Spin Trapping of Methyl Radicals by the Acyl Nitroso Compound PH-C (= O)NO Formed in the Photochemical Reaction Between Benzohydroxamic Acid, Dimethyl Sulfoxide and Hydrogen Peroxide. An Epr Study

By the use of EPR spectroscopy, it has been shown that acyl nitroso compounds can act as spin traps for short-lived radicals with the formation of acyl aminoxyl radicals. The reaction was studied for the system benzohydroxamicacid[Ph-C (= O)N(H)] - dimethyl sulfoxide - hydrogen peroxide. The acyl aminoxyl radicals appeared almost immediately when the reaction mixture was irradiated in situ in the EPR cavity with UV light. The trapping reaction involved two photochemical reactions, i.e. the oxidation of the hydroxamic acid to the acyl nitroso compound Ph-C (= O)NO, and the formation of methyl radicals from dimethyl sulfoxide. The EPR spectra are superpositions of the spectra of two species of acyl aminoxyl radicals, i.e. the radicals Ph-C (= O)N(O·)H formed by oxidation of the parent benzohydrox-amic acid, and the radical Ph-C (= O)N(O·)CH₃, formed by trapping of methyl radicals.     

N-Hydroxy sulfonimidamides as new nitroxyl (HNO) donors

Chlorination and condensation of simple sulfinamides with O-benzyl and O-tert-butyl dimethyl siloxy hydroxylamine gives O-protected N-hydroxy sulfonimidamides. Deprotection of these compounds produces the corresponding sulfinamide and nitrous oxide, which provides evidence for the intermediacy of nitroxyl (HNO) and identifies these compounds as new potential HNO donors.     

Adenosine 3',5'-monophosphate formation by preparations of rat liver soluble guanylate cyclase activated with nitric oxide, nitrosyl ferroheme, S-nitrosothiols, and other nitroso compounds

Cyclic AMP formation from ATP was stimulated by unpurified and partially purified soluble hepatic guanylate cyclase in the presence of nitric oxide (NO) or compounds containing a nitroso moiety such as nitroprusside, N-methyl-N-nitro-N-nitrosoguanidine (MNNG), nitrosyl ferroheme, and S-nitrosothiols. Cyclic AMP formation was undetectable in the absence of NO or nitroso compounds and was not stimulated by fluoride or glucagon, indicating the absence of adenylate cyclase activity. The nitroso compounds failed to activate, whereas fluoride or glucagon activated, adenylate cyclase in washed rat liver membrane fractions. Cyclic GMP formation from GTP was markedly stimulated by the soluble hepatic fraction in the presence of NO or nitroso compounds. Cyclic AMP formation by partially purified guanylate cyclase was competitively inhibited by GTP and cyclic GMP formation is well-known to be competitively inhibited by ATP. Therefore, it appears that activated guanylate cyclase, rather than adenylate cyclase, was responsible for the formation of cyclic AMP from ATP. Formation of cyclic AMP of cyclic GMP was enhanced by thiols, inhibited by hemoproteins and oxidants, and required the addition of either Mg2+ or Mn2+. Further, several nitrosyl ferroheme compounds and S-nitrosothiols stimulated the formation of both cyclic AMP and cyclic GMP by the soluble hepatic fraction. These observations support the view that soluble guanylate cyclase is capable, under certain well-defined conditions, of catalyzing the conversion of ATP to cyclic AMP.     

