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.     

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.     

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.     

Studies on the antioxidant activities of some new chromone compounds

Recent reviews evidence that the naturally occurring compounds containing the chromone skeleton exhibit antiradical activities, providing protection against oxidative stress. The antioxidant activities of 13 new synthesized chromonyl‐2,4‐thiazolidinediones, chromonyl‐2,4‐imidazolidinediones and chromonyl‐2‐thioxoimidzolidine‐4‐ones were evaluated using in vitro antioxidant assays, including superoxide anion radical (), hydroxyl radical (), 2,2‐diphenyl‐1‐picryl‐hydrazyl free radical (DPPH^?) scavenging capacity and total antioxidant capacity ferric ion reducing activity. Superoxide anion radical was produced using potassium superoxide/18‐crown‐6‐ether dissolved in dimethylsulfoxide, and the Fenton‐like reaction (Fe(II) + H₂O₂) was a generator of hydroxyl radicals. Chemiluminescence, spectrophotometry, electron paramagnetic resonance (EPR) and 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) as the spin trap were the measurement techniques. The results showed that the majority of the chromone derivatives tested showed a strong scavenging effect towards free radicals, similar to the chemiluminescence reaction with superoxide anion radical with a high activity, inhibition of the DMPO‐OOH radical EPR signal (24–58%), the DMPO‐OH radical EPR signal (4–75%) and DPPH radical EPR signal (6–100%) at 1 mmol/L. Several of the examined compounds exhibited the high reduction potentials. The results obtained show that the new synthesized chromone derivatives may directly scavenger reactive oxygen species and thus may play a protective role against oxidative damage. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

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.     

6‐Methyl 3‐chromonyl 2,4‐thiazolidinedione/2,4‐imidazolidinedione/2‐thioxo‐imidazolidine‐4‐one compounds: novel scavengers of reactive oxygen species

The benefits of antioxidants on human health are usually ascribed to their potential ability to remove reactive oxygen species providing protection against oxidative stress. In this paper the free radicals scavenging activities of nine 6‐methyl 3‐chromonyl derivatives (CMs) were evaluated for the first time by the chemiluminescence, electron paramagnetic resonance, spin trapping and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH^?) methods. The total antioxidant capacity was also measured using a ferric‐ferrozine reagent. Compounds having a hydrogen atom at the N3‐position of the β‐ring were effective in quenching CL resulted from the KO₂/18‐crown‐6‐ether system (a source of superoxide anion radical, ) in a dose‐dependent manner over the range of 0.05–1 mmol/L [IC₅₀ ranged from 0.353 (0.04) to 0.668 (0.05) mmol/L]. The examined compounds exhibited a significant scavenging effect towards hydroxyl radicals (HO^? HO^?), produced by the Fenton reaction, and this ranged from 24.0% to 61.0%, at the concentration of 2.5 mmol/L. Furthermore, the compounds examined were also found to inhibit DPPH^? and this ranged from 51.9% to 97.4% at the same concentration. In addition, the use of the total antioxidant capacity assay confirmed that CM compounds are able to act as reductants. According to the present study, CM compounds showed effective in vitro free radical scavenging activity and may be considered as potential therapeutics to control diseases of oxidative stress‐related etiology. Copyright © 2013 John Wiley & Sons, Ltd.     

