The role of superoxide anion generation in phagocytic bactericidal activity. Studies with normal and chronic granulomatous disease leukocytes.

The capacity of human phagocytes to generate superoxide anion (O2-), a free radical of oxygen, and a possible role for this radical or its derivatives in the killing of phagocytized bacteria were explored using leukocytes from normal individuals and patients with chronic granulomatous disease (CGD). Superoxide dismutase, which removes O2-, consistently inhibited phagocytosis-associated nitroblue tetrazolium (NBT) reduction indicating the involvement of O2- in this process. Similarly, superoxide dismutase inhibited the luminescence that occurs with phagocytosis, implicating O2- in this phenomenon, perhaps through its spontaneous dismutation into singlet oxygen. Subcellular fractions from homogenates of both normal and CGD leukocytes generated O2- effectively in the presence of NADH as substrate. However, O2- generation by intact cells during phagocytosis was markedly diminished in nine patients with CGD. Leukocytes from mothers determined to be carriers of X-linked recessive CGD by intermediate phagocytic reduction of NBT elaborated O2- to an intermediate extent, further demonstrating the interrelationship between NBT reduction and O2- generation in phagocytizing cells. Activity of superoxide dismutase, the enzyme responsible for protecting the cell from the damaging effects of O2-, was approximately equal in homogenates of normal and CGD granulocytes. Polyacrylamide electrophoresis separated this activity into a minor band that appeared to be the manganese-containing superoxide dismutase associated with mitochondria and a more concentrated, cyanide-sensitive, cytosol form of the enzyme with electrophoretic mobility that corresponded to that of erythrocyte cuprozinc superoxide dismutase. Superoxide dismutase inhibited the phagocytic killing of Escherichia coli, Staphylococcus aureus, and Streptococcus viridans. A similar inhibitory effect was noted with catalase which removes hydrogen peroxide. Neither enzyme inhibited the ingestion of bacteria. Peroxide and O2- are believed to interact to generate the potent oxidant, hydroxyl radical (.OH). A requirement for .OH in the phagocytic bactericidal event might explain the apparent requirement for both O2- and H2O2 for such activity. In agreement with this possibility, benzoate and mannitol, scavengers of .OH, inhibited phagocytic bactericidal activity. Generation of singlet oxygen from O2- and .OH also might explain these findings. It would seem clear from these and other studies that the granulo cyte elaborates O2- as a concomitant of the respiratory burst that occurs with phagocytosis. To what extent the energy inherent in O2- is translated into microbialdeath through O2- itself, hydrogen peroxide, .OH, singlet oxygen, or some other agent remains to be clearly defined.

The purification and properties of superoxide dismutase from a blue-green alga.

Soluble extracts of Plectonema boryanum have been shown to contain a single, electrophoretically distinct, superoxide dismutase. The enzyme has been isolated and has been found to be an iron-containing enzyme similar to that described from the periplasm of Escherichia coli. It contains 1 Fe3+/mole of enzyme. The molecular weight was approximately 36 500, and the enzyme appeared to be composed of two subunits of equal size joined by non-covalent interactions. ESR data are presented, as are the results of amino acid analysis.

Amelioration of postischemic reperfusion injury by antiarrhythmic drugs in isolated perfused rat lung.

Antiarrhythmic drugs, such as lidocaine, quinidine, and procainamide, have been shown to be effective against postischemic reperfusion injury in isolated rat lungs. Rat lungs were perfused at a constant flow with Krebs-Henseilet buffer supplemented with 4% bovine serum albumin and ventilated with air containing 5% CO2. The lungs were subjected to ischemia by stopping perfusion and ventilation for 60 min followed by 30 min of reperfusion. Lung injury was determined by measuring the increase in wet-to-dry lung weight ratio, while pulmonary arterial pressure and peak airway pressure were calculated from the pre- and postischemic differences. Lidocaine, quinidine, and procainamide at doses of 5, 10, and 20 mg/kg body weight, respectively, were found to attenuate the postischemic lung injury significantly (p < 0.0001). The formation of cyclooxygenase products, which were elevated during reperfusion, was also significantly (p < 0.0001) inhibited by these drugs. Since these antiarrhythmic agents are found to be powerful scavengers of hydroxyl radicals and can prevent membrane lipid peroxidation, these findings suggest that the antioxidant properties of these drugs may, in part, be responsible for protecting the lungs against reperfusion injury.