Acyloxy Nitroso Compounds Inhibit LIF Signaling in Endothelial Cells and Cardiac Myocytes: Evidence That STAT3 Signaling Is Redox-Sensitive

We previously showed that oxidative stress inhibits leukemia inhibitory factor (LIF) signaling by targeting JAK1, and the catalytic domains of JAK 1 and 2 have a cysteine-based redox switch. Thus, we postulated that the NO sibling and thiophylic compound, nitroxyl (HNO), would inhibit LIF-induced JAK-STAT3 activation. Pretreatment of human microvascular endothelial cells (HMEC-1) or neonatal rat cardiomyocytes with the HNO donors Angeli's salt or nitrosocyclohexyl acetate (NCA) inhibited LIF-induced STAT3 activation. NCA pretreatment also blocked the induction of downstream inflammatory genes (e.g. intercellular adhesion molecule 1, CCAAT/enhancer binding protein delta). The related 1-nitrosocyclohexyl pivalate (NCP; not a nitroxyl donor) was equally effective in inhibiting STAT3 activation, suggesting that these compounds act as thiolate targeting electrophiles. The JAK1 redox switch is likely not a target of acyloxy nitroso compounds, as NCA had no effect on JAK1 catalytic activity and only modestly affected JAK1-induced phosphorylation of the LIF receptor. However, pretreatment of recombinant human STAT3 with NCA or NCP reduced labeling of free sulfhydryl residues. We show that NCP in the presence of diamide enhanced STAT3 glutathionylation and dimerization in adult mouse cardiac myocytes and altered STAT3 under non-reducing conditions. Finally, we show that monomeric STAT3 levels are decreased in the Gαq model of heart failure in a redox-sensitive manner. Altogether, our evidence indicates that STAT3 has redox-sensitive cysteines that regulate its activation and are targeted by HNO donors and acyloxy nitroso compounds. These findings raise the possibility of new therapeutic strategies to target STAT3 signaling via a redox-dependent manner, particularly in the context of cardiac and non-cardiac diseases with prominent pro-inflammatory signaling.     

Oxidation and nitrosylation of oxyhemoglobin by S-nitrosoglutathione via nitroxyl anion

The reaction between low molecular weight S-nitrosothiols and hemoglobin is often used to synthesize S-nitrosohemoglobin, a form of hemoglobin suggested to be involved in the regulation of vascular oxygen delivery. However, this reaction has not been studied in detail, and several groups have reported a variable co-formation of oxidized methemoglobin (metHb) during synthesis. This study examines the mechanism of metHb formation and shows that nitrosylhemoglobin (HbNO) can also be formed. Generation of metHb and HbNO is largely dependent on the presence of protein thiol groups. We present evidence for a mechanism for the formation of metHb and HbNO involving the intermediacy of nitroxyl anion. Specifically, the reaction of nitroxyl with S-nitrosothiols to liberate nitric oxide and reduced thiol is proposed to be central to the reaction mechanism.     

Nitrosation of uric acid induced by nitric oxide under aerobic conditions.

Uric acid is a well-established scavenger of reactive oxygen and nitrogen species such as hydroxyl radical and peroxynitrite. However, little attention has been paid to the relationship between uric acid and nitric oxide. This paper reports the identification and characterization of a reaction product of uric acid induced by nitric oxide. When uric acid was treated with nitric oxide gas in a neutral solution under aerobic conditions, uric acid was consumed, yielding an unknown product. The product was identified as nitrosated uric acid from mass spectrometric data, although the position of the nitroso group on the molecule was not determined. The nitrosated uric acid decomposed to several compounds including uric acid with a half-life of 2.2 min at pH 7.4 and 37 degrees C. The incubation of nitrosated uric acid with glutathione resulted in the formation of S-nitrosoglutathione. Nitrosated uric acid was also formed in the reaction with nitric oxide donors, but not with peroxynitrite. Nitrosated uric acid was detected in human serum and urine by in vitro treatment with a nitric oxide donor. In the reaction of glutathione with the nitric oxide donor, the addition of uric acid caused an increase in the yield of S-nitrosoglutathione. These results indicate that under aerobic conditions nitric oxide can convert uric acid into its nitroso derivative, which can give a nitroso group to glutathione. Uric acid may act as a vehicle of nitric oxide in humans.     

