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Biomedical subjects

B A Freeman

Publications and source records attributed to B A Freeman.

At least 19 recordsLinked to original sources
PubMed1995

Nitric oxide inhibition of lipoxygenase-dependent liposome and low-density lipoprotein oxidation: termination of radical chain propagation reactions and formation of nitrogen-containing oxidized lipid derivatives.

Lipoxygenase-induced lipid oxidation contributes to plasma lipoprotein oxidation and may be an underlying pathogenic mechanism of atherogenesis. Since inactivation of the vasorelaxant actions of nitric oxide (.NO) plays a critical role in the impaired function of atherosclerotic vessels and because .NO reacts rapidly with other radical species, we assessed the influence of .NO on lipoxygenase-catalyzed oxidation of linoleic and linolenic acid, 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PC) liposomes, hypercholesterolemic rabbit beta-very-low-density lipoprotein, and human low-density lipoprotein. Soybean lipoxygenase (SLO)-induced lipid oxidation was assessed by accumulation of conjugated dienes, formation of lipid hydroperoxides, oxygen consumption, and liquid chromatography-mass spectrometry. Different rates of delivery of .NO to lipid oxidation systems were accomplished either by infusion of .NO gas equilibrated with anaerobic buffer or via .NO released from S-nitrosoglutathione. Nitric oxide alone did not induce lipid peroxidation, while exposure to SLO yielded significant oxidation of fatty acids, PC liposomes, or lipoproteins in a metal ion-independent mechanism. Low concentrations of .NO, which did not significantly inhibit the activity of the iron-containing lipoxygenase, induced potent inhibition of lipid peroxidation in a dose-dependent manner. Mass spectral analysis of oxidation products showed formation of nitrito-, nitro-, nitrosoperoxo-, and/or nitrated lipid oxidation adducts, demonstrating that .NO serves as a potent terminator of radical chain propagation reactions. The formation of Schiff's base fluorescent conjugates between SLO-oxidized linoleic or linolenic acid and bovine serum albumin (BSA) was also inhibited by .NO via reaction with lipid hydroperoxyl radicals (LOO.), thus preventing the reaction of LOO. with polypeptide amino groups. Mass spectrometry analysis showed that both lipid peroxidation products and nitrogen-containing oxidized lipid species decreased in the presence of BSA. We conclude that .NO can play a potent oxidant-protective role in the vessel wall by inhibiting lipoxygenase-dependent lipid and lipoprotein oxidation. This occurs via termination of lipid radical chain propagation reactions catalyzed by alkoxyl (LO.) and LOO. intermediates of lipid peroxidation rather than by inhibition of lipoxygenase-catalyzed initiation reactions.

Animals↗
PubMed1995

Altered expression of type I collagen, TGF-beta 1, and related genes in rat lung exposed to 85% O2.

The gene expressions of type I collagen and transforming growth factor-beta 1 (TGF-beta 1) were studied in lung tissue of rats exposed to air or 85% O2 for 14 days. Peak expression of type I collagen mRNA was observed by 14 days of 85% O2 exposure, at the same time as maximal immunoreactive type I collagen, which was most marked surrounding the major airways and vessels. TGF-beta 1 mRNA also significantly increased after 14, but not 4 or 6 days of 85% O2 exposure. TGF-beta 1 immunoreactivity was only detected on day 14 of 85% O2 exposure and was localized primarily to the pulmonary epithelium. As an increase in immunoreactive type I collagen was evident by day 6 of O2 exposure, the gene expressions of interstitial collagenase (MMP-1), stromelysin, and the tissue inhibitor of the metalloproteinases (TIMP) were also examined. Increased mRNA expressions of interstitial collagenase and TIMP preceded those of type I collagen and TGF-beta 1, occurring at 4-6 days of exposure to 85% O2, while there was no significant change in stromelysin mRNA. These findings are compatible with the initial O2-mediated increase in type I collagen deposition being a result of an altered proteinase/antiproteinase balance in the lung, and the subsequent more marked deposition being a response to increased TGF-beta 1 synthesis.

Animals↗
PubMed1995

Delivery of superoxide dismutase to pulmonary epithelium via pH-sensitive liposomes.

