Hydroxyl Radical Adduct Research Articles

In situ growth of self-supported CuO nanorods from Cu-MOFs for glucose sensing and elucidation of the sensing mechanism.

Herein, we present a simple and mild method to in situ prepare CuO nanostructures for non-enzymatic glucose sensing. A Cu-metal organic framework (Cu-MOF) precursor was first directly grown on a pencil lead electrode with 3D graphene-like surfaces (EPLE) and then in situ transformed into CuO nanorods. The CuO nanorod-modified EPLE (CuO/EPLE) shows high sensitivity (1138.32 μA mM-1 cm-2), fast response time (1.5 s) and low detection limit (0.11 μM) for glucose oxidation. It has been found that NaOH promoted the generation of ˙OH groups and Cu(III) on the CuO surface, which then facilitated the electrochemical oxidation of glucose. Signals characteristic of hydroxyl and carbon-centered radical adducts were detected by EPR. Furthermore, the CuO/EPLE sensor also shows good accuracy in glucose determination in human serum samples.

Formation of free radicals in Chi-aroma Baijiu during aging process with fat pork

Soaking aged fat pork is a special aging process in the production of Chi-aroma Baijiu considered to involve the formation of free radicals. This study aimed to investigate the free radicals’ formation pathway in Chi-aroma Baijiu during aged fat pork soaking by using electron paramagnetic resonance (EPR) and spin trapping with 5,5-dimethyl-1-pyrrolin-n-oxide (DMPO). The alkyl radical adducts (DMPO-R) and hydroxyl radical adducts (DMPO-OH) were detected in Baijiu after soaking the fat pork for aging. During the preparation process of aged fat pork, alkoxy radicals adduct (DMPO-RO) were mainly detected since lipid oxidation. Oleic acid and linoleic acid, the two main unsaturated fatty acids in fat pork, produced alkoxy radicals in the oxidation process. The total amounts of spins in linoleic acid and oleic acid after 4-month oxidation treatment increased by 248.07 ± 26.65% and 34.17 ± 0.72% than 0-month. It indicated that the free radicals in aged Chi-aroma Baijiu were mainly derived from the two main unsaturated fatty acids in aged fat pork and linoleic acid had a stronger ability to produce free radicals than oleic acid. Alkoxy radicals (RO·) from fat pork reacted with ethanol in Baijiu to form alkyl radicals (R·). The peroxide bond of hydroperoxides from the oxidation of unsaturated fatty acid was cleaved to form hydroxyl radicals (·OH) that were transferred to Baijiu. The results provide theoretical guidance for the subsequent work of free radicals scavenging.

Spontaneous Water Radical Cation Oxidation at Double Bonds in Microdroplets.

Spontaneous oxidation of compounds containing diverse X=Y moieties (e.g., sulfonamides, ketones, esters, sulfones) occurs readily in organic-solvent microdroplets. This surprising phenomenon is proposed to be driven by the generation of an intermediate species [M+H2O]+·: a covalent adduct of water radical cation (H2O +· ) with the reactant molecule (M). The adduct is observed in the positive ion mass spectrum while its formation in the interfacial region of the microdroplet (i.e., at the air-droplet interface) is indicated by the strong dependence of the oxidation product formation on the spray distance (which reflects the droplet size and consequently the surface-to-volume ratio) and the solvent composition. Importantly, based on the screening of a ca. 21,000-compound library and the detailed consideration of six functional groups, the formation of a molecular adduct with the water radical cation is a significant route to ionization in positive ion mode electrospray, where it is favored in those compounds with X=Y moieties which lack basic groups. A set of model monofunctional systems was studied and in one case, benzyl benzoate, evidence was found for oxidation driven by hydroxyl radical adduct formation followed by protonation in addition to the dominant water radical cation addition process. Significant implications of molecular ionization by water radical cations for oxidation processes in atmospheric aerosols, analytical mass spectrometry and small-scale synthesis are noted.

Independent Generation and Reactivity of 2'-Deoxyguanosin-N1-yl Radical.

