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Inflammatory agents, microbial products, or stromal factors pre‐activate or prime neutrophils to respond to activating stimuli in a rapid and aggressive manner. Primed neutrophils exhibit enhanced chemotaxis, phagocytosis, and respiratory burst when stimulated by secondary activating stimuli. We previously reported that Triggering Receptor Expressed on Myeloid cells‐1 (TREM‐1) mediates neutrophil effector functions such as increased superoxide generation, transepithelial migration, and chemotaxis. However, it is unclear whether TREM‐1 is required for the process of priming itself or for primed responses to subsequent stimulation. To investigate this, we utilized in vitro and in vivo differentiated neutrophils that were primed with TNF‐α and then stimulated with the particulate agonist, opsonized zymosan (OpZ). Bone marrow progenitors isolated from WT and Trem‐1–/–mice were transduced with estrogen regulated Homeobox8 (ER‐Hoxb8) fusion transcription factor and differentiated in vitro into neutrophils following estrogen depletion. The resulting neutrophils expressed high levels of TREM‐1 and resembled mature in vivo differentiated neutrophils. The effects of priming on phagocytosis and oxidative burst were determined. Phagocytosis did not require TREM‐1 and was not altered by priming. In contrast, priming significantly enhanced OpZ‐induced oxygen consumption and superoxide production in WT but not Trem‐1–/–neutrophils indicating that TREM‐1 is required for primed oxidative burst. TREM‐1‐dependent effects were not mediated during the process of priming itself as priming enhanced degranulation, ICAM‐1 shedding, and IL‐1ß release to the same extent in WT and Trem‐1–/‐neutrophils. Thus, TREM‐1 plays a critical role in primed phagocytic respiratory burst and mediates its effects following priming. TREM‐1 is required for primed neutrophil oxidative burst

Ferumoxytol (FMX) is an iron oxide nanoparticle that is FDA approved for the treatment of iron deficiency anemia. FMX contains an Fe3O4core. Currently, the redox chemistry of Fe3O4nanoparticles remains relatively unexplored. FMX has recently gained interest as an anti-cancer agent. Ionizing radiation (IR) is a treatment modality employed to treat several types of cancer. Utilizing electron paramagnetic resonance (EPR) spectroscopy, we found that the products produced from the radiolysis of water can oxidize the Fe3O4core of FMX. Because of the limited diffusion of the HO2•and HO•produced, these highly oxidizing species have little direct effect on FMX oxidation. We have determined that H2O2is the primary oxidant of FMX. In the presence of labile Fe2+, we found that reducing species generated from the radiolysis of H2O are able to reduce the Fe3+sites of the Fe3O4core. Importantly, we also have shown that IR stimulates the release of ferric iron from FMX. Because of its release of iron, FMX may serve as an adjuvant to enhance radiotherapy.

Deficiency of the Nox2 (gp91phox) catalytic subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a genetic cause of X-linked chronic granulomatous disease, a condition in which patients are prone to infection resulting from the loss of oxidant production by neutrophils. Some studies have suggested a role for superoxide derived from Nox2 NADPH oxidase in platelet activation and thrombosis, but data are conflicting. Using a rigorous and comprehensive approach, we tested the hypothesis that genetic deficiency of Nox2 attenuates platelet activation and arterial thrombosis. Our study was designed to test the genotype differences within male and female mice. Using chloromethyl-dichlorodihydrofluorescein diacetate, a fluorescent dye, as well as high-performance liquid chromatography analysis with dihydroethidium as a probe to detect intracellular reactive oxygen species (ROS), we observed no genotype differences in ROS levels in platelets. Similarly, there were no genotype-dependent differences in levels of mitochondrial ROS. In addition, we did not observe any genotype-associated differences in platelet activation, adhesion, secretion, or aggregation in male or female mice. Platelets from chronic granulomatous disease patients exhibited similar adhesion and aggregation responses as platelets from healthy subjects. Susceptibility to carotid artery thrombosis in a photochemical injury model was similar in wild-type and Nox2-deficient male or female mice. Our findings indicate that Nox2 NADPH oxidase is not an essential source of platelet ROS or a mediator of platelet activation or arterial thrombosis in large vessels, such as the carotid artery.