Respiratory insufficiency, when treated with oxygen supplementation, or exposure to diverse pulmonary toxins can cause lung damage as a result of increased oxygen radical production. Enzymes such as superoxide dismutase (SOD) may attenuate this pathological process, but the intracellular delivery and antioxidant action of SOD is impeded by its inability to cross cellular membranes. One approach for facilitating intracellular delivery of macromolecules is to entrap SOD into liposomes. The delivery of SOD to lung cells was accomplished using pH-sensitive liposomes, made with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1-oleoyl-2-oleoyl-sn-glycero-3-succinate (DOSG), added to cultured fetal rat lung distal epithelial (FRLE) cells. FRLE cells, obtained from fetuses at day 19 gestation, expressed a high-affinity receptor for surfactant protein A (SP-A) with an apparent dissociation constant (Kd) = 3.6 +/- 0.2 micrograms/ml (5.5 x 10(-9) M) and a capacity of 130 +/- 3 ng/10(6) cells (125,000 +/- 3,000 binding sites/cell). This receptor was utilized for targeting liposomes to cells, after incorporating SP-A during liposome membrane formation. Liposomes were uniformly small (180 +/- 77 nm; mean +/- SD) and stable at 4 degrees C for 1 wk, entrapping 10 +/- 4% of initially added SOD. After incubation of pH-sensitive liposomes containing entrapped SOD with cultured FRLE cells, cell-associated SOD activity was increased 5.1-fold from 7.8 +/- 2.5 to 40.1 +/- 3.3 U SOD/mg cell protein. Incorporation of SP-A into liposomes increased by 6.2-fold the delivery of liposomal SOD to cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Basic fibroblast growth factor and growth factor receptor gene expression in 85% O2-exposed rat lung.

Lungs exposed to elevated O2 concentrations suffer an initial loss of type I pneumocytes, followed by a reparative type II pneumocyte hyperplasia. We hypothesized that type II pneumocyte hyperplasia after exposure of young adult rats to 85% O2 in vivo would be temporally related to 1) an increased concentration of intrapulmonary basic fibroblast growth factor (bFGF), a potent stimulator of type II pneumocyte DNA synthesis in vitro, and 2) an upregulation of pneumocyte receptors for bFGF (FGF-R). Increased rat lung bFGF mRNA, relative to air-exposed control animals, was observed at 4 days of exposure, with no increase at days 6 and 14 of exposure. Parallel changes were observed with bFGF receptor (flg) mRNA. Nuclear runoff assays confirmed increased transcription of both bFGF and flg genes in response to 85% O2, whereas increased translation at 6 days of exposure was confirmed by protein immunoanalysis. Immunohistochemistry demonstrated a broad distribution of bFGF throughout the lung, including the alveolar epithelium, which increased after 6 and 14 days of exposure to 85% O2. Our findings are compatible with a role for bFGF in O2-mediated pneumocyte hyperplasia.

Amino Acid Sequence↗
PubMed1995

Pulmonary alveolar epithelial inducible NO synthase gene expression: regulation by inflammatory mediators.

Nitric oxide (.NO) is a short-lived mediator that can be induced by different cytokines and lipopolysaccharide (LPS) in a variety of cell types and produces many physiological and metabolic changes in target cells. In the current study, we show that a combination of cytokines, LPS, and zymosan-activated serum (ZAS; called for convenience cytomix Z) induces production of high concentrations of the NO oxidation products nitrite (NO2-) and nitrate (NO3-) by cultured rat fetal lung epithelial type II cells in a time-dependent fashion. Interferon-gamma and tumor necrosis factor-alpha alone did not significantly affect .NO synthesis, whereas ZAS, LPS, and interleukin-1 beta caused only a modest increase in formation of .NO oxidation products. Production of NO2- and NO3- was inhibited by NG-monomethyl-L-arginine and cyclohexmide. After exposure of these cells to a combination of the above cytokines, Escherichia coli LPS, and ZAS (cytomix Z), enhanced inducible nitric oxide synthase (iNOS) expression was indicated by an elevation in steady-state mRNA specific for iNOS (via Northern blot analysis) and increased immunofluorescence for iNOS after cell permeabilization, incubation with anti-iNOS antibody, and treatment with Cy3.18-conjugated rabbit-specific antibody. The extent of inflammatory mediator-induced.NO production by alveolar epithelium, which exceeds that of other lung cell types, reveals new insight into mechanisms of pulmonary host defense and pathways of free radical-mediated lung injury.

