Mitochondrial permeability transition as a source of superoxide anion induced by the nitroaromatic drug nimesulide in vitro

Nimesulide, a widely used nonsteroidal anti-inflammatory drug containing a nitroaromatic moiety, has been associated with rare but serious hepatic adverse effects. The mechanisms underlying this idiosyncratic hepatotoxicity are unknown; however, both mitochondrial injury and oxidative stress have been implicated in contributing to liver injury in susceptible patients. The aim of this study was, first, to explore whether membrane permeability transition (MPT) could contribute to nimesulide's mitochondrial toxicity and, second, whether metabolism-derived reactive oxygen species (ROS) were responsible for MPT. We found that isolated mouse liver mitochondria readily underwent Ca2+-dependent, cyclosporin A-sensitive MPT upon exposure to nimesulide (at >or=3 microM). Net increases in mitochondrial superoxide anion levels, determined with the fluorescent probe dihydroethidium, were induced by nimesulide only in the presence of Ca2+ and were cyclosporin A-sensitive, indicating that superoxide production was a consequence, rather than the cause, of MPT. In addition, nimesulide caused a rapid dissipation of the inner mitochondrial transmembrane potential (at >or=3 microM), followed by a concentration-dependent decrease in ATP biosynthesis. Because nimesulide, unlike the related nitroaromatic drug nilutamide, did not produce any detectable ROS during incubation with mouse hepatic microsomes, we conclude that mitochondrial uncoupling causes MPT and that ROS production is a secondary effect.     

Inhibition of superoxide anion production by extracellular acidification in neutrophils

Extracellular acidification inhibited formyl-Met-Leu-Phe- or C5a-induced superoxide anion (O₂⁻) production in differentiated HL-60 neutrophil-like cells and human neutrophils. A cAMP-increasing agonist, prostaglandin E₁, also inhibited the formyl peptide-induced O₂⁻ production. The inhibitory action on the O₂⁻ production by extracellular acidic pH was associated with cAMP accumulation and partly attenuated by H89, a protein kinase A inhibitor. A significant amount of mRNAs for T-cell death-associated gene 8 (TDAG8) and other proton-sensing ovarian cancer G-protein-coupled receptor 1 (OGR1)-family receptors is expressed in these cells. These results suggest that cAMP/protein kinase A, possibly through proton-sensing G-protein-coupled receptors, may be involved in extracellular acidic pH-induced inhibition of O₂⁻ production.     

Reduction of iodonitrotetrazolium violet by superoxide radicals

p-Iodonitrotetrazolium (2-(4-iodophenyl-3-(4-nitrophenyl)- 5-phenyltetrazolium; INT) was reduced to a water-soluble product with an absorbance maxima at about 505 nm (reddish pink) by superoxide anion (O2-.) generated by xanthine/xanthine oxidase. The rate of INT reduction was linearly related to the xanthine oxidase activities, and was inhibited by superoxide dismutase. The soluble product may further be converted to an insoluble product, presumably nonionic formazan, with an absorbance maxima of 490 nm (purplish), under certain conditions, and the rate of the formazan formation depended on pH and protein concentration.     

Collagen degradation by superoxide anion in pulse and gamma radiolysis

Delipidated collagen fibrils reconstituted from acid-soluble calf skin collagen, suspended in 50 mM phosphate buffer, pH 7.4, containing 100 mM sodium formate, were submitted to pulse radiolysis in Febetron devices or to gamma radiolysis in a 60Co irradiator. A collagen degradation process was found. The kinetics of this degradation was followed by evaluation of the amount of 4-hydroxyproline present in the small peptides liberated during the irradiation period. The yield of 4-hydroxyproline small peptides was low (0.1 mol/100 eV for an initial collagen concentration 3.2 microM). It increased linearly with the dose of irradiation and the concentration of collagen in suspension. The kinetic competition between O2-. dismutation and O2-. reaction with collagen was studied by pulse radiolysis at several concentrations of collagen. A value of the kinetic constant of k(O2-. + collagen) = 4.8 . 10(6) mol-1.l.s-1 was determined.     

