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Results for “Triacetoneamine-N-Oxyl”

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At least 19 recordsLinked to original sources
PubMed1976

The effect of nitroimidazole and nitroxyl radiosensitizers on the post-irradiation synthesis of DNA.

The modification of DNA damage by three radiosensitizing drugs, present during gamma-irradiation of hypoxic Chinese hamster cells, was investigated. Both 2-methyl-5-nitroimidazole-1-ethanol (metronidazole) and 1-(2-nitro-1-imidazole)-3-methoxy-2-propranol (Ro-07-0582) were found to cause large increases in the yield of DNA single-strand breaks (SSB); triacetoneamine-N-oxyl (TAN) was found to have only a small effect on SSB production. The three drugs tested did not inhibit the rejoining of SSB. A pulse label and chase procedure was used to examine post-irradiation DNA synthesis. TAN present during irradiation under hypoxia was found to cause interruptions in subsequent DNA synthesis. Metronidazole and Ro-07-0582 had no effect on post-irradiation DNA synthesis. In addition, the effects of pre- and post-irradiation exposure to TAN were investigated, since these treatments have shown increased cell-killing in survival studies. TAN pre- and post-treatments were found to have no significant effect on subsequent DNA synthesis.

Cells, Cultured↗
PubMed1986

Murine in vivo L-band ESR spin-label oximetry with a loop-gap resonator.

Small mice (approximately 20 g) were anesthetized and placed in a loop-gap resonator of diameter 25 mm resonating at 1.1 GHz. An oxygen-permeable capsule containing 2.5 X 10(-2) M perdeutero 15N TEMPONE (1-oxyl-2,2,6,6-tetramethyl-4-piperidone) in light paraffin oil was implanted in the peritoneal cavity. Electron spin resonance (ESR) spectra from the capsule are sensitive to the concentration of dissolved oxygen and calibration curves are given. The oxygen concentration was found to rise from a value close to zero when the animal breathes air to a level of 220 microM when the animal breaths pure oxygen. It is speculated that this surprisingly high level is related to the effect of the anesthetic on the cardiovascular system. Encapsulation provides a barrier to spin-label reductants and to paramagnetic metal ions that might confound the spin-label oximetric measurements.

Animals↗
PubMed1990

Theoretical models for the conformations and the protonation of triacetonamine.

In this paper we propose theoretical models for the conformations of triacetonamine and protonated triacetonamine (Vincubine, an anticancer chemotherapeutic agent) developed by quantum and molecular mechanics techniques. We discuss the theoretical factors which are involved in the stabilization of the conformations calculated by the MNDO, MM2 and COPEANE methods and show the relative percent abundance of each molecular shape. Graphic representations of the conformers are depicted.

Antineoplastic Agents↗
PubMed1985

The effect of osmotic pressure of aqueous PEG solutions on red blood cells.

A drastic increase of the intracellular microviscosity of red blood cells in the presence of polyethylene glycol (PEG) was established by electron spin resonance using the small spin label molecule 2,2,6,6-tetramethyl-piperidine-N-oxyl-4-one (TEMPONE). The effective osmotic pressure of PEG solutions stressing the cells was estimated by comparison with those cytoplasmic rotational correlation times of TEMPONE measured in NaCl or sucrose containing media of known osmotic pressure.

Electron Spin Resonance Spectroscopy↗
PubMed1990

ESR measurement of time-dependent and equilibrium volumes in red cells.

Red cell water volumes were measured using ESR methods during transient osmotic perturbation, and under equilibrium conditions. Cell water contents were determined using the spin label Tempone (2,2,6,6-tetramethyl piperidine-N-oxyl) and the membrane impermeable quencher potassium chromium oxalate. With appropriate corrections for intracellular viscosity and changes in cavity sensitivity, equilibrium cell water measured both by electron spin resonance (ESR) and wet minus dry weight methods gave excellent agreement in solutions from 243-907 mOsm. Intracellular viscosities determined from the Tempone correlation times in the same cells gave values ranging from 9-47 centipoise at 21 degrees C. Osmotically induced transient volume changes were measured using Tempone and an ESR stopped-flow configuration. The Tempone response time was estimated at 17 msec compared to 250-350 msec for normal water relaxations. Nonlinear least square solutions to the Kedem-Katchalsky equations including a correction for the finite Tempone permeability gave 0.029 and 0.030 cm/sec for the osmotic permeability of RBCs in swell and shrink experiments, respectively. In stopped-flow experiments accurate water flux data are obtained very soon after challenging cells and do not require baseline subtractions. These results represent significant improvements over conventional light scattering techniques which necessitate corrections for long lasting optical artifacts (200-300 msec), and baseline drifts.

