Fluorescence Quenching Effect of a Highly Active Nitroxyl Radical on 7-amino-4-methylcoumarin and Glutathione Sensing.

Nitroxyl radical compounds, such as 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), are stable radical compounds with a variety of unique characteristics, including fluorescence quenching. In this study, we investigated the fluorescence quenching effect of nortropine N-oxyl (NNO), which is a highly active nitroxyl radical that is more active than TEMPO in oxidation catalysis. The fluorescence intensity of 7-amino-4-methylcoumarin (AMC) was quenched by NNO and TEMPO to 5% and 95% of the initial fluorescence intensity, respectively, indicating highly efficient quenching by NNO. In addition, we used this reaction to measure glutathione concentration. The quenching effect of NNO was abrogated by the chemical reaction with glutathione, resulting in restoration of AMC fluorescence. This response was observed at glutathione concentrations from 10 µM to 1 mM, and good calibration curves were obtained from 10 to 250 µM.

[Mechanistic Analysis of Oxidative Stress-related Diseases Using Nitroxyl Radicals and Electron Spin Resonance].

Excessive production of reactive oxygen species (ROS) causes oxidative stress and is involved in the development and progression of a wide variety of diseases. Therefore, techniques for measuring oxidative stress are indispensable for analysis of the mechanisms of various diseases. The method involving ESR and the durable nitroxyl radical (ESR/spin probe method) is useful for this purpose, because the ESR signal intensity of the spin probe changes on reacting with ROS and other unstable radicals. In this review, the author's research applying the ESR/spin probe method to clarify disease mechanisms in vivo and in vitro is presented. The ESR signal of the probe injected into animals may decay through a few mechanisms besides reaction with ROS; thus, interpretation of the results is complicated. As the first approach to solving this problem, a probe resistant to enzymatic reduction by introducing a bulky group adjacent to the nitroxy group was created. The second approach was the use of a hydroxylamine probe which dominantly oxidized to nitroxyl radicals by reacting with superoxide anion radicals and oxidants. Using acyl-protected hydroxyl amine, it was demonstrated that sepsis model mice are under oxidative stress due to ROS production by activated phagocytes. On the other hand, it was shown in vitro that the UV-induced radical reaction of ketoprofen also occurs in lipid membranes, and that the reaction is related to ROS generation and membrane disruption. We believe that use of the ESR/spin probe method with ingenuity will clarify the mechanisms of various diseases.

Electrochemical Characterization of a Novel Organoelectrocatalyst, 7-Azabicyclo[2.2.1]heptan-7-ol (ABHOL), and Its Application to Electrochemical Sensors.

Electrochemical enzyme sensors are suitable for simple monitoring methods, for example, as glucose sensors for diabetic patients; however, they have several disadvantages arising from the properties of the enzyme. Therefore, non-enzymatic electrochemical sensors using functional molecules are being developed. In this paper, we report the electrochemical characterization of a new hydroxylamine compound, 7-azabicyclo[2.2.1]heptan-7-ol (ABHOL), and its application to glucose sensing. Although the cyclic voltammogram for the first cycle was unstable, it was reproducible after the second cycle, enabling electrochemical analysis of ethanol and glucose. In the first cycle, ABHOL caused complex reactions, including electrochemical oxidation and comproportionation with the generated oxoammonium ions. The electrochemical probe performance of ABHOL was more efficient than the typical nitroxyl radical compound, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), and had similar efficiency to 9-azabicyclo[3.3.1]nonane N-oxyl (ABNO), which is activated by the bicyclic structure. The results demonstrated the advantages of ABHOL, which can be synthesized from inexpensive materials via simple methods.

Photoredox Cascade Catalyst for Efficient Hydrogen Production with Biomass Photoreforming.