2'-Deoxyguanosin-N1-yl radical (dG(N1-H)•) is the thermodynamically favored one-electron oxidation product of 2'-deoxyguanosine (dG), the most readily oxidized native nucleoside. dG(N1-H)• is produced by the formal dehydration of a hydroxyl radical adduct of dG as well as by deprotonation of the corresponding radical cation. dG(N1-H)• were formed as a result of the indirect and direct effects of ionizing radiation, among other DNA damaging agents. dG(N1-H)• was generated photochemically (λmax = 350 nm) from an N-aryloxy-naphthalimide precursor (3). The quantum yield for photochemical conversion of 3 is ∼0.03 and decreases significantly in the presence O2, suggesting that bond scission occurs from a triplet excited state. dG is formed quantitatively in the presence of excess β-mercaptoethanol. In the absence of a reducing agent, dG(N1-H)• oxidizes 3, decreasing the dG yield to ∼50%. Addition of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) as a sacrificial reductant results in a quantitative yield of dG and two-electron oxidation products of 8-oxodGuo. N-Aryloxy-naphthalimide 3 is an efficient and high-yielding photochemical precursor of dG(N1-H)• that will facilitate mechanistic studies on the reactivity of this important reactive intermediate involved in DNA damage.

N-t-Butyl-α-aryl Nitrones as Potent Spin Traps: DFT Analysis of Electron Localization Function Topology, Local Selectivity, Reactivity and Solvent Effects

Density functional theory studies were performed to analyze the reactivity and selectivity of radical capture by N-t-butyl-α-aryl nitrones. Biologically relevant three important radicals viz. hydroxyl, methyl and hydroperoxyl were selected for the study. Topological analysis of the electron localization function (ELF) allows to classify these nitrones as zwitter-ionic type three atom components (TAC). Effects of electron withdrawing and electron donating C-aryl substituents on the electronic chemical potentials, global hardness, electrophilic and nucleophilic indices of nitrones were observed. Radical attack at the carbon atom was predicted by Merz-Kollman algorithm, which is in agreement with the experiments unlike the natural population analysis. Hydroxyl adducts were predicted to be more stable than methyl and hydroperoxyl adducts. cis-Adducts were more stable than the trans-, with the highest differences in stability noted for the methyl adducts. Relative energies of adducts was lowered in non-polar solvents and thus increase in stability was observed along the series from water to heptane.

Water molecules mediate zinc mobility in the bacterial zinc diffusion channel ZIPB

Regulated ion diffusion across biological membranes is vital for cell function. In a nanoscale ion channel, the active role of discrete water molecules in modulating hydrodynamic behaviors of individual ions is poorly understood because of the technical challenge of tracking water molecules through the channel. Here we report the results of a hydroxyl radical footprinting analysis of the zinc-selective channel ZIPB from the Gram-negative bacterium, Bordetella bronchiseptica Irradiating ZIPB by microsecond X-ray pulses activated water molecules to form covalent hydroxyl radical adducts at nearby residues, which were identified by bottom-up proteomics to detect residues that interact either with zinc or water in response to zinc binding. We found a series of residues exhibiting reciprocal changes in water accessibility attributed to alternating zinc and water binding. Mapping these residues to the previously reported crystal structure of ZIPB, we identified a water-reactive pathway that superimposed on a zinc translocation pathway consisting of two binuclear metal centers and an interim zinc-binding site. The cotranslocation of zinc and water suggested that pore-lining residues undergo a mode switch between zinc coordination and water binding to confer zinc mobility. The unprecedented details of water-mediated zinc transport identified here highlight an essential role of solvated waters in driving zinc coordination dynamics and transmembrane crossing.

