vitamin-k-semiquinone-radical and 2--7--dichlorofluorescein

Quercetin may contribute to the protection afforded by fruit- and vegetable-rich diets against diseases for which excess production of reactive oxygen species (ROS) has been implicated as a causal or contributory factor. We examine the effect of short term (90 min) quercetin (1-100 microM) exposure on the progress of menadione induced oxidative stress within HL-60 cells. 2',7'-dichlorofluorescein and rhodamine-123 fluorescence, resulting from oxidation of the ROS-sensitive dyes dichlorodihydrofluorescein and dihydrorhodamine-123 respectively, were utilised as indicators of general ROS levels. Ethidium fluorescence, resulting from oxidation of dihydroethidium, was used as a potentially more specific indicator of O(2)(-). Exposure to quercetin alone induced a decrease in DCF and rhodamine fluorescence. Conversely, ethidium fluorescence was enhanced by treatment with >or=40 microM quercetin. Incubation with 1-100 microM quercetin reduced the extent of menadione-induced increase in DCF and rhodamine fluorescence but the menadione-induced increase in ethidium fluorescence was further elevated for cells treated with >or=25 microM quercetin. Exposure to >or=10 microM quercetin abrogated menadione-induced DNA single-strand breaks but, paradoxically, quercetin exacerbated membrane damage and failed to enhance the viability of menadione-challenged cells. In conclusion, quercetin exerts only site-specific protection against oxidative stress.

Topics: Cell Membrane Permeability; Cell Survival; DNA Damage; Dose-Response Relationship, Drug; Fluoresceins; Fluorescent Dyes; Glutathione; HL-60 Cells; Humans; Oxidative Stress; Quercetin; Reactive Oxygen Species; Rhodamine 123; Vitamin K

In studies into the oxidative burst in RAW 264 monocyte/macrophages, it was observed that capsaicin, a vanilloid receptor agonist, stimulated dichlorofluorescin (DCFH) oxidation in a concentration-dependent manner, which could be blocked by capsazepine, a vanilloid receptor antagonist. However, by use of a number of vanilloid agonists (including N-octyl-3-chloro-4-hydroxyphenylacetamide, 4m), we demonstrated that there was no relationship between vanilloid agonist potency and the capacity to stimulate DCFH oxidation. The oxidative burst stimulators Tween 20 and phorbol myristyl acetate (PMA) also stimulated reactive oxygen species generation, which again was inhibited by capsazepine. Use of the selective inhibitor diphenyliodonium iodide ruled out a role for plasma membrane NAD(P)H oxidase as the site of capsaicin- and 4m-stimulated DCFH oxidation. However, this DCFH oxidation was modulated by a number of inhibitors of mitochondrial respiration. Rotenone enhanced DCFH oxidation induced by capsaicin and 4m, whilst malonic acid and potassium cyanide inhibited this response. 2,4-Dinitrophenol, an inhibitor of oxidative phosphorylation, was without effect. The antioxidant trolox c inhibited DCFH oxidation stimulated by capsaicin, 4m, and PMA, whereas N-acetylcysteine, a precursor of glutathione, was without effect. Capsazepine inhibited DCFH oxidation in unstimulated cells and in cells treated with menadione, a redox-cycling quinone. Capsazepine was also a potent antioxidant when measured in a Fe3+ reduction assay. We concluded that DCFH oxidation stimulated by vanilloid analogues was not mediated via a vanilloid receptor, but rather by impairment of mitochondrial electron transport.

Topics: Acetylcysteine; Animals; Antioxidants; Capsaicin; Carcinogens; Chromans; Fluoresceins; Macrophages; Mice; Monocytes; Oxidation-Reduction; Polysorbates; Reactive Oxygen Species; Receptors, Drug; Surface-Active Agents; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vitamin K