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Sekwon Kim - One of the best experts on this subject based on the ideXlab platform.
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in vitro antioxidant activity of a peptide isolated from nile tilapia oreochromis niloticus scale gelatin in free radical mediated oxidative systems
Journal of Functional Foods, 2010Co-Authors: Dai Hung Ngo, Zhong Ji Qian, Bomi Ryu, Jae W Park, Sekwon KimAbstract:Abstract In the present study, a peptide possessing antioxidant properties was isolated from Nile tilapia ( Oreochromis niloticus ) scale gelatin. Gelatin protein was hydrolyzed using alcalase, pronase E, trypsin and pepsin. Antioxidant efficacy of respective hydrolysates were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical and superoxide radical anion scavenging activities. Moreover, protective effect on DNA damage caused by hydroxyl radicals generated was determined. Further, the level of reactive oxygen species (ROS) was determined using a fluorescence probe, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), which could be converted to highly fluorescent Dichlorofluorescein (DCF) with the presence of intracellular ROS on mouse macrophages, RAW 264.7 cells. Among hydrolysates, alcalase-derived hydrolysate exhibited the highest antioxidant activity compared to other enzymatic hydrolysates. Therefore, it was further analyzed and the sequence of an active peptide present in it was identified as Asp-Pro-Ala-Leu-Ala-Thr-Glu-Pro-Asp-Pro-Met-Pro-Phe (1382.57 Da). This peptide showed no cytotoxic effect on mouse macrophages (RAW 264.7) and human lung fibroblasts (MRC-5). In addition, it scavenged hydroxyl, DPPH and superoxide radicals at the IC 50 values of 7.56, 8.82 and 17.83 μM, respectively. These results suggest that the peptide derived from Nile tilapia ( O. niloticus ) scale gelatin acts as a candidate against oxidative stress and could be used as a potential functional food ingredient.
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in vitro antioxidant activity of a peptide isolated from nile tilapia oreochromis niloticus scale gelatin in free radical mediated oxidative systems
Journal of Functional Foods, 2010Co-Authors: Dai Hung Ngo, Zhong Ji Qian, Bomi Ryu, Jae W Park, Sekwon KimAbstract:Abstract In the present study, a peptide possessing antioxidant properties was isolated from Nile tilapia ( Oreochromis niloticus ) scale gelatin. Gelatin protein was hydrolyzed using alcalase, pronase E, trypsin and pepsin. Antioxidant efficacy of respective hydrolysates were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical and superoxide radical anion scavenging activities. Moreover, protective effect on DNA damage caused by hydroxyl radicals generated was determined. Further, the level of reactive oxygen species (ROS) was determined using a fluorescence probe, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), which could be converted to highly fluorescent Dichlorofluorescein (DCF) with the presence of intracellular ROS on mouse macrophages, RAW 264.7 cells. Among hydrolysates, alcalase-derived hydrolysate exhibited the highest antioxidant activity compared to other enzymatic hydrolysates. Therefore, it was further analyzed and the sequence of an active peptide present in it was identified as Asp-Pro-Ala-Leu-Ala-Thr-Glu-Pro-Asp-Pro-Met-Pro-Phe (1382.57 Da). This peptide showed no cytotoxic effect on mouse macrophages (RAW 264.7) and human lung fibroblasts (MRC-5). In addition, it scavenged hydroxyl, DPPH and superoxide radicals at the IC 50 values of 7.56, 8.82 and 17.83 μM, respectively. These results suggest that the peptide derived from Nile tilapia ( O. niloticus ) scale gelatin acts as a candidate against oxidative stress and could be used as a potential functional food ingredient.
Colin F Chignell - One of the best experts on this subject based on the ideXlab platform.
