FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2034334015> ?p ?o ?g. }

• W2034334015 endingPage "42768" @default.
• W2034334015 startingPage "42761" @default.
• W2034334015 abstract "Despite its negative redox potential, nitroxyl (HNO) can trigger reactions of oxidation. Mechanistically, these reactions were suggested to occur with the intermediate formation of either hydroxyl radical (·OH) or peroxynitrite (ONOO–). In this work, we present further experimental evidence that HNO can generate ·OH. Sodium trioxodinitrate (Na2N2O3), a commonly used donor of HNO, oxidized phenol and Me2SO to benzene diols and ·CH3, respectively. The oxidation of Me2SO was O2-independent, suggesting that this process reflected neither the intermediate formation of ONOO– nor a redox cycling of transition metal ions that could initiate Fenton-like reactions. In solutions of phenol, Na2N2O3 yielded benzene-1,2-diol and benzene-1,4-diol at a ratio of 2:1, which is consistent with the generation of free ·OH. Ethanol and Me2SO, which are efficient scavengers of ·OH, impeded the hydroxylation of phenol. A mechanism for the hydrolysis of Na2N2O3 is proposed that includes dimerization of HNO to cis-hyponitrous acid (HO-N=N-OH) with a concomitant azo-type homolytic fission of the latter to N2 and ·OH. The HNO-dependent production of ·OH was with 1 order of magnitude higher at pH 6.0 than at pH 7.4. Hence, we hypothesized that HNO can exert selective toxicity to cells subjected to acidosis. In support of this thesis, Na2N2O3 was markedly more toxic to human fibroblasts and SK-N-SH neuroblastoma cells at pH 6.2 than at pH 7.4. Scavengers of ·OH impeded the cytotoxicity of Na2N2O3. These results suggest that the formation of HNO may be viewed as a toxicological event in tissues subjected to acidosis. Despite its negative redox potential, nitroxyl (HNO) can trigger reactions of oxidation. Mechanistically, these reactions were suggested to occur with the intermediate formation of either hydroxyl radical (·OH) or peroxynitrite (ONOO–). In this work, we present further experimental evidence that HNO can generate ·OH. Sodium trioxodinitrate (Na2N2O3), a commonly used donor of HNO, oxidized phenol and Me2SO to benzene diols and ·CH3, respectively. The oxidation of Me2SO was O2-independent, suggesting that this process reflected neither the intermediate formation of ONOO– nor a redox cycling of transition metal ions that could initiate Fenton-like reactions. In solutions of phenol, Na2N2O3 yielded benzene-1,2-diol and benzene-1,4-diol at a ratio of 2:1, which is consistent with the generation of free ·OH. Ethanol and Me2SO, which are efficient scavengers of ·OH, impeded the hydroxylation of phenol. A mechanism for the hydrolysis of Na2N2O3 is proposed that includes dimerization of HNO to cis-hyponitrous acid (HO-N=N-OH) with a concomitant azo-type homolytic fission of the latter to N2 and ·OH. The HNO-dependent production of ·OH was with 1 order of magnitude higher at pH 6.0 than at pH 7.4. Hence, we hypothesized that HNO can exert selective toxicity to cells subjected to acidosis. In support of this thesis, Na2N2O3 was markedly more toxic to human fibroblasts and SK-N-SH neuroblastoma cells at pH 6.2 than at pH 7.4. Scavengers of ·OH impeded the cytotoxicity of Na2N2O3. These results suggest that the formation of HNO may be viewed as a toxicological event in tissues subjected to acidosis. The biochemistry of nitroxyl (HNO) has attracted considerable interest in recent years. In cells, the biosynthesis of HNO is believed to proceed via reduction of NO· by superoxide dismutase (1Murphy M.E. Sies H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10860-10864Crossref PubMed Scopus (288) Google Scholar) and cytochrome c (2Sharpe M.A. Cooper C.E. Biochem. J. 1998; 332: 9-19Crossref PubMed Scopus (188) Google Scholar), and reduction of S-nitrosoglutathione by low molecular weight and protein thiols (3Jensen D.E. Belka G.K. Du Bois G.C. Biochem. J. 1998; 331: 659-668Crossref PubMed Scopus (232) Google Scholar, 4Wong P.S. Hyun J. Fukuto J.M. Shirota F.N. DeMaster E.G. Shoeman D.W. Nagasawa H.T. Biochemistry. 