Language：English   Publishing type：Research paper (scientific journal)  

DOI： https://doi.org/10.1248/bpb.b23-00849

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1161/CIRCRESAHA.123.323517

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1080/10715762.2023.2244155

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1172/jci.insight.169756

Language：English   Publishing type：Research paper (scientific journal)  

Objective
Nonalcoholic steatohepatitis is a chronic liver disease caused by the progression of hepatocellular death and inflammation from simple steatosis. However, the pathogenesis of this disease remains unclear. Lipid peroxidation is one of the most critical factors in the development of nonalcoholic steatohepatitis; however, oxidised lipids – the products of lipid peroxidation – are insufficiently analysed. Here, we comprehensively analysed oxidised lipids in the liver during nonalcoholic steatohepatitis development in a choline-deficient, l-amino acid-defined, high-fat diet-fed mouse model.

Methods
Liver from C57BL/6J mice, fed a standard diet or a choline-deficient l-amino acid-defined high-fat diet for 1, 3, or 6 weeks, were collected to evaluate fibrosis, steatosis, inflammation, liver injury, and oxidised lipid production and to observe the suppression of these parameters upon vitamin E administration. In addition, organellar localisation of lipid peroxidation was assessed using fluorescence imaging. Finally, a mitochondria-targeted antioxidant was administered to model mice to investigate the mechanism underlying lipid peroxidation.

Results
We found an accumulation of oxidised triglycerides in the early stages of nonalcoholic steatohepatitis. Furthermore, our data indicate that oxidised triglycerides are generated by lipid peroxidation in lipid droplets due to mitochondria-derived reactive oxygen species.

Conclusion
These results suggest the importance of lipid droplet peroxidation in the progression of nonalcoholic steatohepatitis and may contribute to the development of therapeutic methods for nonalcoholic steatohepatitis in the future.

Significance statement
We demonstrate the specific and early occurrence of lipid peroxidation in nonalcoholic steatohepatitis pathogenesis and propose a previously unknown mechanism of disease progression.

DOI： https://doi.org/10.1530/REM-22-0024

Language：English   Publishing type：Research paper (scientific journal)  

Doxorubicin (DOX) is an effective anti-cancer agent for various malignancies. Nevertheless, it has a side effect of cardiotoxicity, referred to as doxorubicin-induced cardiomyopathy (DIC), that is associated with a poorer prognosis. This cardiotoxicity limits the clinical use of DOX as a therapeutic agent for malignancies. Recently, ferroptosis, a form of regulated cell death induced by the accumulation of lipid peroxides, has been recognized as a major pathophysiology of DIC. Ethoxyquin is a lipophilic antioxidant widely used for food preservation and thus may be a potential therapeutic drug for preventing DIC. However, the efficacy of ethoxyquin against ferroptosis and DIC remains to be fully elucidated. Here, we investigated the inhibitory action of ethoxyquin against GPx4-deficient ferroptosis and its therapeutic efficacy against DOX-induced cell death in cultured cardiomyocytes and cardiotoxicity in a murine model of DIC. In cultured cardiomyocytes, ethoxyquin treatment effectively prevented GPx4-deficient ferroptosis. Ethoxyquin also prevented DOX-induced cell death, accompanied by the suppression of malondialdehyde (MDA) and mitochondrial lipid peroxides, which were induced by DOX. Furthermore, ethoxyquin significantly prevented DOX-induced cell death without any suppression of caspase cleavages representing apoptosis. In DIC mice, ethoxyquin treatment ameliorated cardiac impairments, such as contractile dysfunction and myocardial atrophy, and lung congestion. Ethoxyquin also suppressed serum lactate dehydrogenase and creatine kinase activities, decreased the levels of lipid peroxides such as MDA and acrolein, inhibited cardiac fibrosis, and reduced TUNEL-positive cells in the hearts of DIC mice. Collectively, ethoxyquin is a competent antioxidant for preventing ferroptosis in DIC and can be its prospective therapeutic drug.

DOI： 10.1097/FJC.0000000000001328.

Language：English   Publishing type：Research paper (scientific journal)  

Clinical use of doxorubicin (DOX) is limited because of its cardiotoxicity, referred to as DOX-induced cardiomyopathy (DIC). Mitochondria-dependent ferroptosis, which is triggered by iron overload and excessive lipid peroxidation, plays a pivotal role in the progression of DIC. Here, we showed that DOX accumulated in mitochondria by intercalating into mitochondrial DNA (mtDNA), inducing ferroptosis in an mtDNA content-dependent manner. In addition, DOX disrupted heme synthesis by decreasing the abundance of 5'-aminolevulinate synthase 1 (Alas1), the rate-limiting enzyme in this process, thereby impairing iron utilization, resulting in iron overload and ferroptosis in mitochondria in cultured cardiomyocytes. Alas1 overexpression prevented this outcome. Administration of 5-aminolevulinic acid (5-ALA), the product of Alas1, to cultured cardiomyocytes and mice suppressed iron overload and lipid peroxidation, thereby preventing DOX-induced ferroptosis and DIC. Our findings reveal that the accumulation of DOX and iron in mitochondria cooperatively induces ferroptosis in cardiomyocytes and suggest that 5-ALA can be used as a potential therapeutic agent for DIC.

DOI： 10.1126/scisignal.abn8017.

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1038/s12276-022-00888-9

Language：English   Publishing type：Research paper (scientific journal)  

DOI： doi: 10.1038/s12276-022-00888-9

Language：English   Publishing type：Research paper (scientific journal)  

Vascular complications in β-thalassemia are associated with oxidative modification of lipoproteins under high oxidative stress. The lipid components of lipoproteins are oxidized via lipid peroxidation and produce lipid radicals (L•) as the key initial intermediates. Modification of lipid components, therefore, might result in alterations in the rate and products of lipid peroxidation. In this study, the kinetics of L• formation during the 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH)- and hemin-induced oxidation of low-density and high-density lipoproteins (LDL and HDL) from β-thalassemia patients and healthy volunteers were investigated using a specific and sensitive fluorescence probe for L•. Kinetic parameters, including initial lag time, propagation rate and total L• production, were calculated by monitoring a fluorescence-active NBD-Pen-L• adduct. Oxidation of thalassemia lipoproteins exhibited a significantly shorter lag time but a slower propagation rate of L• formation when compared with healthy lipoproteins. LDL showed higher resistance to oxidation during the initiation phase but higher L• formation than HDL. Our results indicated that the levels of α-tocopherol determined the initial lag time, whereas the levels of core lipids and cholesteryl esters, especially cholesteryl linoleate (CL), determined the propagation rate and total L• production. The difference in potency of AAPH and hemin supported that hemin preferentially targeted core lipids. Moreover, analysis of 13-hydroxyoctadecadienoic acid cholesteryl ester (13-HODE-CE)/CE ratio indicated that thalassemia lipoproteins have higher susceptibility to oxidation than healthy lipoproteins. In conclusion, our findings suggested that CL and α-tocopherol were implicated in the susceptibility of lipoproteins to lipid peroxidation in physiological and pathological conditions of β-thalassemia.

Language：English   Publishing type：Research paper (scientific journal)  

Ferroptosis, a non-apoptotic form of cell death marked by iron-dependent lipid peroxidation1, has a key role in organ injury, degenerative disease and vulnerability of therapy-resistant cancers2. Although substantial progress has been made in understanding the molecular processes relevant to ferroptosis, additional cell-extrinsic and cell-intrinsic processes that determine cell sensitivity toward ferroptosis remain unknown. Here we show that the fully reduced forms of vitamin K—a group of naphthoquinones that includes menaquinone and phylloquinone3—confer a strong anti-ferroptotic function, in addition to the conventional function linked to blood clotting by acting as a cofactor for γ-glutamyl carboxylase. Ferroptosis suppressor protein 1 (FSP1), a NAD(P)H-ubiquinone reductase and the second mainstay of ferroptosis control after glutathione peroxidase-44,5, was found to efficiently reduce vitamin K to its hydroquinone, a potent radical-trapping antioxidant and inhibitor of (phospho)lipid peroxidation. The FSP1-mediated reduction of vitamin K was also responsible for the antidotal effect of vitamin K against warfarin poisoning. It follows that FSP1 is the enzyme mediating warfarin-resistant vitamin K reduction in the canonical vitamin K cycle6. The FSP1-dependent non-canonical vitamin K cycle can act to protect cells against detrimental lipid peroxidation and ferroptosis.

Language：English   Publishing type：Research paper (scientific journal)  

Ischemia-reperfusion (I/R) injury is a promising therapeutic target to improve clinical outcomes after acute myocardial infarction. Ferroptosis, triggered by iron overload and excessive lipid peroxides, is reportedly involved in I/R injury. However, its significance and mechanistic basis remain unclear. Here, we show that glutathione peroxidase 4 (GPx4), a key endogenous suppressor of ferroptosis, determines the susceptibility to myocardial I/R injury. Importantly, ferroptosis is a major mode of cell death in I/R injury, distinct from mitochondrial permeability transition (MPT)-driven necrosis. This suggests that the use of therapeutics targeting both modes is an effective strategy to further reduce the infarct size and thereby ameliorate cardiac remodeling after I/R injury. Furthermore, we demonstrate that heme oxygenase 1 up-regulation in response to hypoxia and hypoxia/reoxygenation degrades heme and thereby induces iron overload and ferroptosis in the endoplasmic reticulum (ER) of cardiomyocytes. Collectively, ferroptosis triggered by GPx4 reduction and iron overload in the ER is distinct from MPT-driven necrosis in both in vivo phenotype and in vitro mechanism for I/R injury. The use of therapeutics targeting ferroptosis in conjunction with cyclosporine A can be a promising strategy for I/R injury.

DOI： 10.1016/j.jacbts.2022.03.012.

Language：English   Publishing type：Research paper (scientific journal)  

Although oxidized phosphatidylcholines (oxPCs) play critical roles in numerous pathological events, the type and production sites of endogenous oxPCs remain unknown because of the lack of structural information and dedicated analytical methods. Herein, a library of 465 oxPCs is constructed using high-resolution mass spectrometry-based non-targeted analytical methods and employed to detect 70 oxPCs in mice with acetaminophen-induced acute liver failure. We show that doubly oxygenated polyunsaturated fatty acid (PUFA)-PCs (PC PUFA;O2), containing epoxy and hydroxide groups, are generated in the early phase of liver injury. Hybridization with in-vivo 18O labeling and matrix-assisted laser desorption/ionization-tandem MS imaging reveals that PC PUFA;O2 are accumulated in cytochrome P450 2E1-expressing and glutathione-depleted hepatocytes, which are the major sites of liver injury. The developed library and visualization methodology should facilitate the characterization of specific lipid peroxidation events and enhance our understanding of their physiological and pathological significance in lipid peroxidation-related diseases.

Language：English  

Although oxidized phosphatidylcholines (oxPCs) play critical roles in numerous pathological events, the type and production sites of endogenous oxPCs remain unknown because of the lack of structural information and dedicated analytical methods. Herein, a library of 465 oxPCs is constructed using high-resolution mass spectrometry-based non-targeted analytical methods and employed to detect 70 oxPCs in mice with acetaminophen-induced acute liver failure. We show that doubly oxygenated polyunsaturated fatty acid (PUFA)-PCs (PC PUFA;O2), containing epoxy and hydroxide groups, are generated in the early phase of liver injury. Hybridization with in-vivo 18O labeling and matrix-assisted laser desorption/ionization-tandem MS imaging reveals that PC PUFA;O2 are accumulated in cytochrome P450 2E1-expressing and glutathione-depleted hepatocytes, which are the major sites of liver injury. The developed library and visualization methodology should facilitate the characterization of specific lipid peroxidation events and enhance our understanding of their physiological and pathological significance in lipid peroxidation-related diseases.

Repository Public URL： https://hdl.handle.net/2324/7172670

Language：English  

Mammalian hibernators endure severe and prolonged hypothermia that is lethal to non-hibernators, including humans and mice. The mechanisms responsible for the cold resistance remain poorly understood. Here, we found that hepatocytes from a mammalian hibernator, the Syrian hamster, exhibited remarkable resistance to prolonged cold culture, whereas murine hepatocytes underwent cold-induced cell death that fulfills the hallmarks of ferroptosis such as necrotic morphology, lipid peroxidation and prevention by an iron chelator. Unexpectedly, hepatocytes from Syrian hamsters exerted resistance to cold- and drug-induced ferroptosis in a diet-dependent manner, with the aid of their superior ability to retain dietary α-tocopherol (αT), a vitamin E analog, in the liver and blood compared with those of mice. The liver phospholipid composition is less susceptible to peroxidation in Syrian hamsters than in mice. Altogether, the cold resistance of the hibernator’s liver is established by the ability to utilize αT effectively to prevent lipid peroxidation and ferroptosis.

DOI： 10.1038/s42003-021-02297-6.

Language：English   Publishing type：Research paper (scientific journal)  

Acetaminophen (APAP) overdose is a major cause of drug-induced acute liver failure. In such cases, free iron is released from lysosomes and is transported to mitochondria where it plays a pivotal role in APAP-induced liver injury. We previously reported that ascorbic acid (Asc) markedly mitigates APAP-induced hepatic damage in aldehyde reductase (Akr1a)-knockout (KO) mice that produce about 10% Asc as wild-type (WT) mice. However, the issue of the protective mechanism of Asc in association with the status of iron remains ambiguous. To gain additional insights into this issue, we examined effects of APAP (500 mg/kg) on female KO mice under conditions of iron loading. While the KO mice without AsA supplementation were more sensitive to APAP toxicity than the WT mice, FeSO4 loading (25 mg/kg) to WT mice aggravated the hepatic injury, which was a similar extent to that of the KO mice. Supplementation of Asc (1.5 mg/ml in the drinking water) ameliorated KO mice irrespective of iron status but did not change the iron-mediated increase in the lethality in the WT mice. Hepatic cysteine and glutathione levels declined to similar extents in all mouse groups at 3 h irrespective of the iron status and largely recovered at 18 h after the APAP treatment when liver damage was evident. Asc prominently mitigated APAP toxicity in KO mice irrespective of the iron status but had no effect on the synergistic action of iron and APAP in the WT mice, suggesting that the mechanism for the deteriorating action of loaded iron is different from that of APAP toxicity.

DOI： 10.1080/10715762.2021.1881500

Language：English  

Aldehyde reductase encoded by the Akr1a gene catalyzes the NADPH-dependent reduction of a variety of aldehyde compounds, and it plays a role in the biosynthesis of ascorbic acid (AsA) by converting D-glucuronate to L-gulonate. Although supplementing drinking water with AsA (1.5 mg/mL) ameliorates the fertility of Akr1a-/- (KO) female mice, litter sizes in the KO mice are typically smaller than those for Akr1a+/+ (WT) mice, and about one-third of the neonates have a reduced stature. Half of the neonates in the smallest, developmentally retarded group died before weaning, and the remaining half (less than 6 g in weight) also barely grew to adulthood. While no difference was found in the number of fetuses between the KO and WT mice at 14.5-embryonic days, the sizes of the KO fetuses had already diverged. Among the organs of these retarded KO neonates at 30 d, the spleen and thymus were characteristically small. While an examination of spleen cells showed the normal proportion of immune cells, apoptotic cell death was increased in the thymus, which would lead to thymic atrophy in the retarded KO neonates. Plasma AsA levels were lower in the small neonates despite the fact that their mothers had received sufficient AsA supplementation, and the corticosterone levels were inversely higher compared to wild-type mice. Thus, insufficient AsA contents together with a defect in corticosterone metabolism might be the cause of the retarded growth of the AKR1A-deficient mice embryos and neonates.

