Details of a Researcher - Mashino, Tadahiko

Papers - Mashino, Tadahiko

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Fullerene derivatives as dual inhibitors of HIV-1 reverse transcriptase and protease

Yasuno T., Ohe T., Kataoka H., Hashimoto K., Ishikawa Y., Furukawa K., Tateishi Y., Kobayashi T., Takahashi K., Nakamura S., Mashino T.

Bioorganic and Medicinal Chemistry Letters (Bioorganic and Medicinal Chemistry Letters) 31 2021.01

ISSN 0960894X

© 2020 Elsevier Ltd In the present study, we newly synthesized three types of novel fullerene derivatives: pyridinium-type derivatives trans-3a and 4a-5b, piperidinium-type derivative 9, and proline-type derivatives 10a-12. Among the assessed compounds, 5a, 10e, 10f, 10i, 11a-d, and 12 were found to inhibit both HIV reverse transcriptase and HIV protease (HIV-PR), with IC50 values in the low micromolar range being observed. Regarding HIV-PR inhibition activity, proline-type derivatives 11a-11d and 12, bearing an alkyl chain between the hydroxylmethylcarbonyl (HMC) moiety and pyrrolidine ring, were more potent than other derivatives. This result might indicate that connecting HMC moieties with proline-type fullerene derivatives through properly sized alkyl chain leads to improved HIV-PR inhibitory activity.

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Development of Novel Diclofenac Analogs Designed to Avoid Metabolic Activation and Hepatocyte Toxicity

Tateishi Y., Ohe T., Ogawa M., Takahashi K., Nakamura S., Mashino T.

ACS Omega (ACS Omega) 5 ( 50 ) 32608 - 32616 2020.12

© Diclofenac (DCF) is widely used as a nonsteroidal anti-inflammatory drug; however, it is associated with severe liver injury. This adverse reaction is thought to be related to the reactive quinone imine (QI) and acyl glucuronide (AG) metabolites of DCF, but it remains controversial which reactive metabolites mainly contribute to DCF-induced toxicity. In this study, we synthesized five types of DCF analogs that were designed to mitigate the formation of reactive QI and/or AG metabolites and evaluated their metabolic stability, cyclooxygenase (COX) inhibitory activity, and toxicity to cryopreserved human hepatocytes. Compounds with fluorine at the 5- A nd 4-positions of aromatic rings exhibited modest and high metabolic stability to oxidation by cytochrome P450, respectively, but induced cytotoxicity comparable to DCF. Replacing the carboxylic group of DCF with its bioisosteres was effective in terms of stability to oxidative metabolism and glucuronidation; however, sulfonic acid and sulfonamide groups were not preferable for COX inhibition, and tetrazole-containing analogs induced strong cytotoxicity. On the other hand, compounds that have fluorine at the benzylic position were resistant to glucuronidation and showed little toxicity to hepatocytes. In addition, among these compounds, those with hydrogen at the 4-position (2a and 2c) selectively inhibited the COX-2 enzyme. Throughout these data, it was suggested that compounds 2a and 2c might be novel safer and more efficacious drug candidates instead of DCF.

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Synthesis and evaluation of nevirapine analogs to study the metabolic activation of nevirapine

Tateishi Y., Ohe T., Yasuda D., Takahashi K., Nakamura S., Kazuki Y., Mashino T.

Drug Metabolism and Pharmacokinetics (Drug Metabolism and Pharmacokinetics) 35 ( 2 ) 238 - 243 2020.04

ISSN 13474367

© 2020 The Japanese Society for the Study of Xenobiotics Nevirapine (NVP) is widely used as a non-nucleoside reverse transcriptase inhibitor of HIV-1, however, it is associated with severe skin and liver injury. The mechanisms of these adverse reactions are not yet clear, but the metabolic activation of NVP is thought to be related to the injury process. Until now, several metabolic activation pathways of NVP have been reported. In this study, in order to identify the reactive metabolite of NVP mainly responsible for CYP inhibition and liver injury, we synthesized five NVP analogs designed to avoid the proposed bioactivation pathway and evaluated their metabolic stabilities, CYP3A4 time-dependent inhibitory activities, and cytotoxicity. As a result, only a pyrimidine analog of NVP, which could avoid the formation of a reactive epoxide intermediate, did not inhibit CYP3A4. Outside of this compound, the other synthesized compounds, which could avoid the generation of a reactive quinone-methide intermediate, inhibited CYP3A4 equal to or stronger than NVP. The pyrimidine analog of NVP did not induce cytotoxicity in HepG2 and transchromosomic HepG2 cells, expressing major four CYP enzymes and CYP oxidoreductase. These results indicated that the epoxide intermediate of NVP might play an important role in NVP-induced liver injury.

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Inhibitors of the protein–protein interaction between phosphorylated p62 and Keap1 attenuate chemoresistance in a human hepatocellular carcinoma cell line

Yasuda D., Ohe T., Takahashi K., Imamura R., Kojima H., Okabe T., Ichimura Y., Komatsu M., Yamamoto M., Nagano T., Mashino T.

Free Radical Research (Free Radical Research) 2020

ISSN 10715762

© 2020, © 2020 Informa UK Limited, trading as Taylor & Francis Group. Resistance to anticancer agents has been an obstacle to developing therapeutics and reducing medical costs. Whereas sorafenib is used for the treatment of human hepatocellular carcinoma (HCC), resistance limits its efficacy. p62, a multifunctional protein, is overexpressed in several HCC cell lines, such as Huh-1 cells. Phosphorylated p62 (p-p62) inhibits the protein–protein interaction (PPI) between Keap1 and Nrf2, resulting in the Nrf2 overactivation that causes drug resistance. We have found a unique Nrf2 inactivator, named K67, that inhibited the PPI between Keap1 and p-p62 and attenuated sorafenib resistance in Huh-1 cells. Herein, we designed and synthesised novel K67 derivatives by modification of the substituent at the 4-position of the two benzenesulfonyl groups of K67. Although these new derivatives inhibited the Keap1-p-p62 PPI to a level comparable to or weaker than that of K67, the isopropoxy derivative enhanced the sensitivity of Huh-1 cells to sorafenib to a greater extent than K67 without any influence on the viability of Huh-7 cells, which is a non-resistant HCC cell line. The isopropoxy derivative also increased the sensitivity of Huh-1 cells to regorafenib, which suggests that this derivative has the potential to be used as an agent to overcome chemoresistance based on Nrf2 inactivation.

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N-Acetyl cysteine prevents activities of STAT3 inhibitors, Stattic and BP-1-102 independently of its antioxidant properties

Uchihara Y., Ohe T., Mashino T., Kidokoro T., Tago K., Tamura H., Funakoshi-Tago M.

Pharmacological Reports (Pharmacological Reports) 71 ( 6 ) 1067 - 1078 2019.12

ISSN 17341140

© 2019 Institute of Pharmacology, Polish Academy of Sciences Background: Inhibitors for signal transducer and activator of transcription 3 (STAT3), Stattic, BP-1-102, and LLL12 significantly induce apoptosis in transformed Ba/F3 cells expressing an oncogenic fusion protein, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) that induces the activation of STAT3. We found that the antioxidant reagent, N-acetyl cysteine (NAC) prevented the abilities of Stattic and BP-1-102, but not LLL12 to induce apoptosis in transformed cells expressing NPM-ALK, providing a novel problem in use of STAT3 inhibitors. We herein investigated the mechanisms how NAC prevented the effects of Sttatic and BP-1-102. Methods: Ba/F3 cells expressing NPM-ALK and SUDHL-1 cells were treated with antioxidants such as NAC, Trolox or edaravone in combination with STAT3 inhibitors. Phosphorylation of STAT3, cell proliferation rate, cell viability, cell cycle, internucleosomal DNA fragmentation and the intracellular accumulation of reactive oxygen species (ROS) was investigated. The binding of STAT3 inhibitors and NAC was analyzed by LC–MS. Results: NAC but not Trolox and edaravone diminished the abilities of Stattic and BP-1-102 to induce apoptosis in cells expressing NPM-ALK. The ROS levels in cells expressing NPM-ALK were not markedly affected by the treatments with Stattic and BP-1-102 in combination with NAC, suggesting that NAC inhibited the activity of Stattic and BP-1-102 independent of its antioxidant activity. LC–MS analysis revealed that NAC directly bound to Stattic and BP-1-102. Furthermore, these NAC adducts exhibited no cytotoxicity, and failed to affect the activity of STAT3. Conclusions: NAC antagonizes the activities of Stattic and BP-1-102, which inhibit STAT3 activation by interacting with cysteine residues in STAT3.

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Synthesis and antitumor activity of novel pyridinium fullerene derivatives

Yasuno T., Ohe T., Ikeda H., Takahashi K., Nakamura S., Mashino T.

International Journal of Nanomedicine (International Journal of Nanomedicine) 14 6325 - 6337 2019

ISSN 11769114

© 2019 Yasuno et al. Purpose: We have previously reported that some cationic fullerene derivatives exhibited anticancer activity, and they are expected to be a potential lead compound for an anti-drug resistant cancer agent. However, they are bis-adducts and a mixture of multiple regioisomers, which cannot be readily separated due to the variability of substituent positions on the fullerene cage. To overcome this issue, we evaluated the antiproliferative activities of a set of mono-adduct derivatives and examined their structure-activity relationship. In addition, the in vivo antitumor activity of selected derivatives was also examined. Methods: Nineteen pyridinium fullerene derivatives were newly designed and synthesized in this study. Their antiproliferative activities were evaluated using several cancer cell lines including drug-resistant cells. Furthermore, in vivo antitumor activity of several derivatives was investigated in mouse xenograft model of human lung cancer. Results: The derivatives inhibited the proliferation of cancer cell lines, including cisplatin-resistant cells and doxorubicin-resistant cells. It was also shown that compound 10 (10 μM), 13 (10 μM) and cis-14 (10 μM) induced the intracellular oxidative stress. In addition, compound 13 (20 mg/kg) and cis-14 (15 mg/kg) significantly exhibited antitumor activity in mouse xenograft model of human lung cancer. Conclusion: We synthesized a novel set of mono-adduct fullerene derivatives functionalized with pyridinium groups and found that most of them show potent antiproliferative activities against cancer cell lines and some of them show significant antitumor activities in vivo. We propose that these fullerene derivatives serve as the lead compounds for a novel type of antitumor agents.

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Synthesis of novel benzbromarone derivatives designed to avoid metabolic activation

Tomoyuki Ohe, Ryutaro Umezawa, Yumina Kitagawara, Daisuke Yasuda, Kyoko Takahashi, Shigeo Nakamura, Akiko Abe, Shuichi Sekine, Kousei Ito, Kentaro Okunushi, Hanae Morio, Tomomi Furihata, Naohiko Anzai, and Tadahiko Mashino*

Bioorg Med Chem Lett 28 3708 - 3711 2018.12

Research paper (scientific journal), Joint Work, Accepted

A 5-hydroxyoxindole derivative attenuates LPS-induced inflammatory responses by activating the p38-Nrf2 signaling axis

Niino Tomomi, Tago Kenji, Yasuda Daisuke, Takahashi Kyoko, Mashino Tadahiko, Tamura Hiroomi, Funakoshi-Tago Megumi

Biochemical Pharmacology 155 182 - 197 2018.09

Research paper (scientific journal), Joint Work, Accepted

A major component of vitamin E, α-tocopherol inhibits the anti-tumor activity of crizotinib against cells transformed by EML4-ALK

Uchihara Y., Kidokoro T., Tago K., Mashino T., Tamura H., Funakoshi-Tago M.

