2,7-DIHYDRODICHLOROFLUORESCEIN DIACETATE AS A FLUORESCENT MARKER FOR PEROXYNITRITE FORMATION

Reactive oxygen species (ROS) have been implicated as an important cau sative 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, Rece nt experiments underline the importance of peroxynitrite, the reaction product of the two potent reactive species nitric oxide and superoxid e, 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-dihydrodichlo rofluorescein (DCF-H) is used for detection of hydrogen peroxide and n itric oxide, We employed a cell free approach to evaluate the specific ity and sensitivity of DCF-H to various oxidizing compounds, Our studi es imply that DCF-H is much more sensitive to peroxynitrite oxidation than any other compound tested. In order to study peroxynitrite genera tion within individual cells, primary glial cultures loaded with DCF-H mere monitored with a laser scanning microscope, Microglia, stimulate d to simultaneously produce the peroxynitrite precursors nitric oxide and superoxide, displayed the greatest increase in DCF fluorescence, w hereas microglia producing either nitric oxide or superoxide alone sho wed a relatively small increase in DCF fluorescence. Tn conclusion, DC F-H was demonstrated to be an excellent peroxynitrite marker with the potential to detect peroxynitrite formation in living cells. (C) 1997 Federation of European Biochemical Societies.