Photocontrollable peroxynitrite generator based on N-methyl-N-nitrosoaminophenol for cellular application

We designed and synthesized a photocontrollable peroxynitrite (ONOO(-)) generator, P-NAP, which has N-methyl-N-nitrosoaminophenol structure with four methyl groups introduced onto the benzene ring to block reaction of the photodecomposition product with ONOO(-) and to lower the semiquinoneimine's redox potential. The semiquinoneimine intermediate generated by photoinduced release of nitric oxide (NO) reduces dissolved molecular oxygen to generate superoxide radical anion (O(2)(?-)), which reacts with NO to afford ONOO(-) under diffusion control (k = 6.7 × 10(9) M(-1) s(-1)). NO release from P-NAP under UV-A (330-380 nm) irradiation was confirmed by ESR spin trapping. Tyrosine nitration, characteristic of ONOO(-), was demonstrated by HPLC analysis of a photoirradiated aqueous solution of P-NAP and N-acetyl-l-tyrosine ethyl ester. ONOO(-) formation was confirmed with a ONOO(-)-specific fluorogenic probe, HKGreen-3, and compared with that from 3-(4-morpholinyl)sydnonimine hydrochloride (SIN-1), which is the most widely used ONOO(-) generator at present. The photoreaction of P-NAP was influenced by superoxide dismutase, indicating that generation of O(2)(?-) occurs before ONOO(-) formation. The quantum yield for formation of duroquinone, the main P-NAP photodecomposition product, was measured as 0.86 ± 0.07 at 334 nm with a potassium ferrioxalate actinometer. Generation of ONOO(-) from P-NAP in HCT-116 cells upon photoirradiation was successfully imaged with HKGreen-3A. This is the first example of a photocontrollable ONOO(-) donor applicable to cultured cells.     

A highly selective, cell-permeable fluorescent nanoprobe for ratiometric detection and imaging of peroxynitrite in living cells

Peroxynitrite (ONOO(-)) is a highly reactive species implicated in the pathology of numerous diseases and there is currently great interest in developing fluorescent probes that can selectively detect ONOO(-) in living cells. Herein, a polymeric micelle-based and cell-penetrating peptide-coated fluorescent nanoprobe that incorporates ONOO(-) indicator dye and reference dye for the ratiometric detection and imaging of ONOO(-) has been developed. The nanoprobe effectively avoids the influences from enzymatic reaction and high-concentration ·OH and ClO(-). The improved ONOO(-) selectivity of the nanoprobe is achieved by a delicate complementarity of properties between the nanomatrix and the embedded molecular probe (BzSe-Cy). This nanoprobe also has other attractive properties, such as good water solubility, photostability, biocompatibility, and near-infrared excitation and emission. Fluorescence imaging experiments by confocal microscopy show that this nanoprobe is capable of visualizing ONOO(-) produced in living cells and it exhibits very low toxicity and good membrane permeability. We anticipate that this technique will be a potential tool for the precise pathological understanding and diagnosis of ONOO(-)-related human diseases.     

Peroxynitrite generation from a NO-releasing nitrobenzene derivative in response to photoirradiation

Photocontrollable ONOO(-) generation from a nitrobenzene derivative was demonstrated. The designed compound released NO in response to photoirradiation, and the resulting semiquinone reduced molecular oxygen to generate O(2)˙(-); reaction of the two generated ONOO(-), as confirmed with an ONOO(-) fluorescent probe, HKGreen-3.     

