Magnetic resonance imaging of organic contrast agents in mice: capturing the whole-body redox landscape

Nitroxides are a class of stable free radicals that have several biomedical applications including radioprotection and noninvasive assessment of tissue redox status. For both of these applications, it is necessary to understand the in vivo biodistribution and reduction of nitroxides. In this study, magnetic resonance imaging was used to compare tissue accumulation (concentration) and reduction of two commonly studied nitroxides: the piperidine nitroxide Tempol and the pyrrolidine nitroxide 3-CP. It was found that 3-CP was reduced 3 to 11 times slower (depending on the tissue) than Tempol in vivo and that maximum tissue concentration varies substantially between tissues (0.6-7.2mM). For a given tissue, the maximum concentration usually did not vary between the two nitroxides. Furthermore, using electron paramagnetic resonance spectroscopy, we showed that the nitroxide reduction rate depends only weakly on cellular pO(2) in the oxygen range expected in vivo. These observations, taken with the marked variation in nitroxide reduction rates observed between tissues, suggest that tissue pO(2) is not a major determinant of the nitroxide reduction rate in vivo. For the purpose of redox imaging, 3-CP was shown to be an optimal choice based on the achievable concentrations and bioreduction observed in vivo.     

Pro-oxidative activity of nitroxides in their reactions with glutathione

Nitroxides are unreactive towards glutathione in vitro. Interaction of nitroxides with peroxynitrite does not lead to a significant loss of their electron paramagnetic resonance (EPR) signal. However, addition of peroxynitrite to a solution containing glutathione and nitroxides induces an irreversible disappearance of EPR signal of nitroxides and augmentation of glutathione oxidation which is a pro-oxidant effect of these compounds. Nitroxide loss leading to the formation of amine derivatives is initiated by products of glutathione oxidation by peroxynitrite. The pro-oxidant action of nitroxides at micromolar concentrations may be important in view of the proposed use of these compounds as antioxidants.     

Dual activity of nitroxides as pro- and antioxidants: catalysis of copper-mediated DNA breakage and H2O2 dismutation

Nitroxide antioxidants can be reduced to hydroxylamines or oxidized to oxoammonium cations. Consequently, nitroxides can modify oxidative damage acting as reducing and/or as oxidizing agents, and in many cases the nitroxides are continuously recycled. They provide protection against oxidative stress via various mechanisms including SOD-mimic activity and detoxification of carbon-, oxygen-, and nitrogen-centered radicals, as well as oxidation of reduced transition metals. In contrast to the common concept, according to which the nitroxides' protective effect takes place via inhibition of the Fenton reaction, there are observations suggesting the opposite. In the present investigation, DNA breakage catalyzed by copper served as an experimental model for studying the anti- and pro-oxidative activity of nitroxides. Nitroxides provided protection in the presence of GSH, which is known to facilitate metal-catalyzed DNA damage. In the absence of a reductant, nitroxides enhanced DNA breakage under aerobic conditions with or without added H(2)O(2) and facilitated H(2)O(2) depletion. The rates of nitroxide-catalyzed DNA breakage and H(2)O(2) depletion increased as the concentrations of copper, H(2)O(2), and nitroxide increased. Although the catalytic activity of nitroxides is low, it is sufficient to induce DNA breakage. The efficacy of DNA breakage by the tested piperidine nitroxides correlated with the nitroxide-induced depletion of H(2)O(2) with the exception of the pyrrolidine nitroxide 3-carbamoylproxyl. The results suggest that the nitroxide and the copper are continuously recycled while catalyzing DNA breakage and depletion of H(2)O(2), which serves both as a source of reducing equivalents and as the electron sink.     

Application of Spin Traps to Biological Systems

Since 1971. when nitroxides were first reported to be bioreduced, several cellular enzymes, in addition to ascorbic acid. have been found to catalyze the reduction of nitroxides to their corresponding hydroxylami-nes. Numerous studies have demonstrated that cellular bioreduction of nitroxides are both dependent upon the structure of the nitroxide and cell type. For example, pyrrolidinyloxyls are considerably more resistant to bioreduction than their corresponding piperidinyloxyls. In addition, cellular levels of reductases present in freshly isolated rat hepatocytes are considerably greater than concentrations found in freshly isolated rat enterocytes. Thus, through the proper selection of a cell type and an appropriate nitroxide. one can study cellular-mediated free radical processes.