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a photochemical study of cells loaded with 2 7 dichlorofluorescin implications for the detection of reactive oxygen species generated during uva irradiation
Free Radical Biology and Medicine, 2003Co-Authors: Colin F Chignell, Robert H SikAbstract:There have been several attempts to implicate reactive oxygen species in UVA-induced damage by loading cells with 2′,7′-dichlorofluorescin (DCFH) and following the appearance of 2′,7′-Dichlorofluorescein (DCF), its highly fluorescent oxidation product. However, both DCF and DCFH have significant absorption in the 300–400 nm range so it is possible that photochemical reactions will occur in cells containing these dyes when they are irradiated with UVA. HaCaT keratinocytes loaded with DCFH were irradiated with 0, 1, 2, or 4 J/cm2 UVA and DCF fluorescence was measured. A dose-dependent increase in DCF fluorescence was observed, with the cells exposed to 4 J/cm2 UVA exhibiting an almost 10-fold increase over dark controls. However, there was no difference in cell viability, as measured by the MTS assay or LDH release, between the dark and the 4 J/cm2 UVA-exposed groups. Furthermore, a large increase in DCF fluorescence was observed when a cell-free system containing DCF, DCFH, and horseradish peroxidase was UVA irradiated. As a control, keratinocytes loaded with DCFH were incubated in the dark with either exogenously added H2O2 or 5-hydroxy-1,4-naphthoquinone (juglone), which redox cycles to generate superoxide (and H2O2). In both cases, the cells showed a concentration-dependent increase in DCF fluorescence and a concomitant decrease in viability. Our findings suggest that DCFH can not be used to detect the UVA-induced generation of reactive oxygen species in cells when the dye is present during exposure.
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photosensitized oxidation of 2 7 dichlorofluorescin singlet oxygen does not contribute to the formation of fluorescent oxidation product 2 7 Dichlorofluorescein
Free Radical Biology and Medicine, 2002Co-Authors: Paweł Bilski, A. G. Belanger, Colin F ChignellAbstract:2′,7′-Dichlorofluorescin (DCFH) is often employed to assess oxidative stress in cells by monitoring the appearance of 2′,7′-Dichlorofluorescein (DCF), its highly fluorescent oxidation product. We have investigated the photosensitized oxidation of DCFH in solution and elucidated the role played by singlet molecular oxygen (1O2) in this reaction. We used rose bengal (RB), protoporphyrin, and DCF as photosensitizers. Irradiation (550 nm) of RB (20 μM) in 50 mM phosphate (pH 7.4) in the presence of DCFH (50 μM) resulted in the rapid formation of DCF, measured as an increase in its characteristic absorbance and fluorescence. The oxidation rate was faster in deoxygenated solution, did not increase in D2O, and even increased in the presence of sodium azide. The presence of antioxidants that react with 1O2, thus removing oxygen, accelerated DCF formation. Such results eliminate any potential direct involvement of 1O2 in DCF formation, even though DCFH is an efficient (physical) quencher of 1O2 (kq = 1.4 × 108 M−1s−1 in methanol). DCF is also a moderate photosensitizer of 1O2 with a quantum yield of circa φ = 0.06 in D2O and φ = 0.08 in propylene carbonate, which unequivocally indicates that DCF can exist in a triplet state upon excitation with UV and visible light. This triplet can initiate photo-oxidization of DCFH via redox-and-radical mechanism(s) similar to those involving RB (vide supra). Our results show that, upon illumination, DCF can function as a moderate photosensitizer initiating DCFH oxidation, which may prime and accelerate the formation of DCF. We have also shown that, while 1O2 does not contribute directly to DCF production, it can do so indirectly via reaction with cellular substrates yielding peroxy products and peroxyl radicals, which are able to oxidize DCFH in subsequent dark reactions. These findings suggest that DCFH should not be regarded as a probe sensitive to singlet molecular oxygen, and that care must be taken when using DCFH to measure oxidative stress in cells as a result of both visible and UV light exposure.