Deficiency of the Nox2 (gp91phox) catalytic subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a genetic cause of X-linked chronic granulomatous disease, a condition in which patients are prone to infection resulting from the loss of oxidant production by neutrophils. Some studies have suggested a role for superoxide derived from Nox2 NADPH oxidase in platelet activation and thrombosis, but data are conflicting. Using a rigorous and comprehensive approach, we tested the hypothesis that genetic deficiency of Nox2 attenuates platelet activation and arterial thrombosis. Our study was designed to test the genotype differences within male and female mice. Using chloromethyl-dichlorodihydrofluorescein diacetate, a fluorescent dye, as well as high-performance liquid chromatography analysis with dihydroethidium as a probe to detect intracellular reactive oxygen species (ROS), we observed no genotype differences in ROS levels in platelets. Similarly, there were no genotype-dependent differences in levels of mitochondrial ROS. In addition, we did not observe any genotype-associated differences in platelet activation, adhesion, secretion, or aggregation in male or female mice. Platelets from chronic granulomatous disease patients exhibited similar adhesion and aggregation responses as platelets from healthy subjects. Susceptibility to carotid artery thrombosis in a photochemical injury model was similar in wild-type and Nox2-deficient male or female mice. Our findings indicate that Nox2 NADPH oxidase is not an essential source of platelet ROS or a mediator of platelet activation or arterial thrombosis in large vessels, such as the carotid artery.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of pain, fever, and inflammation. Long-term use of these drugs is associated with significant gastric injury. Activated neutrophils and oxidative stress seem to play a significant role in NSAID-induced gastric mucosal damage. The objective of our study is to examine the protective effects of an antioxidant and anti-inflammatory enzyme, heme oxygenase-1 (HO-1), in NSAID-induced gastric injury.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of pain, fever, and inflammation. Long-term use of these drugs is associated with significant gastric injury. Activated neutrophils and oxidative stress seem to play a significant role in NSAID-induced gastric mucosal damage. The objective of our study is to examine the protective effects of an antioxidant and anti-inflammatory enzyme, heme oxygenase-1 (HO-1), in NSAID-induced gastric injury. Mice were intraperitoneally injected with indomethacin (10 mg/kg) or sham. A specific inducer of HO-1, cobalt protoporphyrin (5 mg/kg), was given 24 hours before indomethacin to allow for the expression of HO-1. Controls received sham treatment. Twenty-four hours after indomethacin injection, gastric tissue damage was examined with histology. HO-1 expression was measured with immunoblot; cytokine levels were measured with enzyme-linked immunosorbent assay. Neutrophil infiltration was quantified with myeloperoxidase assay. Using electron paramagnetic resonance and desferrioxamine, we measured the labile iron pool in the mouse stomach as a marker of oxidative stress. Indomethacin caused gastric inflammation and ulcers, neutrophil activation, and increased tissue expression of interleukin-6 and tumor necrosis factor-alpha in mice. Inducing HO-1 with cobalt protoporphyrin reduced gastric inflammation, number of stomach ulcers, tissue neutrophil activation, and proinflammatory cytokine expression caused by indomethacin. These findings suggest that the induction of an anti-inflammatory and cytoprotective enzyme HO-1 may be a strategy to overcome the gastrointestinal adverse effects limiting the use of NSAIDs.

Cellular antioxidant enzymes protect against damage caused by exposure to endogenous or exogenous prooxidants. Singlet oxygen (1O2) 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 1O2 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 1O2, 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 1O2 quencher, protected against the inactivation of cellular CuZnSOD, MnSOD, and catalase enzymes induced by photodynamically generated 1O2. The conclusion from these experiments is that the primary cellular antioxidant enzymes CuZnSOD, MnSOD, and catalase can be inactivated by photodynamically generated 1O2 in nucleated mammalian cells. These findings may be useful in the future development of antineoplastic adjuvant therapies that use photodynamic generation of 1O2 to inactivate antioxidant defenses with a goal of sensitizing tumor cells to prooxidant-generating drugs.

Cells oxidize molecules to generate energy and to make the materials to build and support the structures and functions needed for life. However, unwanted oxidations can damage these same structures and impair function. Lipids (the lipids in membranes and lipoproteins) are targets of unwanted oxidations. The primary mechanism of these oxidations is free radical-mediated chain reactions. Here we provide an overview of how electron paramagnetic resonance (EPR) can be used to detect the free radicals formed during lipid peroxidation. Although direct detection of lipid-derived radicals has been accomplished, the approach is not feasible for detecting these radicals in cells. Spin trapping with α-(4-pyridyl-1-oxide)-N-tert-butylnitrone and 5,5-dimethyl-pyrroline-1-oxide has provided the most information on cellular lipid peroxidation. We present some considerations for successful detection of lipid radicals by EPR.

Free radical injury is thought to play a significant role in the pathogenesis of several disease processes in low birth weight premature infants including retinopathy of prematurity and necrotizing enterocolitis. Because iron is a known catalyst in free radical–mediated oxidation reactions, the objectives of the present in vitrostudies were to determine whether after exposure to air 1) iron present in infant formula, or that added to human milk or formula as medicinal iron or as iron contained in human milk fortifier, increases free radical and lipid peroxidation products; and 2) recombinant human lactoferrin added to formula or human milk attenuates iron-mediated free radical formation and lipid peroxidation. Before adding medicinal iron to formula and human milk, significantly more ascorbate and -hydroxyethyl radical production and more lipid peroxidation products (i.e.thiobarbituric acid reactive substances, malondialdehyde, and ethane) were observed in formula. After the addition of medicinal iron to either formula or human milk, further increases were observed in free radical and lipid peroxidation products. When iron-containing human milk fortifier was added to human milk, free radicals also increased. In contrast, the addition of apo-recombinant human lactoferrin to formula or human milk decreased the levels of oxidative products when medicinal iron or human milk fortifier was present. We speculate that the presence of greater concentration of iron and the absence of lactoferrin in formula compared with human milk results in greater in vitrogeneration of free radicals and lipid peroxidation products. Whether iron-containing formula with lactoferrin administered enterally to preterm infants will result in less free radical generation in vivohas yet to be established.

