Nitrogen-centered Free Radicals Research Articles

Effect of Morphology and Concentration on Crossover between Antioxidant and Pro-oxidant Activity of MgO Nanostructures.

The toxicity of nanomaterials can sometimes be attributed to photogenerated reactive oxygen species (ROS), but these ROS can also be scavenged by nanomaterials, yielding opportunities for crossover between the properties. The morphology of nanomaterials also influences such features due to defect-induced properties. Here we report morphology-induced crossover between pro-oxidant activity (ROS generation) and antioxidant activity (ROS scavenging) of MgO. To study this process in detail, we prepared three different nanostructures of MgO (nanoparticles, nanoplates, and nanorods) and characterized them by HRTEM. These three nanostructures effectively generate superoxide anions (O2•-) and hydroxyl radicals (•OH) at higher concentrations (>500 μg/mL) but scavenge O2•- at lower concentrations (40 μg/mL) with successful crossover at 200 μg/mL. Nanorods of MgO generate the highest levels of O2•-, whereas nanoparticles scavenge O2•- to the highest extent (60%). Photoluminescence studies reveal that such crossover is based on the suppression of F2+ and the evolution of F+, F2+, and F23+ defect centers. The evolution of these defect centers reflects the antibacterial activity of MgO nanostructures which is initiated at 200 μg/mL against Gram-positive S. aureus ATCC 29737 and among different bacterial strains including Gram-positive B. subtilis ATCC 6633 and M. luteus ATCC 10240 and Gram-negative E. coli ATCC K88 and K. pneumoniae ATCC 10031. Nanoparticles exhibited the highest antibacterial (92%) and antibiofilm activity (17%) against B. subtilis ATCC 6633 in the dark. Interestingly, the nitrogen-centered free radical DPPH is scavenged (100%) by nanoplates due to its large surface area (342.2 m2/g) and the presence of the F2+ defect state. The concentration-dependent interaction with an antioxidant defense system (ascorbic acid (AA)) highlights nanoparticles as potent scavengers of O2•- in the dark. Thus, our findings establish guidelines for the selection of MgO nanostructures for diverse therapeutic applications.

Nanozymatic Antioxidant System Based on MoS2 Nanosheets

The enzymatic antioxidant system (EAS) protects aerobic cells from oxidative stress. However, it is brittle and susceptible of inactivation of reactive oxygen species (ROS) immoderate production. Here, we demonstrated that MoS2 nanosheets (few-layer MoS2), as a multifunctional nanozyme, possess intrinsic activity of mimicking enzymes of superoxide dismutases (SODs), catalases (CATs), and peroxidases (PODs) under physiological conditions (pH 7.4, 25 °C). Further, MoS2 nanosheets showed POD-like activity by transferring electrons instead of generating ROS. Similar to EAS, a defense termed nanozymatic antioxidant system (NAS) was developed by MoS2 nanosheets, for regulation of oxidative stress. Surprisingly, this NAS can effectively scavenge other free radicals including hydroxyl radicals (•OH), nitrogen-centered free radicals (•DPPH), and nitric oxide (•NO). To evaluate these unique properties of MoS2-based NAS in vivo, Escherichia coli ( E. coli), Staphylococcus aureus ( S. aureus), and A549 cell models were established, respectively. These results showed MoS2 nanosheets superiorly protect bacteria and cells against oxidative injury caused by H2O2. This work makes MoS2 nanosheets promising antioxidants in the pathological processes and expands their application in biocatalysis and nano-biomedicine.

Phenolic composition and prospective anti-infectious properties of Atriplex lindleyi