Vasoactive intestinal polypeptide relaxes pulmonary artery by an endothelium-independent mechanism.

Vasoactive intestinal polypeptide (VIP) is a potent endogenous vasodilator. VIP-induced vasodilation is independent of adrenergic or cholinergic receptors and, for the most part, of arachidonate metabolites, but its mechanism is still unknown. In view of the recently demonstrated essential role of endothelium in the relaxant effect of numerous vasodilators, we have investigated the importance of endothelium in the vascular relaxation induced by VIP. Vascular smooth muscle preparations were circular strips of the pulmonary artery of guinea pig, rat and rabbit, and rat thoracic aorta, previously contracted by a synthetic prostaglandin endoperoxide analog. VIP relaxed pulmonary artery of all species by an endothelium-independent mechanism, but relaxed rat thoracic aorta only in the presence of intact endothelium.

Generation of free radicals during photosensitization of hypocrellin A and their effects on cardiac membranes.

Hypocrellin A (HA), a peryloquinone derivative, has recently been isolated from a fungus Hypocrella bambusae. This lipid soluble pigment, in combination with phototherapy, has been used to treat many skin diseases including the keloids caused by scalding and burns. We have studied the effects of photosensitized HA on biomembranes using pig heart microsomes. Photosensitization of HA was found to peroxidize the membrane lipids in the cardiac microsomes. The photodamage imposed by HA depended not only on the concentration of HA but also on the time of irradiation and pH of the system. Superoxide dismutase (SOD), ascorbic acid, beta-carotene and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) inhibited the lipid peroxidation approximately 50, approximately 50, approximately 30 and approximately 97%, respectively. Spin trapping in combination with EPR spectroscopic techniques was used to identify the reactive free radicals during the photoreaction. Formation of superoxide anion radical, (O2-.), was identified by the SOD-inhibitable DMPO-O2- EPR spectrum. Both SOD and ascorbic acid inhibited the EPR signal intensity in a dose-dependent manner with rate constants of 6.78 x 10(8) M-1 s-1 and 1.82 x 10(4) M-1 s-1, respectively. The lifetime of O2-., under these conditions, was found to be 1.1 s. Photoirradiation of HA yielded a HA free radical with a g = 2.002 which was not suppressed by SOD but in the presence of reductants such as ascorbic acid and catechol the septum was completely suppressed. The increase of the EPR signal intensity and malondialdehyde formation with increasing pH may be due, in part, to the production of predominant *HA- species at high pH which would be more reactive with oxygen to yield O2-.. These results indicate that the lipid peroxidation of the cardiac membranes observed during photooxidation of HA may arise, in part, from the interaction of membrane lipids with reactive species of oxygen and HA free radical produced during the photo-irradiation.

EPR studies of trapped singlet oxygen (1O2) generated during photoirradiation of hypocrellin A.

Hypocrellin A, a peryloquinone derivative, has recently been isolated from the sacs of the fungus Hypocrella bambusae. This pigment, in combination with phototherapy, has been used in human medicine to cure various skin diseases. The generation of singlet oxygen during photoirradiation of Hypocrellin A (HA) was detected as an oxidation product of a sterically hindered amine (tetramethylpiperidine oxide; TEMPO) by electron paramagnetic resonance (EPR) spectroscopic techniques. Azide inhibited the EPR signal intensity in a dose-dependent manner with a quenching rate constant of 3.86 x 10(8) M-1s-1 in ethanol. Deuterated solvents, known to increase the lifetime of singlet oxygen, augmented the EPR signal intensity. The rate of production of singlet oxygen was dependent not only upon the concentration of HA and the time of irradiation but also on the oxygen content of the reaction mixture. The hyperfine splitting constant (aN = 16.3 G) and g-value (g = 2.0056) of the photoproduct of TEMP-singlet oxygen and TEMPO were found to be identical. This indicates that the nitroxide species detected by EPR spectroscopy generated by reacting TEMP with photogenerated 1O2 is TEMPO. The rate constant (kT) for the reaction of singlet oxygen with TEMP to form TEMPO radical was found to be 5.3 x 10(5) M-1s-1.