GC-MS and HPLC methods for peroxynitrite (ONOO- and O15NOO-) analysis: a study on stability, decomposition to nitrite and nitrate, laboratory synthesis, and formation of peroxynitrite from S-nitrosoglutathione (GSNO) and KO2

Nitric oxide (˙NO) and superoxide (O(2)(-)˙) are ubiquitous in nature. Their reaction product peroxynitrite (ONOO(-)) and notably its conjugated peroxynitrous acid (ONOOH) are highly unstable in aqueous phase. ONOO(-)/ONOOH (referred to as peroxynitrite) isomerize and decompose to NO(3)(-), NO(2)(-) and O(2). Here, we report for the first time GC-MS and HPLC methods for the analysis of peroxynitrite in aqueous solution. For GC-MS analysis peroxynitrite in alkaline solution was derivatized to a pentafluorobenzyl derivative using pentafluorobenzyl bromide. O(15)NOO(-) was synthesized from H(2)O(2) and (15)NO(2)(-) and used as internal standard. HPLC analysis was performed on stationary phases consisting of Nucleosil? 100-5C(18)AB or Nucleodur? C(18) Gravity. The mobile phase consisted of a 10 mM aqueous solution of tetrabutylammonium hydrogen sulfate and had a pH value of 11.5. UV absorbance detection at 300 nm was used. HPLC allows simultaneous analysis of ONOO(-), NO(2)(-) and NO(3)(-). The GC-MS and HPLC methods were used to study stability, synthesis, formation from S-[(15)N]nitrosoglutathione (GS(15)NO) and KO(2), and isomerization/decomposition of peroxynitrite to NO(2)(-) and NO(3)(-) in aqueous buffer.     

Mechanism of nitrite formation by nitrate photolysis in aqueous solutions: the role of peroxynitrite, nitrogen dioxide, and hydroxyl radical

Photolysis of aqueous NO3(-) with lambda > or = 195 nm is known to induce the formation of NO2(-) and O2 as the only stable products. The mechanism of NO3- photolysis, however, is complex, and there is still uncertainty about the primary photoprocesses and subsequent reactions. This is, in part, due to photoisomerization of NO3(-) to ONOO(-) at lambda < 280 nm, followed by the formation of *OH and *NO2 through the decomposition of ONOOH (pKa = 6.5-6.8). Because of incomplete information concerning the mechanism of peroxynitrite (ONOOH/ONOO(-)) decomposition, previous studies were unable to account for all observations. In the present study aqueous nitrate solutions were photolyzed by monochromatic light in the range of 205-300 nm. It is shown that the main primary processes at this wavelength range are NO3(-) hv-->*NO2 + O*(-) (reaction 1) and NO3(-) hv--> ONOO(-) (reaction 2). Based on recent knowledge on the mechanisms of peroxynitrite decomposition and its reactions with reactive nitrogen and oxygen species, we determined Phi(1) and Phi(2) using different experimental approaches. Both quantum yields increase with decreasing the excitation wavelength, approaching Phi(1) = 0.13 and Phi(2) = 0.28 at 205 nm. It is also shown that the yield of nitrite increases with decreasing the excitation wavelength. The implications of these results on UV disinfection of drinking water are discussed.     

Carbonate interlayered hydrotalcites-enhanced peroxynitrous acid chemiluminescence for high selectivity sensing of ascorbic acid

In this study, Mg-Al-carbonate layered double hydroxides (denoted as Mg-Al-CO(3) LDHs) were found to catalyze the chemiluminescence (CL) emission from peroxynitrous acid (ONOOH). The enhanced CL signals resulted from the concentration of peroxynitrite (ONOO(-)) onto the LDHs surface by electrostatic attraction, meaning that ONOO(-) can interact with the intercalated carbonate easily and effectively. Moreover, ascorbic acid can react with ONOO(-), or its decomposition products (e.g., ˙OH and ˙NO(2)), resulting in a decrease in the CL intensity from the Mg-Al-CO(3) LDHs-catalyzed ONOOH reaction. Based on these findings, a sensitive, selective and rapid CL method was developed for the determination of ascorbic acid using Mg-Al-CO(3) LDHs-catalyzed ONOOH as a novel CL system. The CL intensity was proportional to the concentration of ascorbic acid in the range from 5.0 to 5000 nM. The detection limit (S/N = 3) was 0.5 nM and the relative standard deviation (RSD) for nine repeated measurements of 0.1 μM ascorbic acid was 2.6%. This method has been successfully applied to determine ascorbic acid in commercial liquid fruit juices with recoveries of 97-107%. This work is not only of importance for a better understanding of the unique properties of LDHs-catalyzed CL but also of great potential for extensive applications in many fields, such as luminescence devices, bioanalysis, and labeling probes.     

