tempo and 5-5-dimethyl-1-pyrroline-1-oxide

Topics: Animals; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Free Radicals; Humans; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Membrane Fluidity; Nitrogen Oxides; Oxidation-Reduction; Pyridines; Sensitivity and Specificity; Skin; Skin Diseases; Spin Labels; Ultraviolet Rays

The aim of this study was to determine the wavelength dependence of light-induced redox reactions in cells, particularly whether there is any contribution by red wavelengths. An additional aim was to assess the potential of 2,2,6,6-tetramethyl piperidine-N-oxyl (TEMPO) as a tool for measuring these redox reactions.. Visible light has been shown to affect cells, and redox reactions, which have been detected previously using spin traps, have been proposed as a mechanism. However, there is little evidence that red light, which is used in most such experiments, is redox active in cells.. Redox activity was observed by measuring the decay of the electron paramagnetic resonance signal of TEMPO that occurs in the presence of illuminated cells. Color filters were used to generate blue, green, and red light, and the decay resulting from these wavelengths was compared to the decay caused by white light.. Shorter wavelengths have a considerably stronger effect than longer wavelengths, although red light has some effect. Creation of reactive oxygen species by red light was confirmed with the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).. Red light can induce redox reactions in illuminated cells. However, shorter wavelengths are more efficient in this regard. In addition, TEMPO was found to be a more sensitive probe than DMPO for detecting light-induced cellular redox reactions.

Topics: Animals; Antioxidants; Color; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Humans; Light; Myocytes, Cardiac; Oxidation-Reduction; Rats; Reactive Oxygen Species; Spin Labels; Spin Trapping

The influence of complexation with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) or beta-cyclodextrin (beta-CD) on the photo-induced production of free radicals by the sunscreen agents octyl-dimethylaminobenzoate (ODAB), oxybenzone (OB) and octyl-methoxycinnamate (OMC) was investigated. The formation of radical species during irradiation was detected by spin-trapping electron paramagnetic resonance (EPR) spectroscopy. 2,2,6,6-tetramethylpiperidine-1-oxyl, nitroxide radical (TEMPO) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were used a spin-traps. Following the 4-h illumination with simulated sunlight, OB did not generate radicals. On the other hand, photoexcitation of solutions containing ODAB or OMC produced a marked decrease (>40%) of the TEMPO signal intensity, demonstrating the formation of carbon-centred radicals. In addition, the results obtained on irradiation of ODAB solutions containing DMPO as spin-trap indicated the generation of oxygen-centred radicals. Complexation of ODAB with HP-beta-CD and OMC with beta-CD markedly inhibited (>64%) the formation of free radicals generated by the sunscreens on exposure to simulated sunlight. Therefore, inclusion of ODAB and OMC into the cyclodextrin cavities minimizes their photosensitising potential.

Topics: Chromatography, High Pressure Liquid; Cinnamates; Cyclic N-Oxides; Cyclodextrins; Electron Spin Resonance Spectroscopy; Free Radicals; Light; Magnetic Resonance Spectroscopy; p-Dimethylaminoazobenzene; Photochemistry; Spectrophotometry, Ultraviolet; Sunscreening Agents

Electron spin resonance (ESR) spectrometry has been adapted to accelerated (light and temperature) storage experiments with processed cheese comparing the spin trapping technique with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as spin trap added to the cheese and the spin labeling technique with 2,2,6, 6-tetramethylpiperidine-1-oxyl radical (TEMPO) as spin label added to the cheese. Both methods showed that light was the more important factor compared to temperature for early stages (up to 11 days) in the formation of radicals in processed cheese. For the spin labeling technique other, unidentified reactions interfered during the early stages of the accelerated storage, indicating that longer reaction times are required for the evaluation, and the spin trapping technique is recommended. As a third method, direct measurement of free radicals formed in processed cheese stored for 15 months and subsequently freeze-dried showed that for longer storage, temperature is more important for the steady-state concentration of radicals than light exposure. In agreement with this result, formation of secondary lipid oxidation products determined as thiobarbituric acid reactive substances was found to correlate with the direct ESR measurement.

