Cadmium-induced mitochondrial membrane-potential dissipation does not necessarily require cytosolic oxidative stress: studies using rhodamine-123 fluorescence unquenching.
The impact of cadmium on the cellular redox state and mitochondrial membrane potential (psi(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 microM 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 psi(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 psi(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 psi(m). We conclude that Cd rapidly induces ROS formation, mainly hydroxyl radical species OH(*), as well as the loss of psi(m). However, psi(m) dissipation does not necessarily require cellular OH(*) and may occur in the absence of apparent oxidative injury.
Départementes de Chimie, Centre TOXEN, Université du Québec à Montréal, Montréal, Québec, Canada H3C 3P8. jumarie.catherine@uqam.ca
J-S
JS
Bolduc
F
F
Denizeau
C
C
Jumarie
D018938:Caco-2 Cells; D002104:Cadmium / Q000494:pharmacology*; D002478:Cells, Cultured; D004785:Environmental Pollutants / Q000494:pharmacology*; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D007413:Intestinal Mucosa / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000737:chemistry / Q000187:drug effects* / Q000502:physiology; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000378:metabolism; D020112:Rhodamine 123
D004785:Environmental Pollutants; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D002104:Cadmium; D020112:Rhodamine 123