Studies on the antioxidant activity of some chromonylrhodanine derivatives

Fifteen chromonylrhodamine derivatives (CRs) were synthesized and the antioxidant activity levels were evaluated for the first time. The antioxidant activity potencies of these chromone derivatives were evaluated towards superoxide anion radicals, hydroxyl radicals and 2,2‐diphenyl‐1‐picrylhydrazyl radicals. Also, the total antioxidant capacity of the tested compounds was measured using the ferric‐ferrozine assay. The antioxidant activities were investigated using a chemiluminescence (CL) assay, spectrophotometry measurements, direct electron paramagnetic resonance (EPR) and the EPR spin‐trapping technique. The 5,5‐dimethyl‐ 1‐pyrroline‐1‐oxide (DMPO) was applied as spin trap. Eleven of the 15 chromone compounds exhibited a decrease in the CL accompanying the superoxide anion radical produced in anhydrous dimethylsulfoxide (DMSO), ranging from 71–94% at concentration of 1 mmol /L; four of these compounds enhanced light emission in the range 231–672%. Similarly, these compounds caused 28–58% inhibition in the intensity of the DMPO‐OOH radical EPR signal and the DMPO‐OH radical (from 12–48%). Furthermore, three of these compounds showed very good antioxidant response towards the DPPH radical (EC₅₀: 0.51–0.56 µmol/L) and the high reduction potentials. These findings demonstrate that the chromone compounds tested may be considered as effective free radicals scavengers, a finding that is of great pharmacological importance. Copyright © 2014 John Wiley & Sons, Ltd.     

Superoxide Dismutase Enhanced the Formation of Hydroxyl Radicals in a Reaction Mixture Containing Xanthone under UVA Irradiation

To clarify the effect of superoxide dismutase (SOD) on the formation of hydroxyl radical in a standard reaction mixture containing 15 μM of xanthone, 0.1 M of 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and 45 mM of phosphate buffer (pH 7.4) under UVA irradiation, electron paramagnetic resonance (EPR) measurements were performed. SOD enhanced the formation of hydroxyl radicals. The formation of hydroxyl radicals was inhibited on the addition of catalase. The rate of hydroxyl radical formation also slowed down under a reduced oxygen concentration, whereas it was stimulated by disodium ethylenediaminetetraacetate (EDTA) and diethyleneaminepentaacetic acid (DETAPAC). Above findings suggest that O₂, H₂O₂, and iron ions participate in the reaction. SOD possibly enhances the formation of the hydroxyl radical in reaction mixtures of photosensitizers that can produce O₂ ^?·.     

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.     

Studies on the antioxidant activity of some thiazolidinedione,imidazolidinedione and rhodanine derivatives having a flavone core

A series of flavonyl‐2,4‐thiazolidinedione, imidazolidinedione and rhodanine derivatives were tested for their antioxidant activity as scavengers of oxygen free radicals. Free radical scavenging activities, including superoxide anion radical , hydroxyl radical (HO^?) and 2,2′‐diphenyl‐1‐picrylhydrazyl free radical have been evaluated using chemiluminescence, electron paramagnetic resonance and spin trapping with 5,5‐dimethyl‐1‐pyrroline‐1‐oxide as a spin trap. Potassium superoxide in dimethylsulfoxide and 18‐crown‐6 ether were used for the production of . Hydroxyl radical was generated using the Fenton reaction. Ten of the eleven examined compounds exhibited decrease in chemiluminescence, but there were large differences in the decrease, ranging from 16% to 89%; also, two of these compounds increased light emission by about 200%. On the contrary, all compounds tested exhibited 30–68% scavenging HO^? and 25–96% scavenging the DPPH^? radical respectively. Possible mechanisms are proposed to explain the results. Copyright © 2014 John Wiley & Sons, Ltd.     

Reactivity of hypotaurine and cysteine sulfinic acid toward carbonate radical anion and nitrogen dioxide as explored by the peroxidase activity of Cu,Zn superoxide dismutase and by pulse radiolysis

