ToxSci Advance Access originally published online on November 4, 2003
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Toxicological Sciences 77, 299-306 (2004)
Copyright © 2004 by the Society of Toxicology
IN VITRO TOXICOLOGY AND ALTERNATIVE TESTING |
Cadmium-Induced Mitochondrial Membrane-Potential Dissipation Does Not Necessarily Require Cytosolic Oxidative Stress: Studies Using Rhodamine-123 Fluorescence Unquenching
* Départementes de Chimie,
des Sciences Biologiques, Centre TOXEN, Université du Québec à Montréal, C.P. 8888, Succ. centre-ville, Montréal, Québec, Canada H3C 3P8
ABSTRACT
The impact of cadmium on the cellular redox state and mitochondrial membrane potential (
m) has been studied by monitoring dichlorofluorescein (DCF), CMXRos (dichlorodihydrofluorescein diacetate, chloromethyl-X-rosamine), and Rh-123 fluorescence in 5-day-old TC7 cells, a highly differentiated clone of the human intestinal Caco-2 cell line. Flow cytometry analyses, using DCFH oxidation to DCF, clearly revealed that a 30-min incubation to 50 µM cadmium (Cd) is sufficient to induce reactive oxygen species (ROS) formation; this effect was completely eliminated by the presence of 50 mM mannitol for the 30-min incubation period, but mannitol only partially scavenged ROS for the longer period of time studied. Imaging studies using fluorescence video microscopy revealed a parallel increase in (DCF) fluorescence in the nuclear and cytoplasmic regions as soon as Cd was added to the exposure medium. Flow cytometry analyses monitoring CMXRos fluorescence clearly showed that Cd also leads to m disruption, but, contrary to what was observed for ROS formation, mannitol was completely inefficient in preventing this effect. Further investigation using fluorescence microscopy and Rh-123 fluorescence unquenching revealed that although mannitol did not protect against Cd-induced dissipation of m, it considerably delayed the process. We found that Rh-123 unquenching, occurring during probe redistribution, is a suitable tool to monitor the decrease of m. We conclude that Cd rapidly induces ROS formation, mainly hydroxyl radical species OH•, as well as the loss of m. However, m dissipation does not necessarily require cellular OH• and may occur in the absence of apparent oxidative injury.
Key Words: cadmium; dichlorodihydrofluorescein diacetate (DCF), chloromethyl-X-rosamine CMXRos, rhodamine-123; reactive oxygen species (ROS); mitochondrial membrane potential; intestinal Caco-2 cells.