DNA Single Strand Breaks by Aromatic Nitroso Compounds in the Presence of Thiols

Aromatic nitroso compounds, nitrosobenzene (NB), N, N-dimethyl-4-nitrosoaniline (DMNA) and 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS), caused DNA single strand breaks in the presence of thiol compounds. The strand breaking was inhibited completely by free radical scavenger ethanol. Electron spin resonance (ESR) studies showed that hydronitroxyl (or sulfur-substituted nitroxyl) radicals were generated in the early stage of the interactions. Formation of these radicals was not inhibited by ethanol, indicating that these radicals did not directly contribute to the strand breaking. The DNA strand breaking was inhibited partially by superoxide dismutase and catalase under the limited conditions, but not by removal of oxygen from or addition of metal chelators to the reaction mixture. By ESR-spin trapping technique using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the DMPO-OH spin adduct was detected. Formation of the spin adduct was inhibited by superoxide dismutase and catalase. The hydronitroxyl (or the sulfur-substituted nitroxyl) radicals may reduce oxygen into active oxygen species and also transformed by themselves into other unidentified free radical species to cause the DNA strand breaks.     

Nitration and nitrosation of N-acetyl-L-tryptophan and tryptophan residues in proteins by various reactive nitrogen species

Proteins are targets of reactive nitrogen species such as peroxynitrite and nitrogen dioxide. Among the various amino acids in proteins, tryptophan residues are especially susceptible to attack by reactive nitrogen species. We carried out experiments on the reactions of peroxynitrite and other reactive nitrogen species with N-acetyl-L-tryptophan under various conditions. Four major products were identified as 1-nitroso-N-acetyl-L-tryptophan, 1-nitro-N-acetyl-L-tryptophan, 6-nitro-N-acetyl-L-tryptophan, and N-acetyl-N'-formyl-L-kynurenine on the basis of their mass and UV spectra. The reactions with SIN-1 (a peroxynitrite generator), Angeli's salt (a nitroxyl donor), and spermine NONOate (a nitric oxide donor) generated the nitroso derivative but not the nitro derivatives. A myeloperoxidase-H(2)O(2)-NO(2)(-) system generated the nitro derivatives but not the nitroso derivative. Under physiological conditions 6-nitro-N-acetyl-L-tryptophan was stable, whereas the 1-nitroso and 1-nitro derivatives decomposed with half-lives of 1.5 and 18 h, respectively. After treatment with various reactive nitrogen species, bovine serum albumin was enzymatically hydrolyzed and analyzed for 6-nitro-L-tryptophan and 3-nitro-L-tyrosine by HPLC with electrochemical detection. Levels of 6-nitro-L-tryptophan and 3-nitro-L-tyrosine were similar in the nitrated protein. 6-Nitro-L-tryptophan in proteins can be measured as an additional biomarker of protein nitration.     

Formation of nitric oxide in animal tissues during inflammatory process

EPR evidence was obtained that more intensive formation of mononitrosyl non-heme iron complexes with diethyl-dithiocarbamate (DETC) took place in mouse liver when inflammation process was initiated in mice by the lipopolysaccharide isolated from Salmonella typhimurium bacterium wall DETC intraperitoneally injected bound with endogenous non-heme iron resulted with DETC-Fe complex formation. These complexes were as a traps of nitric oxide appeared in animal tissues, and NO-Fe-DETC complexes were observed. Phenazone known as a free radical process inhibitor lowered NO production in animal organism. The free radical processes were suggested to intensify under inflammation reactions and to cause the various amino groups oxidation to nitroso groups which were capable to release free nitric oxide.     

Dermal application of nitric oxide releasing acidified nitrite-containing liniments significantly reduces blood pressure in humans

