An integrated imaging approach to the study of oxidative stress generation by mitochondrial dysfunction in living cells.
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 (H2O2)-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.


CONCLUSIONS
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.
Cheng
Wan-Yun
WY
Department of Environmental Sciences and Engineering, University of North Carolina-Chapel Hill, USA.
Tong
Haiyan
H
Miller
Evan W
EW
Chang
Christopher J
CJ
Remington
James
J
Zucker
Robert M
RM
Bromberg
Philip A
PA
Samet
James M
JM
Hofer
Thomas P J
TP
D001562:Benzimidazoles; D002232:Carbocyanines; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000378:metabolism; D004782:Environmental Health / Q000379:methods*; D005456:Fluorescent Dyes; D015386:Hazardous Substances / Q000633:toxicity*; D006801:Humans; D006861:Hydrogen Peroxide / Q000032:analysis / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D028361:Mitochondrial Diseases / Q000139:chemically induced* / Q000503:physiopathology; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D017382:Reactive Oxygen Species / Q000032:analysis* / Q000378:metabolism; D014407:Tumor Cells, Cultured; D015032:Zinc / Q000633:toxicity
D001562:Benzimidazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; D015386:Hazardous Substances; D017382:Reactive Oxygen Species; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D006861:Hydrogen Peroxide; D015032:Zinc