Evidence of reactive oxygen species generation in synovial fluid from patients with temporomandibular disease by electron spin resonance spectroscopy

Reactive oxygen species (ROS) have been implicated in the pathogenesis of temporomandibular disorders. In the present study, we provide the first evidence of ROS generation in the synovial fluid from human temporomandibular disorder patients, as shown by electron spin resonance (ESR) and spin trapping. Three distinct ESR spectra of DMPO spin adducts were observed in the synovial fluid. They corresponded to three free radical species: hydroxyl radical (HO(*)), hydrogen radical (H(*)), and carbon-center radical (R(*)). Among them, the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OH spectrum was the most prominent, suggesting that HO(*) was dominantly generated in the synovial fluid from temporomandibular disorder patients. Desferrioxamine (DFO), an iron chelator, strongly depressed the DMPO-OH signal intensity in the synovial fluid from patients with temporomandibular disorders. We successfully demonstrated ROS-induced oxidative stress in the synovial fluid from temporomandibular disorder patients. ROS generation in the temporomandibular joint could lead to exacerbation of inflammation and activation of cartilage matrix degrading enzymes that proceed to degenerative change of the temporomandibular joint. Thus, iron-dependent generation of HO( *) might have a crucial role in the pathogenesis of temporomandibular disorders.     

Scavenging of reactive oxygen species by N‐substituted indole‐2 and 3‐carboxamides

Indole‐2 and 3‐carboxamides (IDs) are proposed to be selective cyclooxygenase inhibitors. Since cyclooxygenase‐1 may be involved in reactive oxygen species (ROS) production, we hypothesize that these indole derivatives have antioxidative properties. We have employed chemiluminescence (CL) and electron spin resonance (ESR) spin trapping to examine this hypothesis. We report here the results of a study of reactivity of 10 selected indole derivatives towards ROS. The following generators of ROS were applied: potassium superoxide (KO₂) as a source of superoxide radicals (O₂^·?), the Fenton reaction (Co‐EDTA/H₂O₂) for hydroxyl radicals (HO^·), and a mixture of alkaline aqueous H₂O₂ and acetonitrile for singlet oxygen (¹O₂). Hydroxyl radicals were detected as 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) spin adduct, whereas 2,2,6,6‐tetramethyl‐piperidine (TEMP) was used as a detector of ¹O₂. Using the Fenton reaction, 0.5 mmol/L IDs were found to inhibit DMPO‐?H radical formation in the range 7–37%. Furthermore the tested compounds containing the thiazolyl group also inhibited the ¹O₂‐dependent TEMPO radical, generated in the acetonitrile + H₂O₂ system. About 20% inhibition was obtained in the presence of 0.5 mmol/L IDs. 1 mmol/L IDs caused an approximately 13–70% decrease in the CL sum from the O₂^·? generating system (1 mmol/L). The aim of this paper is to evaluate these indole derivatives as antioxidants and their abilities to scavenge ROS. Copyright © 2004 John Wiley & Sons, Ltd.     

Hydroxyl Radical Generation Caused by the Reaction of Singlet Oxygen with a Spin Trap,DMPO, Increases Significantly in the Presence of Biological Reductants

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (¹O²) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of ¹O² with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (?OH) adduct of DMPO (DMPO-OH) resulting from ¹O²-dependent generation of ?OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its ?OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of ?OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of ?OH from ¹O², and that spin trap-mediated ?OH generation hardly occurs with DEPMPO.     

Free radical scavenging activity of novel thiazolidine‐2,4‐dione derivatives

Free radical activity towards superoxide anion radical (), hydroxyl radical (HO^?) and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH^?) of a series of novel thiazolidine‐2,4‐dione derivatives (TSs) was examined using chemiluminescence, electron paramagnetic resonance (EPR) and EPR spin trapping techniques. 5,5‐Dimethyl‐1‐pyrroline‐N‐oxide (DMPO) was applied as the spin trap. Superoxide radical was produced in the potassium superoxide/18‐crown‐6 ether dissolved in dimethyl sulfoxide. Hydroxyl radical was generated in the Fenton reaction (Fe(II) + H₂O₂. It was found that TSs showed a slight scavenging effect (15–38% reduction at 2.5 mmol/L concentration) of the DPPH radical and a high scavenging effect of (41–88%). The tested compounds showed inhibition of HO^? ‐dependent DMPO‐OH spin adduct formation (the amplitude of EPR signal decrease ranged from 20 to 76% at 2.5 mmol/L concentration. Our findings present new group compounds of relatively high reactivity towards free radicals. Copyright © 2012 John Wiley & Sons, Ltd.     