DOI： 10.1016/j.jnutbio.2021.108604

Language：English   Publishing type：Research paper (scientific journal)  

It is well known that lipid carbon radicals (lipid radicals) are the origin of lipid peroxidation and are involved in various diseases such as cancer. Therefore, the in vivo detection of lipid radicals would be expected to lead to early diagnosis of these diseases. However, there are no methods for measuring lipid radicals in vivo. Nitroxides are known to be highly reactive with lipid radicals, but they tend to be reduced in vivo. Focusing on the excellent detection sensitivity of nuclear medical imaging, we have developed a radioiodinated nitroxide derivative with resistance to bioreduction for the in vivo detection of lipid radicals. The desired compound was obtained successfully and was highly stable against bioreduction while maintaining high reactivity toward lipid radicals. The I-125 labeling was efficacious with radiochemical yields of 84-87% and radiochemical purities of >99%. A cellular uptake assay showed that the radioiodinated compound was significantly taken up by cells under lipid radical-producing conditions compared to that in the absence of lipid radical production. A biodistribution study indicated that the radioiodinated compound accumulated more in organs where lipid peroxidation was promoted than the methoxyamine derivative, which lost reactivity to lipid radicals. These results indicated that the developed probe became trapped in cells or organs by reacting with lipid radicals. Thus, the radioiodinated nitroxide is a candidate probe for in vivo detection of lipid radicals.

DOI： 10.1016/j.freeradbiomed.2020.12.028

Language：English  

Emissive push–pull‐type bisnaphthyridylamine derivatives (BNA‐X: X=Me, Et, Bzl, Ph, BuBr, and BuTEMPO) aggregate in aqueous methanol. Furthermore, a two‐step emission and aggregation process is controllable by varying the methanol‐to‐water ratio. At 2:3 MeOH/H2O, crystallization‐induced emission enhancement (CIEE) occurs via formation of an emissive crystal phase, whereas, at 1:9 MeOH/H2O, aggregation‐induced emission enhancement (AIEE) occurs, induced by emissive supramolecular nanoparticles (NPs). For BNA‐Ph, the emission quantum yield was 25 times higher in aqueous methanol than that in pure methanol. Despite the high hydrophobicity of BNA‐X (C log P=6.1–8.0), the spherical NPs were monodisperse (polydispersity indices <0.2). Moreover, the emissive NPs exhibited fluorescence resonance energy transfer (FRET) with pyrene; however, for BNA‐X bearing the TEMPO radical (BNA‐BuTEMPO), no FRET was observed because of quenching. In particular, the BNA‐BuTEMPO NPs have a slow rotational correlation time (1.3 ns), suggesting applications as magnetic resonance imaging contrast agents with large relaxivity.

DOI： 10.1002/chem.202003854

Language：English   Publishing type：Research paper (scientific journal)  

Superoxide dismutase 1 (Sod1) plays pivotal roles in antioxidation via accelerating the conversion of superoxide anion radicals into hydrogen peroxide, thus inhibiting the subsequent radical chain reactions. While Sod1 deficient cells inevitably undergo death in culture conditions, Sod1-knockout (KO) mice show relatively mild phenotypes and live approximately two years. We hypothesized that the presence of abundant levels of ascorbic acid (AsA), which is naturally produced in mice, contributes to the elimination of reactive oxygen species (ROS) in Sod1-KO mice. To verify this hypothesis, we employed mice with a genetic ablation of aldehyde reductase (Akr1a), an enzyme that is involved in the biosynthesis of AsA, and established double knockout (DKO) mice that lack both Sod1 and Akr1a. Supplementation of AsA (1.5 mg/ml in drinking water) was required for the DKO mice to breed, and, upon terminating the AsA supplementation, they died within approximately two weeks regardless of age or gender. We explored the etiology of the death from pathophysiological standpoints in principal organs of the mice. Marked changes were observed in the lungs in the form of macroscopic damage after the AsA withdrawal. Histological and immunological analyses of the lungs indicated oxidative damage of tissue and activated immune responses. Thus, preferential oxidative injury that occurred in pulmonary tissues appeared to be primary cause of the death in the mice. These collective results suggest that the pivotal function of AsA in coping with ROS in vivo, is largely in pulmonary tissues that are exposed to a hyperoxygenic microenvironment.

DOI： 10.1016/j.freeradbiomed.2020.10.023

Language：English  

Acetaminophen (APAP) overdose is a major cause of drug-induced acute liver failure. In such cases, free iron is released from lysosomes and is transported to mitochondria where it plays a pivotal role in APAP-induced liver injury. We previously reported that ascorbic acid (Asc) markedly mitigates APAP-induced hepatic damage in aldehyde reductase (Akr1a)-knockout (KO) mice that produce about 10&#37; Asc as wild-type (WT) mice. However, the issue of the protective mechanism of Asc in association with the status of iron remains ambiguous. To gain additional insights into this issue, we examined effects of APAP (500 mg/kg) on female KO mice under conditions of iron loading. While the KO mice without AsA supplementation were more sensitive to APAP toxicity than the WT mice, FeSO4 loading (25 mg/kg) to WT mice aggravated the hepatic injury, which was a similar extent to that of the KO mice. Supplementation of Asc (1.5 mg/ml in the drinking water) ameliorated KO mice irrespective of iron status but did not change the iron-mediated increase in the lethality in the WT mice. Hepatic cysteine and glutathione levels declined to similar extents in all mouse groups at 3 h irrespective of the iron status and largely recovered at 18 h after the APAP treatment when liver damage was evident. Asc prominently mitigated APAP toxicity in KO mice irrespective of the iron status but had no effect on the synergistic action of iron and APAP in the WT mice, suggesting that the mechanism for the deteriorating action of loaded iron is different from that of APAP toxicity.

DOI： 10.1080/10715762.2020.1819996.

Language：English   Publishing type：Research paper (scientific journal)  

Diversified oxidized-lipid molecules are responsible for inflammation and cell death, including ferroptosis. Lipid radicals are the source of these oxidized lipids, which are the initial key molecules in the lipid peroxidation chain reaction. However, owing to their extremely high reactivity and short half-life, an established detection technique is not available. Here, we propose a high-performance liquid chromatography fluorometry and high-resolution tandem mass spectrometry system combined with a fluorescent probe as a structural analysis method for lipid-derived radicals. We detected 132 lipid-derived radicals, including 111 new species, from five polyunsaturated fatty acids. In addition, a database was constructed for which the initial fatty acid could be determined using the radical structure. Further, 12 endogenous lipid-derived radicals were identified in carcinogen-induced liver cancer mouse models. Therefore, this method and its corresponding database will provide novel insights into mechanisms underlying the lipid peroxidation, including the associated inflammation and ferroptosis.

DOI： 10.1021/acs.analchem.0c00053

Language：English  

For the construction of metal-free magnetic resonance imaging (MRI) contrast agents, radical-based nanoparticles (RNPs) are promising materials because they allow the water-proton longitudinal relaxivity (r₁) to be enhanced not only by paramagnetic resonance effects but also by prolonging the rotational correlation times (τ_R). However, the τ_R effect is limited because the radical units are often located within the central hydrophobic core of oil-in-water (o/w) emulsions, resulting in a lack of water molecules surrounding the radical units. In this study, to construct supramolecular RNPs that have high r₁ values, we designed a liposome-type RNP in which the radical units are located at positions with sufficient surrounding water molecules. Using this strategy, PRO1 with a PROXYL framework was prepared by introducing hydrophilic groups on both sides of the radical unit. The RNP composed of PRO1 formed spherical nanoparticles approximately 100 nm in size and yielded a higher r₁ value (0.26 mM^-1 s^-1) compared to those of small radical species and similar supramolecular o/w emulsion-type nanoparticles (0.17 mM^-1 s^-1 in PRO2).

DOI： 10.1021/acs.langmuir.0c00610

Language：English  

Doxorubicin (DOX), a chemotherapeutic agent, induces a cardiotoxicity referred to as doxorubicin-induced cardiomyopathy (DIC). This cardiotoxicity often limits chemotherapy for malignancies and is associated with poor prognosis. However, the molecular mechanism underlying this cardiotoxicity is yet to be fully elucidated. Here, we show that DOX downregulated glutathione peroxidase 4 (GPx4) and induced excessive lipid peroxidation through DOX-Fe2+ complex in mitochondria, leading to mitochondria-dependent ferroptosis; we also show that mitochondria-dependent ferroptosis is a major cause of DOX cardiotoxicity. In DIC mice, the left ventricular ejection fraction was significantly impaired, and fibrosis and TUNEL+ cells were induced at day 14. Additionally, GPx4, an endogenous regulator of ferroptosis, was downregulated, accompanied by the accumulation of lipid peroxides, especially in mitochondria. These cardiac impairments were ameliorated in GPx4 Tg mice and exacerbated in GPx4 heterodeletion mice. In cultured cardiomyocytes, GPx4 overexpression or iron chelation targeting Fe2+ in mitochondria prevented DOX-induced ferroptosis, demonstrating that DOX triggered ferroptosis in mitochondria. Furthermore, concomitant inhibition of ferroptosis and apoptosis with ferrostatin-1 and zVAD-FMK fully prevented DOX-induced cardiomyocyte death. Our findings suggest that mitochondria-dependent ferroptosis plays a key role in progression of DIC and that ferroptosis is the major form of regulated cell death in DOX cardiotoxicity.

DOI： 10.1172/jci.insight.132747

Language：English   Publishing type：Research paper (scientific journal)  

AIMS: Aldehyde reductase (AKR1A) is involved in the synthesis of ascorbic acid (AsA) as well as the detoxification of aldehydes. AKR1A-/- (KO) mice produce about 10&#37; of the normal amounts of AsA compared to AKR1A+/+ (WT) mice. We investigated physiologic roles of AKR1A in running using the KO mice. MAIN METHODS: The KO mice were subjected to a treadmill test under either restricted AsA production or a sufficiency by supplementation and compared the results with those of WT mice. Contents of glucose, aspartate aminotransferase, AsA and free fatty acids in blood were measured. Glycogen contents were measured in the liver and skeletal muscle, and hepatic proteins were examined by immunoblot analyses. KEY FINDINGS: Running performance was higher in the KO mice than the WT mice irrespective of the AsA status. After the exercise period, blood glucose levels were decreased in the WT mice but were preserved in the KO mice. Liver glycogen levels were also consistently preserved in the KO mice after exercise. Free fatty acid levels tended to be originally high in blood plasma compared to those of the WT mice and were increased to similar extent in them. A key regulator of energy metabolism, PGC-1α, and the products of downstream target genes that encode for glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphatase, were constitutively at high levels in the KO mice. SIGNIFICANCE: The genetic ablation of AKR1A activates the PGC-1α pathway and spare glucose, which would consequently confer exercise endurance.

DOI： 10.1016/j.lfs.2020.117501

Language：English   Publishing type：Research paper (scientific journal)  

Lipid peroxidation (LPO) is reported to be involved in the pathogenesis of several oxidative diseases, and several therapeutic approaches using antioxidants have been proposed. LPO is thought to progress via a complicated series of multistep reactions suggesting that the activity of each antioxidant may be different, and depends on the reacting molecules. Hence, in this study, we evaluated the inhibitory mechanisms of several antioxidants toward arachidonic acid (AA) peroxidation induced by the azo initiator 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) or a lipid hydroperoxide, hydroperoxyoctadecadienoic acid (HpODE)/hemin. Edaravone, ferrostatin-1, TEMPO and trolox effectively inhibited the production of malondialdehyde (MDA) and several oxidised AAs generated in the AAPH-induced LPO because of their scavenging ability toward lipid peroxyl radicals. In contrast, ebselen and ferrostatin-1 showed strong antioxidative activity in the HpODE/hemin-induced peroxidation. Under this condition, ebselen and ferrostatin-1 were thought to reduce HpODE and its derived alkoxyl radicals to the corresponding lipid alcohols. In conclusion, we found that each antioxidant had different antioxidative activities that prevented the progression of LPO. We expect that these findings will contribute to the design of novel therapeutic strategies using an appropriate antioxidant targeted to each step of the development of oxidative stress diseases.

DOI： 10.1080/10715762.2020.1761020

Language：English  

For the construction of metal-free magnetic resonance imaging (MRI) contrast agents, radical-based nanoparticles (RNPs) are promising materials because they allow the water-proton longitudinal relaxivity (r1) to be enhanced not only by paramagnetic resonance effects but also by prolonging the rotational correlation times (τR). However, the τR effect is limited because the radical units are often located within the central hydrophobic core of oil-in-water (o/w) emulsions, resulting in a lack of water molecules surrounding the radical units. In this study, to construct supramolecular RNPs that have high r1 values, we designed a liposome-type RNP in which the radical units are located at positions with sufficient surrounding water molecules. Using this strategy, PRO1 with a PROXYL framework was prepared by introducing hydrophilic groups on both sides of the radical unit. The RNP composed of PRO1 formed spherical nanoparticles approximately 100 nm in size and yielded a higher r1 value (0.26 mM–1 s–1) compared to those of small radical species and similar supramolecular o/w emulsion-type nanoparticles (0.17 mM–1 s–1 in PRO2).

DOI： 10.1021/acs.langmuir.0c00610

Language：English   Publishing type：Research paper (scientific journal)  

Thus far, no accurate measurement technology has been developed to detect lipid alkyl radicals (lipid radicals), which cause lipid peroxidation. Therefore, we aimed to develop a nuclear medical imaging probe that can be taken up in the lipophilic site in cells such as biological membranes, by reacting specifically with the lipid radicals generated there. We designed and synthesized 4-(4-[125I]iodobenzamido)-2,2,6,6-tetramethylpiperidine-1-oxyl, which shows high reactivity to lipid radicals with a high radiochemical yield and purity. Intracellular retention was found to increase significantly when lipid radicals were produced.

DOI： 10.1021/acsmedchemlett.9b00416

Language：English  

Materials possessing electron spin can shorten the T₁ relaxation times in magnetic resonance imaging (MRI). For example, gadolinium (Gd) complexes with seven f-orbital electrons are widely used as contrast agents in clinical applications. However, Gd has severe potential side effects, and thus metal-free alternatives are needed. Toward this end, we synthesized seven NO radicals consisting of a dioxa-azaspiro[4.5]decane framework having various substituents, DAD-X (X = methyl, ethyl, n-propyl, c-propyl, vinyl, phenyl, and 2-pyridyl), that functioned as metal-free MRI contrast agents. The relationship between (i) water-proton relaxivity and log※P and (ii) reactivity for ascorbic acid and the spin density of the NO oxygen atom were established, which provided a basis for the rational design of practical metal-free contrast agents.