Revascularization of the limbs with urokinase and TEC catheter endarterectomy for occluded bypass grafts.

Occlusion of a femoral-popliteal or a femoral-tibial bypass graft on a prior occluded superficial femoral artery or the popliteal artery (in cases of ischemia of the legs) creates a complicated problem that may result in limb loss. The revascularization of such limbs with a repeat femoral-tibial or peroneal bypass is difficult and results in a high rate of limb loss from failure of the repeat grafts. Therefore, attempts were made to re-open the grafts through urokinase administration and thrombolysis for the grafts. To address the distal critical stenosis, or occlusion, the urokinase administration and thrombolysis were followed by endovascular endarterectomy using a thromboembolytic catheter (TEC). From 1990 to 1992, the above protocol was followed with 15 patients. In all patients, the protocol included 24 to 48 hours of urokinase administration with 60,000 to 120,000 U of urokinase per hour. A distal pathology was detected in all 15 patients, of whom 8 had complete occlusion at the site of distal anastomosis, 7 had critical stenosis ranging from 90% to 99%, and 1 was found to have a proximal critical stenosis at the femoral-graft junction. TEC catheter endarterectomy was performed to address the problem of occlusion and critically stenosed distal anastomotic lesions; a 100% success rate was immediately achieved, with flow being re-established to the distal limb. Fourteen of 15 patients remained well revascularized for more than 1 year. One patient experienced re-occlusion after 8 months; the occlusion was re-opened using the same technique of urokinase administration and the use of the TEC catheter, in which case the graft subsequently remained open. The longest follow-up of one patient is more than 2 years, during which time the graft has stayed open. With the aforementioned results, after revascularization of the ischemic limbs from occluded bypass grafts, it is strongly recommended that urokinase administration, along with TEC endovascular endarterectomy, be used to establish the circulation when occluded grafts threaten limb loss.

Elevated concentrations of ascorbate and normoxia suppress testosterone production in cultured guinea pig Leydig cells.

In recent years, several metabolic roles have been proposed for vitamin C. Recent information suggests a strong causal relationship between high endogenous levels of ascorbic acid and changes in normal reproductive biology. Using highly enriched populations of guinea pig Leydig cells, we have found that elevated levels (50 to 500 microM) of ascorbate significantly (P < 0.01) depressed testosterone production in a dose-dependent manner while low levels (0 to 10 microM) were without effect. Leydig cells incubated under hypoxic (3% oxygen) culture conditions produced significantly (P < 0.01) more testosterone than similar cells cultured under normoxic (19% oxygen) conditions. The results of this study suggest that high concentrations of ascorbate and normoxic culture conditions suppress testosterone production in isolated Leydig cells. Thus, it would seem that there exists a delicate balance between normal metabolic requirements for vitamin C and excessive ascorbate levels that might alter normal gonadal reproductive events.

Effects of gossypol on the hepatic drug metabolizing system in rats.

Gossypol, a polyphenolic pigment found in cotton plants, has been implicated as an antifertility agent. This pigment has been shown to alter myriad metabolic pathways. The objective of this study was to determine the effect of gossypol acetic acid on the hepatic microsomal drug metabolizing enzyme system in adult female Fisher 344 rats. Subcutaneous administration in both low (15 mg/kg) and high (30 mg/kg) doses of gossypol acetic acid for five consecutive days significantly (P less than 0.00001) reduced hepatic aniline hydroxylase activity, as well as cytochrome P-450 and b5 levels. Thus, compared to the controls, the low doses of gossypol decreased the hepatic aniline hydroxylase activity and cytochrome P-450 and b5 content by 73, 54, and 43 percent, respectively. The high doses of gossypol decreased the aniline hydroxylase, cytochrome P-450, and b5 levels by 60, 51, and 46 percent, respectively, as compared to controls. These data indicate that alteration of the hepatic metabolizing system may, in part, be responsible for the secondary toxemic effects seen when gossypol is used to provide antifertility action.

A histopathologic study of the effects of gossypol on the female rat.

