Spin-trapping of superoxide by 5,5-dimethyl-1-pyrroline N-oxide: application to isolated perfused organs

Of the available techniques used to identify free radicals, spin-trapping offers the unique opportunity to simultaneously measure and distinguish among a variety of important biologically generated free radicals. For superoxide and hydroxyl radical, the spin trap 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) is most frequently used. However, this nitrone has several drawbacks. For example, its reaction with superoxide is slow, having a second-order rate constant around 10 M-1 s-1. Because of this, high concentrations of DMPO are essential in order to observe the corresponding spin-trapped adduct, 5,5-dimethyl-2-hydroperoxy-1-pyrrolidinyloxy. This may, in some cases, lead to cellular toxicity. In an attempt to circumvent this serious limitation, it has been proposed that an indirect approach be employed to detect and identify free radicals generated as a consequence of ischemia/reperfusion injury. In the direct (most frequently used) approach, the spin trap is first added to an isolated perfused organ under the appropriate experimental conditions. Then, the infusion buffer containing the spin-trap adduct(s) is placed into an quartz flat cell to be inserted into an ESR spectrometer. In the indirect method, the spin trap is added to the perfusate, which had previously exited the organ. Therefore, with this method one can prevent any spin-trap-mediated toxicities to the isolated perfused organ. However, because of the very rapid rate of free radical reactions catalyzed by either superoxide or hydroxyl radical, it is questionable whether ESR spectra recorded using this indirect method result from the actual spin-trapping of free radicals. In this report, we evaluated the indirect spin-trapping technique in light of the kinetic considerations discussed above.     

Formation of reactive sulfite-derived free radicals by the activation of human neutrophils: an ESR study

The objective of this study was to determine the effect of (bi)sulfite (hydrated sulfur dioxide) on human neutrophils and the ability of these immune cells to produce reactive free radicals due to (bi)sulfite oxidation. Myeloperoxidase (MPO) is an abundant heme protein in neutrophils that catalyzes the formation of cytotoxic oxidants implicated in asthma and inflammatory disorders. In this study sulfite (^?SO₃^?) and sulfate (SO₄^??) anion radicals are characterized with the ESR spin-trapping technique using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in the reaction of (bi)sulfite oxidation by human MPO and human neutrophils via sulfite radical chain reaction chemistry. After treatment with (bi)sulfite, phorbol 12-myristate 13-acetate-stimulated neutrophils produced DMPO–sulfite anion radical, –superoxide, and –hydroxyl radical adducts. The last adduct probably resulted, in part, from the conversion of DMPO–sulfate to DMPO–hydroxyl radical adduct via a nucleophilic substitution reaction of the radical adduct. This anion radical (SO₄^??) is highly reactive and, presumably, can oxidize target proteins to protein radicals, thereby initiating protein oxidation. Therefore, we propose that the potential toxicity of (bi)sulfite during pulmonary inflammation or lung-associated diseases such as asthma may be related to free radical formation.     

Spin-trapping and human neutrophils. Limits of detection of hydroxyl radical

Using the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) and an excess of dimethyl sulfoxide, we previously reported that in the absence of an exogenous iron catalyst, human neutrophils will not generate hydroxyl radical, manifested as the catalse-inhibitable methyl radical spin-trapped adduct, 2,2,5-trimethyl-1-pyrrolidinyloxy (DMPO-CH3) (Britigan, B. E., Rosen, G. M., Chai, Y., and Cohen, M. S. (1986) J. Biol. Chem. 261, 4426-4431). However, superoxide destroys the preformed hydroxyl radical spin-trapped adduct, 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxy (DMPO-OH), and DMPO-CH3. The present study was undertaken to better resolve the limits of sensitivity of the spin-trapping method. Photolytically generated DMPO-CH3 and DMPO-OH slowly decomposed in the presence of a low flux (1 microM/min) of enzymatically (xanthine/xanthine oxidase)-generated superoxide, but more rapid decomposition of these adducts occurred with higher superoxide flux (5 microM/min). Inclusion of cysteine markedly increased the rate of DMPO-OH and DMPO-CH3 decomposition, masking the effect of superoxide alone. The addition of varying concentrations of superoxide dismutase did not lead to increased formation of DMPO-OH or DMPO-CH3, as should have occurred if these adducts were being destroyed by superoxide. As a positive control, we employed an iron-supplemented system with phorbol 12-myristate 13-acetate-stimulated neutrophils or xanthine/xanthine oxidase to generate DMPO-CH3. Addition of superoxide dismutase increased the magnitude of DMPO-CH3, primarily by increasing the rate of hydrogen peroxide formation, and to a lesser extent by prolonging the half-life of DMPO-CH3. Although spin-trapped adducts can be destroyed by a high concentration of superoxide, or by lower concentrations of superoxide in the presence of thiol-containing compounds, our results demonstrate that such decomposition does not interfere with the ability of the spin-trapping method to detect hydroxyl radical generated by human neutrophils. These data do not support the capacity of neutrophils to generate hydroxyl radical in the absence of an exogenous Haber-Weiss catalyst.     

