Bactericidal activity of a superoxide anion-generating system. A model for the polymorphonuclear leukocyte.

The acetaldehyde-xanthine oxidase system in the presence and absence of myeloperoxidase (MPO) and chloride has been employed as a model of the oxygen-dependent antimicrobial systems of the PMN. The unsupplemented xanthine oxidase system was bactericidal at relatively high acetaldehyde concentrations. The bactericidal activity was inhibited by superoxide dismutase (SOD), catalase, the hydroxyl radical (OH.) scavengers, mannitol and benzoate, the singlet oxygen (1O2) quenchers, azide, histidine, and 1,4-diazabicyclo[2,2,2]octane (DABCO) and by the purines, xanthine, hypoxanthine, and uric acid. The latter effect may account for the relatively weak bactericidal activity of the xanthine oxidase system when purines are employed as substrate. A white, carotenoid-negative mutant strain of Sarcina lutea was more susceptible to the acetaldehyde-xanthine oxidase system than was the yellow, carotenoid-positive parent strain. Carotenoid pigments are potent 1O2 quenchers. The xanthine oxidase system catalyzes the conversion of 2,5-diphenylfuran to cis-dibenzoylethylene, a reaction which can occur by a 1O2 mechanism. This conversion is inhibited by SOD, catalase, azide, histidine, DABCO, xanthine, hypoxanthine, and uric acid but is only slightly inhibited by mannitol and benzoate. The addition of MPO and chloride to the acetaldehyde-xanthine oxidase system greatly increases bactericidal activity; the minimal effective acetaldehyde concentration is decreased 100-fold and the rate and extent of bacterial killing is increased. The bactericidal activity of the MPO-supplemented system is inhibited by catalase, benzoate, azide, DABCO, and histidine but not by SOD or mannitol. Thus, the acetaldehyde-xanthine oxidase system which like phagocytosing PMNs generates superoxide (O.2-) and hydrogen peroxide, is bactericidal both in the presence and absence of MPO and chloride. The MPO-supplemented system is considerably more potent; however, when MPO is absent, bactericidal activity is observed which may be mediated by the interaction of H2O2 and O.2- to form OH. and 1O2.

Normal functional characteristics of cultured human promyelocytic leukemia cells (HL-60) after induction of differentiation by dimethylsulfoxide.

The HL-60 human promyelocytic leukemia cell line can be induced to terminally differentiate to mature myeloid cells sharing a number of functional characteristics with normal granulocytes including response to chemoattractants, development of complement receptors, phagocytosis, superoxide production, and nitroblue tetrazolium dye reduction. Hence the Me2SO-induced HL-60 cells provide a unique in vitro model for studying various important aspects of human myeloid cell differentiation.

Oxygen-independent killing by alveolar macrophages.

We have found that normal alveolar macrophages can kill an intracellular parasite by a mechanism that does not involve toxic metabolites of oxygen. We studied the interaction between Toxoplasma gondii and rat alveolar macrophages in vitro. We were interested in Toxoplasma because it causes pneumonia in immunosuppressed patients but not in healthy individuals, and we chose the rat because it resembles immunocompetent human subjects in being resistant to T. gondii. Resident rat alveolar macrophages could kill large numbers of T. gondii. This occurred without a respiratory burst as judged by intracellular reduction of nitroblue tetrazolium and quantitative release of superoxide. Furthermore, scavengers of toxic oxygen metabolites had no effect on the toxoplasmacidal activity of the alveolar macrophages, nor did prior exhaustion of their respiratory burst with PMA. Whereas acid pH (e.g., 4.5-6.0) rapidly kills extracellular T. gondii, raising of the intralysosomal acid pH of rat alveolar macrophages by incubating them with weak bases did not inhibit their ability to kill T. gondii. Killing of Toxoplasma occurred within 1 h of initial exposure to the alveolar macrophages. However, there was no evidence that killing preceded ingestion; Toxoplasma attached to the surface of the cell appeared viable, and when phagocytosis was blocked with sodium fluoride the organisms survived. These results indicate that rat alveolar macrophages possess a powerful nonoxidative microbicidal mechanism, which is distinct from acidification of the phagolysosome but which probably involves phagosome formation. This mechanism may be clinically relevant, for we have recently observed that human alveolar macrophages also kill T. gondii by an oxygen-independent process.

Role of reactive oxygen species in reperfusion injury of the rabbit lung.

We have developed a model of reperfusion injury in Krebs buffer-perfused rabbit lungs, characterized by pulmonary vasoconstriction, microvascular injury, and marked lung edema formation. During reperfusion there was a threefold increase in lung superoxide anion (O2-) production, as measured by in vivo reduction of nitroblue tetrazolium, and a twofold increase in the release of O2- into lung perfusate, as measured by reduction of succinylated ferricytochrome c. Injury could be prevented by the xanthine oxidase inhibitor allopurinol, the O2- scavenger SOD, the hydrogen peroxide scavenger catalase, the iron chelator deferoxamine, or the thiols dimethylthiourea or N-acetylcysteine. The protective effect of SOD could be abolished by the anion channel blocker 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, indicating that SOD consumes O2- in the extracellular medium, thereby creating a concentration gradient favorable for rapid diffusion of O2- out of cells. Our results extend information about the mechanisms of reperfusion lung injury that have been assembled by studies in other organs, and offer potential strategies for improved organ preservation, for treatment of reperfusion injury after pulmonary thromboembolectomy, and for explanation and therapy of many complications of pulmonary embolism.