Vascular ischemic diseases, hypertension, and other systemic hemodynamic and vascular disorders may be the result of impaired bioavailability of nitric oxide (NO). NO but also its active derivates like nitrite or nitroso compounds are important effector and signal molecules with vasodilating properties. Our previous findings point to a therapeutical potential of cutaneous administration of NO in the treatment of systemic hemodynamic disorders. Unfortunately, no reliable data are available on the mechanisms, kinetics and biological responses of dermal application of nitric oxide in humans in vivo. The aim of the study was to close this gap and to explore the therapeutical potential of dermal nitric oxide application. We characterized with human skin in vitro and in vivo the capacity of NO, applied in a NO-releasing acidified form of nitrite-containing liniments, to penetrate the epidermis and to influence local as well as systemic hemodynamic parameters. We found that dermal application of NO led to a very rapid and significant transepidermal translocation of NO into the underlying tissue. Depending on the size of treated skin area, this translocation manifests itself through a significant systemic increase of the NO derivates nitrite and nitroso compounds, respectively. In parallel, this translocation was accompanied by an increased systemic vasodilatation and blood flow as well as reduced blood pressure. We here give evidence that in humans dermal application of NO has a therapeutic potential for systemic hemodynamic disorders that might arise from local or systemic insufficient availability of NO or its bio-active NO derivates, respectively.     

Buried S-nitrosocysteine revealed in crystal structures of human thioredoxin

We have determined the 1.65 A crystal structure of human thioredoxin-1 after treatment with S-nitrosoglutathione, providing a high-resolution view of this important protein modification and mechanistic insight into protein transnitrosation. Thioredoxin-1 appears to play an intermediary role in cellular S-nitrosylation and is important in numerous biological and pathobiological activities. S-Nitroso modifications of cysteines 62 and 69 are clearly visible in the structure and display planar cis geometries, whereas cysteines 32, 35, and 73 form intra- and intermolecular disulfide bonds. Surprisingly, the Cys 62 nitroso group is completely buried and pointing to the protein interior yet is the most readily formed at neutral pH. The Cys 69 nitroso group is also protected but requires a higher pH for stable formation. The helix intervening between residues 62 and 69 shifts by approximately 0.5 A to accommodate the SNO groups. The crystallographic asymmetric unit contains three independent molecules of thioredoxin, providing three views of the nitrosated protein. The three molecules are in general agreement but display subtle differences, including both cis and trans conformers for Cys 69 SNO in molecule C, and greater disorder in the Cys 62-Cys 69 helix in molecule B. Possible mechanisms for protein transnitrosation with specific geometric requirements and charge stabilization of the nitroxyl disulfide reaction intermediate are discussed.     

Spin trapping of nitrogen dioxide and of radicals generated from nitrous acid

Spin trapping of nitrogen dioxide radical by several nitrones has been studied. The reaction results in the formation of persistent acyl nitroxides, after the oxidation of the intermediate spin adducts having an -ONO group on C-2 atom. The intermediate is effectively detected when DEPMPO is used as the spin trap. The reaction between PBN or 5,7-di-tert-butyl-3,3-dimethyl indoline N-oxide with nitrous acid gives the corresponding acyl nitroxide only when oxygen is present in the reaction milieu.

On the other hand, nitroso spin traps do not trap ^⋅NO₂ confirming that the unpaired electron of nitrogen dioxide is localized on the oxygen atom.     

Diet-induced endogenous formation of nitroso compounds in the GI tract

Red or processed meat, but not white meat or fish, is associated with colorectal cancer. The endogenous formation of nitroso compounds is a possible explanation, as red or processed meat--but not white meat or fish--causes a dose-dependent increase in fecal apparent total N-nitroso compounds (ATNC) and the formation of nitroso-compound-specific DNA adducts. Red meat is particularly rich in heme and heme has also been found to promote the formation of ATNC. To investigate the underlying mechanism of ATNC formation, fecal and ileal samples of volunteers fed a high red meat or a vegetarian diet were analyzed for nitrosyl iron, nitrosothiols, and heme. To simulate the processes in the stomach, food homogenates and hemoglobin were incubated under simulated gastric conditions. Nitrosyl iron and nitrosothiols were significantly (p < 0.0001) increased in ileal and fecal samples after a high red meat diet compared with a vegetarian diet; significantly more nitrosyl iron than nitrosothiols was detectable in ileal (p < 0.0001) and fecal (p < 0.001) samples. The strong correlation between fecal nitrosyl iron and heme (0.776; p < 0.0001) suggested that nitrosyl heme is the main source of nitrosyl iron, and ESR confirmed the presence of nitrosyl heme in fecal samples after a high red meat diet. Under simulated gastric conditions, mainly nitrosothiols were formed, suggesting that acid-catalyzed thionitrosation is the initial step in the endogenous formation of nitroso compounds. Nitrosyl heme and other nitroso compounds can then form under the alkaline and reductive conditions of the small and large bowel.     