Topics: Cheese; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Food Preservation; Oxidation-Reduction; Spin Labels

1. The effects of reactive oxygen intermediates derived from photoactivated rose bengal on the vascular reactivity have been evaluated in rabbit mesenteric artery ring preparations. The artery rings were exposed to xanthene dye rose bengal (50 nM) illuminated (6,000 lux) at 560 nm for 30 min. Spin trapping studies with 2,2,6,6-tetramethylpiperidine (TEMP) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) with electron spin resonance spectrometry were also conducted in solution (and not within tissues) to determine quantitatively the reactive oxygen species generated from photoactivated rose bengal. 2. Contraction of the ring preparations induced by noradrenaline (10(-8) to 10(-4) M) was attenuated by previous exposure to photolysed rose bengal; the observation that the pD2 decreased without a significant reduction in maximum tension generation is consistent with the view that receptor dysfunction may be involved in the effect of photolysed rose bengal. 3. Prior exposure to photolysed rose bengal of the ring preparations inhibited the endothelium-dependent relaxation evoked by acetylcholine (10(-6) M) and calcium ionophore A23187 (10(-7) M), but not the endothelium-independent relaxation evoked by nitroglycerin (10(-6) M). 4. A variety of scavengers, superoxide dismutase (33 units ml-1), catalase (32 units ml-1) and 1,3-dimethyl-2-thiourea (DMTU, 10 mM), which should eliminate the superoxide anion radical, H2O2 and the hydroxyl radical, had no effect on the attenuated responses to noradrenaline and acetylcholine induced by photolysed rose bengal. In contrast, the inhibition of the observed effect of photolysed rose bengal was obtained with addition of histidine (25 mM), a singlet molecular oxygen quencher. 5. It was found that photolysis of rose bengal from a 1:2:2:1 quartet, characteristic of the hydroxyl radical-DMPO spin adduct, which was effectively blunted by DMTU, superoxide dismutase and catalase whereas histidine was ineffective. The results of the electron spin resonance study also showed that a singlet molecular oxygen was produced by photoactivation of rose bengal; this was detected as singlet oxygen-TEMP product (TEMPO; 2,2,6,6-tetramethylpiperidine-N-oxyl). The formation of the TEMPO signal was strongly inhibited by histidine, but not by DMTU, superoxide dismutase and catalase. 6. It is suggested that the superoxide anion radical, H2O2 and hydroxyl radical are formed in addition to singlet molecular oxygen, and the data obtained from the present stu

Topics: Acetylcholine; Animals; Catalase; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Fluorescent Dyes; Free Radical Scavengers; Hydrogen Peroxide; Hydroxyl Radical; Male; Mesenteric Arteries; Muscle Contraction; Muscle, Smooth, Vascular; Nitroglycerin; Photolysis; Rabbits; Rose Bengal; Spin Labels; Spin Trapping; Superoxide Dismutase; Superoxides; Thiourea; Ultraviolet Rays; Vasodilator Agents

Nitrone/nitroso spin traps are often used for detection of unstable hydroxyl radical giving stable nitroxide radicals with characteristic electron spin resonance (ESR) signals. This technique may be useful only when the nitroxide radicals are kept stable in the reaction system. The aim of the present study is to clarify whether the nitroxide radicals are kept stable in the presence of the hydroxyl radical scavengers. Effect of hydroxyl radical scavengers on the ESR signals of nitroxide radicals, 2,2,6,6-tetramethyl-piperidine- N-oxyl (TEMPO) and the spin adduct (DMPO-OH) of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and hydroxyl radical, was examined. Although the ESR signals of TEMPO and the DMPO-OH spin adduct were unchanged on treatment with ethanol and dimethyl sulfoxide, their intensities were effectively decreased on treatment with 6-hydroxy-2,5,7, 8-tetra-methylchroman-2-carboxylic acid (Trolox), cysteine, glutathione, 2-mercaptoethanol and metallothionein. Hence, the results of the detection of hydroxyl radical in the presence of phenolic and thiol antioxidants by the ESR technique using nitrone/nitroso spin traps may be unreliable.

