Lymphocytes can produce respiratory burst and oxygen radicals as polymorphonuclear leukocytes

The respiratory burst and production of oxygen radicals by lymphocytes stimulated with phorbol myristate acetate (PMA) was studied and compared with that of polymorphonuclear leukocytes (PMN) by electron paramagnetic resonance (EPR) and spin trapping technique. Superoxide anion and hydroxyl radicals spin adducts of DMPO were detected in the stimulated PMN system, but only hydroxyl radical spin adducts of DMPO were detected in the stimulated lymphocyte system. It was proved by Superoxide dismutase (SOD) and catalase that the hydroxyl radicals produced in the stimulated lymphocyte system came from Superoxide anions, just like the hydroxyl radicals produced in the stimulated PMN.     

Lymphocytes can produce respiratory burst and oxygen radicals as polymorphonuclear leukocytes.

The respiratory burst and production of oxygen radicals by lymphocytes stimulated with phorbol myristate acetate (PMA) was studied and compared with that of polymorphonuclear leukocytes (PMN) by electron paramagnetic resonance (EPR) and spin trapping technique. Superoxide anion and hydroxyl radicals spin adducts of DMPO were detected in the stimulated PMN system, but only hydroxyl radical spin adducts of DMPO were detected in the stimulated lymphocyte system. It was proved by superoxide dismutase (SOD) and catalase that the hydroxyl radicals produced in the stimulated lymphocyte system came from superoxide anions, just like the hydroxyl radicals produced in the stimulated PMN.     

The catalytic effect of the carcinogen "4-nitroquinoline-N-oxide" on the oxidation of vitamin C.

The carcinogen 4-nitroquinoline-N-oxide was found to mediate the reaction between ascorbate and oxygen. The oxidation of ascorbate was initiated by the production of the nitro radical anion which reacted with oxygen to produce the oxygen superoxide radical anion, peroxide and hydroxyl radical. The production of partially reduced oxygen intermediates resulted in additional reactions with ascorbate. The consumption of oxygen could be either completely blocked by reacting the nitro radical with ferricytochrome c or partially blocked by the combined effects of superoxide dismutase and catalase. The consumption of oxygen could be enhanced by reducing the hydroxyl radicals with dimethylsulfoxide.     

Luminol-enhanced chemiluminescence of rabbit polymorphonuclear leukocytes: The nature of oxidants that directly cause luminol oxidation

In this study, we investigated the pathways (including the formation of hydroxyl radicals and chloramines) leading to luminol chemiluminescence induced by hypochlorite generated in a suspension of stimulated rabbit polymorphonuclear leukocytes. Chemiluminescence of leukocytes stimulated by phorbol myristate acetate, which was enhanced by luminol (0.02 mM), did not change in the presence of dimethyl sulfoxide at moderate concentrations (0.02–2.6 mM), under which the latter should manifest the specific ability to scavenge hydroxyl radicals. This indicates that stimulation of polymorphonuclear leukocytes is not accompanied by the generation of hydroxyl radicals with the involvement of superoxide anion and hypochlorite synthesized by myeloperoxidase. At high concentrations of dimethyl sulfoxide (260 mM), chemiluminescence markedly declined because dimethyl sulfoxide directly reacts with hypochlorite. The luminol emission intensity considerably increased after its addition to a suspension of leukocytes that were preliminarily stimulated for 10 min. This effect was caused by the accumulation of hydrogen peroxide rather than chloramines. Exogenous amino acids and taurine at high concentrations (3–15 mM) quench chemiluminescence. All these data indicate that chemiluminescence in the system studied is largely determined by the direct initial reaction of hypochlorite with luminol, the emission intensity increasing as a result of oxidation of luminol transformation products by hydrogen peroxide.     

The superoxide anion and singlet molecular oxygen: their role in the microbicidal activity of the polymorphonuclear leukocyte

Superoxide dismutase was found to partially inhibit both chemiluminescence and nitroblue tetrazolium (NBT) reduction from intact human polymorphonuclear leukocytes. This capacity to reduce NBT was lost when the polymorphonuclear leukocytes were sonicated, but could be regained if exogenous NADPH (or NADH) was added to the system. Superoxide dismutase was found to inhibit this NADPH- and NADH-dependent NBT reduction. A mechanism is proposed that relates superoxide anion generation to the univalent reduction of O₂ by the activated NADPH (and NADH) oxidase. The relationship of superoxide anion production to NBT reduction, singlet molecular oxygen generation, and chemiluminescence is discussed.     

