A Kinetic Study of Reactive Oxygen Species in Rainbow Trout Hepatocytes by Fluorometry.

The kinetics of reactive oxygen species (ROS) formation in a primary culture of rainbow trout hepatocytes was investigated using three fluorescent probes: 5-,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), dihydrorhodamine 123 (DHR 123), and dihydroethidium (DHE). The cell cultures were loaded with the three probes, separately. Hepatocytes were then exposed to Cu (0.15-10 mM) in serum-free Leibovitz's medium for 30 min before being quantified by a fluorescence plate reader during 30 min. Membrane integrity and glutathione (GSH) content were quantified using the fluorescent probes 5-carboxyfluorescein diacetate-acetoxymethyl ester (CFDA-AM) and monochlorobimane. Increasing ROS formation with increasing concentrations of Cu was shown using CM-H2DCFDA, whereas DHR 123 fluorescence decreased. Significant differences between control and treatment groups were observed at the highest concentrations (2.5 and 10 mM) for both probes. DHE fluorescence was lower than that of the other two probes and did not appear to be affected by any exposure. Additionally, a dose-dependent depletion of GSH and decreasing membrane integrity with increasing Cu concentrations were demonstrated, with significant effects observed at 2.5 and 10 mM for both endpoints. The results showed that both CMH2DCFDA and DHR 123 detected the development of their target Cu-induced ROS in trout hepatocytes but did so in opposite fashions. DHE was found to be unsuitable for detecting kinetics of ROS formation in this model system.