1998; 37: 5362-5371Crossref PubMed Scopus (327) Google Scholar, 5Hogg N. Singh R.J. Kalyanaraman B. FEBS Lett. 1996; 382: 223-228Crossref PubMed Scopus (246) Google Scholar). It has been suggested that HNO can affect the etiology of various pathophysiological conditions such as inflammation and neurodegenerative diseases, especially when H2O2 and transition metal ions are present (6Chazotte-Aubert L. Oikawa S. Gilibert I. Bianchini F. Kawanishi S. Ohshima H. J. Biol. Chem. 1999; 274: 20909-20915Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 7Vaananen A.J. Moed M. Tuominen R.K. Helkamaa T.H. Wiksten M. Liesi P. Chiueh C.C. Rauhala P. Free Radical Res. 2003; 37: 381-389Crossref PubMed Scopus (26) Google Scholar). Similar to NO· and NO+, HNO is a potent inducer of the antioxidant protein heme oxygenase 1 (8Naughton P. Foresti R. Bains S.K. Hoque M. Green C.J. Motterlini R. J. Biol. Chem. 2002; 277: 40666-40674Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), exhibits vasorelaxant properties (9Ellis A. Li C.G. Rand M.J. Br. J. Pharmacol. 2000; 129: 315-322Crossref PubMed Scopus (82) Google Scholar), and modulates the activity of thiol-containing proteins, such as aldehyde dehydrogenase (10Conway T.T. DeMaster E.G. Lee M.J. Nagasawa H.T. J. Med. Chem. 1998; 41: 2903-2909Crossref PubMed Scopus (19) Google Scholar, 11DeMaster E.G. Redfern B. Quast B.J. Dahlseid T. Nagasawa H.T. Alcohol. 1997; 14: 181-189Crossref PubMed Scopus (42) Google Scholar) and the N-methyl-d-aspartate receptor (12Kim W.K. Choi Y.B. Rayudu P.V. Das P. Asaad W. Arnelle D.R. Stamler J.S. Lipton S.A. Neuron. 1999; 24: 461-469Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 13Choi Y.B. Tenneti L. Le D.A. Ortiz J. Bai G. Chen H.S. Lipton S.A. Nat. Neurosci. 2000; 3: 15-21Crossref PubMed Scopus (379) Google Scholar). In in vivo experiments, Paolocci et al. (14Paolocci N. Katori T. Champion H.C. St. John M.E. Miranda K.M. Fukuto J.M. Wink D.A. Kass D.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5537-5542Crossref PubMed Scopus (280) Google Scholar) observed that HNO exerts positive inotropic and lusitropic action, which unlike NO· and nitrates is independent and additive to β-adrenergic stimulation and increases the release of plasma calcitonin gene-related peptide; these results suggest that donors of HNO are potential prodrugs for the treatment of heart failure (14Paolocci N. Katori T. Champion H.C. St. John M.E. Miranda K.M. Fukuto J.M. Wink D.A. Kass D.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5537-5542Crossref PubMed Scopus (280) Google Scholar). At high doses, HNO has been shown to induce DNA single-strand breakage (15Ohshima H. Gilibert I. Bianchini F. Free Radical Biol. Med. 1999; 26: 1305-1313Crossref PubMed Scopus (80) Google Scholar, 16Bai P. Bakondi E. Szabo E. Gergely P. Szabo C. Virag L. Free Radical Biol. Med. 2001; 31: 1616-1623Crossref PubMed Scopus (43) Google Scholar) and a concentration-dependent cytotoxicity in murine thymocytes (16Bai P. Bakondi E. Szabo E. Gergely P. Szabo C. Virag L. Free Radical Biol. Med. 2001; 31: 1616-1623Crossref PubMed Scopus (43) Google Scholar). This cytotoxicity was associated with activation of the nuclear nick sensor enzyme poly(ADP-ribose)polymerase, perturbation of the mitochondrial membrane potential, and an increased production of superoxide (16Bai P. Bakondi E. Szabo E. Gergely P. Szabo C. Virag L. Free Radical Biol. Med. 2001; 31: 1616-1623Crossref PubMed Scopus (43) Google Scholar).On a molecular level, there are several differences between the reactivity of NO· and HNO that may account for the distinct biological effects of the latter species: in contrast to NO·, HNO directly interacts with thiols (4Wong P.S. Hyun J. Fukuto J.M. Shirota F.N. DeMaster E.G. Shoeman D.W. Nagasawa H.T. Biochemistry. 1998; 37: 5362-5371Crossref PubMed Scopus (327) Google Scholar, 17Doyle M.P. Mahapatro S.N. Broene R.D. Guy J.K. J. Am. Chem. Soc. 1988; 110: 593-599Crossref Scopus (197) Google Scholar), it preferentially binds to FeIII complexes (18Xia Y. Cardounel A.J. Vanin A.F. Zweier J.L. Free Radical Biol. Med. 2000; 29: 793-797Crossref PubMed Scopus (63) Google Scholar, 19Miranda K.M. Nims R.W. Thomas D.D. Espey M.G. Citrin D. Bartberger M.D. Paolocci N. Fukuto J.M. Feelisch M. Wink D.A. J. Inorg. Biochem. 