The purpose of this study was to examine the tissues of female rats treated with gossypol acetic acid for morphologic evidence of an underlying mechanism of infertility. The number of estrous cycles, and body and adrenal weights were also compared. The number of estrous cycles decreased in rats treated with 60 mg/kg gossypol acetic acid for 30 days. Body weights were also reduced in rats treated with 40 mg or 60 mg/kg per day for 30 days when compared to controls. However, no significant differences were found in any group when comparing adrenal weights, adrenal weight/body weight ratios or adrenal histology. The body weight loss was related, at least in part, to diarrhea and dehydration in eight of the treated animals. It is interesting that though the gossypol-treated rats had reduced numbers of estrous cycles, no histopathologic changes were found in their ovaries, uterus or vagina.

Chemiluminescence of canine peripheral blood lymphocytes stimulated by mitogens.

Luminol-dependent chemiluminescence of peripheral blood lymphocytes from dogs stimulated with concanavalin A (Con A) or phytohemagglutinin P (PHA) was measured with a Pico-Lite luminometer. 10 microliter of luminol gave optimal quantum yield from 1 X 10(6) lymphocytes sensitized with either 80 micrograms Con A or 160 micrograms PHA. Addition of superoxide dismutase did not influence the course of chemiluminescence. Whereas catalase produced 41% increase in quantum yield, mannitol caused a 51% inhibition of chemiluminescence. Lymphocytes exposed to varying doses of short term x-irradiation or lymphocytes isolated from dogs kept under continuous exposure through a gamma irradiation source showed dose-related depression of chemiluminescence. Membrane factors may be involved in lymphocyte stimulation to chemiluminescence as pulse experiments with Con A and PHA revealed. It is proposed that chemiluminescence measurements may be useful in monitoring early events in lymphocyte stimulation by antigens and mitogens.

Ulcerogenic mechanism of ethanol and the action of sulphanilyl fluoride on the rat stomach in-vivo.

The effects of ethanol alone and in combination with sulphanilyl fluoride on some of the antioxidant defences in the stomach of rats have been examined. These effects were correlated with lesion formation in the gastric mucosa. Oral administration of ethanol induced gastric lesions which were prevented by sulphanilyl fluoride pre-treatment. N-Ethylmaleimide antagonized the anti-lesion action of sulphanilyl fluoride. Ethanol administration lowered the glucose-6-phosphate dehydrogenase activity in the gastric mucosa, an effect potentiated by N-ethylmaleimide pre-treatment. The total superoxide dismutase activity was unaffected by the drugs used in the present study. Ethanol, however, markedly increased mucosal catalase activity which was reduced by sulphanilyl fluoride pretreatment and reversed by N-ethylmaleimide. It is concluded that the ulcerogenic mechanism of ethanol is mediated at least in part by the depression of the hexose monophosphate shunt and the production of active oxygen species, whereas the anti-lesion action of sulphanilyl fluoride is probably not mediated through these mechanisms.

Lack of formyl-methionyl-leucyl-phenylalanine receptors on porcine neutrophils.

The response of blood neutrophils to the chemotactic peptide formyl-methyl-leucyl-phenylalanine varies among species. Our results indicate that this peptide does not activate the respiratory burst of porcine neutrophils. Specifically, concentrations less than or equal to 10(-6) M did not cause production of either superoxide or hydrogen peroxide. Studies designed to delineate the biochemical deficit responsible for these results indicated that these cells do not express specific chemotactic peptide receptors on the external surface of the plasma membrane. Although these data do not rule out the possibility that internal stores of chemotactic peptide receptor exist, attempts to induce expression of the receptor by priming the cells with either lipopolysaccharide or calcium ionophore were unsuccessful.

Inhibition of superoxide dismutase by nitroprusside and electron spin resonance observations on the formation of a superoxide-mediated nitroprusside nitroxyl free radical.

Nitroprusside appears to inhibit the known types of superoxide dismutases irrespective of their metal prosthetic group and regardless of the source from which the enzymes were isolated. Thus the copper-zinc enzyme from bovine erythrocyte or Neurospora crassa behaved identically as did the manganese enzymes from Escherichia coli or red alga and the iron enzyme from E. coli and a blue-green alga. The inhibition was dose dependent with a Ki = 2.5 X 10(-5) for nitroprusside. Nitroprusside does not bind to the copper moiety of copper-zinc enzyme and seems to compete with O2- for superoxide dismutase. These inhibitions by nitroprusside, which were elicited not only in purified enzymes but also in crude soluble extracts of biological samples, were rapidly reversible. Nitroprusside was found to react with O2- to form a paramagnetic species with three absorption lines of equal width with a separation AN = 15.0 G and a g value of 2.028. The spin adduct appears to be a nitroxide radical and was stable for several minutes.

