Actions of melatonin in the reduction of oxidative stress

Melatonin was discovered to be a direct free radical scavenger less than 10 years ago. Besides its ability to directly neutralize a number of free radicals and reactive oxygen and nitrogen species, it stimulates several antioxidative enzymes which increase its efficiency as an antioxidant. In terms of direct free radical scavenging, melatonin interacts with the highly toxic hydroxyl radical with a rate constant equivalent to that of other highly efficient hydroxyl radical scavengers. Additionally, melatonin reportedly neutralizes hydrogen peroxide, singlet oxygen, peroxynitrite anion, nitric oxide and hypochlorous acid. The following antioxidative enzymes are also stimulated by melatonin: superoxide dismutase, glutathione peroxidase and glutathione reductase. Melatonin has been widely used as a protective agent against a wide variety of processes and agents that damage tissues via free radical mechanisms.     

Does exogenous melatonin influence the free radicals metabolism and pain sensation in rat?

Melatonin has been shown to play a role in antioxidative defence. We therefore studied its effect on oxidative damage to the rat cerebral cortex evoked by painful stimulation and immobilization-induced stress. Moreover, the effect of melatonin on chronic pain perception was examined. Rats were injected with either a high dose of melatonin (100 mg/kg i.p.) or a vehicle for five days and were subjected to painful stimulation or immobilization stress 30 min after the treatment. To determine the degree of oxidative stress, the levels of free radicals, thiobarbituric acid reactive substances (TBARS) as indicators of lipid peroxidation and glutathione peroxidase (GSHPx) were estimated in somatosensory cortex. Pain perception was measured by the tail-flick and plantar test. Melatonin reduced the level of TBARS previously increased by painful stimulation. Melatonin also exhibited a slight analgesic effect in those animals exposed to painful stimulation but its role in free radical scavenging did not contribute to this effect.     

An ESR study of the nitroxide radical of pentastarch-conjugated deferoxamine

At higher concentrations, deferoxamine (DFO) reacts with hydroxyl radicals to produce a stable nitroxide free radical. Formation and decay of this nitroxide radical was investigated and compared with a novel modified pentastarch conjugate of DFO (MPS-DFO). Photolytic generation of hydroxyl radicals from H2O2 in the presence of free DFO produced a nitroxide radical with coupling constants of aN = 8.0 G and aH = 6.5 G. Under the same experimental conditions, equimolar concentrations of MPS-DFO produced an ESR signal of reduced intensity while iron-saturated MPS-DFO produced no signal. Incubation of free DFO with pentastarch (i.e., without conjugation) greatly decreased the intensity of the nitroxide radical signal. Using a spin-trapping technique with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), the pentastarch vehicle was shown to inhibit the DMPO-OH adduct formation. The decay of the DFO nitroxide radical decayed with a second-order rate constant while that of MPS-DFO decayed with a first-order rate constant. Thus, a novel derivative of DFO may provide some additional benefit in limiting DFO nitroxide radical formation and might explain the reported reduced in vivo toxicity of MPS-DFO relative to free DFO.     

Different protective roles in vitro of alpha- and beta-domains of growth inhibitory factor (GIF) on neuron injuries caused by oxygen free radicals.

It was well known that beta-amyloid (Abeta) and tau protein play an important role in pathological procedure of Alzheimer's disease (AD), a senile dementia. The growth inhibitory factor (GIF, also named metallothionein-3, MT-3) had been demonstrated to inhibit the outgrowth of cortex neurons in the medium with extract of the AD patient brain. In our experiments, it was found that the neurons of cortex and the PC12 (pheochromocytoma) cells could be protected from the cytotoxicity of beta-amyloid 25-35 in presence of GIF and its domains. Additionally, GIF can scavenge the hydroxyl radical efficiently in CytC-VitC radical producing system and its alpha-domain shown more effective potentials than its beta-domain. The electron paramagnetic resonance spectra also show that the alpha-domain has more potential ability for eliminating reactive oxygen free radicals than its beta-domain. The results suggest that GIF could act as an efficient scavenger against free radicals in vitro and the alpha-domain in GIF molecule shows more potential in protecting against reactive oxygen species injury than the beta-domain.     

