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PubMed1994

Investigation of oxygen-derived free radical generation in cancellous bone specimens obtained from dogs.

Generation of free radicals and the ability of various antioxidants to attenuate radical production in freshly procured cancellous bone specimens was investigated, using spin-trapping and electron spin resonance (ESR) techniques. Seven core cancellous bone specimens, 10 mm long and 7.9 mm in diameter, were obtained using aseptic technique, from the proximal portion of the humerus of 9 adult mixed-breed dogs. One core cancellous bone specimen from each dog was incubated in spin trap alpha-phenyl-N-tert-butylnitrone in Eagle's minimum essential medium and served as a control. The other 6 specimens from each dog were incubated in alpha-phenyl-N-tert-butylnitrone/Eagle's minimum essential medium plus 1 of the following antioxidants: superoxide dismutase, catalase, superoxide dismutase/catalase, indomethacin, allopurinol, or deferoxamine mesylate. All specimens were incubated at 26 C for 90 minutes, then frozen at -20 C until they were prepared for analysis by ESR spectroscopy. Each specimen was thawed, homogenized, and extracted in a low-dielectric organic solvent prior to obtaining an ESR spectrum which was analyzed for hyperfine splitting constants to identify radicals. Each first-derivative spectrum was digitally double-integrated to obtain an area: these areas were used to compare intensities of the spin. For each treatment group, the areas from the treated specimens were compared with the areas from the control specimens, using a paired t-test. Significance was accepted at P < or = 0.05. Spin adducts were detected in all cancellous bone specimens. Specimens incubated in deferoxamine (P = 0.0017) and superoxide dismutase/catalase (P = 0.0452) had significantly smaller areas than did control specimens.(ABSTRACT TRUNCATED AT 250 WORDS)

Allopurinol↗

PubMed1994

Effect of acute hypertension in the coronary circulation: role of mechanical factors and oxygen radicals.

OBJECTIVE: In previous studies severe, acute hypertension damaged the endothelium in proximal coronary arteries and selectively potentiated constriction of the artery to serotonin. In the present study we investigated the role of several mechanical factors and of oxygen radicals in this response. DESIGN: To test the role of mechanical factors in the response to acute hypertension, the effect of different magnitudes of elevation of perfusion pressure and the rate of the rise in perfusion pressure were studied. Pharmacologically induced increases in blood pressure were produced by infusion of angiotensin II or phenylephrine. The role of oxygen radicals was tested by measuring responses to serotonin before and after increases in perfusion pressure in dogs treated with a combination of superoxide dismutase and catalase or with deferoxamine. METHODS: In open-chest anesthetized dogs the diameter of the left anterior descending coronary artery (LADCA) was measured using sonomicrometer crystals, and the LADCA was perfused at a constant pressure of 80 mmHg from a reservoir. Responses to serotonin were measured at this perfusion pressure before and after an abrupt increase in perfusion pressure. RESULTS: Intracoronary serotonin (5 or 50 micrograms/min) produced a dose-dependent constriction of the LADCA while increasing coronary flow. Abruptly increasing the coronary perfusion pressure from 80 to 120, 150 or 200 mmHg augmented the constriction to serotonin twofold, whereas increases in perfusion pressure to 100 mmHg had no effect. Increasing coronary pressure slowly (over a 4-min period) from 80 to 200 mmHg augmented constriction to serotonin. Inducing acute hypertension (coronary pressure 200 mmHg) pharmacologically with angiotensin II also augmented constriction to serotonin, whereas phenylephrine-induced hypertension did not. Superoxide dismutase, a scavenger of superoxide anions and catalase, a scavenger of hydrogen peroxide, prevented the augmented constriction to serotonin following a pressure increase. Deferoxamine, which prevents generation of hydroxyl radicals from superoxide anions and hydrogen peroxide, also prevented the enhanced constriction to serotonin following an acute pressure increase. CONCLUSIONS: Moderate physiological increases in pressure, induced either mechanically or pharmacologically, can augment the responses to serotonin. Oxygen-derived free radicals, particularly hydroxyl radicals, might be involved in the abnormal response to serotonin following an abrupt increase in coronary pressure.

Angiotensin II↗

PubMed1993

Analysis of monocyte chemoattractant protein 1-mediated lung injury using rat lung organ cultures.