Electron Spin Resonance Spectroscopy↗
PubMed1989

Exchange and shuttling of electrons by nitroxide spin labels.

The ability of nitroxide spin labels to act as oxidizers of reduced nitroxides (hydroxylamines) in biological and model systems was demonstrated. All of the nitroxides tested were able to act as oxidizing agents with respect to hydroxylamine derivatives of nitroxides. The rates of these reactions were first order with respect to nitroxide concentration and with respect to hydroxylamine concentration, making the reaction second order overall. The second-order rate constants are reported for a number of these reactions. These reactions proceeded to an equilibrium state and the equilibrium constants for several combinations of reactants are presented. Both the rate constants and the equilibrium constants were found to be dependent on the ring structure of the nitroxide and hydroxylamine, with piperidines being reduced more easily and pyrrolidines and oxazolidines being oxidized more easily. All of the hydroxylamine derivatives were oxidized by air to their respective nitroxides, with the rate of this oxidation greater for pyrrolidines than for piperidines. Furthermore, hydroxylamines that are permeable to lipid bilayers were able to act as shuttles of reducing equivalents to liposome-encapsulated nitroxides that were otherwise inaccessible to reducing agents. This mechanism of shuttling of electrons was able to explain the relatively rapid reduction by cells of a nonpermeable nitroxide in the presence of a permeable nitroxide.

Animals↗
PubMed1990

Measurement of time-resolved oxygen concentration changes in photosynthetic systems by nitroxide-based EPR oximetry.

The application of recent developments of EPR oximetry to photosynthetic systems is described and used to study rapid processes in isolated thylakoid membranes from spinach and in intact photoautotrophic soybean cells. Using the peak heights of 15N perdeuterated Tempone and two microwave power levels oxygen evolution and consumption were measured. The method measured time-resolved oxygen concentration changes in the micromolar range. Oxygen evolution was linearly proportionate to the chlorophyl concentration of thylakoid membrane over the range studied (0-2 mg/ml). Oxygen evolution associated with single turnover light pulses was consistent with the four state model. The time (t1/2) to reach equilibrium of oxygen concentrations after a single turnover pulse was 0.4-0.5 ms, indicating that the evolution of oxygen coupled to the S4-S0 transition may be shorter than reported previously. The time for equilibrium of oxygen after single turnover pulses in soybean cells was relatively long (400 ms), which suggests that there are significant barriers to the free diffusion of oxygen in this system. The method also was used to study oxygen consumption by the electron transport chain of photosystem I and photosystem II. We conclude that EPR oximetry can provide quantitative and time-resolved data on oxygen concentrations with a sensitivity that is useful for studies of such systems.

Cyclic N-Oxides↗
PubMed1988

Rapid determination of internal volumes of membrane vesicles with electron spin resonance-stopped flow technique.

We have developed an electron spin resonance (ESR)-stopped flow technique and employed it for the simple and rapid determination of internal volumes of biomembrane vesicles and liposomes. A vesicle suspension containing a neutral and membrane-permeable spin label, 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl (TEMPONE), was mixed in the stopped-flow apparatus with an isotonic solution of relatively impermeable line broadening agents, potassium tris(oxalato)chromate(III) or potassium ferricyanide, and an ESR spectrum was recorded. From the relative intensity of the sharp triplet signal due to TEMPONE in the aqueous space within vesicles, the determination of the internal aqueous volume was straightforward. Using this technique, it is possible to measure intravesicular volumes in 0.1 s. The internal volume of sonicated phospholipid vesicles was approximately 0.3 microliter/mg lipid. The light fraction of sarcoplasmic reticulum membrane vesicles isolated from rabbit skeletal muscle was estimated to have an internal volume of 2.2-2.6 microliter/mg protein in its resting state. Activation of Ca2+ pumps in the membrane upon addition of ATP and Ca2+ ions decreased the internal volume by about 10%. This finding supports the hypothesis that the Ca2+ pump is electrogenic and that the efflux of potassium ions compensates for the influx of positive charges. The present technique is widely applicable to the simple and rapid determination of the internal volumes of membrane vesicles.

Animals↗
PubMed1991

Oxidation of specific SH protein of mitochondria by photodynamic action of hematoporphyrin. Relevance to uncoupling of oxidative phosphorylation.