With the discovery that α-hydrogen-containing nitroxides, including 2, Z-dimethyl-S-hydroxy-l-pyrrolidinyloxyl (DMPO-OH) decompose rapidly in the presence of superoxide and thiols, the ability to determine if hydroxyl radical is generated during stimulation of human neutrophils, is in doubt. To explore the limits of spin trapping in this context. we have studied the effect of varying the rates of superoxide production. in the presence and absence of thiols, on the decomposition of DMPO-OH. In parallel studies, we have found that t-butyl α-methyl-4-pyridinyl-N-oxide nitroxide (4-POBN-CH₃) will not degrade in the presence of superoxide and a thiol. From these studies. we have determined that if hydroxyl radicals were generated as an isolated event in the presence of a continual flow of superoxide. spin trapping might not be able to detect its formation. Otherwise. spin trapping should be able to measure hydroxyl radicals. if continually generated, during activation of human neutrophils.     

Different effectiveness of piperidine nitroxides against oxidative stress induced by doxorubicin and hydrogen peroxide

The piperidine nitroxides Tempamine and Tempace have been studied for their effect on doxorubicin (DOX) and hydrogen peroxide (H₂O₂) cytotoxicity in immortalized B14 cells, a model for neoplastic phenotype. The significance for nitroxide performance of the substituent in position 4 of the piperidine ring was evaluated. The cells were exposed to DOX/H₂O₂ alone or in combination with the nitroxides Tempamine or Tempace. Two other piperidine nitroxides, Tempo and Tempol, were used for comparison. All the nitroxides except Tempamine modestly reduced DOX cytotoxicity. Tempamine evoked a biphasic response: at concentrations lower than 200 μmol/L the nitroxide decreased DOX cytotoxicity, while at concentrations higher than 200 μmol/L, it enhanced DOX cytotoxicity. In contrast to Tempo and Tempol, Tempamine and Tempace ameliorated hydrogen peroxide cytotoxicity, but none of the nitroxides influenced TBARS stimulated by hydrogen peroxide. The cytoprotective effect of Tempace, Tempo and Tempol in DOX-treated cells correlated with the inhibition of DOX-induced lipid peroxidation. The bioreduction rates of the investigated nitroxides differed significantly and were variously affected by DOX depending on the nitroxide substituent. In combination with DOX, Tempo and Tempol were reduced significantly more slowly, while no influence of DOX on Tempamine and Tempace bioreduction was observed. Our results suggest that the structure of the 4-position substituent is an important factor for biological activity of piperidine nitroxides. Among the investigated nitroxides, Tempace displayed the best protective properties in vitro but Tempamine was the only nitroxide that potentiated cytotoxicity of DOX and did not influence DOX-induced lipid peroxidation. However, this nitroxide showed different performance depending on its concentration and conditions of oxidative stress.     

Ascorbate- and dehydroascorbic acid-mediated reduction of free radicals in the human erythrocyte

Nitroxides were used as models of persistent free radicals to study the antioxidant function of ascorbic acid in the human erythrocyte. It was concluded that: 1) ascorbate and other reductant(s) derived from dehydroascorbic acid (DHA) in the presence of thiols are the only significant reducing agents for nitroxides, 2) glutathione and DHA reduce nitroxides by a process that cannot be inhibited by ascorbic acid oxidase, 3) erythrocytes can be depleted of ascorbic acid by exhaustive washing in the presence of membrane-permeable cationic nitroxides such as N,N-dimethylamino-Tempo, 4) ascorbate-depleted cells do not reduce nitroxides; however, nitroxide reduction is restored when the cells are incubated with DHA, 5) reduction of nitroxides in ascorbate-depleted, DHA-treated cells is significantly faster than in buffered solutions of DHA and glutathione, 6) several equivalents of nitroxide are reduced relative to the intracellular ascorbate pool, 7) sustained nitroxide reduction is observed even when most of the intracellular ascorbate is oxidized, 8) spin trapping of oxyradicals in tert-butyl hydroperoxide-treated cells is accelerated with ascorbate depletion and inhibited with ascorbate loading, 9) ascorbate can be quantified within intact cells by analyzing the initial reduction rates of membrane-permeable cationic nitroxides, and 10) DHA-stimulated reduction of cationic nitroxides is slower and less extensive in erythrocytes deficient in glucose-6-phosphate dehydrogenase than in normal erythrocytes.     