Using a rat lung organ culture system, we analyzed the role of monocyte chemoattractant protein 1 (MCP 1) in leukocyte to lung adhesive interactions and monocyte-mediated lung injury. Quantitative leukocyte to lung adhesive interactions were examined using an adaptation of the Woodruff-Stamper frozen section binding assay. Pretreatment of organ cultures with recombinant human tumor necrosis factor (rhTNF alpha) resulted in a protein synthesis-dependent increase in the adhesiveness of lung tissue for peripheral blood monocytes. Adhesion of monocytes to lung tissue was not increased above baseline after 7 hours but increased more than twofold by 24 hours and persisted through 48 hours. Binding of monocyte to lung tissue was further increased when recombinant rat MCP 1 was added to monocyte suspensions immediately before being layered onto lung sections derived from either TNF alpha-treated or untreated organ cultures. Addition of antibody directed against rat CD11b/c resulted in a moderate reduction in monocyte binding. TNF or lipopolysaccharide-induced activation of mononuclear cells in the presence of [3H]leucine-labeled organ cultures resulted in lung injury as assessed by radioisotope release. Mononuclear cell-mediated organ culture injury could be partially inhibited with anti-rat MCP 1 antibody, anti-rat CD11b/c antibody, or antioxidants including catalase and deferoxamine. Anti-MCP 1 and anti-CD11b/c increased the absolute numbers of monocytes that could be retrieved from monocyte-lung co-cultures while catalase and deferoxamine did not. In vitro studies revealed that isolated rat peripheral blood monocytes produce O2- in response to MCP 1. These data provide a functional correlate for recent in vitro studies which suggest that MCP 1 may mediate leukocyte adhesive processes by up-regulating beta 2 integrin expression on monocytes. This study provides evidence that monocytes activated by MCP 1 can damage lung tissue through an oxidant-mediated mechanism. Monocyte chemoattractant protein 1 may participate in the pathogenesis of monocyte-mediated lung injury by modulating inflammatory cell adhesion as well as through monocyte activation.

Animals↗

PubMed1993

Yersinia enterocolitica bacteremia in a chronically transfused patient with sickle cell anemia. Case report and review of the literature.

PURPOSE: Yersinia enterocolitica sepsis is rarely encountered in patients without an underlying susceptibility and is most frequently reported in iron-overloaded patients. This is thought to be related to the unusual utilization of iron by this microorganism. We report a case of Y. enterocolitica bacteremia in a chronically transfused adolescent with sickle cell anemia. This type of serious infection in sickle cell disease is previously unreported. A description of the case and the relationship between Y. enterocolitica and iron is discussed. A review of the literature is presented. RESULTS: Y. enterocolitica can cause a severe septicemia, and increased virulence of this organism has been shown to correlate with increased iron burden and/or use of the chelator deferoxamine. It may also occur as a consequence of a contaminated blood transfusion. CONCLUSIONS: We believe our case demonstrates that Y. enterocolitica should be considered a possible pathogen in febrile chronically transfused patients with sickle cell disease. Broad antibiotic coverage should be initiated and deferoxamine discontinued pending results of cultures.

Adolescent↗

PubMedDate not supplied

[Arrhythmia in thalassemia major: evaluation of iron chelating therapy by dynamic ECG].

Arrhythmias and sudden death represent striking features in the natural history of thalassemia major. Antiarrhythmic treatment, however, does not appear to change the clinical course. During recent years the disease's therapeutics approach has undergone a substantial evolution, being more adequate the transfusional regimens as well as more effective the iron chelation therapy through subcutaneous infusion of deferoxamine. The aim of the present study was to determine possible influences exerted by the current treatment upon disease's arrhythmic disorders. Thirty patients of both sexes were enrolled in the study. The age ranged from 9 to 24 years. No congenital or acquired heart diseases were present. Each patient underwent concentrated red cell transfusions (in order to obtain pretransfusional hemoglobin levels of 10-11 g%), and iron-binding therapy through continuous subcutaneous microinjection of deferoxamine 40-50 mg/kg/day (6-8 hours/day, 6 days/week). Patients were divided in 2 groups: the first group (group A) comprising the 16 patients with good therapeutic compliance and regular pharmacological regimen; the second group (Group B) including the remaining poorly compliant 14 patients. The following parameters were analyzed: age, average hemoglobin levels during the last year, total amount of red cell transfusions, ferritin levels, starting age of iron-binding therapy. Moreover, each patient underwent 24-hour ECG Holter monitoring. Age (Group A: 18 +/- 4.6; Group B: 14 +/- 2.7; p < 0.02), total amount of transfusions (Group A: 272 +/- 73; Group B: 211 +/- 44; p < 0.03), and ferritin levels (Group A: 1697 +/- 860; Group B: 2908 +/- 730; p < 0.002) proved to be significantly different in the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1993

Superoxide dismutase and allopurinol improve survival in an animal model of hemorrhagic shock.