Role of free radical processes in stimulated human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes produce large quantities of superoxide when they attack and kill bacteria. However, superoxide is a weak oxidizing and reducing agent, and other more reactive oxygen species derived from reactions of superoxide are suggested to participate in the killing processes. To test the hypothesis that a reactive free radical or singlet oxygen is involved in bactericidal activity, human polymorphonuclear leukocytes were exposed to phagocytozable particles containing lipids that contain the easily autoxidized 1,4-diene moiety. After incubation the preparations were extracted and the extracts reduced with NaBH4 to convert hydroperoxides to stable alcohols. Using gas chromatography/mass spectrometry to analyze the extracts, we were unable to detect products unless iron salts were added to the medium. The products obtained by extraction are those that would be expected if both free radical chain autoxidation and 1O2 oxidation were taking place. In summary, we find that polymorphonuclear leukocytes do not cause peroxidation, implying that formation of strongly oxidizing free radicals is not an intrinsic property of the leukocyte. Added iron catalyzes peroxidation by activated leukocytes yielding an unusual distribution of hydroxylated products.     

Cephaloridine-induced lipid peroxidation initiated by reactive oxygen species as a possible mechanism of cephaloridine nephrotoxicity

Rat kidney microsomes reduced cephaloridine when incubated anaerobically with NADPH. Superoxide anion was generated in a concentration- and time-dependent manner when cephaloridine was incubated with rat kidney microsomes. Cephaloridine increased the in vitro peroxidation of rat kidney microsomal lipids in a concentration- and time-dependent manner. Cephaloridine-induced lipid peroxidation was inhibited by a combination of superoxide dismutase and catalase, by the hydroxyl radical scavengers, mannitol, (+)-cyanidanol-3 and by the singlet oxygen scavenger histidine in a concentration-dependent manner. It is proposed that cephaloridine nephrotoxicity may occur through the transfer of an electron from reduced cephaloridine to oxygen and subsequent formation of the superoxide anion, hydrogen peroxide, the hydroxyl radical and singlet oxygen. These activated oxygen species then are very likely to react with membrane lipids to induce lipid peroxidation and nephrotoxicity.     

Presence of Hydrogen Peroxide,a Source of Hydroxyl Radicals,in Acid Electrolyzed Water

Background

Acid electrolyzed water (AEW), which is produced through the electrolysis of dilute sodium chloride (NaCl) or potassium chloride solution, is used as a disinfectant in various fields because of its potent antimicrobial activity. The hydroxyl radical, an oxygen radical species, is often suggested as a putative active ingredient for AEW antimicrobial activity.

Methodology/Principal Findings

The aim of the present study is to detect hydroxyl radicals in AEW. The hydroxyl radicals in AEW prepared under different conditions were determined using an electron spin resonance (ESR) technique. A signal from 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-OH, an adduct of DMPO and the hydroxyl radical, was detected in AEW prepared by double or triple electrolyses of 1% NaCl but not of 0.1% NaCl solution. Then the presence of hydrogen peroxide as a proposed source of hydroxyl radicals was examined using a combination of ESR and a Fenton reaction. The DMPO-OH signal was clearly detected, even in AEW prepared by single electrolysis of 0.1% NaCl solution, when ferrous sulfate was added to induce a Fenton reaction, indicating the presence of hydrogen peroxide in the AEW. Since sodium formate, a hydroxyl radical scavenger, did not affect the bactericidal activity of AEW, it is concluded that the radical is unlikely to contribute to the antimicrobial activity of AEW, although a small amount of the radical is produced from hydrogen peroxide. Dimethyl sulfoxide, the other hydroxyl radical scavenger used in the present study, canceled the bactericidal activity of AEW, accompanied by complete depletion of free available chlorine, suggesting that hypochlorous acid is probably a major contributor to the antimicrobial activity.

Conclusions

It is strongly suggested that although hydrogen peroxide is present in AEW as a source of hydroxyl radicals, the antimicrobial activity of AEW does not depend on these radicals.     