DOI： 10.1021/acsomega.9b03003

Language：English  

Background: Ferroptosis, nonapoptotic cell death mediated by free radical reactions and driven by the oxidative degradation of lipids, is a therapeutic target because of its role in organ damage, including AKI. Ferroptosis-causing radicals that are targeted by ferroptosis suppressors have not been unequivocally identified. Because certain cytochrome P450 substrate drugs can prevent lipid peroxidation via obscure mechanisms, we evaluated their antiferroptotic potential and used them to identify ferroptosis-causing radicals.

Methods: Using a cell-based assay, we screened cytochrome P450 substrate compounds to identify drugs with antiferroptotic activity and investigated the underlying mechanism. To evaluate radical-scavenging activity, we used electron paramagnetic resonance-spin trapping methods and a fluorescence probe for lipid radicals, NBD-Pen, that we had developed. We then assessed the therapeutic potency of these drugs in mouse models of cisplatin-induced AKI and LPS/galactosamine-induced liver injury.

Results: We identified various US Food and Drug Administration-approved drugs and hormones that have antiferroptotic properties, including rifampicin, promethazine, omeprazole, indole-3-carbinol, carvedilol, propranolol, estradiol, and thyroid hormones. The antiferroptotic drug effects were closely associated with the scavenging of lipid peroxyl radicals but not significantly related to interactions with other radicals. The elevated lipid peroxyl radical levels were associated with ferroptosis onset, and known ferroptosis suppressors, such as ferrostatin-1, also functioned as lipid peroxyl radical scavengers. The drugs exerted antiferroptotic activities in various cell types, including tubules, podocytes, and renal fibroblasts. Moreover, in mice, the drugs ameliorated AKI and liver injury, with suppression of tissue lipid peroxidation and decreased cell death.

Conclusions: Although elevated lipid peroxyl radical levels can trigger ferroptosis onset, some drugs that scavenge lipid peroxyl radicals can help control ferroptosis-related disorders, including AKI.

Keywords: Ferroptosis; Promethazine; Regulated cell death; Rifampicin; acute kidney injury; lipid peroxidation.

DOI： 10.1681/ASN.2019060570

Language：English   Publishing type：Research paper (scientific journal)  

Time courses of the redox status in the brains of mice after X-ray or carbon-ion beam irradiation were observed by magnetic resonance redox imaging (MRRI). The relationship between radiation-induced oxidative stress on the cerebral nervous system and the redox status in the brain was discussed. The mice were irradiated by 8-Gy X-ray or carbon-ion beam (C-beam) on their head under anesthesia. C-beam irradiation was performed at HIMAC (Heavy-Ion Medical Accelerator in Chiba, NIRS/QST, Chiba, Japan). MRRI measurements using a blood-brain-barrier-permeable nitroxyl contrast agent, MCP or TEMPOL, were performed using 7-T scanner at several different times, i.e., 5-10 h, 1, 2, 4, and 8 day(s) after irradiation. Decay rates of the nitroxyl-enhanced T1-weighted MR signals in the brains were estimated from MRRI data sets, and variation in the decay rates after irradiation was assessed. The variation in decay rates of MCP and TEMPOL after X-ray or C-beam irradiation was similar, but different variation patterns were observed between X-ray and C-beam. The apparent decay rate of both MCP and TEMPOL decreased due to the temporal reduction of blood flow in the brain several hours after X-ray and/or C-beam irradiation. After decreasing, the apparent decay rates of nitroxyl radicals in the brain gradually increased during the following days after X-ray irradiation or rapidly increased 1 day after C-beam irradiation. The sequential increase in nitroxyl decay rates may have been due to the oxidative atmosphere in the tissue due to ROS generation. X-ray and C-beam irradiation resulted in different redox responses, which may have been due to time-varying oxidative stress/injury, in the mouse brain. The C-beam irradiation effects were more acute and larger than those of X-ray irradiation.

DOI： 10.1016/j.freeradbiomed.2019.08.020

Language：English  

DOI： 10.3164/jcbn.19-41

Language：English  

DOI： 10.1080/10715762.2019.1660323

Language：English  

Superoxide dismutase 1 (SOD1) suppresses oxidative stress within cells by decreasing the levels of superoxide anions. A dysfunction of the ovary and/or an aberrant production of sex hormones are suspected causes for infertility in SOD1-knockout (KO) mice. We report on attempts to rescue the infertility in female KO mice by providing two antioxidants, ascorbic acid (AsA) and/or coenzyme Q10 (CoQ10), as supplements in the drinking water of the KO mice after weaning and on an investigation of their reproductive ability. On the first parturition, 80&#37; of the untreated KO mice produced smaller litter sizes compared to wild-type mice (average 2.8 vs 7.3 pups/mouse), and supplementing with these antioxidants failed to improve these litter sizes. However, in the second parturition of the KO mice, the parturition rate was increased from 18&#37; to 44-75&#37; as the result of the administration of antioxidants. While plasma levels of progesterone at 7.5 days of pregnancy were essentially the same between the WT and KO mice and were not changed by supplementation of these antioxidants, sizes of corpus luteum cells, which were smaller in the KO mouse ovaries after the first parturition, were significantly ameliorated in the KO mouse with the administration of the antioxidants. Moreover, the impaired vasculogenesis in uterus/placenta was also improved by AsA supplementation. We thus conclude that AsA and/or CoQ10 are involved in maintaining ovarian and uterus/placenta homeostasis against insults that are augmented during pregnancy and that their use might have positive effects in terms of improving female fertility.

DOI： 10.1093/biolre/ioz149.

Language：English   Publishing type：Research paper (scientific journal)  

AIMS: Repeated use of nonsteroidal anti-inflammatory drugs can induce changes in the redox status, including production of reactive oxygen species (ROS), but the specific details of these changes remain unknown. Overhauser-enhanced magnetic resonance imaging (OMRI) has been used in vivo to monitor the redox status in several diseases and map tissue oxygen concentrations. We monitored the intra- and extracellular redox status in the stomach of rats with indomethacin-induced gastric ulcers using OMRI and investigated the relationship with gastric mucosal damage. RESULTS: One hour after oral administration of indomethacin (30 mg/kg), OMRI measurements in the stomach were made following nitroxyl probe administration. OMRI with the membrane-permeable nitroxyl probe, 4-hydroxy-2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPOL), demonstrated a redox change toward oxidation, which was reversed by a membrane-permeable antioxidant. Conversely, imaging with the impermeable probe, 4-trimethylammonium-2,2,6,6-tetramethyl-piperidine-1-oxyl (CAT-1), demonstrated little redox change. Redox imbalance imaging of a live rat stomach with indomethacin-induced gastric ulcers was produced by dual imaging of 15N-labeled TEMPOL and 14N-labeled CAT-1, in addition to imaging with another membrane-permeable 15N-labeled probe, 3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (MC-PROXYL), and 14N-labeled CAT-1. Pretreatment with MC-PROXYL suppressed gastric mucosal damage, whereas pretreatment with CAT-1 did not suppress ulcer formation. INNOVATION: OMRI combined with a dual probe is a less invasive imaging technique for evaluation of intracellular ROS production contributing to the formation of gastric ulcers in the stomach of indomethacin-treated rats, which cannot be done with other methods. CONCLUSION: This method may be a very powerful tool for characterizing the pathogenesis of various diseases and may have medical applications.

DOI： 10.1089/ars.2017.7336

Language：English  

midazole-containing dipeptides (IDPs), such as carnosine and anserine, are found exclusively in various animal tissues, especially in the skeletal muscles and nerves. IDPs have antioxidant activity because of their metal-chelating and free radical-scavenging properties. However, the underlying mechanisms that would fully explain IDP antioxidant effects remain obscure. Here, using HPLC-electrospray ionization-tandem MS analyses, we comprehensively investigated carnosine and its related small peptides in the soluble fractions of mouse tissue homogenates and ubiquitously detected 2-oxo-histidine-containing dipeptides (2-oxo-IDPs) in all examined tissues. We noted enhanced production of the 2-oxo-IDPs in the brain of a mouse model of sepsis-associated encephalopathy. Moreover, in SH-SY5Y human neuroblastoma cells stably expressing carnosine synthase, H2O2 exposure resulted in the intracellular production of 2-oxo-carnosine, which was associated with significant inhibition of the H2O2 cytotoxicity. Notably, 2-oxo-carnosine showed a better antioxidant activity than endogenous antioxidants such as GSH and ascorbate. Mechanistic studies indicated that carnosine monooxygenation is mediated through the formation of a histidyl-imidazole radical, followed by the addition of molecular oxygen. Our findings reveal that 2-oxo-IDPs are metal-catalyzed oxidation products present in vivo and provide a revised paradigm for understanding the antioxidant effects of the IDPs.

DOI： 10.1074/jbc.RA118.006111.

Language：Others  

Non-invasive mapping of glutathione levels in mouse brains by in vivo electron paramagnetic resonance (EPR) imaging: Applied to a kindling mouse model.

DOI： 10.1016/j.neulet.2018.10.001

Other Link： 10.1016/j.neulet.2018.10.001

Language：Others  

Fluorescence Tumor-Imaging Using a Thermo-Responsive Molecule with an Emissive Aminoquinoline Derivative.

DOI： 10.3390/nano8100782

Language：English  

Aldehyde reductase (Akr1a) has been reported to be involved in detoxification of reactive aldehydes as well as in the synthesis of bioactive compounds such as ascorbic acid (AsA). Because Akr1a is expressed at high levels in the liver and is involved in xenobiotic metabolism, our objective was to investigate the hepato-protective role of Akr1a in a thioacetamide (TAA)-induced hepatotoxicity model using Akr1a-deficient (Akr1a-/-) mice. Wild-type (WT) and Akr1a-/- mice were injected intraperitoneally with TAA and the extent of liver injury in the acute phase was assessed. Intriguingly, the extent of TAA-induced liver damage was less in the Akr1a-/- mice than in the WT mice. Biomarkers for the ER stress-induced apoptosis pathway were markedly decreased in the livers of Akr1a-/- mice, whereas AsA levels in plasma did not change significantly in any of the mice. In the liver, TAA is converted to reactive metabolites such as TAA S-oxide and then to TAA S, S-dioxide via the action of CYP2E1. In Akr1a-/- mice, CYP2E1 activity was relatively lower than WT mice at the basal level, leading to reactive TAA metabolites being produced at lower levels after the TAA treatment. The levels of liver proteins that were modified with these metabolites were also lower in the Akr1a-/- mice than the WT mice after the TAA treatment. Furthermore, after a lethal dose of a TAA challenge, the WT mice all died within 36 h, whereas almost all of the Akr1a-/- mice survived. These collective results suggest that Akr1a-/- mice are resistant to TAA-induced liver injury, and it follows that the absence of Akr1a might modulate TAA bioactivation.

DOI： 10.1016/j.toxlet.2018.05.015

Language：Others  

Genotoxic Responses of Mitochondrial Oxygen Consumption Rate and Mitochondrial Semiquinone Radicals in Tumor Cells

DOI： 10.1007/s00723-018-1007-0

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1007/s00723-018-1035-9

Language：English   Publishing type：Research paper (scientific journal)  

Connexin 30 deficiency attenuates A2 astrocyte responses and induces severe neurodegeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride Parkinson's disease animal model.
BACKGROUND: The first pathology observed in Parkinson's disease (PD) is 'dying back' of striatal dopaminergic (DA) terminals. Connexin (Cx)30, an astrocytic gap junction protein, is upregulated in the striatum in PD, but its roles in neurodegeneration remain elusive. We investigated Cx30 function in an acute PD model by administering 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to wild-type (WT) and Cx30 knockout (KO) mice. METHODS: On days 1 and 7 after MPTP administration, we evaluated changes in astrocytic Cx30, Cx43, glial fibrillary acidic protein, and ionised calcium-binding adapter molecule 1 expression by immunostaining and biochemical analysis. Loss of DA neurons was evaluated by tyrosine hydroxylase immunostaining. Gene expression was analysed using A1, A2, pan-reactive astrocyte microarray gene sets, and M1, M2, and M1/M2 mixed microglial microarray gene sets. Real-time PCR and in situ hybridisation were performed to evaluate glial cell-derived neurotrophic factor (Gdnf) and S100a10 expression. Striatal GDNF protein levels were determined by enzyme-linked immunosorbent assay. RESULTS: MPTP treatment induced upregulation of Cx30 and Cx43 levels in the striatum of WT and KO mice. DA neuron loss was accelerated in Cx30 KO compared with WT mice after MPTP administration, despite no change in the striatal concentration of methyl-4-phenylpyridinium+. Astrogliosis in the striatum of Cx30 KO mice was attenuated by MPTP, whereas microglial activation was unaffected. Microarrays of the striatum showed reduced expression of pan-reactive and A2 astrocyte genes after MPTP treatment in Cx30 KO compared with WT mice, while M1, M2, and M1/M2 mixed microglial gene expression did not change. MPTP reduced the number of striatal astrocytes co-expressing Gdnf mRNA and S100β protein or S100a10 mRNA and S100β protein and also reduced the level of GDNF in the striatum of Cx30 KO compared with WT mice. CONCLUSIONS: These findings indicate that Cx30 plays critical roles in astrocyte neuroprotection in an MPTP PD model.

DOI： 10.1186/s12974-018-1251-0

Language：English   Publishing type：Research paper (scientific journal)  

Genotoxic Responses of Mitochondrial Oxygen Consumption Rate and Mitochondrial Semiquinone Radicals in Tumor Cells

DOI： 10.1007/s00723-018-1007-0

Language：English  

In the treatment with anticancer drugs, it is important to deliver an anticancer agent to target site of the tumor at an appropriate concentration. However, it is difficult to directly measure the distribution amount of the agent and effect of anticancer drug is evaluated using its tumor suppression effect. In this study, we report an approach to visualizing an anticancer agent distribution in tumor-bearing mouse model using Overhauser enhanced magnetic resonance imaging (OMRI). The agent, doxorubicin, is one of anthracycline anticancer drugs and can form a free radical at its quinone sites and could be visualized using OMRI. After direct injection into a tumor, doxorubicin free radical was successfully imaged in tumor-bearing mouse, demonstrating practical usefulness of OMRI in the study of pharmacodynamics of free radical compounds. Imaging of antitumor agent would be potentially useful as a guidance tool for image-guided-therapy of cancer local chemotherapy.

