Metabolism of Ethanol to 1-Hydroxyethyl Radicals in Rat Liver Microsomes: Comparative Studies with Three Spin Trapping Agents

Metabolism of ethanol to 1-hydroxyethyl radicals by rat liver microsomes was studied with three nitrone spin trapping agents (POBN, PBN, and DMPO) under essentially comparable conditions. The data indicate that POBN was the superior spin trapping agent for 1-hydroxyethyl radicals, and that DMPO was least efficient. Addition of deferoxamine completely prevented detection of 1-hydroxyethyl radicals with PBN or DMPO, but caused only 50% decrease in EPR signals when POBN was the spin trap. However, superoxide dismutase only decreased 1-hydroxyethyl radical formation when POBN was the spin trap. Other experiments demonstrated that POBN was the most effective of these nitrones for reduction of Fe(III) in aqueous solutions. Furthermore, 1-hydroxyethyl radical adducts were formed when POBN was added to mixtures of ethanol, phosphate buffer, POBN and FeCl₃, but this effect did not occur with either PBN or DMPO. Thus, these data indicate that undesirable effects of POBN on iron chemistry may influence results of spin trapping experiments, and complicate interpretation of the resulting data.     

Metabolism of Ethanol to 1-Hydroxyethyl Radicals in Rat Liver Microsomes: Comparative Studies with Three Spin Trapping Agents

Metabolism of ethanol to 1-hydroxyethyl radicals by rat liver microsomes was studied with three nitrone spin trapping agents (POBN, PBN, and DMPO) under essentially comparable conditions. The data indicate that POBN was the superior spin trapping agent for 1-hydroxyethyl radicals, and that DMPO was least efficient. Addition of deferoxamine completely prevented detection of 1-hydroxyethyl radicals with PBN or DMPO, but caused only 50% decrease in EPR signals when POBN was the spin trap. However, superoxide dismutase only decreased 1-hydroxyethyl radical formation when POBN was the spin trap. Other experiments demonstrated that POBN was the most effective of these nitrones for reduction of Fe(III) in aqueous solutions. Furthermore, 1-hydroxyethyl radical adducts were formed when POBN was added to mixtures of ethanol, phosphate buffer, POBN and FeCl₃, but this effect did not occur with either PBN or DMPO. Thus, these data indicate that undesirable effects of POBN on iron chemistry may influence results of spin trapping experiments, and complicate interpretation of the resulting data.     

New evidence that the Alzheimer beta-amyloid peptide does not spontaneously form free radicals: an ESR study using a series of spin-traps

The direct formation of free radicals from Abeta has been suggested to be a key neurotoxic mechanism in Alzheimer's disease (AD). We have explored the possibility of the spontaneous formation of peptide-derived free radicals during the incubation of Abeta 1-40 by ESR spectroscopy using N-tert-butyl-alpha-phenylnitrone (PBN), 5,5-dimethyl-1-pyrroline N-oxide (DMPO), alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), and 3,5-dibromo-4-nitrosobenzenesulfonic acid sodium salt (DBNBS) as spin traps. Employing PBN, we observed spectra during the incubation of beta-amyloid peptide, at 37 degrees C, which included adducts of 2-methyl-2-nitrosopropane (MNP), despite rigorous purification of the PBN before incubation. The formation of some of these adducts was found to be enhanced by ambient laboratory light. Our experiments have led us to propose a hypothesis that PBN undergoes hydrolysis and decomposition in the presence of oxidants, which explains the origin of all of the PBN and MNP adducts observed (even when the PBN is highly purified). Hydrogen peroxide, formed during incubation, could play a major role as an oxidant in these experiments. Of the other three spin traps, only DMPO gave (very weak) spectra, but these could be assigned to its hydroxyl radical adduct, formed as an artifact by the nucleophilic addition of water to DMPO, catalyzed by trace levels of iron ions. Thus, while spectra are observed during our experiments, none of them can be assigned to adducts of radicals derived from the peptide and, therefore, our data do not support the suggestion that radicals are spontaneously formed from beta-amyloid peptide.     