Photoexcited hematoporphyrin (Hp) induces the uncoupling of oxidative phosphorylation of mitochondria. The uncoupling was inhibited by pre-incubation of mitochondria with a fluorescent SH reagent, eosin-5-maleimide, which has been shown to react specifically with an essential SH group of the Pi/H+ symporter [Houstek and Pedersen, J Biol Chem 260: 6288-6295, 1985]. Eosin-5-maleimide labeled 33, 34.5 and 36 kDa proteins in untreated rat liver mitochondria. When eosin-5-maleimide was added after the treatment with Hp plus light, the proteins were not labeled. Singlet oxygen detection by the ESR spin trapping method during photoradiation of Hp was inhibited by amino acids. Cysteine inhibited it more efficiently than histidine, methionine, tryptophan, tyrosine or alanine under the conditions used. HPLC demonstrated that Hp plus light oxidizes cysteine to cystine together with a smaller amount of cysteinesulfinic acid. These results suggest that Hp plus light oxidizes the SH group of mitochondrial protein, probably the Pi/H+ symporter, with singlet oxygen as a mediator. The possibility of the uncoupling of oxidative phosphorylation through such a modification of the Pi/H+ symporter is discussed.

Amino Acids↗
PubMed1992

The effect of the anoxic radiosensitizing agent TAN on induction of revertants by gamma-rays and helium ions in Salmonella tester strains.

The modification effect of the anoxic radiosensitizer TAN on the mutagenesis in various Salmonella tester strains after gamma-ray and helium ion irradiation was studied. The oxygen enhancement ratios (OER) for all 3 strains on the lethal assay after gamma-irradiation are approximately equal to 2. The induction of reversions in TA98 and TA100 does not modify under anoxia. The value of OER on the mutagenic assay in TA102 equals 1.6. The OER after helium ion irradiation on the lethal and mutagenic assays was less than after gamma-irradiation. The mutagenesis in 3 strains after irradiation under anoxia is enhanced by TAN. The value of the TAN modification effect after gamma-irradiation increases from 2.1 +/- 0.2 for TA102 to 5.2 +/- 0.4 for TA100. However, the TAN influence on mutagenesis in TA100 after helium ion irradiation decreases to 3.1 +/- 0.3. We conclude that peculiarities of mutagenesis in various tester strains under anoxia with TAN can be explained by considering the nature of premutational DNA damages.

Anaerobiosis↗
PubMed1986

Correlation of the internal microviscosity of human erythrocytes to the cell volume and the viscosity of hemoglobin solutions.

The microviscosity of the cytoplasm of human erythrocytes as well as of membrane-free hemoglobin solutions was investigated measuring the rotation of the small spin-label molecule, Tempone. The dependence of the intracellular microviscosity on the extracellular pH and osmotic pressure which was varied by NaCl or sucrose was sufficiently explained on the basis of alterations of the red blood cell volume. The intracellular microviscosity depended exclusively on the hemoglobin concentration. It did not differ from that of comparable membrane-free hemoglobin solutions. It was not necessary to take into account long-range interactions between hemoglobin molecules. The conclusion therefore was that the intracellular viscosity is not modified by cytoplasmic structures or the cell membrane. Above a hemoglobin concentration of 6 mM the viscosity of hemoglobin solutions increased much faster than the microviscosity. From measurements obtained with different spin-labels it followed that also the charge of these molecules is of importance.

Cyclic N-Oxides↗
PubMed1987

Oxidant stress in malaria as probed by stable nitroxide radicals in erythrocytes infected with Plasmodium berghei. The effects of primaquine and chloroquine.

Erythrocytes from normal mice and mice infected with the malarial parasite Plasmodium berghei reduce the water-soluble spin probes 2,2,6,6-tetramethylpiperidine-4-hydroxy-N-oxyl (TEMPOL), 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) and 2,2,6,6-tetramethylpiperidine-4-keto-N-oxyl (TEMPONE) at similar rates under both air and N2 atmospheres. The ESR signal of the lipid-soluble spin probe 5-doxyl-stearate is stable on incorporation into erythrocytes from normal mice. In contrast, parasitized red cells reduce this nitroxide probe, at a rate which increases with the level of parasitemia. Inhibitors of electron transport such as KCN and NaN3, increase the rate of reduction. We propose that nitroxide reduction occurs via the electron transport chain in the parasite. The antimalarial drug primaquine causes reduction of both water-soluble and lipid-soluble spin probes. This action of primaquine is independent of its ability to release H2O2 from oxyhemoglobin, and is ascribed to the ability of primaquine to accelerate flux through the hexose monophosphate shunt. The increased production of NADPH results in increased rates of reduction of the nitroxide radicals. Methylene blue, which also increases flux through the shunt, is even more effective than primaquine at reducing the nitroxides. Chloroquine has no such effect. Parasitized mice treated with chloroquine six hours prior to ESR measurements show less nitroxide reducing capacity than do untreated mice. Chloroquine is known to decrease flux through the hexose monophosphate shunt. The metabolic influences of the two antimalarial drugs are, thus, quite different.