Cell-penetrating nitroxides as molecular sensors for imaging of cancer in vivo, based on tissue redox activity

The present study shows that hydrophobic and cell-penetrating piperidine-type nitroxide radicals SLENU and TEMPOL, but not hydrophilic and partially penetrating or non-penetrating pyrrolidine-type nitroxides carbamoyl-PROXYL and carboxy-PROXYL, are appropriate contrast agents for magnetic resonance imaging (MRI) of cancer, based on its functionality - tissue redox activity. The experiments were conducted on anesthetized mice: healthy and neuroblastoma-bearing in a moderate stage of cancer development. The method is based on the nitroxide redox cycle, coupled with appearance or disappearance of the MRI signal. The half-life (τ(1/2)) of a nitroxide-enhanced MRI signal in the respective tissue was used as a marker to assess tissue redox activity to the nitroxide radical. In the case of SLENU and TEMPOL, there were large differences in the histograms between control and cancer-bearing mice. All tissues (cancer and non-cancer) of cancer-bearing organisms were characterized by a long-lived MRI signal (τ(1/2) > 14 min), indicating a high oxidative activity. The tissues of healthy organisms were characterized by a short-lived MRI signal (τ(1/2) = 1-3 min), indicating a high reducing activity. In the case of carbamoyl-PROXYL and carboxy-PROXYL, there was no difference in the histograms between control and cancer-bearing mice. The data show that the penetration of nitroxide in cells and tissues is obligatory for imaging of cancer, based on its redox activity. The principle of the method is applicable also to biopsy specimens, using MRI or EPR spectroscopy. We provide direct evidence that the nitroxide redox cycle could be used as a sensing platform for functional imaging of different pathologies, based on changes in cellular and tissue redox activity, as in the case of cancer.     

Cellular uptake of electron paramagnetic resonance imaging probes through endocytosis of liposomes

Electron paramagnetic resonance imaging (EPRI) allows detection and localization of paramagnetic spin probes in vivo and in real time. We have shown that nitroxide spin probes entrapped in the intracellular milieu can be imaged by EPRI. Therefore, with the development of a tumor-targetable vehicle that can efficiently deliver nitroxides into cells, it should be possible to use nitroxide spin probes to label and image cells in a tumor. In this study, we assess the potential of liposomes as a delivery vehicle for imaging probes. We demonstrate that liposomes can stably encapsulate nitroxides at very high concentrations (> 100 mM), at which nitroxides exhibit concentration-dependent quenching of their EPR signal—a process analogous to the quenching of fluorescent molecules. The encapsulating liposomes thus appear spectroscopically “dark”. When the liposomes are endocytosed and degraded by cells, the encapsulated nitroxides are liberated and diluted into the much larger intracellular volume. The consequent relief of quenching generates a robust intracellular nitroxide signal that can be imaged. We show that through endocytosis of nitroxide-loaded liposomes, CV1 cells can achieve intracellular nitroxide concentrations of ∼ 1 mM. By using tissue phantom models, we verify that this concentration is more than sufficient for in vivo EPR imaging.     

Effect of complexation with randomly methylated beta-cyclodextrin on the aqueous solubility, photostability and antioxidant activity of an indolinonic nitroxide radical

The interaction between the hydrophobic indolinonic nitroxide radical, 1,2-dihydro-2-methyl-2-phenyl-3H-indole-3-one-1-oxyl and hydrophilic alpha-, beta- and gamma-cyclodextrin derivatives was investigated in water by phase-solubility analysis. Among the studied cyclodextrins, random methyl-beta-cyclodextrin (RM-beta-CD) had the greatest solubilizing activity (1312-fold increase in. the intrinsic aqueous solubility). Solid complexes were prepared by the freeze-drying method and characterized by powder X-ray diffractometry and thermal analysis. Complexation of the nitroxide with RM-beta-CD was also confirmed in solution by electron paramagnetic resonance (EPR) spectroscopy. Photodegradation of the nitroxide was reduced by complexation with RM-beta-CD, this effect being more pronounced in the solid-state (the extent of degradation was 28.0% for the complex vs. 78.8% for uncomplexed nitroxide) than in solution (41.2 vs. 69.1% for uncomplexed nitroxide). The antioxidant activity of the complex was also investigated on the peroxidation of methyl linoleate micelles and on protein oxidation induced by free radical generators, and in both systems the free form of the nitroxide as well as its complex with RM-beta-CD, showed essentially the same degree of protection. Moreover, EPR experiments showed a time-dependent decrease in the EPR signal of both the complexed and uncomplexed nitroxides with the free-radical generators. Therefore, RM-beta-CD complexation of the nitroxide represents an effective strategy to improve its aqueous solubility and photostability, which is essential for certain biological applications, while it does not interfere with its radical scavenging efficiency.     