Features of the metabolism of nitric oxide in normal state and inflammation

Brief analysis of the metabolism of nitric oxide in living cells in normal state and pathology and also the analysis of the causes that hampered the progress of these studies were carried out. It was established that most of physiological fluids, including blood, normally contain nitrite and non-thiolate nitroso compounds in concentration less than 100 nM. Literature data from different researchers on the normal range of nitrite concentration in plasma of healthy people from several hundreds of nM to several μM is probably the result of low selectivity of the methods used. But nitrite and non-thiolate nitroso compounds concentration in blood is dramatically increased in case of inflammatory diseases. It is proposed that the main mechanism for the production of these substances in blood is the nitrosyl iron complexes transformation by active oxygen species but not the activation of NO production as it was considered previously.     

Isoniazid: Radical-induced oxidation and reduction chemistry

Isoniazid is a potent and selective therapeutic prodrug agent used to treat infections by Mycobacterium tuberculosis. Although it has been used clinically for over five decades its full mechanism of action is still being elucidated. Essential to its mechanism of action is the activation of isoniazid to a reactive intermediate, the isonicotinyl acyl radical, by the catalase-peroxidase KatG. The isonicotinyl acyl radical then reacts with NAD producing an inhibitor of the NADH-dependent enoyl ACP reductase responsible for mycolic acid synthesis as its primary target. However, the initial oxidation of isoniazid by KatG has also revealed alternative reaction pathways leading to an array of carbon-, oxygen-, and nitrogen-centered radical intermediates. It has also been reported that isoniazid produces nitric oxide in the presence of KatG and hydrogen peroxide. In this study, the temperature-dependent rate constants for the hydroxyl radical oxidation and solvated electron reduction of isoniazid and two model compounds have been studied. Based on these data the initial oxidation of isoniazid by the hydroxyl radical has been shown to predominantly occur at the primary nitrogen of the hydrazyl moiety, consistent with the postulated mechanism for the formation of the isonicotinyl radical. The hydrated electron reduction occurred mostly at the pyridine ring. Concomitant EPR spin-trap measurements under a variety of oxidizing and reducing conditions did not show any evidence of nitric oxide production as had been previously reported. Finally, examination of the transient absorption spectra obtained for hydrated electron reaction with isoniazid demonstrated for the first time an initial reduced transient identified as the isonicotinyl acyl radical produced from isoniazid.     

Detection of nitrous oxide in the neuronal nitric oxide synthase reaction by gas chromatography-mass spectrometry

Using headspace gas chromatography-mass spectrometry, we detected significant amounts of nitrous oxide in the reaction products of the monooxygenase reaction catalyzed by neuronal nitric oxide synthase. Nitrous oxide is a dimerization product of nitroxyl anion; its presence in the reaction products indicates that the nitroxyl anion is a product of the neuronal nitric oxide synthase-catalyzed reaction.     

Helicobacter pylori does not mediate the formation of carcinogenic N-nitrosamines

