New sensitive agents for detecting singlet oxygen by electron spin resonance spectroscopy.

Free radicals are well-established transient intermediates in chemical and biological processes. Singlet oxygen, though not a free radical, is also a fairly common reactive chemical species. It is rare that singlet oxygen is studied with the electron spin resonance (ESR) technique in biological systems, because there are few suitable detecting agents. We have recently researched some semiquinone radicals. Specifically, our focus has been on bipyrazole derivatives, which slowly convert to semiquinone radicals in DMSO solution in the presence of potassium tert-butoxide and oxygen. These bipyrazole derivatives are dimers of 3-methyl-1-phenyl-2-pyrazolin-5-one and have anti-ischemic activities and free radical scavenging properties. In this work, we synthesized a new bipyrazole derivative, 4,4'-bis(1p-carboxyphenyl-3-methyl-5-hydroxyl)-pyrazole, DRD156. The resulting semiquinone radical, formed by reaction with singlet oxygen, was characterized by ESR spectroscopy. DRD156 gave no ESR signals from hydroxyl radical, superoxide, and hydrogen peroxide. DRD156, though, gives an ESR response with hypochlorite. This agent, nevertheless, has a much higher ability to detect singlet oxygen than traditional agents with the ESR technique.     

A re-evaluation of the antioxidant activity of purified carnosine

The antioxidant activity of carnosine has been re-evaluated due to the presence of contaminating hydrazine in commercial carnosine preparations. Purified carnosine is capable of scavenging peroxyl radicals. Inhibition of the oxidation of phosphatidylcholine liposomes by purified carnosine is greater in the presence of copper than iron, a phenomenon likely to be due to the copper chelating properties of carnosine. Purified carnosine is capable of forming adducts with aldehydic lipid oxidation products. Adduct formation is greatest for alpha,beta-monounsaturated followed by polyunsaturated and saturated aldehydes. While the ability of carnosine to form adducts with aldehydic lipid oxidation products is lower than other compounds such as glutathione, the higher concentrations of carnosine in skeletal muscle are likely to make it the most important molecule that forms aldehyde adducts. Monitoring changes in carnosine concentrations in oxidizing skeletal muscle shows that carnosine oxidation does not occur until the later stages of oxidation suggesting that carnosine may not be as effective free radical scavenger in vivo as other antioxidants like alpha-tocopherol.     

Stable free radical scavenging and antiperoxidative properties of resveratrol compared in vitro with some other bioflavonoids

Stable free radical scavenging and antiperoxidative activities of resveratrol, a component of grapes and red wine, were evaluated and compared with some other known bioflavonoids (quercetin, catechin, kaempferol, myricetin, fisetin, ellagic acid and naringenin) widely present in the plant kingdom. Free radical scavenging activity was measured in an in vitro chemical system (DPPH assay), while for antiperoxidative activity, biological system comprising of hepatic and pulmonary homogenates was employed. Antiradical activity assay showed quercetin and myricetin to be stronger antiradical agents than resveratrol. Structure-activity study revealed that O-dihydroxy group on ring B of flavonoid plays a crucial role. A double bond at 2-3 position conjugated with a 4-oxo function and hydroxy groups at positions 3 and 5 also contribute towards antiradical activity of flavonoids. Resveratrol exhibited stronger antiradical activity than kaempferol and naringenin and was also more efficient than alpha-tocopherol, a known strong endogenous non-flavonoid antioxidant, used for comparison. In vitro antiperoxidative assay showed fisetin as the strongest and kaempferol as the weakest antioxidant. Resveratrol was found to be stronger antioxidant than catechin, myricetin, kaempferol and naringenin, but was weaker than quercetin, fisetin and alpha-tocopherol. Antiradical and antiperoxidative activities of resveratrol may explain its beneficial effects in disease states. Assays exhibited no direct correlation between antiradical and antiperoxidative activities of the phenolics.     

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.     

