A Stable Nonfluorescent Derivative of Resorufin for the Fluorometric Determination of Trace Hydrogen Peroxide: Applications in Detecting the Activity of Phagocyte NADPH Oxidase and Other Oxidases

Zhou, Mingjie; Diwu, Zhenjun; Panchuk-Voloshina, Nataliya; Haugland, Richard P.

doi:10.1006/abio.1997.2391

Cited by 1,248 publications

(978 citation statements)

References 12 publications

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“…In the presence of peroxidase, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) reacts with hydrogen peroxide to produce red fluorescent resorufin. 32 Samples were rinsed with PBS, 0.25 mL of PBS was added to each well, and cells were scraped off the samples using a rubber policeman into the PBS. The scraped cell suspensions were transferred to 1.5 mL vials and sonicated over ice (Sonics, Vibra Cell, 30 sec at 30% power) to release peroxisomes.…”

Section: Bioassaysmentioning

confidence: 99%

Nanofiber scaffolds influence organelle structure and function in bone marrow stromal cells

Tutak

Giri

Bajcsy

et al. 2016

J. Biomed. Mater. Res.

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Tutak, Wojtek; Jyotsnendu, Giri; Bajcsy, Peter; and Simon, Carl G. Jr., "Nanofiber scaffolds influence organelle structure and function in bone marrow stromal cells" (2016 Abstract: Recent work demonstrates that osteoprogenitor cell culture on nanofiber scaffolds can promote differentiation. This response may be driven by changes in cell morphology caused by the three-dimensional (3D) structure of nanofibers. We hypothesized that nanofiber effects on cell behavior may be mediated by changes in organelle structure and function. To test this hypothesis, human bone marrow stromal cells (hBMSCs) were cultured on poly(e-caprolactone) (PCL) nanofibers scaffolds and on PCL flat spuncoat films. After 1 dayculture, hBMSCs were stained for actin, nucleus, mitochondria, and peroxisomes, and then imaged using 3D confocal microscopy. Imaging revealed that the hBMSC cell body (actin) and peroxisomal volume were reduced during culture on nanofibers. In addition, the nucleus and peroxisomes occupied a larger fraction of cell volume during culture on nanofibers than on films, suggesting enhancement of the nuclear and peroxisomal functional capacity. Organelles adopted morphologies with greater 3D-character on nanofibers, where the Z-Depth (a measure of cell thickness) was increased. Comparisons of organelle positions indicated that the nucleus, mitochondria, and peroxisomes were closer to the cell center (actin) for nanofibers, suggesting that nanofiber culture induced active organelle positioning. The smaller cell volume and more centralized organelle positioning would reduce the energy cost of inter-organelle vesicular transport during culture on nanofibers. Finally, hBMSC bioassay measurements (DNA, peroxidase, bioreductive potential, lactate, and adenosine triphosphate (ATP)) indicated that peroxidase activity may be enhanced during nanofiber culture. These results demonstrate that culture of hBMSCs on nanofibers caused changes in organelle structure and positioning, which may affect organelle functional capacity and transport.

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Section: Bioassaysmentioning

confidence: 99%

Nanofiber scaffolds influence organelle structure and function in bone marrow stromal cells

Tutak

Giri

Bajcsy

et al. 2016

J. Biomed. Mater. Res.

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“…H 2 O 2 production was measured using the Amplex red/horseradish peroxidase method [18]. Standard reaction mixes in 100 μl potassium phosphate buffer (50 mM, pH 7.4) contained 0-0.5 mM NADPH, 0-500 μM nitrofurantoin, 25 μM Amplex red, 1 unit/ml horseradish peroxidase, and 1.25 μg/ml cytochrome P450 reductase or 100 μg/ml cell protein.…”

Section: Kinetic Analysis Of H 2 O 2 Productionmentioning

confidence: 99%

Role of cytochrome P450 reductase in nitrofurantoin-induced redox cycling and cytotoxicity