Diaziquone as a potential agent for photoirradiation therapy: formation of the semiquinone and hydroxyl radicals by visible light

When diaziquone was irradiated with 500 nm visible light, hydroxyl free radicals as well as the diaziquone semiquinone were produced. The diaziquone semiquinone is a stable free radical that exhibits a characteristic 5-line electron spin resonance (ESR) spectrum. Since hydroxyl free radicals are short lived, and not observable by conventional ESR, the nitrone spin trap 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) was used to convert hydroxyl radicals into longer lived ESR detectable spin adducts. The formation of hydroxyl radicals was further confirmed by investigating reactions in which hydroxyl radical scavangers, sodium formate and dimethylsulfoxide, compete with the spin traps DMPO or POBN (alpha-(4-Pyridyl-1-oxide)-N- tert-butylnitrone) for hydroxyl free radicals. The products of these scavenging reactions were also trapped with DMPO or POBN. If drug free radicals and hydroxyl free radicals are important in the activity of quinone-containing antitumor agents, AZQ may have a potential in photoirradiation therapy or photodynamic therapy.     

Increase in Swimming Endurance Capacity of Mice by Capsaicin-induced Adrenal Catecholamine Secretion

Increase in endurance swimming capacity caused by capsaicin (CAP), a pungent component of red pepper, -induced increase of fat metabolism in mice was investigated using an adjustable-current water pool. The mice administered CAP via a stomach tube, showed longer swimming time until exhaustion than the control group of mice, in a dose-dependent manner. The maximal effect was observed at a dose of 10 mg/kg while more than 15 mg/kg had no effect. The increase of endurance was observed only when CAP was administered two hours before swimming. After the administration of CAP, the serum glucose concentration rapidly increased and then decreased within 60min, while the concentration of serum-free fatty acids gradually increased through 3 hours. The residual glycogen concentration of the gastrocnemius muscle after 30 min of swimming was significantly higher in the CAP-administered mice than in control mice, suggesting that use of the serum free fatty acids spared muscle glycogen consumption. The serum adrenaline concentration significantly increased with twin peaks at 30 min and two hours after administration of CAP. An experiment using adrenalectomized mice was done to confirm that the effect of CAP is due to increased energy metabolism through the secretion of adrenaline from the adrenal gland. The swimming endurance capacity of the adrenalectomized mice was not increased by CAP administration, although adrenaline injection induced a 58% increase in the endurance time. These results suggest that the increase of swimming endurance induced by CAP in mice is caused by an increase in fatty acid utilization due to CAP-induced adrenal catecholamine secretion.     

ESR characterization of thallium(III)-mediated nitrones oxidation

We characterized the reactions between Tl (III) and the usual nitrone spin traps (DMPO, POBN, and PBN). The ESR signal obtained with DMPO corresponded to DMPOX which was avoided neither by the enzymes superoxide dismutase or catalase, nor by the hydroxyl radical scavengers’ mannitol, ethanol, or methanol. Only when methanol concentration rose to 90% (v/v), a significant decrease in DMPOX formation was observed while no detectable DMPOX signal was present at 100% methanol solution. POBN rendered an unexpected adduct, while no oxidation products were obtained with PBN. Together, the experimental evidence demonstrates that Tl(III)-supported DMPO oxidation is not mediated by oxygen reactive species. Instead, we propose that Tl(III) interacts with the probe, favoring the addition of water. The intermediate is susceptible to be further oxidized by Tl(III) yielding a keto group. Kinetic studies showed that Tl(III) disappearance rate was higher (1.6 times) than DMPOX formation rate, and equal to Tl(I) formation rate, suggesting a stoichiometry 1.5:1 for Tl(III):DMPOX and 1:1 for Tl(III):Tl(I). The process, an example of the Forrester-Hepburn’s mechanism, was demonstrated for DMPO while the reactions of Tl(III) with POBN and PBN are still under elucidation.     