Free radical injury is thought to play a significant role in the pathogenesis of several disease processes in low birth weight premature infants including retinopathy of prematurity and necrotizing enterocolitis. Because iron is a known catalyst in free radical–mediated oxidation reactions, the objectives of the present in vitro studies were to determine whether after exposure to air 1) iron present in infant formula, or that added to human milk or formula as medicinal iron or as iron contained in human milk fortifier, increases free radical and lipid peroxidation products; and 2) recombinant human lactoferrin added to formula or human milk attenuates iron-mediated free radical formation and lipid peroxidation. Before adding medicinal iron to formula and human milk, significantly more ascorbate and a-hydroxyethyl radical production and more lipid peroxidation products (i.e. thiobarbituric acid reactive substances, malondialdehyde, and ethane) were observed in formula. After the addition of medicinal iron to either formula or human milk, further increases were observed in free radical and lipid peroxidation products. When iron-containing human milk fortifier was added to human milk, free radicals also increased. In contrast, the addition of apo-recombinant human lactoferrin to formula or human milk decreased the levels of oxidative products when medicinal iron or human milk fortifier was present. We speculate that the presence of greater concentration of iron and the absence of lactoferrin in formula compared with human milk results in greater in vitro generation of free radicals and lipid peroxidation products. Whether iron-containing formula with lactoferrin administered enterally to preterm infants will result in less free radical generation in vivo has yet to be established.

Matrix metalloproteinases (MMPs) participate in cell migration and remodeling processes by affecting the extracellular matrix. MMP-2 is thought to be involved in cancer cell invasiveness. It has been proposed that the activity of MMP-2 can be modulated by intracellular reactive oxygen species (ROS)/reactive nitrogen species. We hypothesized that manganese superoxide dismutase (MnSOD) could mediate MMP-2 activity by changing the intracellular ROS level and that nitric oxide (⋅NO) may be involved in this process. Human breast cancer MCF-7 cells were stably transfected with plasmids containing MnSOD cDNA. A 2–30-fold increase of MnSOD protein and activity was observed in four clones. Our data demonstrated that overexpression of MnSOD stimulated the activation of MMP-2 with a corresponding elevation of ROS. A decrease in ROS by ebselen, a glutathione peroxidase mimetic, or by transduction of adenovirus containing human catalase or glutathione peroxidase cDNA abolished the effect of MnSOD on MMP-2 activation. Treatment of MCF-7 cells with antimycin A or rotenone increased intracellular ROS production and MMP-2 activation simultaneously. Our data also showed a suppression of endothelial nitric-oxide synthase expression that was accompanied by decreased ⋅NO production in MnSOD-overexpressing cells. However, the changes in endothelial nitric-oxide synthase and⋅NO did not correlate with the MnSOD activity. Corresponding changes of MMP-2 activity after the addition of a NOS inhibitor (NG-amino-l-arginine) or a⋅NO donor ((Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate) to the cells suggested the possibility that ⋅NO may be involved in the MnSOD-mediated MMP-2 activation pathway. These results indicate that MnSOD induces MMP-2 activity by regulation of intracellular ROS and imply that signaling pathways involving ⋅NO may also be involved in the MnSOD mediation of MMP-2 activity.

We hypothesized that premature (PT) infants' mother's milk may provide antioxidant advantages compared with milk from mothers of full-term (FT) infants, and human milk may provide antioxidant properties not seen in infant formulas. We designed three experiments to test these hypotheses. Experiment 1 assessed resistance to oxidative stress of human milk and formulas designed for FT and PT infants. Experiment 2 determined differences in resistance to oxidative stress between milk from mothers of FT and PT infants, including analysis of catalase activity. Experiment 3 examined factors in human milk that may account for increased resistance to oxidative stress. In experiment 1, we induced physiologic oxidative stress in human milk (n = 5) and formula (n = 2) and measured ascorbate radical using electron paramagnetic resonance. Results indicated the following: 1) during oxidative stress, ascorbate may be spared in human milk compared with formula; 2) ascorbate radical production is more intense in formula compared with human milk, with or without oxidative stress; and 3) oxygen consumption in human milk is less than that in formula, with or without oxidative stress. In experiment 2, milk samples were collected from mothers of PT (n = 28) and FT (n = 17) infants at wk 1, 2, and 12 of lactation. No differences in oxygen consumption after oxidative stress appeared between PT and FT milk. Catalase levels in human milk increased with time. In experiment 3, addition of catalase, superoxide dismutase, and glutathione peroxidase to formulas (n = 4) increased resistance to oxidative stress. Denaturing endogenous enzymes did not decrease the ability of human milk to resist oxidative stress. Ferrous sulfate plus vitamin C added to human milk and formulas fortified with iron increased oxidative stress. Addition of iron chelators to formula reduced oxidative stress. In conclusion, human milk has better antioxidant protection than do formulas, perhaps because of the higher iron content of formulas. Milk from mothers of PT and FT infants has equal resistance to oxidative stress.