Topics: Antioxidants; Chromans; Cyclic N-Oxides; Cysteine; Dimethyl Sulfoxide; Electron Spin Resonance Spectroscopy; Ethanol; Free Radical Scavengers; Free Radicals; Glutathione; Hydroxyl Radical; Mercaptoethanol; Metallothionein; Nitrogen Oxides; Phenols; Sulfhydryl Compounds

For greatest efficacy, it is desirable to use spin trapping agents in the highest concentrations possible. Fifty-four male Sprague-Dawley rats were used to explore the relative toxicity of four representative nitronyl spin traps at doses chosen on the basis of earlier lethality studies. Most studies were confined to the 3- to 6-h period following drug injection, because the behavioral signs of toxicity are most evident early after injection and because spin trapping studies would typically be performed within this time frame. Doses of spin trap were dissolved in a corn oil/buffer vehicle and injected intraperitoneally (i.p.). Toxic signs were recorded periodically, and at the time of euthanasia or spontaneous death a blood sample was collected by cardiac puncture for clinical chemistry analysis and a necropsy was performed. Both gross pathology and histopathological examination of the major organs were essentially negative in all cases, with no obvious evidence of cellular damage being observed. Neither DMPO (232 mg/100 g b.wt.) nor PBN (100 mg/100 g b.wt.) were lethal in the present study, while both M4PO (20 and 40 mg/ 100 g b.wt.) and PyOBN (100 and 200 mg/100 g b.wt.) were lethal. Abnormal clinical chemistry findings were generally confined to those animals that died spontaneously or were euthanized early for humane reasons. In most cases, death was associated with marked seizure activity and impaired respiration, and deaths occurred within a few min to a few hours. The mechanism of toxicity was unclear due to the lack of histopathological evidence and the wide range of abnormal serum analytes in those rats killed by either M4PO or PyOBN. In conclusion, during the first 6 h after IP administration there is little indication of tissue damage by the nitrone spin traps until the dose is increased to a lethal level, at which point an acute, rapidly occurring, wide-spread disruption of tissue integrity seems to occur.

Topics: Animals; Blood Glucose; Blood Proteins; Blood Urea Nitrogen; Cyclic N-Oxides; Electrolytes; Enzymes; Free Radicals; Injections, Intraperitoneal; Male; Nitrogen Oxides; Pyridines; Rats; Rats, Sprague-Dawley; Spin Labels

Visible light illumination of solutions of perylenequinonoid pigments generates the corresponding semiquinone radicals, singlet oxygen, superoxide anion radical, hydroxyl radical and hydrogen peroxide. In anaerobic solution, the semiquinone radicals are predominantly photoproduced via the self-electron transfer between the excited and ground state species. In aerobic solution, singlet oxygen is the principal product in the photosensitization of perylenequinonoid pigments. The 3,10-dihydroxy-4,9-perylenequinonoid chromophore was shown to be the necessary structural requirement for the generation of singlet oxygen, and the side-chains of the quinones had little effect on the production of singlet oxygen. This conclusion is useful in the development of more efficient photodynamic therapeutic agents than natural perylenequinonoid pigments themselves. Such agents should ideally contain the 3,10-dihydroxy-4,9-perylenequinonoid chromophore to produce singlet oxygen together with appropriate elaborated side-chains to permit the selective localization of the sensitizer in tumor tissue. In addition to singlet oxygen, superoxide anion radical is generated by the perylenequinones on illumination in aerobic solution, but to a lesser extent than singlet oxygen, via the reduction of oxygen by the corresponding semiquinone radicals. This latter process is significantly enhanced by the presence of electron donors.

Topics: Antineoplastic Agents; Benz(a)Anthracenes; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Light; Molecular Structure; Oxygen; Perylene; Photochemistry; Photochemotherapy; Photosensitizing Agents; Quinones; Singlet Oxygen; Spin Labels; Superoxides

It is well known that oxygen enhances the relaxation of free radical EPR probes through spin lattice and Heisenberg spin-spin interactions with consequent effect on the line height and width. The two relaxation processes have opposing effects on the signal heights and depend on the concentration of oxygen, the incident microwave power, and the presence of other paramagnetic species. During EPR studies of chemical, biochemical, and cellular processes involving free radicals, molecular oxygen has significant magnetic influence on the EPR signal intensity of the free radical species under investigation in addition to affecting the rates of production of the primary species and the stability of the spin adduct nitroxides. These effects are often overlooked and can cause artifacts and lead to erroneous interpretation. In the present study, the effects of oxygen and ferricyanide on the EPR signal height of stable and persistent spin adduct nitroxides at commonly employed microwave powers were examined. The results show that under commonly adopted EPR spectrometer instrumental conditions, artifactual changes in the EPR signal of spin adducts occur and the best way to avoid them is by keeping the oxygen level constant using a gas-permeable cell.

Topics: Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Ferricyanides; Free Radicals; Microwaves; Oxygen; Spin Labels; Triacetoneamine-N-Oxyl