Formation of long-lived hydroxyl free radical adducts of proline and hydroxyproline in a Fenton reaction

Proline and hydroxyproline when exposed to the hydroxyl free radical generating system of ADP-Fe(II)-H2O2 yielded long-lived free radicals. An analysis of the electron paramagnetic resonance spectra of the long-lived hydroxyl free radical adducts of proline and hydroxyproline is consistent with a free electron on a nitroxyl group interacting with the nitrogen atom as well as with three separate protons. In the case of proline, nitroxide formation was observed under the influence of tert-butyl-hydroperoxide, giving a similar EPR spectrum (Lin, J.S., Tom, T.C. and Olcott, H.S. (1974) J. Agr. Food Chem. 22, 526-528); however, the hydroxyl free radical adduct of hydroxyproline has not been described yet. In the case of the proline nitroxide radical, two of the three protons involved interact with the free electron equivalently. The coupling constants for the hydroxyl free radical adduct of proline are AN = 1.58 mT, AH1 beta = AH2 beta = 2.13 mT, AH3 beta = 1.77 mT and for hydroxyproline are AN = 1.54 mT, AH1 beta = 2.56 mT, AH2 beta = 2.03 and AH3 beta = 1.51. The data are consistent with the amine nitrogen of proline and hydroxyproline being oxidized to a nitroxyl group and the free electron of the nitroxyl interacting with the beta-protons of these amino acid hydroxyl free radical adducts.     

The transition metal-mediated formation of the hydroxyl free radical during the reduction of molecular oxygen by ferredoxin-ferredoxin:NADP+ oxidoreductase

The NADPH-supported enzymatic reduction of molecular oxygen by ferredoxin-ferredoxin:NADP+ oxidoreductase was investigated. The ESR spin trapping technique was employed to identify the free radical metabolites of oxygen. The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to trap and identify the oxygen-derived free radicals. [17O]Oxygen was employed to demonstrate that the oxygen-centered radicals arose from molecular oxygen. From the data, the following scheme is proposed: (Formula:see text). The formation of the free hydroxyl radical during the reduction of oxygen was demonstrated with quantitative competition experiments. The hydroxyl radical abstracted hydrogen from ethanol or formate, and the resulting scavenger-derived free radical was trapped with known rate constants. If H2O2 was added to the enzymatic reaction, a stimulation of the production of the hydroxyl radical was obtained. This stimulation was manifested in both the concentration and the rate of formation of the DMPO/hydroxyl radical adduct. Catalase was shown to inhibit formation of the hydroxyl radical adduct, further supporting the formation of hydrogen peroxide as an intermediate during the reduction of oxygen. All three components, ferredoxin, ferredoxin:NADP+ oxidoreductase, and NADPH, were required for reduction. Ferredoxin:NADP+ oxidoreductase reduces ferredoxin, which in turn is responsible for the reduction of oxygen to hydrogen peroxide and ultimately the hydroxyl radical. The effect of transition metal chelators on the DMPO/hydroxyl radical adduct concentration suggests that the reduction of chelated iron by ferredoxin is responsible for the reduction of hydrogen peroxide to the hydroxyl radical via Fenton-type chemistry.     

