Faculty Collaboration Database

Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps. Free Radic Biol Med 2014 Jun;71:281-290

Date

03/26/2014

Pubmed ID

24662195

DOI

10.1016/j.freeradbiomed.2014.03.019

Scopus ID

2-s2.0-84898773391 (requires institutional sign-in at Scopus site)   72 Citations

Abstract

Reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide (H2O2), have a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of their production. For measuring ROS production in cells, the ESR spin trapping technique using cyclic nitrones distinguishes itself from other methods by its specificity for superoxide and hydroxyl radical. However, several drawbacks, such as the low spin trapping rate and the spontaneous and cell-enhanced decomposition of the spin adducts to ESR-silent products, limit the application of this method to biological systems. Recently, new cyclic nitrones bearing a triphenylphosphonium (Mito-DIPPMPO) or a permethylated β-cyclodextrin moiety (CD-DIPPMPO) have been synthesized and their spin adducts demonstrated increased stability in buffer. In this study, a comparison of the spin trapping efficiency of these new compounds with commonly used cyclic nitrone spin traps, i.e., 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and analogs BMPO, DEPMPO, and DIPPMPO, was performed on RAW 264.7 macrophages stimulated with phorbol 12-myristate 13-acetate. Our results show that Mito-DIPPMPO and CD-DIPPMPO enable a higher detection of superoxide adduct, with a low (if any) amount of hydroxyl adduct. CD-DIPPMPO, especially, appears to be a superior spin trap for extracellular superoxide detection in living macrophages, allowing measurement of superoxide production in unstimulated cells for the first time. The main rationale put forward for this extreme sensitivity is that the extracellular localization of the spin trap prevents the reduction of the spin adducts by ascorbic acid and glutathione within cells.

Author List

Abbas K, Hardy M, Poulhès F, Karoui H, Tordo P, Ouari O, Peyrot F

Author

Micael Joel Hardy PhD Visiting Assistant Professor in the Biophysics department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Ascorbic Acid
Cell Line
Cyclic N-Oxides
Electron Spin Resonance Spectroscopy
Glutathione
Hydrogen Peroxide
Hydroxyl Radical
Macrophage Activation
Macrophages
Mice
Nitrogen Oxides
Spin Labels
Spin Trapping
Superoxides
Tetradecanoylphorbol Acetate