2003; 93: 52-60Crossref PubMed Scopus (112) Google Scholar), and acts as a hydroxylating agent (15Ohshima H. Gilibert I. Bianchini F. Free Radical Biol. Med. 1999; 26: 1305-1313Crossref PubMed Scopus (80) Google Scholar, 20Stoyanovsky D.A. Clancy R.M. Cederbaum A.I. J. Am. Chem. Soc. 1999; 121: 5093-5094Crossref Scopus (28) Google Scholar, 21Kirsch M. de Groot H. J. Biol. Chem. 2002; 277: 13379-13388Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Recently, we have reported that HNO can generate ·OH in a pH-dependent manner (20Stoyanovsky D.A. Clancy R.M. Cederbaum A.I. J. Am. Chem. Soc. 1999; 121: 5093-5094Crossref Scopus (28) Google Scholar). Because of its high reactivity, ·OH is one of the most toxic species that can be formed in biological systems. This free radical reacts with most cellular molecules at diffusion-controlled rates; thus it cannot diffuse from its site of generation further than the nearest molecules (22Halliwell B. Gutteridge J.M. Methods Enzymol. 1990; 186: 1-85Crossref PubMed Scopus (4411) Google Scholar). Hence, we hypothesized that the pH-dependent generation of ·OH from HNO can have toxicological significance, particularly because tissue acidification occurs under various pathological conditions, such as hypoxia, inflammation, and cancer (23Gasbarrini A. Borle A.B. Farghali H. Francavilla A. Van Thiel D. J. Biol. Chem. 1992; 267: 7545-7552Abstract Full Text PDF PubMed Google Scholar, 24Stubbs M. McSheehy P.M. Griffiths J.R. Bashford C.L. Mol. Med. Today. 2000; 6: 15-19Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar, 25Waldmann R. Adv. Exp. Med. Biol. 2001; 502: 293-304Crossref PubMed Scopus (85) Google Scholar). In the present work, we provide further experimental evidence that HNO generates ·OH in an oxygen-independent manner. We also report that HNO exhibits a pH-dependent toxicity to normal human fibroblasts and SK-N-SH neuroblastoma cells that could be impeded by scavengers of radical species.EXPERIMENTAL PROCEDURESReagents—All reagents used were purchased from Sigma. The solutions used in the experiments were prepared in deionized and Chelex 100-treated water or potassium phosphate buffer. Sodium trioxodinitrate was either purchased from Calbiochem, Inc. (La Jolla, CA) or synthesized as described in Ref. 26Smith P.A.S. Hein G.E. J. Am. Chem. Soc. 1960; 82: 5731-5740Crossref Scopus (98) Google Scholar.HPLC 1The abbreviations used are: HPLC, high performance liquid chromatography; PBN, N-tert-butyl-α-phenylnitrone; MGD, N-methyl-d-glucamine dithiocarbamate; DMPO, 5,5′-dimethyl-1-pyroline N-oxide. Analysis—HPLC was performed with a Waters liquid chromatograph (Milford, MA). Separation was achieved with a C-18 reverse phase column (Microsorb, 4.6 mm × 25 cm, 5 μm, 100 A Rainin Instrument Co., Inc., Emeryville, CA). The mobile phase was saturated with helium and contained 10 mm lithium perchlorate and either water with 30% (v/v) methanol for analysis of phenol and benzene diols, or 70% methanol for analysis of N-tert-butyl-α-phenylnitrone (PBN) derivatives. All HPLC analyses were conducted at a flow rate of 1 ml/min. Electrochemical detection of PBN-derived adducts was carried out at +0.8 V with a LC-4C/CC5 amperometric system (Bioanalytical Systems, West Lafayette, IN) equipped with glassy carbon electrode and a Ag/AgCl reference electrode (27Stoyanovsky D.A. Cederbaum A.I. Chem. Res. Toxicol. 1999; 12: 730-736Crossref PubMed Scopus (52) Google Scholar). Phenol and benzene diols were analyzed electrochemically at +0.95 V.ESR Measurements—ESR measurements were performed on a Bruker ECS106 spectrometer with 50 kHz magnetic field modulation at room temperature (25 °C). ESR spectrometer settings were: modulation amplitude 0.7 G, scan time 40 s, time constant 0.64 s, microwave power 20 mW, and receiver gain 1 × 103–1 × 105.Cell Experiments—Normal human fibroblasts or SK-N-SH neuroblastoma cells (800–1000 cells per plate) were treated for 30 min at 37 °C with Na2N2O3 in 50 mm phosphate buffer (pH 6.2–7.4) containing 