ObjectiveTo investigate the antiplasmodial, antimicrobial, radical scavenging effects and to identifiy the phenolic constituents of Atriplex lindleyi (A. lindleyi). MethodsA. lindleyi extracts and some isolated compounds were tested in vitro against the chloroquine-resistant strains of Plasmodium falciparum. The radical scavenging activity was quantified by using 2,2-diphenyl-2-picrylhydrazyl nitrogen-centered free radical. The IC50 of each extract was compared with references. The in vitro anti-infectious activity of extracts was evaluated against representative Gram-positive and Gram-negative bacterial strains [Staphylococcus aureus CIP 4.83, Enterococcus hirae CIP 5855, Pseudomonas aeruginosa (P. aeruginosa) CIP 82118, Escherichia coli CIP 53126], and fungal species [(Candida albicans (C. albicans) IP 48.72, Aspergillus niger IP 1431.83]. Ethanol extract was investigated for chemical composition through column and high performance liquid chromatography. The isolated compounds were identified by mass spectrometry and nuclear magnetic resonance. ResultsQuercetin-7-O-arabinopyranoside-3-O-neohesperidosides (1), quercetin-3-O-arabinopyranosyl(1→6) glucopyranoside (2), quercetin-3-O-glucopyranoside-7-O-rhamnopyranoside (3), quercetin-3-O-glucopyranoside-7-O-arabinoside (4), schaftoside (5), quercetin-7-O-glucopyranoside (6) were isolated for the first time from the ethanol extract of A. lindleyi aerial parts in addition to isorhamnetin-3-O-β-glucopyranoside (7) and quercetin (8). The extracts exhibited moderate antiplasmodial activity with IC50≈ranging from 10–50 μg/mL. Quercetin was the most potent compound with IC50 of 9 µg/mL. P. aeruginosa and C. albicans were the most susceptible organisms. ConclusionsThe study implies that A. lindelyi can contribute to the fight against malaria, and be useful as prophylactic against C. albicans and P. aeruginosa.

Synthesis and biological evaluation of primaquine–chloroquine twin drug: a novel heme-interacting molecule prevents free heme and hydroxyl radical-mediated protein degradation

Accumulations of oxidized and degraded proteins are the markers for oxidative stress. Degradation of hemoprotein e.g. hemoglobin during different pathological conditions produces heme, which induces oxidative stress and inflammation through its pro-oxidant nature and leads to protein degradation. Moreover reduced transition-metal ions Fe2+ can generate toxic hydroxyl radicals (˙OH) and leads to protein degradation. Therefore, synthesis of a compound that will detoxify free heme, chelate Fe+2 and show antioxidant activity by scavenging ˙OH would be beneficial against protein degradation. Here, we report the synthesis of a novel heme-interacting primaquine–chloroquine twin drug (PQCL) that could chelate free iron and showed excellent antioxidant activity as evident from ferric reducing antioxidant power. PQCL prevented ˙OH and heme-mediated protein degradation. PQCL also could scavenge nitrogen-centered free radical (2,2-diphenyl-1-picrylhydrazyl). Thus, we have synthesized PQCL, a heme-interacting molecule, which is capable to prevent free heme and ˙OH-mediated protein degradation.

New heteroaryl nitrones with spin trap properties: Identification of a 4-furoxanyl derivative with excellent properties to be used in biological systems

A new series of heteroaryl nitrones, 1– 7, bearing furoxanyl and thiadiazolyl moieties, were evaluated for their free radical-trapping properties. The physicochemical characterization by electron paramagnetic resonance (EPR) demonstrated its capability to trap and stabilize oxygen-, carbon-, sulfur-, and nitrogen-centered free radicals. The 4-furoxanyl nitrone 3 ( FxBN), α( Z)-(3-methylfuroxan-4-yl)- N- tert-butylnitrone, showed appropriate solubility in aqueous solution and taking into account that this physicochemical property is very important for biological applications, we studied it deeply in terms of its trapping and kinetic behaviors. For this, kinetic studies of the hydroxyl adduct decay gave rate constants k ST of 1.22 × 10 10 dm 3 mol −1 s −1 and half-live up to 7200 s at physiological pH, without any artifactual signals. The ability of FxBN to directly traps and stabilizes superoxide free radical, with a half-life of 1620 s at physiological pH, was also demonstrated. Besides, FxBN-hydroxyl and -superoxide adducts exhibited distinct and characteristic EPR spectral patterns. Finally, we confirmed the ability of FxBN to act as spin trap in a specific biological system, that is, in the free radical production of experimental anti-trypanosomatid drugs using Trypanosoma cruzi microsomes as biological system. Moreover, previous observations of low FxBN toxicity transform it in a good candidate for in vivo spin trapping.