Hydroxyl radical production by stimulated neutrophils reappraised

Release of active oxygen species during the human neutrophil respiratory burst is thought to be mandatory for effective defense against bacterial infections and may play an important role in damage to host tissues. Part of the critical bacterial and host tissue damage has been attributed to hydroxyl radicals produced from superoxide and hydrogen peroxide. Because of the short life time of the very reactive hydroxyl radical, direct study of hydroxyl radical production is not possible; therefore, indirect detection methods such as electron spin resonance (ESR) coupled with appropriate spin-trapping agents such as 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) have been used. Superoxide production during the oxidative burst has been unambiguously demonstrated. Recent reports claim that hydroxyl radicals are not made during neutrophil stimulation and offer as an explanation the presence of granular components that interfere with hydroxyl radical production. When using the spin-trap agent DMPO, absence of the relatively long-lived adducts DMPO-OH and DMPO-CH3 has been assumed to be prima facie evidence for lack of hydroxyl radical participation. We show that high superoxide flux produced during stimulation of human neutrophils rapidly destroys both DMPO-OH and DMPO-CH3. In accord with previous implications, our results provide an alternative explanation for the absence of .OH adduct in spin-trapping studies and corroborate results obtained using other methods that implicate hydroxyl radical production during neutrophil stimulation.     

Activation of the superoxide-generating NADPH oxidase of intestinal lymphocytes produces highly reactive free radicals from sulfite.

The objective of this study was to investigate the ability of immune cells of the small intestine to produce highly reactive free radicals from the food additive sulfites. These free radicals were characterized with a spin-trapping technique using the spin traps 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). In the presence of glucose, purified lymphocytes from intestinal Peyer's patches (PP) and mesenteric lymph nodes (MLN) were stimulated with phorbol 12-myristate 13-acetate (PMA) to produce superoxide and hydroxyl DEPMPO radical adducts. The formation of these adducts was inhibited by superoxide dismutase or diphenyleneiodonium chloride, indicating that these cells produced superoxide radical during reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. With the treatment of sodium sulfite, PMA-stimulated PP lymphocytes produced a DEPMPO-sulfite radical adduct and an unknown radical adduct. When DEPMPO was replaced with DMPO, DMPO-sulfite and hydroxyl radical adducts were detected. The latter adduct resulted from DMPO oxidation by sulfate radical, which was capable of oxidizing formate or ethanol. Oxygen consumption rates were further increased after the addition of sulfite to PMA-stimulated lymphocytes, suggesting the presence of sulfiteperoxyl radical. Taken together, oxidants generated by stimulated lymphocytes oxidized sulfite to sulfite radical, which subsequently formed sulfiteperoxyl and sulfate radicals. The latter two radicals are highly reactive, contributing to increased oxidative stress, which may lead to sulfite toxicity, altered functions in intestinal lymphocytes, or both.     

Spin-trapping of superoxide by 5,5-dimethyl-1-pyrroline N-oxide: Application to isolated perfused organs

Of the available techniques used to identify free radicals, spin-trapping offers the unique opportunity to simultaneously measure and distinguish among a variety of important biologically generated free radicals. For superoxide and hydroxyl radical, the spin trap 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) is most frequently used. However, this nitrone has several drawbacks. For example, its reaction with superoxide is slow, having a second-order rate constant around 10 ^{−1 −1}. Because of this, high concentrations of DMPO are essential in order to observe the corresponding spin-trapped adduct, 5,5-dimethyl-2-hydroperoxy-1-pyrrolidinyloxy. This may, in some cases, lead to cellular toxicity. In an attempt to circumvent this serious limitation, it has been proposed that an indirect approach be employed to detect and identify free radicals generated as a consequence of ischemia/reperfusion injury. In the direct (most frequently used) approach, the spin trap is first added to an isolated perfused organ under the appropriate experimental conditions. Then, the infusion buffer containing the spin-trap adduct(s) is placed into an quartz flat cell to be inserted into an ESR spectrometer. In the indirect method, the spin trap is added to the perfusate, which had previously exited the organ. Therefore, with this method one can prevent any spin-trap-mediated toxicities to the isolated perfused organ. However, because of the very rapid rate of free radical reactions catalyzed by either superoxide or hydroxyl radical, it is questionable whether ESR spectra recorded using this indirect method result from the actual spin-trapping of free radicals. In this report, we evaluated the indirect spin-trapping technique in light of the kinetic considerations discussed above.     