We hypothesized that premature (PT) infants’ mother’s milk may provide antioxidant advantages compared with milk from mothers of full-term (FT) infants, and human milk may provide antioxidant properties not seen in infant formulas. We designed three experiments to test these hypotheses. Experiment 1 assessed resistance to oxidative stress of human milk and formulas designed for FT and PT infants. Experiment 2 determined differences in resistance to oxidative stress between milk from mothers of FT and PT infants, including analysis of catalase activity. Experiment 3 examined factors in human milk that may account for increased resistance to oxidative stress. In experiment 1, we induced physiologic oxidative stress in human milk (n5) and formula (n2) and measured ascorbate radical using electron paramagnetic resonance. Results indicated the following 1) during oxidative stress, ascorbate may be spared in human milk compared with formula; 2) ascorbate radical production is more intense in formula compared with human milk, with or without oxidative stress; and 3) oxygen consumption in human milk is less than that in formula, with or without oxidative stress. In experiment 2, milk samples were collected from mothers of PT (n28) and FT (n17) infants at wk 1, 2, and 12 of lactation. No differences in oxygen consumption after oxidative stress appeared between PT and FT milk. Catalase levels in human milk increased with time. In experiment 3, addition of catalase, superoxide dismutase, and glutathione peroxidase to formulas (n4) increased resistance to oxidative stress. Denaturing endogenous enzymes did not decrease the ability of human milk to resist oxidative stress. Ferrous sulfate plus vitamin C added to human milk and formulas fortified with iron increased oxidative stress. Addition of iron chelators to formula reduced oxidative stress. In conclusion, human milk has better antioxidant protection than do formulas, perhaps because of the higher iron content of formulas. Milk from mothers of PT and FT infants has equal resistance to oxidative stress.

Reactive oxygen species have been shown to play important roles in v-Ha-Ras mitogenic signaling. We hypothesized that v-Ha-Ras overexpression would induce superoxide production, and therefore modify expression of the primary antioxidant enzyme system. We have demonstrated that immortal rat kidney epithelial cells stably transduced with constitutively active v-Ha-ras produced significantly larger amounts of superoxide radical than wildtype or vector-transfected control cells. The levels of the primary antioxidant enzymes copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase, and glutathione peroxidase were increased in the superoxide-overproducing cells. DNA-binding activities of the transcription factors activator protein-1, activator protein-2, and nuclear factor-κB were all enhanced in the superoxide-overproducing cells. These v-Ha-ras transduced cells also had a shortened cell doubling time and higher plating efficiency, and displayed greater constitutive levels of phosphorylated mitogen-activated protein kinases. These data demonstrate that v-Ha-Ras overexpression increases superoxide production and this apparently affects a wide variety of cell signaling and redox systems.

OBJECTIVES

Redox state is a term used widely in the research field of free radicals and oxidative stress. Unfortunately, it is used as a general term referring to relative changes that are not well defined or quantitated. In this review we provide a definition for the redox environment of biological fluids, cell organelles, cells, or tissue. We illustrate how the reduction potential of various redox couples can be estimated with the Nernst equation and show how pH and the concentrations of the species comprising different redox couples influence the reduction potential. We discuss how the redox state of the glutathione disulfide-glutathione couple (GSSG/2GSH) can serve as an important indicator of redox environment. There are many redox couples in a cell that work together to maintain the redox environment; the GSSG/2GSH couple is the most abundant redox couple in a cell. Changes of the half-cell reduction potential (Ehc) of the GSSG/2GSH couple appear to correlate with the biological status of the cell: proliferation Ehc≈ −240 mV; differentiation Ehc≈ −200 mV; or apoptosis Ehc≈ −170 mV. These estimates can be used to more fully understand the redox biochemistry that results from oxidative stress. These are the first steps toward a new quantitative biology, which hopefully will provide a rationale and understanding of the cellular mechanisms associated with cell growth and development, signaling, and reductive or oxidative stress.

Because cell-mediated reduction of menadione leads to the generation of reactive oxygen species (ROS), this quinone is widely used to investigate the effects of ROS on cellular functions. We report that A549 human lung epithelial cells exposed to menadione demonstrate a dose-dependent increase in both intracellular calcium ([Ca2+]i) and ROS formation. The concentrations of menadione required to initiate these two events are markedly different, with ROS detection requiring higher levels of menadione. Modulators of antioxidant defences (e.g. buthionine sulphoximine, 3-amino-1,2,4-triazole) have little effect on the [Ca2+]i response to menadione, suggesting that ROS formation does not account for menadione-dependent alterations in [Ca2+]i. Additional evidence suggests that menadione photochemistry may be responsible for the observed [Ca2+]i effects. Specifically: (a) EPR studies with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) show that light exposure (maximum effect at 340nm) stimulates menadione-dependent formation of the DMPO/•OH spin adduct that was not sensitive to antioxidant interventions; (b) DMPO inhibits menadione and light-dependent increases in [Ca2+]i; and (c) light (maximum effect at 340nm) augments the deleterious effects of menadione on cell viability as determined by 51Cr release. These photo effects do not appear to involve formation of singlet oxygen by menadione, but rather are the result of the oxidizing chemistry initiated by menadione in the triplet state. This work demonstrates that menadione species generated by photo-irradiation can exert biological effects on cellular functions and points to the potential importance of photochemistry in studies of menadione-mediated cell damage.

