title: An integrated imaging approach to the study of oxidative stress generation by mitochondrial dysfunction in living cells.
abstract: 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.
authors.affiliation: Department of Environmental Sciences and Engineering, University of North Carolina-Chapel Hill, USA.
authors.forename: Wan-Yun
authors.initials: WY
authors.lastname: Cheng
authors.forename: Haiyan
authors.initials: H
authors.lastname: Tong
authors.forename: Evan W
authors.initials: EW
authors.lastname: Miller
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Chang
authors.forename: James
authors.initials: J
authors.lastname: Remington
authors.forename: Robert M
authors.initials: RM
authors.lastname: Zucker
authors.forename: Philip A
authors.initials: PA
authors.lastname: Bromberg
authors.forename: James M
authors.initials: JM
authors.lastname: Samet
authors.forename: Thomas P J
authors.initials: TP
authors.lastname: Hofer
mesh_terms: 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
chemical_list: 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