Reevaluation of the spin-trapped adduct formed from 5,5-dimethyl-1-pyrroline-1-oxide during the respiratory burst in neutrophils

By employing EPR spectrometry with the aid of a spin-trapping agent, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO), the generation of superoxide anion and hydroxyl radical was reevaluated during the respiratory burst of porcine and human neutrophils. Properly prepared resting neutrophils did not generate any spin-trapped radical, and, when the cells were stimulated with phorbol myristate acetate, only DMPO-OOH, the spin-trapped adduct of superoxide anion, was detected. No formation of DMPO-OH, the spin-trapped adduct of the hydroxyl radical, was observed. DMPO-OOH was also detected principally when the neutrophils were stimulated with opsonized zymosan, a particulate stimulus. In the latter case, however, the formation of DMPO-OOH ceased shortly after the addition of zymosan and subsequent production of DMPO-OH was observed. The production of DMPO-OH was found to be associated with cell injury. DMPO at the concentration usually used for the experiment (0.045-0.09 M) injured phagocytizing neutrophils, causing lysis of the cells. On the other hand, an addition of cell homogenate or glutathione-glutathione peroxidase system to the suspension of intact cells which were producing DMPO-OOH resulted in the formation of DMPO-OH. Thus, DMPO-OH was probably derived from DMPO-OOH by the action of enzymes and/or factor(s) which were released from the lysed cells.     

Catalysis of the Haber-Weiss reaction by iron-diethylenetriaminepentaacetate.

To help settle controversy as to whether the chelating agent diethylenetriaminepentaacetate (DTPA) supports or prevents hydroxyl radical production by superoxide/hydrogen peroxide systems, we have reinvestigated the question by spectroscopic, kinetic, and thermodynamic analyses. Potassium superoxide in DMSO was found to reduce Fe(III)DTPA. The rate constant for autoxidation of Fe(II)DTPA was found (by electron paramagnetic resonance spectroscopy) to be 3.10 M-1 s-1, which leads to a predicted rate constant for reduction of Fe(III)DTPA by superoxide of 5.9 x 10(3) M-1 s-1 in aqueous solution. This reduction is a necessary requirement for catalytic production of hydroxyl radicals via the Fenton reaction and is confirmed by spin-trapping experiments using DMPO. In the presence of Fe(III)DTPA, the xanthine/xanthine oxidase system generates hydroxyl radicals. The reaction is inhibited by both superoxide dismutase and catalase (indicating that both superoxide and hydrogen peroxide are required for generation of HO.). The generation of hydroxyl radicals (rather than oxidation side-products of DMPO and DMPO adducts) is attested to by the trapping of alpha-hydroxethyl radicals in the presence of 9% ethanol. Generation of HO. upon reaction of H2O2 with Fe(II)DTPA (the Fenton reaction) can be inhibited by catalase, but not superoxide dismutase. The data strongly indicate that iron-DTPA can catalyze the Haber-Weiss reaction.     

A comparison of cobalt(II) and iron(II) hydroxyl and superoxide free radical formation

We have employed the electron spin resonance spin-trapping technique to study the reaction of Co(II) with hydrogen peroxide in a chemical system and in a microsomal system. In both cases, we employed the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and were able to detect the formation of DMPO/.OH and DMPO/.OOH. DMPO/.OOH was the predominant radical adduct formed in the chemical system, while the two adducts were of similar concentrations in the microsomal system. The formation of both of these adducts in either reaction system was inhibited by the addition of superoxide dismutase or catalase, and by chelating the cobalt with either ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA). The incorporation of the hydroxyl radical scavengers ethanol, formate, benzoate, or mannitol inhibited the formation of DMPO/.OH in both systems. We also repeated the study using Fe(II) in place of Co(II). In contrast to the Co(II) results, Fe(II) reacted with hydrogen peroxide to yield only DMPO/.OH, and this adduct formation was relatively insensitive to the presence of added superoxide dismutase. In addition, Fe(II)-mediated DMPO/.OH formation increased when the iron was chelated to either EDTA or DTPA rather than being inhibited as for Co(II). Thus, we propose that Co(II) does not react with hydrogen peroxide by the classical Fenton reaction at physiological pH values.     