DOI： 10.1007/s00723-018-1004-3

Language：English   Publishing type：Research paper (scientific journal)  

Oxidative stress causes cell death and induces many kinds of disease, including liver disease. Nitroxides are known to react catalytically with free radicals. In this study, the cell protective activities of nitroxides were compared with those of other antioxidants. Nitroxides showed much greater inhibition of hydrogen peroxide-induced cell death than other antioxidants in a hepatic cell line and in primary hepatocytes. The intracellular oxidative stress level at 24 h after hydrogen peroxide stimulation was significantly decreased by nitroxides, but not by other antioxidants. To clarify the mechanism of cell protection by nitroxides, we investigated whether nitroxides inhibited DNA damage and mitogen-activated protein kinase pathway activation. We found that nitroxides reduced caspase3 activation and may have ultimately inhibited cell death. In conclusion, nitroxides are very useful for attenuating cell damage due to oxidative stress. Nitroxides are thus a potential therapeutic agent for oxidative stress-related diseases.

DOI： 10.3164/jcbn.17-60

Language：English  

Ulcerative colitis is characterized by colonic mucosal bleeding and ulceration, often with repeated active and remission stages. One factor in ulcerative colitis development is increased susceptibility to commensal bacteria and lipopolysaccharide (LPS). LPS activates macrophages to release nitric oxide (NO) through Toll-like receptor 4 (TLR4) signaling. However, whether NO is beneficial or detrimental to colitis remains controversial. In this study, we investigated whether NO enhances the development of colitis in mice treated with dextran sulfate sodium (DSS) and inflammation in cells treated with low-dose LPS. An NO donor, NOC18, induced colitis and increased CD14 protein and nitrotyrosine levels in colonic macrophages from mice treated with DSS for 7d (molecular weight: 5000). In the mouse peritoneal macrophage cell line RAW264.7 stimulated with 3ng/mL LPS, NO activated the CD14-TLR4-nuclear factor kappa B (NF-κB) axis. Low-dose LPS stimulation did not change the levels of signal transducer and activator of transcription (STAT) 3 phosphorylation, CD14, inducible NO synthase, interleukin (IL)-6, or NF-κB. In addition, low-dose LPS increased phosphorylation of src homology protein tyrosine phosphatase 2 (SHP2), a negative regulator of STAT3 phosphorylation. However, NO decreased SHP2 phosphorylation and significantly activated the downstream signaling molecules. NO increased SHP2 nitration in LPS-stimulated RAW264.7 cells and DSS-treated mice. These results indicate that SHP2 nitration in macrophages might be involved in activation of the CD14-TLR4-NF-κB axis through STAT3 signaling in mice with DSS-induced colitis.

DOI： 10.1248/bpb.b18-00558

Language：English   Publishing type：Research paper (scientific journal)  

Oxidized low density lipoprotein (Ox-LDL) is implicated in a variety of oxidative diseases. To clarify the mechanisms involved and facilitate the investigation of therapeutics, we previously developed a detection method for lipid-derived radicals using the fluorescent probe 2,2,6-trimethyl-6-pentyl-4-(4-nitrobenzo[1,2,5]oxadiazol7-ylamino)piperidine-1-oxyl (NBD-Pen). In this study, NBD-Pen was used to detect lipid-derived radicals in OxLDL from in vitro and in vivo samples using an iron overloaded mouse model. By following the timeline of lipid radical generation using this method, the iron overloaded mice could be successfully treated with the antioxidant Trolox, resulting in successful lowering of the plasma lipid peroxidation, aspartate transaminase and alanine transaminase levels. Furthermore, using a combination therapy of the chelating agent deferoxamine (DFX) and Trolox, liver injury and oxidative stress markers were also reduced in iron overloaded mice. The NBD-Pen method is highly sensitive as well as selective and is suitable for targeting minimally modified LDL compared with other existing methods.

DOI： 10.1016/j.freeradbiomed.2017.10.388

Language：English   Publishing type：Research paper (scientific journal)  

Feasibility of Magnetic Resonance Redox Imaging at Low Magnetic Field: Comparison at 1 T and 7 T
The effect of different static magnetic field strengths, 1 T or 7 T, on the quality of nitroxyl radical-based magnetic resonance redox imaging (MRRI) was examined. A stable nitroxyl radical, 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl (MC-PROXYL), was used as a T-1 contrast agent. Phantoms and animals were scanned at 1 T and 7 T using a similar gradient echo sequence. The quality of T-1-weighted images and susceptibility of T-1-weighted signals were compared. The nitroxyl radical-based T-1-weighted signal enhancement ratio was higher at 1 T compared with at 7 T when the identical phantom was scanned using a similar gradient echo sequence. The gradient echo scanning at 7 T was sensitive to movement and/or flux of the sample solution, which could result in the distortion of baseline T-1-weighted signals. No such wobbling of the signal was observed when the experiment was done at 1 T. The detection at the lower field is less affected by voltex flow in the sample, much stable T-1-weighted signal detection is available at the lower field. The visual characteristics of in vivo nitroxyl decay profiles were similar between the 1 T and 7 T experiments, except noises were large at 1 T. The correlation trends of in vivo decay constants among brain regions also similar between 1 T and 7 T experiments. Nitroxyl radical-based MRRI could be an adequate theranostic tool when performed on clinically popular low magnetic field MRI instruments.

Language：English   Publishing type：Research paper (scientific journal)  

Age-related macular degeneration (AMD) is the leading cause of blindness worldwide. Although the cause of AMD remains unknown, lipid peroxidation (LPO) end-products are critical molecules for its development. Herein, we report the imaging of lipid radicals, which are key factors in the LPO reaction, and therapeutic information using animal models

DOI： 10.1039/c7cc03387g.

Language：English   Publishing type：Research paper (scientific journal)  

It has recently been reported that radiation enhances mitochondrial energy metabolism in various tumor cell lines. To examine how this radiation -induced alteration in mitochondrial function influences tumor cell viability, Various lipophilic triphenylphosphonium (TPP+) cation derivatives and related compounds such as 4-hydroxy-2,2,6,6-tetramethyl-l-oxy-piperidin (Tempol) with TPP+ (named."Mito-") were designed to inhibit the mitochondrial electron transport chain. Mito-(CH2)io-Tempol (M10T) and its derivatives, Mito-(CH2)(5)-Tempol (M5T), Mito-(CH2)(10)-Tempol-Methyl (M10T-Me), Mito-C10H21 (M10), and C10H21-Tempol (10T), were prepared. In HeLa human cervical adenocarcinoma cells and A549 human lung carcinoma cells, the fractional uptake of the compound into mitochondria was highest among the TTP+ analogs conjugated with Tempol (M10T, M5T, and 10T). MlOT, M10T-Me, and M10 exhibited strong cytotoxicity and enhanced X-irradiation -induced reproductive cell death, while 10T and M5T did not. Furthermore, M10T, M10T-Me, and M10 decreased basal mitochondrial membrane potential and intracellular ATP. MIOT treatment inhibited X-ray -induced increases in ATP production. These results indicate that the TPP cation and a long hydrocarbon linker are essential for radiosensitization of tumor cells. The reduction in intracellular ATP by lipophilic TP13(+) is partly responsible for the observed radiosensitization. (C) 2017 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.canlet.2017.01.006

Language：English   Publishing type：Research paper (scientific journal)  

Glutathione (GSH) is the most abundant non-protein thiol that buffers reactive oxygen species in the brain. GSH does not reduce nitroxides directly, but in the presence of ascorbates, addition of GSH increases ascorbate-induced reduction of nitroxides. In this study, we used electron paramagnetic resonance (EPR) imaging and the nitroxide imaging probe, 3-methoxycarbonyl-2,2,5,5-tetramethyl-piperidine-1-oxyl (MCP), to non-invasively obtain spatially resolved redox data from mouse brains depleted of GSH with diethyl maleate compared to control. Based on the pharmacokinetics of the reduction reaction of MCP in the mouse heads, the pixel-based rate constant of its reduction reaction was calculated as an index of the redox status in vivo and mapped as a "redox map". The obtained redox maps from control and GSH-depleted mouse brains showed a clear change in the brain redox status, which was due to the decreased levels of GSH in brains as measured by a biochemical assay. We observed a linear relationship between the reduction rate constant of MCP and the level of GSH for both control and GSH-depleted mouse brains. Using this relationship, the GSH level in the brain can be estimated from the redox map obtained with EPR imaging. (C) 2017 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2017.02.134

Language：English   Publishing type：Research paper (scientific journal)  

Aldehyde reductase (Akr1a) is involved in the synthesis of ascorbic acid (AsA) which may play a role in social behavior. In the current study, we performed analyses on Akrla-deficient (Akr1a(-/-)) mice that synthesize about 10% as much AsA as wild-type mice from the viewpoint of intermale aggression. The use of the resident-intruder test revealed that the Akr1a(-/-) mice exhibited more aggressive phenotypes than wild-type control mice. Unexpectedly, however, the oral administration of additional AsA failed to reduce the aggressive behavior of Akr1a(-/-) mice, suggesting that the heightened aggression was independent of AsA biosynthesis. The findings also show that the plasma levels of corticosterone, but not serotonin and testosterone, were increased in the absence of Akr1a in mice, suggesting that the mice were highly stressed. These results suggest that Akr1a might be involved in the metabolism of steroids and other carbonyl-containing compounds and, hence, the absence of Akr1a results in heightened aggression via a malfunction in a metabolic pathway. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbr.2016.11.038

Language：English   Publishing type：Research paper (scientific journal)  

It has recently been reported that radiation enhances mitochondrial energy metabolism in various tumor cell lines. To examine how this radiation-induced alteration in mitochondrial function influences tumor cell viability, various lipophilic triphenylphosphonium (TPP+) cation derivatives and related compounds such as 4-hydroxy-2,2,6,6-tetramethyl-1-oxy-piperidin (Tempol) with TPP+ (named “Mito-”) were designed to inhibit the mitochondrial electron transport chain. Mito-(CH2)10-Tempol (M10T) and its derivatives, Mito-(CH2)5-Tempol (M5T), Mito-(CH2)10-Tempol-Methyl (M10T-Me), Mito-C10H21 (M10), and C10H21-Tempol (10T), were prepared. In HeLa human cervical adenocarcinoma cells and A549 human lung carcinoma cells, the fractional uptake of the compound into mitochondria was highest among the TTP+ analogs conjugated with Tempol (M10T, M5T, and 10T). M10T, M10T-Me, and M10 exhibited strong cytotoxicity and enhanced X-irradiation-induced reproductive cell death, while 10T and M5T did not. Furthermore, M10T, M10T-Me, and M10 decreased basal mitochondrial membrane potential and intracellular ATP. M10T treatment inhibited X-ray-induced increases in ATP production. These results indicate that the TPP cation and a long hydrocarbon linker are essential for radiosensitization of tumor cells. The reduction in intracellular ATP by lipophilic TPP+ is partly responsible for the observed radiosensitization.

DOI： 10

Other Link： http://www.sciencedirect.com/science/article/pii/S0304383517300289

Language：Others  

Ablation of aldehyde reductase aggravates carbon tetrachloride-induced acute hepatic injury involving oxidative stress and endoplasmic reticulum stress.

DOI： 10.1016/j.bbrc.2016.08.022

Language：English   Publishing type：Research paper (scientific journal)  

Purpose: The detailed in vivo T-1-weighted contrasting abilities.. of nitroxyl contrast agents. which have been used as redo)( responsive contrast agents in several magnetic resonance based imaging modalities. in mouse brain were investigated. Methods: Distribution and pharmacokinetics of five types of five-membered-ring nitroxyl radical compound were compared using T-1-weighted MRI. Results: The blood-brain barrier (BBB)-impermeable 3-carboxy-2,2,5.5-tetramethylpyrrolicline-N-oxyl (CxP) could not be distributed in the brain. The slightly lipophilic 3-carbamoy12,2,5,5-tetramethylpyrrolidine oxyl (CmP) showed slight distribution only in the ventricle, but not in the medulla and cortex. The amphiphilic 3-methoxy-carbonyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl (MCP) had good initial uniform distribution in the brain and showed typical 2-phase signal decay profiles. A brain-seeking nitroxyl probe, acetoxyrnethy1-2,2.5,5-tetrarnethylpyrrolidine-N-oxyl-3-carboxylate (CxP-AM), showed an accumulating phase, and then its accumulation was maintained in the medulla and ventricle regions, but not in the cortex. The lipophilic 4-(N-methyl piperidine)-2,2.5,5-tetrarnethylpyrroline-N-oxyl (23c) was well distributed in the cortex and medulla, but slightly in the ventricle, and showed relatively rapid linear signal decay. Conclusion: Nitroxyl contrast agents equipped with a suitable lipophilic substitution group could be BBB-permeable functional contrast agents. MR redox imaging, which can estimate not only the redox characteristics but also the detailed distribution of the contrast agents, is a good candidate for a theranostic tool. (C)2015 Wiley Periodicals, Inc.

DOI： 10.1002/mrm.25918

Language：English   Publishing type：Research paper (scientific journal)  

Lipids and their metabolites are easily oxidized in chain reactions initiated by lipid radicals, forming lipid peroxidation products that include the electrophiles 4-hydroxynonenal and malondialdehyde. These markers can bind cellular macromolecules, causing inflammation, apoptosis and other damage. Methods to detect and neutralize the initiating radicals would provide insights into disease mechanisms and new therapeutic approaches. We describe the first high-sensitivity, specific fluorescence probe for lipid radicals, 2,2,6-trimethyl-4-(4-nitrobenzo[1,2,5]oxadiazol-7-ylamino)-6-pentylpiperidine-1-oxyl (NBD-Pen). NBD-Pen directly detected lipid radicals in living cells by turn-on fluorescence. In a rat model of hepatic carcinoma induced by diethylnitrosamine (DEN), NBD-Pen detected lipid radical generation within 1 h of DEN administration. The lipid radical scavenging moiety of NBD-Pen decreased inflammation, apoptosis and oxidative stress markers at 24 h after DEN, and liver tumor development at 12 weeks. Thus, we have developed a novel fluorescence probe that provides imaging information about lipid radical generation and potential therapeutic benefits in vivo.

DOI： 10.1038/NCHEMBIO.2105

Language：English   Publishing type：Research paper (scientific journal)  

Continuous energy conversion is controlled by reduction-oxidation (redox) processes. NAD(+) and NADH represent an important redox couple in energy metabolism. 4-Hydroxy-2,2,6,6-tetramethylpiperidine-Noxyl (TEMPOL) is a redox-cycling nitroxide that promotes the scavenging of several reactive oxygen species (ROS) and is reduced to hydroxylamine by NADH. TEMPOL is also involved in NAD(+) production in the ascorbic acid-glutathione redox cycle. We utilized the chemical properties of TEMPOL to investigate the effects of antioxidants and NAD(+)/NADH modulators on the metabolic imbalance in obese mice. Increases in the NAD(+)/NADH ratio by TEMPOL ameliorated the metabolic imbalance when combined with a dietary intervention, changing from a high-fat diet to a normal diet. Plasma levels of the superoxide marker dihydroethidium were higher in mice receiving the dietary intervention compared with a control diet, but were normalized with TEMPOL consumption. These findings provide novel insights into redox regulation in obesity. (C) 2016 Published by Elsevier B.V.

