The reaction products of sulfide and S-nitrosoglutathione are potent vasorelaxants.

The chemical interaction of sodium sulfide (Na2S) with the NO-donor S-nitrosoglutathione (GSNO) has been described to generate new reaction products, including polysulfides and nitrosopersulfide (SSNO(-)) via intermediacy of thionitrous acid (HSNO). The aim of the present work was to investigate the vascular effects of the longer-lived products of the Sulfide/GSNO interaction. Here we show that the products of this reaction relax precontracted isolated rings of rat thoracic aorta and mesenteric artery (but to a lesser degree rat uterus) with a >2-fold potency compared with the starting material, GSNO (50 nM), whereas Na2S and polysulfides have little effect at 1-5 µM. The onset of vasorelaxation of the reaction products was 7-10 times faster in aorta and mesenteric arteries compared with GSNO. Relaxation to GSNO (100-500 nM) was blocked by an inhibitor of soluble guanylyl cyclase, ODQ (0.1 and 10 µM), and by the NO scavenger cPTIO (100 µM), but less affected by prior acidification (pH 2-4), and unaffected by N-acetylcysteine (1 mM) or methemoglobin (20 µM heme). By contrast, relaxation to the Sulfide/GSNO reaction products (100-500 nM based on the starting material) was inhibited to a lesser extent by ODQ, only slightly decreased by cPTIO, more markedly inhibited by methemoglobin and N-acetylcysteine, and abolished by acidification before addition to the organ bath. The reaction mixture was found to generate NO as detected by EPR spectroscopy using N-(dithiocarboxy)-N-methyl-D-glucamine (MGD2)-Fe(2+) as spin trap. In conclusion, the Sufide/GSNO reaction products are faster and more pronounced vasorelaxants than GSNO itself. We conclude that in addition to NO formation from SSNO(-), reaction products other than polysulfides may give rise to nitroxyl (HNO) and be involved in the pronounced relaxation induced by the Sulfide/GSNO cross-talk.

Oxidation of quinolones with peracids (an in situ EPR study).

4-Oxoquinoline derivatives (quinolones) represent heterocyclic compounds with a variety of biological activities, along with interesting chemical reactivity. The quinolone derivatives possessing secondary amino hydrogen at the nitrogen of the enaminone system are oxidized with 3-chloroperbenzoic acid to nitroxide radicals in the primary step while maintaining their 4-pyridone ring. Otherwise, N-methyl substituted quinolones also form nitroxide radicals coupled with the opening of the 4-pyridone ring in a gradual oxidation of the methyl group via the nitrone-nitroxide spin-adduct cycle. This was confirmed in an analogous oxidation using N,N-dimethylaniline as a model compound. N-Ethyl quinolones in contrast to its N-methyl analog form only one nitroxide radical without a further degradation.

Stable radical trianions from reversibly formed sigma-dimers of selenadiazoloquinolones studied by in situ EPR/UV-vis spectroelectrochemistry and quantum chemical calculations.

The redox behavior of the series of 7-substituted 6-oxo-6,9-dihydro[1,2,5]selenadiazolo[3,4-h]quinolines and 8-substituted 9-oxo-6,9-dihydro[1,2,5]selenadiazolo[3,4-f]quinolines with R(7), R(8) = H, COOC(2)H(5), COOCH(3), COOH, COCH(3), and CN has been studied by in situ EPR and EPR/UV-vis spectroelectrochemistry in dimethylsulfoxide. All selenadiazoloquinolones undergo a one-electron reduction process to form the corresponding radical anions. Their stability strongly depends on substitution at the nitrogen atom of the 4-pyridone ring. The primary generated radical anions from N-ethyl-substituted quinolones are stable, whereas for the quinolones with imino hydrogen, the initial radical anions rapidly dimerize to produce unusually stable sigma-dimer (σ-dimer) dianions. These are reversibly oxidized to the initial compounds at potentials considerably less negative than the original reduction process in the back voltammetric scan. The dimer dianion can be further reduced to the stable paramagnetic dimer radical trianion in the region of the second reversible reduction step. The proposed complex reaction mechanism was confirmed by in situ EPR/UV-vis cyclovoltammetric experiments. The site of the dimerization in the σ-dimer and the mapping of the unpaired spin density both for radical anions and σ-dimer radical trianions with unusual unpaired spin distribution have been assigned by means of density functional theory calculations.