Cellular metabolism of proxyl nitroxides and hydroxylamines

Previous data from model systems indicated that the proxyl nitroxides should be especially resistant to bioreduction and therefore could be an effective solution to this often problematic characteristic of nitroxides. Therefore, we investigated the rate of reduction by cells and by the usual model system, ascorbate, of four proxyl nitroxides and three reference nitroxides. We found that, while the rate of reduction by ascorbate of the proxyl nitroxides was slower than the rate of a prototypic pyrrolidine nitroxide (PCA), the reverse was true for reduction by cells. We also studied the rate of oxidation of the corresponding hydroxylamines. The rate of oxidation by cells of the proxyl hydroxylamines was relatively fast, especially for the most lipophilic derivative. These results indicate that: (i) proxyl nitroxides may not be unusually resistant to bioreduction by functional biological systems; (ii) accurate knowledge of relative rates of metabolism of nitroxides and hydroxylamines in cells and tissues will require direct studies in these systems because the rates may not closely parallel those observed in model (chemical) systems; and (iii) proxyl nitroxides show potential value as agents to measure oxygen concentrations by the rates of oxidation of their corresponding hydroxylamines.     

Preparation and characterization of spin-labeled derivatives of epidermal growth factor (EGF) for investigations of the interactions of EGF with its receptor by electron paramagnetic resonance spectroscopy.

We prepared, purified, and characterized derivatives of epidermal growth factor (EGF) having a nitroxide spin-label attached covalently at the amino terminus. Characterization of these derivatives with regard to the positions of attachment of the spin-label was accomplished by a combination of peptide mapping, protein sequencing, and fast atom bombardment-mass spectrometry. One derivative was chosen for use in initial investigations by electron paramagnetic resonance (EPR) spectroscopy of receptor-bound EGF and its dissociation kinetics. This derivative was found to be equipotent with the native hormone in competitive binding assays, in activating the EGF receptor kinase, and in stimulating the formation of EGF receptor dimers in solubilized cell extracts. Upon binding to solubilized EGF receptor, the spin-labeled EGF derivative became immobilized, giving rise to a visually distinct slow-motion EPR spectrum. The resulting spectrum showed no detectable dipolar interaction between nitroxides, indicating that the nitroxide moieties of spin-labels reacted at the amino termini of receptor-bound spin-labeled EGF molecules are separated by a distance of at least 16 A. An EPR study of the kinetics of dissociation of spin-labeled EGF in the presence of excess unlabeled EGF revealed a rapid component with a k off approximately 2 x 10(-2) s-1 and a less well resolved slow component.     

Azethoxyl nitroxide spin labels. ESR studies involving thiourea crystals, model membrane systems and chromatophores, and chemical reduction with ascorbate and dithiothreitol.