0.15 m NaCl and 0.2 mm CaCl2. Thereafter, the fluid was removed, the cells were covered with minimal essential medium containing 10% fetal bovine serum and incubated at 37 °C for 4 h. In selected experiments, the incubation medium containing Na2N2O3 also included ascorbic acid (5 mm) plus superoxide dismutase (300 units/ml), catalase (500 units/ml), and EDTA (0.1 mm), or α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (10 mm) or Me2SO (0.2 m). On completion of the incubations, the cell number was determined by the crystal violet method.Anaerobic Experiments—To achieve anaerobic conditions, all solutions were placed in septum-caped vials and purged for 30 min with a stream of nitrogen. Thereafter, additions to the final reaction solution were made through the septum of the corresponding vial using a 0.10-ml gas-tight syringe.RESULTSEPR Analysis of the Hydrolysis of Na2N2O3—In model studies aimed at mimicking the biochemistry of HNO, sodium trioxodinitrate (Na2N2O3; Angeli's salt) is often used as a donor of this species. Depending on the degree of protonation, the stability of this compound in aqueous solutions follows the sequence N2O32-(1c)x003E;HN2O3-(1b)x003E;H2N2O3(1a) (pK 1 = 3.0 and pK 2 = 9.35; Scheme 1) (28Hughes M.N. Wimbledon P.E. J. Chem. Soc. Dalton Trans. 1976; : 703-704Crossref Google Scholar). 1c is relatively stable in alkaline solutions (pH > 10). However, the rate of decomposition of 1b within the pH interval 4–8 is [H+]-independent and leads to the formation of HNO (k1b = 5.1 × 10–4m–1 s–1 (29Bonner F.T. Ravid B. J. Inorg. Chem. 1975; 14: 558-563Crossref Scopus (117) Google Scholar)). The latter species can dimerize to cis-hyponitrous acid (3a), which is unstable and decomposes to N2O and H2O. The decomposition of 3a is especially fast in aqueous solutions with pH 6–13 (30Loechler E.L. Schneider A.M. Schwartz D.B. Hollocher T.C. J. Am. Chem. Soc. 1987; 109: 3076-3087Crossref Scopus (19) Google Scholar, 31Anderson J.H. Analyst. 1963; 88: 494-499Crossref Google Scholar), which most likely reflects shifts in the equilibrium between 3a and 3b in favor of the latter (Scheme 1). Bonner and Ravid (29Bonner F.T. Ravid B. J. Inorg. Chem. 1975; 14: 558-563Crossref Scopus (117) Google Scholar) reported that the hydrolysis of Na2(O15NNO2) at either pH 3.0 or 8.5 yields exclusively 15N2O and NO2-, implying that this process follows the reaction sequence presented in Scheme 1 (29Bonner F.T. Ravid B. J. Inorg. Chem. 1975; 14: 558-563Crossref Scopus (117) Google Scholar). However, these authors noted that the proportions of 15N in the reaction products were different at pH 5.0, suggesting that some modification of pathway cannot be ruled out. At pH < 4, the decomposition rate of 1a increases with increasing acidity with production of NO· (28Hughes M.N. Wimbledon P.E. J. Chem. Soc. Dalton Trans. 1976; : 703-704Crossref Google Scholar, 29Bonner F.T. Ravid B. J. Inorg. Chem. 1975; 14: 558-563Crossref Scopus (117) Google Scholar).We recently reported that 1b can convert primary alcohols to aldehydes via the intermediate formation of ·OH (20Stoyanovsky D.A. Clancy R.M. Cederbaum A.I. J. Am. Chem. Soc. 1999; 121: 5093-5094Crossref Scopus (28) Google Scholar). In these experiments, the formation of ·OH was characterized by EPR spin trapping analysis. However, we could not evaluate the absolute amounts of spin-trapped ·OH as the resulting nitroxides are readily converted to EPR silent hydroxylamines under reductive conditions (32Stoyanovsky D.A. Melnikov Z. Cederbaum A.I. Anal. Chem. 1999; 71: 715-721Crossref PubMed Scopus (42) Google Scholar). Quantitative evaluation of the latter reaction is important because the production of ·OH from HNO may have toxicological implications. Hence, we have carried out EPR/HPLC-UV/EC spin trapping experiments optimized for the quantitation of ·OH under reductive conditions.In the presence of FeIII and N-methyl-d-glucamine dithiocarbamate (MGD), the hydrolysis of 1b was paralleled by the appearance of the characteristic EPR spectrum of ·ON-FeII-MGD formed via the interaction of HNO and FeIII-MGD (Fig. 1A; Scheme 2) (18Xia Y. Cardounel A.J. Vanin A.F. Zweier J.L. Free Radical Biol. Med. 2000; 29: 793-797Crossref PubMed Scopus (63) Google Scholar). The formation of ·ON-FeII-MGD was H+-independent within the pH interval of 4.5 to 7.4 (Fig. 1A), which is in agreement with previous findings (28Hughes M.N. Wimbledon P.E. J. Chem. Soc. Dalton Trans. 