European Journal of Pharmacology (European Journal of Pharmacology) 825 1 - 9 2018.04

ISSN 00142999

© 2018 Elsevier B.V. Crizotinib is an inhibitor of anaplastic lymphoma kinase (ALK) and is of significant therapeutic benefit to patients with non-small cell lung cancer (NSCLC) harboring the EML4-ALK fusion gene. In the present study, we demonstrated that α-tocopherol, a major component of vitamin E, attenuated the effects of crizotinib independently of its anti-oxidant properties. α-Tocopherol significantly inhibited crizotinib-induced apoptosis in cells transformed by EML4-ALK. It also effectively attenuated the crizotinib-induced inhibition of EML4-ALK and its downstream molecules, STAT3 and ERK, and suppressed the inhibitory effects of crizotinib on EML4-ALK-mediated transformation in the focus formation assay. On the other hand, other members of the vitamin E family, namely, β-tocopherol, γ-tocopherol, δ-tocopherol, and α-tocotrienol, and a water-soluble analog of vitamin E, Trolox had no effects on the anti-tumor activity of crizotinib in cells transformed by EML4-ALK. Collectively, these results revealed the risk of the anti-tumor activity of crizotinib being attenuated when it is administrated in combination with vitamin E supplements containing α-tocopherol as a major component.

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Hit-to-lead in academia: Discovery of a protein-protein interaction inhibitor of Keap1-Nrf2

Yasuda D., Obata R., Takahashi K., Ohe T., Mashino T.

Yakugaku Zasshi (Yakugaku Zasshi) 138 ( 8 ) 1059 - 1065 2018

ISSN 00316903

© 2018 The Pharmaceutical Society of Japan. In the process of recent hit-to-lead studies, not only in industry but also in academia, early evaluation of metabolic properties has been one of the key aspects supporting a higher probability of success in drug discovery. In this review, we introduce the development of chemical seeds targeting the Kelch-like ECH-associated protein-1 (Keap1) as an example of an academic hit-to-lead study considering metabolic stability. Keap1 regulates the function of nuclear factor erythroid 2-related factor 2 (Nrf2), which induces various antioxidative or detoxification proteins. An inhibitor of protein-protein interaction (PPI) between Keap1 and Nrf2 to activate Nrf2 is expected to be a novel target for drug discovery. However, Nrf2 is also activated in several cancers, such as human hepatocellular carcinoma, and causes chemoresistance, which is mediated by phosphorylated p62/Sqstm1 (p-p62), an autophagy-related protein that also undergoes a PPI with Keap1. In this case, an Nrf2 suppressor could be used to attenuate drug resistance.We discovered inhibitors against the Nrf2-Keap1 PPI and p-p62-Keap1 PPI using high-throughput screening and established the synthetic routes for the hit compounds and their derivatives. Furthermore, we assessed the metabolic stability of both of the PPI inhibitors in human liver microsomes and identified the metabolic sites.

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Discovery of benzo[g]indoles as a novel class of non-covalent Keap1-Nrf2 protein-protein interaction inhibitor

Yasuda, D., Yuasa, A., Obata, R., Nakajima M., Takahashi, K., Ohe, T., Ichimura, Y., Komatsu, M., Yamamoto, M., Imamura, R., Kojima, H., Okabe, T., Nagano, T., and Mashino, T.

Bioorg Med Chem Lett 27 ( 22 ) 5006 - 5009 2017.11

Research paper (scientific journal), Joint Work, Accepted

alpha-Tocopherol attenuates the anti-tumor activity of crizotinib against cells transformed by NPM-ALK

Funakoshi-Tago, M., Uchihara, Y., Ueda, F., Tago, K., Nakazawa, Y., Ohe, T., Mashino, T., Yokota, S., Kasahara, T., and Tamura, H.

PLOS ONE 12 ( 8 ) e0183003 2017.08

Research paper (scientific journal), Joint Work, Accepted

Synthesis of Keap1-phosphorylated p62 and Keap1-Nrf2 protein-protein interaction inhibitors and their inhibitory activity

MASHINO Tadahiko

Bioorg Med Chem Lett 26 ( 24 ) 5956 - 5959 2016.12

Research paper (scientific journal), Joint Work, Accepted

Potential suppressive effects of two C60 fullerene derivatives on acquired immunity

MASHINO Tadahiko

Nanoscale Res Lett 11 449 2016.11

Research paper (scientific journal), Joint Work, Accepted

Novel fullerene derivatives as dual inhibitors of Hepatitis C virus NS5B polymerase and NS3/4A protease

MASHINO Tadahiko

Bioorg Med Chem Lett, 26 4565 - 4567 2016.10

Research paper (scientific journal), Joint Work, Accepted

p62/Sqstm1 promotes malignancy of HCV-positive hepatocellular carcinoma through Nrf2-dependent metabolic reprogramming

MASHINO Tadahiko

Nat Commun 7 1 - 16 2016

Research paper (scientific journal), Joint Work, Accepted

Preparation and antioxidant/pro-oxidant activities of 3-monosubstituted 5-hydroxyoxindole derivatives

MASHINO Tadahiko

J Clin Biochem Nutr 59 165 - 173 2016

Research paper (scientific journal), Joint Work, Accepted

A proline-type fullerene derivative inhibits adipogenesis by preventing PPARgamma activation

MASHINO Tadahiko

Biochemistry and Biophysics Reports 20 258 - 263 2016

Research paper (scientific journal), Joint Work, Accepted

Novel bioactivation pathway of benzbromarone mediated by cytochrome P450

Kitagawara Yumina, Ohe Tomoyuki, Tachibana Kumiko, Takahashi Kyoko, Nakamura Shigeo, Mashino Tadahiko

Drug Metabolism and Disposition 43 ( 9 ) 1303 - 1306 2015.09

Research paper (scientific journal), ISSN 0090-9556

Benzbromarone (BBR) is a hepatotoxic drug, but the detailed mechanism of its toxicity remains unknown. We identified 2,6-dibromohydroquinone (DBH) and mono-debrominated catechol (2-ethyl-3-(3-bromo-4,5-dihydroxybenzoyl) benzofuran; CAT) as novel metabolites of BBR in rat and human liver microsomal systems by comparison with chemically synthesized authentic compounds, and we also elucidated that DBH is formed by cytochrome P450 2C9 and that CAT is formed mainly by CYP1A1, 2D6, 2E1, and 3A4. Furthermore, CAT, DBH, and the oxidized form of DBH are highly cytotoxic in HepG2 compared with BBR. Taken together, our data demonstrate that DBH, a novel reactive metabolite, may be relevant to BBR-induced hepatotoxicity.

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The human immunodeficiency virus-reverse transcriptase inhibition activity of novel pyridine/pyridinium-type fullerene derivatives

Yasuno Takumi, Ohe Tomoyuki, Takahashi Kyoko, Nakamura Shigeo, Mashino Tadahiko

Bioorganic and Medicinal Chemistry Letters 25 ( 16 ) 3226 - 3229 2015.07

Research paper (scientific journal), ISSN 0960-894X

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Pyrrolidinium fullerene induces apoptosis by activation of procaspase-9 via suppression of Akt in primary effusion lymphoma

Watanabe Tadashi, Nakamura Shigeo, Ono Toshiya, Ui Sadaharu, Yagi Syota, Kagawa Hiroki, Watanabe Hisami, Ohe Tomoyuki, Mashino Tadahiko, Fujimuro Masahiro

Biochemical and Biophysical Research Communications 451 ( 1 ) 93 - 100 2014.08

Research paper (scientific journal), ISSN 0006-291X

Primary effusion lymphoma (PEL) is a subtype of non-Hodgkin's B-cell lymphoma and is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients. In general, PEL cells are derived from post-germinal center B-cells and are infected with KSHV. To evaluate potential novel anti-tumor compounds against KSHV-associated PEL, seven water-soluble fullerene derivatives were evaluated as potential drug candidates for the treatment of PEL. Herein, we discovered a pyrrolidinium fullerene derivative, 1,1,1′,1′-tetramethyl [60]fullerenodipyrrolidinium diiodide, which induced apoptosis of PEL cells via a novel mechanism, the caspase-9 activation by suppressing the caspase-9 phosphorylation, causing caspase-9 inactivation. Pyrrolidinium fullerene treatment reduced significantly the viability of PEL cells compared with KSHV-uninfected lymphoma cells, and induced the apoptosis of PEL cells by activating caspase-9 via procaspase-9 cleavage. Pyrrolidinium fullerene additionally reduced the Ser473 phosphorylation of Akt and Ser196 of procaspase-9. Ser473-phosphorylated Akt (i.e., activated Akt) phosphorylates Ser196 in procaspase-9, causing inactivation of procaspase-9. We also demonstrated that Akt inhibitors suppressed the proliferation of PEL cells compared with KSHV-uninfected cells. Our data therefore suggest that Akt activation is essential for cell survival in PEL and a pyrrolidinium fullerene derivative induced apoptosis by activating caspase-9 via suppression of Akt in PEL cells. In addition, we evaluated whether pyrrolidinium fullerene in combination with the HSP90 inhibitor (geldanamycin; GA) or valproate, potentiated the cytotoxic effects on PEL cells. Compared to treatment with pyrrolidinium fullerene alone, the addition of low-concentration GA or valproate enhanced the cytotoxic activity of pyrrolidinium fullerene. These results indicate that pyrrolidinium fullerene could be used as a novel therapy for the treatment of PEL. © 2014 Elsevier Inc. All rights reserved.

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Effect of chemical modification on the ability of pyrrolidinium fullerene to induce apoptosis of cells transformed by JAK2 V617F mutant

Funakoshi-Tago, Megumi, Tsukada, Masaki, Watanabe, Toshiro, Mameda, Yuka, Tago, Kenji, Ohe, Tomoyuki, Nakamura, Shigeo, Mashino, Tadahiko, Kasahara, Tadashi

INTERNATIONAL IMMUNOPHARMACOLOGY 20 ( 1 ) 258 - 263 2014.05

Research paper (scientific journal), ISSN 1567-5769

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Antioxidant activities of 5-hydroxyoxindole and its 3-hydroxy-3-phenacyl derivatives: The suppression of lipid peroxidation and intracellular oxidative stress.

Yasuda D, Takahashi K, Ohe T, Nakamura S, Mashino T.

Bioorg Med Chem 21 7709-7714 2013.12

Research paper (scientific journal), Joint Work, Accepted

Anti-influenza activity of c60 fullerene derivatives.

Shoji M, Takahashi E, Hatakeyama D, Iwai Y, Morita Y, Shirayama R, Echigo N, Kido H, Nakamura S, Mashino T, Okutani T, Kuzuhara T.

PLoS One 8 1-10 2013.06

Research paper (scientific journal), Joint Work, Accepted

Synthesis, radical scavenging activity and structure-activity relationship of uric acid analogs

Yasuda Daisuke, Takahashi Kyoko, Kakinoki Tomohiro, Tanaka Yoko, Ohe Tomoyuki, Nakamura Shigeo, Mashino Tadahiko

MedChemComm 4 ( 3 ) 527 - 529 2013.03

Research paper (scientific journal), ISSN 2040-2503

Uric acid (UA) is known to play an important role as an endogenous antioxidant. However, its insolubility in the serum is a risk for hyperuricemia. We assume that UA is an equivalent to hydroquinone or p-aminophenol, which can be oxidized to quinone/quinoimine and thus acts as a radical scavenger. Based on this hypothesis, a series of UA analogs was designed and synthesized. In the chemical radical scavenging assay, active compounds were considered as hydroquinone or p-aminophenol equivalents. A highly functionalized UA structure is not essential to have radical scavenging activity. Potent active 5-hydroxyindolinones (1a, 2a, and 3a) showed sufficient activity with high solubility and low cytotoxicity. © 2013 The Royal Society of Chemistry.

