Characterization of 2′,7′-dichlorofluorescin fluorescence in dissociated mammalian brain neurons: estimation on intracellular content of hydrogen peroxide

Oyama, Yasuo; Hayashi, Ayami; Ueha, Toshiko; Maekawa, Keisei

doi:10.1016/0006-8993(94)91429-x

Cited by 165 publications

(78 citation statements)

References 24 publications

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“…Generation of ROS was assessed immediately after treatments, using 2Ј,7Ј-dichlorofluorescein diacetate (Sigma) (33), by a confocal laser scanning microscope (Bio-Rad MRC 1024 ES scanning microscope) equipped with a krypton/argon laser source. The emitted fluorescence was monitored at 488 and 568 nm wavelengths with a Nikon plan Apo ϫ 60 oil-immersion objective.…”

Section: Methodsmentioning

confidence: 99%

“…A, ROS cellular levels were evaluated by confocal microscopy following labeling with the ROS-specific fluorescent indicator immediately after administration of apoptotic stimuli, as described previously (33,36). B, MDA-4-HNE cellular levels (left panel) and SOD activity (right panel) evaluated at the 2nd h after administration of apoptotic stimuli.…”

Section: Detection Of Change In Mitochondrial Transmembrane Potentialmentioning

confidence: 99%

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Coenzyme Q10 Prevents Apoptosis by Inhibiting Mitochondrial Depolarization Independently of Its Free Radical Scavenging Property

Papucci¹,

Schiavone²,

Witort³

et al. 2003

Journal of Biological Chemistry

262

193

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The permeability transition pore (PTP) is a mitochondrial channel whose opening causes the mitochondrial membrane potential (⌬ ) collapse that leads to apoptosis. Some ubiquinone analogues have been demonstrated previously to modulate the PTP open-closed transition in isolated mitochondria and thought to act through a common PTP-binding site rather than through oxidation-reduction reactions. We have demonstrated recently both in vitro and in vivo that the ubiquitous free radical scavenger and respiratory chain coenzyme Q 10 (CoQ 10 ) prevents keratocyte apoptosis induced by excimer laser irradiation more efficiently than other antioxidants. On this basis, we hypothesized that the antiapoptotic property of CoQ 10 could be independent of its free radical scavenging ability and related to direct inhibition of PTP opening. In this study, we have verified this hypothesis by evaluating the antiapoptotic effects of CoQ 10 in response to apoptotic stimuli, serum starvation, antimycin A, and ceramide, which do not generate free radicals, in comparison to control, free radical-generating UVC irradiation. As hypothesized, CoQ 10 dramatically reduced apoptotic cell death, attenuated ATP decrease, and hindered DNA fragmentation elicited by all apoptotic stimuli. This was accompanied by inhibition of mitochondrial depolarization, cytochrome c release, and caspase 9 activation. Because these events are consequent to mitochondrial PTP opening, we suggest that the antiapoptotic activity of CoQ 10 could be related to its ability to prevent this phenomenon.

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Section: Methodsmentioning

confidence: 99%

Section: Detection Of Change In Mitochondrial Transmembrane Potentialmentioning

confidence: 99%

Coenzyme Q10 Prevents Apoptosis by Inhibiting Mitochondrial Depolarization Independently of Its Free Radical Scavenging Property

Papucci¹,

Schiavone²,

Witort³

et al. 2003

Journal of Biological Chemistry

262

193

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“…Furthermore, there are some disagreements in the literature concerning the effect of superoxide dismutase on oxidative stress in cells monitored by the DCF fluorometric assay. Some studies report an inhibitory effect by superoxide dismutase (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15), while almost the same number of studies report no effect (16 -26). A recent paper by LeBel et al (27) mentioned that the interpretation of specific reactive oxygen species involved in the oxidation of DCFH to DCF in biological systems should be approached with caution.…”

mentioning

confidence: 99%

Phenoxyl Free Radical Formation during the Oxidation of the Fluorescent Dye 2′,7′-Dichlorofluorescein by Horseradish Peroxidase