Anodic oxidation of selenadiazoloquinolones in alkaline media.

Newly synthesized derivatives of 6-oxo-6,9-dihydro[1,2,5]selenadiazolo[3,4-h]quinoline variously substituted at position 7 (R = H, COOH, COCH(3), CN, COOC(2)H(5) and COOCH(3)) are established in strongly alkaline aqueous solutions (0.1 M NaOH; pH ∼ 13) as N(9)-deprotonated structures, but in less alkaline solutions (0.001 M NaOH; pH ∼ 11) the N(9)-protonated oxo tautomeric forms dominate. Upon their anodic oxidation in alkaline solutions, the selenadiazole ring is replaced, forming instead the paramagnetic species analogous to the ortho semiquinone radical anions as monitored by in situ EPR spectroscopy. The quantum chemical calculations for two representative selenadiazoloquinolones (R = H and COOH) and their anodic oxidation products presented are in agreement with experiments.

Photoinduced superoxide radical anion and singlet oxygen generation in the presence of novel selenadiazoloquinolones (an EPR Study).

Novel 7-substituted 6-oxo-6,9-dihydro[1,2,5]selenadiazolo[3,4-h]quinoline (SeQ(1-6)) and 8-substituted 9-oxo-6,9-dihydro[1,2,5]selenadiazolo[3,4-f ]quinoline derivatives (SeQN(1-5)) with R(7), R(8) =H, COOC(2) H(5), COOCH(3), COOH, COCH(3) or CN were synthesized and their spectral characteristics were obtained by UV/Vis spectroscopy. Ultraviolet A photoexcitation of the selenadiazoloquinolones in dimethylsulfoxide or acetonitrile resulted in the formation of paramagnetic species coupled with molecular oxygen activation generating the superoxide radical anion or singlet oxygen, evidenced by electron paramagnetic resonance spectroscopy. The cytotoxic/photocytotoxic impact of selenadiazoloquinolones on murine and human cancer cell lines was demonstrated using the derivative SeQ5 (with R(7)=COCH(3)).

Electron transfer: a primary step in the reactions of sodium hydrosulphide, an H2S/HS(-) donor.

Endogenously produced H2S/HS(-), a newly found gasotransmitter, is well represented by NaHS. In deoxygenated media it terminated semi-stable oxidant radicals up to stoichiometric ratios of 1:1. In the presence of oxygen the antioxidant activities of NaHS were impaired considerably due to its competitive reactions with molecular oxygen. The primary reaction steps of NaHS were investigated using two different spin traps, 5,5-dimethylpyrroline-N-oxide and sodium 3,5-dibromo-4-nitrosobenzenesulphonate (DBNBS), in protolytic and aprotic solvents (water and dimethylsulphoxide, DMSO) under argon and oxygen. Sulphhydryl radicals (HS(*)/S(* -)) were primarily formed (S(* -) in water and HS(*) in DMSO), probably coupled to the formation of superoxide radical anions. The DBNBS spin trap acted also as an electron acceptor and formed its radical anions in the presence of NaHS. Hence, one of the primary steps in the reactions of sulphides is the electron transfer from H2S/HS(-) species to a suitable acceptor, which may play a fundamental role in their biological functions.

Thermal generation of stable SO4*- spin trap adducts with super-hyperfine structure in their EPR spectra: an alternative EPR spin trapping assay for radical scavenging capacity determination in dimethylsulphoxide.