Reaction of copper-zinc superoxide dismutase with diethyldithiocarbamate.

Diethyldithiocarbamate reacted with superoxide dismutase from bovine erythrocytes. Changes in both optical and esr spectra, which accompanied this reaction, indicated involvement of the Cu(II). The reaction was accelerated by raising the concentrations of the reactants, elevating the temperature, and lowering the pH, in the range 10.2 to 5.5, and it was independent of the presence of oxygen. During the first phase of this reaction the Cu(II).diethyldithiocarbamate complex remained bound to the enzyme and the catalytic activity did not diminish. There followed a second and slower process which was accompanied by the appearance of colloidal Cu(II).chelate complex and by a loss of activity which could be restored by the addition of CuSO4. All of the observations are accomodated by a model in which 1 diethyldithiocarbamate molecule reacts/copper center, with retention of activity, in Phase I, while a second diethyldithiocarbamate displaces the copper, with a loss of activity, in Phase II.

Alterations of guinea pig alveolar macrophage oxidative metabolism by nicotine.

The influence of nicotine, a cholinergic agonist, on guinea pig pulmonary alveolar macrophage (PAM) oxidative metabolism was examined. Nicotine caused a concentration-dependent enhancement or inhibition of chemiluminescence response and superoxide anion release by zymosan-stimulated PAM. Thus, at 5 x 10(-10) and 5 x 10(-8) M of nicotine the chemiluminescence response was augmented to 132 and 113%, respectively. At higher concentrations, such as 5 x 10(-7) and 1 x 10(-4) M, however, these responses were inhibited to 83 and 51% of the control, respectively. Similarly, at 5 x 10(-10) and 5 x 10(-9) M nicotine, superoxide anion release was enhanced to 226 and 209% of the control, respectively. Higher concentrations of nicotine, 5 x 10(-5) and 5 x 10(-4) M, inhibited this response to 53 and 58% of the control, respectively. Neither the potentiating nor the inhibitory effect of nicotine was affected by a muscarinic (atropine) or nicotinic (hexamethonium) cholinergic antagonist. None of the drugs examined, by themselves, stimulated PAM oxidative metabolism or influenced oxyradical generation by a cell-free system. This study demonstrates that nicotine, a major component of cigarette smoke, may play a significant role in the pathogenesis of some pulmonary diseases.

Pirfenidone inhibits NADPH-dependent microsomal lipid peroxidation and scavenges hydroxyl radicals.

Pirfenidone (Pf), a new broad-spectrum anti-fibrotic agent, is known to offer protection against lung fibrosis in vivo in laboratory animals, and against mitogenesis and collagen formation by human lung fibroblasts in vitro. Because reactive oxygen species are thought to be involved in these events, we investigated the mechanism(s) by which Pf ameliorates oxidative stress and its effects on NADPH-dependent lipid peroxidation. Pf has been shown to cause inhibit NADPH-dependent lipid peroxidation in sheep liver microsomes in a dose-dependent manner. The concentration of Pf required to cause 50% inhibition of lipid peroxidation was approximately 6 mM. Pf was found to be ineffective as a superoxide radical scavenger. Pf was also ineffective in decomposing H2O2 and chelating iron. In deoxyribose degradation assays, Pf was a potent scavenger of hydroxyl radicals with a rate constant of 5.4 x 10(9) M(-1) sec(-1). EPR spectroscopy in combination with spin trapping techniques, using a Fenton type reaction and DMPO as a spin-trapping agent, Pf scavenged hydroxyl radicals in a dose-dependent manner. The concentration of Pf required to inhibit 50% signal height was approximately 2.5 mM. Because iron was used in the Fenton reaction, the ability of Pf in chelating iron was verified in a fluorescent competitive assay using calcein as the fluorescent probe. Pf up to 10 mM concentration was ineffective in chelating either Fe2+ or Fe3+ in this system. We propose that Pf exerts its beneficial effects, at least in part, through its ability to scavenge toxic hydroxyl radicals.