Inhibition of phorbol ester stimulated interleukin 2 production by copper(II) complexes.

Superoxide dismutase mimetic copper(II) complexes, such as copper(II)(3,5-diisopropylsalicylate)2 (CuDIPS), inhibit phorbol ester stimulated tumor promotion in mouse skin. Therefore, CuDIPS was tested as a potential inhibitor of another effect of phorbol esters, induction of interleukin 2 (IL2) synthesis, in the mouse thymoma cell line EL4. CuDIPS inhibited phorbol ester induced IL2 production in a concentration dependent manner with a 50% inhibitory concentration of about 10 microM. However, the ligand 3,5-diisopropylsalicylic acid also inhibited the induction of IL2 by phorbol esters (50% inhibitory concentration, 15 microM). Since the superoxide dismutase mimetic activity of CuDIPS is not stable in the presence of ethylenediaminetetraacetic acid, the effects of CuDIPS could be due to the free ligand and not to the intact metallocomplex. Consequently, a series of extremely stable copper(II) macrocyclic compounds was synthesized, and the reduction potential, superoxide dismutase mimetic activity, and ability to inhibit phorbol ester induced IL2 production were determined for each. Of the copper(II) macrocyclic complexes studied, only the most potent superoxide dismutase mimetic compound was found to inhibit phorbol ester induced IL2 production. Copper(II) complexes had to be added no later than 4 following phorbol ester administration to be effective inhibitors of the IL response, suggesting that these compounds act subsequent to the binding of phorbol esters but prior to the transcription of IL2 messenger RNA. Adherence of EL4 cells to substrate in response to phorbol esters was unaffected by copper(II) compounds. In summary, copper(II) compounds with appropriate reduction potentials can act within a defined time period to inhibit some, but not all, of the effects of phorbol esters on EL4 cells.

Differential role of reactive oxygen intermediates in photofrin-I- and photofrin-II-mediated photoenhancement of lipid peroxidation in epidermal microsomal membranes.

Photoradiation therapy with porphyrins and light offers an alternative approach to the management of certain types of cancer. The mechanism of tissue destruction mediated by this modality is poorly understood. In this study, epidermal microsomes incubated in vitro with Photofrin-I (Pf-I) and Photofrin-II (Pf-II) followed by exposure to radiation (approximately 400 nm) resulted in increased (180%) NADPH-supported (enzymatic) as well as ADP/iron-supported (140%) (nonenzymatic) lipid peroxidative damage as measured by malondialdehyde formation. Lipid peroxidation by Pf-I and Pf-II was found to be differentially affected by quenchers of singlet oxygen (2,5-dimethylfuran, histidine, beta-carotene, ascorbic acid, and sodium azide), superoxide anion (superoxide dismutase), and the hydroxyl radical (sodium benzoate, mannitol, and ethanol). Catalase, a quencher of hydrogen peroxide, afforded significant protection only against Pf-II-enhanced lipid peroxidative damage while it had little effect against the Pf-I-mediated reaction. Deuterium oxide, which is known to increase the half-life of singlet oxygen, was found to enhance Pf-I-mediated lipid peroxidation but produced insignificant effects upon Pf-II-mediated photosensitization. Our results indicate that Pf-I and Pf-II, which are employed for the photodynamic therapy of malignant tumors, evoke membrane damage by generating different reactive oxygen species. The Pf-I-mediated photodestruction mainly involves a type II mechanism via singlet oxygen formation, whereas Pf-II-mediated photodestruction preferentially involves a type I mechanism by generating superoxide anions and hydroxyl radicals. Our data indicate that tumor necrosis evoked by porphyrins and light is likely due to the generation of reactive oxygen species.

Aging of the lungs: speculation on the role of oxidants and proteases.

In most regards, aging of the human lungs mimics the development of emphysema. Various lines of evidence suggest that oxidants and proteases play an important role in the pathogenesis of emphysema. The disease seems to arise through a complex interplay of genetic and environmental factors. This paper explores the possibility that normal aging of the lungs, as it has been defined by various cross-sectional and longitudinal studies, is part of a pathological continuum ending with frank emphysema. It is not clear if these are "normal" aging effects or the result of the accumulation of disease.

Ceruloplasmin: an acute phase reactant that scavenges oxygen-derived free radicals.