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Photosensitized oxidation of 2′,7′-dichlorofluorescin: Singlet oxygen does not contribute to the formation of fluorescent oxidation product 2′,7′-Dichlorofluorescein
Free Radical Biology and Medicine, 2002Co-Authors: Paweł Bilski, A. G. Belanger, Colin F ChignellAbstract:2′,7′-Dichlorofluorescin (DCFH) is often employed to assess oxidative stress in cells by monitoring the appearance of 2′,7′-Dichlorofluorescein (DCF), its highly fluorescent oxidation product. We have investigated the photosensitized oxidation of DCFH in solution and elucidated the role played by singlet molecular oxygen (1O2) in this reaction. We used rose bengal (RB), protoporphyrin, and DCF as photosensitizers. Irradiation (550 nm) of RB (20 μM) in 50 mM phosphate (pH 7.4) in the presence of DCFH (50 μM) resulted in the rapid formation of DCF, measured as an increase in its characteristic absorbance and fluorescence. The oxidation rate was faster in deoxygenated solution, did not increase in D2O, and even increased in the presence of sodium azide. The presence of antioxidants that react with 1O2, thus removing oxygen, accelerated DCF formation. Such results eliminate any potential direct involvement of 1O2 in DCF formation, even though DCFH is an efficient (physical) quencher of 1O2 (kq = 1.4 × 108 M-1s-1 in methanol). DCF is also a moderate photosensitizer of 1O2 with a quantum yield of circa φ = 0.06 in D2O and φ = 0.08 in propylene carbonate, which unequivocally indicates that DCF can exist in a triplet state upon excitation with UV and visible light. This triplet can initiate photo-oxidization of DCFH via redox-and-radical mechanism(s) similar to those involving RB (vide supra). Our results show that, upon illumination, DCF can function as a moderate photosensitizer initiating DCFH oxidation, which may prime and accelerate the formation of DCF. We have also shown that, while 1O2 does not contribute directly to DCF production, it can do so indirectly via reaction with cellular substrates yielding peroxy products and peroxyl radicals, which are able to oxidize DCFH in subsequent dark reactions. These findings suggest that DCFH should not be regarded as a probe sensitive to singlet molecular oxygen, and that care must be taken when using DCFH to measure oxidative stress in cells as a result of both visible and UV light exposure.
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evidence for free radical formation during the oxidation of 2 7 dichlorofluorescin to the fluorescent dye 2 7 Dichlorofluorescein by horseradish peroxidase possible implications for oxidative stress measurements
Free Radical Biology and Medicine, 1999Co-Authors: Cristina Rota, Colin F Chignell, Ronald P MasonAbstract:The oxidation of 2′-7′-dichlorofluorescin (DCFH) to the fluorescent 2′-7′-Dichlorofluorescein (DCF) by horseradish peroxidase (HRP) was investigated by fluorescence, absorption, and electron spin resonance spectroscopy (ESR). As has been previously reported, HRP/H2O2 oxidized DCFH to the highly fluorescent DCF. However, DCF fluorescence was still observed when H2O2 was omitted, although its intensity was reduced by 50%. Surprisingly, the fluorescence increase, in the absence of exogenous H2O2, was still strongly inhibited by catalase, demonstrating that H2O2 was present and necessary for DCF formation. H2O2was apparently formed during either chemical or enzymatic deacetylation of 2′-7′-dichlorofluorescin diacetate (DCFH-DA), probably by auto-oxidation. Spectrophotometric measurements clearly showed that DCFH could be oxidized either by HRP-compound I or HRP-compound II with the obligate generation of the DCF semiquinone free radical (DCF•−). Oxidation of DCF•− to DCF by oxygen would yield superoxide (O2•−). ESR spectroscopy in conjunction with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) revealed the presence of both superoxide and hydroxyl radicals in the DCFH/H2O2/HRP system. Both radicals were also detected in the absence of added H2O2, although the intensities of the resultant adducts were decreased. This work demonstrates that DCF fluorescence cannot be used reliably to measure O2•− in cells because O2•− itself is formed during the conversion of DCFH to DCF by peroxidases. The disproportionation of superoxide forms H2O2 which, in the presence of peroxidase activity, will oxidize more DCFH to DCF with self-amplification of the fluorescence. Because the deacetylation of DCFH-DA, even by esterases, can produce H2O2, the use of this probe to measure H2O2 production in cells is problematic.
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photoreduction of the fluorescent dye 2 7 Dichlorofluorescein a spin trapping and direct electron spin resonance study with implications for oxidative stress measurements
Free Radical Biology and Medicine, 1999Co-Authors: Emanuela Marchesi, Colin F Chignell, Cristina Rota, Yang C Fann, Ronald P MasonAbstract:Abstract The photoreduction of 2′-7′-Dichlorofluorescein (DCF) was investigated in buffer solution using direct electron spin resonance (ESR) and the ESR spin-trapping technique. Anaerobic studies of the reaction of DCF in the presence of reducing agents demonstrated that during visible irradiation (λ > 300 nm) 2′-7′-Dichlorofluorescein undergoes one-electron reduction to produce a semiquinone-type free radical as demonstrated by direct ESR. Spin-trapping studies of incubations containing DCF, 5,5-dimethyl-1-pyrroline N -oxide (DMPO) and either reduced glutathione (GSH) or reduced NADH demonstrate, under irradiation with visible light, the production of the superoxide dismutase-sensitive DMPO/ · OOH adduct. In the absence of DMPO, measurements with a Clark-type oxygen electrode show that molecular oxygen is consumed in a light-dependent process. The semiquinone radical of DCF, when formed in an aerobic system, is immediately oxidized by oxygen, which regenerates the dye and forms superoxide.