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Fullerene derivative prevents cellular transformation induced by JAK2 V617F mutant through inhibiting c-Jun N-terminal kinase pathway

Funakoshi-Tago Megumi, Nagata Tatsuaki, Tago Kenji, Tsukada Masaki, Tanaka Kazuyuki, Nakamura Shigeo, Mashino Tadahiko, Kasahara Tadashi

Cellular Signalling 24 ( 11 ) 2024 - 2034 2012.11

Research paper (scientific journal), ISSN 0898-6568

The constitutively activated mutation (V617F) of tyrosine kinase Janus kinase 2 (JAK2) is found in the majority of patients with myeloproliferative neoplasms (MPNs). The development of a novel chemical compound to suppress JAK2 V617F mutant-induced onset of MPNs and clarification of the signaling cascade downstream of JAK2 V617F mutant will provide clues to treat MPNs. Here we found that a water-soluble pyrrolidinium fullerene derivative, C₆₀-bis (N, N-dimethylpyrrolidinium iodide), markedly induced apoptosis of JAK2 V617F mutant-induced transformed cells through a novel mechanism, inhibiting c-Jun N-terminal kinase (JNK) activation pathway but not generation of reactive oxygen species (ROS). Pyrrolidinium fullerene derivative significantly reduced the protein expression level of apoptosis signal-regulating kinase 1 (ASK1), one of the mitogen-activated protein kinase kinase kinases (MAPKKK), resulting in the inhibition of upstream molecules of JNK, mitogen-activated protein kinase kinase 4 (MKK4) and mitogen-activated protein kinase kinase 7 (MKK7). Strikingly, the knockdown of ASK1 enhanced the sensitivity to pyrrolidinium fullerene derivative-induced apoptosis, and the treatment with a JNK inhibitor, SP600125, also induced apoptosis of the transformed cells by JAK2 V617F mutant. Furthermore, administration of both SP600125 and pyrrolidinium fullerene derivative markedly inhibited JAK2 V617F mutant-induced tumorigenesis in nude mice. Taking these findings together, JAK2 V617F mutant-induced JNK signaling pathway is an attractive target for MPN therapy, and pyrrolidinium fullerene derivative is now considered a candidate potent drug for MPNs. © 2012 Elsevier Inc.

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Water-soluble fullerene derivatives for drug discovery

Shigeo Nakamura; Tadahiko Mashino

Journal of Nippon Medical School 79 ( 4 ) 248 - 254 2012.08

Research paper (scientific journal)

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Impairment of heme biosynthesis induces short circadian period in body temperature rhythms in mice

Reiko Iwadate; Yoko Satoh; Yukino Watanabe; Hiroshi Kawai; Naomi Kudo; Yoichi Kawashima; Tadahiko Mashino; Atsushi Mitsumoto

American Journal of Physiology - Regulatory Integrative and Comparative Physiology 303 ( 1 ) R8 - R18 2012.07

Research paper (scientific journal)

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Isolation and structural identification of a direct-acting mutagen derived from N-nitroso-N-methylpentylamine and Fenton's reagent with copper ion

Miura Motofumi, Inami Keiko, Yoshida Masafumi, Yamaguchi Kentaro, Mashino Tadahiko, Mochizuki Masataka

Bioorganic and Medicinal Chemistry 19 ( 18 ) 5693 - 5697 2011.09

Research paper (scientific journal), ISSN 0968-0896

N-Nitrosodialkylamines show their mutagenicity by forming α-hydroxynitrosamines in the presence of rat S9 mix in the Ames assay. The hydroxyl radical derived from Fe ²⁺-H ₂O ₂ (Fenton's reagent) with Cu ²⁺ activates N-nitrosamines, with an alkyl chain longer than a propyl constituent, to a direct-acting mutagen. The reactivity of Fe ²⁺-Cu ²⁺-H ₂O ₂ on nitrosamines in relation to their metabolic activation is not fully characterized. Here, we report the identification of the direct-acting mutagen derived from N-nitroso-N-methylpentylamine (NMPe) in the presence of Fe ²⁺, Cu ²⁺, H ₂O ₂ and nitric oxide (NO), which is a product of nitrosamine metabolism. A dichloromethane extract of the NMPe reaction mixtures was fractionated by silica gel column chromatography several times and by a preparative high performance liquid chromatography (HPLC); we obtained white crystals as a product. The direct-acting mutagen that was isolated was provisionally identified as 5-ethyl-5-nitro-1-pyrazoline 1-oxide by ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy, infrared (IR) spectroscopy and X-ray crystallography. To confirm the structure of the mutagen, the authentic compound was synthesized from 2-nitrobutene and diazomethane, followed by N-oxidation with m-chloroperoxybenzoic acid. The ¹H NMR spectral data from the direct-acting mutagen that was synthesized was identical to the data from the isolated mutagen. Furthermore, the authentic 5-ethyl-5-nitro-1-pyrazoline 1-oxide was mutagenic in Salmonella typhimurium TA1535. The results showed that 5-ethyl-5-nitro-1-pyrazoline 1-oxide was a direct-acting mutagen derived from the reaction of NMPe and Fe ²⁺-Cu ²⁺-H ₂O ₂-NO. © 2011 Elsevier Ltd. All rights reserved.

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Anti-inflammatory activity of structurally related flavonoids, Apigenin, Luteolin and Fisetin.

Funakoshi-Tago M*, Nakamura K, Tago K, Mashino T, Kasahara T.

Int Immunopharmacol 11(9) 1150-1159 2011.09

Research paper (scientific journal), Joint Work, Accepted

Ipso Substitution of bisphenol A catalyzed by microsomal cytochrome P450 and Enhancement of estrogenic activity.

Nakamura S, Tezuka Y, Ushiyama A, Kawashima C, Kitagawara Yumina Takahashi K, Ohto S, Mashino T.

Toxicology Letters 203 92-95 2011.05

Research paper (scientific journal), Joint Work, Accepted

The fixed structure of Licochalcone A by α, β-unsaturated ketone is necessary for anti-inflammatory activity through the inhibition of NF-κB activation

Funakoshi-Tago Megumi, Nakamura Kei, Tsuruya Rina, Hatanaka Masashi, Mashino Tadahiko, Sonoda Yoshiko, Kasahara Tadashi

International Immunopharmacology 10 ( 5 ) 562 - 571 2010.05

Research paper (scientific journal), ISSN 1567-5769

Glycyrrhiza inflata has been used as a traditional medicine with anti-inflammatory activity. Previously, we reported that a major component, Licochalcone A, potently inhibited TNFα-induced NF-κB activation by inhibiting IKKβ activation. In this study, we investigated whether the fixed structure of Licochalcone A by α, β-unsaturated ketone is required for its inhibitory effect of NF-κB activation. Interestingly, reduced Licochalcone A, which lacks a double bond, failed to inhibit TNFα-induced NF-κB activation. Whereas Licochalcone A potently inhibited TNFα-induced IKK activation, IκBα degradation, nuclear localization of NF-κB and its DNA binding activity, no inhibitory effect was observed by reduced Licochalcone A. In addition, TNFα-induced expression of inflammatory cytokines, CCL2/MCP-1 and CXCL1/KC, was clearly inhibited by Licochalcone A but not reduced Licochalcone A. As a result, culture media pretreated with Licochalcone A but not reduced Licochalcone A following TNFα stimulation significantly inhibited the chemotactic activity of neutrophils. Furthermore, acute carrageenan-induced paw edema in mice was markedly inhibited by administration of Licochalcone A but not reduced Licochalcone A. Taken together, it is suggested that Licochalcone A is a promising anti-inflammatory drug in vivo and its fixed structure is critical for anti-inflammatory activity. © 2010 Elsevier B.V. All rights reserved.

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Biological safety of water-soluble fullerenes evaluated using tests for genotoxicity, phototoxicity, and pro-oxidant activity

Aoshima Hisae, Yamana Shuichi, Nakamura Shigeo, Mashino Tadahiko

Journal of Toxicological Sciences 35 ( 3 ) 401 - 409 2010

Research paper (scientific journal), ISSN 0388-1350

We evaluated the safety of water-soluble polymer-enwrapped fullerenes (PVP/fullerenes) as antioxidants in cosmetic and pharmaceutical preparations by studying the genotoxicity, phototoxicity, and pro-oxidant effects of these fullerenes. These materials were not mutagenic to any of the tested bacterial strains and did not induce chromosomal aberrations in cultured mammalian cells. The PVP/fullerenes did not exhibit cytotoxicity under ultraviolet or sham irradiation in the alternative phototoxicity test. Moreover, they did not show any pro-oxidant effect in the presence of Fe2+ or Cu2+. Thus, we concluded that PVP/fullerenes are safe for use in cosmetic and pharmaceutical applications. This is the first study in which toxicity tests were performed on PVP/fullerenes.

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Pyrrolidinium-type fullerene derivative-induced apoptosis by the generation of reactive oxygen species in HL-60 cells.

Nishizawa C, Hashimoto N, Yokoo S, Funakoshi-Tago M, Kasahara T, Takahashi K, Nakamura S, Mashino T*.

Free Radical Res 43(12) 1240-1247 2009.12

Research paper (scientific journal), Joint Work, Accepted

Licochalcone A potently inhibits tumor necrosis factor alpha-induced nuclear factor-kappaB activation through the direct inhibition of IkappaB kinase complex activation.

Funakoshi-Tago M*, Tanabe S, Tago K, Itoh H, Mashino T, Sonoda Y, Kasahara T.

Molecular Pharmacology 76(4) 745-753 2009.10

Research paper (scientific journal), Joint Work, Accepted

Licochalcone A significantly suppresses LPS signaling pathway through the inhibition of NF-κB p65 phosphorylation at serine 276

Furusawa Jun ichi, Funakoshi-Tago Megumi, Tago Kenji, Mashino Tadahiko, Inoue Hideo, Sonoda Yoshiko, Kasahara Tadashi

Cellular Signalling 21 ( 5 ) 778 - 785 2009.05

Research paper (scientific journal), ISSN 0898-6568

Licorice root, Glycyrrhiza inflata, has been used as a traditional medicine for the treatment of bronchial asthma and inflammation; however, the mechanism of its anti-inflammatory activity has not been clarified. Here, we investigated the effect of Licochalcone A, a major component of G. inflata, on the LPS signaling pathway. We found that Licochalcone A remarkably inhibited LPS-induced NO production, and TNFα expression and MCP-1 expression in both RAW264.7 cells and primary macrophages. Furthermore, when injected with Licochalcone A prior to injection of LPS, the serum level of TNFα and MCP-1 in C57BL/6 mice was clearly decreased, indicating that Licochalcone A has a potent anti-inflammatory effect both in vitro and in vivo. Strikingly, Licochalcone A significantly inhibited LPS-induced NF-κB transcriptional activation; however, it had no effect on not only the phosphorylation and degradation of IκBα but also nuclear translocation and DNA binding activity of NF-κB p65. Interestingly, Licochalcone A markedly inhibited the phosphorylation of p65 at serine 276. As a result, it reduced NF-κB transactivation by preventing the interaction of p65 with p300. Taken together, Licochalcone A might contribute to the potent anti-inflammatory effect of G. inflata through the unique mechanism of NF-κB inhibition. © 2009 Elsevier Inc. All rights reserved.