Synergistic cytotoxic effect of tetrachlorocatechol and sodium azide in Escherichia coli: Toxicity, metabolism, and mechanistic aspects

Pentachlorophenol (PCP) is used in industrial and domestic applications, including as a biocide and a wood preservative. Metabolism of PCP undergoes oxidative dechlorination, forming tetrachlorocatechol (TCC) and tetrachlorohydroquinone (TCHQ). Both sodium azide (NaN(3)) and TCC appear naturally in soil. None of them are cytotoxic by themselves or facilitate autooxidation. Here, we show that their combination leads to synergistic cytotoxicity (>6 log bacterial killing) to Escherichia coli. The rate of oxygen consumption in a cell-free system showed that NaN(3) increases TCC oxidation by 520-fold. The synergism coefficient to cells was calculated as 96 or greater, and we have shown the formation of a new compound. It is suggested that the intermediate species, o-tetrachlorosemiquinine, and an unknown, nitrogen-centered free radical, both visualized by electron-spin resonance, are harmful species responsible for the synergistic cytotoxicity of TCC/NaN(3), rather than the endproduct formed during the reaction. Desferrioxamine and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide offered nearly complete protection, but through radical scavenging rather than through chelating properties. The mechanism of damage for TCC compared to its analogue, TCHQ, were investigated, and whereas the cellular damage of TCHQ/NaN(3) is through a site-specific mechanism, in the case of TCC/NaN(3) it is through the accumulation of the component(s) in the bacterial cell membrane, eventually leading to dysfunction, as evidenced by electron microscopy.

Identification of a Novel Glutathione Conjugate of Flutamide in Incubations with Human Liver Microsomes

Flutamide, a nonsteroidal antiandrogen drug widely used in the treatment of prostate cancer, has been associated with rare incidences of hepatotoxicity in patients. It is believed that bioactivation of flutamide and subsequent covalent binding to cellular proteins is responsible for its toxicity. Current in vitro studies were undertaken to probe the cytochrome P450 (P450)-mediated bioactivation of flutamide and identify the possible reactive species using reduced glutathione (GSH) as a trapping agent. NADPH- and GSH-supplemented human liver microsomal incubations of flutamide gave rise to a novel GSH conjugate where GSH moiety was conjugated to the flutamide molecule via the amide nitrogen, resulting in a sulfenamide. The structure of the conjugate was characterized by liquid chromatography-tandem mass spectrometry and NMR experiments. The conjugate formation was primarily catalyzed by heterologously expressed CYP2C19, CYP1A2, and, to a lesser extent, CYP3A4 and CYP3A5. The mechanism for the formation of this conjugate is unknown; however, a tentative bioactivation mechanism involving a P450-catalyzed abstraction of hydrogen atom from the amide nitrogen of flutamide and the subsequent trapping of the nitrogen-centered radical by GSH or oxidized glutathione (GSSG) was proposed. Interestingly, the same adduct was formed when flutamide was incubated with human liver microsomes in the presence of GSSG and NADPH. This finding suggests that P450-mediated oxidation of flutamide via a nitrogen-centered free radical could be one of the several bioactivation pathways of flutamide. Even though the relationship of the GSH conjugate to flutamide-induced toxicity is unknown, the results have revealed the formation of a novel, hitherto unknown, GSH adduct of flutamide.

Prostaglandin H synthase‐catalyzed bioactivation of amphetamines to free radical intermediates that cause CNS regional DNA oxidation and nerve terminal degeneration 1

Reactive oxygen species (ROS) are implicated in amphetamine-initiated neurodegeneration, but the mechanism is unclear. Here, we show that amphetamines are bioactivated by CNS prostaglandin H synthase (PHS) to free radical intermediates that cause ROS formation and neurodegenerative oxidative DNA damage. In vitro incubations of purified PHS-1 with 3,4-methylenedioxyamphetamine (MDA) and methamphetamine (METH) demonstrated PHS-catalyzed time- and concentration-dependent formation of an amphetamine carbon- and/or nitrogen-centered free radical intermediate, and stereoselective oxidative DNA damage, evidenced by 8-oxo-2'-deoxyguanosine (8-oxo-dG) formation. Similarly in vivo, MDA and METH caused dose- and time-dependent DNA oxidation in multiple brain regions, remarkably dependent on the regional PHS levels, including the striatum and substantia nigra, wherein neurodegeneration of dopaminergic nerve terminals was evidenced by decreased immunohistochemical staining of tyrosine hydroxylase. Motor impairment using the rotarod test was evident within 3 wk after the last drug dose, and persisted for at least 6 months. Pretreatment with the PHS inhibitor acetylsalicylic acid blocked MDA-initiated DNA oxidation and protected against functional motor impairment for at least 1.5 months after drug treatment. This is the first direct evidence for PHS-catalyzed bioactivation of amphetamines causing temporal and regional differences in CNS oxidative DNA damage directly related to structural and functional neurodegenerative consequences.