Hydroxyl and 1-hydroxyethyl free radical detection using spin traps followed by derivatization and gas chromatography—mass spectrometry

Summary

Detection of hydroxyl free radicals is frequently performed by electron spin resonance (ESR) following spin trapping of the radical using 5,5-dimethylpyrroline N-oxide (DMPO) to generate a stable free radical having a characteristic ESR spectrum. The necessary ESR equipment is expensive and not readily available to many laboratories. In the present study, a specific and sensitive gas chromatography—mass spectrometry (GC/MS) method for detection of hydroxyl and hydroxyethyl free radicals is described. The DMPO or N-t-butyl—α—phenylnitrone (PBN) radical adducts are extracted and derivatized by trimethylsylilation and analyzed by GC/MS. To standardize the method, ^.OH and 1-hydroxyethyl radicals were generated in two different systems: 1) a Fenton reaction in a pure chemical system in the absence or presence of ethanol and 2) in liver microsomal suspensions where ethanol is metabolized in the presence of NADPH. In the Fenton system both radicals were easily detected and specifically identified using DMPO or PBN. In microsomal suspensions DMPO proved better for detection of ^.OH radicals and PBN more suitable for detection of 1-hydroxyethyl radicals. The procedure is specific, sensitive and potentially as useful as ESR.     

Atypical effect of some spin trapping agents: reversible inhibition of acetylcholinesterase

N-tert-butyl-alpha-phenylnitrone (PBN), a widely used nitrone-based free radical trap was recently shown to prevent acetylcholinesterase (AChE) inhibitors induced muscle fasciculations and brain seizures while being ineffective against glutamergic or cholinergic receptor agonist induced seizures. In the present study we compared the effects on AChE activity of four free radical spin traps PBN, alpha-(4-pyridil-1)-N-tert-butyl nitrone (POBN), N-tert-butyl-alpha-(2-sulfophenyl)-nitrone (S-PBN) and 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). The kinetics of AChE inhibition were studied in vitro using a spectrophotometric kinetic assay with AChE from rat brain, diaphragm, electric eel and mouse brain. Spin trapping compounds S-PBN and DEPMPO, in concentrations up to 3 mM did not inhibit hydrolysis of ACh, while PBN and POBN inhibited hydrolysis of ACh in a reversible and concentration-dependent manner. Double reciprocal plots of the reaction velocity against varying ACh concentrations at each inhibitor concentration were linear and generally indicated mixed type inhibition. PBN was the most potent inhibitor of mouse AChE with Ki and Ki' of 0.58 and 2.99 mM, respectively, and the weakest inhibitor of electric eel AChE. In contrast, POBN showed the highest affinity for electric eel enzyme, with Ki and Ki' values of 1.065 and 3.15 mM, respectively. These findings suggest that the effect of PBN and POBN on AChE activity does not depend on trapping of damaging reactive oxygen and that in addition to their antioxidant action other pharmacological effects of these compounds should be considered when neuroprotective actions of PBN or POBN are investigated.     

Alpha-Phenyl-Tert-Butyl-Nitrone (PBN) Attenuates Hydroxyl Radical Production During Ischemia-Reperfusion Injury of Rat Brain: An EPR Study

-phenyl-tert-butyl-nitrone (PBN) a spin adduct forming agent is believed to have a protective action in ischemia-reperfusion injury of brain by forming adducts of oxygen free radicals including ±OH radical. Electron paramagnetic resonance (EPR) has been used to both detect and monitor the time course of oxygen free radical formation in the in vivo rat cerebral cortex. Cortical cups were placed over both cerebral hemispheres of methoxyflurane anesthetized rats prepared for four vessel occlusion-evoked cerebral ischemia. Prior to the onset of sample collection, both cups were perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent -(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN 100 mM) for 20 min. In addition 50 mg/kg BW of POBN was administered intraperitoneally (IP) 20 min prior to ischemia in order to improve our ability to detect free radical adducts. Cup fluid was subsequently replaced every 15 min during ischemia and every 10 min during reperfusion with fresh POBN containing CSF and the collected cortical superfusates were analyzed for radical adducts by EPR spectroscopy. After a basal 10 min collection, cerebral ischemia was induced for 15 or 30 min (confirmed by EEG flattening) followed by a 90 min reperfusion. -OH radical adducts (characterized by six line EPR spectra) were detected during ischemia and 90 min reperfusion. No adduct was detected in the basal sample or after 90 min of reperfusion. Similar results were obtained when diethylenetriaminepenta-acetic acid (100 μM; DETAPAC) a chelating agent was included in the artificial CSF. Systemic administration of PBN (100 mg/kg BW) produced a significant attenuation of radical adduct during reperfusion. A combination of systemic and topical PBN (100 mM) was required to suppress -OH radical adduct formation during ischemia as well as reperfusion. PBN free radical adducts were detected in EPR spectra of the lipid extracts of PBN treated rat brains subjected to ischemia/reperfusion. Thus this study suggests that PBN's protective action in cerebral ischemia/reperfusion injury is related to its ability to prevent a cascade of free radical generation by forming spin adducts.     