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Glycyrrhiza inflata-derived chalcones, Licochalcone A, Licochalcone B and Licochalcone D, inhibit phosphorylation of NF-κB p65 in LPS signaling pathway

Furusawa Jun ichi, Funakoshi-Tago Megumi, Mashino Tadahiko, Tago Kenji, Inoue Hideo, Sonoda Yoshiko, Kasahara Tadashi

International Immunopharmacology 9 ( 4 ) 499 - 507 2009.04

Research paper (scientific journal), ISSN 1567-5769

Licorice root has been used as a traditional medicine for the treatment of gastric ulcer, bronchial asthma and inflammation. Licochalcone A is a major component of Xinjiang licorice, Glycyrrhiza inflata. Previously we showed that Licochalcone A significantly inhibited LPS-induced NF-κB transcriptional activation by abrogating the phosphorylation of NF-κB p65 at serine 276. Glycyrrhiza inflata contains not only Licochalcone A but also Licochalcone B, Licochalcone C, Licochalcone D, Echinatin and Isoliquiritigenin, harboring the common structure of chalcones. No chalcones had any effect on LPS-induced IκB degradation, nuclear translocation and DNA binding activity of NF-κB p65; however, we observed that Licochalcone B and Licochalcone D significantly inhibited LPS-induced phosphorylation at serine 276 and transcriptional activation of NF-κB, the same as Licochalcone A. Interestingly, we also found that Licochalcone A, Licochalcone B and Licochalcone D effectively inhibited LPS-induced activation of PKA, which is required for the phosphorylation of NF-κB p65 at serine 276. Consequently, Licochalcone B and Licochalcone D significantly reduced the LPS-induced production of NO, TNFα and MCP-1. On the other hand, Licochalcone C, Echinatin and Isoliquitigenin failed to inhibit LPS-induced NF-κB activation. These findings suggest that the anti-inflammatory effect of Glycyrrhiza inflata is ascribable to the potent inhibition of NF-κB by Licochalcone A, Licochalcone B and Licochalcone D. © 2009 Elsevier B.V. All rights reserved.

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Glycyrrhiza inflata-derived chalcones, Licochalcone A, Licochalcone B and Licochalcone D, inhibit phosphorylation of NF-kappaB p65 in LPS signaling pathway.

Furusawa J, Funakoshi-Tago M*, Mashino T, Tago K, Inoue H, Sonoda Y, Kasahara T.

International Immunopharmacology 9(4) 499-507 2009.04

Research paper (scientific journal), Joint Work, Accepted

Fullerene nanowhiskers and related fullerene nanomaterials.

Miyazawa K*, Kato R, Saito K, Kizuka T, Mashino T, Nakamura S.

J Phys Conf Series 159 012007 2009.04

Research paper (scientific journal), Joint Work, Accepted

Biological activities of water-soluble fullerene derivatives

S. Nakamura; T. Mashino

Journal of Physics: Conference Series 159 2009.01

Research paper (scientific journal)

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Fullerene nanowhiskers and related fullerene nanomaterials

Miyazawa K., Kato R., Saito K., Kizuka T., Mashino T., Nakamura S.

Journal of Physics: Conference Series 159 2009

Research paper (scientific journal), ISSN 1742-6588

Since the discovery of C₆₀ nanowhiskers in a colloidal solution of PZT in 2001, various low-dimensional fullerene nanomaterials such as fullerene nanotubes and fullerene nanosheets have been synthesized by the liquid-liquid interfacial precipitation method (LLIP method) as well as the C₆₀ and C₇₀ nanowhiskers. The LLIP method is an excellent process to synthesize the quasi one-dimensional fullerene nanomaterials. This paper first reviews the typical fullerene nano and micro materials that have been prepared by the LLIP method and then show that the LLIP method is also applicable to the synthesis of composite C₆₀ nano and micro whiskers containing wide compositions of C₆₀ derivative molecules. © 2009 IOP Publishing Ltd.

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In vitro free radical scavenging activity of platinum nanoparticles

Watanabe Aki, Kajita Masashi, Kim Juewon, Kanayama Atsuhiro, Takahashi Kyoko, Mashino Tadahiko, Miyamoto Yusei

Nanotechnology 20 ( 45 ) 2009

Research paper (scientific journal), ISSN 0957-4484

A polyacrylic acid (PAA)-protected platinum nanoparticle species (PAA-Pt) was prepared by alcohol reduction of hexachloroplatinate. The PAA-Pt nanoparticles were well dispersed and homogeneous in size with an average diameter of 2.0 0.4nm (n = 200). We used electron spin resonance to quantify the residual peroxyl radical () generated from 2,2-azobis (2-aminopropane) dihydrochloride (AAPH) by thermal decomposition in the presence of O₂ and a spectrophotometric method to quantify the residual 1,1-diphenyl-2- picrylhydrazyl (DPPH) radical. PAA-Pt scavenged these two radicals in a dose-dependent manner. Platinum was the functional component. PAA-Pt reduced the rate of oxygen consumption required for linoleic acid peroxidation initiated by generated from AAPH, indicating inhibition of the propagation of linolate peroxidation. A thiobarbituric acid test also revealed dose-dependent inhibition of the linolate peroxidation by PAA-Pt. Fifty micromolar platinum, as PAA-Pt, completely quenched 250 νM DPPH radical for 5min. Even when twice diluted in half, the PAA-Pt still quenched 100% of the 250νM DPPH radical. The scavenging activity of PAA-Pt is durable. These observations suggest that PAA-Pt is an efficient scavenger of free radicals. © IOP Publishing Ltd.

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Biological activities of water-soluble fullerene derivatives

Nakamura S., Mashino T.

Journal of Physics: Conference Series 159 2009

Research paper (scientific journal), ISSN 1742-6588

Three types of water-soluble fullerene derivatives were synthesized and their biological activities were investigated. C₆₀-dimalonic acid, an anionic fullerene derivative, showed antioxidant activity such as quenching of superoxide and relief from growth inhibition of E. coli by paraquat. C ₆₀-bis(7V,7V-dimethylpyrrolidinium iodide), a cationic fullerene derivative, has antibacterial activity and antiproliferative effect on cancer cell lines. The mechanism is suggested to be respiratory chain inhibition by reactive oxygen species produced by the cationic fullerene derivative. Proline-type fullerene derivatives showed strong inhibition activities on HIV-reverse transcriptase. The IC₅₀ values were remarkably lower than nevirapine, a clinically used anti-HIV drug. Fullerene derivatives have a big potential for a new type of lead compound to be used as medicine. © 2009 IOP Publishing Ltd.

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Pyrrolidinium-type fullerene derivative-induced apoptosis by the generation of reactive oxygen species in HL-60 cells

Nishizawa Chiho, Hashimoto Noriyuki, Yokoo Sachiko, Funakoshi-Tago Megumi, Kasahara Tadashi, Takahashi Kyoko, Nakamura Shigeo, Mashino Tadahiko

Free Radical Research 43 ( 12 ) 1240 - 1247 2009

Research paper (scientific journal), ISSN 1071-5762

The biological activities of C60-bis(N,N-dimethylpyrrolidinium iodide), a water-soluble cationic fullerene derivative, on human promyeloleukaemia (HL-60) cells were investigated. The pyrrolidinium fullerene derivative showed cytotoxicity in HL-60 cells. The characteristics of apoptosis, such as DNA fragmentation and condensation of chromatin in HL-60 cells, were observed by exposure to the pyrrolidinium fullerene derivative. Caspase-3 and-8 were activated and cytochrome c was also released from mitochondria. The generation of reactive oxygen species (ROS) by the pyrrolidinium fullerene derivative was observed by DCFH-DA, a fluorescence probe for the detection of ROS. Pre-treatment with α-tocopherol suppressed cell death and intracellular oxidative stress caused by the pyrrolidinium fullerene derivative. The apoptotic cell death induced by the pyrrolidinium fullerene derivative was suggested to be mediated by ROS generated by the pyrrolidinium fullerene derivative.

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Licochalcone A is a potent inhibitor of TEL-Jak2-mediated transformation through the specific inhibition of Stat3 activation.

Funakoshi-Tago M*, Tago K, Nishizawa C, Takahashi K, Mashino T, Iwata S, Inoue H, Sonoda Y, Kasahara T.

Biochem Pharmacol 76(12) 1681-1693 2008.12

Research paper (scientific journal), Joint Work, Accepted

2,2′-Pyridoin derivatives protect HL-60 cells against oxidative stress

Hatanaka Masashi, Nishizawa Chiho, Kakinoki Tomohiro, Takahashi Kyoko, Nakamura Shigeo, Mashino Tadahiko

Bioorganic and Medicinal Chemistry Letters 18 ( 19 ) 5290 - 5293 2008.10

Research paper (scientific journal), ISSN 0960-894X

Focusing on 2,2′-pyridoin (1, 1,2-di(2-pyridyl)-1,2-ethenediol) and its synthetic derivatives as the lead compound of the potent antioxidative enediol, their protective effect against oxidative stress was evaluated on the HL-60 cell system. 2,2′-Pyridoins showed no remarkable cytotoxic effect on HL-60 cells. The derivatives 1, 2, 3, 5, and 6 inhibited H₂O₂-induced cell death and intracellular oxidative stress more significantly than ascorbic acid. Since 2,2′-pyridoins are oxidized to the diketones, 2,2′-pyridils, in a protic solvent, the antioxidant activity of 2,2′-pyridils was also investigated. 2,2′-Pyridils showed antioxidant activity in the cell; however, the activity was lower than that of 2,2′-pyridoins. These results suggested that 2,2′-pyrdoin derivatives can be good cytoprotective agents against oxidative stress. © 2008 Elsevier Ltd. All rights reserved.

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C₆₀ nanowhisker field-effect-transistor application for nano-electronics

Ogawa Ken Ichi, Aoki Nobuyuki, Miyazawa Kun'ichi, Nakamura Shigeo, Mashino Tadahiko, Bird Jonathan P., Ochiai Yuichi

Japanese Journal of Applied Physics 47 ( 1 PART 2 ) 501 - 504 2008.01

Research paper (scientific journal), ISSN 0021-4922

Various kinds of field-effect transistor (FET) have been fabricated with C₆₀ nanowhisker (NW) and also studied for nanoelectronics application. Especially, pure and solvated C₆₀ NWs have been synthesized in N2 environment so as to clarify the best device performance of C₆₀ NW-FET. The FET works not only under vacuum but also in N2 environment when kept in the solvated condition. The solvated C₆₀ NW-FET shows a clear improvement of their on/off ratio in the solvated condition, and the highest electron mobility after annealing. Although further study is needed, our results demonstrate the possibility, by appropriate choice of the solvent, of achieving good improvements in FET performance. Moreover, new kinds of C₆₀ NW, such as derivative-based and nanotube-type one, have also been studied with regards to their fundamental FET characteristics. © 2008 The Japan Society of Applied Physics.