Vitamin K-dependent carboxylase: Evidence for a semiquinone radical intermediate

Nanosecond laser flash photolysis has been used to produce and identify the vitamin K semiquinone (radical) from vitamin K dihydroquinone and to observe its formation and decay in the presence of vitamin K-dependent carboxylase (epoxidase). The activity of vitamin K-dependent carboxylase is not decreased by exposure to the laser. Absorbance of the semiquinone is proportional to enzyme concentration and is stimulated by a synthetic substrate, PheLeuGluGluIle. Stabilization of the semiquinone is observed in the presence of the enzyme. The semiquinone is rapidly destroyed in the presence of inhibitors of vitamin K-dependent carboxylase and vitamin K epoxidase.     

Free radical scavenging activity of propolis

We investigated the radical scavenging activity of propolis by ESR spectroscopy using spin trapping method. In addition, we examined the influence of a diet of 2% propolis on mice under oxidative stress. At low concentrations, the methanolic extract of propolis exhibited strong scavenging activity in vitro towards both the superoxide anion radical, generated by the hypoxanthine-xanthine oxidase reaction, and the NO radical, generated from the mixture of NOC-7 (NO generator) and carboxy-PTIO (spin trapping agent). An inhibitory effect of propolis on lipid peroxidation in vivo was observed, as determined by measurement of thiobarbituric acid-reactive substances in mouse liver homogenate. The level of vitamin C in the brain of mice under oxidative stress significantly increased compared with control mice under atmosphere, which was not observed in the mice given 2% propolis. The level of alpha-tocopherol in the brain of mice given 2% propolis significantly increased compared with control mice under atmosphere, which was not observed in mice under oxidative stress. SOD activity in the brain and plasma of mice given 2% propolis significantly decreased under atmosphere and oxidative stress compared with control mice. These results suggest that propolis possesses potent antioxidant activity in vitro and in vivo.     

Thiol oxidation induced by oxidative action of adriamycin

To clarify the mechanism of the cardiotoxic action of adriamycin (ADM), the participation of free radicals from ADM in cardiotoxicity was investigated through the protective action of glutathione (GSH) or by using electron spin resonance (ESR). Oxidation of ADM by horseradish peroxidase and H₂O₂ (HRP-H₂O₂) was blocked by GSH concentration dependently. Inactivation of creatine kinase (CK) induced during interaction of ADM with HRP-H₂O₂ was also protected by GSH. Other anthracycline antitumor drugs that have a p-hydroquinone structure in the B ring also inactivated CK, and GSH inhibited the inactivation of CK. These results suggest that ADM was activated through oxidation of the p-hydroquinone in the B ring by HRP-H₂O₂. Although ESR signals of the oxidative ADM B ring semiquinone were not detected, glutathionyl radicals were formed during the interaction of ADM with HRP-H₂O₂ in the presence of GSH. ADM may be oxidized to the ADM B ring semiquinone and then reacts with the SH group. However, ESR signals of ADM C ring semiquinone, which was reductively formed by xanthine oxidase (XO) and hypoxanthine (HX) under anaerobic conditions, were not diminished by GSH, but they completely disappeared with ferric ion. These results indicate that oxidative ADM B ring semiquinones oxidized the SH group in CK, but reductive ADM C ring semiquinone radicals may participate in the oxidation of lipids or DNA and not of the SH group.     

Oxidation mechanism of vitamin E analogue (Trolox C, 6-hydroxy-2,2,5,7,8-pentamethylchroman) and vitamin E by horseradish peroxidase and myoglobin.

The oxidation of 6-hydroxy-2,2,5,7,8-pentamethylchroman, Trolox C, and alpha-tocopherol by horseradish peroxidase was examined by stopped-flow and ESR experiments. The catalytic intermediate of horseradish peroxidase during the oxidation of vitamin E analogues and vitamin E was invariably Compound II, and rate constants for the rate-determining step decreased in the order 6-hydroxy-2,2,5,7,8-pentamethylchroman > Trolox C > alpha-tocopherol. The formation of phenoxyl radicals from substrates was verified with ESR and was followed optically. Resulting 6-hydroxy-2,2,5,7,8-pentamethylchroman and Trolox C radicals decayed through a dismutation reaction, followed by formation of the quinoid form via a transient intermediate. The sequence of events after formation of 6-hydroxy-2,2,5,7,8-pentamethylchroman and Trolox C radicals was similar to that observed by pulse radiolysis (Thomas, M. J., and Bielski, B. H. J. (1989). J. Am. Chem. Soc. 111, 3315-3319). Final oxidation products of 6-hydroxy-2,2,5,7,8-pentamethylchroman and Trolox C were identified as the quinoid forms and were obtained quantitatively whether or not the analogue had a carboxyl or methyl group at the 2-position of chroman ring. In contrast, enzymatic oxidation of alpha-tocopherol gave alpha-tocopherol quinone in very low yield. Conversion of 6-hydroxy-2,2,5,7,8-pentamethylchroman, Trolox C, and alpha-tocopherol to the corresponding quinones was also catalyzed by metmyoglobin in a reaction completely inhibited by ascorbate.     

