Menadione-induced oxidative stress in bovine heart microvascular endothelial cells.
OBJECTIVE
Oxidative stress from increased production of reactive oxygen species or decreased efficiency of inhibitory and scavenger systems may contribute to vascular injury. In this study, we developed an in vitro model of vascular injury by menadione-induced oxidative stress in bovine heart microvascular endothelial cells.


METHODS
Oxidative stress was induced by exposure to menadione. Superoxide, hydrogen peroxide and hydroxyl radical formation was measured by superoxide dismutase-inhibitable cytochrome c reduction, the dichlorofluorescin technique and the salicylate method, respectively. Electron paramagnetic-spin resonance spectroscopy employing 5-5'-dimethyl-l-pyrroline-N-oxide for superoxide trapping was used. Endothelial cell cytotoxicity was assessed by lactate dehydrogenase release.


RESULTS
Superoxide and hydroxyl radical were produced in a time- and concentration-dependent fashion. Fluorescence in the presence of dichlorofluorescin confirmed hydrogen peroxide formation. Endothelial cell cytotoxicity became evident after 5 h of menadione treatment at concentrations of 100 microM. 3-Aminobenzamide, a poly(ADP-ribose)polymerase inhibitor, and dimethylthiourea, a hydrogen peroxide and hydroxyl radical scavenger, decreased menadione cytotoxicity, whereas deferoxamine, an inhibitor of hydroxyl radical formation, did not.


CONCLUSIONS
The results suggest that menadione toxicity is mediated by poly(ADP-ribose)polymerase activation via hydrogen peroxide formation and that menadione-treated bovine heart microvessel endothelial cells provide a suitable in vitro model to study oxidative stress in endothelial cells.
Kossenjans
W
W
Department of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, OH, USA.
Rymaszewski
Z
Z
Barankiewicz
J
J
Bobst
A
A
Ashraf
M
M
D000818:Animals; D002417:Cattle; D016923:Cell Death / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects; D003326:Coronary Circulation / Q000187:drug effects*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects*; D004789:Enzyme Activation; D016166:Free Radical Scavengers / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism; D017665:Hydroxyl Radical; D008833:Microcirculation / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects*; D011065:Poly(ADP-ribose) Polymerases / Q000819:agonists; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism; D014812:Vitamin K / Q000494:pharmacology*
D016166:Free Radical Scavengers; D017382:Reactive Oxygen Species; D013481:Superoxides; D014812:Vitamin K; D017665:Hydroxyl Radical; D006861:Hydrogen Peroxide; D011065:Poly(ADP-ribose) Polymerases