We studied the efficacy of resuscitation with antioxidants in an animal model of hemorrhagic shock. Male Sprague-Dawley rats were anesthetized, and 27 mL/kg of blood was withdrawn from the carotid artery over 2 minutes. The animals remained in hemorrhagic shock for 45 minutes, followed by 1 hour of resuscitation. Experimental groups were as follows: 1) 15,000 u/kg superoxide dismutase (SOD) in 54 mL/kg lactated Ringer (LR); 2) 175,000 u/kg catalase (CAT) in LR; 3) 15,000 u/kg SOD+175,000 u/kg CAT in LR; 4) allopurinol in LR; 5) deferoxamine bound to pentafraction (DFO), 27 mL/kg; 6) pentafraction alone; and 7) LR alone. Compared with resuscitation with LR alone, SOD and allopurinol improved survival over 72 hours, P < 0.05. Survival with SOD+CAT was not different from LR alone. Deferoxamine bound to pentafraction did not increase survival over that with pentafraction alone. CAT had increased mortality compared to LR, P < 0.01. The efficacy of both SOD and allopurinol in decreasing mortality suggests the importance of superoxide radicals after hemorrhagic shock and resuscitation. These and other antioxidants are potential therapeutic agents in the clinical setting of trauma and hemorrhagic shock.

Allopurinol↗

PubMedDate not supplied

Serum ferritin, free erythrocyte protoporphyrin, and urinary iron excretion in patients with iron disorders.

Tests to evaluate body iron stores were compared in patients with iron deficiency and the anemia of chronic disease. The serum ferritin assay separated these disorders in 20 of 22 patients. One discrepancy was explained by the concomitant association of both disorders. From this study and review of literature a low serum ferritin level is a good indication for iron therapy. The serum ferritin assay is a clinically useful test in lieu of bone marrow estimation of body iron stores to detect patients with iron deficiency. Total iron binding capacity levels when high-normal or elevated are sometimes helpful as a screening test in separating iron deficiency from the anemia of chronic disorders. Free erythrocyte protoporphyrin values were elevated in both conditions but were higher in iron deficiency than in the anemia of chronic disorders with considerable overlap of values. Urinary iron excretion with deferoxamine was not helpful in separating these disorders but is a useful test to establish iron overload. An elevated serum ferritin level is usually found with disease of iron overload but serum iron levels, deferoxamine iron excretion tests, and liver biopsy for estimation of iron stores are still beneficial diagnostic aids.

Anemia↗

PubMed1995

Synthesis, pharmacokinetics, and biodistribution of 67GA deferoxamineacetyl-cysteinylbiotin.

The exceptionally high affinity of streptavidin for biotin may be exploited for two-step in vivo approaches for delivering radiolabelled biotin derivatives to lesion-bound streptavidin-conjugated monoclonal antibodies. A radiolabeled biotin derivative was prepared, and its characterization, stability, pharmacokinetics, and biodistribution studies are presented. This derivative contains deferoxamine, a chelating moiety with high affinity for trivalent metals suitable for imaging and therapy. Deferoxamineacetyl-cysteinylbiotin (DACB) was synthesized in three steps: nucleophilic reaction of deferoxamine with N-hydroxysuccinimide iodoacetate, aminolysis of N-hydroxysuccinimide biotin by L-cysteine, followed by coupling of cysteinylbiotin with N-iodoacetyldeferoxamine. DACB was characterized by matrix-assisted laser desorption/ionization MS. Radiolabeling of DACB with 67Ga led to a labeling efficiency of > 95%. Pharmacokinetics of 67Ga DACB exhibited rapid blood clearance, with < 10% circulating at 30 min and < 1% at 6 hr. Plasma samples collected at various time intervals showed > 95% binding with streptavidin, indicating in vivo stability of 67Ga DACB. Urinalysis showed > 80% of the administered dose excreted at 6 hr. Biodistribution data at 6 hr showed < 1% radioactivity remaining per organ.

