Superoxide-mediated inactivation of nitric oxide and peroxynitrite formation by tobacco smoke in vascular endothelium: studies in cultured cells and smokers

Am J Physiol Heart Circ Physiol. 2009 Jun;296(6):H1781-92. doi: 10.1152/ajpheart.00930.2008. Epub 2009 Apr 10.


Tobacco smoke is known to cause nitric oxide ((*)NO) inactivation and endothelial dysfunction. In this work we evaluated the interplay between (.)NO and superoxide (O(2)(*-)) radicals and the consequent impact on (*)NO bioavailability and nitroxidative stress in bovine aortic endothelial cells exposed to cigarette smoke extract (CSE) and in smokers. Bovine aortic endothelial cells in the presence of CSE triggered O(2)(*-) production as indicated by spin-trapping electron paramagnetic resonance experiments. O(2)(*-) was produced both extracellulary (3.4 vs. 1.0 nmol.h(-1)*mg(-1); CSE vs. control; cytochrome c(3+) reduction assay) and intracellularly (40% inhibition of cytosolic aconitase). CSE also led to the production of peroxynitrite as evaluated by dihydrorhodamine oxidation and protein tyrosine nitration on cells. O(2)(*-) and peroxynitrite formation were decreased by ascorbate and alpha-tocopherol. Additionally, CSE led to the oxidation of endothelial nitric oxide synthase increasing the monomeric inactive form of endothelial nitric oxide synthase. Smokers and age-matched healthy volunteers were supplemented orally with 500 mg ascorbate plus 400 IU all-rac-alpha-tocopherol every 12 h for 165 days. Smokers had endothelial dysfunction compared with control subjects (95% confidence interval: 2.5, 8.3 vs. 10.6, 14.2; P < 0.05) as assessed by flow-mediated dilation of the brachial artery, and plasma levels of protein 3-nitrotyrosine were 1.4-fold higher. The loss of flow-mediated dilation in smokers reverted after a long-term antioxidant supplementation (95% confidence interval: 13.9, 19.9; P < 0.05), reaching values comparable with the control population. Our data indicate that elements on tobacco smoke, most likely through redox cycling, divert (*)NO toward peroxynitrite by inducing O(2)(*-) production in vascular endothelial cells both in vitro and in vivo.

Publication types

  • Clinical Trial
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Animals
  • Antioxidants / administration & dosage*
  • Antioxidants / metabolism
  • Antioxidants / pharmacokinetics
  • Aorta / cytology
  • Ascorbic Acid / administration & dosage
  • Ascorbic Acid / blood
  • Ascorbic Acid / pharmacokinetics
  • Brachial Artery / physiology
  • Cattle
  • Cells, Cultured
  • Endothelial Cells / cytology
  • Endothelial Cells / metabolism*
  • Female
  • Humans
  • Male
  • Nitric Oxide / metabolism*
  • Nitric Oxide Synthase Type III / metabolism
  • Oxidative Stress / drug effects
  • Oxidative Stress / physiology
  • Peroxynitrous Acid / metabolism*
  • Smoking / adverse effects
  • Smoking / metabolism*
  • Superoxides / metabolism*
  • Young Adult
  • alpha-Tocopherol / administration & dosage
  • alpha-Tocopherol / blood
  • alpha-Tocopherol / pharmacokinetics


  • Antioxidants
  • Superoxides
  • Peroxynitrous Acid
  • Nitric Oxide
  • NOS3 protein, human
  • Nitric Oxide Synthase Type III
  • alpha-Tocopherol
  • Ascorbic Acid