Repair of Oxidizing Hydroxyl Adduct Radicals of DNA Bases by Hydroxyl‐trans‐Stilbenes via Single Electron Transfer

AbstractRepair of oxidizing hydroxyl adduct radicals of DNA bases (base‐OH‐adduct) by eleven hydroxyl‐trans‐stilbenes via single electron transfer under physiological conditions are investigated using density functional theory. For eleven hydroxyl‐trans‐stilbenes (PSOHs), their anions make all the contributions to repairing oxidizing base‐OH‐adduct radicals. Ten PSOHs show better beneficial antioxidant effect than trans‐resveratrol. 3, 3′, 4, 4′, 5, 5′‐Hexahydroxy‐trans‐stilbene (PSOHd) performs the best repair activity to oxidizing base‐OH‐adduct radicals, and 3′, 4′, 5, 5′‐tetrahydroxy‐trans‐stibene has the smallest pKa value. The rate constants for the reactions of PSOHd with oxidizing base‐OH‐adduct radicals are about 105∼109 dm3 mol−1 s−1. Main factors affecting the repair reactions are the vertical ionization potential, the pKa of PSOHs and the vertical electron affinity of oxidizing base‐OH‐adduct radicals. This study gives information on repairing oxidizing base‐OH‐adduct radicals by PSOHs in biological environment for the first time and finds more beneficial antioxidant effect polyphenol than trans‐resveratrol or piceatannol.

Enhanced degradation of sulfamethoxazole by Fe-Mn binary oxide synergetic mediated radical reactions.

In this study, a novel Fe-Mn binary oxide (FMBO), which combined the oxidation capability of iron and manganese oxides, was constructed to remove sulfamethoxazole (SMX) effectively using the simultaneous co-precipitation and oxidation methods, and the reaction products were probed by liquid chromatography-mass spectrometry (LC/MS). Particularly, FMBO-mediated transformation mechanisms of SMX were explored using radical scavengers and electron paramagnetic resonance (EPR). Results indicated that the best removal efficiency was obtained at a pH of 4.0, with the H2O2 of 6.0mmol/L and the FMBO dosage of 2.0g/L, giving 97.6% removal of 10mg/L SMX within 60min. More importantly, we found that the hydroxyl (•OH) radicals generated by FMBO through Fenton-like reaction were responsible for the SMX oxidation. EPR studies were confirmed that the peak intensities of hydroxyl adduct decreased remarkably with increasing pH values. Moreover, the four SMX degradation intermediate products were detected by LC/MS and a reaction pathway for the possible mineralization of SMX, with •OH radicals as the main oxidant, was proposed. These findings provide a novel insight into the removal of SMX by FMBO-mediated radical reactions in aquatic environments. Moreover, this research suggested that FMBO can act as an efficient catalyst to remove SMX in hospital wastewater.

Photocatalytic Performance of Titania Nanosheets Templated by Graphene Oxide

Abstract A facile process was developed to fabricate two dimensional titania nanosheets (t-NS) by using graphene oxide (GO) as template. In-situ growth of anatase TiO 2 on GO suspended in butanol was achieved by combining sol-gel and solvothermal process without further calcination. Stoichiometric amounts of acetic acid were added to introduce esterification with butanol to release sufficient water for the rate-controlled hydrolysis of titanium butoxide. The two-dimensional GO prevented the agglomeration of TiO 2 particles, and simultaneously established the interaction between two phases. For comparison, a following thermal treatment in air was applied to partially remove GO and increase the crystalline size of t-NS. TiO 2 -decorated samples showed better charge transfer efficiency and thus produced more photoinduced radicals and weaker photoluminescence. Electron paramagnetic resonance (EPR) spectra were utilized to in-situ identify the concentrations and types of photo-induced radicals by mixing various nitroxide spin traps with t-NS suspensions under ambient conditions. In aqueous suspension, hydroxyl radicals produced from illuminated t-NS and trapped by 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) immediately within 30 second. The following decay in the hydroxyl adducts of DMPO suggested the presence of strong reductants in the illuminated sample suspension. A sample with a higher production rate of spin adduct (DMPO-OH) or a higher reduction rate of DMPO-OH demonstrated a more efficient decoloration on methylene blue (MB). Spin trapping results indicated that the hydroxyl radicals were produced from both photoinduced holes and electrons. Hence, an optimum rate of hole titration by ethylenediaminetetraacetic acid was essential to efficiently reduce the recombination of charge carriers and consequently to produce more active radicals to decolor MB. The strong interaction between graphitic and titania structures, rather than the crystallite size of anatase, dominated the photoreduction capability of t-NS.