We examined the mechanism of H2O2-induced cytotoxicity and its relationship to oxidation in human leukemia cells. The HL-60 promyelocytic leukemia cell line was sensitive to H2O2, and at concentrations up to about 20–25 μm, the killing was mediated by apoptosis. There was limited evidence of lipid peroxidation, suggesting that the effects of H2O2do not involve hydroxyl radical. When HL-60 cells were exposed to H2O2in the presence of the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), we detected a 12-line electron paramagnetic resonance spectrum assigned to the POBN/POBN⋅N-centered spin adduct previously described in peroxidase-containing cell-free systems. Generation of this radical by HL-60 cells had the same H2O2concentration dependence as initiation of apoptosis. In contrast, studies with the K562 human erythroleukemia cell line, which is often used for comparison with the HL-60, and with high passaged HL-60 cells (spent HL-60) studied under the same conditions failed to generate POBN⋅. Cellular levels of antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase did not explain the differences between these cell lines. Interestingly, the K562 and spent HL-60 cells, which did not generate the radical, also failed to undergo H2O2-induced apoptosis. Based on this we reasoned that the difference in H2O2-induced apoptosis might be due to the enzyme myeloperoxidase. Only the apoptosis-manifesting HL-60 cells contained appreciable immunoreactive protein or enzymatic activity of this cellular enzyme. When HL-60 cells were incubated with methimazole or 4-aminobenzoic acid hydrazide, which are inhibitors of myeloperoxidase, they no longer underwent H2O2-induced apoptosis. Hypochlorous acid stimulated apoptosis in both HL-60 and spent HL-60 cells, indicating that another oxidant generated by myeloperoxidase induces apoptosis and that it may be the direct mediator of H2O2-induced apoptosis. Taken together these observations indicate that H2O2-induced apoptosis in the HL-60 human leukemia cell is mediated by myeloperoxidase and is linked to a non-Fenton oxidative event marked by POBN⋅.

Direct current (DC) shocks to the heart cause morphologic and functional myocardial damage. Previous studies have suggested that acute DC shock injury is free radical mediated and that the administration of antioxidant enzymes superoxide dismutase and catalase can reduce the level of DC shock-induced free radicals. Angiotensin-converting enzyme (ACE) inhibitors are clinically used drugs that may scavenge free radicals or reduce free radical generation. The objective of our study was to determine whether the ACE inhibitor captopril lowers free radicals after DC shocks.

Nitric oxide (•NO) can protect cells against the detrimental effects of reactive oxygen species. Using low-density lipoprotein as well as model systems, it has been demonstrated that •NO can serve as a chain-breaking antioxidant to blunt lipid peroxidation. To test the hypothesis that •NO can serve as a chain-breaking antioxidant in cell membranes, we examined the effect of •NO on iron-induced lipid peroxidation in human leukemia cells. We exposed HL-60 cells to an oxidative stress (20 μM Fe2+) and monitored the consumption of oxygen as a measure of lipid peroxidation. Oxygen consumption was arrested by the addition of •NO as a saturated aqueous solution. The duration of inhibition of oxygen consumption by •NO was concentration-dependent in the 0.4–1.8 μM range. The inhibition ended upon depletion of •NO. The addition of •NO prior to initiation of peroxidation delayed the onset of peroxidation; the nearer in time it was before Fe2+addition, the longer the inhibition. Depletion of cellular glutathione levels by d,l-buthionine-S,R-sulfoximine prior to Fe2+addition resulted in a more rapid initial rate of oxygen depletion and a shorter time for the •NO-induced inhibition of oxygen consumption. Complementary studies of this iron-induced lipid peroxidation, using thiobarbituric acid reactive substances as a marker, also demonstrated the protective effects of •NO. This protection of cells against lipid peroxidation also manifested itself as a reduction in trypan blue uptake, an observation demonstrating the protective effects of •NO on membrane integrity. We conclude that •NO protects HL-60 human leukemia cells from lipid peroxidation and that this protection ameliorates the toxicity of the oxidation processes initiated by Fe2+and dioxygen.

The reaction of superoxide with the spin trap DMPO (5,5-dimethylpyrroline-N-oxide) is widely used to study superoxide production as well as issues associated with the superoxide-related formation of hydroxyl radical. However, the interpretation of observed intensities of DMPO/•OOH and DMPO/•OH signals in electron paramagnetic resonance spin trapping experiments is not without its difficulties. In this paper, I report experiments that demonstrate: 1. That the flux of superoxide formation in a DMPO spin trapping experiment can alter the apparent importance of weak DMPO/•OH signals; 2. That iron can influence the DMPO/•OOH spin trapping results; 3. That there is very little spontaneous breakdown of DMPO/•OOH to form DMPO/•OH.

