salicylates and 5-5-dimethyl-1-pyrroline-1-oxide

Constitutive production of hydroxyl radicals from four established cancer cell lines was detected as spin adducts of 5,5-dimethyl-l-pyroline-N-oxide (DMPO), using an electron spin resonance spectrometer. The generated hydroxyl radicals was decreased in three out of four cancer cell lines when incubated in vitro for 3 h with TNF-alpha No direct scavenging effect of TNF-alpha on hydroxyl radicals or superoxide anions was observed in the in vitro radical generation system. The modulation of intracellular reactive oxygen species of these cancer cells by adding menadione or CuDIPS to the culture medium changed the antiproliferative effect of TNF-alpha on the cells. The ultrastructural localization of the radical-generating sites in cancer cells was visualized using the diaminobenzidine/horseradish peroxide histochemical system at the electron microscopic level. The hydrogen peroxide-dependent formation of electron-dense materials localized at the mitochondrial membranes was decreased after the treatment of the cancer cells with TNF-alpha. These data indicate that the reduction of radical generation in cancer cells by TNF-alpha may be an early mechanism that contributes to the antiproliferative effect of this cytokine on some cancer cells.

Topics: Cell Division; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Free Radicals; Humans; Hydroxyl Radical; Microscopy, Electron; Reactive Oxygen Species; Salicylates; Spin Labels; Superoxides; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Vitamin K

The photosensitive organic hydroperoxide, NP-III, which produces hydroxyl radicals on illumination by UVA light, was used to examine the antioxidant activity of alpha-lipoic acid and its derivatives toward hydroxyl radical. Apolipoprotein (apo-B) of human low density lipoprotein (LDL) and bovine serum alubumin (BSA) were irradiated with UVA in the presence of NP-III and alpha-lipoic acid. The oxidation of BSA and the apo-B protein of LDL by NP-III was completely suppressed by alpha-lipoic acid. ESR studies using dimethylpyrroline oxide (DMPO) as a spin trapping reagent also revealed that in the presence of alpha-lipoic acid, the DMPO-OH adduct produced from the irradiation of NP-III and DMPO completely disappeared. DMPO-OH quenching experiments were performed in the presence or absence of desferoxamine but no change in the signal intensity was found. Hence, the quenching activity of alpha-lipoic acid is not due to its chelating activity toward transition metals (ferrous ions). The results lead us to conclude that alpha-lipoic acid is an efficient hydroxyl radical quencher owing to the disulfide bond in the dithiolane ring.

Topics: Antioxidants; Apolipoproteins B; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Humans; Hydroxyl Radical; Kinetics; Naphthalenes; Radiation-Sensitizing Agents; Salicylates; Salicylic Acid; Serum Albumin, Bovine; Spin Labels; Thioctic Acid; Ultraviolet Rays

Topics: Chromatography, High Pressure Liquid; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Hydroxyl Radical; Hydroxylation; Indicators and Reagents; Salicylates; Salicylic Acid; Spectrophotometry; Spin Labels

In our search to establish a reference .OH production system with respect to which the reactivity of copper(II) complexes could then be tested, the influence of free Cu2+ ions on the Cu+/H2O2 reaction has been investigated. This influence depends on the CCu2+/CCu+ ratio. At low Cu2+ concentrations, .OH damage to various detector molecules decreases with increasing Cu2+ concentrations until CCu2+/CCu+ reaches unity. Above this value, .OH damage increases sharply until CCu2+/CCu+ becomes equal to 5 with salicylate and 2 with deoxyribose, ratios for which the protective effect of Cu2+ cancels. Finally, at higher concentrations, Cu2+ ions logically add their own .OH production to that normally expected from Cu+ ions. The possible origin of this unprecedented alternate effect has been discussed. The possible influence of Cu+ ions on the generation of .OH radicals by water gamma radiolysis has also been tested and, as already established for Cu2+ in a previous work, shown to be nonexistent. This definitely confirms that either form of ionised copper cannot scavenge .OH radicals in the absence of a ligand.