Abstract

Hypotaurine and cysteine sulfinic acid are known to be readily oxidized to the respective sulfonates, taurine and cysteic acid, by several oxidative agents that may be present in biological systems. In this work, the relevance of both the carbonate anion and nitrogen dioxide radicals in the oxidation of hypotaurine and cysteine sulfinic acid has been explored by the peroxidase activity of Cu,Zn superoxide dismutase (SOD) and by pulse radiolysis. The extent of sulfinate oxidation induced by the system SOD/H₂O₂ in the presence of bicarbonate (CO₃^?– generation), or nitrite (^?NO₂ generation) has been evaluated. Hypotaurine is efficiently oxidized by the carbonate radical anion generated by the peroxidase activity of Cu,Zn SOD. Pulse radiolysis studies have shown that the carbonate radical anion reacts with hypotaurine more rapidly (k = 1.1 × 10⁹ M^?1s^?1) than nitrogen dioxide (k = 1.6 × 10⁷ M^?1s^?1). Regarding cysteine sulfinic acid, it is less reactive with the carbonate radical anion (k = 5.5 × 10⁷ M^?1s^?1) than hypotaurine. It has also been observed that the one-electron transfer oxidation of both sulfinates by the radicals is accompanied by the generation of transient sulfonyl radicals (RSO₂^?). Considering that the carbonate radical anion could be formed in vivo at high level from bicarbonate, this radical can be included in the oxidants capable of performing the last metabolic step of taurine biosynthesis. Moreover, the protective effect exerted by hypotaurine and cysteine sulfinate on the carbonate radical anion-mediated tyrosine dimerization indicates that both sulfinates have scavenging activity towards the carbonate radical anion. However, the formation of transient reactive intermediates during sulfinate oxidation by carbonate anion and nitrogen dioxide radical may at the same time promote oxidative reactions.     

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.     

Epr Spectra of Dmpo Spin Adducts of Superoxide and hydroxyl Radicals in Pyridine

Electron spin resonance spectroscopy and the spin trapping technique were used to study the formation of the superoxide radical in pyridine. 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was employed as a trapping agent. Superoxide radical was generated using chemical (potassium superoxide) and photochemical methods with anthralin, benzanthrone, rose bengal, 1,8-dihydroxyanthraquinone and zinc tetraphenylporphyrine as photoactive pigments. Hyperfine coupling (hf) constants for DMPO/O₂- were determined to be a_N = 12.36 G, a^β_H= 9.85G, a^γ_H = 1.34 G. The a_N and a^β_H constants are in good agreement with values calculated from a previously determined relationship between hf constants and solvent acceptor number (Reszka et al., (1992) Free Radical Res. Commun., in press). When concentrated hydrogen peroxide was added to DMPO in pyridine a similar EPR spectrum was observed. It is suggested that in this case the DMPO/'O₂H adduct is formed by nucleophilic addition of H₂O₂ to DMPO to give a hydroxylamine, followed by oxidation to the respective nitroxide. The EPR spectrum observed when tetrapropylammonium hydroxide and H₂O₂ were added to DMPO in pyridine had hf couplings a_N = 13.53 G, a^β_H = 11.38 G, a^γ_H = 0.79 G and it was assigned to a DMPO/'OH adduct. This assignment was based on similarity of this spectrum to the one produced by UV photolysis of hydrogen peroxide and DMPO in aqueous solution and subsequent transfer to pyridine.     

DNA Binding,Antioxidant Activity,and DNA Damage Protection of Chiral Macrocyclic Mn(III) Salen Complexes

We are reporting the synthesis, characterization, and calf thymus DNA binding studies of novel chiral macrocyclic Mn(III) salen complexes S‐1 , R‐1 , S‐2 , and R‐2 . These chiral complexes showed ability to bind with DNA, where complex S‐1 exhibits the highest DNA binding constant 1.20 × 10⁶ M^?1. All the compounds were screened for superoxide and hydroxyl radical scavenging activities; among them, complex S‐1 exhibited significant activity with IC₅₀ 1.36 and 2.37 μM, respectively. Further, comet assay was used to evaluate the DNA damage protection in white blood cells against the reactive oxygen species wherein complex S‐1 was found effective in protecting the hydroxyl radicals mediated plasmid and white blood cells DNA damage. Chirality 24:1063–1073, 2012.© 2012 Wiley Periodicals, Inc.     