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Novel metabolic pathways of p-n-nonylphenol catalyzed by cytochrome P450 and estrogen receptor binding activity of new metabolites

Tezuka Yoshito, Takahashi Kyoko, Suzuki Tomoharu, Kitamura Shigeyuki, Ohta Shigeru, Nakamura Shigeo, Mashino Tadahiko

Journal of Health Science 53 ( 5 ) 552 - 561 2007.10

Research paper (scientific journal), ISSN 1344-9702

Nonylphenol, which is used industrially as a surfactant, is an endocrine-disrupting chemical (EDC) which has estrogenic activity. The novel biotransformation of nonylphenol was investigated, based on our previously reported ipso-metabolism of para-substituted phenols by cytochrome P450 (P450). Three novel metabolites of nonylphenol, i.e., nonylquinol, 4′- hydroxynonanophenone (CO-NP) as benzyl-oxidized nonylphenol, and hydroquinone, were detected in a rat liver microsome reaction mixture. On the other hand, production of 1-(4′-hydroxyphenyl)nonan-1-ol (OH-NP), namely benzyl-hydroxylated nonylphenol, was detected in a human liver microsome reaction mixture. The formation of all these metabolites was suppressed by the addition of P450 inhibitor. This showed that all nonylphenol metabolism was catalyzed by P450. To identify which P450 isoenzyme is involved in each reaction, fourteen human P450 (CYP) isozymes, CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, 3A7, and CYP4A11, were examined. CYP1A1, 1A2, and CYP2B6 effectively catalyzed the production of nonylquinol. CYP2B6 also catalyzed the benzyl-hydroxylation to give OH-NP. Hydroquinone was formed mainly from OH-NP, not via CO-NP. We examined the estrogenic activity of these new metabolites by estrogen receptor (ER)-binding reporter gene assay. Nonylquinol, OH-NP and hydroquinone have no ER-binding activity. However, CO-NP showed the same level of estrogen receptor binding activity as nonylphenol. Moreover, the amount of CONP formed was small. Therefore, the novel metabolic pathways led overall to metabolic inactivation, as concerns the estrogenic activity of nonylphenol through the ER.

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Solubilization of dipropofol, an antibacterial agent, using saccharide and ascorbic acid

Masahiro Ogata; K. Oka; Masako Seki; Midori Hoshi; Hirokatsu Takatsu; Tadahiko Mashino; Shiro Urano; Toyoshige Endo

Biological and Pharmaceutical Bulletin 30 ( 8 ) 1565 - 1568 2007.08

Research paper (scientific journal)

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Iron-chelating agents never suppress Fenton reaction but participate in quenching spin-trapped radicals.

Li L, Abe Y, Kanagawa K, Shoji T, Mashino T, Mochizuki M, Tanaka M, Miyata N.

Analytica Chimica Acta 599 315-319 2007.08

Research paper (conference, symposium, etc.), Joint Work, Accepted

Novel Metabolic Pathways of p-n-Nonylphenol Catalyzed by Cytochrome P450 and Estrogen Receptor Binding Activity of New Metabolites.

Tezuka Y, Takahashi K, Suzuki T, Kitamura S, Ohta S, Nakamura S, Mashino T*.

J Health Sci 53(5) 552-561 2007.07

Research paper (conference, symposium, etc.), Joint Work, Accepted

DMPO-OH Radical Formation form 5,5-Dimethyl-1-pyrroline N-Oxide (DMPO) in Hot Water.

Shoji T, Li L, Abe Y*, Ogata M, Ishimoto Y, Gonda R, Mashino T, Mochizuki M, Uemoto M, Miyata N.

Analytical Sciences 23 219-221 2007.02

Research paper (conference, symposium, etc.), Joint Work, Accepted

TEM and Raman spectroscopy analyses of fullerene nanowhiskers and nanotubes

Miyazawa K., Ringor C., Nakamura K., Tachibana M., Mashino T., Nakamura S.

124 - 127 2007

Research paper (international conference proceedings), Joint Work

Crystalline precipitates composed of C₆₀ and C ₆₀(6,7-dimethoxytetralin) were prepared by the liquid-liquid interfacial precipitation method and examined by Raman spectroscopy and transmission electron microscopy (TEM). The as-prepared C₆₀ nanotubes (C₆₀NTs) dried in air exhibited a Raman spectrum showing a linearly polymerized structure, and a part of them turned to van der Waals polymers after drying in air. On the other hand, the C₆₀NTs dried in air turned to a polymerized structure by irradiation of UV light. The whiskers prepared by use of C₆₀ (6,7- dimethoxytetralin) showed an A_g(2) peak of 1460 cm^-1 suggesting a polymerization of C₆₀ and a Raman spectrum with sharp and clearly splitted peaks in the range of 200-800 cm ^-1, indicating a good crystallinity.

Structural characterization of room-temperature synthesized fullerene nanowhiskers.

Miyazawa K*, Minato J, Mashino T, Nakamura S, Fujino M, Suga T.

NUKLEONIKA 51 S41−48 2006.06

Research paper (conference, symposium, etc.), Joint Work, Accepted

Synthesis of novel amino acid-type fullerenes and their inhibition activity of HIV reverse transcriptase and HCV RNA polymerase.

Nakamura S, Ikegami N, Harada M, Shimotohno K, Takahashi K, Mashino T*.

J Kyoritsu Univ Pharm 1 77-84 2006.03

Research paper (conference, symposium, etc.), Joint Work, Accepted

Metabolic conversion of 24-methyl-D25-cholesterol to 24-methylcholesterol in higher plants.

Takahashi K*, Nasu K, Mashino T, Morisaki M, Hara N, Fujimoto Y.

Bioorg Med Chem 14 732-738 2006.01

Research paper (conference, symposium, etc.), Joint Work, Accepted

Structural characterization of room-temperature synthesized fullerene nanowhiskers

Miyazawa Kun'ichi, Minato Jun Ichi, Mashino Tadahiko, Nakamura Shigeo, Fujino Masahisa, Suga Tadatomo

Nukleonika 51 ( SUPPL. 1 ) 2006

Research paper (scientific journal), ISSN 0029-5922

Structural properties of various kinds of room-temperature synthesized C₆₀ fullerene nanowhiskers were investigated by TEM and XRD. The C₆₀ nanowhiskers prepared by use of toluene lost their initial solvated hexagonal structure faster than the C₆₀ nanowhiskers prepared by use of m-xylene. The hexagonal structure of C₆₀ nanowhiskers prepared by use of toluene was stabilized by adding C ₆₀[C(COOC₂H₅)₂]. These results suggest that large solute molecules in the C₆₀ nanowhisker matrix stabilize their initially solvated hexagonal structure. Furthermore, the addition of a sufficient amount of C₆₀(2-methoxycarbonyl-N- methylpyrrolidine) into the m-xylene solution of C₆₀ produced the C₆₀ nanowhiskers indicating the formation of ordered solid solution of C₆₀ and C₆₀(2-methoxycarbonyl-N-methylpyrrolidine).

Preparation and antioxidant activity of α-pyridoin and its derivatives

Hatanaka Masashi, Takahashi Kyoko, Nakamura Shigeo, Mashino Tadahiko

Bioorganic and Medicinal Chemistry 13 ( 24 ) 6763 - 6770 2005.12

Research paper (scientific journal), ISSN 0968-0896

Focusing on α-pyridoin (1, 1,2-di(2-pyridyl)-1,2-ethenediol) as the lead compound of the novel antioxidative enediol, we synthesized 5,5′- or 6,6′-bis-substituted derivatives of 1 from disubstituted pyridines. The antioxidant activity of 1 and its synthetic derivatives 2-7 was evaluated by DPPH (1,1-diphenyl-2-picrylhydrazyl radical) scavenging assay and inhibition of lipid peroxidation. In the DPPH assay, 1 exhibited an activity stronger than that of ascorbic acid, and 5,5′-dimethyl-(5) or 5,5′-dimethoxy- substituted derivatives (6) exhibited more potent activity than 1. The DPPH scavenging activities of α-pyridoins were correlated with their oxidation potential and thus the electron density of enediol. 5 and 6 effectively inhibited lipid peroxidation in the rat liver microsome/tert-butyl hydroperoxide system. Therefore, 5 and 6 serve as good candidates for a pharmacologically useful enediol antioxidant. © 2005 Elsevier Ltd. All rights reserved.

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Biodegradable polymers in chondrogenesis of human articular chondrocytes

Banu Nasreen, Banu Yasmin, Sakai Masamune, Mashino Tadahiko, Tsuchiya Toshie

Journal of Artificial Organs 8 ( 3 ) 184 - 191 2005.09

Research paper (scientific journal), ISSN 1434-7229

The aim of this study was to evaluate the potential role of polyglycolic acid (PGA), poly(glycolic acid-ε-caprolactone) (PGCL), poly(l-lactic acid-glycolic acid) (PLGA), poly(l-lactic acid-ε-caprolactone, 75:25 (w/w)) [P(LA-CL)25], poly-ε-caprolactone (tetrabutoxy titanium) [PCL(Ti)], and fullerene C-60 dimalonic acid (DMA) in cartilage transplants. After 4 weeks of culture of human articular cartilage, the levels of cell proliferation and differentiation and the expression of cartilage-specific matrix genes were estimated. The relationship between cell differentiation and gap junction protein connexin 43 (Cx43) was also evaluated. All materials except PCL(Ti) retained cell proliferation activities similar to the controls. Cell differentiation levels from the highest to the lowest were in the following order: PGA >> PLGA > PGCL > Control = DMSO > P(LA-CL)25 = PCL(Ti) >> fullerene C-60 DMA. Expression of the collagen type II gene was selectively upregulated for PGA, PGCL, and PLGA and slightly increased for P(LA-CL)25 polymers but was downregulated for fullerene C-60 DMA. Aggrecan gene expression was strongest with PGA and was consistently expressed with other matrices, especially with PGCL and PLGA. However, the expression patterns of the connexin 43 gene were different from the former two genes. Multiple regression analysis revealed a high correlation between cartilage proteoglycans production and expression levels of these three genes. © The Japanese Society for Artificial Organs 2005.

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Human immunodeficiency virus-reverse transcriptase inhibition and hepatitis C virus RNA-dependent RNA polymerase inhibition activities of fullerene derivatives.

Mashino T*, Shimotohno K, Ikegami N, Nishikawa D, Okuda K, Takahashi K, Nakamura S, Mochizuki M.

Bioorg Med Chem Lett 15 1107-1109 2005.01

Research paper (conference, symposium, etc.), Joint Work, Accepted

An HPLC assay of hydroxyl radicals by the hydroxylation reaction of terephthaｌic acid.

Li L, Abe Y*, Nagasawa Y, Kudo R, Usui N, Imai K, Mashino T, Mochizuki M, Miyata N.

Biomedical Chromatography 18 470-474 2004.09

Research paper (conference, symposium, etc.), Single Work, Accepted

Distinguishing the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-OH radical quenching effect from the hydroxyl radical scavenging effect in the ESR spin-trapping method.

Li L, Abe Y*, Kanagawa K, Usui N, Imai K, Mashino T, Mochizuki M, Miyata N.

Analytica Chimica Acta 512 121-124 2004.05

Research paper (conference, symposium, etc.), Joint Work, Accepted

Antibacterial and antiproliferative activity of cationic fullerene derivatives.

Mashino T, Nishikawa D, Takahashi K, Usui N, Yamori T, Seki M, Endo T, Mochizuki M.