Free Radical-mediated Formation of Ethylene from 1-Aminocyclopropane-1-carboxylic Acid: A Spin-frap Study

The ability of free radicals to convert l-aminocyclopropane-l-carboxylicacid (ACC) to ethylene under strictly chemical conditions hasbeen investigated using the aerobic xanthine/xanthine oxidasereaction and the Fenton reaction. Ethylene is formed when 1mM ACC is added to either of these reactions. Ethylene productionby the xanthine/xanthine oxidase system can be stimulated byH₂O₂ and inhibited by both catalase and superoxide dismutase,suggesting that the hydroxyl radical (OH?) formed by the Haber-Weissreaction is reacting with ACC to form ethylene. Ethylene productionfrom ACC by the Fenton reagent, which also produces OH?, showsa strong dependence upon H₂O₂. Involvement of the OH? radicalwas confirmed by spin-trap studies using 5,5-dimethyl-l-pyrroline-l-oxide(DMPO). Only the hydroxyl adduct of DMPO was detectable in boththe xanthine/xanthine oxidase reaction and the Fenton reaction.When ACC was added to the Fenton reaction, an additional adductof DMPO was detectable, which, on the basis of its hyperfinesplitting constants, can be tentatively identified as the DMPOadduct of a carbon-centered free radical. The data are consistentwith the view that formation of ethylene from ACC entails attackby OH? and the resultant formation of a carbon-centered radical,possibly of ACC. The chemical conversion of ACC to ethyleneis less efficient than that characteristic of senescing tissues,in which the reaction is enzymatically mediated. (Received October 1, 1981; Accepted November 17, 1981)     

Inhibition of the microsomal oxidation of ethanol and 1-butanol by the free-radical, spin-trapping agent 5,5-dimethyl-1-pyrroline-1-oxide

The free-radical, spin-trapping agent, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO), inhibited the microsomal oxidation of ethanol and 1-butanol as well as the metabolism of 2-keto-4-thiomethylbutyrate. DMPO also blocked the stimulation of ethanol oxidation caused by the addition of iron-EDTA to microsomes. Previous electron spin resonance studies with DMPO by others had shown that the free radical produced by rat liver microsomes and augmented by iron-EDTA was the hydroxyl radical. In the current study, DMPO had no effect on microsomal oxygen consumption with NADPH as substrate, nor on NADPH-cytochrome c reductase activity, nor on the demethylation of aminopyrine or the hydroxylation of aniline. Therefore, a general toxic action of DMPO on microsomes is excluded. DMPO also failed to inhibit the catalase-dependent pathway of ethanol oxidation by microsomes. Experiments with Chelex 100 resin and the chelating agent, diethylenetri-aminepentaacetic acid, rule out any major role for contaminating iron in the reagents. These results tend to identify the free radical responsible for microsomal metabolism of ethanol, 1-butanol, and 2-keto-4-thiomethylbutyrate as the radical trapped by DMPO, namely the hydroxyl radical.     

Detection of free radicals during the cellular metabolism of adriamycin

Experiments were conducted to determine which free radicals are generated during the metabolism of adriamycin (ADM) by canine tracheal epithelial (CTE) cells, guinea pig enterocytes, and rat hepatocytes. The technique employed in this study was spin trapping; the spin trap utilized was 5,5-dimethyl-1-pyrroline-1-oxide (DMPO). The spin adduct 2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxyl (DMPO-OH) was observed during the metabolism of ADM by CTE cells. However, the addition of dimethyl sulfoxide to the in vitro system suggested that superoxide is initially spin trapped by the nitrone, and that the adduct 2-hydroperoxy-5,5-dimethyl-1-pyrrolidinyloxyl (DMPO-OOH) is rapidly bioreduced to afford DMPO-OH. The addition of superoxide dismutase to the system indicated that superoxide generation was primarily intracellular. The adriamycin semiquinone free radical (ADM-SQ) was produced during the metabolism by enterocytes and hepatocytes. The rate of the production of ADM-SQ was enhanced under anaerobic conditions, suggesting that molecular oxygen was responsible for the degradation of this carbon-centered free radical. However, spin trapping of oxygen radicals was not observed; this observation suggests that these reactive intermediates are not produced at concentrations sufficient for detection by spin-trapping experiments.     