Animals↗

PubMed1997

Iron deprivation and cancer: a view beginning with studies of monoclonal antibodies against the transferrin receptor.

This review provides a perspective on the potential utility of iron deprivation treatments as components of cancer therapy. The perspective began to develop with investigations of the selective inhibitory effects on lymphocyte activation which were produced by monoclonal antibodies against the transferrin receptor. Those investigations led to the unexpected discovery that such antibodies would produce synergistic inhibition of lymphoid tumor growth in vitro when used in combination with the iron chelator deferoxamine. The perspective was further developed when additional studies in vivo indicated that combination iron deprivation treatment could prevent initial tumor outgrowth and cause regressions of established tumors in the 38C13 murine lymphoma model. The anti-tumor effects were accompanied by significant toxicities, however, and the analysis of the causes of those toxicities is now an important issue. The opportunities and problems which these results present are interpreted in the broader context of currently available information concerning the anti-tumor effects of deferoxamine and gallium nitrate in the pre-clinical and clinical settings, and questions for future research are presented.

Animals↗

PubMedDate not supplied

Role of hydroxyl radical in silica-induced NF-kappa B activation in macrophages.

Nuclear transcription factor kappa B (NF-kappa B) is a multiprotein complex that regulates a variety of genes important for immunity and inflammation. The present study investigates the silica-induced activation of this transcription factor in mouse macrophage cell line RAW 264.7 cells, the role of free radical reactions in the mechanism of the activation, and its possible inhibition. Tetrandrine, a benzylisoquinoline alkaloid, which has been used as an antifibrotic drug to treat the lesions of silicosis and has been characterized as a hydroxyl radical (.OH) scavenger, inhibited the NF-kappa B activation induced by silica, lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA). Catalase, metal chelator, deferoxamine, and the silanol group (SiOH) blocker, poly(2-vinylpyridine-N-oxide) (PVPNO), also inhibited silica-induced NF-kappa B activation. Electron spin resonance (ESR) spin trapping measurements show that both deferoxamine and PVPNO decreased silica-mediated .OH radical generation from H2O2. It is shown that Fe(II) and not Fe(III) is able to cause NF-kappa B activation. The antioxidant, ascorbate, attenuated the NF-kappa B activation induced by silica but not by LPS. The .OH radical scavenger, sodium formate, inhibited NF-kappa B activation induced by silica but had only a minor effect on NF-kappa B activation induced by LPS. The results indicate that silica-mediated free radical generation via the Fenton or Fenton-like reaction (M(n)+ + H2O2-->M(n + 1)+ + OH- + .OH) and silanol groups on the silica surface play an important role in silica-induced NF-kappa B activation.

Alkaloids↗

PubMed1997

Carbon monoxide contributes to the cytokine-induced inhibition of surfactant synthesis by human type II pneumocytes.

BACKGROUND: An increase in cyclic guanosine 3',5'-monophosphate (cGMP) due to nitric oxide generation is known to participate in the mediation of the tumor necrosis factor alpha (TNF-alpha) effect in type II cells. Because guanylyl cyclase can be activated also by carbon monoxide (CO), in this study we examined the ability of human type II pneumocytes to produce CO in the presence of cytokines and the relative contribution of this molecule to the TNF-alpha and interleukin 1 effects. DESIGN: Type II pneumocytes were isolated from cadaveric multiple-organ donors by enzymatic digestion, adherence separation of macrophages, and gradient purification. After preculture for 24 hours, cells were cultured for 24 hours in the presence or absence of TNF-alpha, interleukin 1, sodium nitroprusside, N(omega)-nitro-1-arginine, CO, hemin, zinc-protoporphyrin type IX, deferoxamine mesylate, S-adenosyl-L-methionine, alpha-tocopherol, methylene blue (a guanylyl cyclase inhibitor), 8-bromine-cGMP, and combinations of these reagents. Both CO (picomole per microgram of protein) and nitric oxide release to the medium and the cGMP (picomole per microgram of protein) content of the cells were measured. In a different set of experiments, D-glucose labeled with radioactive carbon (14C) was added to the medium, and the labeling of several lipid fractions was determined (picomole per microgram of protein). RESULTS: D-[14C]glucose incorporation into phosphatidylcholine, the main surfactant component, was selectively inhibited in the presence of cytokines, CO, sodium nitroprusside, or 8-bromine-cGMP. The inhibitory effect of TNF-alpha was partially reversed by N(omega)-nitro-L-arginine, deferoxamine, or alpha-tocopherol and totally reversed by methylene blue. Tumor necrosis factor alpha induced an increase in cGMP cell content and in the CO and nitric oxide release to the medium. Hemin increased CO and cGMP production and decreased phosphatidylcholine synthesis. Zinc-protoporphyrin type IX, an inhibitor of heme oxygenase, and all 3 antioxidants, which inhibited CO production, also antagonized the TNF-alpha effect on cGMP and phosphatidylcholine synthesis. CONCLUSIONS: Intracellular cGMP increase due to an endogenous generation of both CO and nitric oxide mediates the cytokine-induced inhibition of surfactant synthesis by type II pneumocytes. Both lipid peroxidation and heme oxygenase activity are sources for the observed CO production.