Rota

Fann

Mason

1999

Journal of Biological Chemistry

205

105

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The oxidation of the fluorescent dye 2,7-dichlorofluorescein (DCF) by horseradish peroxidase was investigated by optical absorption, electron spin resonance (ESR), and oxygen consumption measurements. Spectrophotometric measurements showed that DCF could be oxidized either by horseradish peroxidase-compound I or -compound II with the obligate generation of the DCF phenoxyl radical (DCF ⅐ ). This one-electron oxidation was confirmed by ESR spin-trapping experiments. DCF ⅐ oxidizes GSH, generating the glutathione thiyl radical (GS ⅐ ), which was detected by the ESR spin-trapping technique. In this case, oxygen was consumed by a sequence of reactions initiated by the GS ⅐ radical. Similarly, DCF ⅐ oxidized NADH, generating the NAD ⅐ radical that reduced oxygen to superoxide (O 2 . ), which was also detected by the ESR spin-trapping technique. Superoxide dismutated to generate H 2 O 2 , which reacted with horseradish peroxidase, setting up an enzymatic chain reaction leading to H 2 O 2 production and oxygen consumption. In contrast, when ascorbic acid reduced the DCF phenoxyl radical back to its parent molecule, it formed the unreactive ascorbate anion radical. Clearly, DCF catalytically stimulates the formation of reactive oxygen species in a manner that is dependent on and affected by various biochemical reducing agents. This study, together with our earlier studies, demonstrates that DCFH cannot be used conclusively to measure superoxide or hydrogen peroxide formation in cells undergoing oxidative stress.2Ј,7Ј-Dichlorofluorescin (DCFH) 1 is widely used to measure oxidative stress in cells. The diacetate form of DCFH enters the cell and is hydrolyzed by intracellular esterases to liberate DCFH. Upon reaction with oxidizing species, the highly fluorescent compound 2Ј,7Ј-dichlorofluorescein (DCF) is formed. The fluorescence intensity can be measured and is the basis of the popular cellular assay for oxidative stress (1).Although this assay is commonly used, there are many controversies regarding its real validity. It is not clear which oxidative species is responsible for the oxidation of DCFH. Furthermore, there are some disagreements in the literature concerning the effect of superoxide dismutase on oxidative stress in cells monitored by the DCF fluorometric assay. Some studies report an inhibitory effect by superoxide dismutase (2-15), while almost the same number of studies report no effect (16 -26). A recent paper by LeBel et al. (27) mentioned that the interpretation of specific reactive oxygen species involved in the oxidation of DCFH to DCF in biological systems should be approached with caution. It has also been demonstrated that the photoreduction of DCF results in the formation of the DCF semiquinone free radical (DCF . ), which, under aerobic conditions, is oxidized by oxygen to its parent dye, DCF, concomitantly forming superoxide radical (28). Moreover, it was reported that DCFH, upon reacting with horseradish peroxidase-compound I or -compound II, was oxidized to DCF . , which was subsequently air-ox...

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“…Estimation of ROS in live 100 mg brain cortical tissue was based on several methods with some modifications. 21,22 Cytoplasmic and mitochondrial ROS level was measured by using the fluorescent dihydrorhodamine 123. DHR localizes to mitochondria and fluoresces when oxidized by ROS, particularly peroxynitrite, to the positively charged Rhodamine 123 derivate.…”

Section: Cortical Mitochondria Preparationmentioning

confidence: 99%

Involvement of attenuated antioxidant and Bcl2 signalling property in UV-R/ sunlight irradiated piperine treated ischemia/reperfusion rat model. Highlights

et al. 2014

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Characterization of 2′,7′-dichlorofluorescin fluorescence in dissociated mammalian brain neurons: estimation on intracellular content of hydrogen peroxide

Cited by 165 publications

References 24 publications

Coenzyme Q10 Prevents Apoptosis by Inhibiting Mitochondrial Depolarization Independently of Its Free Radical Scavenging Property

Coenzyme Q10 Prevents Apoptosis by Inhibiting Mitochondrial Depolarization Independently of Its Free Radical Scavenging Property

Phenoxyl Free Radical Formation during the Oxidation of the Fluorescent Dye 2′,7′-Dichlorofluorescein by Horseradish Peroxidase

Involvement of attenuated antioxidant and Bcl2 signalling property in UV-R/ sunlight irradiated piperine treated ischemia/reperfusion rat model. Highlights

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