In the presence of ascorbate, bleomycin (BLM) is converted to a redox-inactive form that is incapable of inducing DNA strand scission. We have employed EPR spin trapping with 5,5-dimethylpyrroline-1-oxide (DMPO) to examine free radical production during this process. The introduction of ascorbate to an Fe(III)BLM-DMPO system results in the formation of three EPR observable free radicals. One of these radicals is the resonance-stabilized ascorbate free radical (aH= 1.8 G) that is not spin trapped by DMPO; the other two are the result of DMPO spin trapping. These radicals appear to be two carbon-centered radicals, DMPO/•CR1, (aN1= 15.75 G, aH1= 22.30 G, aN/aH= 0.706) and DMPO/•CR2(aN2= 15.20 G, aH2= 19.20 G, aN/aH= 0.79). Although it is not possible to identify the exact structures of the carbon-centered radicals that are spin trapped, the hyperfine splittings, as well as the aN/aHvalues, are characteristic of the presence of electron-withdrawing groups, such as the oxygen atom when attached to the carbon atom. In fact, these parameters are characteristic of DMPO spin trapping results obtained when sugars are subjected to oxidative insult from HO.. Thus, these BLM-ascorbate produced radicals may well be derived from the sugar moiety of BLM.

Hematoporphyrin derivative, used in photodynamic therapy of cancer, was found to generate the cysteinyl free radical as seen by spin-trapping. Oxygen appears to be an absolute requirement for thiyl radical production. Singlet oxygen may be the initiating species since azide inhibits oxygen uptake and radical production. In addition, the hydroxyl radical, or a radical with similar reactivity, is also observed and is proposed as the precursor for thiyl free radical production.

Objectives.We sought to demonstrate that direct current (DC) shocks to the heart generate free radicals.

Transition metals such as iron and copper are present in the myocardium and can act as catalysts for the formation of oxygen free radicals during reperfusion after myocardial ischemia. Previous studies suggested that transition metal chelators such as desferrioxamine reduce the production of such radicals and may thereby attenuate postischemic myocardial dysfunction. These studies used spin trapping agents, commonly nitrone compounds, which may themselves influence the severity of the ischemia and reperfusion events being studied. We evaluated two transition metal chelators, desferrioxamine, an iron chelator, and bathocuproine, a copper chelator, by using a new electron paramagnetic resonance technique that does not require the administration of spin traps. We measured ascorbate free radical, an index of free radical production, in the great cardiac vein effluent. Twenty-eight open-chest dogs underwent 20 min of coronary artery occlusion and 30 min of reperfusion. Ten dogs received no drug, 10 dogs received 750 mg bathocuproine, i.v., and eight dogs received 700 mg desferrioxamine, i.v. Both bathocuproine and desferrioxamine blunted the postreperfusion increase in ascorbate free radical generation: no drug, 36 ± 8% increase; desferrioxamine, 13 ± 5% increase; bathocuproine, 21 ± 6% increase (p < 0.05 vs. baseline). Thus direct free radical measurements indicate that chelation of the transition metals iron and copper reduces free radical generation during reperfusion.

Hematoporphyrin derivativeCysteineThiyl free radicalSinglet oxygenHydroxyl radicalSpin‐trapping

Nitric oxide is a paramagnetic molecule that exhibits high affinity for heme proteins, forming a mixture of nitrosylated valency hybrids that can be precisely characterized using low-temperature electron paramagnetic resonance spectroscopy. In red blood cells, nitric oxide will form paramagnetic complexes with deoxyhemoglobin or be oxidized to nitrite and nitrate by oxyhemoglobin with formation of paramagnetic methemoglobin. We have developed a technique to monitor temporal changes in local nitric oxide productionin vivo,by sampling arterial and venous blood across a tissue bed and measuring changes in hemoglobin–nitric oxide and methemoglobin concentrationex vivousing electron paramagnetic resonance spectroscopy. Changes in local nitric oxide concentration are further clarified by quantifying plasma nitrite/nitrate levels. With these techniques, we have observed that whole body hyperthermia increases portal venous concentrations of hemoglobin–nitric oxide and organic radicals that are consistent with enhanced nitric oxide synthase activity, cytotoxic leukocyte activity, and cellular oxidative stress in splanchnic tissues.

To gain an understanding of the mechanism by which the hydroxyl free radical can arise in superoxide generating systems and learn how different chelaters of iron can inhibit this reaction, a pulse radiolysis kinetic study of the reaction of O −2with Fe(III)EDTA, Fe(III)HEDTA and Fe(III)DETAPAC (or DTPA) was undertaken. Superoxide reacts readily with Fe(III)EDTA and Fe(III)HEDTA with a pH-dependent second-order rate constant having values of 1.9 × 10 6M −1.s −1and 7.6 × 10 5M −1.s −1at pH 7, respectively. However, the rate constant for the reaction of O −2with Fe(III)DETAPAC was found to be much slower, the upper limit for the rate constant being 10 4M −1.s −1. These results in conjunction with spin-trapping experiments with Fe(II)EDTA, Fe(II)HEDTA, Fe(II)DETAPAC and H 2O 2suggests that DETAPAC inhibits the formation of OH by slowing the reduction of Fe(III) to Fe(II) and not by inhibiting the Fenton reaction.