Radiolysis of spin-labeled DNA: an electron spin resonance investigation

The reactions of free and DNA-bound 2,2,5,5-tetramethylpyrrolidine-N-oxyl (PROXYL) probes with radicals generated during radiolysis of dilute aqueous solutions of DNA were examined. For the free PROXYL probe in deaerated solution with each of the four nucleotides (dAMP, dCMP, dGMP, and TMP) it was found that the pyrimidine radicals were more reactive toward the probe than were the purine radicals. Reactions of the electron adduct of TMP and the hydroxyl radical adducts of dAMP, dGMP, and TMP with the probe resulted in little or no reduction of the probe. For TMP these results are consistent with the fact that both the protonated electron and hydroxyl radical adducts of TMP will covalently bind to the nitroxide function of the probe. Reduction of the PROXYL probe was observed in reactions with the hydroxyl radical adduct of dCMP and with the electron adducts of dAMP, dCMP, and dGMP. Results of the radiolysis of the free PROXYL probe in deaerated dilute solution of DNA suggest that the PROXYL probe protects the DNA from water radical attack as the ratio of DNA bases to PROXYL probe increases above 50:1. Reactions of DNA-bound probes are dependent on the depth of the nitroxide function in relation to the major groove of the DNA helix. Two probes with tether lengths which are less than the depth of the major groove show an expected increase in reactions with DNA base radicals as compared to a probe with a tether that extends beyond the groove. The longer probe is involved largely in reactions with sugar and water radicals along the periphery of the DNA helix. In the presence of oxygen, there is a dramatic decrease in the loss of both the free and DNA-bound probes due to the lack of reaction of these probes with peroxyl radicals formed by the addition of molecular oxygen to DNA radicals.     

Melatonin oxidative stress and neurodegenerative diseases

Oxidative Stress is implicated as one of the primary factors that contribute to the development of neurodegenerative diseases like Alzheimer's Disease, Parkinsonism and neurological conditions like epileptic seizures, stroke, brain damage, neurotrauma etc. The increased formation and release of oxygen free radicals coupled with the rather low antioxidative potential of the central nervous system are the major reasons that account for the enhanced oxidative stress seen in neuronal cells. In addition to this, brain is also enriched with polyunsaturated fatty acids that render neuronal cells easily vulnerable to oxidative attack. The fact that there is increased incidence of neurodegenerative disorders in aged individuals, has prompted many investigators to search for a common factor whose progressive decline with increase in age could account for increased oxidative stress resulting in senescence and age associated degenerative diseases. Since melatonin, the hormone secreted from the pineal gland has a remarkable anti-oxidant property and whose rate of production declines with increase in age, has prompted many to suggest that this hormone plays a crucial role in the genesis of neurodegenerative diseases. Melatonin cannot only scavenges oxygen free radicals like super oxide radical (O2-), hydroxyl radical (*OH), peroxyl radical (LOO*) and peroxynitrite anion (ONOO-), but can also enhance the antioxidative potential of the cell by stimulating the synthesis of antioxidative enzymes like super oxide dismutase (SOD), glutathione peroxidase (GPX), and also the enzymes that are involved in the synthesis of glutathione. In many instances, melatonin increases the expression of m RNA's of the antioxidative enzymes. Melatonin administration has been shown to be effective in counteracting the neurodegenerative conditions both in experimental models of neurodegenerative diseases and in patients suffering from such diseases. A disturbance of melatonin rhythm and secretion also has been noted in patients suffering from certain neurodegenerative diseases. From all these, it is evident that melatonin has a neuroprotective role.     

Different protective roles in vitro of α- and β-domains of growth inhibitory factor (GIF) on neuron injuries caused by oxygen free radicals

It was well known that β-amyloid (Aβ) and tau protein play an important role in pathological procedure of Alzheimer’s disease (AD), a senile dementia. The growth inhibitory factor (GIF, also named metallothionein-3, MT-3) had been demonstrated to inhibit the outgrowth of cortex neurons in the medium with extract of the AD patient brain. In our experiments, it was found that the neurons of cortex and the PC12 (pheochromocytoma) cells could be protected from the cytotoxicity of β-amyloid 25–35 in presence of GIF and its domains. Additionally, GIF can scavenge the hydroxyl radical efficiently in CytC–VitC radical producing system and its α-domain shown more effective potentials than its β-domain. The electron paramagnetic resonance spectra also show that the α-domain has more potential ability for eliminating reactive oxygen free radicals than its β-domain. The results suggest that GIF could act as an efficient scavenger against free radicals in vitro and the α-domain in GIF molecule shows more potential in protecting against reactive oxygen species injury than the β-domain.     

The role of free radicals in asbestos-induced diseases.