In vitro generation of superoxide anion by the hemocytes of Macrobrachium rosenbergii: possible mechanism and pathways

The hemocytes from the giant freshwater prawn Macrobrachium rosenbergii were examined for their ability to generate superoxide anion (O(2) (-)) in vitro upon exposure to various components derived from microbial cell wall components. Among the test molecules, laminarin (a polymer of beta-1, 3 glucans), mannan and LPS from five different bacterial species produced a differential response in terms of stimulated O(2) (-) production in prawn hemocytes, suggesting the ability of the hemocytes to differentiate non-self. This response was almost completely inhibited by superoxide dismutase (SOD) suggesting SOD-inhibitable O(2) (-) generation by prawn hemocytes. Phorbol 12-myristate 13-acetate (PMA) and Ca ionophore led to enhanced O(2) (-) generation by the hemocytes and this suggests the possible role of protein kinase C and Ca(2+) ions in such generation. Cytochemical analysis using nitroblue tetrazolium (NBT)-staining revealed the importance of granular hemocytes in O(2) (-) generation in these prawns. Inhibition of O(2) (-) generation by inhibitors of NADPH-oxidase and phenoloxidase pathways clearly reveal the involvement of two different pathways in non-self stimulated O(2) (-) generation by the prawn hemocytes. These findings demonstrate the importance of O(2) (-) generation and role of possible pathways in hemocyte mediated cellular immune response of a crustacean.     

Uptake of metallic mercury and mercuric ion by human erythrocytes

Uptake of metallic mercury (Hg degrees) and mercuric ion (Hg2+) by erythrocytes was studied by incubating erythrocytes with various concentrations of radioactive metallic mercury and mercuric ion in phosphate-buffered saline (pH 6.8) or plasma at 25 degrees C for 30 min. Radioactivity taken up in the cytosol (endsome) and stroma were determined with a gamma scintillation counter. The radioactivity ratio of the mercury recovered in the cytosol fraction to metallic mercury incubated in the saline was significantly higher than the ratio of that to mercuric ion. Similar findings were observed in erythrocytes incubated with metallic mercury and mercuric ion in plasma, although the recovered radioactivity of mercury in the cytosol of erythrocytes incubated with metallic mercury or mercuric ion in plasma was less than that incubated in phosphate-buffered saline. Thus, erythrocytes incubated with metallic mercury took up a larger amount of mercury than those incubated with mercuric ion. Discussion is made on these findings.     

Osmotic stress stimulates generation of superoxide anion by spermatozoa in horses

The objective of this study was to examine the interplay between osmotic and oxidative stress as well as to determine mechanisms by which osmotic stress increases superoxide generation in spermatozoa of horses. Superoxide production, as measured by dihydroethidium (DHE), increased when spermatozoa of horses were incubated under either hyperosmotic or hyposmotic conditions. This increase in superoxide production was inhibited by the MAP kinase p38 inhibitor, SB203580, and by the superoxide scavenger, tiron. Incubation of spermatozoa under hyperosmotic conditions increased overall protein tyrosine phosphorylation as measured by western blotting techniques; however, a similar increase was not detected when spermatozoa were incubated under hyposmotic conditions. The general protein kinase C (PKC) and protein tyrosine kinase (PTK) inhibitor staurosporine inhibited (P < 0.05) tyrosine phosphorylation in samples from cells under hyperosmotic conditions. In addition, the NADPH oxidase inhibitor diphenyleneiodonium (DPI) also inhibited (P < 0.05) protein tyrosine phosphorylation in cells under hyperosmotic conditions. In summary, these data indicate that incubation of equine spermatozoa under both hyposmotic and hyperosmotic conditions can increase superoxide anion generation. Under hyperosmotic conditions, this increased generation of superoxide anion was accompanied by increased protein tyrosine phosphorylation.     

Generation of superoxide anion by equine spermatozoa as detected by dihydroethidium