Background. Both N‐nitroso compounds and colonization with Helicobacter pylori represent known risk‐factors for the development of gastric cancer. Endogenous formation of N‐nitroso compounds is thought to occur predominantly in acidic environments such as the stomach. At neutral pH, bacteria can catalyze the formation of N‐nitroso compounds. Based on experiments with a noncarcinogenic N‐nitroso compound as end product, and using only a single H. pylori strain, it was recently reported that H. pylori only displays a low nitrosation capacity. As H. pylori is a highly diverse bacterial species, it is reasonable to question the generality of this finding. In this study, several genetically distinct H. pylori strains are tested for their capacity to form carcinogenic N‐nitrosamines. Materials and Methods. Bacteria were grown in the presence of 0–1000 µM morpholine and nitrite (in a 1 : 1 molar ratio), at pH 7, 5 and 3. Results. Incubation of Neisseria cinerea (positive control) with 500 µM morpholine and 500 µM nitrite, resulted in a significant increase in formation of N‐nitrosomorpholine, but there was no significant induction of N‐nitrosomorpholine formation by any of the H. pylori strains, at any of the three pH conditions. Conclusion. H. pylori does not induce formation of the carcinogenic N‐nitrosomorpholine in vitro. The previously reported weak nitrosation capacity of H. pylori is not sufficient to nitrosate the more difficultly nitrosatable morpholine. This probably also holds true for other secondary amines. These results imply that the increased incidence of gastric cancer formation that is associated with gastric colonization by H. pylori is unlikely to result from the direct induced formation of carcinogenic nitrosamines by H. pylori. However, this has to be further confirmed in in vivo studies.     

The pathway of nitrogen and reductive enzymes of denitrification

Some recent studies on the pathway of nitrogen and the reductases of denitrification are reviewed. The available evidence suggests that while the intermediates of denitrification can remain enzyme-bound (presumably to nitrite reductase) prior to formation of N₂O, NO and nitroxyl (HNO) can be released in part by certain bacteria. Release of NO is recognized by a nitrite/NO?¹⁵N exchange reaction and isotopic scrambling in product N₂O; release of nitroxyl by Pseudomonas stutzeri is recognized by isotopic scrambling of nitrite and NO in product N₂O in absence of exchange and affords evidence that the first N?N bond forms in denitrification at the N¹⁺ redox level. The recent purification and partial characterization of nitrous oxide reductase are described. The ability of the dissimilatory nitrite reductase to activate nitrite for nitrosyl transfer affords a new chemical probe into the mechanism of action of this central enzyme. It would appear that reduction of nitrite is subject to electrophilic catalysis. ¹⁸O studies show that dissociation of nitrite from nitrite reductase can be slow relative to competing reduction or nitrosyl transfer.     

Ingress and reactive chemistry of nitroxyl-derived species within human cells

The mechanisms that control the biological signaling and toxicological properties of the nitrogen oxide species nitroxyl (HNO) are largely unknown. The ingress and intracellular reactivity of nitroxyl-derived species were examined using Angeli's salt (AS), which decomposes initially to HNO and nitrite at physiologic pH. Exposure of 4,5-diaminofluorescein (DAF) to AS resulted in fluorescent product formation only in the presence of molecular oxygen. Kinetic analysis and the lack of signal from a nitric oxide (NO)-sensitive electrode suggested that these processes did not involve conversion of HNO to NO. On an equimolar basis, bolus peroxynitrite (ONOO(-)) exposure generated only 15% of fluorescent product formation observed from AS decomposition. Moreover, infusion of synthetic ONOO(-) at a rate comparable to AS decomposition resulted in only 4% of the signal. Quenching of AS-mediated product formation within intact human MCF-7 breast carcinoma cells containing DAF by addition of urate to buffer suggested involvement of an oxidized intermediate formed from reaction between HNO and oxygen. Conversely, intact cells competitively sequestered the HNO-derived species from reaction with DAF in solution. These data show this intermediate to be a long-lived diffusible species. Relative product yield from intracellular DAF was decreased 5- to 8-fold when cells were lysed immediately prior to AS addition, consistent with the partitioning of HNO and/or derived species into the cellular membrane, thereby shielding these reactive intermediates from either hydrolysis or cytoplasmic scavenger pools. These findings establish that oxygen-derived species of nitroxyl can readily penetrate and engage the intracellular milieu of cells and suggest this process to be independent of NO and ONOO(-) intermediacy. The substantial facilitation of oxygen-dependent nitroxyl chemistry by intact lipid bilayers supports a focusing role for the membrane in modulation of cellular constituents proteins by this unique species.