DOI： 10.1016/j.redox.2016.02.007

Language：English   Publishing type：Research paper (scientific journal)  

Aldehyde reductase (AKR1A) plays a role in the biosynthesis of ascorbic acid (AsA), and AKR1A-deficient mice produce about 10-15% of the AsA that is produced by wild-type mice. We found that acetaminophen (AAP) hepatotoxicity was aggravated in AKR1A-deficient mice. The pre-administration of AsA in the drinking water markedly ameliorated the AAP hepatotoxicity in the AKR1A-deficient mice. Treatment of the mice with AAP decreased both glutathione and AsA levels in the liver in the early phase after AAP administration, and an AsA deficiency delayed the recovery of the glutathione content in the healing phase. While in cysteine supply systems; a neutral amino acid transporter ASCT1, a cystine transporter xCT, enzymes for the transsulfuration pathway, and autophagy markers, were all elevated in the liver as the result of the AAP treatment, the AsA deficiency suppressed their induction. Thus, AsA appeared to exert a protective effect against AAP hepatotoxicity by ameliorating the supply of cysteine that is available for glutathione synthesis as a whole. Because some drugs produce reactive oxygen species, resulting in the consumption of glutathione during the metabolic process, the intake of sufficient amounts of AsA would be beneficial for protecting against the hepatic damage caused by such drugs. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.abb.2016.06.004

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10

Language：English   Publishing type：Research paper (scientific journal)  

Aims. Recently various dipeptidyl peptidase-4 (DPP-4) inhibitors have emerged because of their high effectiveness and safety. In spite of their common effect of DPP-4 inhibition, the chemical structures are diverse. Here we show that the structure of teneligliptin, a novel DPP-4 inhibitor, has a scavenging activity on hydroxyl radical (center dot OH).
Methods. center dot OH and superoxide (O-2(-)) were detected by electron spin resonance (ESR) spectroscopy. center dot OH and O-2(-) were generated in vitro by the Fenton reaction and a hypoxanthine-xanthine oxidase system, respectively. The level of free radicals was estimated from the ESR signal intensity. The product via teneligliptin and center dot OH reaction was identified by thin layer chromatography and mass spectrometry analysis. In vivo effect was also evaluated using DPP-4 deficient rats with streptozotocin-induced diabetes.
Results. ESR spectroscopy analysis showed that teneligliptin did not scavenge O-2(-), but scavenged center dot OH in a dose dependent manner. Its activity was greater than that of glutathione. The reaction product appeared to have an oxygen-atom added structure to that of teneligliptin, which was identical to the most abundant metabolite of teneligliptin in human plasma. Furthermore, using DPP-4 deficient rat, teneligliptin did not affect plasma glucose levels or body weight, but normalized increased levels of 8-hydroxy-2'-deoxyguanosine in urine, kidney and aorta of diabetic rats, supporting that teneligliptin may have a center dot OH scavenging activity in vivo independently of DPP-4 inhibition.
Conclusions. Teneligliptin is not only effective as DPP-4 inhibitor, but may also be beneficial as center dot OH scavenger, which may be useful in the prevention of diabetic complications. (C) 2015 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.metabol.2015.10.030

Language：English   Publishing type：Research paper (scientific journal)  

The reactive species generated in aqueous 3,3',5,5'-tetrabromobisphenol A (TBBPA)/humic acid (HA) suspensions above the TBBPA pKa (similar to 7.4), under various light-irradiation conditions, namely ambient and ultraviolet light, were investigated using electron paramagnetic resonance (EPR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS). We confirmed that singlet oxygen and OH radicals are the key reactive oxygen species generated at wavelengths greater than 400 and 300 nm, respectively. The amount of 2,6-dibromo-p-benzosemiquinone anion radicals (2,6-DBSQ(center dot-)) formed under irradiation at 400 nm increased linearly with respect to irradiation time; the initial reaction rate was 7.03 x 10(-9) mol g(-1) HA s(-1). The rate increased with increasing pH and light intensity. LC-MS and EPR spectroscopy showed that tribromohydroxybisphenol A was formed under irradiation at 300 nm via reaction of OH radicals with TBBPA. This study, for the first time, shows that the main byproducts formed during irradiation at wavelengths above 300 nm are 2,6-DBST center dot- and tribromohydroxybisphenol A, generated from singlet oxygen (O-1(2)) and OH radicals, respectively. Photodecomposition of TBBPA in the environment may occur by formation of O-1(2) and OH radicals. (C) 2015 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.chemosphere.2015.12.072

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10

Language：English   Publishing type：Research paper (scientific journal)  

Ascorbic acid is an important antioxidant that plays an essential role in the biosynthesis of numerous bioactive substances. The detection of ascorbic acid has traditionally been achieved using high-performance liquid chromatography and absorption spectrophotometry assays. However, the development of fluorescence probes for this purpose is highly desired because they provide a much more convenient and highly sensitive technique for the detection of this material. OFF-ON-type fluorescent probes have been developed for the detection of non-fluorescent compounds. Photo-induced electron transfer and fluorescence resonance energy transfer are the two main fluorescence quenching mechanisms for the detection of ascorbic acid, and several fluorescence probes have been reported based on redox-responsive metals and quantum dots. Profluorescent nitroxide compounds have also been developed as non-metal organic fluorescence probes for ascorbic acid. These nitroxide systems have a stable unpaired electron and can therefore react with ascorbic acid and a strong fluorescence quencher. Furthermore, recent synthetic advances have allowed for the synthesis of alpha-substituted nitroxides with varying levels of reactivity towards ascorbic acid. In this review, we have discussed the design strategies used for the preparation of fluorescent probes for ascorbic acid, with particular emphasis on profluorescent nitroxides, which are unique radical-based redox-active fluorescent probes.

DOI： 10.3164/jcbn.15-105

Language：English   Publishing type：Research paper (scientific journal)  

Much evidence supports the idea that oxidative stress is involved in the pathogenesis of epilepsy, and therapeutic interventions with antioxidants are expected as adjunct antiepileptic therapy. The aims of this study were to non-invasively obtain spatially resolved redox data from control and pentylenetetrazole (PTZ)-induced kindled mouse brains by electron paramagnetic resonance (EPR) imaging and to visualize the brain regions that are sensitive to oxidative damage. After infusion of the redox-sensitive imaging probe 3-methoxycarbony1-2,2,5,5-tetramethyl-piperidine-1-oxyl (MCP), a series of EPR images of PTZ-induced mouse heads were measured. Based on the pharmacokinetics of the reduction reaction of MCP in the mouse heads, the pixel-based rate constant of its reduction reaction was calculated as an index of redox status in vivo and mapped as a redox map. The obtained redox map showed heterogeneity in the redox status in PTZ-induced mouse brains compared with control. The co-registered image of the redox map and magnetic resonance imaging (MRI) for both control and PTZ-induced mice showed a clear change in the redox status around the hippocampus after PTZ. To examine the role of antioxidants on the brain redox status, the levels of antioxidants were measured in brain tissues of control and PTZ-induced mice. Significantly lower concentrations of glutathione in the hippocampus of PTZ-kindled mice were detected compared with control. From the results of both EPR imaging and the biochemical assay, the hippocampus was found to be susceptible to oxidative damage in the PTZ-induced animal model of epilepsy. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.neulet.2015.10.008

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Background
Mitochondrial DNA (mtDNA) copy number decreases in animal and human heart failure (HF), yet its role in cardiomyocytes remains to be elucidated. Thus, we investigated the cardioprotective function of increased mtDNA copy number resulting from the overexpression of human transcription factor A of mitochondria (TFAM) or Twinkle helicase in volume overload (VO)-induced HF.
Methods and Results
Two strains of transgenic (TG) mice, one overexpressing TFAM and the other overexpressing Twinkle helicase, exhibit an approximately 2-fold equivalent increase in mtDNA copy number in heart. These TG mice display similar attenuations in eccentric hypertrophy and improved cardiac function compared to wild-type (WT) mice without any deterioration of mitochondrial enzymatic activities in response to VO, which was accompanied by a reduction in matrix-metalloproteinase (MMP) activity and reactive oxygen species after 8 weeks of VO. Moreover, acute VO-induced MMP-2 and MMP-9 upregulation was also suppressed at 24 h in both TG mice. In isolated rat cardiomyocytes, mitochondrial reactive oxygen species (mitoROS) upregulated MMP-2 and MMP-9 expression, and human TFAM (hTFAM) overexpression suppressed mitoROS and their upregulation. Additionally, mitoROS were equally suppressed in H9c2 rat cardiomyoblasts that overexpress hTFAM or rat Twinkle, both of which exhibit increased mtDNA copy number. Furthermore, mitoROS and mitochondrial protein oxidation from both TG mice were suppressed compared to WT mice.
Conclusions
The overexpression of TFAM or Twinkle results in increased mtDNA copy number and facilitates cardioprotection associated with limited mitochondrial oxidative stress. Our findings suggest that increasing mtDNA copy number could be a useful therapeutic strategy to target mitoROS in HF.

DOI： 10.1371/journal.pone.0119687

Language：Others   Publishing type：Research paper (scientific journal)  

Electron spin resonance (ESR) studies were carried out for 14N-labeled deuterated 3-methoxy-carbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (MC-PROXYL) and 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidin-1-oxyl (carboxy-PROXYL) in pure water and various concentrations of liposomal solutions by using 300 MHz ESR spectrometer. The ESR parameters such as the line width, hyperfine coupling constant, rotational correlation time, g-factor, partition parameter and permeability were reported for the samples. The line width broadening was observed for MC-PROXYL and carboxy-PROXYL in liposomal solution. The hyperfine coupling constant was observed for both nitroxyl spin probes. The permeable and impermeable nature of nitroxyl spin probes was demonstrated. The rotational correlation time increases with increasing concentration of liposome. The partition parameter increases with increasing concentration of liposome for MC-PROXYL, which indicates that the nitroxyl spin probes diffuse into lipid membrane. The permeability value decreases with increasing concentration of liposome, which reveals an increase in membrane permeability. The peaks corresponding to the lipid phase were observed for MC-PROXYL in liposomal solution, but not resolved for carboxy-PROXYL. These results confirm the permeable and impermeable nature of nitroxyl spin probes.

Language：English   Publishing type：Research paper (scientific journal)  

In response to sustained damage to a kidney, fibrosis that can be characterized as the deposition of a collagenous matrix occurs and consequently causes chronic kidney failure. Because most animals used in experiments synthesize ascorbic acid (AsA) from glucose, the roles of AsA in fibrotic kidney diseases are largely unknown. Unilateral ureteric obstruction (UUO) mimics the complex pathophysiology of chronic obstructive nephropathy and is an ideal model for the investigation of the roles of AsA in kidney failure. We examined the impact of a deficiency of Akr1a, a gene that encodes aldehyde reductase and is responsible for the production of AsA, on fibrotic damage caused by UUO in mice. Oxidatively modified DNA was elevated in wild-type and Akr1a-deficient kidneys as a result of UUO to a similar extent, and was only slightly suppressed by the administration of AsA. Even though Akr1a-deficient mice could produce only about 10&#37; of the AsA produced by wild-type mice, no difference was observed in collagen I synthesis under pathological conditions. The data implied either a low demand for AsA or the presence of another electron donor for collagen I production in the mouse kidney. Next, we attempted to elucidate the potential causes for oxidative damage in kidney cells during the fibrotic change. We found decreases in mitochondrial proteins, particularly in electron transport complexes, at the initial stage of the kidney fibrosis. The data imply that a dysfunction of the mitochondria leads to an elevation of ROS, which results in kidney fibrosis by stimulating cellular transformation to myofibroblasts.

DOI： 10.3109/10715762.2014.915031

Language：English   Publishing type：Research paper (scientific journal)  

Aldehyde reductase (AKR1A), a member of the aldo-keto reductase superfamily, suppresses diabetic complications via a reduction in metabolic intermediates; it also plays a role in ascorbic acid biosynthesis in mice. Because primates cannot synthesize ascorbic acid, a principle role of AKR1A appears to be the reductive detoxification of aldehydes. In this study, we isolated and immortalized mouse embryonic fibroblasts (MEFs) from wild-type (WT) and human Akr1a-transgenic (Tg) mice and used them to investigate the potential roles of AKR1A under culture conditions. Tg MEFs showed higher methylglyoxal- and acrolein-reducing activities than WT MEFs and also were more resistant to cytotoxicity. Enzymatic analyses of purified rat AKR1A showed that the efficiency of the acrolein reduction was about 20&#37; that of glyceraldehyde. Ascorbic acid levels were quite low in the MEFs, and while the administration of ascorbic acid to the cells increased the intracellular levels of ascorbic acid, it had no affect on the resistance to acrolein. Endoplasmic reticulum stress and protein carbonylation induced by acrolein treatment were less evident in Tg MEFs than in WT MEFs. These data collectively indicate that one of the principle roles of AKR1A in primates is the reductive detoxification of aldehydes, notably acrolein, and protection from its detrimental effects.

DOI： 10.1016/j.bbrc.2014.08.072

Language：English   Publishing type：Research paper (scientific journal)  

Ulcerative colitis is an inflammatory bowel disease characterized by acute inflammation, ulceration, and bleeding of the colonic mucosa. Its cause remains unknown. Increases in adhesion molecules in vascular endothelium, and activated neutrophils releasing injurious molecules such as reactive oxygen species, are reportedly associated with the pathogenesis of dextran sodium sulfate (DSS)-induced colitis. Nitric oxide (NO) production derived from inducible NO synthase (iNOS) via activation of nuclear factor kappa B (NF-kappa B) has been reported. It is also reported that stimulation of Toll-like receptor 4 (TLR4) by lipopolysaccharide can activate NF-kappa B. In this study, we investigated the involvement of NO production in activation of the TLR4/NF-kappa B signaling pathway in mice with DSS-induced colitis. The addition of 5&#37; DSS to the drinking water of male ICR mice resulted in increases in TLR4 protein in colon tissue and NF-kappa B p65 subunit in the nuclear fraction on day 3, increases in colonic tumor necrosis factor-alpha on day 4, and increases in P-selectin, intercellular adhesion molecule-1, NO2-/NO3-, and nitrotyrosine in colonic mucosa on day 5. These activated inflammatory mediators and pathology of colitis were completely suppressed by treatment with a NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, as well as an iNOS inhibitor, aminoguanidine. Conversely, a NO-releasing compound, NOC-18, increased TLR4 levels and nuclear translocation of NF-kappa B p65 and exacerbated mucosal damage induced by DSS challenge. These data suggest that increases in TLR4 expression induced by drinking DSS-treated water might be directly or indirectly associated with NO overproduction. (C) 2014 Published by Elsevier Inc.

DOI： 10.1016/j.freeradbiomed.2014.06.020

Language：English   Publishing type：Research paper (scientific journal)  

Dendritic poly(L-lysine) polymers were modified with stable nitroxyl radicals through the substrate peptides of a protease specifically expressed in tumors. Cleavage of the peptides by the protease affected the Overhauser-enhanced MRI (OMRI) signal from the radicals. This system could be the basis of a functional tumor-imaging technique by OMRI.