Low-level production of the superoxide anion (O2*-) is an important signal transduction event in sperm function including capacitation; however, excessive production of O2*- can be detrimental to sperm function. The objective of this study was to assess dihydroethidium (DHE) as a probe for O2*- in equine spermatozoa. Ejaculated spermatozoa were separated by centrifugation over a Percoll gradient (40:80), and loaded with DHE (2.0 microM) as well as with calcein-acetoxymethylester (CAM, 7.8 nM) to determine cell viability. In Experiment 1, cells were incubated with the xanthine-xanthine oxidase (X, 0.1 mM; XO, 0.01 U/mL) generating system for the production of O2*-, with or without the addition of superoxide dismutase (SOD, 150 U/mL) or the SOD mimetic, Tiron (0.1, 1.0 or 5.0 mM) for 1h. Changes in fluorescence of DHE were determined for the live cell population (calcein-positive cells) by flow cytometry. The DHE fluorescence increased with the X-XO incubation; this increase was inhibited by SOD or Tiron, indicating that DHE is specific for O2*- detection. In Experiment 2, spermatozoa were loaded with DHE/CAM, treated with calcium ionophore A23187 (0, 0.8, or 8.0 microM), and incubated for 15 min. Cell fluorescence was again determined by flow cytometry. Calcium ionophore A23187 increased O2*- production in a dose-dependent manner. In Experiment 3, cells were loaded with DHE/CAM, treated with NADPH (0.0, 0.25, 0.5, or 1 mM) with or without 0.5% Triton X-100, and incubated for 15 min prior to flow cytometry. Cells treated with NADPH with or without 0.5% Triton X-100 did not have O2*- levels that were significantly different from the control. In Experiment 4, spermatozoa loaded with DHE/CAM were incubated under capacitating conditions (1.2 mM dibutryl-cAMP+1.0 mM caffeine) or in control media for 3h. Although O2*- generation increased over time in control and capacitated treatments, spermatozoa incubated under capacitating conditions had higher O2*- production than those incubated in control media. Therefore, DHE was a useful probe for the detection of O2*- in equine spermatozoa and elevation in intracellular calcium as well as capacitation in vitro were associated with increased generation of O2*-.     

Reduction of lactoperoxidase by the dithionite anion monomer

The reduction of lactoperoxidase with sodium dithionite has been studied by means of stopped-flow spectrophotometry in an anaerobic system. Under pseudo-first-order conditions the rate constant was found to be linearly dependent on the square root of the dithionite concentration, which confirms the monomeric radical, SO2- as the reducing species. The second-order rate constant is moderately influenced by increased ionic strength but drastically increased at lower pH. The pH dependence supports the previously suggested existence of a carboxyl group, essential to the different enzymatic functions of lactoperoxidase. The second-order rate constant for the reduction of lactoperoxidase at pH 7.0 (kappa 1 = 1.3 X 10(5) M-1 s-1) was about three times higher than the rate constant for the reduction of cyanide-bound lactoperoxidase and two times the rate constant for the reduction of the fluoride-lactoperoxidase complex.     

Effects of in vitro and in vivo supplementation with zinc on superoxide anion production in leukocytes

The effects of zinc on the rate of production of bactericidal O2- of polymorphonuclear leukocytes (PMN) in response to three different types of stimulating agents (serum-treated zymosan (STZ), Con A, and myristate) were studied. The percentage reduction of O2- production of PMN stimulated by STZ, Con A, and myristate were all reduced in response to Zn, irregardless of whether Zn was added to the reaction mixture immediately before SZT addition or following a prior 20 min. incubation of PMN in the presence of Zn. However, when Zn was introduced intraperitonially into guinea pigs before the collection of PMN from the animal, zinc treatment produced inhibition only in STZ-activated PMN; it produced no effect in O2- production of PMN stimulated by myristate, and it further augmented the O2- production stimulated by Con A.     

Production of superoxide anion by phagocytosing human and pig leukocytes

The production of Superoxide anion during phagocytosis of zymosan particles and E. coli 055 by human polymorphonuclear leukocytes was found to be 2.2 –3 times higher than by pig leukocytes.     

Reduction of methemerythrin by dithionite ion

The reduction of methemerythrin (Hr⁺) by dithionite produces deoxyhemerythrin (Hr^o) in multi, possibly three, stages. The kinetics were examined at pH 8·2 and 25 °C. The first stage is reduction of methemerythrin to an intermediate A by SO₂^- (k = 1.3 × 10⁵m^?1s^?1). The much slower second and third stages have rates independent of dithionite concentrations. Reaction is completed after about 10 h. The kinetics of reactions of A with N₃^-, H₂O₂, and O₂ were examined, as well as the conversion of A to intermediate B (k = 4·4 × 10^?4s^?1). It is concluded that A is an (Fe(II)Fe(III))₈ species, and that in B the unit (Fe(II)Fe(II))₈ is well developed, judging by its unreactivity towards N₃^?, its reaction with H₂O₂, and its reversible uptake of O₂ (85–90% of the final product). There is little effect of adjusting the pH to 6·3 on the rates of the processes examined.     

Priming of superoxide anion in polymorphonuclear neutrophils by brain natriuretic peptide.