Catalase Expression Is Modulated by Vancomycin and Ciprofloxacin and Influences the Formation of Free Radicals in Staphylococcus aureus Cultures

Detection of free radicals in biological systems is challenging due to their short half-lives. We have applied electron spin resonance (ESR) spectroscopy combined with spin traps using the probes PBN (N-tert-butyl-α-phenylnitrone) and DMPO (5,5-dimethyl-1-pyrroline N-oxide) to assess free radical formation in the human pathogen Staphylococcus aureus treated with a bactericidal antibiotic, vancomycin or ciprofloxacin. While we were unable to detect ESR signals in bacterial cells, hydroxyl radicals were observed in the supernatant of bacterial cell cultures. Surprisingly, the strongest signal was detected in broth medium without bacterial cells present and it was mitigated by iron chelation or by addition of catalase, which catalyzes the decomposition of hydrogen peroxide to water and oxygen. This suggests that the signal originates from hydroxyl radicals formed by the Fenton reaction, in which iron is oxidized by hydrogen peroxide. Previously, hydroxyl radicals have been proposed to be generated within bacterial cells in response to bactericidal antibiotics. We found that when S. aureus was exposed to vancomycin or ciprofloxacin, hydroxyl radical formation in the broth was indeed increased compared to the level seen with untreated bacterial cells. However, S. aureus cells express catalase, and the antibiotic-mediated increase in hydroxyl radical formation was correlated with reduced katA expression and catalase activity in the presence of either antibiotic. Therefore, our results show that in S. aureus, bactericidal antibiotics modulate catalase expression, which in turn influences the formation of free radicals in the surrounding broth medium. If similar regulation is found in other bacterial species, it might explain why bactericidal antibiotics are perceived as inducing formation of free radicals.     

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.     

Fibrinogen is an efficient antioxidant

Fibrinogen has been included among the risk factors for vascular disease. Fibrinogen belongs with albumin, ceruloplasmin and transferrin to an acute phase protein group in the plasma. Albumin, ceruloplasmin and transferrin are already recognized as natural antioxidants. In the present study we used three different oxygen generating systems in order to test whether fibrinogen is able to act as an antioxidant in an in vitro system. We used 1) pyrogallol auto-oxidation, 2) the reaction catalysed by xanthine oxidase coupled with the reduction of ferricytochrome c and 3) chemiluminescence. We found that in a dose-dependent manner fibrinogen inhibited superoxide generation (pyrogallol and xanthine-xanthine oxidase reactions), ferrous ion oxidation and hydroxyl radical dependent degradation (of deoxyribose). Fibrinogen also inhibited LDL oxidation (copper and azo compound-induced), hydrogen peroxide oxidation and chemiluminescence produced by polymorphonuclear leukocytes. Fibrinogen, albumin, ceruloplasmin and transferrin act as a supplementary antioxidant defense mechanism against oxidative stress arising from inflammatory conditions.     

A picomolar spectrophotometric assay for superoxide dismutase

During the aerobic xanthine oxidase reaction, O₂^? is produced and accumulates to a steady state determined by a balance between the rate of production of this radical and its rate of dismutation. Addition of ferricytochrome c then results in a biphasic reduction, the very rapid phase of which reflects reaction of the accumulated O₂^?, while the slower phase corresponds to the continuing production of this radical. Superoxide dismutase suppresses the accumulation of O₂^? during the xanthine oxidase reaction and thus diminishes the burst of reduction seen upon addition of ferricytochrome c. This effect has been utilized, at pH 10.2, as the basis of an assay that permits measurement of picomolar levels of superoxide dismutase. The theory and practice of this ultrasensitive assay are described.     

Cytochrome c-catalyzed oxidation of organic molecules by hydrogen peroxide.

Cytochrome c catalyzed the oxidation of various electron donors in the presence of hydrogen peroxide (H2O2), including 2-2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 4-aminoantipyrine (4-AP), and luminol. With ferrocytochrome c, oxidation reactions were preceded by a lag phase corresponding to the H2O2-mediated oxidation of cytochrome c to the ferric state; no lag phase was observed with ferricytochrome c. However, brief preincubation of ferricytochrome c with H2O2 increased its catalytic activity prior to progressive inactivation and degradation. Superoxide (O2-) and hydroxyl radical (.OH) were not involved in this catalytic activity, since it was not sensitive to superoxide dismutase (SOD) or mannitol. Free iron released from the heme did not play a role in the oxidative reactions as concluded from the lack of effect of diethylenetriaminepentaacetic acid. Uric acid and tryptophan inhibited the oxidation of ABTS, stimulation of luminol chemiluminescence, and inactivation of cytochrome c. Our results are consistent with an initial activation of cytochrome c by H2O2 to a catalytically more active species in which a high oxidation state of an oxo-heme complex mediates the oxidative reactions. The lack of SOD effect on cytochrome c-catalyzed, H2O2-dependent luminol chemiluminescence supports a mechanism of chemiexcitation whereby a luminol endoperoxide is formed by direct reaction of H2O2 with an oxidized luminol molecule, either luminol radical or luminol diazoquinone.     