Thermal decomposition of potassium persulphate (K2S2O8) was studied in detail by the EPR spin trapping technique in dimethylsulphoxide (DMSO), employing 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 5-ethoxycarbonyl-5-methyl-1-pyrroline-N-oxide (EMPO) and 5-(diisopropoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DIPPMPO) as spin traps. DMPO and/or its derivatives exclusively trapped the primary formed SO4*- radical anions producing stable spin adducts with half-lives exceeding 2 h at room temperature. High-resolution EPR spectra of these adducts showed unusually rich hyperfine structure due to the interaction of the unpaired electron with all magnetically active nuclei of the spin trap moiety. In contrast to aprotic DMSO solvent, *DMPO-OH adducts dominated in mixed DMSO/water solutions with water content higher than 50%. The thermal decomposition of K2S2O8 in DMSO represents an effective source of free radicals for the radical scavenging capacity (RSC) determination assay, applicable to hydrophilic as well as hydrophobic antioxidants. Efficiency of the assay is demonstrated with a series of cereal samples.

H(2)S and HS(-) donor NaHS releases nitric oxide from nitrosothiols, metal nitrosyl complex, brain homogenate and murine L1210 leukaemia cells.

Nitrosoglutathione [(GSNO), 500 nmol/l] relaxed the norepinephrine precontracted rat aortic rings. The relaxation effect was pronouncedly enhanced by H(2)S- and HS(-)-donor NaHS (30 micromol/l) at 7.5 pH but not at 6.3 pH. To study molecular mechanism of this effect, we investigated whether NaHS can release NO from NO donors. Using an electron paramagnetic resonance spectroscopy method of spin trap and by measuring the NO oxidation product, which is nitrite, by the Griess reaction, we report that NaHS released NO from nitrosothiols, namely from GSNO, S-nitroso-N-acetyl-DL: -penicillamine (SNAP), from metal nitrosyl complex nitroprusside (SNP) and from rat brain homogenate and murine L1210 leukaemia cells. From the observation that the releasing effect was more pronounced at 8.0 pH than 6.0 pH, we suppose that HS(-), rather than H(2)S, is responsible for the NO-releasing effect. Since in mammals, H(2)S and HS(-) are produced endogenously, we assume that their effect to release NO from nitrosothiols and from metal nitrosyl complexes are responsible for some of their biological activities and that this mechanism may be involved in S-nitrosothiol-signalling reactions.

Antioxidant and radical-scavenging activities of Slovak honeys - An electron paramagnetic resonance study.

The antioxidant properties of 15 honey samples from different floral sources and various Slovak regions were investigated by means of electron paramagnetic resonance spectroscopy. Cation radical of ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) diammonium salt), DPPH (1,1-diphenyl-2-picrylhydrazyl) and hydroxyl radicals generated by the photochemical decomposition of hydrogen peroxide were used as oxidants. The antioxidant activities found with ABTS(+), expressed as trolox equivalent antioxidant capacity (TEAC), ranged from 0.15 to 1.14mmolkg(-1), and those determined with DPPH, from 0.04 to 0.32mmolkg(-1). TEAC values correlated well with results found by elimination of DPPH, and both values revealed a linear relationship with the concentration of phenolics obtained with the Folin-Ciocalteu phenol test (expressed as gallic acid equivalents, GAE). The colour coordinates (CIE L(∗)a(∗)b(∗)), as well as reflectance spectra determined for original honeys using a white background, demonstrated that the colour difference (ΔE(∗)) and coordinate b(∗) interrelate with TEAC values. The radical-scavenging capacities (RSC) of the honey samples determined in the experiments with photochemically decomposed hydrogen peroxide, generating reactive OH radicals in the presence of spin trapping agent, differ from those found with ABTS(+) and DPPH. Here, probably, the reactive OH radicals, having higher redox potential, are scavenged by a variety of compounds not effective with ABTS(+) and DPPH (e.g., saccharides, proteins).

The potential pitfalls of using 1,1-diphenyl-2-picrylhydrazyl to characterize antioxidants in mixed water solvents.