Bioorg Med Chem Lett 13 4395-4397 2003.12

Research paper (conference, symposium, etc.), Joint Work, Accepted

Structural characterization of the C60[C(COOC2H5)2] wiskers prepared by the liquid-liquid interfacial precipitation method.

Miyazawa K, Mashino T, and Suga T.

J Mater Res 18(11) 2730-2735 2003.11

Research paper (conference, symposium, etc.), Joint Work, Accepted

Structural characterization of the C₆₀[C(COOC₂H ₅)₂] whiskers prepared by the liquid-liquid interfacial precipitation method

Miyazawa Kun'ichi, Mashino Tadahiko, Suga Tadatomo

Journal of Materials Research 18 ( 11 ) 2730 - 2735 2003.11

Research paper (scientific journal), ISSN 0884-2914

Fine whiskers of a C₆₀ derivative, i.e., C₆₀ monosubstituted by ethyl ester of malonic acid, C₆₀[C(COOC ₂H₅)₂], were fabricated for the first time using a system of toluene with concentrated C₆₀[C(COOC ₂H₅)₂] and isopropyl alcohol. The center-to-center distance of C₆₀[C(COOC₂H ₅)₂] molecules along the close-packed direction of the C₆₀[C(COOC₂H₅)₂] whiskers was in the same range as that of the C₆₀ nanowhiskers prepared by the same method. The C₆₀[C(COOC₂H₅)₂] whiskers were assumed to take body-centered tetragonal or hexagonal close-packed crystal structures. The C₆₀[C(COOC₂H ₅)₂] whiskers showed a high density of stacking faults. The C₆₀[C(COOC₂H₅)₂] whiskers with the growth direction different from the C₆₀ whiskers were observed.

Signal enhancement in ESR spin-trapping for hydroxyl radicals.

Li L, Abe Y*, Mashino T, Mochizuki M, Miyata N.

Analytical Sciences 19 1083-1084 2003.07

Research paper (conference, symposium, etc.), Joint Work, Accepted

Respiratory chain inhibition by fullerene derivatives: Hydrogen peroxide production caused by fullerene derivatives and respiratory chain system.

Mashino T, Usui N, Okuda K, Hirota T, Mochizuki M.

Bioorg Med Chem 11 1433-1438 2003.04

Research paper (conference, symposium, etc.), Joint Work, Accepted

Synthesis of water-soluble C60-porphyrin hybrid compounds.

Okuda K, Abeta C, Hirota T, Mochizuki M, Mashino T*.

Chemical Pharmaceutical Bullitein 50 985-987 2002.07

Research paper (conference, symposium, etc.), Joint Work, Accepted

Effect of hexasulfobutylated C60 on the isolated aortic ring of Guinea pig.

Huang SS, Mashino T, Mochizuki M, Chiang LY, Chih LH, Hsieh HM, Teng CM, Okuda K, Hirota T, Tsai MC*.

Pharmacology 64 91-97 2002.02

Research paper (conference, symposium, etc.), Joint Work, Accepted

Effect of hexasulfobutylated C₆₀ on the isolated aortic ring of guinea pig

Huang Shiang Suo, Mashino Tadahiko, Mochizuki Masataka, Chiang Long Y., Chih Lan Hui, Hsieh Hung Ming, Teng Chen Ming, Okuda Kensuke, Hirota Takasi, Tsai Ming Cheng

Pharmacology 64 ( 2 ) 91 - 97 2002

Research paper (scientific journal), ISSN 0031-7012

The effects of hexasulfobutylated C₆₀ (FC₄S) and monomalonic acid C₆₀ (MMA C₆₀), the fullerene C₆₀ derivatives, on isolated endothelium-containing or endothelium-denuded aorta of guinea pig were studied pharmacologically in vitro. In the endothelium-containing preparation of the aortic rings, phenylephrine (PHE) elicited contracture and acetylcholine (ACh) elicited a relaxing effect on the PHE-precontracted preparation. In the PHE-precontracted preparation, MMA C₆₀ (10 μmol/l) did not elicit a relaxing effect on the PHE-precontracted preparation. However, MMA C₆₀ (10 μmol/l) significantly reduced the maximum response of the relaxation elicited by ACh. FC₄S itself significantly relaxed the PHE-precontracted aortic rings. ACh-induced relaxation in PHE-precontracted endothelium-containing strips of the aortic rings was significantly potentiated if pretreated with FC₄S (10 μmol/l). In the denuded aortic rings, FC₄S did not elicit the relaxing effect in the PHE-precontracted preparation. The relaxing effect of FC₄S on the PHE-precontracted preparation was not altered when superoxide dismutase (250 units/ml) was pretreated. However, the relaxing effect was reduced when N(G)-nitro-L-arginine methyl ester (L-NAME, 1 mmol/l) or methylene blue (1 μmol/l) was pretreated. These results demonstrated that the vasorelaxation effect of FC₄S on the PHE-precontracted aortic ring is partly dependent on the release of nitric oxide (NO) or an NO-derived substance from the vascular endothelium. Copyright © 2002 S. Karger AG, Basel.

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Effects of hexasulfobutylated C60 on the gastric circular muscle of Gunia pig.

Huang SS, Chih LH, Lin CH, Chiang LY, Mashino T, Mochizuki M, Okuda K, Hirota T, Tsai MC*.

Fullerene Sci Tech 9(3) 375-395 2001.09

Research paper (conference, symposium, etc.), Joint Work, Accepted

Inhibitory effects of fullerene C60 derivatives on endothelium-derived relaxation in rabbit thoracic aorta.

Satoh M*, Mashino T, Nagano T, Hirobe M, Takayanagi I, and Koike K.

Fullerene Sci Tech 9 141-151 2001.04

Research paper (conference, symposium, etc.), Joint Work, Accepted

Inhibitory effect of fullerene derivatives on glutathione reductase.

Mashino T*, Okuda K, Hirota T, Hirobe M, Nagano T, Mochizuki M.

Fullerene Sci Tech 9 191-196 2001.04

Research paper (conference, symposium, etc.), Joint Work, Accepted

Inhibitory effect of fullerene derivatives on glutathione reductase

Mashino T., Okuda K., Hirota T., Hirobe M., Nagano T., Mochizuki M.

Fullerene Science and Technology 9 ( 2 ) 191 - 196 2001

Research paper (scientific journal), Joint Work, ISSN 1064-122X

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Inhibitory effects of fullerene C₆₀ derivatives on endothelium-derived relaxation in rabbit thoracic aorta

Satoh M., Mashino T., Nagano T., Hirobe M., Takayanagi I., Koike K.

Fullerene Science and Technology 9 ( 2 ) 141 - 151 2001

Research paper (scientific journal), Joint Work, ISSN 1064-122X

Inhibitory effects of newly synthesized fullerene C₆₀ derivatives 1 (C₆₀-bis(N,N-dimethylpyrrolidinium iodide)), 2 (C₆₀-proline-N-acetic acid) and 3 (C₆₀-ethylenediamine-N, N′-diacetic acid) on acetylcholine-induced relaxation in endothelium-intact rabbit thoracic aorta precontracted by phenylephrine (10^-6 M) were studied. Fullerene C₆₀ derivative 1 (3 × 10^-6 M), 2 (10^-5 M) and 3 (10^-5 M) reduced the maximum amplitude of the acetylcholine-induced relaxation without significantly changing the pD₂ values obtained from the concentration - response curves. In the presence of fullerene C₆₀ derivative 1 (10^-5 M) the acetylcholine-induced relaxation was eliminated and an acetylcholine-induced contraction was observed. These results suggest that fullerene C₆₀ derivative 1 strongly inhibits endothelium (nitric oxide)-dependent acetylcholine-induced relaxation in thoracic aorta of rabbit.

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Antioxidant Activity and Xanthine Oxidase Inhibition Activity of Reductic Acid; Ascorbic Acid Analog.

Mashino T, Takigawa Y, Saito N, Wong L Q, Mochizuki M.

Bioorg Med Chem Lett 10 2783-2785 2000.12

Research paper (conference, symposium, etc.), Joint Work, Accepted

Synthesis of various water-soluble C60 derivatives and their superoxide-quenching activity.

Okuda K, Hirota T, Hirobe M, Nagano T, Mochizuki M, Mashino T.

Fullerene Sci Tech 8 89-104 2000.09

Research paper (conference, symposium, etc.), Joint Work, Accepted

Novel metabolic pathway of estrone and 17b-estradiol catalyzed by cytochrome P450.

Ohe T, Hirobe M, Mashino T.

Drug Metab Dispos 28 110-112 2000.01

Research paper (scientific journal), Joint Work, Accepted

Novel metabolic pathway of estrone and 17β-estradiol catalyzed by cytochrome P-450

Mashino Tadahiko, Ohe Tomoyuki, Hirobe Masaaki

Drug Metabolism and Disposition 28 ( 2 ) 110 - 112 2000

Research paper (scientific journal), Joint Work, ISSN 0090-9556

We have already reported that the quinol formation from some para- alkylphenols, which is a novel metabolic pathway catalyzed by cytochrome P- 450, occurs in a rat liver microsomal system (Ohe et al., 1997). In the present study, we investigated whether estrone and 17β-estadiol, each of which contains a p-alkylphenol moiety, are also oxidized into the corresponding quinols by cytochrome P-450. Six recombinant human cytochrome P-450 enzymes, CYP1A1, CYP1A2, CYP2B6, CYP2C9, CYP2E1, and CYP3A4, were tested. The results show that estrone and 17β-estadiol were converted into the corresponding quinols by CYP1A1, CYP2B6, and CYP2E1.

Inhibition of E. coli growth by fullerene derivatives and inhibition mechanism.

Mashino T, Okuda K, Hirota T, Hirobe M, Nagano T, and Mochizuki M.

Bioorg Med Chem Lett 9 2959-2962 1999.07

Research paper (conference, symposium, etc.), Joint Work, Accepted

Inhibitory effect of a fullerene derivative, monomalonic acid C₆₀, on nitric oxide-dependent relaxation of aortic smooth muscle

Satoh Mitsutoshi, Matsuo Kazuhiro, Kiriya Hitoshi, Mashino Tadahiko, Hirobe Masaaki, Takayanagi Issei

General Pharmacology 29 ( 3 ) 345 - 351 1997.09

Research paper (scientific journal), Joint Work, ISSN 0306-3623

1. Effects of a fullerene C₆₀, derivative, monomalonic acid C₆₀ (MMA C₆₀), on endothelium-containing or denuded aorta of rabbit, trachea and ileum of guinea pig, and stomach (fundus), vas deferens and uterus of rat were studied pharmacologically. 2. MMA C₆₀ (10^-5 M) significantly reduced the maximum response of the relaxation induced by acetylcholine in endothelium containing thoracic aorta of rabbit, and the acetylcholine induced relaxation was recovered in the presence of superoxide dismutase (SOD, 250 units/ml). 3. Nitric oxide-generating agent, S-nitroso-N-acetylpenicillamine, caused the relaxation of aorta without endothelium in a concentration dependent manner, and the concentration-response curve was shifted to the right in the presence of MMA C₆₀. This inhibitory effect of the derivative was also masked in the presence of superoxide dismutase (SOD). 4. Sodium nitroprusside-induced relaxation was not affected by either MMA C₆₀ Or SOD. In the other tissues, this C₆₀ derivative had no effect on the responses induced by any agonist. 5. These observations indicate that MMA C₆₀ inhibits the endothelium dependent relaxation induced by acetylcholine but does not affect the agonist-induced contractile response of smooth muscle.

