vitamin-k-semiquinone-radical and 1-10-phenanthroline

Protection by the flavonoids, quercetin and rutin, against tert-butylhydroperoxide (tert-BOOH)- and menadione-induced DNA single strand breaks was investigated in Caco-2 cells. Both tert-BOOH and menadione induced DNA single strand breaks in a concentration-dependent manner. Pre-incubation of Caco-2 cells with either quercetin or rutin for 24 h significantly decreased the formation of DNA single strand breaks evoked by tert-BOOH (P <.05). Iron chelators, 1,10-phenanthroline (o-Phen) and deferoxamine mesylate (DFO), also protected against tert-BOOH-induced DNA damage, whereas butylated hydroxytoluene (BHT) had no effect. Quercetin, and not rutin, decreased the extent of menadione-induced DNA single strand breaks. DFO and BHT, and not o-Phen, protected against menadione-induced DNA strand break formation (P <.05). From the results of this study, iron ions were involved in tert-BOOH-induced DNA single strand break formation in Caco-2 cells, whereas DNA damage evoked by menadione was far more complex. We demonstrated that the flavonoids, quercetin and rutin, protected against tert-BOOH-induced DNA strand breaks by way of their metal ion chelating mechanism. However, quercetin, and not rutin, protected against menadione-induced DNA single strand breaks by acting as both a metal chelator and radical scavenger.

Topics: Butylated Hydroxytoluene; Caco-2 Cells; Cell Survival; Deferoxamine; DNA Damage; Free Radical Scavengers; Humans; Iron; Iron Chelating Agents; Phenanthrolines; Quercetin; Rutin; tert-Butylhydroperoxide; Vitamin K

Endothelial cells (ECs) are subjected to oxidative stress during many pathological processes, including ischemia/reperfusion and general inflammation. In the present study, we examined the effects of oxidative stress on rates of apoptosis in EC cultures. We treated large and microvessel ECs with menadione for 1 h in vitro to simulate the most common physiological form of oxidative stress, exposure to O2*-. Capillary ECs were resistant to menadione-induced apoptosis when compared with large-vessel ECs. Treatment with 35 microM menadione resulted in an apoptotic rate of approximately 5% in capillary EC cultures compared with approximately 45% in large-vessel EC cultures. At higher concentrations of menadione (35-75 microM), both types of ECs exhibited a concentration-related increase in apoptosis. Necrotic cell death only became evident at menadione concentrations ranging from 75-100 microM for both cell types. The timing of the apoptotic response to a 1 h menadione exposure was very specific. For both EC types, peaks of apoptosis occurred in two distinct waves, at 6-8 and 18-22 h after treatment. Analysis of the events leading up to the first peak of apoptosis indicated that specific matrix metalloproteinases (MMPs) were activated, suggesting that MMPs may be involved in initiating the apoptotic process.

Topics: Animals; Apoptosis; Cattle; Cells, Cultured; Culture Media, Conditioned; Dose-Response Relationship, Drug; Endothelium, Vascular; Glutathione; Matrix Metalloproteinases; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Oxidative Stress; Phenanthrolines; Phenylmercuric Acetate; Superoxides; Time Factors; Vitamin K

The model quinone compound menadione has been used to study the effects of oxidative stress in mammalian cells, and to investigate the mechanism of action of the quinone nucleus which is present in many anti-cancer drugs. We have used the alkaline single cell gel electrophoresis assay (comet assay) to investigate the effects of low doses of this compound on isolated human lymphocytes. We found that concentrations of menadione as low as 1 microM were sufficient to induce strand breaks in these cells. Pre-incubation with the NAD(P)H quinone oxidoreductase inhibitor dicoumarol, enhanced the production of menadione-induced strand breaks. In contrast, the metal ion chelator 1,10-phenanthroline inhibited formation of strand breaks, although prolonged incubation with 1,10-phenanthroline in combination with menadione resulted in an increase in a population of very severely damaged nuclei. A marked variation in the response of lymphocytes from different donors to menadione, and in different samples from the same donor was also observed.

Topics: Antioxidants; Ascorbic Acid; Cell Survival; Chelating Agents; Dicumarol; Dimethyl Sulfoxide; DNA Damage; DNA Fragmentation; Electrophoresis, Agar Gel; Enzyme Inhibitors; Humans; Lymphocytes; Male; NAD(P)H Dehydrogenase (Quinone); Oxidative Stress; Phenanthrolines; Salicylates; Salicylic Acid; Vitamin K