Pro-carcinogenic activity of beta-carotene, a putative systemic photoprotectant.

Beta-carotene is a strong singlet oxygen quencher and antioxidant. Epidemiologic studies have implied that an above average intake of the carotenoid might reduce cancer risks. Earlier studies found that the carotenoid, when added to commercial closed-formula rodent diets, provided significant photoprotection against UV-carcinogenesis in mice. Clinical intervention trials found that beta-carotene supplementation evoked no change in incidence of nonmelanoma skin cancer. However, when smokers were supplemented with the carotenoid a significant increase in lung cancer resulted. Recently, employing a beta-carotene supplemented semi-defined diet, not only was no photoprotective effect found, but significant exacerbation of UV-carcinogenesis occurred. Earlier, a mechanism, based upon redox potential of interacting antioxidants, was proposed in which beta-carotene participated with vitamins E and C to efficiently repair oxy radicals and, thus, thought to provide photoprotection. In this schema, alpha-tocopherol would first intercept an oxy radical. In terminating the radical-propagating reaction, the tocopherol radical cation is formed which, in turn, is repaired by beta-carotene to form the carotenoid radical cation. This radical is repaired by ascorbic acid (vitamin C). As the carotenoid radical cation is a strongly oxidizing radical, unrepaired it could contribute to the exacerbating effect on UV-carcinogenesis. Thus, vitamin C levels could influence the levels of the pro-oxidant carotenoid radical cation. However, when hairless mice were fed beta-carotene supplemented semi-defined diet with varying levels of vitamin C (0-5590 mg kg(-1) diet) no effect on UV-carcinogenesis was observed. Lowering alpha-tocopherol levels did result in further increase of beta-carotene exacerbation, suggesting beta-carotene and alpha-tocopherol interaction. It was concluded that the non-injurious or protective effect of beta-carotene found in the closed-formula rations might depend on interaction with other dietary factors that are absent in the semi-defined diet. At present, beta-carotene use as a dietary supplement for photoprotection should be approached cautiously.     

Spin-trapping and direct electron spin resonance investigations of the redox metabolism of quinone anticancer drugs

The superoxide free radical has been spin trapped in microsomal incubations containing adriamycin, daunorubicin, and mitomycin C. The time sequence of the appearance of the spin-trapped superoxide and the semiquinone radical metabolite of these quinone-containing anticancer drugs indicates that air oxidation of the semiquinone is responsible for the superoxide formation. Superoxide dismutase prevents the formation of the superoxide spin adducts. Microsomal incubations containing anthracyclines intercalated in DNA produce much less superoxide than incubations free of DNA. The first unambiguous ESR evidence for the semiquinone metabolite of mitomycin C in a biological system is also presented.     

HPLC analysis of vitamin E isoforms in human epidermis: correlation with minimal erythema dose and free radical scavenging activity

