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, Zn²⁺ 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 Zn²⁺ ionophore pyrithione of A431 skin carcinoma cells preloaded with the H₂O₂-specific indicator PG1 resulted in a significant increase in H₂O₂ production that could be significantly inhibited with the mitochondrial inhibitor carbonyl cyanide 3-chlorophenylhydrazone. Mitochondria were further implicated as the source of zinc-induced H₂O₂ 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, H₂O₂ 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.