Menadione produces DNA strand breaks (DNA sb) in cultured Chinese hamster fibroblasts which are, to a great extent, mediated by OH radical. A reasonable hypothesis is that H2O2, a product of menadione metabolism, reacts with nuclear iron and produces OH radical in situ. Consistent with that, 1,10-phenanthroline (PHEN) prevents menadione-induced DNA sb at low (< 200 microM) concentrations of the chelator. However, at higher PHEN concentrations, the effect is reversed and an enhancement of DNA sb is observed. The PHEN-induced enhancement of DNA sb becomes more evident at high (> 60 microM) menadione concentrations and is strongly prevented by neocuproine (NEO), an efficient copper chelator. However, NEO offers only a slight protection against DNA sb caused by menadione alone. The results are consistent with the following events: (i) the products of menadione metabolism causes copper ion release from some cellular compartment; (ii) in the presence of PHEN, a Cu(PHEN)2 complex is formed; (iii) the Cu(PHEN)2 complex is known to be very clastogenic, inducing DNA damage in a reducing environment. Evidence is also presented that menadione metabolism causes an increase in intracellular chelatable iron: in the presence of a constant 2,2'-dipyridyl concentration, the DNA sb produced by increasing concentrations of menadione become progressively less susceptible to inhibition by the chelator. Therefore the DNA damage originated from menadione metabolism seems to be caused by two conjugated and synergistic events, viz., the production of reactive oxygen species and the release of copper and iron from a cellular storage site into a 'free' form pool, capable of catalyzing DNA damaging reactions.

Topics: Animals; Cells, Cultured; Copper; Cricetinae; DNA Damage; Fibroblasts; Homeostasis; Hydrogen Peroxide; Iron; Iron Chelating Agents; Oxidation-Reduction; Phenanthrolines; Vitamin K

In rat hepatocytes exposed to the quinones menadione and 2,3-dimethoxy-1,4-naphthoquinone (2,3-diOMe-1,4-NQ) a decrease in NAD+ is observed. DNA damage and activation of poly(ADP-ribose)polymerase are often associated with a decrease in NAD+. Using rat hepatocytes and human myeloid leukaemic cells (K562), we examined the extent of DNA damage induced by these quinones at non-toxic concentrations, i.e. at concentrations at which the cells completely exclude the dye trypan blue. Both quinones caused significant DNA damage at very low concentrations (5-100 microM). With 2,3-diOME-1,4-NQ (15 microM) or menadione (15 microM) single strand breaks (SSB) were observed at very early time points (less than 5 min), reaching a maximum between 20 and 30 min. Most SSB were repaired within 45 min of the removal of the quinones. Whilst extensive repair was observed within 4 hr of the removal of 2,3-diOMe-1,4-NQ (15 microM), only partial repair was observed following exposure to menadione (15 microM). SSB induced by 2,3-diOMe-1,4-NQ (15 microM) were completely inhibited by the iron chelator 1,10-phenanthroline (25 microM), whereas in cells exposed to menadione (15 microM) they were only partially inhibited. Finally, although the membrane integrity of K562 cells was unaffected by exposure to high concentrations of both quinones (less than or equal to 400 microM), cytostasis was observed at much lower concentrations (50 microM). Our results demonstrate that at very low concentrations these quinones induce extensive DNA damage possibly caused by hydroxyl radicals. The DNA damage was accompanied by an early cytostasis but no loss of membrane integrity.

Topics: Animals; Cell Division; DNA Damage; DNA, Single-Stranded; Dose-Response Relationship, Drug; Humans; Liver; NAD; Naphthoquinones; Phenanthrolines; Rats; Trypan Blue; Tumor Cells, Cultured; Vitamin K

When Chinese hamster fibroblasts were exposed to hydrogen peroxide or to a system consisting of xanthine oxidase and hypoxanthine, which generates superoxide anion plus hydrogen peroxide, sister-chromatid exchanges (SCEs) were formed in a dose-dependent manner. When the iron-complexing agent o-phenanthroline was present in the medium, however, the production of these SCEs was completely inhibited. This fact indicates that the Fenton reaction: Fe2+ + H2O2----OH0 + OH- + Fe3+ is responsible for the production of SCEs. When O2- and H2O2 were generated inside the cell by incubation with menadione, the production of SCE was prevented by co-incubation with copper diisopropylsalicylate, a superoxide dismutase mimetic agent. The most likely role of O2- is as a reducing agent of Fe3+: O2- + Fe3+----Fe2+ + O2, so that the sum of this and the Fenton reaction, i.e., the iron-catalyzed Haber-Weiss reaction, provides an explanation for the active oxygen species-induced SCE: H2O2 + O2(-)----OH- + OH0 + O2. According to this view, the OH radical thus produced is the agent which ultimately causes SCE. These results are discussed in comparison with other mechanisms previously proposed for induction of SCE by active oxygen species.

Topics: Animals; Cell Cycle; Cell Line; Cricetinae; Drug Synergism; Free Radicals; Hydrogen Peroxide; Hydroxides; Iron; Phenanthrolines; Salicylates; Sister Chromatid Exchange; Superoxides; Vitamin K