In acute coronary syndromes such as unstable angina and myocardial infarction, serum concentration of brain natriuretic peptide, a cardiac hormone with potent vasodilatatory, natriuretic and diuretic activities, is elevated. Little is known about the effect of elevated BNP plasma concentration on free radical-mediated tissue damage in these states. We investigated the influence of human BNP 32 and its fragment BNP 7-32 on the production of superoxide anion by PMN, a major cause for myocardial damage. Although BNP showed itself no stimulatory potential on superoxide anion release in PMN, it enhanced significantly the stimulatory potential of cell stimuli such as fMLP or phorbol 12-myristate 13-acetate (PMA) in PMN. Thus our data show that the cardiac-derived hormone BNP influences an important function of PMN. This 'priming' effect of BNP on PMN may contribute to the tissue damage occuring during acute coronary syndromes.     

Production of superoxide anion during the oxidation of hemoglobin by menadione

Menadione in the presence of oxyhemoglobin will accelerate the formation of methomoglobin and result in the generation of superoxide anion. Menadione appears to oxidize slowly ferrohemoglobin to ferrihemoglobin, while forming menadione semiquinone in the process. Menadione semiquinone is known to react with molecular oxygen to yield superoxide anion. The superoxide anion appears to be the source of hydrogen peroxide which accounts for most of the observed methemoglobin formation when hemoglobin is reacted with menadione.     

NPGB-induced inhibition of superoxide anion production by normal Lewis rat macrophages

The treatment of Lewis rat peritoneal macrophages with p¹-nitrophenyl p-guanidinobenzoate (NPGB) inhibited the superoxide anion production stimulated with phorbol myristate acetate (PMA). The addition of NPGB at the time of maximum superoxide generation was still able to block the superoxide release. It appears from these findings that NPGB may block either the activation process of the membrane bound NAD(P)H oxidase or directly on the active enzyme. Other protease inhibitors such as, epsilon-amino caproic acid (EACA), pepstatin, trans aminomethyl cyclohexane carboxylic acid (AMCA), aprotinin, and leupeptin did not inhibit the superoxide release. The superoxide anion release by the xanthine-xanthine oxidase system was not inhibited by NPGB. This finding indicates that NPGB does not itself react with superoxide. It has been also demonstrated that NPGB is a good reactant toward sulfhydryl group. The relevance of these finding to experimental allergic encephalomyelitis (EAE) is discussed.     

Cyanobacteria as a biosorbent for mercuric ion

The biosorption of Hg(2+) by two strains of cyanobacteria, Spirulina platensis and Aphanothece flocculosa, was studied under a batch stirred reaction system. Essential process parameters, including pH, biomass concentration, initial metal concentration, and presence of co-ions were shown to influence the Hg(2+) uptake. Hg(2+) uptake was optimal at pH 6.0 for both strains. The maximum loading capacities per gram of dry biomass were found to be 456 mg Hg(2+) for A. flocculosa and 428 mg Hg(2+) for S. platensis. At an initial concentration of 10 ppm Hg(2+), A. flocculosa was able to remove more than 98% of the mercury ion from solution. The biosorption kinetics of both strains showed that the metal uptake is bi-phasic, exhibiting a rapid initial uptake followed by a slower absorption process. The presence of dissolved Co(2+), Ni(2+), and Fe(3+) were found to play a synergistic role for Hg(2+) uptake by both strains. Regeneration of the biomass was examined by treating Hg(2+)-loaded samples with HCl and NH(4)Cl over four cycles of sorption and desorption.     

In vitro effects of different steroid hormones on superoxide anion production of human neutrophil granulocytes

Neutrophil granulocytes play an important role in atherogenesis also through their free radical generation. According to recent studies, a point of action by which estrogens can provide protection against atherosclerosis is their inhibiting effect on superoxide anion production. The aim of our study was to test whether this means a common effect of steroids on superoxide production, or whether various steroid hormones have different action on superoxide generation of human granulocytes. Neutrophils were separated from the blood samples of twelve healthy volunteers. Isolated cells were incubated with different concentrations (10(-9), 10(-8), 10(-7) M) of hydrocortisone, aldosterone, cortexolone, 17-beta-estradiol, progesterone, and testosterone. Superoxide anion production was determined by photometry using the reduction of ferricytochrome-C. Compared to that of control cells neutrophils incubated with 17-beta-estradiol, progesterone, testosterone and hydrocortisone showed significantly reduced superoxide production. No significant alteration of superoxide anion production was found after the incubation of cells with aldosterone and cortexolone. It is concluded that similarly to estradiol other sex steroids and cortisol can inhibit the free radical production of human granulocytes, but mineralocorticoid aldosterone and Reichstein's substance S do not show such activity. Our results provide new evidence supporting the theory that certain types of steroid hormones have antioxidant capacity. This may give further reasons for investigating the molecular background of the existence or absence of this property and thus might lead to the development of new free radical scavengers.