Topics: Cations, Divalent; Copper; Cyclic N-Oxides; Deoxyribose; DNA; Electron Spin Resonance Spectroscopy; Gamma Rays; Hydroxyl Radical; Hydroxylation; Iron; Kinetics; Salicylates; Salicylic Acid; Spin Labels

Prior to comparative studies on the reactivity of various copper complexes with respect to OH. radicals, the influence of free Cu2+ ions on the superoxide-independent generation of OH. radicals through Fenton assays and water gamma radiolysis has been tested in the present work. Cu2+ ions have been shown to behave in a distinct manner towards each of these two production systems. As was logically expected from the noninvolvement of copper in OH. radical production through gamma radiolysis, no influence of Cu2+ ions has been observed on the amount of radicals detected in that case. In contrast, Cu2+ ions do influence OH. radical generation through iron-driven Fenton reactions, but differently depending on copper concentration. When present in high concentrations, Cu2+ ions significantly contribute to OH. radical production, which confirms previous observations on the reactivity of these in the presence of hydrogen peroxide. At lower levels corresponding to copper/iron ratios below unity on the contrary, Cu2+ ions behave as inhibitors of the OH. production in a pH-dependent manner over the 1-6 range investigated: the lower the pH, the greater the inhibition. The possible origin of this previously unreported inhibitory effect is discussed.

Topics: Chromatography, High Pressure Liquid; Copper; Cyclic N-Oxides; Deoxyribose; Free Radicals; Gamma Rays; Hydrogen-Ion Concentration; Hydroxides; Hydroxyl Radical; Hydroxylation; Iron; Ligands; Salicylates; Salicylic Acid; Spin Labels; Superoxides

Activated oxygen species produced during merocyanine 540 (MC540)-mediated photosensitization have been examined by electron spin resonance (ESR) spin trapping and by trapping reactive intermediates with salicylic acid using HPLC with electrochemical detection (HPLC-EC) for product analysis. Visible light irradiation of MC540 associated with dilauroylphosphatidylcholine liposomes in the presence of the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) gave an ESR spectrum characteristic of the DMPO-hydroxyl radical spin adduct (DMPO/.OH). Addition of ethanol or methanol produced additional hyperfine splittings due to the respective hydroxyalkyl radical adducts, indicating the presence of free.OH.DMPO/.OH formation was not significantly inhibited by Desferal, catalase, or superoxide dismutase (SOD). Production of DMPO/.OH was strongly inhibited by azide and enhanced in samples prepared with deuterated phosphate buffer (PB-D2O), suggesting that singlet molecular oxygen (1O2) was an important intermediate. When MC540-treated liposomes were irradiated in the presence of salicylic acid (SA), HPLC-EC analysis indicated almost exclusive formation of 2,5-dihydroxybenzoic acid (2,5-DHBA), with production of very little 2,3-DHBA, in contrast to .OH generated by uv photolysis of H2O2, which gave nearly equimolar amounts of the two products. 2,5-DHBA production was enhanced in PB-D2O and inhibited by azide, again consistent with 1O2 intermediacy. 2,5-DHBA formation was significantly reduced in samples saturated with N2 or argon, and such samples showed no D2O enhancement. Ethanol had no effect on 2,5-DHBA production, even when present in large excess. Catalase and SOD also had no effect, and only a small inhibition was observed with Desferal. DMPO inhibited 2,5-DHBA production in a concentration-dependent fashion and enhanced formation of 2,3-DHBA. We propose that 1O2 reacts with DMPO to give an intermediate which decays to form DMPO/.OH and free.OH, and that the reaction between 1O2 and SA preferentially forms the 2,5-DHBA isomer. This latter process may provide the basis for a sensitive analytical method to detect 1O2 intermediacy. Singlet oxygen appears to be the principle activated oxygen species produced during MC540-mediated photosensitization.

Topics: Chromatography, High Pressure Liquid; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Free Radicals; Hydroxides; Hydroxyl Radical; Oxygen; Photochemistry; Pyrimidinones; Radiation-Sensitizing Agents; Salicylates