Addition of DMTU also decreased H2O2-mediated injury to bovine pulmonary artery endothelial cells (as reflected by LDH release) and DMTU disappeared according to both added amounts of H2O2 and corresponding degrees of injury. DMTU disappearance was also relatively specific for reaction with H2O2 in suspensions of endothelial cells where it was prevented by addition of catalase, but not AMT-catalase or SOD and did not occur following sonication or treatment with elastase, trypsin or leukotrienes. Addition of washed human erythrocytes (RBC) also prevented both H2O2 mediated injury and corresponding DMTU decreases in suspensions of endothelial cells. In addition, phorbol myristate acetate (PMA) and normal neutrophils, but not O2 metabolite deficient neutrophils from patients with chronic granulomatous disease (CGD), caused DMTU disappearance in vitro which was decreased by simultaneous addition of catalase, but not SOD, sodium benzoate or DMSO. Finally, addition of normal neutrophils (but not CGD neutrophils) and PMA caused DMTU disappearance and increased the concentrations of the stable prostacyclin derivative (PGF1 alpha) in supernatants of endothelial cell suspensions. In parallel, DMTU also decreased PMA and neutrophil-mediated PGF1 alpha increases in supernatants from endothelial cell monolayers. Our results indicate that DMTU can decrease H2O2 or neutrophil mediated injury to endothelial cells and that simultaneous measurement of DMTU disappearance can be used to improve assessment of the presence and toxicity of H2O2 as well as the H2O2 inactivating ability of scavengers, such as RBC, in biological systems.     

Inhibition of organic anion transport in endothelial cells by hydrogen peroxide.

ATP loss is a prominent feature of cellular injury induced by oxidants or ischemia. How reduction of cellular ATP levels contributes to lethal injury is still poorly understood. In this study we examined the ability of H2O2 to inhibit in a dose-dependent manner the extrusion of fluorescent organic anions from bovine pulmonary artery endothelial cells. Extrusion of fluorescent organic anions was inhibited by probenecid, suggesting an organic anion transporter was involved. In experiments in which ATP levels in endothelial cells were varied by treatment with different degrees of metabolic inhibition, it was determined that organic anion transport was ATP-dependent. H2O2-induced inhibition of organic anion transport correlated well with the oxidant's effect on cellular ATP levels. Thus H2O2-mediated inhibition of organic anion transport appears to be via depletion of ATP, a required substrate for the transport reaction. Inhibition of organic anion transport directly by probenecid or indirectly by metabolic inhibition with reduction of cellular ATP levels was correlated with similar reductions of short term viability. This supports the hypothesis that inhibition of organic anion transport after oxidant exposure or during ischemia results from depletion of ATP and may significantly contribute to cytotoxicity.     

Involvement of the CD11b/CD18 integrin, but not of the endothelial cell adhesion molecules ELAM-1 and ICAM-1 in tumor necrosis factor-alpha-induced neutrophil toxicity.

TNF-alpha can incite neutrophil-mediated endothelial cell damage and neutrophil H2O2 release. Both effects require adherent neutrophils. Using specific mAb, we showed in this in vitro study that the CD18 beta 2-chain and the CD11b alpha M-chain of the CD11/CD18 integrin heterodimer have a major role in both TNF-alpha-induced neutrophil-mediated detachment of human umbilical vein endothelial cells and H2O2 release by TNF-alpha-activated human neutrophils. In contrast to anti-CD18 mAb, which consistently prevented neutrophil activation, anti-CD11a mAb and two of three anti-CD11b mAb did not reduce endothelial cell detachment and neutrophil H2O2 release, although they decreased neutrophil adhesion to human umbilical vein endothelial cells. mAb 904, directed against the bacterial LPS binding region of CD11b, reduced endothelial cell detachment for about 40% and neutrophil H2O2 release for more than 50%, demonstrating that CD11b/CD18 is engaged in TNF-induced neutrophil activation. Dependence on CD11b/CD18 could not be overcome by CD18-independent anchoring of neutrophils via PHA. Additionally, neither induction of increased expression of the endothelial cell adhesion molecules ICAM-1 and ELAM-1, nor subsequent addition of specific mAb, influenced endothelial cell injury or H2O2 release by TNF-activated neutrophils. Interaction with ICAM-1 and ELAM-1 therefore appears not to induce additional activation of TNF-stimulated neutrophils. These studies suggest that a specific, CD11b/CD18-mediated signal, instead of adherence only, triggers toxicity of TNF-activated neutrophils.     

Cellular sites of H2O2-induced damage and their protection by nitroxides

While the exact mechanism of H2O2-induced cytotoxicity is unknown, there is considerable evidence implicating DNA as a primary target. A recent study showed that a cell-impermeable nitroxide protected mammalian cells from H2O2-induced cell killing and suggested that the protection was mediated through cell membrane-bound or extracellular factors. To further define the protective properties of nitroxides, Chinese hamster V79 cells were exposed to H2O2 with or without cell-permeable and impermeable nitroxides and selected metal chelators. EPR spectroscopy and paramagnetic line broadening agents were used to distinguish between intra- and extracellular nitroxide distribution. To study the effectiveness of nitroxide protection, in the absence of a cell membrane, H2O2-mediated damage to supercoiled plasmid DNA was evaluated. Both deferrioxamine and Tempol cross the cell membrane, and inhibited H2O2-mediated cell killing, whereas the cell-impermeable DTPA and nitroxide, CAT-1, failed to protect. Similar protective effects of the chelators and nitroxides were observed when L-histidine, which enhances intracellular injury, was added to H2O2. In contrast, when damage to plasmid DNA was induced (in the absence of a cell membrane), both nitroxides were protective. Collectively, these results do not support a role for membrane-bound or extracellular factors in mediating H2O2 cytotoxicity in mammalian cells.