Electron Paramagnetic Resonance Spectroscopy Investigation of Radical Production by Gold Nanoparticles in Aqueous Solutions Under X-ray Irradiation.

Nanomaterials can enhance the effect of X-rays, but the mechanisms of enhancement can be complicated. Electron paramagnetic resonance (EPR) was used here to shed light on enhancement mechanisms by detecting the originally proposed physical enhancement of the effect of X-rays by as-made large gold nanoparticles. Specifically spin trap reagent 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO) was used to trap radicals produced in aqueous solutions under X-ray irradiation. Even though only BMPO hydroxyl adducts were detected at the time of EPR measurement, both hydroxyl and superoxide radicals were found to contribute to the enhancement. The measured total enhancement was 0.7-fold per weight percent (wp) of Au in water using unfiltered X-rays. The theoretically predicted physical enhancement is 0.49 fold per wp of gold in water. This information, together with scavenging experimental results and the fact that the G-values are close for both radicals, suggest that hydroxyl and superoxide radicals contributing almost equally to the total measured enhancement. Further, the enhancement was found to be linearly dependent on the amount of large gold nanoparticles in water and no additional radical was produced beyond the amount predicted by type 1 physical enhancement, indicating that hydroxyl or superoxide radicals were not produced via catalytic pathways.

Alleviation of glutamate mediated neuronal insult by piroxicam in rodent model of focal cerebral ischemia: a possible mechanism of GABA agonism.

Neurotransmitter imbalance is an inevitable outcome in cerebral ischemia that leads to neuronal death. In the present study, we evaluated the effects of piroxicam, a nonsteroidal anti-inflammatory drug (NSAID), on extracellular brain glutamate and γ-aminobutyric acid (GABA) release, survival time, and neuronal cell death. Transient focal cerebral ischemia in male Charles Foster rat led to neuronal infarction and compromised intrinsic antioxidant status. Thirty-minute preadministration of piroxicam (10 mg/kg b.w.) showed a significant (P < 0.01) reduction in cerebral infarct volume and potentiation of the intrinsic antioxidant status. High-performance liquid chromatography of brain cortex and striatum revealed changes in extracellular concentrations of neurotransmitters which were found to be 0.519 ± 0.44 pmole/mg (GABA); 1.18 ± 0.28 pmole/mg (glutamate), and 0.63 ± 0.21 pmole/mg (serotonin), respectively. Hydroxyl radical (·OH) adduct of salicylate in the frontal cortex and striatum in control, untreated, and treated groups was found to be 0.261 ± 0.06, 0.68 ± 0.52, and 0.401 ± 0.68 pmole/mg, respectively. After stroke, the extracellular level of glutamate in rat brain increases continuously as compared to that of control group. However, piroxicam administration in stroke rat significantly reduced (P < 0.05) elevated extracellular cerebral glutamate. This indicates that piroxicam attenuates extracellular glutamate release and also reduces neuronal cell death due to reduction in oxidative stress in cerebral ischemia. Our results also indicate a consequent increase of extracellular GABA in brain regions administered with piroxicam, which hints that piroxicam alleviates glutamate excitotoxicity possibly by GABA agonism.

Chemical repair activity of free radical scavenger edaravone: reduction reactions with dGMP hydroxyl radical adducts and suppression of base lesions and AP sites on irradiated plasmid DNA.

Reactions of edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) with deoxyguanosine monophosphate (dGMP) hydroxyl radical adducts were investigated by pulse radiolysis technique. Edaravone was found to reduce the dGMP hydroxyl radical adducts through electron transfer reactions. The rate constants of the reactions were greater than 4 × 108 dm3 mol−1 s−1 and similar to those of the reactions of ascorbic acid, which is a representative antioxidant. Yields of single-strand breaks, base lesions, and abasic sites produced in pUC18 plasmid DNA by gamma ray irradiation in the presence of low concentrations (10–1000 μmol dm−3) of edaravone were also quantified, and the chemical repair activity of edaravone was estimated by a method recently developed by the authors. By comparing suppression efficiencies to the induction of each DNA lesion, it was found that base lesions and abasic sites were suppressed by the chemical repair activity of edaravone, although the suppression of single-strand breaks was not very effective. This phenomenon was attributed to the chemical repair activity of edaravone toward base lesions and abasic sites. However, the chemical repair activity of edaravone for base lesions was lower than that of ascorbic acid.