Objective. This study demonstrated that magnesium (Mg) reduces free radicals after a brief coronary occlusion-reperfusion sequence.

Much of what is known about the antioxidant mechanism of vitamin E has been learned from studies of lipid dispersions, solutions, or subcellular organelles. We have investigated the effect of vitamin E supplementation on intact live eucaryotic cells. L1210 murine leukemia cells were exposed to an oxidative stress induced by 20 μMFe2+and 100 μMascorbic acid introduced immediately before oxidative measurements were begun, and the kinetics of the generation of lipid-derived free radicals, as measured by EPR spin trapping (a product) and O2consumption (a reactant) were measured. Cells grown for 24 h with supplemental (5–100 μM) vitamin E in their media had a slower rate of lipid radical generation compared to cells grown without vitamin E supplementation; this inhibition in the rate of oxidation was generally dependent upon the amount of vitamin E supplementation. In complementary studies measuring O2consumption, 5–100 μMvitamin E slowed the rate of oxidation (10-fold with 100 μMsupplemental vitamin E) consistent with the EPR studies. The membrane active drug edelfosine accentuated the vitamin E effects; vitamin E introduced a discernible lag phase (time delay) in both lipid radical generation and O2consumption that was not seen in the absence of edelfosine. Vitamin E supplementation of cells also altered the kinetics of ascorbate free radical formation. We conclude that vitamin E inhibits lipid peroxidation in cells by slowing the rate of lipid peroxidation; but with iron/ascorbate as the initiating system, vitamin E does not delay the onset of peroxidation. Of special interest is that these free radical peroxidation events parallel cell membrane damage as detected using trypan blue exclusion. These observations are consistent with the free radical events preceding and causing the observed membrane damage.

Some plastic syringes introduce a nitroxide free radical into the fluids they contain. The EPR spectrum shows a triplet with aN= 17.0 ±± 0.1 G in plasma. We report here that this nitroxide/hydroxylamine couple serves as an antioxidant system, whose redox potential appears to be between that of vitamin C and vitamin E. This adventitious antioxidant can alter the course of free radical processes.

It has been suggested that ultraviolet light induces free radical formation in skin, leading to photoaging and cancer. We have demonstrated by electron paramagnetic resonance that the ascorbate free radical is naturally present in unexposed skin at a very low steady state level. When a section of SKH‐1 hairless mouse skin in an EPR cavity is exposed to UV light (4,500 J m−2−1, Xe lamp, 305 nm cutoff and IR filters), the ascorbate free radical signal intensity increases. These results indicate that UV light increases free radical oxidative stress, consistent with ascorbate's role as the terminal, small‐molecule antioxidant. The initial radicals produced by UV light would have very short lifetimes at room temperature; thus, we have applied EPR spin trapping techniques to detect these radicals. Using α‐[4‐pyridyl 1‐oxide]‐N‐tert‐butyl nitrone (POBN), we have for the first time spin trapped a UV light‐produced carbon‐centered free radical from intact skin. The EPR spectra exhibited hyperfine splittings that are characteristic of POBN/alkyl radicals, aN= 15.56 G and aH= 2.70 G, possibly generated from membrane lipids as a result of β‐scission of lipid alkoxyl radicals. Iron can act as a catalyst for free radical oxidative reactions; chronic exposure of skin to UV radiation causes increased iron deposition. Using our spin trapping system, we have shown that topical application of the iron‐chelator, Desferal, to a section of skin reduces the UV light‐induced POBN adduct radical signal. These results provide direct evidence for free radical generation and a role for iron in UV light‐induced dermatopathology. We suggest that iron chelators can serve as photoprotective agents by preventing these oxidations.

To gain an understanding of the mechanism by which the hydroxyl free radical can arise in superoxide generating systems and learn how different chelaters of iron can inhibit this reaction, a pulse radiolysis kinetic study of the reaction of O −2with Fe(III)EDTA, Fe(III)HEDTA and Fe(III)DETAPAC (or DTPA) was undertaken. Superoxide reacts readily with Fe(III)EDTA and Fe(III)HEDTA with a pH‐dependent second‐order rate constant having values of 1.9 × 106M−1.s−1and 7.6 × 105M−1.s−1at pH 7, respectively. However, the rate constant for the reaction of O −2with Fe(III)DETAPAC was found to be much slower, the upper limit for the rate constant being 104M−1.s−1. These results in conjunction with spin‐trapping experiments with Fe(II)EDTA, Fe(II)HEDTA, Fe(II)DETAPAC and H2O2suggests that DETAPAC inhibits the formation of OH by slowing the reduction of Fe(III) to Fe(II) and not by inhibiting the Fenton reaction.