Trans- and cis-azethoxyl nitroxides 1, 2, 3 and 4 can be trapped in the cavities of thiourea crystals. The presence of a single gauche conformation on either side of the pyrrolidine ring within the crystals was indicated by the ESR spectra. Rotation about the long molecular axis then corresponds approximately to y-axis motion of the nitroxide moiety. Proxyl nitroxides in which the nitroxide group is located on the penultimate carbon of long chain lipids can also be trapped and were shown to adopt the azethoxyl conformation in the thiourea crystals. The measured deltaA values (A parallel to - A perpendicular) of oriented egg lecithin multilayers containing trans- and cis-azethoxyl nitroxides 1 and 2 were quite small, consistent with the unique orientation of the nitroxide principal axes with respect to the long axis of the molecule. The deltaA values for a series of lipids bearing a label near the terminus of the chain were very similar to that of 1, showing that the azethoxyl conformation is likely the predominant one in these labels in orienting systems. Computer simulations of the ESR spectra of 1 and 2 in egg lecithin vesicles provided values for molecular orientation and motion parameters consistent with those expected from a consideration of molecular models in the extended (all trans) conformation. Azethoxyl nitroxides have also proven useful in the investigation of motion restricted (boundary) lipid in a lipid-protein system. Thus, the values (69 +/- 10%) for the amount of boundary lipid in the chromatophore membranes from Rhodopseudomonas sphaeroides as determined using trans- 2 and cis- 2 are in good agreement with values using 16-doxylstearic acid (64 +/- 3%). The fact that all three labels show about the same fraction of boundary lipid in this system indicates that the lipid binding sites are relatively insensitive to the geometry of the lipid chain. Also, both 1 and 2 appear to be able to detect a third lipid environment not seen with the doxyl fatty acid. The apparent fluidity of this component lies between that of boundary and bilayer lipid. The unique orientation of the nitroxide principal axes with respect to the long molecular axis in azethoxyl nitroxides 1 and 2 allows detection of hindrance to rotation about the long molecular axis, in contrast to the analogous doxyl and proxyl fatty acids. Comparative reduction studies using ascorbate and dithiothreitol indicate that azethoxyl nitroxides are slightly more resistant toward reduction than proxyl nitroxides and much more resistant than doxyl nitroxides.     

A novel ascorbic acid-resistant nitroxide in fat emulsion is an efficient brain imaging probe for in vivo EPR imaging of mouse

Abstract

The loss of paramagnetism of nitroxide radicals due to reductant reactions in biological systems, places a fundamental time constraint on their application as an imaging probe in in vivo EPR imaging studies. However, in vitro studies of the newly synthesized tetraethyl-substituted piperidine nitroxide radical demonstrated high resistivity to paramagnetic reduction when exposed to ascorbic acid, a common reduction agent in biological systems. In this work we investigated the use of these nitroxides as an imaging probe in EPR imaging of small rodents. 2,2,6,6-Tetraethyl-piperidine nitroxide (TEEPONE) is not highly soluble in aqueous media, thus a lipid-based emulsion system of lecithin was used to solubilize TEEPONE. The obtained solution was homogenous and with low viscosity, allowing smooth intravenous injection into mice tail vein. Acquired three dimensional (3D) EPR images of mouse head clearly showed TEEPONE distributed in all tissues including brain tissues, with an average measurable signal half-life of more than 80 min, thus demonstrating high resistivity to reduction due to ascorbic acid in in vivo animal studies, and the potential for use of this compound in in vivo studies of animal model systems.     

Inhibition of myeloperoxidase- and neutrophil-mediated oxidant production by tetraethyl and tetramethyl nitroxides

The powerful oxidant HOCl (hypochlorous acid and its corresponding anion, ⁻OCl) generated by the myeloperoxidase (MPO)–H₂O₂–Cl⁻ system of activated leukocytes is strongly associated with multiple human inflammatory diseases; consequently there is considerable interest in inhibition of this enzyme. Nitroxides are established antioxidants of low toxicity that can attenuate oxidation in animal models, with this ascribed to superoxide dismutase or radical-scavenging activities. We have shown (M.D. Rees et al., Biochem. J. 421, 79–86, 2009) that nitroxides, including 4-amino-TEMPO (4-amino-2,2,6,6-tetramethylpiperidin-1-yloxyl radical), are potent inhibitors of HOCl formation by isolated MPO and activated neutrophils, with IC₅₀ values of ~1 and ~6 µM respectively. The utility of tetramethyl-substituted nitroxides is, however, limited by their rapid reduction by biological reductants. The corresponding tetraethyl-substituted nitroxides have, however, been reported to be less susceptible to reduction. In this study we show that the tetraethyl species were reduced less rapidly than the tetramethyl species by both human plasma (89–99% decreased rate of reduction) and activated human neutrophils (62–75% decreased rate). The tetraethyl-substituted nitroxides retained their ability to inhibit HOCl production by MPO and activated neutrophils with IC₅₀ values in the low-micromolar range; in some cases inhibition was enhanced compared to tetramethyl substitution. Nitroxides with rigid structures (fused oxaspiro rings) were, however, inactive. Overall, these data indicate that tetraethyl-substituted nitroxides are potent inhibitors of oxidant formation by MPO, with longer plasma and cellular half-lives compared to the tetramethyl species, potentially allowing lower doses to be employed.     