Selective addition of secondary amines to C₆₀: formation of penta- and hexaamino[60]fullerenes.

Secondary amines are well-known to add to [60]fullerene to form the tetraamino epoxy adduct C60(O)(NR1R2)4 under both photolysis and thermal conditions in the presence of oxygen. We have now found that pentaamino hydroxyl adduct C60(OH)(NR1R2)5 and hexaamino adduct C60(NR1R2)6 can be formed as the major products in the dark in the presence of oxygen. Key steps of the reaction mechanism probably involve repeated oxygen oxidation of the radical ion pair between fullerene and amines.

Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps

Reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide (H2O2), have a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of their production. For measuring ROS production in cells, the ESR spin trapping technique using cyclic nitrones distinguishes itself from other methods by its specificity for superoxide and hydroxyl radical. However, several drawbacks, such as the low spin trapping rate and the spontaneous and cell-enhanced decomposition of the spin adducts to ESR-silent products, limit the application of this method to biological systems. Recently, new cyclic nitrones bearing a triphenylphosphonium (Mito-DIPPMPO) or a permethylated β-cyclodextrin moiety (CD-DIPPMPO) have been synthesized and their spin adducts demonstrated increased stability in buffer. In this study, a comparison of the spin trapping efficiency of these new compounds with commonly used cyclic nitrone spin traps, i.e., 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and analogs BMPO, DEPMPO, and DIPPMPO, was performed on RAW 264.7 macrophages stimulated with phorbol 12-myristate 13-acetate. Our results show that Mito-DIPPMPO and CD-DIPPMPO enable a higher detection of superoxide adduct, with a low (if any) amount of hydroxyl adduct. CD-DIPPMPO, especially, appears to be a superior spin trap for extracellular superoxide detection in living macrophages, allowing measurement of superoxide production in unstimulated cells for the first time. The main rationale put forward for this extreme sensitivity is that the extracellular localization of the spin trap prevents the reduction of the spin adducts by ascorbic acid and glutathione within cells.

Filter strip as a method of choice for apoplastic fluid extraction from maize roots

Apoplastic fluid was extracted from maize (Zea mays L.) roots using two procedures: collection from the surface of intact plant roots by filter paper strips (AF) or vacuum infiltration and/or centrifugation from excised root segments (AWF). The content of cytoplasmic marker (glucose-6-phosphate, G-6-P) and antioxidative components (enzymes, organic acids, phenolics, sugars, ROS) were compared in the extracts. The results obtained demonstrate that AF was completely free of G-6-P, as opposed to AWF where the cytoplasmic constituent was detected even at mildest centrifugation (200×g). Isoelectric focusing of POD and SOD shows the presence of cytoplasmic isoforms in AWF, and HPLC of sugars and phenolics a much more complex composition of AWF, due to cytoplasmic contamination. Organic acid composition differed in the two extracts, much higher concentrations of malic acid being registered in AF, while oxalic acid due to intracellular contamination being present only in AWF. EPR spectroscopy of DEPMPO spin trap in the extracts showed persistent generation of hydroxyl radical adduct in AF. The results obtained argue in favor of the filter strip method for the root apoplastic fluid extraction, avoiding the problems of cytoplasmic contamination and dilution and enabling concentration measurements in minute regions of the root.

Apelin-12 and its structural analog enhance antioxidant defense in experimental myocardial ischemia and reperfusion.