The content and composition of different vitamin E isoforms was analyzed in normal human skin. Interestingly the epidermis contained 1% alpha-tocotrienol, 3% gamma-tocotrienol, 87% alpha-tocopherol, and 9% gamma-tocopherol. Although the levels of tocotrienol in human epidermis appear to be considerably lower than reported in the hairless mouse, the presence of significant amounts of tocotrienol levels leads to speculation about the physiological function of tocotrienols in skin. Besides antioxidant activity and photoprotection, tocotrienols may have skin barrier and growth-modulating properties. A good correlation was found for epidermal alpha-tocopherol (r = 0.7909, p <.0003), gamma-tocopherol (r = 0.556, p <.025), and the total vitamin E content (r = 0.831, p <.0001) with the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging in epidermis, as assessed by electron paramagnetic resonance (EPR) spectroscopy. In human epidermis, alpha-tocopherol is quantitatively the most important vitamin E isoform present and comprises the bulk of first line free radical defense in the lipid compartment. Epidermal tocotrienol levels were not correlated with DPPH scavenging activity. The minimal erythema dose (MED), an individual measure for sun sensitivity and a crude indicator for skin cancer susceptibility, did not correlate with the epidermal content of the vitamin E isoforms. Hence it is concluded that vitamin E alone is not a determinant of individual photosensitivity in humans.     

Thiol Oxidation Induced by Oxidative Action of Adriamycin

To clarify the mechanism of the cardiotoxic action of adriamycin (ADM), the participation of free radicals from ADM in cardiotoxicity was investigated through the protective action of glutathione (GSH) or by using electron spin resonance (ESR). Oxidation of ADM by horseradish peroxidase and H₂O₂ (HRP-H₂O₂) was blocked by GSH concentration dependently. Inactivation of creatine kinase (CK) induced during interaction of ADM with HRP-H₂O₂ was also protected by GSH. Other anthracycline antitumor drugs that have a p-hydroquinone structure in the B ring also inactivated CK, and GSH inhibited the inactivation of CK. These results suggest that ADM was activated through oxidation of the p-hydroquinone in the B ring by HRP-H₂O₂. Although ESR signals of the oxidative ADM B ring semiquinone were not detected, glutathionyl radicals were formed during the interaction of ADM with HRP-H₂O₂ in the presence of GSH. ADM may be oxidized to the ADM B ring semiquinone and then reacts with the SH group. However, ESR signals of ADM C ring semiquinone, which was reductively formed by xanthine oxidase (XO) and hypoxanthine (HX) under anaerobic conditions, were not diminished by GSH, but they completely disappeared with ferric ion. These results indicate that oxidative ADM B ring semiquinones oxidized the SH group in CK, but reductive ADM C ring semiquinone radicals may participate in the oxidation of lipids or DNA and not of the SH group.     

Electron spin resonance studies on a flavoprotein in neutrophil plasma membranes. Redox potentials of the flavin and its participation in NADPH oxidase

Plasma membrane fractions of stimulated and resting cells were isolated from pig blood neutrophils. The midpoint redox potential (Em) of the membrane-bound flavin was determined potentiometrically by analysis of the flavin free-radical signal by electron spin resonance (ESR) spectroscopy. In both stimulated and resting cells, a peak position of the titration curve gave an Em value of -280 mV at pH 7.0 (Em7). The flavin free radical showed an ESR spectrum at g = 2.004 with a peak to peak width of 19 G, which indicates that the redox intermediate is a neutral semiquinone. Redox titrations were anaerobically examined at 25 degrees C with NADPH in place of dithionite. Addition of NADPH to plasma membranes of stimulated cells resulted in a rapid change in potential, accompanied by the formation of the ESR signal of flavin free radical. Computer simulation of the titration points gave an ambient midpoint potential of -280 mV (Em7). In contrast, those of resting cells showed a very slow change in potential and no g = 2.00 signal formation. Power saturation behavior of the ESR signal showed a marked difference between those of stimulated and resting cells. ESR characteristics of the flavin are discussed in relation to the membrane-bound NADPH oxidase.     

Alpha-tocopherol supplementation does not affect monocyte endothelial adhesion or C-reactive protein levels but reduces soluble vascular adhesion molecule-1 in the plasma of healthy subjects