We hypothesized that exposure of cells to H2O2plus Fe2+would increase formation of cell-derived lipid peroxides that would inactivate prostaglandin H synthase, resulting in decreased prostaglandin synthesis. Therefore, we treated human endothelial cells with 0–100 μmH2O2followed immediately by addition of 0–200 μmFe2+. After oxidant exposure, cells were stimulated with 20 μmarachidonic acid to induce prostaglandin I2(PGI2) synthesis. Adding 100 μmH2O2prior to arachidonic acid decreased PGI2synthesis more than 80%. However, to our surprise, the addition of Fe2+, in increasing amounts, progressively protected PGI2synthesis against the harmful effects of H2O2. A ratio of one part H2O2to two parts Fe2+offered almost complete protection, whereas Fe3+did not protect PGI2synthesis from H2O2. We found that 100 μmH2O2was not cytolytic; however, 250 μmH2O2was cytolytic; Fe2+protected against this cytotoxicity. In addition, extracellular Fe2+prevented the rise in intracellular calcium caused by H2O2and extracellular Fe2+preserved intracellular glutathione in H2O2-exposed cells. Electron paramagnetic resonance spin trapping demonstrated that extracellular Fe2+generated the hydroxyl free radical, HO•, outside the cell. We speculate that extracellular Fe2+protects the intracellular space from H2O2by initiating the Fenton reaction outside the cell. This reductive cleavage of H2O2generates HO•in the extracellular space, where much of the HO•will react with noncellular components, thereby protecting the cell interior.

Phagocytes secrete the heme protein myeloperoxidase, which is present and active in human atherosclerotic tissue. These cells also generate hydrogen peroxide (H2O2), thereby allowing myeloperoxidase to generate a range of oxidizing intermediates and stable end products. When this system acts on l-tyrosine in vitro, it forms o, o′-dityrosine, which is enriched in atherosclerotic lesions. Myeloperoxidase, therefore, may oxidize artery wall proteins in vivo, cross-linking theirl-tyrosine residues. In these studies, we used electron paramagnetic resonance (EPR) spectroscopy to identify an oxidizing intermediate in this reaction pathway and in parallel reactions catalyzed by horseradish peroxidase and lactoperoxidase. Using an EPR flow system to rapidly mix and examine solutions containing horseradish peroxidase, H2O2, and l-tyrosine, we detected free tyrosyl radical (a2,6H= 6.3 G,a3,5H= 1.6 G, and aβH= 15.0 G). We then used spin trapping techniques with 2-methyl-2-nitrosopropane (MNP) to further identify this intermediate. The resulting three-line spectrum (aN= 15.6 G) was consistent with an MNP/tyrosyl radical spin adduct. Additional MNP spin trapping studies with ring-labeledl-[13C6]tyrosine yielded a characteristic eight-line EPR spectrum (aN= 15.6 G, a13C(2) = 8.0 G, a13C(1) = 7.1 G, a13C(1) = 1.3 G), indicating that the MNP adduct resulted from trapping a carbon-centered radical located on the aromatic ring ofl-tyrosine. This same eight-line spectrum was observed when human myeloperoxidase or bovine lactoperoxidase was substituted for horseradish peroxidase. Furthermore, a partially immobilized MNP/tyrosyl radical spin adduct was detected when we exposed a synthetic polypeptide composed of glutamate and l-tyrosine residues to the myeloperoxidase-H2O2-l-tyrosine system. The broadened EPR signal resulting from this MNP/polypeptide adduct was greatly narrowed by proteolytic digestion with Pronase, confirming that the initial spin-trapped radical was protein-bound. Collectively, these results indicate that peroxidases use H2O2to convert l-tyrosine to free tyrosyl radical. They also support the idea that free tyrosyl radical initiates cross-linking of l-tyrosine residues in proteins. We suggest that this pathway may play an important role in protein and lipid oxidation at sites of inflammation and in atherosclerotic lesions.

Host animals in algal-invertebrate endosymbiotic associations are exposed to photosynthetically generated hyperoxia while in sunlight, conditions conducive to photodynamic excitations and production of cytotoxic oxygen-derived radicals such as the superoxide anion and the hydroxyl radical (.OH). All previous evidence of oxyradical production in symbiotic associations has been circumstantial. We here present direct evidence, from electron paramagnetic resonance studies on tissue homogenates of the photosymbiont-containing sea anemone Anthopleura elegantissima (Brandt), of substantial light-dependent .OH and production that is abolished by dichlorophenyldimethylurea (DCMU), an inhibitor of photosynthesis. Shade-adapted A. elegantissima lacking endosymbiotic algae likewise show .OH production upon illumination. The latter flux is not dependent on photosynthesis, and DCMU has no effect. Rather, .OH production in apozooxanthellate anemones is via direct photoexcitations. The selective reaction of dimethyl sulfoxide (DMSO) with .OH to form methane sulfinic acid allows quantification of .OH produced in vivo. Such in vivo measurements confirm the production of .OH in both host and algae in illuminated zooxanthel-late anemones, where the amount of .OH in the zooxanthellae is disproportionately large relative to their fractional contribution to the biomass of the symbiosis. In vivo studies using DMSO also suggest a photochemical production of .OH in apozooxanthellate anemones exposed to simulated sunlight enriched in ultraviolet (UV) wavelengths, and the enhancement by UV light of .OH production in zooxanthellate individuals. Such chronic radical exposure necessitates defenses against photooxidative stress, a cost that is seldom considered in these mutualistic symbioses.