This study investigated the effects of peptide apelin-12 (H-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe-OH, A12) and its novel structural analog (H-(NαMe)Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Nle-Pro-Phe-OH, AI) on myocardial antioxidant enzyme activities, lipid peroxidation, and reactive oxygen species formation in ex vivo and in vivo models of myocardial ischemia/reperfusion (I/R) injury. Isolated working rat hearts were subjected to global ischemia and reperfusion. Infusion of 140 μM A12 or AI before global ischemia improved cardiac function recovery; increased the activity of Cu,Zn superoxide dismutase (Cu,Zn SOD), catalase (CAT), and glutathione peroxidase (GSH-Px); decreased malondialdehyde (MDA) content in reperfused heart; and reduced the formation of hydroxyl radical adduct of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide in the myocardial effluent during early reperfusion compared with these indices in control. Anesthetized open-chest rats were subjected to the left anterior descending coronary artery occlusion and coronary reperfusion. Peptide A12 or its analog AI was injected intravenously at the onset of reperfusion at a dose of 0.35 μmol/kg. Treatment with A12 or AI significantly limited infarct size and reduced the activity of lactate dehydrogenase and creatine kinase MB isoenzyme in blood plasma at the end of reperfusion compared with control. These effects were accompanied by complete recovery of Cu,Zn SOD, CAT, and GSH-Px activities; and decrease in MDA content in the area at risk by the end of reperfusion. The study concluded that C-terminal fragment of native peptide apelin-12 and its synthesized analog is involved in the upregulation of cardiac antioxidant defense systems and attenuation of lipid peroxidation in myocardial I/R injury.

Radical chemistry of glucosamine naphthalene acetic acid and naphthalene acetic acid: a pulse radiolysis study

Free radical‐induced oxidation reactions of glucosamine naphthalene acetic acid (GNaa) and naphthalene acetic acid (Naa) have been studied using pulse radiolysis. GNaa was synthesized by covalently attaching Naa on glucosamine. Hydroxyl adduct (from the reaction of hydroxyl radicals (●OH) at the naphthalene ring) was identified as the major transient intermediate (suggesting that the ●OH reaction is on the naphthalene ring) and is characterized by its absorption maxima of 340 and 400 nm. Both GNaa and Naa undergo similar reaction pattern. The bimolecular rate constants determined for the reactions are 4.8 × 109 and 8.9 × 109 dm3 mol−1 s−1 for GNaa and Naa respectively. The mechanism of reaction of ●OH with GNaa was further confirmed using steady‐state method. Radical cation of GNaa was detected as an intermediate during the reaction of sulfate radical (SO4●−) with GNaa (k2 = 4.52 × 109 dm3 mol−1 s−1). This radical cation transforms to a ●OH adduct at higher pH. The radical cation of GNaa is comparatively long lived, and a cyclic transition state by neighboring group participation accounts for its stability. The oxy radical anion (O●−) reacts with GNaa (k2 = 1.12 × 109 dm3 mol−1 s−1) mainly by one‐electron transfer mechanism. The reduction potential values of Naa and GNaa were determined using cyclic voltammetric technique, and these are 1.39 V versus NHE for Naa and 1.60 V versus NHE for GNaa. Copyright © 2014 John Wiley &amp; Sons, Ltd.

Optimization of photocatalytic performance of TiO2 coated glass microspheres using response surface methodology and the application for degradation of dimethyl phthalate

Hollow glass microspheres coated with photocatalytic TiO2 (HGM-TiO2), recently became commercially available and have the distinct advantages of easy separation and recovery after treatment. With this in mind, we determined the optimum conditions for hydroxyl radical generation from HGM-TiO2 photocatalysis using response surface methodology (RSM). The hydroxyl radical yield and its average generation rate are critical parameters for practical applications of TiO2 photocatalysis. In this study, terephthalic acid was used as a hydroxyl radical trap because of the selective formation of the readily detectable hydroxyl radical adduct, 2-hydroxy terephthalic acid. Three independent variables, including loading of HGM-TiO2, concentration of terephthalic acid and irradiation time, were investigated. The 3D response surface graphs of hydroxyl radical yield and average hydroxyl radical generation rate indicated that optimum conditions of loading of HGM-TiO2, concentration of terephthalate acid and irradiation time were 8.0 g/L, 4.0 mM, and 20 min, respectively. Under these optimized conditions, we measured the photocatalysis employing HGM-TiO2 for the remediation of dimethyl phthalate (DMP), as a representative compound for problematic phthalate acid esters. HGM-TiO2 photocatalysis leads to the rapid destruction of DMP and there is a linear correlation between the DMP destruction and hydroxyl radical production. The results of our study demonstrate RSM can be used to readily determine the optimal conditions for hydroxyl radical production and the subsequent treatment of target compounds may be correlated to the hydroxyl radical production during HGM-TiO2 photocatalysis.