Vascular monocyte retention in the subintima is pivotal to the development of cardiovascular disease and is facilitated by up-regulation of adhesion molecules on monocytes/endothelial cells during oxidative stress. Epidemiological studies have shown that cardiovascular disease risk is inversely proportional to plasma levels of the dietary micronutrients, vitamin C and vitamin E (alpha-tocopherol). We have tested the hypothesis that alpha-tocopherol supplementation may alter endothelial/monocyte function and interaction in subjects with normal ascorbate levels (> 50 microM), as ascorbate has been shown to regenerate tocopherol from its oxidised tocopheroxyl radical form in vitro. Healthy male subjects received alpha-tocopherol supplements (400 IU RRR-alpha-tocopherol/day for 6 weeks) in a placebo-controlled, double-blind intervention study. There were no significant differences in monocyte CD11b expression, monocyte adhesion to endothelial cells, plasma C-reactive protein or sICAM-1 concentrations post-supplementation. There was no evidence for nuclear translocation of NF-kappaB in isolated resting monocytes, nor any effect of alpha-tocopherol supplementation. However, post-supplementation, sVCAM-1 levels were decreased in all subjects and sE-selectin levels were increased in the vitamin C-replete group only; a weak positive correlation was observed between sE-selectin and alpha-tocopherol concentration. In conclusion, alpha-tocopherol supplementation had little effect on cardiovascular disease risk factors in healthy subjects and the effects of tocopherol were not consistently affected by plasma vitamin C concentration.     

The reduction of anti-tumour diaziridinyl benzoquinones

The properties of the semiquinone radicals produced for 2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone (AZQ) and 2,5-bis(2-hydroxyethylamino)-3,6-diaziridinyl-1,4-benzoquinone (BZQ), have been investigated. AZQ semiquinone radicals can be produced from the reduction of AZQ by superoxide radicals, whereas BZQ semiquinone radicals are unstable in the presence of oxygen. The one-electron reduction potentials of the couples Q/Q-. at pH 7.0 were determined as -70 +/- 10 mV for AZQ and -376 +/- 15 mV for BZQ. The difference in these potentials is explained. As a consequence of ESR studies on the enzymatically produced radicals, we have considered the factors which determine the detection of ESR signals for reduced quinones produced in a biological system.     

Hydroxyl radical scavenging and antipsoriatic activity of benzoic acid derivatives

The hydroxyl radical scavenging and antipsoriatic activity of a number of lipophilic and hydrophilic benzoic acid derivatives was investigated. To quantify antioxidative effects, a newly introduced test system based on the diminution of the ESR signal of DMPO-OH (generated by Fenton's reagent) by the tested compounds was applied. It was found that the in vitro antioxidative (toward hydroxyl radical) activity of benzoic acid esters decreases with increasing chain length whereas the antipsoriatic activity increases. This effect is discussed in terms of a larger lipophilicity of long-chain esters. Propyl gallate was found to be the most active OH scavenger since it is some orders of magnitude more efficient than "model" antioxidants like alpha-tocopherol or mannitol. The highest antipsoriatic activity was exhibited by hydroxy benzoic acid decyl ester.     

Detection of a paramagnetic product developing during oxidative phosphorylation in mitochondria

It was shown by ESR technique using flow system combined with ESR-spectrometer that paramagnetic product appearing in the course of oxidative phosphorylation was directly associated with mitochondrial ATPase operation. A decrease of ESR signal intensity and the changes of its form observed on mitochondria uncoupled by 2,4-dinitrophenol as compared with those inhibited by olygomicin suggest that in the case of olygomycin block a free radical ATPase linked intermediate can be recorded, the ESR signal of which seems to be partly due to flavin semiquinone of ATPsynthetase itself.     

Reaction of beta-alkannin (shikonin) with reactive oxygen species: detection of beta-alkannin free radicals

beta-Alkannin (shikonin), a compound isolated from the root of Lithospermum erythrorhizon Siebold Zucc., has been used as a purple dye in ancient Japan and is known to exert an anti-inflammatory activity. This study aimed to understand the biological activity in terms of physico-chemical characteristics of beta-alkannin. Several physico-chemical properties including proton dissociation constants, half-wave potentials and molecular orbital energy of beta-alkannin were elucidated. This compound shows highly efficient antioxidative activities against several types of reactive oxygen species (ROS), such as singlet oxygen ((1)O2). superoxide anion radical (.O2), hydroxyl radical (.OH) and tert-butyl peroxyl radical (BuOO.) as well as iron-dependent microsomal lipid peroxidation. During the reactions of beta-alkannin with 1O2, .O2- and BuOO., intermediate organic radicals due to beta-alkannin were detectable by ESR spectrometry. Compared with the radicals due to naphthazarin, the structural skeleton of beta-alkannin, the beta-alkannin radical observed as an intermediate in the reactions with (1)O2, and .O2- was concluded to be a semiquinone radical. On the other hand, during the reactions of beta-alkannin and naphthazarin with BuOO., ESR spectra different from the semiquinone radical were observed, and proposed to result from the abstraction of hydrogen atoms from phenolic hydroxyl groups of beta-alkannin by BuOO.. Based on the ROS-scavenging abilities of beta-alkannin, the compound was concluded to react directly with ROS and exhibits antioxidative activity, which in turn exerts anti-inflammatory activity.     