Asbestos exposure causes pulmonary fibrosis and malignant neoplasms by mechanisms that remain uncertain. In this review, we explore the evidence supporting the hypothesis that free radicals and other reactive oxygen species (ROS) are an important mechanism by which asbestos mediates tissue damage. There appears to be at least two principal mechanisms by which asbestos can induce ROS production; one operates in cell-free systems and the other involves mediation by phagocytic cells. Asbestos and other synthetic mineral fibers can generate free radicals in cell-free systems containing atmospheric oxygen. In particular, the hydroxyl radical often appears to be involved, and the iron content of the fibers has an important role in the generation of this reactive radical. However, asbestos also appears to catalyze electron transfer reactions that do not require iron. Iron chelators either inhibit or augment asbestos-catalyzed generation of the hydroxyl radical and/or pathological changes, depending on the chelator and the nature of the asbestos sample used. The second principal mechanism for asbestos-induced ROS generation involves the activation of phagocytic cells. A variety of mineral fibers have been shown to augment the release of reactive oxygen intermediates from phagocytic cells such as neutrophils and alveolar macrophages. The molecular mechanisms involved are unclear but may involve incomplete phagocytosis with subsequent oxidant release, stimulation of the phospholipase C pathway, and/or IgG-fragment receptor activation. Reactive oxygen species are important mediators of asbestos-induced toxicity to a number of pulmonary cells including alveolar macrophages, epithelial cells, mesothelial cells, and endothelial cells. Reactive oxygen species may contribute to the well-known synergistic effects of asbestos and cigarette smoke on the lung, and the reasons for this synergy are discussed. We conclude that there is strong evidence supporting the premise that reactive oxygen species and/or free radicals contribute to asbestos-induced and cigarette smoke/asbestos-induced lung injury and that strategies aimed at reducing the oxidant stress on pulmonary cells may attenuate the deleterious effects of asbestos.     

Nitroxides as redox probes of melanins: dark-induced and photoinduced changes in redox equilibria

The interaction of nitroxide free radicals and their reduced products (hydroxylamines) with synthetic and natural melanins has been studied. Electron spin resonance spectroscopy was used to measure changes in radical concentration in the dark and during irradiation with visible or uv light. Some reduction of nitroxide occurs in the dark, and is reversible: the nitroxide can be completely regenerated by the one-electron oxidant ferricyanide. The kinetics of the process depend strongly on radical charge and pH. For positively charged nitroxides the rate is much faster than for either neutral or anionic radicals. At pH 10 the rate is about 20 times faster than at pH 5. Oxidation of hydroxylamine also can occur so that a redox equilibrium is established. The equilibrium constant has been estimated for the reaction between a nitroxide and melanin from autoxidation of 3,4-dihydroxyphenylalanine. Results are also dependent upon the type of melanin used and chemical modification (oxidation or reduction) of the melanin. Redox equilibria are altered during irradiation with either visible or uv light. Rapid oxidation of hydroxylamine to nitroxide is apparent, together with a slower reduction of nitroxide. Action spectra for these processes are related to those for melanin radical production and oxygen consumption in nitroxide-free melanin systems. Reduction of nitroxide is inhibited by oxygen, suggesting a competition between nitroxide and oxygen for photoinduced reducing equivalents.     

The effect of melatonin on antioxidant enzymes in human diabetic skin fibroblasts

Melatonin plays several important physiological functions in mammals, such as immune enhancement and regulation of dark-light signal transduction. Melatonin is also known to be an endogenous free radical scavenger and an efficient antioxidant. It detoxifies a variety of free radicals and reactive oxygen intermediates, including the hydroxyl radical, singlet oxygen and nitric oxide. These radicals participate in many diseases, for example diabetes. This study determined the effect of melatonin on the antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and the level of glutathione (GSH) in human diabetic (C2 line) skin fibroblasts. Confluent monolayers of control (S2 line) and diabetic (C2 line) skin fibroblasts were incubated with different concentrations of melatonin: 10, 50, 100 and 1000 micromol/l at 37 degrees C for 24 h. Next, the GSH level and SOD, CAT and GPx activities were measured colorimetrically. The activities of the antioxidant enzymes and the GSH level were lower in diabetic skin fibroblasts than in the control S2 line. Concentrations of melatonin of 100 and 1000 micromol/l caused a significant increase in the enzymes' activities and GSH level.     