On-chip multi-electrochemical sensor array platform for simultaneous screening of nitric oxide and peroxynitrite

In this work we report on the design, microfabrication and analytical performances of a new electrochemical sensor array (ESA) which allows for the first time the simultaneous amperometric detection of nitric oxide (NO) and peroxynitrite (ONOO(-)), two biologically relevant molecules. The on-chip device includes individually addressable sets of gold ultramicroelectrodes (UMEs) of 50 μm diameter, Ag/AgCl reference electrode and gold counter electrode. The electrodes are separated into two groups; each has one reference electrode, one counter electrode and 110 UMEs specifically tailored to detect a specific analyte. The ESA is incorporated on a custom interface with a cell culture well and spring contact pins that can be easily interconnected to an external multichannel potentiostat. Each UME of the network dedicated to the detection of NO is electrochemically modified by electrodepositing thin layers of poly(eugenol) and poly(phenol). The detection of NO is performed amperometrically at 0.8 V vs. Ag/AgCl in phosphate buffer solution (PBS, pH = 7.4) and other buffers adapted to biological cell culture, using a NO-donor. The network of UMEs dedicated to the detection of ONOO(-) is used without further chemical modification of the surface and the uncoated gold electrodes operate at -0.1 V vs. Ag/AgCl to detect the reduction of ONOOH in PBS. The selectivity issue of both sensors against major biologically relevant interfering analytes is examined. Simultaneous detection of NO and ONOO(-) in PBS is also achieved.     

Lacidipine,a potential peroxynitrite scavenger: investigation of activity by liquid chromatography and mass spectrometry

Inflamed tissues are often characterised by the production of *NO and O(2)(-) radicals, which are known to react at an extremely fast rate to produce peroxynitrite (ONOO(-)). This highly oxidising entity reacts with protein-bound tyrosine to give 3-nitrotyrosine, which is considered a biochemical marker of peroxynitrite-induced damage. Lacidipine is a calcium antagonist indicated for the treatment of mild to moderate hypertension. In the present work, electrospray mass spectrometry with and without liquid chromatography was used to evaluate the capability of lacidipine and two other related molecules as ONOO(-) scavengers. This capability is compared with that associated with a number of commercial polyphenols described in the literature as efficient scavengers of this cytotoxic agent. The use of mass spectrometry provided rapid quantitative assessment of both the nitration and its reduction, and showed that lacidipine possesses a reasonable capability for reducing in vitro nitration of superoxide dismutase.     

The rate of homolysis of adducts of peroxynitrite to the C=O double bond.

Nucleophilic addition of the peroxynitrite anion, ONOO(-), to the two prototypical carbonyl compounds, acetaldehyde and acetone, was investigated in the pH interval 7.4-14. The process is initiated by fast equilibration between the reactants and the corresponding tetrahedral adduct anion, the equilibrium being strongly shifted to the reactant side. The adduct anion also undergoes fast protonation by water and added buffers. Consequently, the rate of the bimolecular reaction between ONOO(-) and the carbonyl is strongly dependent on the pH and on the concentration of the buffer. The pK(a) of the carbonyl-ONOO adduct was estimated to be approximately 11.8 and approximately 12.3 for acetone and acetaldehyde, respectively. It is shown that both the anionic and the neutral adducts suffer fast homolysis along the weak O-O bond to yield free alkoxyl and nitrogen dioxide radicals. The yield of free radicals was determined to be about 15% with both carbonyl compounds at low and high pH, while the remainder collapses to molecular products in the solvent cage. The rate constants for the homolysis of the adducts vary from ca. 3 x 10(5) to ca. 5 x 10(6) s(-1), suggesting that they cannot act as oxidants in biological systems. This small variation around a mean value of about 10(6) s(-1) suggests that the O-O bond in the adduct is rather insensitive to its protonation state and to the nature of its carbonyl precursor. An overall reaction scheme was proposed, and all the corresponding rate constants were evaluated. Finally, thermokinetic considerations were employed to argue that the formation of dioxirane as an intermediate in the reaction of ONOO(-) with acetone is an unlikely process.     