Air↗
PubMed1991

Measurement of bioreduction rates of cells with distinct responses to ionizing radiation and cisplatin.

A low frequency electron paramagnetic resonance (EPR) spectrometer has been used to measure the bioreduction rate of an exogenously added nitroxide free radical species. Measurements have been made in a well controlled, in vitro system using an X-ray and cisplatin sensitive Chinese hamster ovary (CHO) cell line, xrs-5, and partial revertants which display wild-type levels of sensitivity to X-rays but retain xrs-5 levels of cisplatin sensitivity. The xrs-5 cells reduce this radical species at a rate which is approx. 50% that of the wild-type CHO cell line, K1. The partial revertants maintain this defect in bioreduction despite their decrease in radiosensitivity. However, the bioreduction rate observed in these cells correlates with their sensitivity to the chemotherapeutic drug cisplatin. Low frequency EPR allows measurements and imaging of living tissue and may be of value as a predictive assay of human tumor response to chemotherapy.

Animals↗
PubMed1989

The products of the reduction of doxyl stearates in cells are hydroxylamines as shown by oxidation by 15N-perdeuterated Tempone.

The use of nitroxides in functional biological systems has increased greatly as it has become evident that such studies can provide valuable biophysical and metabolic data. This has led to a need to understand the nature of the metabolism of nitroxides and their products. This paper presents data indicating the value of 15N-perdeuterated Tempone specifically to indicate the amount of hydroxylamines that are present in a cellular system. Using this technique, we found that in the mammalian cells that we studied the principal or only products of reduction of doxyl stearates were the corresponding hydroxylamines.

Animals↗
PubMed1990

Activation of the anticancer drug CCNU into a free radical intermediate via UV irradiation--an ESR study.

We studied the formation of a free radical induced by UV irradiation of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in benzene. It was determined a stable nitroxide radical by ESR spectroscopy. We confirmed that sterically hindered cyclic amine 2,2,6,6-tetramethyl-4-piperidone (TMPone) was transformed into a corresponding stable free nitroxyl radical during UV irradiation. When CCNU was present, the rate of free radical formation from TMPone increased considerably.

Benzene↗
PubMed1992

Oxoammonium cation intermediate in the nitroxide-catalyzed dismutation of superoxide.

Dismutation of superoxide has been shown previously to be catalyzed by stable nitroxide compounds. In the present study, the mechanism of superoxide (.O2-) dismutation by various five-membered ring and six-membered ring nitroxides was studied by electron paramagnetic resonance spectrometry, UV-visible spectrophotometry, cyclic voltammetry, and bulk electrolysis. Electron paramagnetic resonance signals from the carbocyclic nitroxide derivatives (piperidinyl, pyrrolidinyl, and pyrrolinyl) were unchanged when exposed to enzymatically generated .O2-, whereas, in the presence of .O2- and reducing agents such as NADH and NADPH, the nitroxides underwent reduction to their respective hydroxylamines. The reaction of 4-hydroxy-2,2,6,6-tetramethyl-1-hydroxypiperidine (Tempol-H) with .O2- was measured and, in agreement with earlier reports on related compounds, the rate was found to be too slow to be consistent with a mechanism of .O2- dismutation involving the hydroxylamine as an intermediate. Voltammetric analyses of the carbocyclic nitroxide derivatives revealed a reversible one-electron redox couple at positive potentials. In contrast, oxazolidine derivatives were irreversibly oxidized. At negative potentials, all of the nitroxides studied exhibited a broad, irreversible reductive wave. The rate of .O2- dismutation correlated with the reversible midpoint redox potential. Bulk electrolysis at positive potentials was found to generate a metastable oxidized form of the nitroxide. The results indicate that the dismutation of .O2- is catalyzed by the oxoammonium/nitroxide redox couple for carbocyclic nitroxide derivatives. In addition to the one-electron mitochondrial reduction pathway, the present results suggest the possibility that cellular bioreduction by a two-electron pathway may occur subsequent to oxidation of stable nitroxides. Furthermore, the cellular destruction of persistent spin adduct nitroxides might also be facilitated by a primary univalent oxidation.

Cyclic N-Oxides↗