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Inhibitory effects of a fullerene derivative, dimalonic acid C60, on nitric oxide-induced relaxation of rabbit aorta.

Satoh M., Matsuo K., Kiriya H., Mashino T., Nagano T., Hirobe M., Takayanagi I.

European Journal of Pharmacology 327 ( 2-3 ) 175 - 181 1997.05

Research paper (scientific journal), Joint Work, ISSN 0014-2999

Dimalonic acid C60 (10(-5) M), a new fullerene derivative, produced an augmentation of phenylephrine-induced tone and reduced both the acetylcholine-induced maximum relaxation and the amplitude of substance P (10(-8) M)-induced relaxation in endothelium-containing thoracic aorta of rabbit; the acetylcholine- and substance P-induced relaxation was restored in the presence of superoxide dismutase (250 U/ml). Dimalonic acid C60 (10(-5) M) did not influence the phenylephrine-induced contractile response in the absence of endothelium, but the acetylcholine-induced relaxation was eliminated by removal of the endothelium. Superoxide anion generation, using hypoxanthine (1 mM)/xanthine oxidase (16 mU/ml), reduced the acetylcholine-induced relaxation and produced an augmentation of phenylephrine-induced tone in endothelium-containing strips; these effects were negated by the addition of superoxide dismutase (250 U/ml). A nitric oxide-generating agent, S-nitroso-N-acetylpenicillamine, caused relaxation of aorta without endothelium in a concentration-dependent manner, and the concentration-response curve was shifted to the right in the presence of dimalonic acid C60. This inhibitory effect of dimalonic acid C60 was also masked in the presence of superoxide dismutase. Sodium nitroprusside-induced relaxation was not affected by either dimalonic acid C60 or superoxide dismutase. These observations suggest that dimalonic acid C60 inhibits endothelium (nitric oxide)-dependent agonist-induced relaxation through the production of superoxide.

Substituent elimination from p-substituted phenols by cytochrome P450 ipso-substitution by the oxygen atom of the active species

Ohe Tomoyuki, Mashino Tadahiko, Hirobe Masaaki

Drug Metabolism and Disposition 25 ( 1 ) 116 - 122 1997.01

Research paper (scientific journal), Joint Work, ISSN 0090-9556

When various p-substituted phenols (substituent = NO₂, CN, CH₂OH, COCH₃, COPh, COOH, F, Cl, and Br) were incubated with rat liver microsomes, the substituent was eliminated to produce hydroquinone, and the reaction was inhibited by CO and a cytochrome P450-specific inhibitor. In the case of p- cresol (substituent = CH₃), p-toluquinol was formed instead of hydroquinone. Experiments using ¹⁸O₂ proved that the elimination is accompanied with ipso-substitution by the oxygen atom of the active species in cytochrome P450. These results are similar to those in a cytochrome P450 chemical model system (Ohe, T., et al., Tetrahedron Lett. 42, 7681-7684, 1995), implying that the model is a good mimic of cytochrome P450. Substrates that lack a hydroxy group, namely p-substituted toluenes, did not undergo the reaction, thus indicating that a hydroxy group at the p-position to the eliminated substituent is necessary for this pathway. This is the same as the result obtained with the cytochrome P450 model. Finally, to elucidate how the substituent is eliminated, we attempted to detect the product derived from the eliminated group with several substrates. Results indicated that the mechanism of the substituent elimination can be divided into two types: the substituent is eliminated as an anion or as a cation.

Singlet oxygen production from fullerene derivatives

Hamano Takeshi, Okuda Kensuke, Mashino Tadahiko, Hirobe Masaaki, Arakane Kumi, Ryu Akemi, Mashiko Shinrou, Nagano Tetsuo

Chemical Communications ( 1 ) 21 - 22 1997

Research paper (scientific journal), Joint Work, ISSN 1359-7345

Superoxide radical quenching and cytochrome C peroxidase-like activity of C₆₀-dimalonic acid, C₆₂(COOH)₄

Okuda Kensuke, Mashino Tadahiko, Hirobe Masaaki

Bioorganic and Medicinal Chemistry Letters 6 ( 5 ) 539 - 542 1996.03

Research paper (scientific journal), Joint Work, ISSN 0960-894X

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Novel oxidative pathway of para-substituted phenols in cytochrome P450 chemical model

Ohe Tomoyuki, Mashino Tadahiko, Hirobe Masaaki

Tetrahedron Letters 36 ( 42 ) 7681 - 7684 1995.10

Research paper (scientific journal), Joint Work, ISSN 0040-4039

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Hydroxylation of benzene by horseradish peroxidase and immobilized horseradish peroxidase in an organic solvent

Akasaka Reiko, Mashino Tadahiko, Hirobe Masaaki

Bioorganic and Medicinal Chemistry Letters 5 ( 16 ) 1861 - 1864 1995.08

Research paper (scientific journal), Joint Work, ISSN 0960-894X

Horseradish peroxidase (HRP) catalyzed hydroxylation of benzene with an oxidant when benzene was used as the reaction solvent. HRP immobilized on poly(γ-methyl-L-glutamate) also catalyzed the reaction with higher activity than that of free HRP. The results of the reaction using [¹⁸O]hydrogen peroxide show that free and immobilized HRP incorporated the oxygen atom of the reactive species into the product, indicating that both forms of HRP can catalyze hydroxylation of benzene in an analogous manner to that of cytochrome P450. © 1995.

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Oxidation of [60]fullerene by cytochrome P450 chemical models

Hamano Takeshi, Mashino Tadahiko, Hirobe Masaaki

Journal of the Chemical Society, Chemical Communications ( 15 ) 1537 - 1538 1995

Research paper (scientific journal), Joint Work, ISSN 0022-4936

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Novel Metabolic Pathway of Arylethers by Cytochrome P450

Ohe T., Mashino T., Hirobe M.

Archives of Biochemistry and Biophysics 310 ( 2 ) 402 - 409 1994.05

Research paper (scientific journal), Joint Work, ISSN 0003-9861

We have found a novel metabolic pathway of arylethers, involving the cleavage of the oxygen-aromatic ring bond. When p-(p-nitrophenoxy)phenol was utilized as a substrate, cleaved products, p-nitrophenol and p-benzoquinone, were formed in two cytochrome P450 model systems, meso-tetraphenylporphinatoiron(III) chloride-NaBH₄/O₂ system and meso-tetrakis (2,6-difluorophenyl)porphinatoiron(III) chloride-m-chloroperoxybenzoic acid (mCPBA) system. Rat liver microsomes also catalyzed this reaction, which was inhibited by a cytochrome P450-specific inhibitor, and it was confirmed that this cleavage proceeded in vivo. Further, experiments using [¹⁸O]mCPBA and ¹⁸O₂ proved that the cleavage reaction is accompanied with the ipso-substitution by the oxygen atom of the active species in both cytochrome P450 model system and cytochrome P450. When the microsomal reactions of p-(p-nitrophenoxy)phenol analogues which lack a hydroxy group, namely p-nitrophenoxybenzene, p-(p-nitrophenoxy) anisole, and p-(p-nitrophenoxy) toluene, were investigated, the cleavage reaction occurred via p-(p-nitrophenoxy)phenol in the cases of p-nitrophenoxybenzene and p-(p-nitrophenoxy)anisole, indicating that a hydroxy group at the p-position to the ether bond is necessary for this pathway. This metabolic pathway appears to be important, because a diarylether linkage, which is very stable and has generally been thought to resist metabolism, is cleaved and benzoquinone, a highly toxic metabolite, is formed. © 1994 Academic Press. All rights reserved.

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Hydroxylation of benzene by immobilized cytochrome c in an organic solvent

Akasaka Reiko, Mashino Tadahiko, Hirobe Masaaki

Journal of the Chemical Society, Perkin Transactions 1 ( 13 ) 1817 - 1821 1994

Research paper (scientific journal), Joint Work, ISSN 1472-7781

Recently we showed that cytochrome c (cyt.c) has some cytochrome P450 (P450)-like substrate oxidation activity, which was enhanced by immobilization on poly-γ-methyl-L-glutamate. We now report that immobilized cyt.c catalysed hydroxylation of benzene when benzene containing a small amount of water was used as the reaction solvent, whereas free cyt.c did not catalyse the reaction. The EPR spectrum of immobilized cyt.c showed that the immobilization had caused a change in the protein conformation, and this might have led cyt.c to acquire the new activity. The results of the reaction using [ ¹⁸O]m-chloroperbenzoic acid (mCPBA) or [¹⁸O] molecular dioxygen showed that 59% of the oxygen atom of the product, phenol, was derived from the oxidant, and 33% from molecular dioxygen. When [18O]mCPBA was used as an oxidant, the value of deuterium isotope effect (k_H/k _D)was 1.08 when ¹⁸O was incorporated into the product, and 1.48 when ¹⁸O was not incorporated. These results indicate that immobilized cyt.c catalysed hydroxylation of benzene via two major pathways, of which one resembles that of P450.

Application of Chemical Cytochrome P450 Model Systems to Studies on Drug Metabolism .VI

Doi T., Mori T., Mashino T., Hirobe M.

Biochemical and Biophysical Research Communications 191 ( 2 ) 737 - 743 1993.03

Research paper (scientific journal), Joint Work, ISSN 0006-291X

Polypeptide-bound porphyrinatoiron(III) effectively catalyzed the N,N coupling reaction of N-methylaniline in the presence of NaBH₄/O₂/tetramethyl-ammonium hydroxide or cumenehydroperoxide, whereas the corresponding non-bound monomer porphyrinatoiron(III) had little catalytic activity. Rat liver microsomes also catalyzed the N,N-coupling which was inhibited by cytochrome P-450 specific inhibitors, SKF-525A and metyrapone. The polypeptide-bound porphinatoiron(III) appears to be an excellent cytochrome P450 model for drug metabolism studies. © 1993 Academic Press. All rights reserved.

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Cytochrome P450-like Substrate Oxidation Catalyzed by Cytochrome c and Immobilized Cytochrome c

Akasaka R., Mashino T., Hirobe M.

Archives of Biochemistry and Biophysics 301 ( 2 ) 355 - 360 1993.03

Research paper (scientific journal), Joint Work, ISSN 0003-9861

Cytochrome c (cyt.c) was shown to catalyze cytochrome P450 (P450)-like oxidative reactions, such as N-, and 0-demethylation, S-oxidation, and epoxidation of olefins. A more detailed examination showed that (i) N-methylcarbazole and thioanisole oxidation with H₂¹⁸O₂ catalyzed by cyt.c resulted in introduction of ¹⁸O into the product, and (ii) during the epoxidation of cis-stilbene catalyzed by cyt.c, the stereochemistry of the substrate was retained and ¹⁸O was introduced when H₂¹⁸O₂ was used as an oxidant. These results show that cyt.c catalyzed N-demethylation, S-oxidation, and epoxidation in the same manner as P450. To utilize these P450-like reactivities effectively, cyt.c was immobilized on poly-γ-methyl-L-glutamate. Up to 99% of the cyt.c used was immobilized. This immobilized cyt.c catalyzed N-demethylation, S-oxidation, and epoxidation in the same manner as both P450 and free cyt.c, and the activities of these reactions were increased by the immobilization. In N-demethylation of N,N-dimethylaniline with cumene hydroperoxide (CHP) catalyzed by cyt.c, the V_max for CHP was increased by 4.4-fold by the immobilization of the enzyme, while the K_m remained unchanged. Since P450 is involved in the metabolism of many xenobiotics, the above results suggest that immobilized cyt.c may be useful in drug metabolism research. © 1993 Academic Press. All rights reserved.