1976; : 703-704Crossref Google Scholar) that the rate of 1b hydrolysis at these proton concentrations is constant. The substitution of FeIII-MGD with 5,5′-dimethyl-1-pyroline N-oxide (DMPO) resulted in the appearance of the typical EPR spectrum of DMPO/·OH (6; Fig. 1B) (33Buettner G.R. Free Radical Biol. Med. 1987; 3: 259-303Crossref PubMed Scopus (1511) Google Scholar), suggesting that the hydrolysis of 1b resulted in the formation of ·OH. In contrast to the formation of HNO, the generation of 6 from 1b was strongly affected by the acidity of the reaction solutions. The latter implies that ·OH was not directly derived from 1b but rather followed the release of HNO (Fig. 1, panel A compared with panel B; Fig. 4D).Fig. 1EPR spectra of ·ON-FeII-MGD and DMPO/·OH formed in solutions of Na2N2O3, FeIII-MGD, and DMPO. All reactions were carried out at 20 °C in either 0.1 m Tris (panel A) or 0.1 m phosphate buffer (panel B). The reactions were initiated by an addition of Na2N2O3 and EPR spectra were recorded 4 min thereafter. A, FeCl3, 0.3 mm; MGD, 1 mm; Na2N2O3, 0.1 mm. B, DMPO, 100 mm; Na2N2O3, 0.3 mm. For each experiment, a fresh stock solution of Na2N2O3 was prepared in 10 mm NaOH).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Scheme 2View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Kinetics of formation of the nitroxide and hydroxylamine forms of PBN/CH3 in solutions of Na2N2O3, PBN, and Me2SO. All reactions were carried out for 30 min in 0.1 m phosphate buffer (pH 5; 40 °C) containing PBN (50 mm), Me2SO (0.5 m), and Na2N2O3 (panels A, C, and D, 10 mm). In selected experiments, the acidity of the reaction solutions was adjusted with either NaOH or HCl (panel D). The total production of PBN/CH3 (Scheme 2, 4 plus 5) in the presence (closed circles) or absence (open circles) of oxygen is presented in panel A. Anaerobic conditions were achieved as described under “Experimental Procedures.” Each experimental point represents the mean of three experiments ± S.E.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The low stability of ·OH-derived nitroxides (t ½ ∼30 s to 15 min (34Janzen E.G. Kotake Y. Hinton R.D. Free Radical Biol. Med. 1992; 12: 169-173Crossref PubMed Scopus (172) Google Scholar, 35Towell J. Kalyanaraman B. Anal. Biochem. 1991; 196: 111-119Crossref PubMed Scopus (35) Google Scholar)) is a limiting factor for quantification of ·OH. To solve this experimental difficulty, we used an HPLC protocol for quantification of ·OH that is based on the oxidation of Me2SO (32Stoyanovsky D.A. Melnikov Z. Cederbaum A.I. Anal. Chem. 1999; 71: 715-721Crossref PubMed Scopus (42) Google Scholar). The latter is oxidized by ·OH to ·CH3, which forms relatively stable nitroxides (t ½ > 48 h) with PBN. The hydrolysis of 1b in the presence of Me2SO and PBN produced the typical EPR spectrum of PBN/·CH3 (4; Fig. 2 trace 2) (33Buettner G.R. Free Radical Biol. Med. 1987; 3: 259-303Crossref PubMed Scopus (1511) Google Scholar). However, both 1b and HNO could act as reductants (36Al-Ajlouni A.M. Gould E.S. Inorg. Chem. 1999; 38: 1592-1595Crossref Scopus (9) Google Scholar, 37Bartberger M.D. Liu W. Ford E. Miranda K.M. Switzer C. Fukuto J.M. Farmer P.J. Wink D.A. Houk K.N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10958-10963Crossref PubMed Scopus (286) Google Scholar), suggesting that the EPR spectrum of 4 may not reflect the real amount of ·OH and ·CH3 formed in this reaction system. In the presence of reductants, nitroxides can be readily reduced to the corresponding EPR silent hydroxylamines (32Stoyanovsky D.A. Melnikov Z. Cederbaum A.I. Anal. Chem. 1999; 71: 715-721Crossref PubMed Scopus (42) Google Scholar). In support of the latter assumption, the addition of K3[Fe(CN)6] to an extract of a reaction solution consisting of 1b, Me2SO, and PBN resulted in a pronounced increase of the EPR signal