Amino Acid Oxidoreductases↗
PubMed1995

Evidence for enhanced vascular superoxide anion production in nitrate tolerance. A novel mechanism underlying tolerance and cross-tolerance.

We sought to examine mechanisms underlying nitroglycerin (NTG) tolerance and "cross-tolerance" to other nitrovasodilators. Rabbits were treated for 3 d with NTG patches (0.4 mg/h) and their aortic segments studied in organ chambers. Relaxations were examined after preconstriction with phenylephrine. In NTG tolerant rabbit aorta, relaxations to cGMP-dependent vasodilators such as NTG (45 +/- 6%), SIN-1 (69 +/- 7%), and acetylcholine (ACh, 64 +/- 5%) were attenuated vs. controls, (90 +/- 2, 94 +/- 3, and 89 +/- 2% respectively, P < 0.05 for all), while responses to the cAMP-dependent vasodilator forskolin remained unchanged. In tolerant aorta, endothelial removal markedly enhanced relaxations to NTG and SIN-1 (82 +/- 4 and 95 +/- 3%, respectively). Other studies were performed to determine how the endothelium enhances tolerance. Vascular steady state .-O2 levels (assessed by lucigenin chemiluminescence) was increased twofold in tolerant vs. control vessels with endothelium (0.31 +/- 0.01 vs. 0.61 +/- 0.01 nmol/mg per minute). This difference was less in vessels after denudation of the endothelium. Diphenylene iodonium, an inhibitor of flavoprotein containing oxidases, and Tiron a direct .-O2 scavenger normalized .-O2 levels. In contrast, oxypurinol (1 mM) an inhibitor of xanthine oxidase, rotenone (50 microM) an inhibitor of mitochondrial electron transport and NG-nitro-L-arginine (100 microM) an inhibitor of nitric oxide synthase did not affect the chemiluminescence signals from NTG-tolerant aortas. Pretreatment of tolerant aorta with liposome-entrapped, pH sensitive superoxide dismutase (600 U/ml) significantly enhanced maximal relaxation in response to NTG, SIN-1, and ACh, and effectively reduced chemiluminescence signals. These studies show that continuous NTG treatment is associated with increased vascular .-O2-production and consequent inhibition of NO. mediated vasorelaxation produced by both exogenous and endogenous nitrovasodilators.

Acetylcholine↗
PubMed1995

Antioxidant therapy in critical care medicine.

Antioxidant therapies are currently undergoing clinical trials to determine their benefit in a number of diseases seen in the critical care unit. It is particularly likely that their use will become standard therapy in a number of situations in which there is reperfusion injury following an ischemic episode. During the lag time from the conception of antioxidant interventions to their introduction into clinical practice, our understanding of how oxidant injuries are mediated has changed considerably. This understanding has allowed the development of more rational approaches to antioxidant therapy while exposing the limitations of some of the approaches currently being evaluated in a clinical setting. In addition, some existing therapeutic agents have previously unrecognized antioxidant and pro-oxidant effects. An inherent requirement of an effective antioxidant therapy is that such therapy is effective against the radicals being generated, and that sufficient quantities of the antioxidants used reach all sites of radical generation in time to limit tissue injury. Recent observations suggest that few, if any, of the currently known individual antioxidants can adequately meet these goals. However, combination therapy, or the development of synthetic compounds that can combine the properties of current antioxidants, may come close.

Acute Disease↗
PubMed1994

Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogen-containing oxidized lipid derivatives.