UV-induced free radicals in the skin detected by ESR spectroscopy and imaging using nitroxides

Reactive free radicals and reactive oxygen species (ROS) induced by ultraviolet irradiation in human skin are strongly involved in the occurrence of skin damages like aging and cancer. In the present work an ex vivo method for the detection of free radicals/ROS in human skin biopsies during UV irradiation is presented. This method is based on the Electron Spin Resonance (ESR) spectroscopy and imaging and uses the radical trapping properties of nitroxides. The nitroxides 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO), 3-Carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCM), and 3-Carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCA), were investigated for their applicability of trapping reactive free radicals and reactive oxygen species in skin during UV irradiation. As a result of the trapping process the nitroxides were reduced to the EPR silent hydroxylamins. The reduction rate of TEMPO was due to both the UV radiation and the enzymatic activity of the skin. The nitroxides PCM and PCA are sufficiently stable in the skin and are solely reduced by UV-generated free radicals/ROS. The nitroxide PCA was used for imaging the spatial distribution of UV-generated free radicals/ROS. As a result of the homogeneous distribution of PCA in the skin, it was possible to estimate the penetration of UVA and UVB irradiation: The UV irradiation decreased the PCA intensity corresponding to its irradiance and penetration into the skin. This reduction was shown to be caused mainly by UVA radiation (320-400 nm).     

The oxidant/antioxidant network: role of melatonin

Melatonin is now known to be a multifaceted free radical scavenger and antioxidant. It detoxifies a variety of free radicals and reactive oxygen intermediates including the hydroxyl radical, peroxynitrite anion, singlet oxygen and nitric oxide. Additionally, it reportedly stimulates several antioxidative enzymes including glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and superoxide dismutase; conversely, it inhibits a prooxidative enzyme, nitric oxide synthase. Melatonin also crosses all morphophysiological barriers, e.g., the blood-brain barrier, placenta, and distributes throughout the cell; these features increase the efficacy of melatonin as an antioxidant. Melatonin has been shown to markedly protect both membrane lipids and nuclear DNA from oxidative damage. In every experimental model in which melatonin has been tested, it has been found to resist macromolecular damage and the associated dysfunction associated with free radicals.     

Radicals from "Good's" buffers

Oxidative deposition of iron in ferritin or the autoxidation of iron in the absence of protein produces radicals from Good's buffers. Radical species are formed from the piperazine ring-based buffers Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Epps 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, and Pipes 1,4-piperazinediethanesulfonic acid, but not from Mes (4-morpholineethanesulfonic acid) which contains a morpholine ring. The radicals all have half-lives around 10 min and display very similar electron paramagnetic resonance spectra consisting of at least 30 lines. The Hepes radical can be formed by the addition of potassium superoxide directly to the buffer and its production during iron(II) autoxidation is inhibited by superoxide dismutase (EC 1.15.1.1). Catalase (EC 1.11.1.6) accelerates the decay of the EPR spectrum. Thus the buffer radicals are secondary radical species produced from oxygen radicals formed during the iron catalyzed Haber-Weiss process. The deoxyribose/thiobarbituric acid assay for hydroxyl radical production shows that Hepes is an effective hydroxyl radical scavenging agent. The Hepes radical can also be formed electrolytically at a potential of +0.8 V (vs standard hydrogen electrode). Oxidation of Hepes at pH 10 during the autoxidation of iron(II) or by the addition of hydrogen peroxide produces a nitroxide radical. These results indicate that piperazine ring Good buffers should be avoided in studies of redox processes in biochemistry.     