Wang

Gray

Mishin

et al. 2008

Free Radical Biology and Medicine

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The one-electron reduction of redox-active chemotherapeutic agents generates highly toxic radical anions and reactive oxygen intermediates (ROI). A major enzyme catalyzing this process is cytochrome P450 reductase. Because many tumor cells highly express this enzyme, redox cycling of chemotherapeutic agents in these cells may confer selective antitumor activity. Nitrofurantoin is a commonly used redox-active antibiotic that possesses antitumor activity. In the present studies we determined whether nitrofurantoin redox cycling is correlated with cytochrome P450 reductase activity and cytotoxicity in a variety of cell lines. Recombinant cytochrome P450 reductase was found to support redox cycling of nitrofurantoin and to generate superoxide anion, hydrogen peroxide, and, in the presence of redox-active iron, hydroxyl radicals. This activity was NADPH dependent and inhibitable by diphenyleneiodonium, indicating a requirement for the flavin cofactors in the reductase. Nitrofurantoin-induced redox cycling was next analyzed in different cell lines varying in cytochrome P450 reductase activity including Chinese hamster ovary cells (CHO-OR) constructed to overexpress the enzyme. Nitrofurantoin-induced hydrogen peroxide production was 16-fold greater in lysates from CHO-OR cells than from control CHO cells. A strong correlation between cytochrome P450 reductase activity and nitrofurantoin-induced redox cycling among the cell lines was found. Unexpectedly, no correlation between nitrofurantoininduced ROI production and cytotoxicity was observed. These data indicate that nitrofurantoininduced redox cycling and subsequent generation of ROI are not sufficient to mediate cytotoxicity and that cytochrome P450 reductase is not a determinant of sensitivity to redox-active chemotherapeutic agents.

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“…The Amplex® Red (AR) Hydrogen Peroxide/Peroxidase Assay Kit (Molecular Probes, Eugene, OR) was used to detect ROS generation in mesencephalic cell culture (Zhou et al, 1997). The AR reagent (10-acetyl-3,7-dihydroxyphenoxazine), in the presence of horseradish peroxidase (HP), reacts with peroxides released into the extracellular environment by cells and generates a fluorescent product.…”

Section: Extracellular Ros Measurementmentioning

confidence: 99%

Reactive oxygen species generation by the ethylene-bis-dithiocarbamate (EBDC) fungicide mancozeb and its contribution to neuronal toxicity in mesencephalic cells

Domico

Cooper

Bernard³

et al. 2007

NeuroToxicology

122

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Previous in vitro studies in our laboratory have shown that mancozeb (MZ) and maneb (MB), both widely used EBDC fungicides, are equipotent neurotoxicants that produce cell loss in mesencephalic dopaminergic and GABAergic cells after an acute 24 h exposure. Mitochondrial uncoupling and inhibition were associated with fungicide exposure. Inhibition of mitochondrial respiration is known to increase free radical production. Here the mechanism(s) of neuronal damage associated with MZ exposure was further explored by determining the role that reactive oxygen species (ROS) played in toxicity. Damage to mesencephalic dopamine and GABA cell populations were significantly attenuated when carried out in the presence of ascorbate or SOD indicative of a free radical mediated contribution to toxicity. ROS generation monitored by H 2 O 2 production using Amplex Red increased in a dose-dependent manner in response to MZ. Inhibition of intracellular catalase with aminotriazole had little effect on H 2 O 2 generation, whereas exogenously added catalase significantly reduced H 2 O 2 production demonstrating a large extracellular contribution to ROS generation. Conversely, cells preloaded with the ROS indicator dye DCF showed significant MZ-induced ROS production, demonstrating an increase in intracellular ROS. Both the organic backbone of MZ as well as its associated Mn ion, but not Zn ion were responsible and required for H 2 O 2 generation. The functionally diverse NADPH oxidase inhibitors, diphenylene iodonium chloride, apocynin, and 4-(2-aminoethyl)benzene-sulfonyl fluoride hydrochloride significantly attenuated H 2 O 2 production by MZ. In growth medium lacking cells, MZ produced little H 2 O 2 , but enhanced H 2 O 2 generation when added with xanthine plus xanthine oxidase whereas, in cultured cells, allopurinol partially attenuated H 2 O 2 production by MZ. Minocycline, an inhibitor of microglial activation, modestly reduced H 2 O 2 formation in mesencephalic cells. In contrast, neuronal enriched cultures or cultures treated with MAC-1-SAP to kill microglia, did not show an attenuation of ROS production. These findings demonstrate that Mn-containing EBDC fungicides such as MZ and MB can produce robust ROS generation that likely occurs via redox cycling with extracellular and intracellular oxidases. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeurotoxicology. Author manuscript; available in PMC 2008 November 1. The findings further show that microglia may contribute to but are not required for ROS production by MZ.

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A Stable Nonfluorescent Derivative of Resorufin for the Fluorometric Determination of Trace Hydrogen Peroxide: Applications in Detecting the Activity of Phagocyte NADPH Oxidase and Other Oxidases

Cited by 1,248 publications

References 12 publications

Nanofiber scaffolds influence organelle structure and function in bone marrow stromal cells

Nanofiber scaffolds influence organelle structure and function in bone marrow stromal cells

Role of cytochrome P450 reductase in nitrofurantoin-induced redox cycling and cytotoxicity

Reactive oxygen species generation by the ethylene-bis-dithiocarbamate (EBDC) fungicide mancozeb and its contribution to neuronal toxicity in mesencephalic cells

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