Delineation of the chemical pathways underlying nitric oxide-induced homologous recombination in mammalian cells

Inflammation is an important risk factor for cancer. During inflammation, macrophages secrete nitric oxide (NO*), which reacts with superoxide or oxygen to create ONOO- or N2O3, respectively. Although homologous recombination causes DNA sequence rearrangements that promote cancer, little was known about the ability of ONOO- and N2O3 to induce recombination in mammalian cells. Here, we show that ONOO- is a potent inducer of homologous recombination at an integrated direct repeat substrate, whereas N2O3 is relatively weakly recombinogenic. Furthermore, on a per lesion basis, ONOO(-)-induced oxidative base lesions and single-strand breaks are significantly more recombinogenic than N2O3-induced base deamination products, which did not induce detectable recombination between plasmids. Similar results were observed in mammalian cells from two different species. These results suggest that ONOO(-)-induced recombination may be an important mechanism underlying inflammation-induced cancer.     

Evaluation of the anti-oxidant properties of a SOD-mimic Mn-complex in activated macrophages

SOD-mimics are small complexes that reproduce the activity of superoxide dismutases, natural proteins that catalytically dismutate the superoxide anion. Activated macrophages, which produce ROS and RNS fluxes, constitute a relevant model to challenge antioxidant activity in a cellular context and were used to test a Mn-complex which was shown to efficiently alter the flow of O(2)(-), ONOO(-) and H(2)O(2).     

Carbon dioxide in the nitrosation of amine: catalyst or inhibitor?

Nitrosamines are a class of carcinogenic, mutagenic, and teratogenic compounds generally produced from the nitrosation of amine. This paper investigates the mechanism for the formation of nitrosodimethylamine (NDMA) from the nitrosation of dimethylamine (DMA) by four common nitrosating agents (NO(2)(-), ONOO(-), N(2)O(3), and ONCl) in the absence and presence of CO(2) using the DFT method. New insights are provided into the mechanism, emphasizing that the interactions of CO(2) with amine and nitrosating agents are both potentially important in influencing the role of CO(2) (catalyst or inhibitor). The role of CO(2) as catalyst or inhibitor mainly depends on the nitrosating agents involved. That is, CO(2) shows the catalytic effect when the weak nitrosating agent NO(2)(-) or ONOO(-) is involved, whereas it is an inhibitor in the nitrosation induced by the strong nitrosating agent N(2)O(3) or ONCl. To conclude, CO(2) serves as a "double-edged sword" in the nitrosation of amine. The findings will be helpful to expand our understanding of the pathophysiological and environmental significance of CO(2) and to develop efficient methods to prevent the formation of carcinogenic nitrosamines.     

A new pentacyclic triterpenoid glucoside from Prunus serrulata var. spontanea

A new triterpenoid, 2alpha,3alpha,24-trihydroxyurs-12-en-28-oic acid-28-O-beta-D-glucopyranosyl ester (4) along with four known triterpenoids, ursolic acid (1), 2alpha-hydroxyursolic acid (2), 2alpha,3alpha,24-trihydroxyurs-12-en-28-oic acid (3), and 2alpha,3alpha,19alpha,24-tetrahydroxyurs-12-en-28-oic acid-28-O-beta-D-glucopyranosyl ester (5), were isolated from the leaves of Prunus serrulata var. spontanea (Rosaceae). Compounds 3-5 showed ONOO(-) scavenging activity, whereas compounds 1 and 2 were virtually inactive.     

