title: Cellular sites of H2O2-induced damage and their protection by nitroxides.
abstract: While the exact mechanism of H2O2-induced cytotoxicity is unknown, there is considerable evidence implicating DNA as a primary target. A recent study showed that a cell-impermeable nitroxide protected mammalian cells from H2O2-induced cell killing and suggested that the protection was mediated through cell membrane-bound or extracellular factors. To further define the protective properties of nitroxides, Chinese hamster V79 cells were exposed to H2O2 with or without cell-permeable and impermeable nitroxides and selected metal chelators. EPR spectroscopy and paramagnetic line broadening agents were used to distinguish between intra- and extracellular nitroxide distribution. To study the effectiveness of nitroxide protection, in the absence of a cell membrane, H2O2-mediated damage to supercoiled plasmid DNA was evaluated. Both deferrioxamine and Tempol cross the cell membrane, and inhibited H2O2-mediated cell killing, whereas the cell-impermeable DTPA and nitroxide, CAT-1, failed to protect. Similar protective effects of the chelators and nitroxides were observed when L-histidine, which enhances intracellular injury, was added to H2O2. In contrast, when damage to plasmid DNA was induced (in the absence of a cell membrane), both nitroxides were protective. Collectively, these results do not support a role for membrane-bound or extracellular factors in mediating H2O2 cytotoxicity in mammalian cells.
authors.affiliation: Radiation Biology Branch, Division of Clinical Sciences, Bldg. 10 Rm B3B69, National Cancer Institute, NIH, Bethesda, MD 20892-1002, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Samuni
authors.forename: W
authors.initials: W
authors.lastname: DeGraff
authors.forename: M C
authors.initials: MC
authors.lastname: Krishna
authors.forename: J B
authors.initials: JB
authors.lastname: Mitchell
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000493:pharmacokinetics / Q000494:pharmacology; D002460:Cell Line; D002463:Cell Membrane Permeability; D002470:Cell Survival / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D006224:Cricetinae; D003497:Cyclic N-Oxides / Q000493:pharmacokinetics / Q000494:pharmacology; D004249:DNA Damage; D006639:Histidine / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000633:toxicity*; D009589:Nitrogen Oxides / Q000493:pharmacokinetics / Q000494:pharmacology*; D013113:Spin Labels
chemical_list: D000975:Antioxidants; D002614:Chelating Agents; D003497:Cyclic N-Oxides; D009589:Nitrogen Oxides; D013113:Spin Labels; D006639:Histidine; D006861:Hydrogen Peroxide; C001803:tempol