Characterization of the high-resolution ESR spectra of superoxide radical adducts of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Analysis of conformational exchange

It has been previously reported that the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) can form stable radical adducts with superoxide radical. However, the presence of diastereomers of DEPMPO radical adducts and the appearance of superhyperfine structure complicates the interpretation of the ESR spectra. It has been suggested that the superhyperfine structure in the ESR spectrum of DEPMPO/^?OOH is a result of conformational exchange between conformers. The analysis of the temperature dependence of the ESR spectrum of DEPMPO/^?OOH and of its structural analog DMPO/^?OOH have demonstrated that both ESR spectra contain exchange effects resulting from conversion between two conformers. Computer simulation calculates a conformer lifetime on the order of 0.1?μs for DMPO/^?OOH at room temperature. However, temperature dependence of the ESR spectrum of DEPMPO/^?OOH suggests that superhyperfine structure does not depend on the conformational exchange. We have now found that the six-line ESR spectrum with superhyperfine structure should be assigned to a DEPMPO-superoxide-derived decomposition product. Therefore, ESR spectra previously assigned to DEPMPO/^?OOH contain not only the two diastereomers of DEPMPO/^?OOH but also the decomposition product, and these spectra should be simulated as a combination of four species: two conformers of the first diastereomer, one conformer of the second diastereomer and the superoxide-derived decomposition product. The presence of four species has been supported by the temperature dependence of the ESR spectra, nucleophilic synthesis of radical adducts, and isotopic substitution experiments. It is clear that to correctly interpret DEPMPO spin trapping of superoxide radicals, one must carefully consider formation of secondary radical adducts.     

Scavenging of superoxide anion radical and hydroxyl radical by novel thiazolyl‐thiazolidine‐2,4‐dione compounds

The antioxidant behavior of a series of new synthesized substituted thiazolyl‐thiazolidine‐2,4‐dione compounds (TZDs) was examined using chemiluminescence and electron paramagnetic resonance spin trapping techniques. 5,5‐Dimethyl‐1‐pyrroline‐N‐oxide (DMPO) was used as the spin trap. The reactivity of TZDs with superoxide anion radical (O) and hydroxyl radical (HO^?) was evaluated using potassium superoxide/18‐crown‐6 ether dissolved in dimethylsulfoxide, and the Fenton‐like reaction (Fe²⁺ + H₂O₂), respectively. The results showed that TZDs efficiently inhibited light emission from the O generating system at a concentration of 0.05–1 mmol L^?1 (5–94% reductions were found at 1 mmol L^?1 concentration). The TZD compounds showed inhibition of HO^?‐dependent DMPO–OH spin adduct formation from DMPO (the amplitude decrease ranged from 8 to 82% at 1 mmol L^?1 concentration). The findings showed that examined TZDs had effective activities as radical scavengers. Copyright © 2009 John Wiley & Sons, Ltd.     

Radical scavenging ability of some compounds isolated from Piper cubeba towards free radicals

The purpose of this study was to identify the antioxidant activity of 16 compounds isolated from Piper cubeba (CNCs) through the extent of their capacities to scavenge free radicals, hydroxyl radical (HO^?), superoxide anion radical () and 2,2‐diphenyl‐1‐picrylhydrazyl radical (DPPH^?), in different systems. Electron paramagnetic resonance (EPR) and 5,5‐dimethyl‐1‐pyrroline‐N‐oxide, DMPO, as the spin trap, and chemiluminescence techniques were applied. Using the Fenton‐like reaction [Fe(II) + H₂O₂], CNCs were found to inhibit DMPO? OH radical formation ranging from 5 to 57% at 1.25 mmol L^?1 concentration. The examined CNCs also showed a high DPPH antiradical activity (ranging from 15 to 99% at 5 mmol L^?1 concentration). Furthermore, the results indicated that seven of the 16 tested compounds may catalyse the conversion of superoxide radicals generated in the potassium superoxide/18‐crown‐6 ether system, thus showing superoxide dismutase‐like activity. The data obtained suggest that radical scavenging properties of CNCs might have potential application in many plant medicines. Copyright © 2010 John Wiley & Sons, Ltd.     