On the Reaction of Superoxide With DMPO/*OOH

A kinetic model has been used to estimate the rate constant for the reaction of superoxide (O₂/OOH) with the superoxide spin adduct of 5.5-dimethylpyrroline-N-oxide. DMPO/OOH. This rate constant is estimated to be 4.9 (± 2.2) × 10⁶ M^?1 s^?1, pH 7.4 and 25°C.     

New Aspects in the Free-Radical Chemistry of Pyrimidine Nucleobases

The contribution will cover three aspects:

i) It has been known for some time that OH radicals and H atoms react with the pyrimidines by adding to the C(5)-C(6) double bond, but only the u.v.-spectra of the sum of these radicals have been reported so far. It will be shown how to arrive at the individual spectra of the C(5) and the C(6) adduct radicals.

ii) α-Hydroxyalkyl radicals are known to inactivate biologically active DNA. In contrast to the electrophilic radicals H and OH they are nucleophilic and the hydroxymethyl radicals add exclusively at the C(6) position of 1,3-dimethyluracil (k ～ 10⁴dm³ mol^?1 s^?1). In the corresponding thymine derivative this reaction also occurs, but one third of the hydroxymethyl radicals abstract an H-atom from the C(5)-methyl group thereby forming an allylic radical. In the course of these reactions pyrimidines with an exocyclic double bond are formed. These products react much more rapidly with hydroxymethyl radicals than the starting material leading to highly hydroxymethylated material at very low doses.

iii) The direct effect of ionizing radiation which would produce a pyrimidine base radical cation can be mimicked by reacting the pyrimidine with SO₄^?, a very good electron acceptor. In water, the radical cation of 1,3-dimethyluracil is rapidly (t_1/2 2μs) converted into the C(5) OH adduct radical. In the presence of peroxodisulphate a chain reaction sets in which leads to the cis-glycol.

The relevance of these findings to radiobiological aspects of nucleic acid research will be discussed.     

Fast repair of purine deoxynucleotide radical cations by rutin and quercetin

Repair effects of rutin and quercetin on purine deoxynucleotide radical cations were studied using pulse radiolysis technique. On electron pulse irradiation of N2 saturated deoxynucleotide aqueous solution containing 20 mmol/L K2S2O8, 200 mmol/L f-BuOH and rutin or quercetin, the transient absorption spectra of the deoxynucleotide radical cations decayed quickly. At the same time, the spectra of flavonoid phenoxyl radicals formed within several dozen microseconds. The results indicated that deoxynucleotide radical cations can be repaired by flavonoids. The rate constants of the repair reactions were 3.8 × 108-4.4×108 mol-1 · L · s-1 and 1.3×108-1.8×108 mol-1 · L · s-1 for dAMP and dGMP radical cations, respectively.     

Detection and identification of oxidants formed during •NO/O2•– reaction: A multi-well plate CW-EPR spectroscopy combined with HPLC analyses

Abstract

New techniques and probes are routinely emerging for detecting short-lived free radicals such as superoxide radical anion (O₂^?–), nitric oxide (^?NO), and transient oxidants derived from peroxynitrite (ONOO^–/ONOOH). Recently, we reported the profiles of oxidation products (2-hydroxyethidium, ethidium, and various dimeric products) of the fluorogenic probe hydroethidine (HE) in the ^?NO/O₂^?– system (Zielonka et al. 2012). In this study, we used HPLC analyses of HE oxidation products in combination with continuous wave electron paramagnetic resonance (CW-EPR) spin trapping with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) to define the identity of the oxidizing species formed in the ^?NO/O₂^?– system. EPR spin-trapping technique is still considered as the gold standard for characterization of free radicals and their intermediates. We monitored formation of BMPO-superoxide (BMPO-^?OOH) and BMPO-hydroxyl (BMPO-^?OH) radical adducts. Simultaneous analyses of results from EPR spin-trapping and HPLC measurements are helpful in the interpretation of the mechanism of formation of products of HE oxidation.