Adolescent↗

PubMed1992

Role of iron and iron chelation in dopaminergic-induced neurodegeneration: implication for Parkinson's disease.

Recent studies in Parkinson's disease suggest that the degeneration of the nigrostriatal melanin-containing dopaminergic neurons results from toxic effects of free radicals, which are generated during dopamine metabolism in the substantia nigra (SN). This has been linked to the selective accumulation of iron, a known catalyst of radical formation, in the zona compacta of the SN. We have shown that interaction of iron with melanin may result in a high affinity binding of iron to melanin (KD = 13.0 +/- 0.15 nM). Indeed, x-ray analysis of melanized dopamine neurons of parkinsonian SN has shown an interaction of iron with melanin that is absent in control brains. In the presence of excess Fe3+, melanin potentiates iron-induced lipid peroxidation. Since iron chelators prevent lipid peroxidation, we have ascertained the ability of the iron chelator deferoxamine to prevent the lesion of the nigrostriatal dopamine neuron induced by 6-hydroxy dopamine (6-OHDA). Our results demonstrated that intraventricular injection of 130 ng deferoxamine to rats prior to 250 micrograms of 6-OHDA partially prevented the decrease in striatal dopamine content caused by 6-OHDA (56% reduction vs 90%, respectively). This protection was sufficient to produce normal dopamine-related behavioral responses. These results suggest that iron and iron chelators play a crucial role in the process of dopaminergic neurodegeneration and neuroprotection. The latter is further supported by our recent findings that intranigral injection of iron (50 micrograms) resulted in a substantial selective decrease of striatal dopamine (95%) and impaired dopamine-related responses.

Animals↗

PubMed2001

Genotoxicity of iron chelators in L5178Y mouse lymphoma cells.

To further study the mechanism of observed iron mutagenicity and cellular toxicity, a number of different iron chelators were evaluated to select a compound that was not mutagenic and had limited toxicity to mouse lymphoma cells. A series of iron chelators including those used clinically, those under development for clinical applications, and those used in nonclinical applications were evaluated. The mutagenic activity of the iron chelators was assessed in L5178Y mouse lymphoma cells. Eight of the 12 iron chelators that were tested induced mutagenic responses both with and without the addition of S9. Among those chelators used clinically or developed for clinical use, the only compound that did not induce a mutagenic response was the starch deferoxamine conjugate. In contrast, deferoxamine mesylate showed the highest toxicity in this group of chemicals and the concentrations leading to toxicity and mutagenicity between the activated and nonactivated assays were not significantly different. The other three chelators that were not mutagenic were Na2EDTA, phytic acid, and ferrozine.

Animals↗

PubMed1992

Role of cytochrome P-450 2E1 in ethanol-, carbon tetrachloride- and iron-dependent microsomal lipid peroxidation.