Dai Hung Ngo - One of the best experts on this subject based on the ideXlab platform.
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in vitro antioxidant activity of a peptide isolated from nile tilapia oreochromis niloticus scale gelatin in free radical mediated oxidative systems
Journal of Functional Foods, 2010Co-Authors: Dai Hung Ngo, Zhong Ji Qian, Bomi Ryu, Jae W Park, Sekwon KimAbstract:Abstract In the present study, a peptide possessing antioxidant properties was isolated from Nile tilapia ( Oreochromis niloticus ) scale gelatin. Gelatin protein was hydrolyzed using alcalase, pronase E, trypsin and pepsin. Antioxidant efficacy of respective hydrolysates were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical and superoxide radical anion scavenging activities. Moreover, protective effect on DNA damage caused by hydroxyl radicals generated was determined. Further, the level of reactive oxygen species (ROS) was determined using a fluorescence probe, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), which could be converted to highly fluorescent Dichlorofluorescein (DCF) with the presence of intracellular ROS on mouse macrophages, RAW 264.7 cells. Among hydrolysates, alcalase-derived hydrolysate exhibited the highest antioxidant activity compared to other enzymatic hydrolysates. Therefore, it was further analyzed and the sequence of an active peptide present in it was identified as Asp-Pro-Ala-Leu-Ala-Thr-Glu-Pro-Asp-Pro-Met-Pro-Phe (1382.57 Da). This peptide showed no cytotoxic effect on mouse macrophages (RAW 264.7) and human lung fibroblasts (MRC-5). In addition, it scavenged hydroxyl, DPPH and superoxide radicals at the IC 50 values of 7.56, 8.82 and 17.83 μM, respectively. These results suggest that the peptide derived from Nile tilapia ( O. niloticus ) scale gelatin acts as a candidate against oxidative stress and could be used as a potential functional food ingredient.
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in vitro antioxidant activity of a peptide isolated from nile tilapia oreochromis niloticus scale gelatin in free radical mediated oxidative systems
Journal of Functional Foods, 2010Co-Authors: Dai Hung Ngo, Zhong Ji Qian, Bomi Ryu, Jae W Park, Sekwon KimAbstract:Abstract In the present study, a peptide possessing antioxidant properties was isolated from Nile tilapia ( Oreochromis niloticus ) scale gelatin. Gelatin protein was hydrolyzed using alcalase, pronase E, trypsin and pepsin. Antioxidant efficacy of respective hydrolysates were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical and superoxide radical anion scavenging activities. Moreover, protective effect on DNA damage caused by hydroxyl radicals generated was determined. Further, the level of reactive oxygen species (ROS) was determined using a fluorescence probe, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), which could be converted to highly fluorescent Dichlorofluorescein (DCF) with the presence of intracellular ROS on mouse macrophages, RAW 264.7 cells. Among hydrolysates, alcalase-derived hydrolysate exhibited the highest antioxidant activity compared to other enzymatic hydrolysates. Therefore, it was further analyzed and the sequence of an active peptide present in it was identified as Asp-Pro-Ala-Leu-Ala-Thr-Glu-Pro-Asp-Pro-Met-Pro-Phe (1382.57 Da). This peptide showed no cytotoxic effect on mouse macrophages (RAW 264.7) and human lung fibroblasts (MRC-5). In addition, it scavenged hydroxyl, DPPH and superoxide radicals at the IC 50 values of 7.56, 8.82 and 17.83 μM, respectively. These results suggest that the peptide derived from Nile tilapia ( O. niloticus ) scale gelatin acts as a candidate against oxidative stress and could be used as a potential functional food ingredient.