Photoilluminated riboflavin/riboflavin-Cu(II) inactivates trypsin: Cu(II) tilts the balance

Riboflavin (RF) upon irradiation with fluorescent light generates reactive oxygen species like superoxide anion, singlet and triplet oxygen, flavin radicals and substantial amounts of hydrogen peroxide (H2O2). H2O2 can freely penetrate cell membrane and react with a transition metal ion like Cu(ll), generating hydroxyl radical via the modified metal-catalyzed Haber-Weiss reaction. Earlier, it was reported that trypsin-chymotrypsin mixture served as an indirect antioxidant and decreased free radical generation. Thus, in the present study, we used photoilluminated RF as a source of ROS to investigate the effect of free radicals on the activity of trypsin. We also compared the damaging effect of photoilluminated RF and RF-Cu(ll) system using trypsin as a target molecule. RF caused fragmentation of trypsin and the effect was further enhanced, when Cu(II) was added to the reaction. Results obtained with various ROS scavengers suggested that superoxide radical, singlet and triplet oxygen were predominantly responsible for trypsin damage caused by photoilluminated RF. On the other hand, when Cu(ll) was added to the reaction, hydroxyl radical was mainly responsible for trypsin damage. A mechanism of generation of various ROS in the reaction is also proposed. Trypsin did not show any antioxidant effect with RF alone or with RF-Cu(II) combination.     

Significance of melatonin in antioxidative defense system: reactions and products

Melatonin is a potent endogenous free radical scavenger, actions that are independent of its many receptor-mediated effects. In the last several years, hundreds of publications have confirmed that melatonin is a broad-spectrum antioxidant. Melatonin has been reported to scavenge hydrogen peroxide (H(2)O(2)), hydroxyl radical (HO(.)), nitric oxide (NO(.)), peroxynitrite anion (ONOO(-)), hypochlorous acid (HOCl), singlet oxygen ((1)O(2)), superoxide anion (O(2)(-).) and peroxyl radical (LOO(.)), although the validity of its ability to scavenge O(2)(-). and LOO(.) is debatable. Regardless of the radicals scavenged, melatonin prevents oxidative damage at the level of cells, tissues, organs and organisms. The antioxidative mechanisms of melatonin seem different from classical antioxidants such as vitamin C, vitamin E and glutathione. As electron donors, classical antioxidants undergo redox cycling; thus, they have the potential to promote oxidation as well as prevent it. Melatonin, as an electron-rich molecule, may interact with free radicals via an additive reaction to form several stable end-products which are excreted in the urine. Melatonin does not undergo redox cycling and, thus, does not promote oxidation as shown under a variety of experimental conditions. From this point of view, melatonin can be considered a suicidal or terminal antioxidant which distinguishes it from the opportunistic antioxidants. Interestingly, the ability of melatonin to scavenge free radicals is not in a ratio of mole to mole. Indeed, one melatonin molecule scavenges two HO. Also, its secondary and tertiary metabolites, for example, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine, N-acetyl-5-methoxykynuramine and 6-hydroxymelatonin, which are believed to be generated when melatonin interacts with free radicals, are also regarded as effective free radical scavengers. The continuous free radical scavenging potential of the original molecule (melatonin) and its metabolites may be defined as a scavenging cascade reaction. Melatonin also synergizes with vitamin C, vitamin E and glutathione in the scavenging of free radicals. Melatonin has been detected in vegetables, fruits and a variety of herbs. In some plants, especially in flowers and seeds (the reproductive organs which are most vulnerable to oxidative insults), melatonin concentrations are several orders of magnitude higher than measured in the blood of vertebrates. Melatonin in plants not only provides an alternative exogenous source of melatonin for herbivores but also suggests that melatonin may be an important antioxidant in plants which protects them from a hostile environment that includes extreme heat, cold and pollution, all of which generate free radicals.     

Electron paramagnetic resonance evidence that cellular oxygen toxicity is caused by the generation of superoxide and hydroxyl free radicals