Hydroxyl and superoxide radical scavenging abilities of chromonyl‐thiazolidine‐2,4‐dione compounds

The oxygen free radical scavenging activities of 15 chromonyl‐thiazolidine‐2,4‐dione compounds (CTDs) were examined in chemical systems producing superoxide anion radicals, O (potasium superoxide–18‐crown‐6 ether–DMSO), and hydroxyl radicals, HO^? (a Fenton reaction: Fe(II)–H₂O₂–sodium trifluoroacetate, pH 6.15). Chemiluminescence and electron spin resonance (ESR) spectroscopy using 5,5‐dimethyl‐1‐pyrroline‐1‐oxide (DMPO) as spin trap were applied to evaluate antioxidant behaviour of CTDs towards the oxygen radicals. The results indicated that 11 of the 15 tested compounds showed a significant inhibitory effect on the chemiluminescence generated from the O‐generating system, ranging from 41 to 86%, and 13 CTDs quenched the ESR signal of the DMPO–OH spin adduct by 33–86%, at a concentration of 1 mmol L^?1. Our findings demonstrate that CTDs could be good free radical scavengers. Copyright © 2008 John Wiley & Sons, Ltd.     

Role of reactive oxygen species in cardiac preconditioning: Study with photoactivated rose bengal in isolated rat hearts

Oxygen radical scavengers have been shown to prevent the development of ischemic preconditioning, suggesting that reactive oxygen species (ROS) might be involved in this phenomenon. In the present study, we have investigated whether direct exposure to ROS produced by photoactivated Rose Bengal (RB) could mimic the protective effects of ischemic preconditioning.

Methods In vitro generation of ROS from photoactivated RB in a physiological buffer was first characterised by ESR spectroscopy in the presence of 2,2,6,6-tetramethyl-1-piperidone (oxoTEMP) or 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). In a second part of the study, isolated rat hearts were exposed for 2.5 min to photoactivated RB. After 5 min washout, hearts underwent 30 min no-flow normothermic ischemia followed by 30 min of reperfusion.

Results and Conclusions The production of singlet oxygen (¹O₂) by photoactivated RB in the perfusion medium was evidenced by the ESR detection of the nitroxyl radical oxoTEMPO. Histidine completely inhibited oxoTEMPO formation. In addition, the use of DMPO has indicated that (i) superoxide anions (O^·-₂) are produced directly and (ii) hydroxyl radicals (HO^·) are formed indirectly from the successive O^·-₂ dismutation and the Fenton reaction. In the perfusion experiments, myocardial post-ischemic recovery was dramatically impaired in hearts previously exposed to the ROS produced by RB photoactivation (¹O₂, O^·-₂, H₂O₂ and HO^·) as well as when ¹O₂ was removed by histidine (50 mM) addition. However, functional recovery was significantly improved when hearts were exposed to photoactivated RB in presence of superoxide dismutase (10⁵ IU/L) and catalase (10⁶ IU/L).

Further studies are now required to determine whether the cardioprotective effects of Rose Bengal in presence of O^·-₂ and H₂O₂ scavengers are due to singlet oxygen or to other species produced by Rose Bengal degradation.     