Biomass photoreforming is a promising method to provide both a clean energy resource in the form of hydrogen (H2 ) and valuable chemicals as the results of water reduction and biomass oxidation. To overcome the poor contact between heterogeneous photocatalysts and biomass substrates, we fabricated a new photoredox cascade catalyst by combining a homogeneous catalyst, 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), and a heterogeneous dual-dye sensitized photocatalyst (DDSP) composed of two Ru(II)-polypyridine photosensitizers (RuP6 and RuCP6 ) and Pt-loaded TiO2 nanoparticles. During blue-light irradiation (λ=460±15 nm; 80 mW), the DDSP photocatalytically reduced aqueous protons to form H2 and simultaneously oxidized TEMPO• radicals to generate catalytically active TEMPO+ . It oxidized biomass substrates (water-soluble glycerol and insoluble cellulose) to regenerate TEMPO• . In the presence of N-methyl imidazole as a proton transfer mediator, the photocatalytic H2 production activities for glycerol and cellulose reforming reached 2670 and 1590 µmol H2 (gTiO2 )-1  h-1 , respectively, which were comparable to those of state-of-the-art heterogeneous photocatalysts.

Determination of antibiotics by amperometry using nortropine N-oxyl, a highly active nitroxyl radical.

Nitroxyl radical compounds oxidize hydroxy groups and some amino groups upon application of an electric potential. The resulting anodic current depends on the concentration of these functional groups in solution. Thus, it is possible to quantify compounds containing these functional groups by electrochemical methods. Cyclic voltammetry has been used to evaluate the catalytic activity of nitroxyl radicals, and the ability of such radicals to sense biological and other compounds. In this study, we evaluated a method for quantifying compounds using constant-potential electrolysis (amperometry) of nitroxyl radicals for application in flow injection analysis and high-performance liquid chromatography as an electrochemical detector. When amperometry was performed using 2,2,6,6-tetramethylpiperidine 1-oxyl, a common nitroxyl radical compound, little change was observed even with 100 mM glucose due to its low reactivity in neutral aqueous solutions. In contrast, 2-azaadamantane N-oxyl and nortropine N-oxyl, which are highly active nitroxyl radicals, showed a concentration-dependent response in neutral aqueous solution. Responses of 33.8 and 125.9 µA, respectively, were observed. By recognition of hydroxy and amino groups, we have succeeded in the electrochemical detection of some drugs by amperometry. Streptomycin, an aminoglycoside antibiotic, was quantifiable in the range of 30-1000 µM.

Structural Features Governing the Metabolic Stability of Tetraethyl-Substituted Nitroxides in Rat Liver Microsomes.

Nitroxides are potent tools for studying biological systems by electron paramagnetic resonance (EPR). Whatever the application, a certain stability is necessary for successful detection. Since conventional tetramethyl-substituted cyclic nitroxides have insufficient in vivo stability, efforts have recently been made to synthesize more stable, tetraethyl-substituted nitroxides. In our previous study on piperidine nitroxides, the introduction of steric hindrance around the nitroxide moiety successfully increased the resistance to reduction into hydroxylamine. However, it also rendered the carbon backbone susceptible to modifications by xenobiotic metabolism due to increased lipophilicity. Here, we focus on a new series of three nitroxide candidates with tetraethyl substitution, namely with pyrrolidine, pyrroline, and isoindoline cores, to identify which structural features afford increased stability for future probe design and application in in vivo EPR imaging. In the presence of rat liver microsomes, pyrrolidine and pyrroline tetraethyl nitroxides exhibited a higher stability than isoindoline nitroxide, which was studied in detail by HPLC-HRMS. Multiple metabolites suggest that the aerobic transformation of tetraethyl isoindoline nitroxide is initiated by hydrogen abstraction by P450-FeV = O from one of the ethyl groups, followed by rearrangement and further modifications by cytochrome P450, as supported by DFT calculations. Under anaerobic conditions, only reduction by rat liver microsomes was observed with involvement of P450-FeII.

[Electrochemical Analysis Using Organic Nitroxyl Radicals].

Organic nitroxyl radicals represented by 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) are known to be compounds that catalyze alcohol oxidation reactions. These catalytic reactions can be applied to a wide range of compounds with hydroxy and amino groups. It is also possible to selectively oxidize primary alcohols by designing the skeleton around the nitroxyl radical moiety for use in organic synthesis. Reactions can also be carried out by electrochemical methods, and the electrical current measured during the reaction can be used to quantify the substrates. Therefore, the combination of reactions catalyzed by nitroxyl radicals and electrochemical techniques is expected to be applied as a new analytical method. However, since the reaction does not proceed rapidly in neutral aqueous solutions, it has mostly been applied in basic aqueous solutions or organic solvents, and there have been no reports on sensor applications under physiological conditions. Herein, we have developed a novel catalyst, nortropine N-oxyl (NNO), which is highly active even in neutral aqueous solutions, and have found that it can be used for the analysis of biological components and drugs under physiological conditions. The combination of this method with enzymatic reactions made it possible to specifically detect certain compounds. In this review, we describe a novel analytical method that combines these nitroxyl radicals with electrochemical methods.