DOI： 10.1246/cl.140209

Language：English   Publishing type：Research paper (scientific journal)  

Oxidative stress is associated with both healthy aging and age-related disease states. In connection with oxidative stress, immunity is also a major component as a result of the chronic, low-grade inflammation associated with the development of tissue aging. Here we show that long-term treatment with the antioxidant tempol extends life-span in mice. Tempol-treated mice exhibited a reduction in mortality at 20 months. Tempol drinking did not have any effect on body weight, amount of visceral adipose tissue, or plasma biochemical parameters in aged mice. Body temperature of aged control mice (which drank only water) was significantly lower than young mice, but this reduction of body temperature was partially restored in aged mice which drank tempol. Plasma thiobarbituric acid-reactive substances and C-reactive protein were significantly increased in the control aged mice compared with young mice, but levels of both were normalized by tempo! drinking. One of the endogenous antioxidants, ascorbic acid, was significantly increased in the plasma of mice which consumed tempol. The proportion of CD4 lymphocytes in the blood of aged tempol-treated mice was partially increased in comparison to aged control mice. These results suggest that the reduction of mortality by tempol is due to amelioration of chronic inflammation and improved function of the immune system through antioxidant effects.

DOI： 10.3164/jcbn.14-4

Language：English  

DOI： 10.1016/j.freeradbiomed.2014.02.011

Language：English   Publishing type：Research paper (scientific journal)  

Aims: Aldehyde reductase (AKR1A), a member of the aldo-lceto reductase superfamily, is highly expressed in the liver and is involved in both the detoxification of carbonyl compounds and ascorbic acid biosynthesis. By comparison with wild-type mice, Akr1a-knockout (Akr1a(-/-)) mice and human Akr1a-transgenic (Akr1a(tg/+)) mice experience different anesthetic actions from pentobarbital-prolonged in Akr1a-knockout (Akr1a(-/-)) mice and shortened in human Ala1a-transgenic (Akr1a(tg/+)) mice.
Main methods: We investigated this alteration in the anesthetic efficacy of pentobarbital in Akrl a genetically modified mice.
Key findings: Neither the cytosolic protein of wild-type mouse liver nor purified rat AKR1A directly reduced pentobarbital. Ascorbic acid administration neutralized the prolonged duration of the loss of the righting reflex (LORR) in Akr1a(-/-) mice, but preincubation of pentobarbital with ascorbic acid prior to administration did not change the anesthetic effect. Those results indicated that ascorbic acid does not directly reduce pentobarbital. Enzymatic activities and levels of the proteins of some cytochrome P450s that make up a potent detoxification system for pentobarbital showed no changes in the genetically modified mice examined. Thus, ascorbic acid also had no effect on the detoxification system in the liver. The prolonged duration of LORR in the Akr1a(-/-) mice caused by pentobarbital and the neutralization of the anesthetic effect by ascorbic acid together with other results imply that ascorbic acid alters the responses of the neuronal system to anesthetics.
Significance: Pentobarbital action is increased under conditions of ascorbic acid deficiency, and this may have to be taken into account when anesthetizing malnourished patients. (C) 2013 Published by Elsevier Inc.

DOI： 10.1016/j.lfs.2013.12.004

Language：Others  

Non-invasive Redox Imaging of Cisplatin-Induced Nephrotoxicity Using Overhauser-MRI

DOI： 10.1016/j.freeradbiomed.2013.10.707

Language：English   Publishing type：Research paper (scientific journal)  

Parkinson's disease (PD) is a common neurodegenerative disorder that is characterized by the degeneration of dopaminergic neurons in the substantia nigra and dopamine depletion in the striatum. Although the motor symptoms are still regarded as the main problem, non-motor symptoms in PD also markedly impair the quality of life. Several non-motor symptoms, such as sleep disturbances and depression, are suggested to be implicated in the alteration in circadian clock function. In this study, we investigated circadian disruption and the mechanism in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. MPTP-treated mice exhibited altered 24-h rhythms in body temperature and locomotor activity. In addition, MPTP treatment also affected the circadian clock system at the genetic level. The exposure of human neuroblastoma cells (SH-SY5Y) to 1-metyl-4-phenylpyridinium (MPP+) increased or decreased the mRNA levels of several clock genes in a dose-dependent manner. MPP+-induced changes in clock genes expression were reversed by Compound C, an inhibitor of AMP-activated protein kinase (AMPK). Most importantly, addition of ATP to the drinking water of MPTP-treated mice attenuated neurodegeneration in dopaminergic neurons, suppressed AMPK activation and prevented circadian disruption. The present findings suggest that the activation of AMPK caused circadian dysfunction, and ATP may be a novel therapeutic strategy based on the molecular clock in PD.

DOI： 10.1007/s12017-012-8214-x

Language：English   Publishing type：Research paper (scientific journal)  

Nitroxides have recently been used as redox-sensitive contrast agents for both MRI and EPR imaging. However, the rapid in vivo reduction in paramagnetism of nitroxides due to reductants such as ascorbic acid (AsA) has limited their use as contrast agents. This study developed a formulation of a newly synthesized AsA-resistive nitroxide (2,2,6,6-tetraethylpiperidine-4-one-1-oxyl (TEEPONE)) with a lipid emulsion system and examined the in vivo stability of TEEPONE by magnetic resonance imaging (MRI). MRI of mouse heads after administration of TEEPONE clearly indicated that TEEPONE has a remarkable in vivo stability and is a blood-brain barrier (BBB) permeable nitroxide. MRI also showed that TEEPONE is preferentially localized in the mouse brain. The distribution of TEEPONE in the mouse head can be controlled by the lipid content in the emulsion system used to solubilize TEEPONE.

DOI： 10.1016/j.neulet.2013.04.044

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1002/ajoc.201300011

Language：English   Publishing type：Research paper (scientific journal)  

Electron spin resonance (ESR) studies were carried out for 2 mM N-14-labeled deuterated 3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (MC-PROXYL) and 3-carboxy-2,2,5,5,-tetramethyl-pyrrolidin-1-oxyl (carboxy-PROXYL) in pure water and various concentrations of liposomal solution by using an L-band ESR spectrometer. The ESR parameters, such as the line width, hyperfine coupling constant, g-factor, rotational correlation time and partition parameter, were reported for the samples. The changes in the line width were observed for N-14-labeled deuterated MC-PROXYL and carboxy-PROXYL in liposomal solution. The hyperfine coupling constant was observed for both nitroxyl spin probes. The permeable and impermeable nature of nitroxyl radicals was demonstrated using the ESR L-band spectra. The rotational correlation time increases with increasing concentration of liposome. The partition parameter for N-14-labeled deuterated MC-PROXYL in liposomal solution increases with increasing concentration of liposome, which reveals that the nitroxyl spin probe permeates into lipid membrane. The lipid peaks were observed for 2 mM N-14-labeled deuterated MC-PROXYL in 200, 300 and 400 mM liposomal concentration. The lipid peaks were not observed for N-14-labeled deuterated carboxy-PROXYL. These results indicate the permeable and impermeable nature of N-14-labeled deuterated nitroxyl spin probe.

DOI： 10.1007/s00723-012-0383-0

Language：English   Publishing type：Research paper (scientific journal)  

Nitric oxide (NO) is thought to be a key molecule in the progression of ulcerative colitis and experimental colitis induced by dextran sodium sulfate (DSS). However, the detrimental effect of DSS-induced NO production on the colonic mucosa is incompletely understood. Increases in the expression of adhesion molecules in the vascular endothelium and activated neutrophils (thereby releasing injurious molecules such as reactive oxygen species) are reportedly associated with the pathogenesis of DSS-induced colitis. We investigated if the detrimental effect of NO production on the colonic mucosa was attributable to the activation of neutrophil infiltration by NO in mice with DSS-induced colitis. NO2-/NO3- content in the middle and distal colon was increased on days 5 and 7, but alterations in the proximal colon were not observed. Myeloperoxidase (MPO) activity and expression of P-selectin and intercellular adhesion molecule-1 (ICAM-1) were significantly increased in the entire colon, whereas TNF-alpha levels were significantly increased only in the middle and distal colon on day 7. The pathology of colitis and increases in colonic MPO activity, P-selectin, ICAM-1, and TNF-alpha levels were suppressed by the inducible NO synthase (iNOS)-specific inhibitor aminoguanidine and NO scavenger c-PTIO, whereas all but TNF-alpha levels were increased by the non-specific NOS inhibitor L-NAME. These findings suggest that iNOS-derived NO increases TNF-alpha levels in the middle and distal colon and increased TNF-alpha levels induce expression of P-selectin and ICAM-1, thereby promoting the infiltration of activated neutrophils, which leads to damage to colonic tissue.

DOI： 10.3109/10715762.2012.732698

Language：English   Publishing type：Research paper (scientific journal)  

Ascorbic acid is a small-molecule reductant with multiple functions in vivo. Reducing ascorbic acid intake leads to a lack of hydroxylation of prolines and lysines, causing a looser triple helix and resulting in scurvy. Ascorbic acid also acts as an antioxidant to prevent oxidative stress. Because ascorbic acid is related to disease states, rapid and convenient detection of ascorbic acid should be useful in diagnosis. Nitroxide is reduced to the corresponding hydroxylamine by ascorbic acid and a sensitive and novel approach to its detection employs covalent coupling of nitroxide with a fluorophore, leading to intramolecular quenching of fluorescence emission by electron-exchange interactions. Here, we developed a new fluorophore-nitroxide probe. Naph-DiFy nitroxide, for ascorbic acid. Naph-DiFy nitroxide rapidly reacted with ascorbic acid and showed fluorescence enhancement, but not in response to other reductants or reactive oxygen species. To confirm the practical usefulness of the fluorophore-nitroxide probe, we demonstrated the use of Naph-DiFy nitroxide for the measurement of ascorbic acid in the plasma of osteogenic disorder Shionogi rats when fed an ascorbic acid-deficient diet. The results suggest that this novel fluorophore-nitroxide probe could sensitively and easily detect ascorbic acid and be useful as a tool for the diagnosis of disease states. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.freeradbiomed.2012.09.032

Language：English   Publishing type：Research paper (scientific journal)  

Overhauser-enhanced MRI (OMRI) enables visualization of free radicals in animals based on dynamic nuclear polarization. Real-time data of tissue redox status gathered from kinetic images of redox-sensitive nitroxyl radical probes using OMRI provided both anatomic and physiological information. Phantom experiments demonstrated the linear correlation between the enhancement factor and the concentration of a membrane-impermeable probe, carboxy-PROXYL (3-carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl). Whole-body OMRI images illustrated the in vivo kinetics of carboxy-PROXYL for 25 min. Initial distribution was observed in lung, heart, liver, and kidney, but not brain, corresponding to its minimal lipophilicity. Based on these images (pixel size, 1.33 x 1.33 min: slice thickness, 50 mm), a time-concentration curve with low coefficient of variance ( < 0.21) was created to assess pharmacokinetic behaviors. A biexponential curve showed a distribution phase from 1 to 10 min and an elimination phase from 15 to 25 min. The alpha rate constant was greater than the beta rate constant in ROIs, confirming that its pharmacokinetics obeyed a two-compartment model. As a noninvasive technique, combining OMRI imaging with redox probes to monitor tissue redox status may be useful in acquiring valuable information regarding organ function for preclinical and clinical studies of oxidative diseases. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.freeradbiomed.2012.04.026

Language：English   Publishing type：Research paper (scientific journal)  

Tumor necrosis factor-alpha (TNF-alpha) is one of the main mediators of inflammatory response activated by fatty acids in obesity, and this signaling through TNF-alpha receptor (TNFR) is responsible for obesity-associated insulin resistance. Recently, TNF-alpha has shown to affect lipid metabolism including the regulation of lipase activity and bile acid synthesis. However, there is scanty in vivo evidence for the involvement of TNF-alpha in this process, and the mechanistic role of TNFR remains unclear. In this study, TNFR2 knockout mice (R2KO) and wild-type (WT) mice were fed commercial normal diet (ND) or high-fat diet (HFD) for 8 weeks. In R2KO/HFD mice, the increase in body weight and the accumulation of fat were significantly ameliorated compared with WT/HFD mice in association with the decrease in plasma total cholesterol (137.7 +/- A 3.1 vs. 98.6 +/- A 3.1 mg/dL, P < 0.005), glucose (221.9 +/- A 14.7 vs. 167.3 +/- A 8.1 mg/dL, P < 0.01), and insulin (5.1 +/- A 0.3 vs. 3.4 +/- A 0.3 ng/mL, P < 0.05). Fecal excretion of lipid contents was significantly increased in R2KO mice. In R2KO/HFD mice, the decrease in hepatic cholesterol-7a-hydroxylase activity, the rate-limiting enzyme in bile acid synthesis, was inhibited (1.7 +/- A 0.2 vs. 8.1 +/- A 1.0 pmol/min/mg protein, P < 0.01). These results suggested that HFD-induced obesity with metabolic derangements could be ameliorated in mice lacking TNF-alpha receptor 2 via increasing fecal bile acid and lipid content excretion. Therefore, TNF-alpha signaling through TNFR2 is essentially involved in the bile acid synthesis and excretion of lipids, resulting in its beneficial effects.

DOI： 10.1007/s11010-011-1010-3

Language：Others  

Nitroxides prevent exacerbation of indomethacin-induced gastric damage in adjuvant arthritis rats.

DOI： 10.1016/j.freeradbiomed.2011.08.010

Language：Others  

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

DOI： 10.3109/10715762.2011.618499

Language：English   Publishing type：Research paper (scientific journal)  

Oxidative stress may be involved in the dopaminergic neurodegenerations seen in 6-OHDA-lesioned rats through its production of free radicals and through mitochondrial dysfunction. In this study, we noninvasively demonstrate brain redox alterations in 6-OHDA-lesioned rats using Overhauser-enhanced magnetic resonance imaging (OMRI). The reduction rate of 3-methoxycarbony1-2,2,5,5-tetramethylpyrrolidine-1-oxyl (methoxycarbonyl-PROXYL), a redox-sensitive contrast agent, was used as an index of the redox status in vivo. The methoxycarbonyl-PROXYL reduction rate, calculated from continuous images, decreased significantly in lesioned hemispheres compared to their corresponding contralateral hemispheres. The reduction rates in cellular fractions obtained from the striatum were estimated by X-band electron spin resonance (ESR) and calculated by assuming first-order kinetics for their time-dependent decreases. When methoxycarbonyl-PROXYL was mixed with cytoplasm fractions, the reduction rates were the same in both hemispheres. However, the ESR signal of methoxycarbonyl-PROXYL in the mitochondrial fraction of the lesioned hemispheres decayed more slowly than that of the corresponding contralateral hemispheres. Concordantly, biochemical assays showed that the activity of mitochondrial complex I also decreased more slowly in lesioned hemispheres. Thus, this method of noninvasively imaging brain redox alterations faithfully reflects changes in mitochondrial complex I activity in 6-OHDAlesioned rats. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.neuint.2011.08.010

Language：Others  

Oxygen concentration dependence of lipid peroxidation and lipid-derived radical generation: application of profluorescent nitroxide switch.