Scavenging of hydroxyl radical by catecholamines

The direct effects of the four catecholamines (CATs), adrenaline (A), noradrenaline (NA), dopamine (D) and isoproterenol (I), on free radicals were investigated using the free radical 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH^?) and hydroxyl radial (HO^?). The CATs examined were found to inhibit the ESR signal intensity of DPPH^? in a dose‐dependent manner over the range 0.1–2.5 mmol/L in the following order: NA > A > I > D, with IC₅₀ = 0.30 ± 0.03 for noradrenaline and IC₅₀ = 0.86 ± 0.02 for dopamine. Hydroxyl radicals were produced using a Fenton reaction in the presence of the spin trap 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO), and ESR technique was applied to detect the CATs reactivity toward the radicals. The reaction rates constant (k_r) of CATs with HO^? were found to be in the order of 10⁹ L/mol/s, and the k_r value for noradrenaline was the highest (k_r = 8.4 × 10⁹ L/mol/s). The CATs examined exhibited also a strong decrease in the light emission (62–73% at 1 mmol/L concentration and 79–89% at 2 mmol/L concentration) from a Fenton‐like reaction. These reactions may be relevant to the biological action of these important polyphenolic compounds. Copyright © 2012 John Wiley & Sons, Ltd.     

Hydroxyl and 1-hydroxyethyl free radical detection using spin traps followed by derivatization and gas chromatography—mass spectrometry

Summary

Detection of hydroxyl free radicals is frequently performed by electron spin resonance (ESR) following spin trapping of the radical using 5,5-dimethylpyrroline N-oxide (DMPO) to generate a stable free radical having a characteristic ESR spectrum. The necessary ESR equipment is expensive and not readily available to many laboratories. In the present study, a specific and sensitive gas chromatography—mass spectrometry (GC/MS) method for detection of hydroxyl and hydroxyethyl free radicals is described. The DMPO or N-t-butyl—α—phenylnitrone (PBN) radical adducts are extracted and derivatized by trimethylsylilation and analyzed by GC/MS. To standardize the method, ^.OH and 1-hydroxyethyl radicals were generated in two different systems: 1) a Fenton reaction in a pure chemical system in the absence or presence of ethanol and 2) in liver microsomal suspensions where ethanol is metabolized in the presence of NADPH. In the Fenton system both radicals were easily detected and specifically identified using DMPO or PBN. In microsomal suspensions DMPO proved better for detection of ^.OH radicals and PBN more suitable for detection of 1-hydroxyethyl radicals. The procedure is specific, sensitive and potentially as useful as ESR.     

Formation of reactive sulfite-derived free radicals by the activation of human neutrophils: an ESR study

The objective of this study was to determine the effect of (bi)sulfite (hydrated sulfur dioxide) on human neutrophils and the ability of these immune cells to produce reactive free radicals due to (bi)sulfite oxidation. Myeloperoxidase (MPO) is an abundant heme protein in neutrophils that catalyzes the formation of cytotoxic oxidants implicated in asthma and inflammatory disorders. In this study sulfite (^?SO₃^?) and sulfate (SO₄^??) anion radicals are characterized with the ESR spin-trapping technique using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in the reaction of (bi)sulfite oxidation by human MPO and human neutrophils via sulfite radical chain reaction chemistry. After treatment with (bi)sulfite, phorbol 12-myristate 13-acetate-stimulated neutrophils produced DMPO–sulfite anion radical, –superoxide, and –hydroxyl radical adducts. The last adduct probably resulted, in part, from the conversion of DMPO–sulfate to DMPO–hydroxyl radical adduct via a nucleophilic substitution reaction of the radical adduct. This anion radical (SO₄^??) is highly reactive and, presumably, can oxidize target proteins to protein radicals, thereby initiating protein oxidation. Therefore, we propose that the potential toxicity of (bi)sulfite during pulmonary inflammation or lung-associated diseases such as asthma may be related to free radical formation.     

Evaluation of the antioxidant activity of tetracycline antibiotics in vitro

Tetracyclines are the second most common antibiotic family in medicine usage. These antibiotics exhibit antioxidant potential; however, the exact mechanism remains unclear. The antiradical activity of the seven tetracyclines (TCs; tetracycline, chlortetracycline, oxytetracycline, doxocycline, methacycline, demeclocycline, minocycline) was determined using the free radical 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH^?) and hydroxyl radicals (HO^?) generated in a Fenton reaction. Electron spin resonance (ESR), ESR spin‐trapping, chemiluminescence and spectrophotometry techniques were applied. It was found that the TCs showed high DPPH antiradical activity in the range 26–96% at 2.5 mmol/L concentration. The second‐order rate constants for the reaction between HO^? and TCs were calculated, in the range (3.6–9.6) × 10⁹ L/mol/s. The tetracycline compounds also exhibited a strong decrease in light emission (range 61–85% at concentration of 1 mmol/L). This study also showed that TCs promote the generation of singlet oxygen in the presence of and Fe(II)/Fe(III) ions. Our findings suggest direct scavenging activity of the examined tetracyclines towards free radicals, and may be relevant to therapeutic strategy. Copyright © 2011 John Wiley & Sons, Ltd.     