Approaching living systems, aqueous solutions are appropriate to characterize antioxidants, whereas the frequently used standard 1,1-diphenyl-2-picrylhydrazyl (DPPH) is insoluble in water. Therefore, mixed water-ethanol solvents were investigated using the electron paramagnetic resonance (EPR) spectroscopy. Two forms of DPPH were identified: at higher ethanol ratios a quintet spectrum characteristic of solutions, and at lower ratios, a singlet spectrum typical for solid DPPH, were found. Mixed solvents with 0-50% (v/v) water reproduced the same antioxidant equivalent points well and the reaction rate between DPPH and the antioxidant may increase considerably with increasing water ratios, as demonstrated using vitamin E as an antioxidant. But at still higher water ratios (70-90% (v/v)) the antioxidant activities dropped, since a part of the DPPH in the aggregated form does not react sufficiently with the antioxidants. Characteristics of the most common antioxidants were determined in ethanol or its 50% (v/v) aqueous solution.

Reactive oxygen species produced upon photoexcitation of sunscreens containing titanium dioxide (an EPR study).

Commercial sunscreen products containing titanium dioxide were irradiated with lambda>300 nm and the formation of oxygen- (.OH, O2.-/.OOH) and carbon-centered radicals was monitored by EPR spectroscopy and spin trapping technique using 5,5-dimethyl-1-pyrroline N-oxide, alpha-phenyl-N-tert-butylnitrone (PBN), alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone as spin traps, and free nitroxide radical 4-hydroxy-2,2,6,6-tetramethylpiperidine N-oxyl. The photoinduced production of singlet oxygen was shown by 4-hydroxy-2,2,6,6-piperidine. The generation of reactive oxygen radical species upon irradiation of sunscreens significantly depends on their composition, as the additives present (antioxidants, radical-scavengers, solvents) can transform the reactive radicals formed to less harmful products. The continuous in situ irradiation of titanium dioxide powder, recommended for cosmetic application, investigated in different solvents (water, dimethyl sulfoxide, isopropyl myristate) resulted in the generation of oxygen-centered reactive radical species (superoxide anion radical, hydroxyl and alkoxyl radicals).

Antioxidant properties of tea investigated by EPR spectroscopy.

The antioxidant properties of green, black and mixed (fruit) tea samples of different origin were investigated by means of EPR spectroscopy. A six line EPR spectrum of solid tea samples indicates the presence of Mn(II) ions and it is superimposed with a sharp singlet line attributed to semiquinone radical species (Delta H(pp)=1 mT; g=2.0022). Antioxidant properties of aqueous tea extracts in H(2)O(2)/NaOH/dimethylsulfoxide system generating reactive radicals (*OH, O(2)*-), *CH(3)) were followed by spin trapping technique. In addition, antioxidant capacity of these samples was assessed using stable radicals 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPOL). Typically, the highest antioxidant potential to terminate superoxide radicals was found in green teas, followed by black and fruity teas. The pro-oxidant activity of green teas evidenced by spin traps was promoted in samples with higher Mn(II) and ascorbic acid concentrations. Various sources of free radicals used in the antioxidant tests due to their specific action show different termination rates in the presence of the individual tea samples.

The influence of additives on beer stability investigated by EPR spectroscopy.

In thermally-accelerated aging followed by EPR spectroscopy of beer samples of various stabilities, free radical 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPOL) was shown to be an effective indicator of the breakdown of a sample's stability, comparable to the commonly used spin trapping agent alpha-phenyl-N-tert-butylnitrone (PBN). Both indicators were then employed to investigate the influence of additives on beer stability. The addition of L-ascorbic acid (ASC) to the beer samples accelerated the radical processes and a lower stability was found. DL-alpha-tocopherol (alpha-TOC) did not influence beer stability significantly (probably due to its limited solubility). Na2SO3, described as a very effective stabilizer in experiments with the PBN spin trap, was found not to be effective using the TEMPOL indicator. This is probably due to inhibition in the formation of spin adducts or their degradation by Na2SO3.