DOI： 10.3109/10715762.2011.595410

Language：English   Publishing type：Research paper (scientific journal)  

Abuse of the powerfully addictive psychostimulant, methamphetamine, occurs worldwide. Recent studies have suggested that methamphetamine-induced dopaminergic neurotoxicity is related to oxidative stress. In response to nerve activation, the mitochondrial respiratory chain is rapidly activated. The enhancement of mitochondrial respiratory chain activation may induce oxidative stress in the brain. However, there is little experimental evidence regarding the mitochondrial function after methamphetamine administration in vivo. Here, we evaluated whether a single administration of methamphetamine induces ATP consumption and overactivation of mitochondria. We measured mitochondrial function in two different ways: by monitoring oxygen partial pressure using an oxygen-selective electrode, and by imaging of redox reactions using a nitroxyl radical (i.e., nitroxide) coupled with Overhauser-enhanced magnetic resonance imaging (OMRI). A single administration of methamphetamine to Wistar rats induced dopaminergic nerve activation, ATP consumption and an increase in mitochondrial respiratory chain function in both the striatum and cortex. Furthermore, antioxidant TEMPOL prevented the increase in mitochondrial oxidative damage and methamphetamine-induced sensitization. These findings suggest that energy-supplying reactions after dopaminergic nerve activation are associated with oxidative stress in both the striatum and cortex, leading to abnormal behavior. (c) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.neuroimage.2011.05.041

Language：Others  

Nifedipine treatment reduces brain damage after transient focal ischemia, possibly through its antioxidative effects.

DOI： 10.1038/hr.2011.51

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Structural concept of nitroxide as a lipid peroxidation inhibitor.
Nitroxides have antioxidative activities toward lipid peroxidation, but the influence of steric factors is not known. We synthesized alkyl-substituted nitroxides at the α-position of the N-O moiety to enhance lipophilicity and the bulk effect. There was good correlation between the IC(50) and lipophilicity (log P(o/w)) of nitroxides with use of the thiobarbituric acid-reactive substances (TBARS) assay. Furthermore, an inhibitory effect on the TBARS assay was dependent upon the number and length of alkyl groups, though nitroxides had almost identical lipophilicity.

DOI： 10.1021/jo200361p

Language：English   Publishing type：Research paper (scientific journal)  

We have synthesized several nitroxides with different substituents which vary the steric and electronic environment around the N-O moiety and have systematically investigated the role of substituents on the stability of the radicals. Our results demonstrated the reactivity toward ascorbate correlates with the redox potential of the derivatives. Furthermore, ab initio calculations also indicated a correlation between the reduction rate and the computed singly occupied molecular orbital-lowest unoccupied molecular orbital energy gap, but not with solvent accessible surface area of the N-O moiety, supporting the experimental results and suggesting that the electronic factors largely determine the radicals' stability. Hence, it is possible to perform virtual screening of nitroxides to optimize their stability, which can help to rationally design novel nitroxides for their potential use in vivo.

DOI： 10.1021/jo101961m

Language：Others  

In vivo evaluation of novel nitroxyl radicals with reduction stability.

DOI： 10.1016/j.freeradbiomed.2010.08.027

Language：English   Publishing type：Research paper (scientific journal)  

Overhauser-enhanced magnetic resonance imaging (OMRI) studies of a membrane-permeable nitroxyl spin probe, 2H-enriched 3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (MC-PROXYL), used in simultaneous molecular imaging is reported. Phantom imaging was performed with liposomal solutions of MC-PROXYL at varying spin probe and liposome concentrations using a field-cycle mode, custom-built OMRI scanner. Dynamic nuclear polarization (DNP) spectra of the liposomal solution of the spin probe, measured at 14.529 mT using a 5 mT sweep of the electron paramagnetic resonance (EPR) irradiation field showed splitting of the low and high filed hyperfine lines. Spectral measurements using D2O and a spin broadening agent, K3Fe(CN)6 confirmed that these peaks originated from water molecules in two different environments, compartmentalized with liposomes. The nuclear Overhauser enhancement measured at different EPR irradiation times and power levels showed reduction in water nuclear magnetic resonance (NMR) signal enhancement in liposomal membrane due to the reduction in the coupling constant, ρ. This study illustrates that OMRI can be used to differentiate between the intra- and extra- membrane water by loading the liposome vesicles with a lipid-permeable nitroxyl spin probe. © 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.jmr.2010.02.016

Language：English  

Language：English   Publishing type：Research paper (scientific journal)  

Nitroxyl radicals (nitroxides) with unpaired electron are widely used as antioxidants, contrast agents, and spin probes. Although piperidine nitroxyl radicals have many applications, these are mainly tetramethylpiperidine compounds, and only a few reports consider the substitution of N-O surround as a reaction site, such as 2,2,6,6-tetrasubstituted piperidine nitroxyl radicals. Our results revealed that the 2,6-position of the 2,2,6,6-tetramethylpiperidin-4-one compound was substituted by cyclohexyl groups to produce 2,2,6,6-tetrasubstituted piperidin-4-one derivatives under mild reaction conditions. An interesting result was obtained by using (15)N-labeled NH(4)Cl instead of (14)NH(4)Cl: it gave (15)N-labeled 2,2,6,6-tetrasubstituted piperidin-4-one-1-oxyls with a high (15)N content. In conclusion, the new method for the synthesis of nitroxyl radicals readily yields 2,2,6,6-tetrasubstituted piperidin-l-one under mild conditions. (C) 2009 Published by Elsevier Ltd.

DOI： 10.1016/j.tet.2010.02.004

Language：English   Publishing type：Research paper (scientific journal)  

Oxidative stress has been implicated in the cell death that occurs after ischemia-reperfusion of the brain, which causes the production of reactive oxygen species and a decrease in antioxidants, leading to mitochondrial dysfunction. However, the invasive methods used to collect much of this evidence are themselves stress inducing, which could skew the results. In this study, we aimed at demonstrating brain redox alterations after ischemia-reperfusion noninvasively, using Overhauser-enhanced magnetic resonance imaging. The reduction rate of 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-L-oxyl (methoxycarbonyl-PROXYL), a redox-sensitive contrast agent, was used as an index of the redox status in vivo. No changes were observed in the antioxidant concentration, the mitochondrial complex activity, or in the redox status image intensity after 3 h of reperfusion, following transient middle cerebral artery occlusion; however, after 24 h of reperfusion, the methoxycarbonyl-PROXYL reduction rate, calculated from continuous images, had decreased significantly. Concordantly, biochemical assays showed that the concentration of ascorbic acid in the ischemic hemisphere and the activity of mitochondrial complex II had also decreased. Thus, the noninvasive imaging of the brain redox alterations faithfully reflected changes in antioxidant levels and in mitochondrial complex II activity after ischemia-reperfusion. Journal of Cerebral Blood Flow & Metabolism (2009) 29, 1655-1664; doi:10.1038/jcbfm.2009.84; published online 24 June 2009

DOI： 10.1038/jcbfm.2009.84

Language：Others  

Development of novel nitroxyl radicals for controlling reactivity with ascorbic acid.

DOI： 10.1080/10715760902914575

Language：English   Publishing type：Research paper (scientific journal)  

Language：Others  

Synthesis of nitroxyl radicals for Overhauser-enhanced magnetic resonance imaging.

DOI： 10.1002/ardp.200800053

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Nitroxyl radicals are important antioxidants that have been used to protect animal tissues from oxidative damage. Their reaction with hydroxyl radical ((OH)-O-center dot) is generally accepted to be the mechanism of antioxidant function. However, the direct interaction of nitroxyl radicals with (OH)-O-center dot does not always provide a satisfactory explanation in various pH, because the concentration of hydrogen ion may affect the generation of secondary (OH)-O-center dot-derived radicals. In the present study, it was confirmed that the reaction between 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) and (OH)-O-center dot generated TEMPOL-hydroxylamine, 4-oxo-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPON) and TEMPON-hydroxylamine using HPLC coupled with electrochemical detection. In the absence of NADH, TEMPOL-H may be generated by the reaction with secondary (OH)-O-center dot-derived radicals in acidic condition. In the presence of NADH, a large proportion of the nonparamagnetic products was TEMPOL-H. Finally, it was clarified that TEMPOL-H was generated during dopamine metabolism, which is believed to be one of the (OH)-O-center dot sources in pathological processes such as Parkinson's disease.

DOI： 10.1080/10715760802112809

Language：Japanese   Publishing type：Research paper (scientific journal)  

Language：Others  

Advantageous application of a surface coil to EPR irradiation in overhauser-enhanced MRI.

DOI： 10.1002/mrm.21198

Language：English  

Reactive oxygen species (ROS) are thought to play a significant role in the development of diabetic retinopathy; however, no direct evidence supports ROS generation in vivo. This study used in vivo electron spin resonance (ESR) spectroscopy with a surface resonator to detect local free radical reactions. The ESR signal decay of carbamoyl-PROXYL was enhanced in the eyes of streptozotocin (STZ)-induced diabetic mice. This enhanced signal decay was suppressed by the administration of SOD or the pretreatment with aminoguanidine. We demonstrate, for the first time, specific free radical reactions in the eyes of mice with STZ-induced diabetes.

DOI： 10.1089/ars.2006.1502

Language：English   Publishing type：Research paper (scientific journal)  

DNP parameters relevant to Overhauser-enhanced magnetic resonance imaging (OMRI) are reported for a few nitroxyl radicals and their corresponding N-15 and H-2 enriched analogues, used in simultaneous imaging by OMRI. DNP enhancement was measured at 14.529 mT, using a custom-built scanner operating in a field-cycled mode, for different concentrations, ESR irradiation times and RF power levels. DNP enhancements increased with agent concentration up to 2.5 mM and decreased above 3 mM, in tune with ESR line broadening measured at X-band as a function of the agent concentration. The proton spin-lattice relaxation times (T-1) measured at very low Zeeman field (14.529 mT) and the longitudinal relaxivity parameters were estimated. The relaxivity parameters were in good agreement with those independently computed from the linear region of the concentration dependent enhancement. The leakage factor showed an asymptotic increase with increasing agent concentration. The coupling parameters of N-14- and N-15-labeled carbamoyl-PROXYL showed the interaction between the electron and nuclear spins to be mainly dipolar in origin. Upon H-2 labeling, about 70&#37; and 40&#37; increases in enhancement for N-15- and N-14-labeled nitroxyl agents were observed, respectively. It is envisaged that the results reported here may enable better understanding of the factors determining DNP enhancement to design suitable 'beacons' for simultaneous molecular imaging by OMRI. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.jmr.2006.06.025

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Diethylnitrosamine (DEN) is a well-known carcinogenic substance that requires microsomal activation before it can react with DNA to cause mutations and cancer. The aim of this study was to use in vivo spin trapping and spin probe techniques to investigate whether free radicals are generated in rat liver tissue during DEN activation. We used α-phenyl-n-tert-butylnitrone (PBN) as the spin trapping agent, which was delivered through an intraperitoneal injection before DEN administration. One hour after DEN administration, multicomponent PBN adducts in the bile were detected, and the intensities were diminished by the cytochrome P450 inhibitor SKF-525A. A computer simulation of the ESR signals revealed the presence of a lipid-derived radical. Using the in vivo spin probe/ESR technique, the signal decay rate of methoxycarbonyl-PROXYL was significantly increased in the DEN-treated group compared with the rate in the vehicle group. The enhanced signal decay rate was restored with PBN and/or SKF-525A pretreatment. These results suggested that lipid-derived free radicals were generated in the liver within 1 h after DEN administration. © 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.freeradbiomed.2006.01.031

Language：English   Publishing type：Research paper (scientific journal)  

One of the reasons of rheumatoid arthritis (RA) development is widely recognized the relation of free radical reactions in tissue injuries. The aim of this study was to evaluate the location where in vivo free radical reactions was enhanced in adjuvant arthritis (AA) model rats using in vivo electron spin resonance (ESR)/nitroxyl spin probe technique. The signal decay after intravenous injection of spin probe was enhanced in AA than that in control and suppressed by the pre-treatment of dexamethasone (DXT). Interestingly, the decay in joint cavity occurred prior to paw swelling of AA and suppressed by a simultaneous injection of free radical scavengers, indicating that the enhancement of free radical reactions in joint cavity of AA rats. This technique would be useful tool to determine the location of the enhanced free radical reactions and evaluate the activity of antioxidant medicine with non-invasive real-time measurement. © 2006 Taylor &amp
Francis.

DOI： 10.1080/10715760500440536

Language：English   Publishing type：Research paper (scientific journal)  

Reactive oxygen species (ROS) are thought to be involved in the gastric ulcer formation induced by indomethacin, a typical nonsteroidal anti-inflammatory drug. However, the location and the time course of ROS generation remain unknown. To assess the sites of ROS generation, we applied the noninvasive measurement of ROS to indomethacin-treated rats. By giving orally a membrane-permeable or impermeable probe, the spectra were collected as a function of time by in vivo 300-MHz electron spin resonance (ESR) spectroscopy. The ESR signal-decay rates of membrane-permeable probes, hydroxy-2,2,6,6- tetramethylpiperidine-N-oxyl (TEMPO) and 3-methoxycarbonyl-2,2,5,5-tetramethyl- pyrrolidine-1-oxyl, in the gastric mucosal region were significantly enhanced 1 h after indomethacin treatment, and they both caused the protection of ulcer formation
however, membrane-impermeable probes, carboxy- and trimethylammonium-TEMPO, which did not exhibit the enhanced signal decay, had no effect on ulcer formation. The enhanced signal decay in the gastric mucosa was suppressed by coadministration of the antioxidants tiron or dimethylthiourea with the nitroxyl probe. The results suggest that the enhanced signal decay rates in the gastric ulcers observed by in vivo ESR are associated with protective effects. The enhanced signal decay caused by ROS generation in stomach, contributing to the ulcer formation induced by indomethacin, is also suggested to occur at the gastric mucus layer or the interface or the intracellular compartment of epithelial cells. Overall, these results show the potentials of noninvasive assessment of ROS production and the sites of damage by in vivo ESR using nitroxyl probes directed to specific subcellular regions. Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics.