Stephen C Bondy - One of the best experts on this subject based on the ideXlab platform.
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evaluation of the probe 2 7 dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress
Chemical Research in Toxicology, 1992Co-Authors: Carl P Lebel, Harry Ischiropoulos, Stephen C BondyAbstract:The use of dichlorofluorescin (DCFH) as a measure of reactive oxygen species was studied in aqueous media. Hydrogen peroxide oxidized DCFH to fluorescent Dichlorofluorescein (DCF), and the oxidation was amplified by the addition of ferrous iron. Hydrogen peroxide-induced DCF formation in the presence of ferrous iron was completely inhibited by deferoxamine and partially inhibited by ethylenediaminetetraacetic acid, but was augmented by diethylenetriaminepentaacetic acid. Iron-peroxide-induced oxidation of DCFH was partially inhibited by catalase but not by horseradish peroxidase. Nonchelated iron-peroxide oxidation of DCFH was partially inhibited by several hydroxyl radical scavengers, but was independent of the scavenger concentration, and this suggests that free hydroxyl radical is not involved in the oxidation of DCFH in this system. Superoxide anion did not directly oxidize DCFH. Data suggest that H2O2-Fe(2+)-derived oxidant is mainly responsible for the nonenzymatic oxidation of DCFH. In addition, peroxidase alone and oxidants formed during the reduction of H2O2 by peroxidase oxidize DCFH. Since DCFH oxidation may be derived from several reactive intermediates, interpretation of specific reactive oxygen species involved in biological systems should be approached with caution. However, DCFH remains an attractive probe as an overall index of oxidative stress in toxicological phenomena.
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reactive oxygen species formation as a biomarker of methylmercury and trimethyltin neurotoxicity
Neurotoxicology, 1992Co-Authors: Carl P Lebel, Stephen C BondyAbstract:Abstract Reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, and hydroxyl radicals are believed to be initiators of peroxidative cell damage. This paper focused on the use of 2',7'-Dichlorofluorescein-diacetate (DCFH-DA) to quantitate cerebral ROS as an index for neurotoxicity. This technique employs an assay of Dichlorofluorescein (DCF), the fluorescent product of Dichlorofluorescein (DCFH). Data from studies using various free radical generating systems, several iron chelators and hydroxyl radical scavengers suggest that DCFH oxidation may result in several reactive intermediates. In a biological system (synaptosomes isolated from untreated rats) DCF fluorescence was stimulated by ascorbate or FeSO4, while deferoxamine inhibited the ascorbate/FeSO4-induced stimulation of DCF formation. Two organometals, methylmercury (MeHg) and trimethyltin (TMT), known to produce neurotoxicity were tested. In vitro exposure to MeHg (10-20 microM) increased the rate of formation of ROS while TMT (5-40 microM) had no effect. In vivo, 48 hr and 1 week after a single injection of MeHg (1 mg/kg, i.p.) in mice and 1 week after a single injection of MeHg (5 mg/kg, i.p.) in rats, the rate of formation of ROS in both rat and mouse cerebellum was significantly increased. Pretreatment with deferoxamine, a potent iron-chelator, prevented MeHg-induced increase of ROS. In hippocampus and frontal cortex, ROS formation rates were also elevated 48 hr after TMT injection (3 mg/kg, i.p.) in mice. These results demonstrate that DCF fluorescence provides a good measure of overall ROS formation in synaptosomes of both in vitro as well as in vivo systems. Since ROS formation was selectively increased in areas known to be specifically vulnerable to organometals (cerebellum in the case of MeHg and hippocampus in the case of TMT), these studies further support that oxidative damage may be the primary mechanism underlying the neurotoxicity induced by these organometals.
Ronald P Mason - One of the best experts on this subject based on the ideXlab platform.