Alpha-Phenyl-Tert-Butyl-Nitrone (PBN) Attenuates Hydroxyl Radical Production During Ischemia-Reperfusion Injury of Rat Brain: An EPR Study

α-phenyl-tert-butyl-nitrone (PBN) a spin adduct forming agent is believed to have a protective action in ischemia-reperfusion injury of brain by forming adducts of oxygen free radicals including ±OH radical. Electron paramagnetic resonance (EPR) has been used to both detect and monitor the time course of oxygen free radical formation in the in vivo rat cerebral cortex. Cortical cups were placed over both cerebral hemispheres of methoxyflurane anesthetized rats prepared for four vessel occlusion-evoked cerebral ischemia. Prior to the onset of sample collection, both cups were perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent α-(4-pyridyl-1-oxide)-N-tert butylnitrone (POBN 100 mM) for 20 min. In addition 50 mg/kg BW of POBN was administered intraperitoneally (IP) 20 min prior to ischemia in order to improve our ability to detect free radical adducts. Cup fluid was subsequently replaced every 15 min during ischemia and every 10 min during reperfusion with fresh POBN containing CSF and the collected cortical superfusates were analyzed for radical adducts by EPR spectroscopy. After a basal 10 min collection, cerebral ischemia was induced for 15 or 30 min (confirmed by EEG flattening) followed by a 90 min reperfusion. -OH radical adducts (characterized by six line EPR spectra) were detected during ischemia and 90 min reperfusion. No adduct was detected in the basal sample or after 90 min of reperfusion. Similar results were obtained when diethylenetriaminepenta-acetic acid (100 μM; DETAPAC) a chelating agent was included in the artificial CSF. Systemic administration of PBN (100 mg/kg BW) produced a significant attenuation of radical adduct during reperfusion. A combination of systemic and topical PBN (100 mM) was required to suppress -OH radical adduct formation during ischemia as well as reperfusion. PBN free radical adducts were detected in EPR spectra of the lipid extracts of PBN treated rat brains subjected to ischemia/reperfusion. Thus this study suggests that PBN's protective action in cerebral ischemia/reperfusion injury is related to its ability to prevent a cascade of free radical generation by forming spin adducts.     

Capsiate, a nonpungent capsaicin analog, increases endurance swimming capacity of mice by stimulation of vanilloid receptors

We investigated the effect of capsiate, a nonpungent natural capsaicin analog, on the swimming capacity of mice in an adjustable-current water pool. Male BALB/c mice orally given capsiate (10 mg/kg) were able to keep swimming longer before exhaustion than the control mice. After 30 min of swimming, the residual glycogen in the gastrocnemius muscle was higher, the serum free fatty acid concentration tended to be higher, and the serum lactic acid concentration was significantly lower in the capsiate-administered mice. The value for the respiratory exchange ratio of the capsiate group was significantly lower during both resting and treadmill running. These physiological differences were abolished by administering the vanilloid receptor antagonist, capsazepin (0.17 mmol/kg, i.p.). The mice were not averse to the capsiate solution during a 4-h two-bottle choice test. These results suggest that the oral administration of capsiate enhanced fat oxidation and spared carbohydrate utilization, and consequently increased the endurance swimming capacity of the mice via stimulation of their vanilloid receptors. Practical application of capsiate is expected.     

