The plasma membrane of HT29 human colon carcinoma cells was characterized by EPR spectroscopy as the site for redox activation of 3,6-difluoro-2,5-bis(aziridinyl)-1,4-benzoquinone (F-DZQ). Supplementation of HT29 cells with F-DZQ yielded an EPR signal ascribed to the semiquinone species; the hyperfine splitting constants of the 11-line spectrum were 1.4 and 1.35 G for a(N) and a(F), respectively. The intensity of the EPR signal was inhibited competitively by potassium ferricyanide, a compound which has no access to the intracellular milieu and used to evaluate transmembrane NADH-ferricyanide reductase activity. The extracellular localization of the signal was confirmed by using chromium trioxalate, a membrane-impermeant spin-broadening agent, which abolished in a concentration-dependent manner the semiquinone signal originating from the metabolism of F-DZQ by HT29 cells. The intensity of the semiquinone signal was decreased by agents which block sulfhydryl groups upon alkylation, fluorodinitrobenzene and p-chloromercuribenzoate, presumably acting on plasma membrane dehydrogenases. Other flavin dehydrogenase inhibitors, such as allopurinol, deprenyl or clorgyline, and d-arginine or N(G)-methyl-l-arginine did not affect the EPR signal. Conversely, the intensity of the semiquinone signal was increased upon supplementation of HT29 cells with glucose and insulin, which may enhance the intracellular levels of electron donors for the transplasma membrane dehydrogenase activity. The extracellular semiquinone signal was abolished by superoxide dismutase by a mechanism implying displacement of the equilibrium of the autoxidation reaction. Formation of oxygen-centered radicals during this redox activity was evaluated by EPR in conjunction with the spin trap 4-POBN. A composite signal consisting of the spin adducts of methyl, hydroxyl and superoxide radicals was observed (the former arising from hydroxyl radical attack on the quinone solvent, dimethylsulfoxide). The formation of these spin adducts was abolished by superoxide dismutase and their detection became impossible in the presence of the line broadening agent chromiun trioxalate, thus indicating their extracellular formation and localization, respectively. The occurrence of a redox site at the plasma membrane of HT29 cells for the activation of this halogenated aziridinylbenzoquinone is discussed in terms of its significance for intracellular processes and a build-up of oxyradicals in the extracellular milieu. Copyright (C) 1998 Elsevier Science Ireland Ltd.
- Cancer cells
- Electron paramagnetic resonance
- NADH-ferricyanide reductase
- Oxygen radicals
- Plasma membrane
- Plasma membrane dehydrogenase
ASJC Scopus subject areas