An electron spin resonance study on alkylperoxyl radical in thin-sliced renal tissues from ferric nitrilotriacetate-treated rats: the effect of alpha-tocopherol feeding.

Formation of excess free radical causes cellular oxidative stress, which has been shown to be associated with a variety of pathologic conditions. While electron spin resonance (ESR) spectroscopy has been the only method to demonstrate the presence of free radicals, its application to tissue samples has been challenging. We report here the successful ESR detection in thin-sliced fresh tissues or frozen sections in a rat model. Ferric nitrilotriacetate (Fe-NTA) induces oxidative renal tubular damage that ultimately leads to high incidence of renal carcinoma in rodents. Twenty minutes after administration of 5 mg iron/kg Fe-NTA to rats, a thin-slice of the kidney was mounted on a tissue-type cell and analyzed by ESR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). An ESR signal from alkylperoxyl radical adduct was obtained, and the signal was inversely proportional to renal alpha-tocopherol content which was modulated through diet. Furthermore, we undertook ex vivo study using frozen sections. Fe-NTA (1 mM) was added to a rat kidney frozen section for 10 min. After washing the specimen was mounted on a tissue-type cell and analyzed with ESR spin trapping using DMPO. Alkylperoxyl radical signal was dependent on thickness, incubation time and renal tissue levels of alpha-tocopherol, and was reduced by preincubation with catalase or dimethyl sulfoxide but not with alpha-tocopherol outside tissue. This versatile method facilitates identification of free radicals in pathologic conditions, and may be useful for selection of antioxidants.     

Generation and recycling of radicals from phenolic antioxidants

Hindered phenols are widely used food preservatives. Their pharmacological properties are usually attributed to high antioxidant activity due to efficient scavenging of free radicals. Butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) also cause tissue damage. Their toxic effects could be due to the production of phenoxyl radicals. If phenoxyl radicals can be recycled by reductants or electron transport, their potentially harmful side reactions would be minimized. A simple and convenient method to follow phenoxyl radical reactions in liposomes and rat liver microsomes based on an enzymatic (lipoxygenase + linolenic acid) oxidation system was used to generate phenoxyl radicals from BHT and its homologues with substitutents in m- and p-positions. Different BHT-homologues display characteristic ESR signals of their radical species. In a few instances the absence of phenoxyl radical ESR signals was found to be due to inhibition of lipoxygenase by BHT-homologues. In liposome or microsome suspensions addition of ascorbyl palmitate resulted in disappearance of the ESR signal of phenoxyl radicals with concomittant appearance of the ascorbyl radical signal. After exhaustion of ascorbate, the phenoxyl radical signal reappears. Comparison of the rates of ascorbyl radical decay in the presence or absence of BHT-homologues showed that temporary elimination of the phenoxyl radical ESR signal was due to their reduction by ascorbate. Similarly, NADPH or NADH caused temporary elimination of ESR signals as a result of reduction of phenoxyl radicals in microsomes. Since ascorbate and NADPH might generate superoxide in the incubation system used, SOD was tested. SOD shortened the period, during which the phenoxyl radicals ESR signal could not be observed. Both ascorbyl palmitate and NADPH exerted sparing effects on the loss of BHT-homologues during oxidation. These effects were partly diminished by SOD. These data indicate that reduction of phenoxyl radicals was partly superoxide-dependent. It is concluded that redox recycling of phenoxyl radicals can occur by intracellular reductants like ascorbate and microsomal electron transport.