Electrochemical reactions of highly active nitroxyl radicals with thiol compounds.

Nitroxyl radicals are known to electrochemically oxidize thiols as well as alcohols and amines. In this study, a preliminary investigation of the electrochemical reaction of thiols with 9-azabicyclo[3.3.1]nonane N-oxyl (ABNO), 2-azaadamantane N-oxyl (AZADO), and nortropine N-oxyl (NNO), which are highly active due to their bicyclo structures, for use in electrochemical analysis was performed and the results were compared with those for a typical nitroxyl radical compound, 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO). Mercaptopropane sulfonic acid (MPS) was used as a model compound to investigate the electrochemical response in aqueous solution. In addition, electrochemical detection of glutathione, a biological thiol molecule, was performed.

Noninvasive Diagnosis of the Mitochondrial Function of Doxorubicin-Induced Cardiomyopathy Using In Vivo Dynamic Nuclear Polarization-Magnetic Resonance Imaging.

Doxorubicin (DOX) induces dose-dependent cardiotoxicity via oxidative stress and abnormal mitochondrial function in the myocardium. Therefore, a noninvasive in vivo imaging procedure for monitoring the redox status of the heart may aid in monitoring diseases and developing treatments. However, an appropriate technique has yet to be developed. In this study, we demonstrate a technique for detecting and visualizing the redox status of the heart using in vivo dynamic nuclear polarization-magnetic resonance imaging (DNP-MRI) with 3-carbamoyl-PROXYL (CmP) as a molecular imaging probe. Male C57BL/6N mice were administered DOX (20 mg/kg) or saline. DNP-MRI clearly showed a slower DNP signal reduction in the DOX group than in the control group. Importantly, the difference in the DNP signal reduction rate between the two groups occurred earlier than that detected by physiological examination or clinical symptoms. In an in vitro experiment, KCN (an inhibitor of complex IV in the mitochondrial electron transport chain) and DOX inhibited the electron paramagnetic resonance change in H9c2 cardiomyocytes, suggesting that the redox metabolism of CmP in the myocardium is mitochondrion-dependent. Therefore, this molecular imaging technique has the potential to monitor the dynamics of redox metabolic changes in DOX-induced cardiomyopathy and facilitate an early diagnosis of this condition.

Oxazolidine Nitroxide Transformation in a Coordination Sphere of the Ln3+ Ions.

Upon the interaction of the hydrated lanthanide(III) salts found in acetonitrile solution with a tripodal paramagnetic compound, 4,4-dimethyl-2,2-bis(pyridin-2-yl)-1,3-oxazolidine-3-oxyl (Rad), functionalized by two pyridyl groups, three neutral, structurally characterized complexes with diamagnetic polydentate ligands-[Dy(RadH)(hbpm)Cl2], [Yb2(ipapm)2(NO3)4], and [Ce2(ipapm)2(NO3)4(EtOAc)2]-were obtained. These coordination compounds are minor uncolored crystalline products, which were formed in a reaction mixture due to the Rad transformation in a lanthanide coordination sphere, wherein the processes of its simultaneous disproportionation, hydrolysis, and condensation proceed differently than in the absence of Ln ions. The latter fact was confirmed by the formation of the structurally characterized product of the oxazolidine nitroxide transformation during its crystallization in toluene solution. Such a conversion in the presence of 4f elements ions is unique since no similar phenomenon was observed during the synthesis of the 3d-metal complexes with Rad.

Detection of singlet oxygen by EPR: The instability of the nitroxyl radicals.

