title: Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps.
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.
authors.affiliation: Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques (UMR CNRS 8601), Université Paris Descartes, Sorbonne Paris Cité, 75006 Paris, France.
authors.forename: Kahina
authors.initials: K
authors.lastname: Abbas
authors.affiliation: Aix-Marseille Université, CNRS, ICR UMR 7273, 13397 Marseille Cedex 20, France.
authors.forename: Micael
authors.initials: M
authors.lastname: Hardy
authors.affiliation: Aix-Marseille Université, CNRS, ICR UMR 7273, 13397 Marseille Cedex 20, France.
authors.forename: Florent
authors.initials: F
authors.lastname: Poulhès
authors.affiliation: Aix-Marseille Université, CNRS, ICR UMR 7273, 13397 Marseille Cedex 20, France.
authors.forename: Hakim
authors.initials: H
authors.lastname: Karoui
authors.affiliation: Aix-Marseille Université, CNRS, ICR UMR 7273, 13397 Marseille Cedex 20, France.
authors.forename: Paul
authors.initials: P
authors.lastname: Tordo
authors.affiliation: Aix-Marseille Université, CNRS, ICR UMR 7273, 13397 Marseille Cedex 20, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Ouari
authors.affiliation: Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques (UMR CNRS 8601), Université Paris Descartes, Sorbonne Paris Cité, 75006 Paris, France; ESPE de l׳Académie de Paris, Université Paris Sorbonne, 75016 Paris, France. Electronic address: fabienne.peyrot@parisdescartes.fr.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Peyrot
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism; D002460:Cell Line; D003497:Cyclic N-Oxides / Q000138:chemical synthesis*; D004578:Electron Spin Resonance Spectroscopy; D005978:Glutathione / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D017665:Hydroxyl Radical / Q000378:metabolism; D008262:Macrophage Activation; D008264:Macrophages / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D009589:Nitrogen Oxides / Q000138:chemical synthesis*; D013113:Spin Labels; D019097:Spin Trapping / Q000379:methods*; D013481:Superoxides / Q000032:analysis* / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D003497:Cyclic N-Oxides; D009589:Nitrogen Oxides; D013113:Spin Labels; C015388:nitrones; D013481:Superoxides; D017665:Hydroxyl Radical; D006861:Hydrogen Peroxide; D005978:Glutathione; D013755:Tetradecanoylphorbol Acetate; D001205:Ascorbic Acid
keywords: Cyclic nitrones; EPR spectroscopy; ESR spectroscopy; Free radical; Hydroxyl radical; RAW macrophages; Spin trapping; Superoxide