2,7‐Dihydrodichlorofluorescein diacetate as a fluorescent marker for peroxynitrite formation

Abstract

Reactive oxygen species (ROS) have been implicated as an important causative factor in cell damage, including apoptosis and necrosis. Their proposed actions comprise lipid peroxidation, DNA damage, destruction of the mitochondrial respiratory chain and protein modifications. Recent experiments underline the importance of peroxynitrite, the reaction product of the two potent reactive species nitric oxide and Superoxide. Several fluorogenic compounds have been used in order to determine ROS formation in living cells. Besides dihydrorhodamine-123 (DHR-123), at present mostly applied to monitor peroxynitrite, 2,7-dihydrodichlorofluorescein (DCF-H) is used for detection of hydrogen peroxide and nitric oxide. We employed a cell free approach to evaluate the specificity and sensitivity of DCF-H to various oxidizing compounds. Our studies imply that DCF-H is much more sensitive to peroxynitrite oxidation than any other compound tested. In order to study peroxynitrite generation within individual cells, primary glial cultures loaded with DCF-H were monitored with a laser scanning microscope. Microglia, stimulated to simultaneously produce the peroxynitrite precursors nitric oxide and Superoxide, displayed the greatest increase in DCF fluorescence, whereas microglia producing either nitric oxide or Superoxide alone showed a relatively small increase in DCF fluorescence. In conclusion, DCF-H was demonstrated to be an excellent peroxynitrite marker with the potential to detect peroxynitrite formation in living cells. © 1997 Federation of European Biochemical Societies.

Oxidative stress in cells infected with bovine viral diarrhoea virus: a crucial step in the induction of apoptosis.

“…the extrusion of cytosolic glutathione (Bustamante et al, 1997 ;Slater et al, 1996) or the downregulation of the transcription of the antioxidant enzyme manganese superoxide dismutase (Briehl & Baker, 1996). DCF, the marker used in this study to analyse the intracellular redox state, may be oxidized by a variety of ROS, among them peroxides, nitric oxide and peroxynitrite (Possel et al, 1997). However, since the latter two compounds were not found to be produced by BT cells (unpublished observations) it seems likely that peroxides may be responsible for the change in DCF fluorescence observed in BT cells infected with cp BVDV.…”

7-Dehydrocholesterol enhances ultraviolet A-induced oxidative stress in keratinocytes: Roles of NADPH oxidase, mitochondria, and lipid rafts

“…2A). The probe used, carboxy-H 2 DCFDA, is not specific for a single type of ROS but reacts with several ROS in cells including H 2 O 2 , peroxynitrite, and peroxides [46,47], and consequently can detect different ROS from mitochondria, lipid oxidation and other sources after NADPH oxidase is no longer active.…”

Neurotoxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in cerebellar granule cells

“…This is a sensitive and rapid method, which is well suited for detecting overall oxidative stress after stimulation with ROS-forming compounds in low concentrations (Possel et al, 1997;Myhre et al, 2000). TCDD induced production of ROS in a dose-dependent manner.…”