Isolation of tetraprenyltoluquinols from the brown alga Sargassum thunbergii

Thunbergols A (4) and B (5), tetraprenyltoluquinols, along with three known compounds (1-3) have been isolated from the brown alga Sargassum thunbergii. The structures of these two new compounds were determined to be 9-(3,4-dihydro-2,8-dimethyl-6-hydroxy-2H-1-benzopyran-2-yl)-6-methyl-2-(4-methyl-3-pentenyl)-(2E,6E)-nonadienoic acid (4) and 10-(2,3-dihydro-5-hydroxy-7-methyl-1-benzofuran-2-yl)-10-hydroxy-6-methyl-2-(4-methyl-3-pentenyl)-(2E,6E)-undecadienoic acid (5), respectively, by combined spectroscopic methods. Both of them exhibited significant scavenging activities on radical and potently inhibited generation of ONOO(-) from morpholinosydnonimine (SIN-1).     

Fluorimetric determination of peroxynitrite based on an enzymatic reaction.

A novel fluorimetric method for the determination of peroxynitrite (ONOO-) using hemoglobin (Hb) as a catalyst is described. The method employs the reaction of ONOO with thiamine (TM), a colorless, non-fluorescent reagent in a glycine-NaCl-NaOH buffer solution (pH 12.7), to generate a highly fluorescent product, thiochrome (TC). The fluorescent product was monitored by fluorimetry. A linear calibration graph was obtained over an ONOO- concentration range from 4.95 x 10(-7) mol L(-1) to 2.97 x 10(-5) mol L(-1), with a detection limit of 9.78 x 10(-9) mol L(-1) ONOO-. The relative standard deviation at an ONOO- concentration of 2.11 x 10(-6) mol L(-1) was 4.15% (n = 9).     

Study the oxidative injury of yeast cells by NADH autofluorescence

Autofluorescence has an advantage over the extrinsic fluorescence of an unperturbed environment during investigation, especially in complex system such as biological cells and tissues. NADH is an important fluorescent substance in living cells. The time courses of intracellular NADH autofluorescence in the process of yeast cells exposed to H(2)O(2) and ONOO(-) have been recorded in detail in this work. In the presence of different amounts of H(2)O(2) and ONOO(-), necrosis, apoptosis and reversible injury are initiated in yeast cells, which are confirmed by acridine orange/ethidum bromide and Annexin V/propidium iodide staining. It is found that intracellular NADH content increases momently in the beginning of the apoptotic process and then decreases continually till the cell dies. The most remarkable difference between the apoptotic and the necrotic process is that the NADH content in the latter case changes much more sharply. Further in the case of reversible injury, the time course of intracellular NADH content is completely different from the above two pathways of cell death. It just decreases to some degree firstly and then resumes to the original level. Based on the role of NADH in mitochondrial respiratory chain, the time course of intracellular NADH content is believed to have reflected the response of mitochondrial redox state to oxidative stress. Thus, it is found that the mitochondrial redox state changes differently in different pathways of oxidative injury in yeast cells.     

Separation and detection of peroxynitrite and its metabolites by capillary electrophoresis with UV detection

A method for the separation and direct detection of peroxynitrite (ONOO(-)) and two of its degradation products, nitrite (NO(2)(-)) and nitrate (NO(3)(-)), using capillary electrophoresis with ultraviolet detection is described. The separation parameters were optimized and included electrokinetic injection, a run buffer consisting of 25 mM K(2)HPO(4) 7.5 mM DTAB, pH 12, and a field strength of -323 V/cm. A diode array UV detector was employed in these studies as it allowed the determination of all three species simultaneously. Nitrate and nitrite provided the maximum response at 214 nm while peroxynitrite generated the best response at 302 nm. All three species could be detected at 214 nm, while simultaneous detection at 214 and 302 nm positively identified each peak.     

Protective effect of Castanea sativa and Quercus robur leaf extracts against oxygen and nitrogen reactive species