This study investigated the role of cytochrome P-450 2E1 in enhanced microsomal lipid peroxidation in experimental alcoholic liver disease. We also examined the contribution of this isoform to the increased microsomal injury in alcoholic liver disease caused by carbon tetrachloride-induced or iron-induced oxidant stress. Adult male Wistar rats were intragastrically infused with a high-fat diet and ethanol or glucose for 16 wk; this resulted in hepatic lipid peroxidation and fibrogenesis in the ethanol-fed animals. Microsomes were isolated by differential centrifugation in the presence of 100 mumol/L deferoxamine, washed twice in buffer without deferoxamine and incubated in the absence or presence of ethanol (50 mmol/L), carbon tetrachloride (150 mumol/L), ferric citrate (50 mumol/L) or ferric citrate plus ethanol at 37 degrees C for 30 min in an NADPH-generating system. The basal rate of lipid peroxidation in microsomes isolated from ethanol-fed rats was increased by 52% compared with that in microsomes from controls. Carbon tetrachloride-induced and ferric citrate-induced lipid peroxidation were also accentuated in microsomes from ethanol-fed rats, by 76% and 108%, respectively. Ethanol added in vitro significantly reduced basal (-58%) and ferric citrate-induced (-48%) lipid peroxidation in microsomes from ethanol-fed rats, whereas it had an insignificant effect on that in control microsomes. In fact, this protective effect of ethanol on microsomes from ethanol-fed rats resulted in attenuation of the difference in the level of microsomal lipid peroxidation between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2002

The antifungal drug ciclopirox inhibits deoxyhypusine and proline hydroxylation, endothelial cell growth and angiogenesis in vitro.

The hypusine biosynthetic steps represent novel targets for intervention in cell proliferation. Hypusine is a rare amino acid, formed posttranslationally in one cellular protein, eIF5A, and is essential for cell proliferation. Deoxyhypusine hydroxylase, the metalloenzyme catalyzing the final step in hypusine biosynthesis, and prolyl 4-hydroxylase, a non-heme iron enzyme critical for collagen processing, can be inhibited by small chelating molecules that target their essential metal atom. We examined the effects of 5 compounds (ciclopirox, deferiprone, deferoxamine, mimosine and 2,2'-dipyridyl) on these protein hydroxylases in HUVECs, on cell proliferation and on angiogenesis using 2 model assays: tube-like vessel formation on Matrigel and the chick aortic arch sprouting assay. These compounds inhibited cellular deoxyhypusine hydroxylase in a concentration-dependent manner, but their efficacy varied widely in the following order: ciclopirox--> deferoxamine-->2,2'-dipyridyl-->deferiprone-->mimosine (IC(50) 5-200 microM). Inhibition of DNA synthesis, following the same order (IC(50) 10-450 microM), correlated with G(1) arrest of the cell cycle. These compounds also inhibited proline hydroxylation and maturation of collagen in HUVECs and caused inhibition of angiogenesis in vitro. Of the compounds tested, ciclopirox was by far the most effective inhibitor of HUVEC proliferation and angiogenesis. The strong antiangiogenic activity of this readily available antifungal drug along with its antiproliferative effects suggests a new potential application for ciclopirox in the treatment of solid tumors.

Antifungal Agents↗

PubMed1997

Secretion and inactivation of myeloperoxidase by isolated neutrophils.

Neutrophils prevent infection by ingesting and killing microorganisms but oxidants and proteases released by neutrophils damage host tissues. Our aim was to identify factors that regulate oxidant production by the enzyme myeloperoxidase (MPO) following secretion of MPO into the medium. Cells stimulated with phorbol myristate acetate (PMA) or opsonized zymosan particles secreted MPO and released superoxide free radicals (.O2-). Dismutation of .O2- produced hydrogen peroxide (H2O2) and MPO catalyzed the oxidation of chloride ion by H2O2 to produce the toxic oxidant hypochlorous acid (HOCl). Adding the enzyme superoxide dismutase (SOD) to increase the rate of conversion of .O2- to H2O2 had pH-dependent effects on HOCl production. From pH 6.0 to 7.4, SOD promoted HOCl production by up to 500% but SOD had no effect at pH 7.6 and inhibited by 40 +/- 10% at pH 7.8. In further experiments at pH 7.0, MPO activity in the cells decreased by 25 +/- 2 and 44 +/- 4% during 1-h incubations with PMA and zymosan. Only 1 +/- 0 and 3 +/- 1% of the total activity was found in the medium, indicating that most of the secreted MPO was inactivated. Loss of activity was not accompanied by proteolytic destruction of the MPO protein, which was measured with anti-MPO antibodies. SOD raised the amount of active MPO in the medium two- to sevenfold, but adding deferoxamine to chelate iron or adding ferric ion had no effect. The ionophore A23187 was as effective as zymosan as a stimulus for MPO secretion but .O2- production by ionophore-stimulated cells was less than 4% of that of PMA- or zymosan-stimulated cells and most of the secreted MPO was found active in the medium. When PMA-stimulated cells were incubated with purified MPO, the added MPO activity was lost from the medium. Binding or proteolysis did not account for loss of activity as indicated by recovery of added radioiodinated MPO from the medium. The visible absorption spectrum of MPO was lost, indicating destruction of the iron-containing prosthetic group. Loss of activity and loss of the MPO spectrum were blocked by SOD but not by deferoxamine or catalase. The results indicate that, in the physiological pH range, inactivation of MPO in the medium suppressed HOCl production. Inactivation required O2- but not HOCl, H2O2, or free iron. Inactivation of secreted MPO may limit MPO-mediated damage to host tissues by stimulated neutrophils.