Glutathionyl- and hydroxyl radical formation coupled to the redox transitions of 1,4-naphthoquinone bioreductive alkylating agents during glutathione two-electron reductive addition.

The kinetic parameters of the redox transitions subsequent to the two-electron transfer implied in the glutathione (GSH) reductive addition to 2- and 6-hydroxymethyl-1,4-naphthoquinone bioalkylating agents were examined in terms of autoxidation, GSH consumption in the arylation reaction, oxidation of the thiol to glutathione disulfide (GSSG), and free radical formation detected by the spin-trapping electron spin resonance method. The position of the hydroxymethyl substituent in either the benzenoid or the quinonoid ring differentially influenced the initial rates of hydroquinone autoxidation as well as thiol oxidation. Thus, GSSG- and hydrogen peroxide formation during the GSH reductive addition to 6-hydroxymethyl-1,4-naphthoquinone proceeded at rates substantially higher than those observed with the 2-hydroxymethyl derivative. The distribution and concentration of molecular end products, however, was the same for both quinones, regardless of the position of the hydroxymethyl substituent. The [O2]consumed/[GSSG]formed ratio was above unity in both cases, thus indicating the occurrence of autoxidation reactions other than those involved during GSSG formation. EPR studies using the spin probe 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) suggested that the oxidation of GSH coupled to the above redox transitions involved the formation of radicals of differing structure, such as hydroxyl and thiyl radicals. These were identified as the corresponding DMPO adducts. The detection of either DMPO adduct depended on the concentration of GSH in the reaction mixture: the hydroxyl radical adduct of DMPO prevailed at low GSH concentrations, whereas the thiyl radical adduct of DMPO prevailed at high GSH concentrations. The production of the former adduct was sensitive to catalase, whereas that of the latter was sensitive to superoxide dismutase as well as to catalase. The relevance of free radical formation coupled to thiol oxidation is discussed in terms of the thermodynamic and kinetic properties of the reactions involved as well as in terms of potential implications in quinone cytotoxicity.     

Use of high-performance liquid chromatography to detect hydroxyl and superoxide radicals generated from mitomycin C

Distinguishing between short-lived reactive oxygen species like hydroxyl and superoxide radicals is difficult; the most successful approaches employ electron spin resonance (ESR) spin-trapping techniques. Using the spin trap 5,5-dimethyl-l-pyrroline N-oxide (DMPO) to selectively trap various radicals in the presence and absence of ethanol, an HPLC system which is capable of separating the hydroxyl- and superoxide-generated DMPO adduct species has been developed. The radical-generated DMPO adducts were measured with an electrochemical detector attached to the HPLC system and confirmed by spin-trapping techniques. The HPLC separation was carried out on an ODS reverse-phase column with a pH 5.1 buffered 8.5% acetonitrile mobile phase. The advantage of the HPLC system described is that it permits the separation and detection of hydroxyl and superoxide radicals without requiring ESR instrumentation. The antineoplastic bioreductive alkylating agent mitomycin C, when activated by NADPH-cytochrome c reductase, was shown to generate both hydroxyl and superoxide radicals.     

Formation of superoxide and hydroxyl radicals from 1-methyl-4-phenylpyridinium ion (MPP+): reductive activation by NADPH cytochrome P-450 reductase

Formation of free radical intermediates from 1--methyl-4-phenylpyridinium ion(MPP+) has been studied using spin-trapping techniques. Incubation of MPP+ with purified NADPH cytochrome P-450 reductase and NADPH under anaerobic conditions failed to produce any detectable radical intermediates. However, in the presence of air and a spin-trap, a significant stimulation of superoxide and hydroxyl radicals was detected. Formation of these toxic radicals from MPP+ was inhibited by superoxide dismutase, catalase, and ethanol. Under identical conditions, however, considerably less of these radicals were formed with MPP+ in comparison to paraquat, a lung toxin containing two pyridinium moieties.     

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.     

Hydroxyl radical generation by red tide algae.