The use of spin traps and redox probes coupled with electron paramagnetic resonance (EPR) is a method frequently applied in the evaluation of the efficiency of photosensitizers and photocatalysts in phototherapeutic and photocatalytic processes that involve reactive oxygen species. In this way, the method helps to clarify the mechanism behind photo-induced reactions. Hydroxy-TEMP is a very specific redox probe for selectively identifying and quantifying singlet oxygen (1O2). In this work, the kinetics of radical generated by the oxidation products of the Hydroxy-TEMP redox probe was analyzed from EPR spectra in aqueous solutions of several water-soluble porphyrins ([H2T4MPyP](OTs)4, Na4[H2T4SPP], [H2T2MPyP](OTs)4, [ZnT4MyPyP](OTs)4, [MnT4MyPyP](OTs)5, H2T4CPP, and [H2T4TriMAPP](OTs)4) under white light illumination. Different factors such as the concentration of the redox probe, pH of the medium, and photostability of the porphyrins were evaluated. A systematic study was carried out to reveal the factors associated with stable radical degradation (TEMPOL) by illumination in the visible spectral region in systems containing photosensitizer (porphyrin) and redox probe (Hydroxy-TEMP). With the aid of EPR and gas chromatography coupled with mass spectroscopy (GC-MS) techniques, the mechanism of the radical degradation and the photobleaching of porphyrins were investigated. After successive interactions with the porphyrin in its excited state, in alkaline aqueous solution (pH > 10), the free radical TEMPOL is transformed into TEMPONE until the final diamagnetic product Phorone. A protocol was elaborated to identify and quantify the generation of 1O2 by Hydroxy-TEMP reliably, to avoid possible errors in the interpretation of efficiency of photosensitizers.

Redox-Based Theranostics of Gastric Ulcers Using Nitroxyl Radicals.

Significance: Redox-based theranostics involves redox monitoring and therapeutics that normalize redox imbalance. It may be a promising approach to markedly improve a patient's quality of life through streamlined treatment. Nitroxyl radicals are useful for both redox monitoring and treating gastric ulcers in rodents. Recent Advances: Redox monitoring using in vivo electron paramagnetic resonance (EPR) spectroscopy in a gastric ulcer rat model showed the production of reactive oxygen species in the whole stomach. A combination of Overhauser-enhanced magnetic resonance imaging (MRI) and nitroxyl radicals provided high-resolution images of redox imbalance in the stomach of rats with a gastric ulcer. Treatment with nitroxyl radicals was effective to treat ulcers that were formed using model experiments of Helicobacter pylori and mental stress as well as nonsteroidal anti-inflammatory drugs. Critical Issues: For redox monitoring using Overhauser-enhanced MRI, the EPR irradiation power that is delivered to subjects must be within the range of the specific absorption rate regulation to protect against microwave damage regardless of a decrease in image contrast. The effect of long-term treatment with a nitroxyl radical in patients with a gastric ulcer remains unclear. Future Directions: Further research on redox-based theranostics in redox-related diseases, including gastric ulcers, would be accelerated by improving the redox imager and by developing functional nitroxyl radicals that localize in the target organ, tissue, or cell and that have specific reactivity for the redox-related biomolecule.

Redox-Sensitive Mapping of a Mouse Tumor Model Using Sparse Projection Sampling of Electron Paramagnetic Resonance.

Aims: This work aimed to establish an accelerated imaging system for redox-sensitive mapping in a mouse tumor model using electron paramagnetic resonance (EPR) and nitroxyl radicals. Results: Sparse sampling of EPR spectral projections was demonstrated for a solution phantom. The reconstructed three-dimensional (3D) images with filtered back-projection (FBP) and compressed sensing image reconstruction were quantitatively assessed for the solution phantom. Mouse xenograft models of a human-derived pancreatic ductal adenocarcinoma cell line, MIA PaCa-2, were also measured for redox-sensitive mapping with the sparse sampling technique. Innovation: A short-lifetime redox-sensitive nitroxyl radical (15N-labeled perdeuterated Tempone) could be measured to map the decay rates of the EPR signals for the mouse xenograft models. Acceleration of 3D EPR image acquisition broadened the choices of nitroxyl radical probes with various redox sensitivities to biological environments. Conclusion: Sparse sampling of EPR spectral projections accelerated image acquisition in the 3D redox-sensitive mapping of mouse tumor-bearing legs fourfold compared with conventional image acquisition with FBP. Antioxid. Redox Signal. 36, 57-69.

Nitroxyl Radical as a Theranostic Contrast Agent in Magnetic Resonance Redox Imaging.