Scavenging capacities of some thiazolyl thiazolidine‐2,4‐dione compounds on superoxide radical,hydroxyl radical,and DPPH radical

The scavenging effects of eighteen thiazolyl thiazolidine‐2,4‐dione compounds (TTCs) on superoxide radical , hydroxyl radical HO^?, and 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH^?) radical were evaluated by the chemiluminescence technique, electron spin resonance spectrometry (ESR) and visible spectrophotometry, respectively. The examined compounds were shown to have 27–59% scavenging ability, 19–69% HO^? scavenging activity and 2–32% DPPH^? scavenging ability. This property of the tested compound seems to be important in the prevention of various diseases of free radicals etiology. Copyright © 2009 John Wiley & Sons, Ltd.     

Mechanism of Hydroxyl Radical Scavenging by Rebamipide: Identification of Mono-hydroxylated Rebamipide as a Major Reaction Product

Rebamipide, an antiulcer agent, is known as a potent hydroxyl radical (^?OH) scavenger. In the present study, we further characterized the scavenging effect of rebamipide against ^?OH generated by ultraviolet (UV) irradiation of hydrogen peroxide (H²O²), and identified the reaction products to elucidate the mechanism of the reaction. Scavenging effect of rebamipide was accessed by ESR using DMPO as a ^?OH-trapping agent after UVB exposure (305?nm) to H²O² for 1?min in the presence of rebamipide. The signal intensity of ^?OH adduct of DMPO (DMPO-OH) was markedly reduced by rebamipide in a concentration-dependent fashion as well as by dimethyl sulfoxide and glutathione as reference radical scavengers. Their second order rate constant values were 5.62?×?10¹⁰, 8.16?×?10⁹ and 1.65?×?10¹⁰?M^-1?s^-1, respectively. As the rebamipide absorption spectrum disappeared during the reaction, a new spectrum grew due to generation of rather specific reaction product. The reaction product was characterized by LC-MS/MS and NMR measurements. Finally, a hydroxylated rebamipide at the 3-position of the 2(1H)-quinolinone nucleus was newly identified as the major product exclusively formed in the reaction between rebamipide and the ^?OH generated by UVB/H²O². Specific formation of this product explained the molecular characteristics of rebamipide as a potential ^?OH scavenger.     

Photoreduction of the fluorescent dye 2′-7′-dichlorofluorescein: a spin trapping and direct electron spin resonance study with implications for oxidative stress measurements

The photoreduction of 2′-7′-dichlorofluorescein (DCF) was investigated in buffer solution using direct electron spin resonance (ESR) and the ESR spin-trapping technique. Anaerobic studies of the reaction of DCF in the presence of reducing agents demonstrated that during visible irradiation (λ > 300 nm) 2′-7′-dichlorofluorescein undergoes one-electron reduction to produce a semiquinone-type free radical as demonstrated by direct ESR. Spin-trapping studies of incubations containing DCF, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and either reduced glutathione (GSH) or reduced NADH demonstrate, under irradiation with visible light, the production of the superoxide dismutase-sensitive DMPO/^·OOH adduct. In the absence of DMPO, measurements with a Clark-type oxygen electrode show that molecular oxygen is consumed in a light-dependent process. The semiquinone radical of DCF, when formed in an aerobic system, is immediately oxidized by oxygen, which regenerates the dye and forms superoxide.     

Hydroxyl radical generation caused by the reaction of singlet oxygen with a spin trap, DMPO, increases significantly in the presence of biological reductants

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (₁O₂) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of ₁O₂ with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (•OH) adduct of DMPO (DMPO-OH) resulting from ₁O₂-dependent generation of •OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its •OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of •OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of •OH from ₁O₂, and that spin trap-mediated •OH generation hardly occurs with DEPMPO.     