Effects of oxygen free radical scavengers on uranium-induced acute renal failure in rats

Study was made to determine whether oxygen free radicals mediate uranium-induced acute renal failure (ARF). Superoxide dismutase (SOD), a superoxide anion scavenger, did not prevent uranium acetate (UA) (5 mg/kg, i.v.)-induced renal injury 48 h after injection. In contrast, dimethylthiourea (DMTU), a hydroxyl radical scavenger, significantly attenuated UA-induced rise in serum creatinine concentration (1.11 ± 0.05 (DMTU) vs. 1.40 ± 0.06 mg/dl (control), p < .05), and tubular necrosis. Dimethyl sulfoxide (DMSO), a hydroxyl radical scavenger, decreased UA-induced tubular damage. UA injection caused no increase in renal cortical malondialdehyde (MDA) content. DMTU and DMSO did not modify intrarenal MDA content. UA administration brought about significant increase in plasma renin activity but not in renal cortical renin content. Treatment with DMTU and DMSO had no effect on plasma renin activity or intrarenal renin content. It follows from these findings that DMTU and DMSO may attenuate UA-induced renal injury. Such a protective effect would not be mediated through modulation of lipid peroxidation or renin activity.     

Sphingolipid depletion suppresses UPR activation and promotes galactose hypersensitivity in yeast models of classic galactosemia

Classic galactosemia is an inborn error of metabolism caused by deleterious mutations on the GALT gene, which encodes the Leloir pathway enzyme galactose-1-phosphate uridyltransferase. Previous studies have shown that the endoplasmic reticulum unfolded protein response (UPR) is relevant to galactosemia, but the molecular mechanism behind the endoplasmic reticulum stress that triggers this response remains elusive. In the present work, we show that the activation of the UPR in yeast models of galactosemia does not depend on the binding of unfolded proteins to the ER stress sensor protein Ire1p since the protein domain responsible for unfolded protein binding to Ire1p is not necessary for UPR activation. Interestingly, myriocin – an inhibitor of the de novo sphingolipid synthesis pathway – inhibits UPR activation and causes galactose hypersensitivity in these models, indicating that myriocin-mediated sphingolipid depletion impairs yeast adaptation to galactose toxicity. Supporting the interpretation that the effects observed after myriocin treatment were due to a reduction in sphingolipid levels, the addition of phytosphingosine to the culture medium reverses all myriocin effects tested. Surprisingly, constitutively active UPR signaling did not prevent myriocin-induced galactose hypersensitivity suggesting multiple roles for sphingolipids in the adaptation of yeast cells to galactose toxicity. Therefore, we conclude that sphingolipid homeostasis has an important role in UPR activation and cellular adaptation in yeast models of galactosemia, highlighting the possible role of lipid metabolism in the pathophysiology of this disease.     

Are Free Radicals Involved in the Pathobiology of Human Essential Hypertension?

Possible involvement of reactive oxygen species and nitric oxide in the pathogenesis of human essential hypertension was investigated. It was observed that both superoxide anion and hydrogen peroxide production by polymorphonuclear leukocytes and the plasma levels of lipid peroxides are higher in uncontrolled essential hypertension compared with normal controls. Nitric oxide levels measured as its stable metabolite nitrite, as an index of nitric oxide synthesis, revealed its levels to be low in hypertensive patients. Superoxide anion, hydrogen peroxide, lipid peroxides and nitric oxide levels reverted to normal values after the control of hypertension by drugs. The concentrations of anti-oxidants such as vitamin E and superoxide dismutase were found to be decreased in patients with uncontrolled hypertension. Several anti-hypertensive drugs inhibited lipid peroxidation in vitro. Angiotensin-II, a potent vasoconstrictor, stimulated free radical generation in normal leukocytes which could be blocked by calmodulin antagonists. These results suggest that an increase in free radical generation and a simultaneous decrease in the production of nitric oxide and anti-oxidants such as SOD and vitamin E occurs in essential hypertension. This increase in free radical generation can inactivate prostacyclin and nitric oxide and decrease their half life which can lead to an increase in peripheral vascular resistance and hypertension.     