Significance:In vivo assessment of paramagnetic and diamagnetic conversions of nitroxyl radicals based on cyclic redox mechanism can be an index of tissue redox status. The redox mechanism of nitroxyl radicals, which enables their use as a normal tissue-selective radioprotector, is seen as being attractive on planning radiation therapy. Recent Advances:In vivo redox imaging using nitroxyl radicals as redox-sensitive contrast agents has been developed to assess tissue redox status. Chemical and biological behaviors depending on chemical structures of nitroxyl radical compounds have been understood in detail. Polymer types of nitroxyl radical contrast agents and/or nitroxyl radical-labeled drugs were designed for approaching theranostics. Critical Issues: Nitroxyl radicals as magnetic resonance imaging (MRI) contrast agents have several advantages compared with those used in electron paramagnetic resonance (EPR) imaging, while support by EPR spectroscopy is important to understand information from MRI. Redox-sensitive paramagnetic contrast agents having a medicinal benefit, that is, nitroxyl-labeled drug, have been developed and proposed. Future Directions: A development of suitable nitroxyl contrast agent for translational theranostic applications with high reaction specificity and low normal tissue toxicity is under progress. Nitroxyl radicals as redox-sensitive magnetic resonance contrast agents can be a useful tool to detect an abnormal tissue redox status such as disordered oxidative stress. Antioxid. Redox Signal. 36, 95-121.

[Oxidative Deprotection of p-Methoxybenzyl Ethers by a Nitroxyl Radical Catalyst].

The oxidation of p-methoxybenzyl (PMB) ethers was achieved using a nitroxyl radical catalyst 1, which contains electron-withdrawing ester groups adjacent to the nitroxyl group. The oxidative deprotection of the PMB moieties on the hydroxy groups was observed upon treatment of 1 with one equivalent of the co-oxidant phenyl iodonium bis(trifluoroacetate) (PIFA). This system showed an excellent chemoselectivity profile for the deprotection of PMB ethers from a broad range of functional groups including diverse oxidation-sensitive moieties. The corresponding carbonyl compounds were obtained by treating the PMB-protected alcohols with 1 and an excess amount of PIFA.

Nitroxyl Radical/Copper-Catalyzed Electrooxidation of Alcohols and Amines at Low Potentials.

Nitroxyl radicals, such as 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO), can catalyze the electrochemical oxidation of alcohols and amines. Because the oxidation current obtained in this process depends on the concentration of alcohols and amines, this process can be applied to their sensing. However, the relatively high oxidation potentials required by nitroxyl radicals can induce interfering oxidation currents from various reductive substances in biological samples, which affects the accuracy of analyte measurements. In this study, we examined the electrooxidation of alcohols and amines at a low potential by applying cooperative oxidation catalysis using a nitroxyl radical and a copper salt. Nortropine N-oxyl (NNO), which showed higher catalytic activity than TEMPO was used as the nitroxyl radical. An increase in the oxidation current was observed at the low potential, and this increase depended on the alcohol concentration. In the case of the electrooxidation of amines, a positive correlation between oxidation current and amine concentration was observed at low amine concentrations. Therefore, low-potential cooperative catalysis can be applied to alcohol and amine electrooxidation for the development of accurate sensors suitable for clinical settings.

Expansion of Substrate Scope for Nitroxyl Radical/Copper-Catalyzed Aerobic Oxidation of Primary Alcohols: A Guideline for Catalyst Selection.

Four distinctive sets of optimum nitroxyl radical/copper salt/additive catalyst combinations have been identified for accommodating the aerobic oxidation of various types of primary alcohols to their corresponding aldehydes. Interestingly, less nucleophilic catalysts exhibited higher catalytic activities for the oxidation of particular primary allylic and propargylic alcohols to give α,ß-unsaturated aldehydes that function as competent Michael acceptors. The optimum conditions identified herein were successful in the oxidation of various types of primary alcohols, including unprotected amino alcohols and divalent-sulfur-containing alcohols in good-to-high yields. Moreover, N-protected alaninol, an inefficient substrate in the nitroxyl radical/copper-catalyzed aerobic oxidation, was oxidized in good yield. On the basis of the optimization results, a guideline for catalyst selection has been established.

Development of 20 cm sample bore size dynamic nuclear polarization (DNP)-MRI at 16 mT and redox metabolic imaging of acute hepatitis rat model.