Superoxide (O2-.), nitric oxide (.NO), and their reaction product peroxynitrite (ONOO-) have all been shown to independently exert toxic target molecule reactions. Because these reactive species are often generated in excess during diverse inflammatory and other pathologic circumstances, we assessed the influence of .NO on membrane lipid peroxidation induced by O2-., H2O2, and .OH derived from xanthine oxidase (XO) and by ONOO-. Experimental conditions in lipid oxidation systems were adjusted to yield different rates of delivery of .NO, relative to rates of O2-. and H2O2 generation, by infusion of either .NO or via .NO released from S-nitroso-N-acetylpenicillamine or S-nitrosoglutathione. Peroxidation of phosphatidylcholine liposomes was assessed by formation of thiobarbituric acid-reactive products and by liquid chromatography-mass spectrometry. Liposomes exposed to XO-derived reactive species in the presence of .NO exhibited both stimulation and inhibition of lipid peroxidation, depending on the ratio of the rates of reactive oxygen species production and .NO introduction into reaction systems. Nitric oxide alone did not induce lipid peroxidation. Linolenic acid emulsions peroxidized by XO-derived reactive species showed similar dose-dependent regulation of lipid peroxidation by .NO. Mass spectral analysis of oxidation products showed formation of nitrito-, nitro-, nitrosoperoxo-, and/or nitrated lipid oxidation adducts, demonstrating that .NO serves as a potent terminator of radical chain propagation reactions. Electron spin resonance (ESR) analysis of incubation mixtures provided no evidence for formation of paramagnetic iron-lipid-nitric oxide complexes in reaction systems. Peroxynitrite-dependent lipid peroxidation, which predominantly occurs by metal-independent mechanisms, was also inhibited by .NO. Peroxynitrite-mediated benzoate hydroxylation was partially inhibited by .NO, inferring reaction between .NO and ONOOH. It is concluded that .NO can both stimulate O2-./H2O2/.OH-induced lipid oxidation and mediate oxidant-protective reactions in membranes at higher rates of .NO production, with the prooxidant versus antioxidant outcome critically dependent on relative concentrations of individual reactive species. Prooxidant reactions of .NO will occur after O2-. reaction with .NO to yield potent secondary oxidants such as ONOO- and the antioxidant effects of .NO a consequence of direct reaction with alkoxyl and peroxyl radical intermediates during lipid peroxidation, thus terminating lipid radical chain propagation reactions.

Hydroxyl Radical↗
PubMed1992

Responses of vascular endothelial oxidant metabolism to lipopolysaccharide and tumor necrosis factor-alpha.

Quantification of intracellular and extracellular levels and production rates of reactive oxygen species is crucial to understanding their contribution to tissue pathophysiology. We measured basal rates of oxidant production and the activity of xanthine oxidase, proposed to be a key source of O2- and H2O2, in endothelial cells. Then we examined the influence of tumor necrosis factor-alpha and lipopolysaccharide on endothelial cell oxidant metabolism, in response to the proposal that these inflammatory mediators initiate vascular injury in part by stimulating endothelial xanthine oxidase-mediated production of O2- and H2O2. We determined a basal intracellular H2O2 concentration of 32.8 +/- 10.7 pM in cultured bovine aortic endothelial cells by kinetic analysis of aminotriazole-mediated inactivation of endogenous catalase. Catalase activity was 5.72 +/- 1.61 U/mg cell protein and glutathione peroxidase activity was much lower, 8.13 +/- 3.79 mU/mg protein. Only 0.48 +/- 0.18% of total glucose metabolism occurred via the pentose phosphate pathway. The rate of extracellular H2O2 release was 75 +/- 12 pmol.min-1.mg cell protein-1. Intracellular xanthine dehydrogenase/oxidase activity determined by pterin oxidation was 2.32 +/- 0.75 microU/mg with 47.1 +/- 11.7% in the oxidase form. Intracellular purine levels of 1.19 +/- 1.04 nmol hypoxanthine/mg protein, 0.13 +/- 0.17 nmol xanthine/mg protein, and undetectable uric acid were consistent with a low activity of xanthine dehydrogenase/oxidase. Exposure of endothelial cells to 1000 U/ml tumor necrosis factor (TNF) or 1 microgram/ml lipopolysaccharide (LPS) for 1-12 h did not alter basal endothelial cell oxidant production or xanthine dehydrogenase/oxidase activity. These results do not support a casual role for H2O2 in the direct endothelial toxicity of TNF and LPS.

Amitrole↗
PubMed1992

The contribution of vascular endothelial xanthine dehydrogenase/oxidase to oxygen-mediated cell injury.

The conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) and the reaction of XO-derived partially reduced oxygen species (PROS) have been suggested to be important in diverse mechanisms of tissue pathophysiology, including oxygen toxicity. Bovine aortic endothelial cells expressed variable amounts of XDH and XO activity in culture. Xanthine dehydrogenase plus xanthine oxidase specific activity increased in dividing cells, peaked after achieving confluency, and decreased in postconfluent cells. Exposure of BAEC to hyperoxia (95% O2; 5% CO2) for 0-48 h caused no change in cell protein or DNA when compared to normoxic controls. Cell XDH+XO activity decreased 98% after 48 h of 95% O2 exposure and decreased 68% after 48 h normoxia. During hyperoxia, the percentage of cell XDH+XO in the XO form increased to 100%, but was unchanged in air controls. Cell catalase activity was unaffected by hyperoxia and lactate dehydrogenase activity was minimally elevated. Hyperoxia resulted in enhanced cell detachment from monolayers, which increased 112% compared to controls. Release of DNA and preincorporated [8-14C]adenine was also used to assess hyperoxic cell injury and did not significantly change in exposed cells. Pretreatment of cells with allopurinol for 1 h inhibited XDH+XO activity 100%, which could be reversed after oxidation of cell lysates with potassium ferricyanide (K3Fe(CN)6). After 48 h of culture in air with allopurinol, cell XDH+XO activity was enhanced when assayed after reversal of inhibition with K3Fe(CN)6, and cell detachment was decreased. In contrast, allopurinol treatment of cells 1 h prior to and during 48 h of hyperoxic exposure did not reduce cell damage. After K3Fe(CN)6 oxidation, XDH+XO activity was undetectable in hyperoxic cell lysates. Thus, XO-derived PROS did not contribute to cell injury or inactivation of XDH+XO during hyperoxia. It is concluded that endogenous cell XO was not a significant source of reactive oxygen species during hyperoxia and contributes only minimally to net cell production of O2- and H2O2 during normoxia.

Animals↗
PubMed1992

Platelet-derived growth factor and growth-related genes in rat lung. II. Effect of exposure to 85% O2.

The expression of platelet-derived growth factor (PDGF), its receptor, and related genes was studied in the lung tissue of rats exposed to air or 85% O2. PDGF-B chain mRNA was increased by 6 days and PDGF B-type receptor mRNA was increased by 4 and 6 days of exposure to 85% O2. Despite a continued increase of cell division, both PDGF-B chain and B-type receptor mRNAs had returned to control values by 14 days of exposure to 85% O2. PDGF-A chain mRNA was unaffected by exposure to 85% O2. Nuclear runoff transcription analysis confirmed increased transcription of PDGF-B chain mRNA, whereas Western blot analysis of lung homogenates suggested consequent increased translation of PDGF-B chain mRNA to PDGF-BB at 7 days of exposure to 85% O2. Combined immunocytochemistry and autoradiography localized PDGF-BB to the major site of cell division, the pulmonary interstitium. We speculate that the early pulmonary fibroblast hyperplasia observed following exposure to 85% O2 is mediated by increased PDGF-B chain gene expression and may also be mediated by changes in PDGF B-type receptor gene expression.

Animals↗
PubMed1992

Quantitation of alveolar distribution of liposome-entrapped antioxidant enzymes.

Liposome-encapsulated Cu,Zn superoxide dismutase (Cu,Zn SOD) and catalase (CAT) were instilled intratracheally in rabbits, and the temporal and spatial distribution of Cu,Zn SOD and CAT within the lung was assessed at the organ and cellular levels. Specific activities of Cu,Zn SOD and CAT were increased in both lung homogenates and isolated alveolar type II pneumocytes. Peak Cu,Zn SOD activities in lung homogenates and alveolar type II cells were observed 4 h after liposome instillation and returned to control levels by 24 h, whereas CAT activities remained significantly above controls. There were no significant differences in liposome distribution or antioxidant enzyme uptake among lung lobes. The distribution of fluorescently labeled Cu,Zn SOD and CAT was assessed with the use of epifluorescence microscopy and digital image processing to determine patterns of cellular incorporation of liposome-entrapped Cu,Zn SOD and CAT within the lung. Although the mean fluorescence intensity of alveoli from rabbits instilled with liposomes containing labeled Cu,Zn SOD and CAT was greater than autofluorescence observed with either no liposome or empty liposome instillation, fluorescence intensity varied between adjacent alveoli. Both fluorescently labeled Cu,Zn SOD and CAT were located cytosolically, and uptake was not limited to alveolar type II pneumocytes. These results demonstrate that a single intratracheal instillation of liposomes can effect increases in Cu,Zn SOD and CAT activities in distal lung cells, including alveolar type I and type II cells and macrophages.