of 4. This effect most likely reflected the oxidation of the EPR silent hydroxylamine 5 to 4 (Scheme 2). When the reaction solution was analyzed by HPLC with electrochemical detection, the predominant formation of 5 was observed (Fig. 3); an addition of K3[Fe(CN)6] to the analyzed solutions resulted in the inter-conversion of the HPLC peaks reflecting the elution of 5 and 4, respectively. The identity of compounds 4 and 5 was confirmed by coinjections of authentic HPLC standards as described previously (32Stoyanovsky D.A. Melnikov Z. Cederbaum A.I. Anal. Chem. 1999; 71: 715-721Crossref PubMed Scopus (42) Google Scholar, 38Novakov C.P. Feierman D. Cederbaum A.I. Stoyanovsky D.A. Chem. Res. Toxicol. 2001; 14: 1239-1246Crossref PubMed Scopus (16) Google Scholar).Fig. 2EPR spectra of PBN/·CH3 formed in solutions of Na2N2O3, PBN, and Me2SO. All reactions were carried out at 20 °C in 0.5 m phosphate buffer (pH 5.5). EPR spectra 1–3 and spectrum 4 were recorded at a gain of 1 × 105 and 1 × 103, respectively. Spectrum 1, PBN (70 mm) and Me2SO (1 m). Spectrum 2 was recorded after a 30-min incubation of a solution containing PBN, Me2SO, and Na2N2O3 (0.25 m). Thereafter, the reaction products were extracted with ethyl acetate (2 × 6 ml), the extract was diluted 10 times in methanol and EPR spectra were recorded in the absence (spectrum 3) or presence of 1 mm K3[Fe(CN)6] (spectrum 4; incubation time, 10 min).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3HPLC-EC chromatograms of the nitroxide and hydroxylamine forms of PBN/CH3 formed in solutions of Na2N2O3, PBN, and Me2SO. The HPLC-EC profile of 0.1 m phosphate buffer (pH 5) containing Na2N2O3 (0.5 mm), PBN (25 mm), and Me2SO (0.5 m) was obtained after an incubation of the reaction solution for 30 min at 40 °C (solid lines). At the end of the incubation, addition of K3[Fe(CN)6] (1 mm) followed by an incubation of 10 min resulted in the conversion of hydroxylamine 5 into nitroxide 4 (dashed lines; Scheme 2).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The formation of 4 and 5 in solutions of 1b, PBN, and Me2SO was well controlled and with an yield of 7.5% of the initial concentration of 1b (Fig. 4). The actual production of ·OH in this reaction system, however, cannot be estimated as the efficiency of the ·OH-dependent oxidation of Me2SO and the subsequent spin trapping of ·CH3 are undefined. Under anaerobic conditions, the reaction profile remained unchanged (Fig. 4A , open circles), which attests that the generation of ·OH from HNO was O2-independent and reflected neither the intermediate formation of ONOO– (Scheme 4) (21Kirsch M. de Groot H. J. Biol. Chem. 2002; 277: 13379-13388Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 39Miranda K.M. Espey M.G. Yamada K. Krishna M. Ludwick N. Kim S. Jourd'heuil D. Grisham M.B. Feelisch M. Fukuto J.M. Wink D.A. J. Biol. Chem. 2001; 276: 1720-1727Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) nor the occurrence of Fenton-like reactions. Maximal production of 4 and 5 was observed within the pH interval of 4 to 6. At pH 6, the production of 4 and 5 was 1 order of magnitude higher than that at pH 7.4.Scheme 4View Large Image Figure ViewerDownload Hi-res image Download (PPT)Hydroxylation of Phenol in Aqueous Solutions of Na2N2O3— Phenol is often used as a molecular probe to discriminate free ·OH from other oxidizing species. For example, radiolytically generated ·OH reacts with phenol via either abstraction of the hydrogen atom from its -OH function (kH = 2.1 × 109m–1 s–1) or aryl addition (k Ar = 6.6 × 109m–1 s–1) to give a phenol phenoxyl (10) or dihydroxycyclohexadienyl (8) radical, respectively (40Field R.J. Raghavan N.V. Brummer J.G. J. Am. Chem. Soc. 1982; 86: 2443-2449Google Scholar, 41Hage J.P. Llobet A. Sawyer D.T. Bioorg. Med. Chem. 1995; 3: 1383-1388Crossref PubMed Scopus (46) Google Scholar). Under aerobic conditions, 8 interacts with O2 to form benzene diols (9) and the superoxide anion radical; benzene-1,2-diol and benzene-1,4-diol, forming at a ratio of 2:1, are the main isomers generated in this reaction (41Hage J.P. Llobet A. Sawyer D.T. Bioorg. Med. Chem. 1995; 3: 