The unicellular marine phytoplankton Chattonella marina is known to have toxic effects against various living marine organisms, especially fishes. However, details of the mechanism of the toxicity of this plankton remain obscure. Here we demonstrate the generation of superoxide and hydroxyl radicals from a red tide unicellular organism, C. marina, by using ESR spectroscopy with the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and N-t-butyl-alpha-phenylnitrone (PBN), and by using the luminol-enhanced chemiluminescence response. The spin-trapping assay revealed productions of spin adduct of superoxide anion (O2-) (DMPO-OOH) and that of hydroxyl radical (.OH) (DMPO-OH) in the algal suspension, which was not observed in the ultrasonic-ruptured suspension. The addition of superoxide dismutase (500 U/ml) almost completely inhibited the formation of both DMPO-OOH and DMPO-OH, and carbon-centered radicals were generated with the disappearance of DMPO-OH after addition of 5% dimethyl sulfoxide (Me2SO) and 5% ethanol. Furthermore, the generation of methyl and methoxyl radicals, which are thought to be produced by the reaction of hydroxyl radical and Me2SO under aerobic condition, was identified using spin trapping with a combination of PBN and Me2SO. Luminol-enhanced chemiluminescence assay also supported the above observations. These results clearly indicate that C. marina generates and releases the superoxide radical followed by the production of hydroxyl radical to the surrounding environment. The velocity of superoxide generation by C. marina was about 100 times faster than that by mammalian phagocytes per cell basis. The generation of oxygen radical is suggested to be a pathogenic principle in the toxication of red tide to susceptible aquaculture fishes and may be directly correlated with the coastal pollution by red tide.     

Photosensitization by antitumor agents 3: spectroscopic evidence for superoxide and hydroxyl radical production by anthrapyrazole-sensitized oxidation of nadh

EPR and spin-trapping techniques were employed to study the oxidation of the dihydronicotinamide adenine dinucleotide (NADH) photosensitized by an anthrapyrazole-antitumor agent. The superoxide radical was detected as a DMPO adduct upon illumination of the system with visible light. Photoinduced generation of hydroxyl radicals is demonstrated by detection of DMPO adducts of OH scavengers, such as ethyl alcohol, sodium formate, and sodium azide. The dependence of the production of these spin adducts on the presence of catalase implies the involvement of hydrogen peroxide in that process. The production of hydrogen peroxide is demonstrated independently during oxygen consumption measurements with the Clark electrode technique.     

Toxic Effects and Detection of Oxygen Free Radicals on Cultured Articular Chondrocytes Generated by Menadione

The aim of this work was to study the proliferation pathological perturbations of cultured chondrocytes in response to menadione, an oxygen free radicals producing drug. Rabbit articular chondrocytes in monolayer culture were treated with 10^-5, 1.5.M^-5 and 2.10^-5M of menadione during three days. A dose dependent decrease of the proliferative capacity was observed. Flow cytometry analysis revealed a perturbation of the cell cycle progression consisting in an accumulation of cells in the S and G2 + M phases. This growth perturbation was due to oxygen radicals production since a treatment with catalase suppressed these toxic effects. Furthermore, to identify oxygen derived radicals in the cellular suspension of cultures treated with menadione, we used a technique of spin-trapping coupled with electron spin resonance (ESR). The ESR signal corresponding to the DMPO hydroxyl radical adduct (DMPO-OH) has been detected. The spectra observation indicated the actual production of hydroxyl radical. However, superoxide anions have not been identified; this fact can be explained by the low reactivity of these anions with DMPO and by the decomposition of signal DMPO-OOH to DMPO-OH.     

Mechanisms of generation of oxygen radicals and reductive mobilization of ferritin iron by lipoamide dehydrogenase.

The oxidase reaction of lipoamide dehydrogenase with NADH generates superoxide radicals and hydrogen peroxide under aerobic conditions. ESR spin trapping using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was applied to characterize the oxygen radical species generated by lipoamide dehydrogenase and the mechanism of their generation. During the oxidase reaction of lipoamide dehydrogenase, DMPO-OOH and DMPO-OH signals were observed. The DMPO-OOH signal disappeared on addition of superoxide dismutase. These results demonstrate that the DMPO-OOH adduct was produced from the superoxide radical generated by lipoamide dehydrogenase. In the presence of dimethyl sulfoxide, a DMPO-CH3 signal appeared at the expense of the DMPO-OH signal, indicating that the DMPO-OH adduct was produced directly from the hydroxyl radical rather than by decomposition of the DMPO-OOH adduct. The DMPO-OH signal decreased on addition of superoxide dismutase, catalase, or diethylenetriaminepentaacetic acid, indicating that the hydroxyl radical was generated via the metal-catalyzed Haber-Weiss reaction from the superoxide radical and hydrogen peroxide. Addition of ferritin to the NADH-lipoamide dehydrogenase system resulted in a decrease of the DMPO-OOH signal, indicating that the superoxide radical interacted with ferritin iron.