Ascorbic acid induced degradation of beta-glucan: Hydroxyl radicals as intermediates studied by spin trapping and electron spin resonance spectroscopy

The formation of hydroxyl radicals in beta-glucan solutions treated with ascorbic acid and iron(II) was demonstrated by ESR spin trapping based methods. Two different spin traps were tested, namely DMPO which is commonly used to detect hydroxyl radicals, and POBN often used to detect carbon centered radicals. The experiments performed showed that the presence of iron(II) with DMPO led to low DMPO-OH adduct stability and further to DMPO dimerization. The level of hydroxyl radicals formed during the beta-glucan radical mediated degradation was evaluated using two ESR spin trapping methods based on the use POBN together with either 2% (v/v) EtOH or DMSO. The addition of ascorbic acid together with iron(II) in beta-glucan solution led to an immediate maximal production of hydroxyl radicals while the presence of ascorbic acid alone led to a progressive production of radical. Further hydroxyl radicals were found to be formed when iron(II) was added alone in beta-glucan solutions. The viscosity loss observed in the three last mentioned beta-glucan solutions were found to relate with the formation of hydroxyl radicals. These data confirm the involvement of hydroxyl radical in the beta-glucan degradation.     

Caffeine effect on respiratory muscle endurance and sense of effort during loaded breathing

The present study examined respiratory muscle endurance and the magnitude of the sense of effort during inspiratory threshold loading following a dose of caffeine (600 mg) previously observed to increase diaphragm strength. Experiments were performed on 12 normal subjects. Respiratory muscle endurance at a given level of load was assessed from the time of exhaustion and from the time course of the change in the power spectrum (centroid frequency) of the diaphragm electromyogram (EMG). The intensity of the sense of effort during loaded breathing was evaluated using a category (Borg) scale. Increasingly severe loads were associated with more rapid onset of fatigue. At a given load, caffeine prolonged the time to exhaustion and decreased the rate of fall of the centroid frequency of the diaphragm EMG. Caffeine also decreased the sense of effort during loaded breathing in 9 of 11 subjects. Changes in respiratory muscle endurance after caffeine administration were not explained by changes in the pressure-time index of the respiratory muscles or the pattern of thoracoabdominal movement. We conclude that caffeine enhances inspiratory muscle endurance, while concomitantly reducing the sense of effort associated with fatiguing inspiratory muscle contractions.     

Spin Trapping of Ibuprofen Radicals: Evidence That Ibuprofen is a Hydroxyl Radical Scavenger

The purpose of this study was to use electron paramagnetic resonance (EPR) spectroscopy to determine if ibuprofen, [2–(4-isobutylphenyl) propanoic acid], a potent nonsterodial anti-inflammatory agent, could modify hydroxyl radicals generation in vim. Ibuprofen (IBU; 0.1–50 mM) in water or water alone was added to EPR tubes containing ferrous sulfate (0.5–2.0mM). and either 5.5-dimethyl-l-pyrroline-N-oxide (DMPO; 40mM) or a-phenyl N-tert-butyl nitrone (PBN; 48 mM). Hydrogen peroxide (l mM) was added to inititate the Fenton reaction, and the systems were then analyzed by EPR spectroscopy to determine the type and relative quantity of free radical(s) produced. IBU caused a dose-dependent decrease of signal intensity of the hydroxyl radical adduct of DMPO (DMPO-OH) which is an indication that IBU either scavenges the hydroxyl radical and/or chelates iron. In addition, other radicals (presumably IBU radicals) produced in these systems were trapped by both DMPO (a_N = 16.1G, a^H_β = 24.0G) and PBN (a_N = 15.7G. a^H_β = 4.4G and a_N = 17.0G, a^H_β = 2.1 G). The signal height of these IBU radicals increased in systems containing ferrous sulfate (l mM), hydrogen peroxide (lmM), PBN (48mM), and increasing IBU concentrations. Therefore. we conclude that IBU scavenges the hydroxyl radical. If IBU chelated iron, then less hydroxyl radicals would be generated, less IBU radicals formed and the signal height of IBU radicals trapped by PBN would have decreased. However, these data do not fully exclude the possiblity that IBU may, to some extent. also chelate iron. Scavenging of hydroxyl radicals may be one of the mechanisms responsible for the beneficial action of IBU during the management of several rheumatic diseases. However, the IBU radicals produced when IBU scavenges hydroxyl radicals are reactive. and may be associated with the reported toxicity of this therapeutic agent.     