Animals↗
PubMed1992

Distribution of lung cell numbers and volumes between alveolar and nonalveolar tissue.

Although total cell number has been determined for the alveolar region of the lungs of many species, it has not been calculated for the nonalveolar lung tissues. The oriented structure of airways and vessels makes the numerical assessment of cells in nonalveolar tissues difficult. This has led many investigators to use the number of cells in the alveolar region as a direct estimate of total lung cell number. To determine the number of cells in the nonalveolar lung tissues, the lungs of eight rats weighing 230 to 380 g were inflation-fixed and embedded in araldite, and 1.5-microns serial sections of the entire left lobe were cut and stained with methylene blue for light microscopy. The sections were then uniformly point-counted using computer-controlled distances between the fields to determine the fraction of points falling on air, blood, cellular tissue, and noncellular tissue for both the alveolar and the nonalveolar regions. The total volume of cell nuclei in each compartment was determined, and the total number of cells was calculated by dividing the total nuclear volume by the mean cell nuclear volume. It was found that 87% of the lung volume was alveolar, of which 6% was tissue and contained 725 x 10(6) cells. The nonalveolar region constituted 13% of the lung volume, of which 23% was tissue and contained 250 x 10(6) cells. The average rat lung therefore contains 975,000,000 cells, of which 74% was in alveolar tissues and 26% in nonalveolar tissues. On the basis of assays of isolated lung cells, there is an average of 7 pg DNA/cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Detection of catalase in rat heart mitochondria.

The presence of heme-containing catalase in rat heart mitochondria (20 +/- 5 units/mg) was demonstrated by biochemical and immunocytochemical analysis. Intact rat heart mitochondria efficiently consumed exogenously added H2O2. The rate of H2O2 consumption was not influenced by succinate, glutamate/malate, or N-ethylmaleimide but was significantly inhibited by cyanide. Hydrogen peroxide decomposition by mitochondria yielded molecular oxygen in a 2:1 stoichiometry, consistent with a catalytic mechanism. Mitochondrial fractionation studies and quantitative electron microscopic immunocytochemistry revealed that most catalase was matrix-associated. Electrophoretic analysis and Western blotting of the mitochondrial matrix fraction indicated the presence of a protein with similar electrophoretic mobility to bovine and rat liver catalase and immunoreactive to anti-catalase antibody. Myocardial tissue has a lower catalase-specific activity and a greater mitochondrial H2O2 production/g of tissue than most organs. Thus catalase, representing 0.025% of heart mitochondrial protein, is important for detoxifying mitochondrial derived H2O2 and represents a key antioxidant defense mechanism for myocardial tissue.

Animals↗
PubMed1991

Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide.

Endothelial cells, macrophages, neutrophils, and neuronal cells generate superoxide (O2-) and nitric oxide (.NO) which can combine to form peroxynitrite anion (ONOO-). Peroxynitrite, known to oxidize sulfhydryls and to yield products indicative of hydroxyl radical (.OH) reaction with deoxyribose and dimethyl sulfoxide, is shown herein to induce membrane lipid peroxidation. Peroxynitrite addition to soybean phosphatidylcholine liposomes resulted in malondialdehyde and conjugated diene formation, as well as oxygen consumption. Lipid peroxidation was greater at acidic and neutral pH, with no significant lipid peroxidation occurring above pH 9.5. Addition of ferrous (Fe+2) or ferric (Fe+3) iron did not enhance lipid peroxide formation over that attributable to peroxynitrite alone. Diethylenetetraminepentacetic acid (DTPA) or iron removal from solutions by ion-exchange chromatography decreased conjugated diene formation by 25-50%. Iron did not play an essential role in initiating lipid peroxidation, since DTPA and iron depletion of reaction systems were only partially inhibitory. In contrast, desferrioxamine had an even greater concentration-dependent inhibitory effect, completely abolishing lipid peroxidation at 200 microM. The strong inhibitory effect of desferrioxamine on lipid peroxidation was due to direct reaction with peroxynitrous acid in addition to iron chelation. We conclude that the conjugate acid of peroxynitrite, peroxynitrous acid (ONOOH), and/or its decomposition products, i.e., .OH and nitrogen dioxide (.NO2), initiate lipid peroxidation without the requirement of iron. These observations demonstrate a potential mechanism contributing to O2-(-)and .NO-mediated cytotoxicity.