DNA Cleavage by Good’s Buffers in the Presence of Au(III)

Abstract DNA (pBluescript plasmid, 2.96 kbp) cleavage by Good’s buffers, which are extensively used in laboratories of chemistry, biochemistry, or biology, was studied by gel electrophoresis, CD spectra, ESR spectra, and CV in the presence of Au(III). Incubation of plasmid DNA with Good’s buffers and Au(III) resulted in DNA cleavage. DNA cleavage occurred due to the formation of nitrogen-centered cationic free radicals from the Good’s buffers in the presence of Au(III). The formation of the nitrogen-centered free radicals was confirmed by ESR spectroscopy. Gel electrophoresis results indicated that Form I and Form II were converted into Form III and a DNA fragment. The molecular weight of the DNA fragment was estimated ca. 1.47 kbp by DNA marker gel electrophoresis. ESR spectra were not observed from the Good’s buffers in the presence of other metal ions, such as Mn(II), Fe(III), Co(II), Ni(II), Zn(II), Pd(II), Cd(II), Hg(II), and Pb(II), resulting in no DNA cleavage. Gel electrophoresis results indicated a partial DNA cleavage during the incubation of DNA in the presence of Good’s buffers and Au(III). This is because the DNA cleavage reaction stopped at 15 min, since the radical lifetime is ca. 15 min. The intensities of the CD spectra at 270 nm decreased with time by 15 min upon the addition of Au(III), and a constant CD spectrum was observed after 15 min. Further addition of Au(III) showed an alternation of the CD spectra from positive ellipticity to negative ellipticity at 270 nm, suggesting significant DNA cleavage. The mechanism of DNA cleavage is discussed based on the gel electrophoresis, ESR, CD, and CV results.

Determination of the Free Radical Scavenging Activity of Lycium Extracts

Lycium species growing in Turkey have not so far been studied sufficiently. For this reason, non-polar and polar extracts obtained from the fruits of Lycium barbarum L. and L. Ruthenicum Murray (Solanaceae) were assessed both in vitro for their potential as free radical scavenger crude extracts and their phenolic composition. Fruits of Lycium species were sequentially extracted with petroleum ether, ethyl acetate, methanol, n-butanol, and water in a Soxhlet extractor. All the extracts were assessed for the scavenging of the nitrogen-centered free radical 1,1′-diphenyl-2-picrylhydrazyl (DPPH.) by in vitro method. Furthermore, the composition of each extract was investigated both in terms of its Folin-Ciocalteau reactive components and its qualitative content. The phenolic compounds within the extracts were determined as benzoic acid and hydroxycinnamic acid derivatives, flavonoids, and anthocyanins according to their retention time and UV spectral data by HPLC-DAD system.

Reduction of Genotoxic Effects of the Carcinogen N-Methyl-N'-Nitro-N-Nitrosoguanidine by Dietary Lignin in Mammalian Cells Cultured In Vitro

In the present study the protective effect of several lignin polymers against the genotoxic effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was tested in hamster lung V79 cells and human colon Caco-2 cells. Preculturing of cells with sublethal, nongenotoxic concentrations of the lignins A, B, and C (50 mg/ml) was found to decrease significantly the level of DNA strand breaks in both hamster and human cells treated with MNNG. Lignin A also reduced MNNG-induced gene mutations in V79 cells. As in addition to alkyl lesions MNNG gives rise to hydroxyl free radicals (�OH) and nitrogen-centered free radicals (�NR), we tried to determine whether antimutagenicity of lignin A was connected only with the well-known ability of lignin to bind MNNG molecules or also with its antioxidative effects. The use of the modified comet assay technique proved that preculturing of hamster V79 cells with lignin A resulted in a significant decrease of the level of DNA strand breaks originating from oxidized DNA bases. Therefore, we suggest that the antimutagenic effect of lignin A against MNNG is associated with both adsorptive and antioxidative action. This study also showed that the presence of lignin A neither helped to renew DNA replication nor influenced the kinetics of DNA rejoining in MNNG-treated V79 cells.