Free radicals-mediated induction of oxidized DNA bases and DNA-protein cross-links by nickel chloride

Using the comet assay, we showed that nickel chloride at 250-1000 microM induced DNA damage in human lymphocytes, measured as the change in comet tail moment, which increased with nickel concentration up to 500 microM and then decreased. Observed increase might follow from the induction of strand breaks or/and alkali-labile sites (ALS) by nickel, whereas decrease from its induction of DNA-DNA and/or DNA-protein cross-links. Proteinase K caused an increase in the tail moment, suggesting that nickel chloride at 1000 microM might cross-link DNA with nuclear proteins. Lymphocytes exposed to NiCl(2) and treated with enzymes recognizing oxidized and alkylated bases: endonuclease III (Endo III), formamidopyrimidine-DNA glycosylase (Fpg) and 3-methyladenine-DNA glycosylase II (AlkA), displayed greater extent of DNA damage than those not treated with these enzymes, indicating the induction of oxidized and alkylated bases by nickel. The incubation of lymphocytes with spin traps, 5,5-dimethyl-pyrroline N-oxide (DMPO) and PBN decreased the extent of DNA damage, which might follow from the production of free radicals by nickel. The pre-treatment with Vitamin C at 10 microM and Vitamin E at 25 microM decreased the tail moment of the cells exposed to NiCl(2) at the concentrations of the metal causing strand breaks or/and ALS. The results obtained suggest that free radicals may be involved in the formation of strand breaks or/and ALS in DNA as well as DNA-protein cross-links induced by NiCl(2). Nickel chloride can also alkylate DNA bases. Our results support thesis on multiple, free radicals-based genotoxicity pathways of nickel.     

-phenyl-tert-butyl-nitrone inhibits free radical release in brain concussion

Traumatic brain injury (TBI) is one of the important causes of mortality and morbidity. The pathogenesis of the underlying brain dysfunction is poorly understood. Recent data have suggested that oxygen free radicals play a key role in the primary and secondary processes of acute TBI. We report direct electron spin resonance (ESR) evidence of hydroxyl (·OH) radical generation in closed-head injury of rats. Moderate brain concussion was produced by controlled and reproducible mechanical, fixed, closed-head injury. A cortical cup was placed over one cerebral hemisphere within 20 min of the concussion, perfused with artificial cerebrospinal fluid (aCSF) containing the spin trap agent pyridyl-N-oxide-tert-butyl nitrone (POBN, 100 mM), and superfusate samples collected at 10 min intervals for a duration up to 130 min post brain trauma. In addition, POBN was administered systematically (50 mg/kg body wt.) 10 min pretrauma and 20 min posttrauma to improve our ability to detect free radicals. ESR analysis of the superfusate samples revealed six line spectra (_N = 15.4 and ^β_H = 2.5 G) characteristic of POBN-OH radical adducts, the intensity of which peaked 40 min posttrauma. The signal was undetectable after 120 min. Administration of -phenyl-tert-butyl-nitrone (PBN), a spin adduct forming agent systemically (100 mg/kg body wt. IP 10 min prior to concussion) alone or along with topical PBN (100 mM PBN in aCSF),6significantly (P< 0.001) attenuated the ESR signal, suggesting its possible role in the treatment of TBI.     