Free Radicals↗
PubMed1991

Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide.

Peroxynitrite anion (ONOO-) is a potent oxidant that mediates oxidation of both nonprotein and protein sulfhydryls. Endothelial cells, macrophages, and neutrophils can generate superoxide as well as nitric oxide, leading to the production of peroxynitrite anion in vivo. Apparent second order rate constants were 5,900 M-1.s-1 and 2,600-2,800 M-1.s-1 for the reaction of peroxynitrite anion with free cysteine and the single thiol of albumin, respectively, at pH 7.4 and 37 degrees C. These rate constants are 3 orders of magnitude greater than the corresponding rate constants for the reaction of hydrogen peroxide with sulfhydryls at pH 7.4. Unlike hydrogen peroxide, which oxidizes thiolate anion, peroxynitrite anion reacts preferentially with the undissociated form of the thiol group. Peroxynitrite oxidizes cysteine to cystine and the bovine serum albumin thiol group to an arsenite nonreducible product, suggesting oxidation beyond sulfenic acid. Peroxynitrous acid was a less effective thiol-oxidizing agent than its anion, with oxidation presumably mediated by the decomposition products, hydroxyl radical and nitrogen dioxide. The reactive peroxynitrite anion may exert cytotoxic effects in part by oxidizing tissue sulfhydryls.

Animals↗
PubMed1991

Characterization of cultured alveolar epithelial cell xanthine dehydrogenase/oxidase.

Conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) and the toxic reactions of subsequent XO-derived superoxide, hydrogen peroxide and hydroxyl radical, have been suggested to be critical factors in several mechanisms of tissue pathophysiology. In the lung, intracellular XO-derived products may modulate type II pneumocyte surfactant turnover and barrier function, jeopardizing the pulmonary air-blood barrier. We characterized total cellular XDH/XO enzymatic activity in freshly isolated and cultured rat pulmonary type II epithelial cells. Type II cells were isolated and cultured on fibronectin-pretreated dishes, with a plating efficiency after 36 h in culture of 40% or 14% when quantified via cellular protein or DNA, respectively. Over the subsequent 96 h in culture, monolayer DNA was unchanged, whereas protein per cell increased continuously. Alterations in different cellular enzymatic activities were also detected in these cultured cells. In culture, total cellular XDH/XO and catalase activities decreased in a logarithmical fashion with respect to time, whether normalized for cellular protein or DNA. The rate of loss of these enzymes was greatest when normalized for cell protein, but was also significant when the activities were normalized for DNA. When compared to freshly isolated type II cells, catalase and total XDH/XO activities normalized for protein decreased 78% and 72%, respectively, during the first 36 h of culture. After 132 h in culture, XDH/XO and catalase activities normalized for protein decreased 93% and 84%, respectively, when compared to freshly isolated cell values. Total cellular XDH/XO activity in the oxidase form (% XO) was initially 31% in freshly isolated type II cells and increased to 67% during the 132 h culture period. In contrast to the loss of total cellular XDH/XO and catalase, no significant change in lactate dehydrogenase (LDH) activity occurred during culture of the type II cells. In type II cells the conversion of XDH to XO, the cytotoxic potential of XO, and the activity of the hydrogen peroxide scavenger, catalase, is expected to be strongly influenced by in vitro culture. Thus, strong consideration should be made before transposing information obtained from cultured type II cells to in vivo situations.

Analysis of Variance↗