Development of novel antioxidants: design, synthesis, and reactivity.

We are attempting to develop novel synthetic antioxidants aimed at retarding the effects of free-radical induced cell damage. In this paper we discuss the design strategy and report the synthesis of seven novel antioxidants, including six catechols and a benzylic phenol. The bond dissociation enthalpy (BDE) for the most active (weakest) OH bond in each molecule was calculated by theoretical methods, as well as the BDE for the semiquinone radical. Reaction rates with the nitrogen-centered free radical DPPH(*) were measured in ethyl acetate. The log of k(DPPH) for bimolecular reaction correlated well with the primary BDE. The correlation between rate constants and calculated BDEs shows that the BDE is a good predictor of antioxidant activity with DPPH(*), suggesting that our design criteria are useful and that these compounds should undergo further testing in cell cultures and in animal models.

Poly(adenosine 5'-diphosphate) ribose polymerase activation as a cause of metabolic dysfunction in critical illness.

Poly(adenosine 5'-diphosphate) ribose polymerase is a nuclear enzyme activated in response to genotoxic stress induced by a variety of DNA damaging agents. Several oxygen and nitrogen-centered free radicals, notably peroxynitrite, are strong inducers of DNA damage and poly(adenosine 5'-diphosphate) ribose polymerase activation in vitro and in vivo. Activation of this nuclear enzyme depletes the intracellular stores of its substrate nicotinamide adenine dinucleotide, slowing the rate of glycolysis, mitochondrial electron transport and adenosine triphosphate formation. This process triggers a severe energetic crisis within the cell, leading to acute cell dysfunction and cell necrosis. Poly(adenosine 5'-diphosphate) ribose polymerase also plays an important role in the regulation of inflammatory cascades, through a functional association with various transcription factors and transcription co-activators. Recent works identified this enzyme as a critical mediator of cellular metabolic dysfunction, inflammatory injury, and organ damage in conditions associated with overwhelming oxidative stress, including systemic inflammation, circulatory shock, and ischemia-reperfusion. Accordingly, pharmacological inhibitors of poly(adenosine 5'-diphosphate) ribose polymerase protect against cell death and tissue injury in such conditions, and may therefore represent novel therapeutic tools to limit multiple organ damage and dysfunction in critically ill patients.

The critical role of reactive nitrogen species in lung ischemia reperfusion injury

Objectives: Peroxynitrite (ONOO-) is a nitrogen centered free radical released by nitric oxide production. Significant tissue concentrations have been detected in a variety of inflammatory conditions including oxidative stress. The purpose of the study was to study the role of this reactive nitrogen species in lung ischemia reperfusion injury.

NAD(P)H, a directly operating antioxidant?

Endogenous oxygen- and nitrogen-centered free radicals are considered to play a decisive role in a variety of diseases such as neurodegenerative disorders, atherosclerosis, or cancer. Directly operating antioxidants limit the action of freely diffusing radicals by scavenging the attacking, oxidizing radical and re-reducing the oxidized biomolecule, i.e., the biomolecule-derived radical. From textbooks of biochemistry it is understood that NAD(P)H acts as a hydride (hydrogen anion) donor in a variety of enzymatic processes. One example is the re-reduction of GSSG to GSH, catalyzed by glutathione reductase. Because of this reaction, NADPH has been suggested to also act as an indirectly operating antioxidant, thus maintaining the antioxidative power of glutathione. To the best of our knowledge, however, neither NADPH nor NADH has been considered to be directly operating antioxidants. Based on recently published data, new experiments, and theoretical considerations, we propose that NAD(P)H represents a decisive, directly operating antioxidant that should be considered of major importance in the mitochondrial compartment. NAD(P)H fulfills this task both by scavenging toxic free radicals and repairing biomolecule-derived radicals.

