Detection of picomole levels of hydroperoxides using a fluorescent dichlorofluorescein assay

Cathcart, Richard T.; Schwiers, Elizabeth; Ames, Bruce N.

doi:10.1016/0003-2697(83)90270-1

Cited by 786 publications

(406 citation statements)

References 12 publications

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“…ROS formation in isolated rat hepatocytes was assessed by using 2´,7´-dichlorofluorescein (DCFH) as a fluorophore; its non-fluorescent, parent compound, 2´,7´-dichlorofluorescin diacetate (DCF-DA), readily crosses the hepatocyte membrane and is metabolized by nonspecific esterases and oxidized to DCFH by intracellular ROS (Cathcart et al 1983). DCFH was detected by flow cytometric analysis.…”

Section: Methodsmentioning

confidence: 99%

Physiological concentrations of unconjugated bilirubin prevent oxidative stress-induced hepatocanalicular dysfunction and cholestasis

et al. 2013

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Bilirubin is an endogenous antioxidant with cytoprotective properties, and several studies highlight its potential in the treatment of pro-oxidant diseases. We demonstrated that oxidative stress (OS), a key feature in most hepatopathies, induces cholestasis by actin cytoskeleton disarrangement and further endocytic internalization of key canalicular transporters, such as the bile salt export pump (Bsep) and the multidrug resistance-associated protein 2 (Mrp2). Here, we evaluated the capability of physiological concentrations of unconjugated bilirubin (UB)

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Section: Methodsmentioning

confidence: 99%

Physiological concentrations of unconjugated bilirubin prevent oxidative stress-induced hepatocanalicular dysfunction and cholestasis

et al. 2013

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“…Mitochondria were incubated with the fluorescent probe, 2 M, in the absence or presence of ceramide or other electron transport inhibitors (see figure legends). Fluorescence was determined at 529 nm for emission and 503 nm for excitation, with slit widths of 10 and 5 nm, respectively (27,29). Fluorescence of DCF was correlated with increasing concentrations of hydrogen peroxide allowing determination of hydrogen peroxide as described (27).…”

Section: Methodsmentioning

confidence: 99%

Direct Effect of Ceramide on the Mitochondrial Electron Transport Chain Leads to Generation of Reactive Oxygen Species

Garcı́a-Ruiz¹,

Colell²,

Marı́³

et al. 1997

Journal of Biological Chemistry

768

555

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Ceramide is a sphingolipid that is generated in the signaling of inflammatory cytokines such as tumor necrosis factor (TNF), which exerts many functional roles depending on the cell type where it is produced. Since TNF cytotoxicity is mediated by overproduction of reactive oxygen species from mitochondria, we have examined the role of ceramide in generation of oxidative stress in isolated rat liver mitochondria. The present studies demonstrate that addition of N-acetylsphingosine (C 2 -ceramide) to mitochondria led to an increase of fluorescence of dihydrorhodamine 123 or dichlorofluorescein-stained mitochondria, indicating formation of hydrogen peroxide. Such effect was significant at 0.25 M and maximal at 1-5 M C 2 , decreasing at greater concentrations. This inductive effect of ceramide was mimicked by N-hexanoylsphingosine at the same concentration range, whereas the immediate precursor of C 2 , C 2 -dihydroceramide increased hydrogen peroxide at 1-5 M. Sphingosine generated hydrogen peroxide at concentrations 10 M, whereas diacylglycerol failed to increase hydrogen peroxide. The increase in hydrogen peroxide induced by C 2 was not triggered by mitochondrial permeability transition as C 2 did not induce mitochondrial swelling. Blocking electron transport chain at complex I and II prevented the increase in hydrogen peroxide induced by C 2 ; however, interruption of electron flow at complex III by antimycin A potentiated the inductive effect of C 2 . Depletion of matrix GSH prior to exposure to ceramide resulted in a potentiated increase (2-fold) of hydrogen peroxide generation, leading to lipid peroxidation and loss of activity of respiratory chain complex IV compared with GSH-repleted mitochondria. Mitochondria isolated from TNF-treated cells showed an increase (2-3-fold) in the amount of ceramide compared with mitochondria from untreated cells. These results suggest that mitochondria are a target of ceramide produced in the signaling of TNF whose effect on mitochondrial electron transport chain leads to overproduction of hydrogen peroxide and consequently this phenomena may account for the generation of reactive oxygen species during TNF cytotoxicity.

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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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Detection of picomole levels of hydroperoxides using a fluorescent dichlorofluorescein assay

Cited by 786 publications

References 12 publications

Physiological concentrations of unconjugated bilirubin prevent oxidative stress-induced hepatocanalicular dysfunction and cholestasis

Physiological concentrations of unconjugated bilirubin prevent oxidative stress-induced hepatocanalicular dysfunction and cholestasis

Direct Effect of Ceramide on the Mitochondrial Electron Transport Chain Leads to Generation of Reactive Oxygen Species

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

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