Humans↗

PubMed1992

In situ detection of oxidative stress in rat hepatocytes.

In rat hepatocytes in primary culture incubated with nitro blue tetrazolium, formazan content was increased by addition of t-butyl hydroperoxide, a potent oxidant, in a dose-related manner, but not by addition of valinomycin, which kills hepatocytes through mitochondrial damage. This increment after t-butyl hydroperoxide addition was not seen in hepatocytes preincubated with deferoxamine mesylate, a ferric iron chelator which inhibits radical formation. Liver perfusion with nitro blue tetrazolium and t-butyl hydroperoxide in rats produced formazan deposition faintly on the surface of hepatocytes throughout the liver and prominently in the cytoplasm of some hepatocytes, which was attenuated when performed following deferoxamine mesylate perfusion. When liver perfusion with nitro blue tetrazolium was performed in carbon tetrachloride-intoxicated rats, formazan deposition appeared diffusely in hepatocytes in the centrilobular areas. Similar deposition was also observed on the surface and in the cytoplasm of hepatocytes in the periportal and mid-zonal areas in rats undergoing post-ischaemic reperfusion. Liver perfusion with nitro blue tetrazolium can detect in situ oxidative stress in hepatocytes and may be a useful tool for studying the role of lipid peroxidation in rat liver injury.

Animals↗

PubMed1995

Detection of hydroxyl radicals upon interaction of ozone with aqueous media or extracellular surfactant: the role of trace iron.

As part of a study on mechanisms modulating ozone-induced surfactant perturbations, we used the electron paramagnetic resonance (EPR) spin trapping technique to determine the type and origin of radicals generated following interaction of ozone with aqueous solutions and cell-free bronchoalveolar lavage fluid (BAL) fractions. All aqueous media were exposed to ozone at 25 degrees C with or without added chelator, 1 mM diethylenetriaminepentaacetic acid, and spintrap, 100 mM 5,5'-dimethyl-1-pyrroline-1-oxide (DMPO). Exposure of distilled water to 0.5, 1.0, 2.0, and 3.0 ppm ozone for 1 h yielded four-line spectra, 1:2:2:1, consistent with hydroxyl radical adduct formation (DMPO-OH), the amplitudes of which increased with the ozone concentration. No signals were obtained from air-exposed samples. Similar four-line spectra were also produced following interaction of 3 ppm ozone with Hank's balanced salt solution (HBSS) alone or containing BAL fractions. Addition of the hydroxyl radical scavenger dimethyl sulfoxide (DMSO) to the incubation medium strongly inhibited formation of DMPO-OH adduct during ozone exposure. As an alternate method of demonstrating the generation of hydroxyl radicals, aqueous solutions of 1 mM L-phenylalanine were exposed to high concentrations of ozone and shown, using ion-exchange chromatography, to contain small amounts of L-tyrosine. Production of hydroxyl radicals upon interaction of ozone and water was further substantiated using the spintrap PBN (phenyl-N-tert-butylnitrone) in the presence of DMSO which reacts with the hydroxyl radical resulting in the formation of methyl radical. The methyl radical subsequently reacts with spintrap PBN, yielding PBN-methyl adduct. In the absence of DMSO there was no detectable formation of methyl radical adduct. EPR double distilled water containing DMPO showed a small amount of DMPO-OH adduct upon exposure to ozone. Addition of 10 microM ferrous sulfate to this mixture produced a 10-fold increase of the signal, which was attenuated in the presence of 1500 U catalase, strongly attenuated with 50-500 microM deferoxamine or 8000 U catalase and abolished by higher concentration of deferoxamine (1 mM). The signal was not influenced by 1000 U superoxide dismutase. These results indicate that hydroxyl radicals are produced via iron-dependent reactions during the initial interaction of ozone with aqueous media, including bronchoalveolar fluid.

Animals↗