OH radical formation by photolysis of aqueous porphyrin solutions. A spin trapping and e.s.r. study

Radical production during the photolysis of deaerated aqueous alkaline solutions (pH 11) of some water-soluble porphyrins was investigated. Metal-free and metallo complexes of tetrakis (4-N-methylpyridyl)porphyrin (TMPyP) and tetra (4-sulphonatophenyl)porphyrin (TPPS4) were studied. Evidence for the formation of OH radicals during photolysis at 615, 545, 435, 408 and 335 nm of Fe(III) TPPS4 is presented. Fe(III) TMPyP, Mn(III) TPPS4 and Mn(III) TMPyP also gave OH radicals but only during photolysis at 335 nm. The method of spin trapping with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and 4-pyridyl-1-oxide-N-tert-butylnitrone (POBN) combined with e.s.r. was used for the detection of OH, H and hydrated electrons. With the spin trap DMPO, photolysis generated DMPO-OH adducts under certain conditions but no DMPO-H adducts could be observed. With POBN, no POBN-H adducts were found. The formation of OH was confirmed by studying competition reactions for OH between the spin traps and OH scavengers (formate, isopropanol) and the concomitant formation of the CO-2 adduct and the (CH3)2COH adduct with both DMPO and POBN. The photochemical generation of OH radicals was pH dependent; at pH 7.5 no OH radicals could be detected. Photolysis (615-335 nm) of dicyanocomplexes of the Fe(III) porphyrins did not produce OH radicals. When corresponding Cu(II), Ni(II), Zn(II) and metal-free porphyrins were photolysed at 615 and 335 nm, no OH radicals could be spin trapped. These results tend to associate the well-known phenomenon of photoreduction of Fe(III) and Mn(III) porphyrins with the formation of OH radicals. This process is described mainly as the photoreduction of the metal ion by the ligand-bound hydroxyl ion via an intramolecular process.     

Kinetics of anthracycline antibiotic free radical formation and reductive glycosidase activity

Adriamycin free radical anion concentrations have been correlated with production of 7-deoxyadriamycin aglycone in a reaction catalyzed by NADPH-cytochrome c reductase. The free radical species is detected by electron spin resonance (ESR) spectroscopy and quantified by double integrations. The 7-deoxyaglycone product is isolated by thin-layer chromatography (TLC) and quantified by fluorometry. As the concentration of adriamycin increases, a concomitant increase in aglycone and free radical levels occurs. These results as well as those with inhibitors Vitamin K₃, Vitamin E, and 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) point to an obligatory free radical intermediate in the metabolism of adriamycin. DMPO inhibits the reaction under aerobic conditions only, and shows no effect under anaerobiosis at the concentrations studied here. Vitamin E and aerobic DMPO act as free radical scavangers, while Vitamin K₃ competes for the reducing power of NADPH in the NADPH-cytochrome c reductase system.     

Carotenoids as antioxidants: spin trapping EPR and optical study.

The role of several natural and synthetic carotenoids as scavengers of free radicals was studied in homogeneous solutions. A set of free radicals: *OH, *OOH, and *CH(3) were generated by using the Fenton reaction in dimethyl sulfoxide. It was shown that the spin trapping technique is more informative than optical methods for the experimental conditions under study. 5,5-Dimethyl-pyrroline-N-oxide (DMPO) and N-tert-butyl-alpha-phenylnitrone (PBN) were used as spin traps for the EPR studies. The results show that the scavenging ability of the carotenoids towards radical *OOH correlates with their redox properties.     

Carotenoids as scavengers of free radicals in a Fenton reaction: antioxidants or pro-oxidants?

The spin trapping EPR technique was used to study the influence of carotenoids (beta-carotene, 8'-apo-beta-caroten-8'-al, canthaxanthin, and ethyl 8'-apo-beta-caroten-8'-oate) on the yield of free radicals in the Fenton reaction (Fe(2+) + H(2)O(2) --> Fe(3+) + .OH + -OH) in the organic solvents, DMSO, and methanol. DMPO and PBN were used as spin trapping agents. It was demonstrated that carotenoids could increase or decrease the total yield of free radicals depending on the oxidation potential of the carotenoids and the nature of the radicals. A reaction mechanism is suggested which includes the reduction of Fe(3+) to Fe(2+) by carotenoids. The effectiveness of this carotenoid-driven Fenton reaction increases with a decrease of the scavenging rates for free radicals and with decreasing oxidation potentials of carotenoids.