Oxidation pathways for the intracellular probe 2′,7′-dichlorofluorescin

Zhu, Honglan; Bannenberg, Gerard; Moldéus, Peter; Shertzer, Howard G.

doi:10.1007/s002040050118

Cited by 258 publications

(155 citation statements)

References 30 publications

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“…This assay is a reliable method for the measurement of intracellular ROS such as hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OHϪ), and hydroperoxides. [20][21][22] DCFH-DA was dissolved in absolute ethanol at a concentration of 5 mmol/L. Hepatocytes were grown on collagen-coated glass coverslips in 6-well culture plates.…”

Section: Methodsmentioning

confidence: 99%

Oxidative Mitochondrial DNA Damage and Deletion in Hepatocytes of Rejecting Liver Allografts in Rats: Role of TNF-α *

et al. 2005

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An orthotopic liver transplant model in the rat was used to evaluate the role of tumor necrosis factor alpha (TNF-␣) in liver transplant rejection. There were significantly increased levels of TNF-␣ mRNA and parallel increases in 8-hydroxy-2 deoxyguanosine (8-OHdG) indicative of oxidative DNA damage present 7 to 12 days after transplantation. Cells staining positively for 8-OHdG were localized to the cytoplasm of hepatocytes adjacent to the TNF-␣ expressing inflammatory cells in the portal areas or in patches surrounded by inflammatory cells in the hepatic sinusoids. Significantly more cells staining for 8-OHdG were found in the allogeneic grafts that were strongly rejected than in the syngeneic controls or in the grafts placed in species that accepted the allograft permanently after a rejection episode. TUNEL reactivity lagged 2 days behind peak reactivity for 8-OHdG. On day 12 after transplantation, many cells stained for both 8-OHdG and TUNEL, indicating that the cells suffering oxidative DNA injury were undergoing apoptosis or death. Oxidative injury resulted in mtDNA deletion consisting of 4,834 base-pairs. Studies of hepatocytes cultured from normal rats displayed dose-dependent relationships between TNF-␣ concentration and 8-OHdG and mtDNA mutation. Repetitive intraperitoneal injection of Enbrel, a TNF receptor blocker, significantly decreased hepatocyte 8-OHdG levels and the frequency of deleted mtDNA while greatly extending graft survival time. In conclusion, the data presented implicate TNF-␣ as being capable of causing oxidative DNA damage and mtDNA mutation in hepatocytes. (HEPATOLOGY 2005;42:208-215.)

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Section: Methodsmentioning

confidence: 99%

Oxidative Mitochondrial DNA Damage and Deletion in Hepatocytes of Rejecting Liver Allografts in Rats: Role of TNF-α *

et al. 2005

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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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“…The formation of hydrogen peroxide has been observed in other cell types in response to higher energy light, such as that which occurs with epifluorescence. To determine whether embryos exhibit a similar response to irradiation, we utilized a derivative of dihydrofluorescein (DHF), a dye that fluoresces in the presence of reactive oxygen [27][28][29][30] . Embryos exposed for 5 s to irradiation with epifluorescence ( Fig.…”

Section: Peroxide Is Produced In Lscm-imaged Embryosmentioning

confidence: 99%

“…2C, H, and K). Because DHF is thought to be primarily sensitive to the production of hydrogen peroxide 29,33 , we determined if this was also the case in these epifluorescent-irradiated embryos. When embryos were pretreated with catalase to promote the degradation of hydrogen peroxide, the increase in DHF fluorescence was attenuated compared with embroyos not treated with catalase (Fig.…”

Section: Peroxide Is Produced In Lscm-imaged Embryosmentioning

confidence: 99%

Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability

et al. 1999

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A major challenge for fluorescence imaging of living mammalian cells is maintaining viability following prolonged exposure to excitation illumination. We have monitored the dynamics of mitochondrial distribution in hamster embryos at frequent intervals over 24 h using two-photon microscopy (1,047 nm) while maintaining blastocyst, and even fetal, developmental competence. In contrast, confocal imaging for only 8 h inhibits development, even without fluorophore excitation. Photo-induced production of H 2 O 2 may account, in part, for this inhibition. Thus, twophoton microscopy, but not confocal microscopy, has permitted long-term fluorescence observations of the dynamics of three-dimensional cytoarchitecture in highly photosensitive specimens such as mammalian embryos.Keywords two-photon microscopy; laser scanning confocal microscopy; live cell fluorescence imaging; embryo; mitochondrial dynamics; mammal; hamster The detection of specific cellular components by imaging techniques such as wide-field epifluorescence or laser scanning confocal microscopy (LSCM) requires exposure to high intensity light that can cause cellular damage 1 . Consequently, the quantity or quality of images that can be collected is limited or, even worse, the reliability of the images may be compromised. This is a particular problem when imaging events that occur over periods of time ranging from hours to days, such as embryonic development. For this reason, much of our current understanding of subcellular morphological changes during mammalian embryonic development is based on images of fixed or static specimens at different developmental stages [2][3][4][5][6] . Thus, it can be difficult to interpret dynamic processes accurately, because the continuity of events must be inferred. The establishment of long-term fluorescence imaging methods that maintain the viability of live specimens is critical for advancing our understanding of cell biology and embryonic development in areas such as ion dynamics 7 , cytoplasmic reorganization, compaction and blastocoel formation, embryonic development in exotic species (where specimens are heterogeneous and difficult * Corresponding author (jsquirre@students.wisc.edu). to obtain), use of fluorescent tags for the preselection of embryos for subsequent embryo transfer 8 , and studies of protein expression in living cells using green fluorescent protein 9 . HHS Public AccessEmbryos of some mammals, particular hamsters, are very sensitive to culture conditions 10 . Furthermore, studies suggest that mammalian oocytes and embryos are adversely affected by exposure to visible light [11][12][13] . Because of this sensitivity, mammalian embryos are ideal to test live-cell imaging techniques. In addition, there are obvious morphological changes associated with differentiation, namely compaction and blastocoel formation, which can be used to assess viability. The embryo must undergo cell division during and after imaging as well as maintain a level of developmental competence that allows it to initiate dif...

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Oxidation pathways for the intracellular probe 2′,7′-dichlorofluorescin

Cited by 258 publications

References 30 publications

Oxidative Mitochondrial DNA Damage and Deletion in Hepatocytes of Rejecting Liver Allografts in Rats: Role of TNF-α *

Oxidative Mitochondrial DNA Damage and Deletion in Hepatocytes of Rejecting Liver Allografts in Rats: Role of TNF-α *

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

Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability

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