Protection against peroxynitrite-induced DNA damage by mesalamine: implications for anti-inflammation and anti-cancer activity

Mesalamine (5-aminosalicylic acid, 5-ASA) is known to be the first-line medication for treatment of patients with ulcerative colitis. Studies have demonstrated that ulcerative colitis patients treated with 5-ASA have an overall decrease in the risk of developing colorectal carcinoma. However, the mechanisms underlying 5-ASA-mediated anti-inflammatory and anti-cancer effects are yet to be elucidated. Because peroxynitrite has been critically involved in inflammatory stress and carcinogenesis, this study was undertaken to investigate the effects of 5-ASA in peroxynitrite-induced DNA strand breaks, an important event leading to peroxynitrite-elicited cytotoxicity. Incubation of φX-174 plasmid DNA with the peroxynitrite generator 3-morpholinosydnonimine (SIN-1) led to the formation of both single- and double-stranded DNA breaks in a concentration-dependent manner. The presence of 5-ASA at 0.1 and 1.0 mM was found to significantly inhibit SIN-1-induced DNA strand breaks in a concentration-dependent manner. The consumption of oxygen induced by SIN-1 was found to not be affected by 5-ASA at 0.1-50 mM, indicating that 5-ASA at these concentrations is not involved in the auto-oxidation of SIN-1 to form peroxynitrite. It is observed that 5-ASA at 0.1-1 mM showed considerable inhibition of peroxynitrite-mediated luminol chemiluminescence in a dose-dependent fashion, suggesting that 5-ASA is able to directly scavenge the peroxynitrite. Electron paramagnetic resonance (EPR) spectroscopy in combination with spin-trapping experiments, using 5,5-dimethylpyrroline-N-oxide (DMPO) as spin trap resulting in the formation of DMPO-hydroxyl radical adduct from peroxynitrite, and 5-ASA only at higher concentration (1 mM) inhibited the hydroxyl radical adduct while shifting EPR spectra, indicating that 5-ASA at higher concentrations may generate a more stable free radical species rather than acting purely as a hydroxyl radical scavenger. Taken together, these studies demonstrate for the first time that 5-ASA can potently inhibit peroxynitrite-mediated DNA strand breakage, scavenge peroxynitrite, and affect peroxynitrite-mediated radical formation, which may be responsible, at least partially, for its anti-inflammatory and anti-cancer effects.

Method for assessing X-ray-induced hydroxyl radical-scavenging activity of biological compounds/materials

A method for correctly assessing hydroxyl radical scavenging activity of antioxidative chemicals and/or biological compounds/materials was proposed. This method can simultaneously assess two factors, i.e. hydroxyl radical-scavenging and 5,5-dimethyl-2-hydroxy-1-pyrrolidine-N-oxide (hydroxyl radical adduct of 5,5-dimethyl-1-pyrroline-N-oxide)-reducing ability, as antioxidative properties. In this paper, some biologically common hydrophilic molecules, cell culture media, and rat plasma were tested. X-ray-induced hydroxyl radical can be detected using the electron paramagnetic resonance spin trapping technique. Using X-ray irradiation of the reaction mixture as the hydroxyl radical source, the true hydroxyl radical-scavenging ability of the subjected antioxidant can be assessed. In addition, the method simultaneously measures the reduction of 5,5-dimethyl-2-hydroxy-1-pyrrolidine-N-oxide, to estimate the reducing ability of the antioxidant. Biological materials, such as sugars and proteins, could abolish hydroxyl radical at the biological concentration. Ascorbic acid showed reducing ability at the biological concentration. The simultaneous assessment of hydroxyl radical-scavenging and reducing ability of antioxidants can be an informative index for antioxidants.