Hydroxyl radical generation and lipid peroxidation in C2C12 myotube treated with iodoacetate and cyanide.
To mimic exercise-induced events such as energetic impairment, free radical generation, and lipid peroxidation in vitro, mouse-derived C2C12 myotubes were submitted to the inhibition of glycolytic and/or oxidative metabolism with 1 mM iodoacetate (IAA) and/or 2 mM sodium cyanide (CN), respectively, under 5% CO2/95% air up to 180 min. Electron spin resonance (ESR) analysis with a spin-trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) revealed time-course increases in spin adducts from hydroxyl radical (DMPO-OH) and carbon-centered radical (DMPO-R) in the supernatant of C2C12 myotubes treated with the combination of IAA + CN. In this condition, malondialdehyde (MDA) and lactate dehydrogenase (LDH) were released into the supernatant. By the addition of iron-chelating 1 mM deferoxamine to the C2C12 preparation with IAA + CN, both ESR signals of DMPO-OH and DMPO-R were completely abolished, and the release of MDA and LDH were significantly reduced, while cyanide-resistant manganese superoxide dismutase had negligible effects on these parameters. Hence, a part of the injury of C2C12 myotube under IAA + CN was considered to result from the lipid peroxidation, which was induced by hydroxyl radical generated from iron-catalyzed systems such as the Fenton-type reaction. This in vitro model would be a helpful tool for investigating the free radical-related muscle injury.
Matsuki
N
N
Department of Veterinary Clinical Pathobiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan.
Takanohashi
A
A
Boffi
F M
FM
Inanami
O
O
Kuwabara
M
M
Ono
K
K
D000818:Animals; D002460:Cell Line; D003497:Cyclic N-Oxides; D003676:Deferoxamine / Q000494:pharmacology; D004351:Drug Resistance; D004578:Electron Spin Resonance Spectroscopy; D017665:Hydroxyl Radical / Q000378:metabolism*; D007461:Iodoacetates / Q000494:pharmacology*; D007502:Iron Chelating Agents / Q000494:pharmacology; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D015227:Lipid Peroxidation; D008315:Malondialdehyde / Q000378:metabolism; D051379:Mice; D018482:Muscle, Skeletal / Q000187:drug effects* / Q000378:metabolism*; D012966:Sodium Cyanide / Q000494:pharmacology*; D013113:Spin Labels; D013482:Superoxide Dismutase / Q000494:pharmacology
D003497:Cyclic N-Oxides; D007461:Iodoacetates; D007502:Iron Chelating Agents; D013113:Spin Labels; D017665:Hydroxyl Radical; D008315:Malondialdehyde; C017245:5,5-dimethyl-1-pyrroline-1-oxide; D007770:L-Lactate Dehydrogenase; D013482:Superoxide Dismutase; D003676:Deferoxamine; D012966:Sodium Cyanide