Interaction between fulvic acids of different origins and active oxygen radicals

Using the spin trapping technique, the interaction between fulvic acids (FAs) of different origins and the active oxygen radicals was studied. The active oxygen radicals under study included superoxide anion (O2 · -) produced by xanthine oxidase (XOD) and stimulated polymorphonuclear leukocytes (PMN) of human being and hydroxyl radical ( ·OH) produced from Fenton's reaction. It has been found that the FAs from both Kaschin-Beck disease (KBD) region and non-KBD region can accelerate the production of ·OH and scavenge O2 ·- . FA from peat can scavenge both O2·- and ·OH. The results show that the behavior of KBD and non-KBD FAs differs clearly from peat FA. It has been concluded that the superoxidation damage of KBD induced by FA is mainly due to hydroxyl radical reaction initiated in biological system.     

Nitrofuran enhancement of microsomal electron transport, superoxide anion production and lipid peroxidation

Addition of nifurtimox (a nitrofuran derivative used for the treatment of Chagas' disease) to rat liver microsomes produced an increase of (a) electron flow from NADPH to molecular oxygen, (b) generation of both superoxide anion radical (O₂^?) and hydrogen peroxide, and (c) lipid peroxidation. The nifurtimox-stimulated NADPH oxidation was greatly inhibited by NADP⁺ and p-chloromercuribenzoate, and to a lesser extent by SKF-525-A and metyrapone. These inhibitions reveal the function of both the NADPH-cytochrome P-450 (c) reductase and cytochrome P-450 in nifurtimox reduction. Superoxide dismutase, catalase (in the presence of superoxide dismutase), and hydroxyl radical scavengers (mannitol, 5,5-dimethyl-1-pyrroline-1-oxide) inhibited the nifurtimox-stimulated NADPH oxidation, in accordance with the additional operation of a reaction chain including the hydroxyl radical. Further evidence supporting the role of superoxide anion and hydroxyl radicals in the nifurtimox-induced NADPH oxidation resulted from the effect of specific inhibitors on NADPH oxidation by O₂^? (generated by the xanthine oxidase reaction) and by OH. (generated by an iron chelate or the Fenton reaction). Production of O₂^? by rat kidney, testes and brain microsomes was significantly stimulated by nifurtimox in the presence of NADPH. It is postulated that enhanced formation of free radicals is the basis for nifurtimox toxicity in mammals, in good agreement with the postulated mechanism of the trypanocide effect of nifurtimox on Trypanosoma cruzi.     

Oxygen Enhancement of bactericidal activity of rifamycin SV on Escherichia coli and aerobic oxidation of rifamycin SV to rifamycin S catalyzed by manganous ions: the role of superoxide

Oxygen enhanced the bactericidal activity of rifamycin SV to Escherichia coli K12. Anaerobically grown cells, which had a low level of superoxide dismutase, were more susceptible to the bactericidal activity than aerobically grown cells, which contained a high level of superoxide dismutase. Oxygen also enhanced the inhibition of RNA polymerase activity of rifamycin SV, when Mn2+ was used as a cofactor. Rifamycin S was reduced to rifamycin SV by NADPH catalyzed by cell-free extracts of Escherichia coli K12. These results indicate that the inhibition of bacterial growth by rifamycin SV is due to the production of active species of oxygen resulting from the oxidation-reduction cycle of rifamycin SV in the cells. The aerobic oxidation of rifamycin SV to rifamycin S was induced by metal ions, such as Mn2+, Cu2+, and Co2+. The most effective metal ion was Mn2+. In the presence of Mn2+, accompanying the consumption of 1 mol of oxygen and the oxidation of 1 mol of rifamycin SV, 1 mol of hydrogen peroxide and 1 mol of rifamycin S were formed. Superoxide was generated during the autoxidation of rifamycin SV. Superoxide dismutase inhibited the formation of rifamycin S, but scavengers for hydrogen peroxide and the hydroxyl radical did not affect the oxidation. A mechanism of Mn2+-catalyzed oxidation of rifamycin SV is proposed and its relation to bactericidal activity is discussed.     

ESR studies on active oxygen radicals produced in the respiratory burst of human polymorphonuclear leukocytes

The mechanism and process of production of active oxygen radicals in the respiratory burst of polymorphonuclear leukocytes (PMN) stimulated with PMA (phorbol myristate acetate) was studied in this paper. The experimental results indicate that when the PMA was dilute enough or at the beginning of stimulation even when the PMA concentration was high, the spectrum of hydroxyl radical spin adducts, DMPO-OH, was dominant in the ESR spectra. However, at the maximum level of the respiratory burst, the spectrum of superoxide anion spin adducts, DMPO-OOH, was dominant.