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evidence for free radical formation during the oxidation of 2 7 dichlorofluorescin to the fluorescent dye 2 7 Dichlorofluorescein by horseradish peroxidase possible implications for oxidative stress measurements
Free Radical Biology and Medicine, 1999Co-Authors: Cristina Rota, Colin F Chignell, Ronald P MasonAbstract:The oxidation of 2′-7′-dichlorofluorescin (DCFH) to the fluorescent 2′-7′-Dichlorofluorescein (DCF) by horseradish peroxidase (HRP) was investigated by fluorescence, absorption, and electron spin resonance spectroscopy (ESR). As has been previously reported, HRP/H2O2 oxidized DCFH to the highly fluorescent DCF. However, DCF fluorescence was still observed when H2O2 was omitted, although its intensity was reduced by 50%. Surprisingly, the fluorescence increase, in the absence of exogenous H2O2, was still strongly inhibited by catalase, demonstrating that H2O2 was present and necessary for DCF formation. H2O2was apparently formed during either chemical or enzymatic deacetylation of 2′-7′-dichlorofluorescin diacetate (DCFH-DA), probably by auto-oxidation. Spectrophotometric measurements clearly showed that DCFH could be oxidized either by HRP-compound I or HRP-compound II with the obligate generation of the DCF semiquinone free radical (DCF•−). Oxidation of DCF•− to DCF by oxygen would yield superoxide (O2•−). ESR spectroscopy in conjunction with the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) revealed the presence of both superoxide and hydroxyl radicals in the DCFH/H2O2/HRP system. Both radicals were also detected in the absence of added H2O2, although the intensities of the resultant adducts were decreased. This work demonstrates that DCF fluorescence cannot be used reliably to measure O2•− in cells because O2•− itself is formed during the conversion of DCFH to DCF by peroxidases. The disproportionation of superoxide forms H2O2 which, in the presence of peroxidase activity, will oxidize more DCFH to DCF with self-amplification of the fluorescence. Because the deacetylation of DCFH-DA, even by esterases, can produce H2O2, the use of this probe to measure H2O2 production in cells is problematic.
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photoreduction of the fluorescent dye 2 7 Dichlorofluorescein a spin trapping and direct electron spin resonance study with implications for oxidative stress measurements
Free Radical Biology and Medicine, 1999Co-Authors: Emanuela Marchesi, Colin F Chignell, Cristina Rota, Yang C Fann, Ronald P MasonAbstract:Abstract The photoreduction of 2′-7′-Dichlorofluorescein (DCF) was investigated in buffer solution using direct electron spin resonance (ESR) and the ESR spin-trapping technique. Anaerobic studies of the reaction of DCF in the presence of reducing agents demonstrated that during visible irradiation (λ > 300 nm) 2′-7′-Dichlorofluorescein undergoes one-electron reduction to produce a semiquinone-type free radical as demonstrated by direct ESR. Spin-trapping studies of incubations containing DCF, 5,5-dimethyl-1-pyrroline N -oxide (DMPO) and either reduced glutathione (GSH) or reduced NADH demonstrate, under irradiation with visible light, the production of the superoxide dismutase-sensitive DMPO/ · OOH adduct. In the absence of DMPO, measurements with a Clark-type oxygen electrode show that molecular oxygen is consumed in a light-dependent process. The semiquinone radical of DCF, when formed in an aerobic system, is immediately oxidized by oxygen, which regenerates the dye and forms superoxide.
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Photoreduction of the fluorescent dye 2′-7′-Dichlorofluorescein: a spin trapping and direct electron spin resonance study with implications for oxidative stress measurements
Free Radical Biology and Medicine, 1998Co-Authors: Emanuela Marchesi, Colin F Chignell, Cristina Rota, Yang C Fann, Ronald P MasonAbstract:Abstract The photoreduction of 2′-7′-Dichlorofluorescein (DCF) was investigated in buffer solution using direct electron spin resonance (ESR) and the ESR spin-trapping technique. Anaerobic studies of the reaction of DCF in the presence of reducing agents demonstrated that during visible irradiation (λ > 300 nm) 2′-7′-Dichlorofluorescein undergoes one-electron reduction to produce a semiquinone-type free radical as demonstrated by direct ESR. Spin-trapping studies of incubations containing DCF, 5,5-dimethyl-1-pyrroline N -oxide (DMPO) and either reduced glutathione (GSH) or reduced NADH demonstrate, under irradiation with visible light, the production of the superoxide dismutase-sensitive DMPO/ · OOH adduct. In the absence of DMPO, measurements with a Clark-type oxygen electrode show that molecular oxygen is consumed in a light-dependent process. The semiquinone radical of DCF, when formed in an aerobic system, is immediately oxidized by oxygen, which regenerates the dye and forms superoxide.