Lactoperoxidase-catalysed oxidation of indomethacin, a nonsteroidal antiinflammatory drug, through the formation of a free radical

Lactoperoxidase (LPO, EC 1.11.1.7; donor-H 2O 2 oxidoreductase) catalyses the oxidation of indomethacin, a nonsteroidal antiinflammatory drug by H 2O 2 as measured by time-dependent decay of indomethacin extinction at 280 nm and concurrent appearance of stable oxidation product(s) at 412 nm. From a plot of log V max against varying pH of indomethacin oxidation, involvement of an ionizable group of the enzyme having pka = 5.7 could be ascertained for controlling the oxidation process. Spectral studies revealed that LPO-compound II oxidises indomethacin through one-electron transfer and is reduced to the native ferric state as shown by its spectral shift from 430 nm to 412 nm through an isosbestic point at 421 nm. The one-electron oxidation product is a nitrogen-centered free radical detected as a 5,5-dimethyl-l-pyrroline N-oxide (DMPO) adduct ( a N = 15 G, a H β = 16 G) in electron spin resonance spectroscopy. The free radical is scavenged by reaction with O 2 as shown by O 2 consumption sensitive to the free-radical trap, DMPO. Binding studies by optical difference spectroscopy indicate that indomethacin binds to LPO with an apparent K D value of 24.5 μM. The free energy change, ΔG′, for the binding is −26.3 KJ mol −1, suggesting that the interaction is favourable for oxidation. Indomethacin binding remains unaltered by a change of pH from 5.25 to 7.5, presumably because of hydrophobic interaction. The binding is competitive with resorcinol, an aromatic electron donor, showing the K D value to be as high as 100 μM. We suggest that indomethacin interacts at the aromatic donor binding site and is oxidised by one-electron transfer by LPO catalytic intermediates to stable oxidation product(s) through the formation of a free radical. BIOCHEM PHARMACOL 52;8:1169–1175, 1996.

Identification of 3-MI-derived N-centerred radicals obtained from incubation of 3-MI with microsomal-NADPH system by EPR-HPLC spin trapping

3-Methylindole (3-MI) is a metabolite of tryptophan that causes acute pulmonary edema and emphysema in ruminants when administered orally or intravenously. Electron paramagnetic resonance (EPR) spin-trapping techniques have been used to investigate the in vitro and in vivo formation of free radicals during 3-MI metabolism by goat lung. Utilizing C-phenyl-N-tertbutyl nitrone (PBN), a nitrogen-centered free radical has been detected from 3-MI in goat lung muicrosomal incubation. The EPR spectrum of the spin adduct is identical to the observed when 3-MI is irradiated with ultraviolet light. The formation of a nitrogen-centered 3-MI free radical is followed by the appearance of a carbon-centered radical in microsomal preparations. The objective of the present study is to prove that the nitrogen-centered radical generated from the 3-MI incubation systems is a 3-MI radical utilizing [ 14C]-3 MI and the EPR-HPLC technique. The HPLC chromatogram includes three peaks that give EPR signals. These peaks are assigned to nitrogen-, oxygen- and carbon-centered radical adducts. The polarity of the three peaks follows the order: carbon-centered radical adduct > oxygen-centered radical adduct > nitrogen-centered radical adduct. The last has a polarity that is weaker tha 3-MI. Only the nitrogen-centered peak and the 3-MI peak possessef radioactivity. The retention time of the nitrogen centered radical is the same as the spin adduct generated by 3-MI irradiation with ultraviolet light. These results demonstrate that the nitrogen-centered radical is a 3-MI-PBN spin adduct, and supports the hypothesis that 3-MI-induced lung damage results from activation of 3-MI to a free radical. Also, in this study the stability of the radical spin adducts and the best conditions to produce the radicals in the incubation system was investigated.

Production of Hydroxyl Free Radical by Exposure of N-Methyl-N′-Nitro-N-Nitrosoguanidine to Visible Light in the Absence of Hydrogen Peroxide