Superoxide anion radicals have been implicated recently as mediators of inflammation and tissue injury. Protection from superoxide anion radicals is provided primarily by a copper-containing, intracellular enzyme (superoxide dismutase) (SOD) that catalyzes the dismutation of superoxide to hydrogen peroxide and oxygen. We have found that the action of cytoplasmic SOD to scavenge superoxide and thereby to inhibit superoxide-mediated reactions can be mimicked by the copper-containing plasma protein and acute-phase reactant, ceruloplasmin. Ceruloplasmin, at concentrations present in normal plasma, inhibited reduction of both cytochrome c and nitroblue tetrazolium (NBT) mediated by the aerobic action of xanthine oxidase on hypoxanthine (a superoxide-generating system). Ceruloplasmin neither inhibited formation of uric acid by xanthine oxidase nor accelerated autooxidation of cytochrome c. Furthermore, in an experimental system in which contact between ceruloplasmin and indicator was prevented by a relatively impermeable lipid membrane barrier, ceruloplasmin inhibited reduction of NBT trapped within liposomes exposed to xanthine oxidase and hypoxanthine. Ceruloplasmin also inhibited reduction of cytochrome c and NBT mediated by the aerobic action of xanthine oxidase on acetaldehyde (another superoxide-generating system) and mimicked the activity of purified human erythrocyte SOD by inhibiting photoreduction of NBT and by accelerating aerobic photooxidation of dianisidine. Ceruloplasmin could be separated from purified human erythrocyte SOD by electrophoresis on alkaline 12% polyacrylamide gels and identified by its superoxide-scavenging activity. These results suggest that ceruloplasmin may function as a circulating scavenger of oxygen-derived free radicals.

Effect of polyethylene glycol-superoxide dismutase and catalase on endotoxemia in pigs.

We hypothesized that superoxide anion (O2-.) and hydrogen peroxide (H2O2) might be important mediators of endotoxin-induced acute respiratory failure (ARF) in pigs. As specific scavengers of O2-. and H2O2, we infused polyethylene glycol-superoxide dismutase (PEG-SOD; 2,000 IU/kg) and PEG-catalase (CAT; 15,000 IU/kg), respectively. Escherichia coli endotoxin (055-B5) was infused intravenously into anesthetized 10- to 14-wk-old pigs at 5 micrograms/kg the 1st h, followed by 2 micrograms.kg-1.h-1 for 3.5 h. During phase 1 (i.e., 0-2 h) and 2 (i.e., 2-4.5 h), endotoxin decreased cardiac index (CI) and lung dynamic compliance, and increased mean pulmonary arterial pressure (Ppa), pulmonary vascular resistance (PVR), total peripheral resistance (TPR), alveolar-arterial O2 gradient, and hematocrit. Endotoxemia also caused granulocytopenia and increased the postmortem bronchoalveolar lavage fluid (BALF) albumin concentration and wet-to-dry ratio of bloodless lung. During endotoxemia, PEG-SOD failed to significantly alter any measured or calculated parameter. On the other hand, PEG-CAT attenuated the early (i.e., 0-1 h) endotoxin-induced decrease in CI and increases in Ppa, PVR, and TPR, but failed to modify these parameters during phase 2. PEG-CAT also attenuated the endotoxin-induced granulocytopenia and the increased BALF albumin concentration. In the presence of inactivated PEG-CAT, these protective effects were reversed. We conclude that O2-. does not directly contribute to endotoxin-induced lung injury and that H2O2 (or a subsequent metabolite) contributes to the early endotoxin-induced hemodynamic changes, granulocytopenia, and increased permeability of the alveolar-capillary membrane.

Cell division in normal and transformed cells: the possible role of superoxide and hydrogen peroxide.

A unified theory of cell division is presented. This theory explains the division of both tumor cells and normal cells and involves superoxide radicals, hydrogen peroxide, glucose, cAMP, and cGMP.

[Superoxide radical and superoxide dismutase in the free-radical theory of aging (a review)]. / Superoksidnyi radikal i superoksiddismutaza v svobodnoradikal'noi teorii stareniia (obzor).

Review is presented on the role of superoxide radicals and other intermediates of single electron reduction of oxygen in aging as well as on geroprotective importance of superoxide dismutase. These factors are considered in relation with age. Significance of direct and indirect methods for estimation of total O2-radicals is analyzed. Possible role of O2-radicals and superoxide dismutase in regulation of cell division and differentiation is also discussed. Life span of cells in peripheric circulation correlates with superoxide dismutase content in these cells and their ability to generate exogenous O2-.

Superoxide, iron, vascular endothelium and reperfusion injury.

It is proposed that vascular endothelium has an intrinsic capacity to generate O2- for regulatory purposes such as inactivation of endothelium-derived relaxing factor. Ischaemia can disrupt the functioning of this oxidant-generating system, resulting in greater O2- generation when O2 is restored. Ischaemia-induced cellular injury can also lead to release of iron ions, that, upon reperfusion, cause conversion of O2- and H2O2 to powerfully-oxidizing species (such as .OH) that further injure the endothelium.