1383-1388Crossref PubMed Scopus (46) Google Scholar). In contrast, oxidation of phenol by Fenton-type reagents such as L x FeII-OOH leads to the formation of benzene-1,2-diol and benzene-1,4-diol at a ratio that depends on the ligand of the corresponding iron complex and ranges from 11 to ∞ (41Hage J.P. Llobet A. Sawyer D.T. Bioorg. Med. Chem. 1995; 3: 1383-1388Crossref PubMed Scopus (46) Google Scholar). It should be noted that 8 and 10 can follow several reaction pathways, implying that the formation of 9 can only be a qualitative marker for free ·OH. For example, the hydroxylation of phenol by ·OH in the absence of oxygen (or other electron acceptors, such as K3[Fe(CN)6] and quinones (42Raghavan N.V. Steenken S. J. Am. Chem. Soc. 1980; 102: 3495-3499Crossref Scopus (200) Google Scholar)) predominantly leads to the formation of biphenyl diols (11 (41Hage J.P. Llobet A. Sawyer D.T. Bioorg. Med. Chem. 1995; 3: 1383-1388Crossref PubMed Scopus (46) Google Scholar, 43Ito S. Mitarai A. Hikino M. Hirama M. Sasaki K. J. Org. Chem. 1992; 57: 6937-6941Crossref Scopus (57) Google Scholar)) (Scheme 3).Scheme 3View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5 depicts the HPLC-EC profile of a reaction system consisting of 1b and phenol in 0.1 m phosphate buffer (pH = 4.0). 1b caused a time- and dose-dependent hydroxylation of phenol to 9 with an overall yield of 80% (Fig. 6, A and B). Within the pH interval of 4 to 7 (ΔpH = 0.5), the ratio between benzene-1,2-diol and benzene-1,4-diol was 2.2 + 0.24 (n = 5; mean ± S.E.; Fig. 6C), which further supports the notion for the generation of free ·OH in solutions of 1b. In the absence of 1b, no formation of 9 was observed (Fig. 6A , open circles). The 1b-dependent hydroxylation of phenol proceeded with two pH optimums (Fig. 7). Because the pK of phenol is 9.98, the effects of H+ on the formation of 9 most likely reflected changes in the rates of reactions 8 → 9 and 8 → 10, respectively. Hence, pH variations in this reaction system may affect the formation of ·OH and 9 to different extents. The production of 9 increased with increasing temperatures up to 35 °C, whereas at higher temperatures a considerable autoxidation of the reaction products was observed (data not shown). The impeded formation of 9 under anaerobic conditions (Fig. 8A) was attributed to a shift in the equilibrium between 8 and 10, which ultimately resulted in a predominant formation of 11 (41Hage J.P. Llobet A. Sawyer D.T. Bioorg. Med. Chem. 1995; 3: 1383-1388Crossref PubMed Scopus (46) Google Scholar). In contrast, the formation of 9 was markedly increased when the reaction solutions were purged with a stream of air. The latter effect was most likely because of a more efficient oxidation of 8 to 9 (40Field R.J. Raghavan N.V. Brummer J.G. J. Am. Chem. Soc. 1982; 86: 2443-2449Google Scholar, 42Raghavan N.V. Steenken S. J. Am. Chem. Soc. 1980; 102: 3495-3499Crossref Scopus (200) Google Scholar, 43Ito S. Mitarai A. Hikino M. Hirama M. Sasaki K. J. Org. Chem. 1992; 57: 6937-6941Crossref Scopus (57) Google Scholar, 44Stoyanovsky D.A. Osipov A.N. Quinn P.J. Kagan V.E. Arch. Biochem. Biophys. 1995; 323: 343-351Crossref PubMed Scopus (145) Google Scholar). Desferrioxamine did not affect the formation of 9 to any significant extent, indicating that metal ions did not participate in the overall reaction mechanism. However, the hydroxylation of phenol was inhibited by scavengers of ·OH such as ethanol and Me2SO (Fig. 8B) that are known to interact with this radical species at appreciable rates (k Me2SO = 7 × 109m–1 s–1 and k EtOH = 2.2 × 109m–1 s–1, respectively (45Veltwisch D. Janata E. Asmus K.D. Saito Y. J. Chem. Soc. Perkin Trans. 1980; 2: 146-153Crossref Scopus (222) Google Scholar, 46Motohashi N. Saito Y. Chem. Pharm. Bull. 1993; 41: 1842-1845Crossref Scopus (76) Google Scholar)). In these experiments, the observed" @default.
• W2034334015 created "2016-06-24" @default.
• W2034334015 creator A5009759231 @default.
• W2034334015 creator A5018452221 @default.
• W2034334015 creator A5018579654 @default.
• W2034334015 creator A5025283915 @default.
• W2034334015 creator A5086611091 @default.