The production of hydroxyl free radical (.OH) by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in the absence of H2O2 was investigated by electron spin resonance with the use of the spin-trapping agent 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). .OH was detected as well as a nitrogen-centered free radical (.NR) on exposure of 1.25 mM MNNG solution (pH 6.0) containing 25 mM DMPO to visible light from a tungsten-halogen lamp at an intensity of 6.68 mW/cm2. Production of .OH depended on the concentration of MNNG and on the intensity of light exposure, indicating that .OH production resulted from the photolysis of MNNG solution. Manganese-superoxide dismutase and catalase did not inhibit the production of .OH, excluding the possibility that .OH might be produced from O2-. or H2O2. When the concentration of DMPO was increased from 25 mM to 125 mM, the yield of .OH decreased significantly while that of .NR increased over twice that at 25 mM. When the concentration of DMPO was further increased to over 200 mM, only .NR was detected and its yield was the same as that at 125 mM, which indicates that DMPO inhibited .OH production but not through the trapping of .NR. From these results we suggest that nitric oxide (NO) derived with .NR from the photolysis of MNNG resulted in .OH production which was inhibited by DMPO in aqueous solution.

ESR studies of nitrogen-centered free radicals. 40. Exceptionally persistent nitrogen-centered free radicals. Preparation, isolation, and molecular structure of N-(arylthio)-2,4,6-triphenylanilino radicals

ESR studies of nitrogen-centered free radicals. 40. Exceptionally persistent nitrogen-centered free radicals. Preparation, isolation, and molecular structure of N-(arylthio)-2,4,6-triphenylanilino radicals

Metabolism of trinitrobenzene sulfonic acid by the rat colon produces reactive oxygen species

Recent studies have shown that intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in ethanol or intramural injection of TNBS in saline produces an acute and possibly chronic colitis in rats. It has been assumed that interstitial TNBS initiates the inflammatory response via macrophage-mediated recognition and degradation of TNBS-modified mucosal cells and proteins. However, it is known that certain flavoproteins and/or reductants interact with compounds containing the nitro functional group to generate pro-inflammatory, nitrogen-centered free radicals and reactive oxygen metabolites. The objective of this study was to assess the ability of the rat colon, using either colon homogenates, isolated colonocytes, or intestinal interstitial fluid, to produce reactive oxygen species via enzymatic and/or nonenzymatic metabolism of TNBS. It was found that the addition of TNBS (1 mmol/L) to the 10,000 × g supernatant of rat colon homogenates increased the rate of superoxide production from normally undetectable levels to 2.6 ± 0.23 nmol · min−1 · mg protein−1. Addition of nicotinamide adenine dinucleotide, reduced form (NADH, 1 mmol/L) to colon homogenates containing TNBS significantly enhanced superoxide production to 10.4 ± 0.9 nmol · min−1 · mg−1. Similarly, addition of nicotinamide adenine dinucleotide phosphate, reduced form (NADPH, 1 mmol/L) to colon extracts containing TNBS produced an even further increase in the rate of superoxide formation to 25.2 ± 1.1 nmol · min−1 · mg−1. Addition of NADH or NADPH to the colon homogenate in the absence of TNBS produced no detectable superoxide formation, suggesting that TNBS was required for the enhanced oxidative metabolism. In a separate series of experiments, it was found that isolated colonocytes produced small but significant amounts of superoxide (3.15 ± 0.6 nmol/2 × 106 cells) that were significantly increased in the presence of ethanol to 6.55 ± 1.14 nmol/2 × 106 cells. Using purified preparations of two flavoproteins found in the rat colon, it was shown that the addition of TNBS (1 mmol/L) to purified NADH dehydrogenase or glutathione reductase increased the rate of superoxide formation by these enzymes from normally undetectable levels to 1.6 nmol/min and 1.2 nmol/min, respectively. In addition, it was found that intestinal interstitial fluid (lymph) initiated redox cycling of TNBS such that 28.1 ± 1.6 nmol of oxygen was consumed per minute per milliliter of lymph. This increase in oxygen consumption was inhibited by the addition of superoxide dismutase and catalase. One possible metabolite involved in both mucosal and lymph-mediated metabolism of TNBS is ascorbic acid. Data obtained in this study showed that ascorbic acid (1 mmol/L) interacts with TNBS to produce a dramatic increase in oxygen consumption (92 ± 14 nmol/min) that was inhibited by the addition of catalase, superoxide dismutase, or both. Taken together, the data show that TNBS may be metabolized by enzymes and/or metabolites found in the rat colon to yield superoxide and hydrogen peroxide. These data also suggest that some of the inflammation associated with the intrarectal or intramural administration of TNBS may be mediated by pro-inflammatory, reactive oxygen metabolites generated by the oxidative metabolism of this hapten.