DOI： 10.1124/jpet.105.095166

Language：English   Publishing type：Research paper (scientific journal)  

MRI has provided significant clinical utility in the diagnosis of diseases and will become a powerful tool to assess phenotypic changes in genetically engineered animals. Overhauser enhanced MRI (OMRI), which is a double resonance technique, creates images of free radical distributions in small animals by enhancing the water proton signal intensity by means of the Overhauser effect. Several studies have demonstrated noninvasive assessment of reactive oxygen species generation in small animals by using low frequency electron spin resonance (ESR) spectroscopy imaging and nitroxyl radicals. In vivo ESR signal intensities of nitroxyl radicals decrease with time after injection
and the decreases are enhanced by reactive oxygen species, generated in oxidative disease models in a site-specific manner. In this study, we show images of nitroxyl radicals with different isotopes by changing the external magnetic field for ESR irradiation between 14N and 15N nuclei in field-cycled OMRI. OMRI simultaneously obtained dual images of two individual chemical processes. Oxidation and reduction were monitored in a rate-dependent manner at nanometer scale by labeling membrane-permeable and -impermeable nitroxyl radicals with 14N and 15N nuclei. Phantom objects containing ascorbic acid-encapsulated liposomes with membrane-permeable radicals but not membrane-impermeable ones show a time-dependent decrease of the OMRI image intensity. The pharmacokinetics in mice was assessed with OMRI after radical administration. This OMRI technique with dual probes should offer significant applicability to nanometer scale molecular imaging and simultaneous assessment of independent processes in gene-modified animals. Thus, it may become a powerful tool to clarify mechanisms of disease and to monitor pharmaceutical therapy. © 2006 by The National Academy of Sciences of the USA.

DOI： 10.1073/pnas.0510670103

Language：English   Publishing type：Research paper (scientific journal)  

Diesel exhaust particles (DEP) are reactive oxygen species (ROS)-inducing toxic agents that damage lungs. Thioredoxin-1 (Trx-1) is a thiol protein with antioxidant and redox-regulating effects. In this study, we demonstrate that Trx-1 scavenges ROS generated by DEP and attenuates the lung injury. Intratracheal instillation of DEP resulted in the generation of more hydroxyl radicals in control mice than in human Trx-1 (hTrx-1)-transgenic mice as measured by noninvasive L-band in vivo electron spin resonance. DEP caused acute lung damage with massive infiltration of inflammatory cells in control mice, but much less damage in hTrx-1-transgenic mice. The hTrx-1 transgene protected the mice against DEP toxicity. To investigate further the molecular mechanism of the protective role of Trx-1 against DEP-induced lung injury, we used hTrx-1-transfected L-929 cells and recombinant hTrx-1 (rhTrx-1)-pretreated A-549 cells. DEP-induced ROS generation was suppressed by hTrx-1 transfection or pretreatment with rhTrx-1. Endogenous Trx-1 expression was induced by DEP in control cells. The downregulation of Akt phosphorylation by DEP resulted in apoptosis, which was prevented by Trx-1. Moreover, an Akt inhibitor canceled this protective effect of Trx-1. Collectively, the results suggest that Trx-1 exerts antioxidant effects in vivo and in vitro and that this plays a role in protection against DEP-induced lung damage by regulating Akt-mediated antiapoptotic signaling. © 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.freeradbiomed.2005.07.016

Language：English   Publishing type：Research paper (scientific journal)  

We used the membrane-impermeable nitroxyl radical 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-oxyliodide (CAT-1) as a model drug encapsulated in liposomes in order to separately map the 2D distribution of both liposomal-encapsulated CAT-1 and free CAT-1. Phantoms were prepared with a CAT-1 solution and a liposomal CAT-1 suspension. Spectral-spatial images were obtained along several polar-arranged spatial axes through the phantom. The 1D spatial distributions (projections) of each signal component, reflecting the concentration of CAT-1, were then extracted from the spectral-spatial images. 2D EPR images of liposomal-encapsulated CAT-1 and free CAT-1 were separately reconstructed from the resulting projection data sets. 2D mapping of each component exhibited good agreement with respect to the phantom. Separate maps were generated from separate injections of free CAT-1 and liposomal CAT-1 injected into the femoral muscle of a living mouse. The EPR signal of the free CAT-1 gradually decreased during data acquisition. Because of this decay, we calibrated the image intensity by extrapolating the signal intensity to that detected at the beginning of data sampling. Both the position and size of the individual images were in very good agreement with those of the mouse thigh obtained by MRI.

Language：English   Publishing type：Research paper (scientific journal)  

A composition resonator assembly suitable for EPR/NMR co-registration imaging

Language：Others   Publishing type：Research paper (scientific journal)  

The redox dynamics of the rat liver in the whole body hyperthermia was measured by the decay rate of the ESR spectra used carbamoyl-PROXYL as a spin probe. Fifty-two Wistar male rats were randomly assigned to four groups and each group was subjected to only one of the following treatments: heat stress (HS group), anesthesia without hyperthermia (control group), pretreatment with MPG (MPG + HS group) and pretreatment with saline (No-MPG group). The solution of carbamoyl PROXYL was added to rat liver homogenate from 0, 1, 2, 6 and 24 h after heating at concentration of 0.1 mmol/L. sequentially the ESR spectra of the samples were immediately recorded by X-band ESR spectrometer. Compared with the control group, the signal decay rate was increased gradually after heat treatment, and attained the maximum at 2 h, and then returned to the normal level at 24 h. This signal intensity effect was reduced by MPG pretreatment. Generation of reactive oxygen species in liver may be induced by heat stress.

Language：Others   Publishing type：Research paper (scientific journal)  

AIM: The redox dynamics of the rat liver in the whole body hyperthermia was measured by the decay rate of the ESR spectra used carbamoyl-PROXYL as a spin probe. METHODS: Fifty-two Wistar male rats were randomly assigned to four groups and each group was subjected to only one of the following treatments: heat stress (HS group), anesthesia without hyperthermia (control group), pretreatment with MPG (MPG + HS group) and pretreatment with saline (No-MPG group). The solution of carbamoyl PROXYL was added to rat liver homogenate from 0, 1, 2, 6 and 24 h after heating at concentration of 0.1 mmol/L. sequentially the ESR spectra of the samples were immediately recorded by X-band ESR spectrometer. RESULTS: Compared with the control group, the signal decay rate was increased gradually after heat treatment, and attained the maximum at 2 h, and then returned to the normal level at 24 h. This signal intensity effect was reduced by MPG pretreatment. CONCLUSION: Generation of reactive oxygen species in liver may be induced by heat stress.

Language：English   Publishing type：Research paper (scientific journal)  

Free radicals are widely recognized as harmful chemical species in oxidative tissue injury. However, there have been no satisfying methods to visualize free radicals in vivo non-invasively with information of their localization and amount. In vivo electron spin resonance (ESR) spectroscopy was recently developed to measure free radicals generated in rodents. Several kinds of stable nitroxyl radicals were used as spin probes to detect free radicals. ESR signal intensities reflecting the accumulation of nitroxyl probes in each organ decreases time-dependently and reduction decay rates are increased in the presence of free radicals. Such increase in signal decay rates is suppressed by prior administration of antioxidants or antioxidant enzymes. Thus, in vivo ESR techniques are useful in estimating not only in vivo free radical reactions but also the effects of antioxidants, and furthermore, in combination with other tomographic techniques, permits non-invasive localization of free radicals. Application of this technique to animal models will be described.
PMID: 12859975

Other Link： https://www.ncbi.nlm.nih.gov/pubmed/12859975

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Venue：つくば   Country：Japan  

Event date： 2019.4

Language：Japanese  

Venue：京都   Country：Japan  

Event date： 2019.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2019.3

Language：Japanese  

Venue：千葉   Country：Japan  

Event date： 2019.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：千葉   Country：Japan  

Event date： 2019.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：千葉   Country：Japan  

Event date： 2019.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：千葉   Country：Japan  

Event date： 2019.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：千葉   Country：Japan  

Event date： 2019.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Event date： 2019.2

Language：Japanese  

Venue：神戸   Country：Japan  

Event date： 2019.1

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Event date： 2019.1

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2019.1

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：仙台   Country：Japan  

Event date： 2018.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2018.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Event date： 2018.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Event date： 2018.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Event date： 2018.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Event date： 2018.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Event date： 2018.11

Language：Japanese  

Venue：北海道大学   Country：Japan  

Event date： 2018.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京都国際会議場   Country：Japan  

Event date： 2018.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：長崎ブリックホール国際会議場（長崎市）   Country：Japan  

Event date： 2018.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：九州大学（福岡市）   Country：Japan  

Event date： 2018.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：九州大学（福岡市）   Country：Japan  

Event date： 2018.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：北里大学（東京都港区）   Country：Japan  

Event date： 2018.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2018.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2018.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：北里大学（東京都港区）   Country：Japan  

Event date： 2018.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：北里大学（東京都港区）   Country：Japan  

Event date： 2018.7

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京都   Country：Japan  

Event date： 2018.6 - 2018.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京工業大学大岡山キャンパス   Country：Japan  

Event date： 2018.6

Language：English  

Venue：Lisbon   Country：Portugal  

Event date： 2018.5

Language：Japanese  

Venue：京都   Country：Japan  

Event date： 2018.4 - 2018.3

Language：English  

Venue：Mahidol University   Country：Thailand  

Event date： 2018.3

Language：Japanese  

Venue：金沢（石川県）   Country：Japan  

Event date： 2018.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：金沢（石川県）   Country：Japan  

Event date： 2018.3

Language：Japanese  

Venue：金沢（石川県）   Country：Japan  

Event date： 2018.3

Language：Japanese  

Venue：金沢（石川県）   Country：Japan  

Event date： 2018.3

Language：Japanese  

Venue：金沢（石川県）   Country：Japan  

Event date： 2018.3

Language：Japanese  

Venue：千葉（船橋）   Country：Japan  

Event date： 2018.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京大学（東京）   Country：Japan  

Event date： 2018.2

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：名古屋   Country：Japan  

Event date： 2017.12

Language：English  

Venue：東京工科大学（東京）   Country：Japan  

Event date： 2017.11 - 2017.12

Language：English  

Venue：Baltimore Maryland   Country：United States  

Event date： 2017.11

Language：English   Presentation type：Symposium, workshop panel (public)  

Venue：NIH Bethesda   Country：United States  

Event date： 2017.11

Language：Japanese  

Venue：崇城大学（熊本）   Country：Japan  

Event date： 2017.11

Language：Japanese  

Venue：崇城大学（熊本）   Country：Japan  

Event date： 2017.11

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2017.10

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：長崎   Country：Japan  

Event date： 2017.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：大阪   Country：Japan  

Event date： 2017.7

Language：English   Presentation type：Oral presentation (general)  

Venue：Queensland   Country：Australia  

Event date： 2017.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：つくば   Country：Japan  

Event date： 2017.6

Language：English   Presentation type：Oral presentation (general)  

Venue：京都   Country：Thailand  

Event date： 2017.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京都   Country：Japan  

Event date： 2017.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：札幌   Country：Japan  

Event date： 2017.5

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：お台場   Country：Japan  

Event date： 2017.3

Language：Japanese  

Venue：仙台   Country：Japan  

Event date： 2017.3

Language：Japanese  

Venue：仙台   Country：Japan  

Event date： 2017.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：奈良   Country：Japan  

Event date： 2017.3

Language：English   Presentation type：Oral presentation (general)  

Venue：Bangkok   Country：Thailand  

Event date： 2017.1

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：九大西新プラザ   Country：Japan  

Event date： 2016.12

Language：English   Presentation type：Oral presentation (general)  

Venue：Lausanne   Country：Switzerland  

Event date： 2016.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：鹿児島   Country：Japan  

Event date： 2016.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：鹿児島   Country：Japan  

Event date： 2016.11

Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Taipei   Country：Taiwan, Province of China  

Event date： 2016.11

Language：English  

Venue：San Francisco   Country：United States  

Event date： 2016.9

Language：English  

Venue：New York   Country：United States  

Event date： 2016.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京都   Country：Japan  

Event date： 2016.9

Language：Japanese  

Venue：京都   Country：Japan  

Event date： 2016.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：仙台   Country：Japan  

Event date： 2016.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：京都   Country：Japan  

Event date： 2016.5

Language：English  

Venue：仙台   Country：Japan  

Event date： 2016.5

Language：English  

Venue：仙台   Country：Japan  

Event date： 2016.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：横浜   Country：Japan  

Event date： 2016.3

Language：Japanese  

Venue：横浜   Country：Japan  

Event date： 2016.3

Language：Japanese  

Venue：横浜   Country：Japan  

Event date： 2016.3

Language：Japanese  

Venue：横浜   Country：Japan  

Event date： 2016.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：横浜   Country：Japan  

Event date： 2015.12

Language：English   Presentation type：Oral presentation (general)  

Venue：Hawaii   Country：United States  

Event date： 2015.12

Language：English  

Venue：Hawaii   Country：United States  

Event date： 2015.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：鹿児島   Country：Japan  

Event date： 2015.12

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：鹿児島   Country：Japan  

Event date： 2015.11 - 2015.12

Language：English  

Venue：Chiang Mai   Country：Thailand  

Event date： 2015.8

Language：Japanese  

Venue：長崎   Country：Japan  

Event date： 2015.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：長崎   Country：Japan  

Event date： 2015.8

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：長崎   Country：Japan  

Event date： 2015.6

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2015.5

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2015.5

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東京   Country：Japan  

Event date： 2015.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Event date： 2015.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：神戸   Country：Japan  

Event date： 2015.3

Language：Japanese   Presentation type：Symposium, workshop panel (public)  

Venue：神戸   Country：Japan  

Event date： 2014.8

Language：English   Presentation type：Oral presentation (general)  

Venue：Pusan National University   Country：Korea, Republic of  

Event date： 2014.7

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：名古屋   Country：Japan  

Event date： 2014.4

Language：Japanese  

Venue：熊本大学   Country：Japan  

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：鹿児島   Country：Japan  

Language：English   Presentation type：Oral presentation (general)  

Venue：Kyoto   Country：Japan  

Language：English   Presentation type：Oral presentation (general)  

Venue：Zelenogradsk   Country：Russian Federation  

Language：English  

Venue：Seattle, Washington   Country：United States  

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Language：English   Presentation type：Oral presentation (general)  

Venue：Mahidol University   Country：Thailand  

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：福岡   Country：Japan  

Language：Japanese  

Venue：鹿児島   Country：Japan  

Language：Japanese  

Venue：鹿児島   Country：Japan  

Language：Japanese  

Language：English  

DOI： 10.1007/s00723-018-1035-9

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：Japanese  

Language：English  

Language：English  

Language：Japanese  

Language：English  

Language：Japanese  

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Type：Competition, symposium, etc. 

Number of participants：200

Type：Competition, symposium, etc. 

Number of participants：200

Type：Competition, symposium, etc. 

Number of participants：200

Type：Competition, symposium, etc. 

Number of participants：400

Type：Competition, symposium, etc. 

Number of participants：200

Type：Competition, symposium, etc. 

Number of participants：400

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：8

Type：Competition, symposium, etc. 

Number of participants：200

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：8

Type：Competition, symposium, etc. 

Number of participants：200

Type：Competition, symposium, etc. 

Number of participants：400

Type：Academic society, research group, etc. 

Type：Peer review 

Number of peer-reviewed articles in foreign language journals：3

Type：Competition, symposium, etc. 

Number of participants：200