Formation of artifactual DMPO-OH spin adduct in acid solutions containing nitrite ions

We investigated aqueous solutions containing nitrite ions and DMPO (5,5-dimethyl-1-pyrroline-N-oxide) by electron spin resonance (ESR) in the pH range from 1 to 6. A DMPO-OH signal was observed below pH 3.0 in the presence of nitrite ions, whereas in the absence of nitrite ion, an extremely weak signal was observed below pH 1.5. Addition of methanol, a hydroxyl radical scavenger, to this system did not lead to the appearance of a detectable DMPO-CH₂OH signal. The possibility of this DMPO-OH signal being due to a genuine spin trapping process with hydroxyl radical was, therefore, ruled out. The reactivities of reactive nitrogen species (RNS) in this system with DMPO have also been investigated by density functional theory (DFT) at the IEFPCM (water)/B3LYP/6–311?+?G ** level of theory. On the basis of the pH dependence of the signal intensity and the redox potential E° (versus SHE) calculated by DFT theory, we propose that the origin of this signal is “inverted spin trapping” via one-electron oxidation of DMPO by H₂ONO⁺, followed by the nucleophilic addition of water. Prevention of these false-positive results when detecting hydroxyl radical using ESR spin trapping requires an awareness of both the presence of nitrite ions in the solution and the solution pH.     

STUDIES ON NITRIC OXIDE FREE RADICALS GENERATED FROM POLYMORPHONUCLEAR LEUKOCYTES (PMN) STIMULATED BY PHORBOL MYRISTATE ACETATE (PMA)

The interaction of NO and O^?₂free radicals generated from PMA (phorbol myristate acetate)-stimulated PMN (polymorphonuclear leukocytes) was studied by a nitroxide spin trap, DMPO (5,5-dimethyl-1-pyrroline-1-oxide). It was found that addition of L-arginine to the system would significantly decrease the trapped O^?₂by DMPO and addition of N^G-monomethyl-arginine (NGMA) would significantly increase the trapped O^?₂by DMPO. It was proved that the formation of ONOO^?by the reaction of NO and O^?₂was the main reason for the decrease of trapped O^?₂in the experiment with xanthine/xanthine oxidase and irradiation of riboflavin systems. The yield of NO during this process was calculated. The generation dynamic of NO was studied by a luminol-dependent chemiluminescence technique and it was found that after stimulation of PMN by PMA, there would be an immediate, significant chemi-luminescence, which came mainly from the active oxygen free radicals generated by PMN. If L-arginine was added to this system, the chemiluminescence would increase about 100-fold, but NGMA inhibited the increase of the chemiluminescence. Ten minutes after addition of L-arginine, this increase did not change, the chemiluminescence peak decreased gradually, but the half life increased. The ESR and chemiluminescence properties of NO and ONOO^?synthesized were also studied in model systems.     

Reactive oxygen species generation in gingival fibroblasts of Down syndrome patients detected by electron spin resonance spectroscopy

Oral manifestations of Down syndrome include high susceptibility to gingival inflammation with early onset and rapidly progressive periodontitis. The influence of reactive oxygen species (ROS) on periodontitis of Down syndrome is unclear. The aim of this study was to characterize ROS formation in Down syndrome-gingival fibroblasts (DS-GF) using electron spin resonance (ESR) spin trapping with 5,5-dimetyl-1-pyrolline-N-oxide (DMPO), and to determine whether ROS generation plays a role in the pathogenesis of periodontitis in Down syndrome patients. We observed formation of the DMPO-OH spin adduct, indicating HO* generation from cultured DS-GF and non-DS-GF. The increased HO* generation in cultured DS-GF was strongly decreased in the presence of the H2O2 scavenger, catalase, or the iron chelator, desferal. This may due to the enzymatic ability of over-expressed CuZn-superoxide dismutase in Down syndrome to catalyze the formation of H2O2 from O2*-, thereby increasing the availability of substrate H2O2 for the iron-dependent generation of HO* via the Fenton reaction, suggesting that HO* generated from DS-GF may be involved in progressive periodontitis of Down syndrome.