Identification of nitroxide radioprotectors.

The nitroxide Tempol, a stable free radical, has recently been shown to protect mammalian cells against several forms of oxidative stress including radiation-induced cytotoxicity. To extend this observation, six additional water-soluble nitroxides with different structural features were evaluated for potential radioprotective properties using Chinese hamster V79 cells and clonogenic assays. Nitroxides (10 mM) were added 10 min prior to radiation exposure and full radiation dose-response curves were determined. In addition to Tempol, five of the six nitroxides afforded in vitro radioprotection. The best protectors were found to be the positively charged nitroxides, Tempamine and 3-aminomethyl-PROXYL, with protection factors of 2.3 and 2.4, respectively, compared with Tempol, which had a protection factor of 1.3. 3-Carboxy-PROXYL, a negatively charged nitroxide, provided minimal protection. DNA binding characteristics as studied by nonequilibrium dialysis of DNA with each of the nitroxides demonstrated that Tempamine and 3-amino-methyl-PROXYL bound more strongly to DNA than did Tempol. Since DNA is assumed to be the target of radiation-induced cytotoxicity, differences in protection may be explained by variabilities in affinity of the protector for the target. This study establishes nitroxides as a general class of new nonthiol radioprotectors and suggests other parameters that may be exploited to find even better nitroxide-induced radioprotection.     

Kinetics of superoxide-induced exchange among nitroxide antioxidants and their oxidized and reduced forms.

Nitroxide stable radicals generally serve for probing molecular motion in membranes and whole cells, transmembrane potential, intracellular oxygen and pH, and are tested as contrast agents for magnetic resonance imaging. Recently nitroxides were found to protect against oxidative stress. Unlike most low molecular weight antioxidants (LMWA) which are depleted while attenuating oxidative damage, nitroxides can be recycled. In many cases the antioxidative activity of nitroxides is associated with switching between their oxidized and reduced forms. In the present work, superoxide radicals were generated either radiolytically or enzymatically using hypoxanthine/xanthine oxidase. Electron paramagnetic resonance (EPR) spectrometry was used to follow the exchange between the nitroxide radical and its reduced form; whereas, pulse radiolysis was employed to study the kinetics of hydroxylamine oxidation. The results indicate that: a) The rate constant of superoxide reaction with cyclic hydroxylamines is pH-independent and is lower by several orders of magnitude than the rate constant of superoxide reaction with nitroxides; b) The oxidation of hydroxylamine by superoxide is primarily responsible for the non-enzymatic recycling of nitroxides; c) The rate of nitroxides restoration decreases as the pH decreases because nitroxides remove superoxide more efficiently than is hydroxylamine oxidation; d) The hydroxylamine reaction with oxidized nitroxide (comproportionation) might participate in the exchange among the three oxidation states of nitroxide. However, simulation of the time-dependence and pH-dependence of the exchange suggests that such a comproportionation is too slow to affect the rate of non-enzymatic nitroxide restoration. We conclude that the protective activity of nitroxides in vitro can be distinguished from that of common LMWA due to hydroxylamine oxidation by superoxide, which allows nitroxide recycling and enables its catalytic activity.     

Can nitroxides evoke the Keap1–Nrf2–ARE pathway in skin?

Nitroxides are stable cyclic radicals of diverse size, charge, and lipophilicity. They are cell-permeative, which effectively protects cells, tissues, isolated organs, and laboratory animals from radical-induced damage. The mechanisms of activity through which nitroxides operate are diverse, including superoxide dismutase-mimetic activity, oxidation of semiquinone radicals, oxidation of reduced metal ions, procatalase-mimetic activity, interruption of radical chain reactions, and indirect modulation of NO levels. Nitroxides possess both a nucleophilic (reducing properties) and an electrophilic (oxidizing properties) nature and, therefore, they may affect different cellular pathways. In the current study, a novel mechanism of action by which nitroxides provide skin protection based on their electrophilic nature is suggested. This study shows that nitroxides may act as electrophiles, directly or indirectly, capable of activating the Keap1–Nrf2–ARE pathway in human keratinocytes (HaCaT) and in human skin (human organ culture model). The high potency of oxoammonium cations versus hydroxylamines in activating the system is demonstrated. The mechanism of action by which nitroxides activate the Keap1–Nrf2–ARE pathway is discussed. Understanding the mechanism of activity may expand the usage of nitroxides as a skin protection strategy against oxidative stress-related conditions.     

