An Integrated Imaging Approach to the Study of Oxidative Stress Generation by Mitochondrial Dysfunction in Living Cells

作者：Cheng Wan Yun; Tong Haiyan; Miller Evan W; Chang Christopher J; Remington James; Zucker Robert M; Bromberg Philip A; Samet James M^*; Hofer Thomas P J

来源：Environmental Health Perspectives, 2010, 118(7): 902-908.

DOI：10.1289/ehp.0901811

• biosensors
• confocal microscopy
• hydrogen peroxide
• mitochondrial dysfunction
• oxidative stress
• real-time imaging
• ROS

BACKGROUND: The mechanisms of action of many environmental agents commonly involve oxidative stress resulting from mitochondrial dysfunction. Zinc is a common environmental metallic contaminant that has been implicated in a variety of oxidant-dependent toxicological responses. Unlike ions of other transition metals such as iron, copper, and vanadium, Zn(2+) does not generate reactive oxygen species (ROS) through redox cycling.
OBJECTIVE: To characterize the role of oxidative stress in zinc-induced toxicity.
METHODS: We used an integrated imaging approach that employs the hydrogen peroxide (H(2)O(2))-specific fluorophore Peroxy Green 1 (PG1), the mitochondrial potential sensor 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), and the mitochondria-targeted form of the redox-sensitive genetically encoded fluorophore MTroGFP1 in living cells.
RESULTS: Zinc treatment in the presence of the Zn(2+) ionophore pyrithione of A431 skin carcinoma cells preloaded with the H(2)O(2)-specific indicator PG1 resulted in a significant increase in H(2)O(2) production that could be significantly inhibited with the mitochondrial inhibitor carbonyl cyanide 3-chlorophenylhydrazone. Mitochondria were further implicated as the source of zinc-induced H(2)O(2) formation by the observation that exposure to zinc caused a loss of mitochondrial membrane potential. Using MTroGFP1, we showed that zinc exposure of A431 cells induces a rapid loss of reducing redox potential in mitochondria. We also demonstrated that zinc exposure results in rapid swelling of mitochondria isolated from mouse hearts.
CONCLUSION: Taken together, these findings show a disruption of mitochondrial integrity, H(2)O(2) formation, and a shift toward positive redox potential in cells exposed to zinc. These data demonstrate the utility of real-time, live-cell imaging to study the role of oxidative stress in toxicological responses.

• 出版日期2010-7