Tissue redox metabolism is involved in various diseases, and an understanding of the spatio-temporal dynamics of tissue redox metabolism could be useful for diagnosis of progression and treatment. In in vivo dynamic nuclear polarization (DNP)-MRI, electron paramagnetic resonance (EPR) irradiation at the resonance frequency of nitroxyl radicals administered as a redox probe for induction of DNP, increases the intensity of MRI signals. For electron spin, it is necessary to apply a resonant frequency 658 times higher than that required for nuclear spin because of the higher magnetic moment of unpaired electrons. Previous studies using a disease model of small animals and in vivo DNP-MRI have revealed that an abnormal redox status is involved in many diseases, and that it could be used to visualize the dynamics of alterations in redox metabolism. To use the current methods in clinical practice, the development of a prototype DNP-MRI system for preclinical examinations of large animals is indispensable for clarifying the problems peculiar to the increase in size of the DNP-MRI device. Therefore, we developed a in vivo DNP-MRI system with a sample bore size of 20 cm and a 16-mT magnetic field using a U-shaped permanent magnet. Because the NMR frequency is very low, we adopted a digital radiofrequency transmission/reception system with excellent filter and dynamic range characteristics and equipped with a digital eddy current compensation system to suppress large eddy currents. The pulse sequence was based on the fast spin-echo sequence, which was improved for low frequency and large-eddy current equipment. The in vivo DNP-MRI system developed was used to non-invasively image the redox reaction of a carbamoyl-PROXYL probe in the livers of large rats weighing 800 g. Furthermore, DNP-MRI analysis was able to capture significant changes in redox metabolism in hepatitis-model rats.

Differences in pharmacokinetic behaviors of two lipophilic 3-substituted 2,2,5,5-tetramethylpyrrolidine-N-oxyl radicals, in vivo probes to assess the redox status in the brain using magnetic resonance techniques.

PURPOSE: The pharmacokinetics of 3-methoxycarbonyl- and 3-hydroxymethyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl radicals (MCP and HMP, respectively), magnetic resonance probes to assess the brain redox status, were examined in healthy mouse brains. METHODS: The time course of the concentration of the radical form of the probe in the brain was examined by signal enhancements on T1 -weighted MR image after an intravenous injection. The distribution of the total probe (sum of radical and reduced forms) was investigated using brain homogenates. RESULTS: MCP distributed to the brain more than HMP. MCP exhibited biphasic decay with fast and slow components, whereas HMP exhibited monophasic decay with a similar rate constant to the slow component of MCP. Similar profiles were observed in various regions of the brain. The total probe for MCP exhibited monophasic decay at a similar rate constant to the slow component of the radical form; however, the initial content of the total probe was similar to its radical form. For HMP, decay of the total probe coincided with that of the radical form. CONCLUSION: The decay of MCP needs to consider the reduction of the probe in and its elimination from the brain, while the decay of HMP may mainly result from its elimination from the brain.

In vivo ESR imaging of redox status in mice after X-ray irradiation, measured by acyl-protected hydroxylamine probe, ACP.

More detailed investigations on the in vivo redox status are needed to elucidate the mechanisms contributing to damage caused by ionizing radiation. In the present study, the in vivo redox status of mice was examined using in vivo electron spin resonance (ESR) imaging after an intraperitoneal injection of 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) as a probe. ACP is easily hydrolyzed to its hydroxylamine form in the mouse body, and the interconversion between hydroxylamine and the corresponding nitroxyl radical reflects the biological redox status. Liver damage, based on changes in liver weight and plasma aspartate aminotransferase levels, was detected in mice 4 days after X-ray irradiation at 7.5 Gy. ESR imaging showed that the signal intensity of the nitroxyl radical was high at the liver area in both damaged and healthy mice after administration of ACP. Whereas the signal decayed at the liver area for healthy mouse, the decay was negligible in damaged mice. Unlike healthy mouse, signal in the chest for damaged mouse increased with time. The distribution of the sum of hydroxylamine and the nitroxyl radical was similar in damaged and healthy mice. X-ray irradiation slightly lowered the reduction activity of the liver microsomal fraction for the nitroxyl radical. Thiobarbituric acid reactive substances in the liver were higher in damaged mice than in healthy mice; however, no significant differences were noted in reduced glutathione. The present results indicate that the redox status of mice exposed to X-ray irradiation is more oxidative than that in healthy mice.