Quantitation of intracellular oxidation in a renal epithelial cell line

Scott, James A.; Homcy, Charles J.; Khaw, Ban‐An; Rabito, C

doi:10.1016/0891-5849(88)90067-6

Cited by 82 publications

(32 citation statements)

References 14 publications

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“…DCFH has been proved to have a high sensitivity that is capable of detecting picomole levels of hydroperoxides (Cathcart et al 1983). Scott et al (1988) and Zhu et al (1994) explored DCFH response to xanthine oxidase and iron/H 2 O 2 exposures respectively under various conditions and suggested DCF formation from the superoxide, H 2 O 2 , and the OH radical oxidation as the reason for observed results. DCFH oxidized products induced by peroxyl radicals have also been documented (Adom and Liu 2005).…”

Section: Reagentsmentioning

confidence: 99%

Characterization of Wintertime Reactive Oxygen Species Concentrations in Flushing, New York

Venkatachari

Hopke

Brune

et al. 2007

Aerosol Science and Technology

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One of the main hypotheses for the species causing the observed health effects of ambient particulate matter is peroxides and other reactive oxygen species (ROS). However, there is currently very little data available on the concentrations of particle-bound ROS or their behavior in different physical locations and seasons. The concentrations of particle-bound ROS were determined for various size fractions of the aerosol, ranging from 10 nm to 18 μm, in Flushing, New York during the period of January and early February 2004. Sampling was carried out at 3-hour intervals using a MOUDI TM cascade impactor. The collected particles were treated with the non-fluorescent probe dichlorofluorescin (DCFH) that fluoresces when oxidized by the presence of ROS. The measured fluorescent intensities were converted into equivalent hydrogen peroxide concentrations, which were used as indicators of ROS reactivity, by calibrations using H 2 O 2 standards. Diurnal profiles of the ROS concentrations were obtained. Correlations of the particulate ROS concentrations with the intensity of photochemical reaction, estimated secondary organic carbon (SOC) and gas phase OH and HO 2 radical concentrations were explored. The intensity of photochemical reactions and gas phase radical concentrations were found to be moderate factors affecting particulate ROS concentrations. The concentrations of ROS were found to be higher in the submicron size particles of the ambient aerosol.

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Section: Reagentsmentioning

confidence: 99%

Characterization of Wintertime Reactive Oxygen Species Concentrations in Flushing, New York

Venkatachari

Hopke

Brune

et al. 2007

Aerosol Science and Technology

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“…The polar, reactive DCFH liberated intracellularly, as well as the DCF produced in the reaction with ROI, remain trapped in the cytoplasm (Bass et al, 1983). The reaction can be inhibited by catalase and ⅐ OH scavengers, indicating that H 2 O 2 and ⅐ OH can participate in the oxidation of DCFH (Bass et al, 1983;Cathcart et al, 1983;Scott et al, 1988;Simontacchi et al, 1993). We have adapted this procedure for the in vivo measurement of extraprotoplasmic ROI by preparing the substrate DCFH from DCFH-diacetate with esterase in the reaction mixture before adding the plant material to be analyzed.…”

Section: Release Of Roi During Germinationmentioning

confidence: 99%

Release of Reactive Oxygen Intermediates (Superoxide Radicals, Hydrogen Peroxide, and Hydroxyl Radicals) and Peroxidase in Germinating Radish Seeds Controlled by Light, Gibberellin, and Abscisic Acid

2001

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Germination of radish (Raphanus sativus cv Eterna) seeds can be inhibited by far-red light (high-irradiance reaction of phytochrome) or abscisic acid (ABA). Gibberellic acid (GA 3 ) restores full germination under far-red light. This experimental system was used to investigate the release of reactive oxygen intermediates (ROI) by seed coats and embryos during germination, utilizing the apoplastic oxidation of 2Ј,7Ј-dichlorofluorescin to fluorescent 2Ј,7Ј-dichlorofluorescein as an in vivo assay. Germination in darkness is accompanied by a steep rise in ROI release originating from the seed coat (living aleurone layer) as well as the embryo. At the same time as the inhibition of germination, far-red light and ABA inhibit ROI release in both seed parts and GA 3 reverses this inhibition when initiating germination under far-red light. During the later stage of germination the seed coat also releases peroxidase with a time course affected by far-red light, ABA, and GA 3 . The participation of superoxide radicals, hydrogen peroxide, and hydroxyl radicals in ROI metabolism was demonstrated with specific in vivo assays. ROI production by germinating seeds represents an active, developmentally controlled physiological function, presumably for protecting the emerging seedling against attack by pathogens.

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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

204

104

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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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Quantitation of intracellular oxidation in a renal epithelial cell line

Cited by 82 publications

References 14 publications

Characterization of Wintertime Reactive Oxygen Species Concentrations in Flushing, New York

Characterization of Wintertime Reactive Oxygen Species Concentrations in Flushing, New York

Release of Reactive Oxygen Intermediates (Superoxide Radicals, Hydrogen Peroxide, and Hydroxyl Radicals) and Peroxidase in Germinating Radish Seeds Controlled by Light, Gibberellin, and Abscisic Acid

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

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