Topical natural antioxidants are a useful strategy for the prevention of photoaging and oxidative stress mediated skin diseases. In view of this underlying principle, the screening of natural plant extracts with scavenging activity for pro-oxidant reactive species is a primary requirement for the development of new topical antioxidant formulations. In the present study, an ethanol:water (7:3) extract from Castanea sativa leaves and a ethanol:water (2:3) extract from Quercus robur leaves were evaluated for their putative in vitro scavenging effects on reactive oxygen species (ROS) namely superoxide radical (O(2)(-)), hydroxyl radical (HO()), peroxyl radical (ROO()), hydrogen peroxide (H(2)O(2)) and singlet oxygen ((1)O(2)) as well as on reactive nitrogen species (RNS) namely nitric oxide (()NO) and peroxynitrite (ONOO(-)). The extracts presented a high potency to scavenge the tested reactive species, all the IC(50)s being found at the microg/mL level. IC(50)s (mean+/-SE) for the ROS O(2)(-),HO(),H(2)O(2) and (1)O(2) were 13.6+/-1.8; 216+/-4; 410+/-8; 12.3+/-0.7 microug/mL, respectively, for C. sativa, and 11.0+/-0.5; 285+/-22; 251+/-32; 7.90+/-0.56 microg/mL, respectively, for Q. robur. The ORAC values obtained for ROO() were 1.24+/-0.13 for C. sativa and 1.09+/-0.06 for Q. robur. The IC(50)s (mean+/-SE) for ()NO and ONOO(-) were 3.10+/-0.14 and 1.49+/-0.10 microg/mL, respectively, for C. sativa and 3.13+/-0.11 and 0.95+/-0.02 microg/mL, respectively, for Q. robur. The content of total phenolics for C. sativa and Q. robur were 284+/-9 and 346+/-4 mg of gallic acid equivalents (GAE)/g of lyophilized extract respectively. The observed effects might be of relevance considering the putative interest of these extracts as topical antioxidants.     

Mechanistic studies on the reaction between cob(II)alamin and peroxynitrite: evidence for a dual role for cob(II)alamin as a scavenger of peroxynitrous acid and nitrogen dioxide

Peroxynitrite/peroxynitrous acid (ONOO(-)/ONOOH; pK(a(ONOOH)) =6.8) is implicated in multiple chronic inflammatory and neurodegenerative diseases. Both mammalian B(12)-dependent enzymes are inactivated under oxidative stress conditions. We report studies on the kinetics of the reaction between peroxynitrite/peroxynitrous acid and a major intracellular vitamin B(12) form, cob(II)alamin (Cbl(II)), using stopped-flow spectroscopy. The pH dependence of the reaction is consistent with peroxynitrous acid reacting directly with Cbl(II) to give cob(III)alamin (Cbl(III)) and (.)NO(2) , followed by a subsequent rapid reaction between (.)NO(2) and a second molecule of Cbl(II) to primarily form nitrocobalamin. In support of this mechanism, a Cbl(II)/ONOO(H) stoichiometry of 2:1 is observed at pH 7.35 and 12.0. The final major Cbl(III) product observed (nitrocobalamin or hydroxycobalamin) depends on the solution pH. Analysis of the reaction products in the presence of tyrosine-a well-established (.)NO(2) scavenger-reveals that Cbl(II) reacts with (.)NO(2) at least an order of magnitude faster than tyrosine itself. Given that protein-bound Cbl is accessible to small molecules, it is likely that enzyme-bound and free intracellular Cbl(II) molecules are rapidly oxidized to inactive Cbl(III) upon exposure to peroxynitrite or (.)NO(2).     

Seminaphthofluorescein-based fluorescent probes for imaging nitric oxide in live cells

Fluorescent turn-on probes for nitric oxide based on seminaphthofluorescein scaffolds were prepared and spectroscopically characterized. The Cu(II) complexes of these fluorescent probes react with NO under anaerobic conditions to yield a 20-45-fold increase in integrated emission. The seminaphthofluorescein-based probes emit at longer wavelengths than the parent FL1 and FL2 fluorescein-based generations of NO probes, maintaining emission maxima between 550 and 625 nm. The emission profiles depend on the excitation wavelength; maximum fluorescence turn-on is achieved at excitations between 535 and 575 nm. The probes are highly selective for NO over other biologically relevant reactive nitrogen and oxygen species including NO(3)(-), NO(2)(-), HNO, ONOO(-), NO(2), OCl(-), and H(2)O(2). The seminaphthofluorescein-based probes can be used to visualize endogenously produced NO in live cells, as demonstrated using Raw 264.7 macrophages.