In vivo measurement of oxygen concentration using sonochemically synthesized microspheres.

Proteinaceous microspheres filled with nitroxides dissolved in an organic liquid have been synthesized for the first time using high intensity ultrasound; these were used to measure oxygen concentrations in living biological systems. The microspheres have an average size of 2.5 microns, and the proteinaceous shell is permeable to oxygen. Encapsulation of the nitroxides into the microsphere greatly increased the sensitivity of the electron paramagnetic resonance signal line width to oxygen because of the higher solubility of oxygen in organic solvents. The encapsulation also protected the nitroxide from bioreduction. No decrease in intensity of the electron paramagnetic resonance signal was observed during 70 min after intravenous injection of the microspheres into a mouse. Measurement of the changes in oxygen concentration in vivo by means of restriction of blood flow, anesthesia, and change of oxygen content in the respired gas were made using these microspheres.     

Cellular metabolism of water-soluble nitroxides: Effect on rate of reduction of cell/nitroxide ratio,oxygen concentrations and permeability of nitroxides

In order to interpret more accurately studies that have used nitroxides and to improve the efficacy of the use of nitroxides in both basic studies of cells and as contrast agents for in vivo NMR, we have initiated a systematic study of the distribution and metabolism of nitroxides in biological systems. Overall, the results provide a reasonably coherent picture of some aspects of the interactions between nitroxides and cells. Reduction of the nitroxides appears to be an intracellular process, so that one of the principal variables that affects the rate of reduction is the ability of a nitroxide to enter cells. The entrance of nitroxides into cells shows considerable variability and ranges from essentially no penetration (e.g., 2,2,6,6-tetramethylpiperidine-N-oxyl-4-trimethylamine) through rates that are comparable to rates of reduction (e.g., 2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-carboxylic acid), to rates that are so fast that there is complete equilibrium between intracellular and extracellular compartments (e.g., Tempone). The presence of a charged group on the nitroxide appears to be the important variable that affects their ability to enter cells. Once a nitroxides enters the cell, the structure of the nitroxide, e.g., piperidine vs. pyrrolidine ring, is major factor that affects the rate of reduction. The rates of reduction increase with increasing concentrations of nitroxides. This indicates that the principal mechanism(s) of reduction do not saturate in the concentration range we studied. We observed no abrupt changes in the rates of reduction over the entire concentration range of cells and nitroxides that we studied, which suggests that the mechanism(s) of nitroxide reduction did not change. The presence of oxygen decreased the observed rate of reduction of many of the nitroxides and this effect was independent of the concentration of nitroxide.     

Optimization of labile esters for esterase-assisted accumulation of nitroxides into cells: a model for in vivo EPR imaging

Nitroxide-based electron paramagnetic resonance (EPR) imaging agents are useful quantitative probes of O2 concentration in vivo in real time. Lipophilic, labile alkanoyloxymethyl esters of nitroxides can cross the blood-brain barrier, and after hydrolysis, the corresponding anionic nitroxide is intracellularly entrapped at levels sufficient to permit O2 measurements. The utility of nitroxides as EPR imaging agents depends critically on their ability to accumulate in the brain to high levels. In this study, we systematically investigated the relationship between the structure of the alkanoyl moiety and the ability of the corresponding labile ester to deliver nitroxide intracellularly. We demonstrate, in a cultured cell model, that for nitroxide labile esters with unbranched alkanoyl chains, increasing the chain length improves intracellular loading. Moreover, by studying an isomeric series of labile esters, we conclude that branching of the alkanoyl chain drastically reduces intracellular loading. These structural insights improve our general ability to use labile esters to deliver carboxylates intracellularly, and suggest a strategy for enhancing delivery of nitroxide imaging agents across the blood-brain barrier in a living animal.