Cells require molecular oxygen for the generation of energy through mitochondrial oxidative phosphorylation; however, high concentrations of oxygen are toxic and can cause cell death. A number of different mechanisms have been proposed to cause cellular oxygen toxicity. One hypothesis is that reactive oxygen free radicals may be generated; however free radical generation in hyperoxic cells has never been directly measured and the mechanism of this radical generation is unknown. In order to determine if cellular oxygen toxicity is free radical mediated, we applied electron paramagnetic resonance, EPR, spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide, DMPO, to measure free radical generation in hyperoxic pulmonary endothelial cells. Cells in air did not give rise to any detectable signal. However, cells exposed to 100% O2 for 30 min exhibited a prominent signal of trapped hydroxyl radical, DMPO-OH, while cell free buffer did not give rise to any detectable radical generation. This cellular radical generation was demonstrated to be derived from the superoxide radical since the observed signal was totally quenched by superoxide dismutase, but not by equal concentrations of the denatured enzyme. It was confirmed that the hydroxyl radical was generated since in the presence of ethanol the CH3 CH(OH) radical was formed. Loss of cell viability as measured by uptake of trypan blue dye was observed paralleling the measured free radical generation. Thus, superoxide and hydroxyl radicals are generated in hyperoxic pulmonary endothelial cells and this appears to be an important mechanism of cellular oxygen toxicity.     

Conversion of Nitroxide Radicals by Phenolic and Thiol Antioxidants

Nitrone/nitroso spin traps are often used for detection of unstable hydroxyl radical giving stable nitroxide radicals with characteristic electron spin resonance (ESR) signals. This technique may be useful only when the nitroxide radicals are kept stable in the reaction system. The aim of the present study is to clarify whether the nitroxide radicals are kept stable in the presence of the hydroxyl radical scavengers. Effect of hydroxyl radical scavengers on the ESR signals of nitroxide radicals, 2,2,6,6-tetramethyI-piperi-dine-N-oxyl (TEMPO) and the spin adduct (DMPO-OH) of 5,5-dimethyl-l-pyrroline N-oxide (DMPO) and hydroxyl radical, was examined. Although the ESR signals of TEMPO and the DMPO-OH spin adduct were unchanged on treatment with ethanol and dimethyl sulfoxide, their intensities were effectively decreased on treatment with 6-hydroxy-2,5,7,8-tetra-methylchroman-2-carboxylic acid (Trolox), cysteine, glutathione, 2-mercaptoethanol and metallothionein. Hence, the results of the detection of hydroxyl radical in the presence of phenolic and thiol antioxidants by the ESR technique using nitrone/nitroso spin traps may be unreliable.     

Metabolic activation of sulfur mustard leads to oxygen free radical formation

We recently published electron paramagnetic resonance (EPR) spin trapping results that demonstrated the enzymatic reduction of sulfur mustard sulfonium ions to carbon-based free radicals using an in vitro system containing sulfur mustard, cytochrome P450 reductase, NADPH, and the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) in buffer (A.A. Brimfield et al., 2009, Toxicol. Appl. Pharmacol. 234:128-134). Carbon-based radicals have been shown to reduce molecular oxygen to form superoxide and, subsequently, peroxyl and hydroxyl radicals. In some cases, such as with the herbicide paraquat, a cyclic redox system results, leading to magnified oxygen free radical concentration and sustained tissue damage. Low mustard carbon radical concentrations recorded by EPR in our in vitro system, despite a robust (4.0mM) sulfur mustard starting concentration, led us to believe a similar oxygen reduction and redox cycling process might be involved with sulfur mustard. A comparison of the rate of mustard radical-POBN adduct formation in our in vitro system by EPR at atmospheric and reduced oxygen levels indicated a sixfold increase in 4-POBN adduct formation (0.5 to 3.0 μM) at the reduced oxygen concentration. That result suggested competition between oxygen and POBN for the available carbon-based mustard radicals. In parallel experiments we found that the oxygen radical-specific spin trap 5-tert-butoxycarbonyl-5-methylpyrroline-N-oxide (BMPO) detected peroxyl and hydroxyl radicals directly when it was used in place of POBN in the in vitro system. Presumably these radicals originated from O(2) reduced by carbon-based mustard radicals. We also showed that reactive oxygen species (ROS)-BMPO EPR signals were reduced or eliminated when mustard carbon radical production was impeded by systematically removing system components, indicating that carbon radicals were a necessary precursor to ROS production. ROS EPR signals were completely eliminated when superoxide dismutase and catalase were included in the complete in vitro enzymatic system, providing additional proof of oxygen radical participation. The redox cycling hypothesis was supported by density functional theory calculations and frontier molecular orbital analysis.