• W2034334015 creator A5089626875 @default.
• W2034334015 date "2003-10-01" @default.
• W2034334015 modified "2023-10-12" @default.
• W2034334015 title "Formation of Nitroxyl and Hydroxyl Radical in Solutions of Sodium Trioxodinitrate" @default.
• W2034334015 cites W140380104 @default.
• W2034334015 cites W1543601456 @default.
• W2034334015 cites W1593547522 @default.
• W2034334015 cites W1734082729 @default.
• W2034334015 cites W1750720387 @default.
• W2034334015 cites W1844185706 @default.
• W2034334015 cites W1857559494 @default.
• W2034334015 cites W1926953922 @default.
• W2034334015 cites W1963630858 @default.
• W2034334015 cites W1964058023 @default.
• W2034334015 cites W1969384983 @default.
• W2034334015 cites W1971883112 @default.
• W2034334015 cites W1975021973 @default.
• W2034334015 cites W1976555322 @default.
• W2034334015 cites W1976645176 @default.
• W2034334015 cites W1978100205 @default.
• W2034334015 cites W1980146663 @default.
• W2034334015 cites W1981561121 @default.
• W2034334015 cites W1987265340 @default.
• W2034334015 cites W1991431198 @default.
• W2034334015 cites W1992281852 @default.
• W2034334015 cites W1994116537 @default.
• W2034334015 cites W1997208169 @default.
• W2034334015 cites W1999441551 @default.
• W2034334015 cites W2002364325 @default.
• W2034334015 cites W2004342910 @default.
• W2034334015 cites W2006169220 @default.
• W2034334015 cites W2006172842 @default.
• W2034334015 cites W2010239907 @default.
• W2034334015 cites W2015738576 @default.
• W2034334015 cites W2015752494 @default.
• W2034334015 cites W2018748912 @default.
• W2034334015 cites W2021516438 @default.
• W2034334015 cites W2023741837 @default.
• W2034334015 cites W2024921655 @default.
• W2034334015 cites W2026981616 @default.
• W2034334015 cites W2036585283 @default.
• W2034334015 cites W2041177052 @default.
• W2034334015 cites W2045429393 @default.
• W2034334015 cites W2045744719 @default.
• W2034334015 cites W2048467721 @default.
• W2034334015 cites W2048673047 @default.
• W2034334015 cites W2048839667 @default.
• W2034334015 cites W2050264954 @default.
• W2034334015 cites W2053686180 @default.
• W2034334015 cites W2055695959 @default.
• W2034334015 cites W2059456707 @default.
• W2034334015 cites W2062356433 @default.
• W2034334015 cites W2063031193 @default.
• W2034334015 cites W2065281025 @default.
• W2034334015 cites W2065494054 @default.
• W2034334015 cites W2066568699 @default.
• W2034334015 cites W2074767838 @default.
• W2034334015 cites W2083354474 @default.
• W2034334015 cites W2089990423 @default.
• W2034334015 cites W2090147611 @default.
• W2034334015 cites W2091130738 @default.
• W2034334015 cites W2105928417 @default.
• W2034334015 cites W2172134825 @default.
• W2034334015 cites W2314801013 @default.
• W2034334015 cites W23222670 @default.
• W2034334015 cites W2323300972 @default.
• W2034334015 cites W2953038280 @default.
• W2034334015 cites W3139190200 @default.
• W2034334015 cites W4251272780 @default.
• W2034334015 doi "https://doi.org/10.1074/jbc.m305544200" @default.
• W2034334015 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12920123" @default.
• W2034334015 hasPublicationYear "2003" @default.
• W2034334015 type Work @default.
• W2034334015 sameAs 2034334015 @default.
• W2034334015 citedByCount "24" @default.
• W2034334015 countsByYear W20343340152013 @default.
• W2034334015 countsByYear W20343340152015 @default.
• W2034334015 countsByYear W20343340152016 @default.
• W2034334015 countsByYear W20343340152020 @default.
• W2034334015 countsByYear W20343340152022 @default.
• W2034334015 crossrefType "journal-article" @default.
• W2034334015 hasAuthorship W2034334015A5009759231 @default.
• W2034334015 hasAuthorship W2034334015A5018452221 @default.
• W2034334015 hasAuthorship W2034334015A5018579654 @default.
• W2034334015 hasAuthorship W2034334015A5025283915 @default.
• W2034334015 hasAuthorship W2034334015A5086611091 @default.
• W2034334015 hasAuthorship W2034334015A5089626875 @default.
• W2034334015 hasBestOaLocation W20343340151 @default.
• W2034334015 hasConcept C139066938 @default.
• W2034334015 hasConcept C178790620 @default.
• W2034334015 hasConcept C185592680 @default.

• next