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Oxygen activation by iron(III)-porphyrin/NaBH₄/Me₄N·OH system as cytochrome P-450 model. Oxygenation of olefin, N-dealkylation of tertiary amine, oxidation of sulfide, and oxidative cleavage of ether bond

Mori T., Santa T., Higuchi T., Mashino T., Hirobe M.

Chemical and Pharmaceutical Bulletin 41 ( 2 ) 292 - 295 1993

Research paper (scientific journal), Joint Work, ISSN 0009-2363

Oxygenation of olefin, N-dealkylation of tertiary amine, oxidation of sulfide, and oxidative cleavage of ether bond were conducted with tetraphenylporphyrinatoiron(III) (Fe³⁺TPPCl), NaBH₄, Me₄N·OH, and molecular dioxygen in benzene-methanol solution. Fe³⁺TPPCl, NaBH₄, and molecular dioxygen were essential for these reactions and the yields were decreased when Me₄N·OH was absent. Olefins were converted to alcohols, which were not produced from the corresponding epoxides under the same conditions. In styrene oxygenation, an electron-donating substituent on the substrate decreased the reactivity, whereas in N,N-dimethylaniline demethylation, it enhanced the reactivity. Despite the use of the same reagents, the key intermediates of these two reactions are different. Fe²⁺TPP-σ-alkyl complexes produced from Fe³⁺TPPCl, olefin, and NaBH₄ were identified as intermediates under anaerobic conditions. Fe²⁺TPP-σ-alkyl complex reacted with molecular dioxygen to give oxygenated products. Examination of the relative reactivities of p-substituted N,N-dimethylanilines in the NaBH₄ reaction system revealed first, that the demethylation proceeded via one-electron abstraction, and second, that the reactive species of the demethylation reactions seems to be an iron-oxenoid.

¹⁸O incorporation from H₂ ¹⁸O₂ in the oxidation of N-methylcarbazole and sulfides catalyzed by microperoxidase-11

Nakamura Shigeo, Mashino Tadahiko, Hirobe Masaaki

Tetrahedron Letters 33 ( 37 ) 5409 - 5412 1992.09

Research paper (scientific journal), Joint Work, ISSN 0040-4039

Microperoxidase-11 (MP-11), prepared by pepsin digestion of cytochrome c, contains protoheme IX and one proximal histidine ligand. MP-11 oxygenated N-methylcarbazole, methyl phenyl sulfide, and phenacyl phenyl sulfide with 80%, 100%, and 96% ¹⁸O atom incorporation from H₂
¹⁸O₂, respectively. These results indicate that the oxygen atom on heme iron of MP-11 immediately rebinds to one-electron-oxidized intermediates of the substrates and that MP-11 catalyzes N-demethylation through a cytochrome P-450-like mechanism rather than a peroxidase-like mechanism. © 1992.

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Substrate oxidation catalyzed by heme-undecapeptide, microperoxidase-11 in the presence of iodosylbenzene and cumene hydroperoxide

Nakamura S., Mashino T., Hirobe M.

Journal of Pharmacobio-Dynamics 14 ( 11 ) 1991

Research paper (scientific journal), Joint Work, ISSN 0386-846X

Metabolism and penetration through blood-brain barrier of parkinsonism-related compounds. 1,2,3,4-Tetrahydroisoquinoline and 1-methyl-1,2,3,4-tetrahydroisoquinoline

Kikuchi K., Nagatsu Y., Makino Y., Mashino T., Ohta S., Hirobe M.

Drug Metabolism and Disposition 19 ( 1 ) 257 - 262 1991

Research paper (scientific journal), Joint Work, ISSN 0090-9556

¹⁴C-Labeled 1,2,3,4-tetrahydroisoquinoline (TIQ) and 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) were synthesized, and their metabolism and tissue distribution were studied. Both compounds showed similar metabolic patterns. In 24 hr after po administration (50 mg/kg) to rats, 76% of TIQ and 72% of 1MeTIQ were excreted unchanged, and 2.7 and 8.7% were excreted as the 4-hydroxyl derivatives, 4-hydroxy-TIQ and 4-hydroxy-1MeTIQ, respectively. Small amounts of N-methylated metabolites, 2-methyl-TIQ (0.4%) and 2-methyl-1MeTIQ (0.7%) were detected. Isoquinoline (2.5%) also was found as a metabolite of TIQ and 1-methyl-3,4-dihydroisoquinoline (1.0%) was found as a metabolite of 1MeTIQ. The concentration of labeled compounds in the brain was about 4.5-fold higher than the blood concentration at 4 hr after dosing, and over 90% was unchanged TIQ or 1MeTIQ. These data indicate that TIQ and 1MeTIQ easily passed through the blood-brain barrier and were concentrated in the brain. Thus, it appears that TIQ and 1MeTIQ as endogenous or exogenous amines may accumulate in the brain and may be related to the onset of Parkinson's disease.

Sulfide oxidation, amine N-demethylation, and olefin oxidation by heme-undecapeptide, microperoxidase-11, in the presence of hydrogen peroxide

Mashino Tadahiko, Nakamura Shigeo, Hirobe Masaaki

Tetrahedron Letters 31 ( 22 ) 3163 - 3166 1990

Research paper (scientific journal), Joint Work, ISSN 0040-4039

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Metabolism and blood-brain barrier permeability of tiqs

Ohta S., Tachikawa O., Makino Y., Kikuchi K., Mashino T., Hirobe M.

Journal of Pharmacobio-Dynamics 12 ( 3 ) 1989

Research paper (scientific journal), Joint Work, ISSN 0386-846X

The reactivity of superoxide

Yamamoto Hiroshi, Mashino Tadahiko, Nagano Tetsuo, Hirobe Masaaki

Tetrahedron Letters 30 ( 31 ) 4133 - 4136 1989

Research paper (scientific journal), Joint Work, ISSN 0040-4039

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Reactions of hypochlorite with catalase

Mashino Tadahiko, Fridovich Irwin

Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular 956 ( 1 ) 63 - 69 1988.08

Research paper (scientific journal), Joint Work, ISSN 0167-4838

OCl^-/HOCl imposed a rapid inactivation of catalase (hydrogen-peroxide: hydrogen-peroxide oxidoreductase, EC 1.11.1.6), some of which was slowly reversible upon subsequent exposure to H₂O₂. Ethanol accelerated this restoration of activity by H₂O₂. OCl^- caused biphasic changes in the visible absorption spectrum of catalase, which were partially reversed by dithionite. A scheme of reactions involving axial ligation of one or two OCl^- to heme iron, followed by heterolytic or homolytic cleavages of the OCl bond, is proposed to account for the behavior of the system. © 1988.

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NADPH mediates the inactivation of bovine liver catalase by monochloroamine

Maskino Tadahiko, Fridovich Irwin

Archives of Biochemistry and Biophysics 265 ( 2 ) 279 - 285 1988

Research paper (scientific journal), Joint Work, ISSN 0003-9861

Bovine liver catalase suffered a biphasic inactivation when exposed to NH₂Cl. A rapid and irreversible phase of activity loss was followed by a much slower and reversible inactivation. Removal of tightly bound NADPH from the enzyme decreased the extent of the rapid phase; whereas addition of NADPH augmented it. The catalase from Aspergillus niger, which does not contain bound NADPH, was not nearly as sensitive toward NH₂Cl as was the liver enzyme and was not sensitized by added NADPH. NADPH is oxidized by NH₂Cl, as evidenced by loss of the 340-nm absorption band, but the product of this oxidation was not NADP⁺. The rapid inactivation of liver catalase by NH₂Cl was accompanied by some bleaching of the bands in the visible, while the slow inactivation was coincident with the appearance of a new band at 570 nm. A tentative explanation for these observations is proposed. © 1988.

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Effects of urea and trimethylamine-N-oxide on enzyme activity and stability

Mashino Tadahiko, Fridovich Irwin

Archives of Biochemistry and Biophysics 258 ( 2 ) 356 - 360 1987.11

Research paper (scientific journal), Joint Work, ISSN 0003-9861

The interactions of urea, trimethylamine-N-oxide (TMAO), and related solutes on a number of enzymes were examined. Urea inhibited enzymatic activity and accelerated the thermal inactivation of catalase, whereas TMAO activated some enzymes but inhibited others. The effects of urea and of TMAO, whether parallel or in opposition, were exerted independently. Thus, in those cases where TMAO increases enzymatic activity, it did so to the same relative degree, whether or not urea was present. TMAO markedly decreased the rate of thermal inactivation of catalase, indicating that it does favor compact protein structures. The assumption that TMAO factors compaction of protein structure, whereas urea has the contrary effect, does not lead to the expectation that TMAO must always oppose the effect of urea on enzymatic activity, since the most compact form of an enzyme may not always be the most active form. © 1987.

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Superoxide radical initiates the autoxidation of dihydroxyacetone

Mashino Tadahiko, Fridovich Irwin

Archives of Biochemistry and Biophysics 254 ( 2 ) 547 - 551 1987.05

Research paper (scientific journal), Joint Work, ISSN 0003-9861

The aerobic xanthine oxidase reaction causes the cooxidation of dihydroxyacetone in a process which is strongly inhibited by superoxide dismutase but not by catalase, HO · scavengers, or iron-inactivating chelating agents. Several molecules of the sugar can be oxidized per O₂^- introduced. A free radical chain mechanism, in which O₂^- acts both as an initiator and as a chain propagator, is proposed. Simple sugars capable of tautomerizing to enediols may now be added to the list of biologically relevant targets for O₂^-. © 1987.

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Base liberation from nucleotides by superoxide and intramolecular enhancement effect of phosphate group

Yamane Hiroshi, Yada Nobuhisa, Katori Etsu, Mashino Tadahiko, Nagano Tetsuo, Hirobe Masaaki

Biochemical and Biophysical Research Communications 142 ( 3 ) 1104 - 1110 1987.02

Research paper (scientific journal), Joint Work, ISSN 0006-291X

The reaction of nucleotides with superoxide gave the corresponding nucleobases in good yield. The base-release reaction of nucleotides due to superoxide was examined and compared to that of nucleosides. Hence, the phosphate moiety greatly enhances the yields, in particular those of adenine from adenosine monophosphates. Superoxide in combination with the phosphate moiety has been revealed to be more active than superoxide alone. The phosphate peroxy radical, generated in situ from superoxide and phosphate, seems to be the species responsible for the formation of the free bases. © 1987.

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Mechanism of the cyanide-catalyzed oxidation of α-ketoaldehydes and α-ketoalcohols

Mashino Tadahiko, Fridovich Irwin

Archives of Biochemistry and Biophysics 252 ( 1 ) 163 - 170 1987

Research paper (scientific journal), Joint Work, ISSN 0003-9861

Cyanide catalyzes the reduction of dioxygen or of ferricytochrome c by dihydroxyacetone phosphate. The rapid initial phase of these reactions, but not the subsequent slow phase, was augmented by incubating the triose phosphate aerobically or anaerobically at pH 9.0 prior to adding the cyanide. The aerobic incubation, which was most effective, was associated with a decline in enediol, whereas the less effective anaerobic incubation was accompanied by an increase in enediol content. This suggested that the α-ketoaldehyde product of autoxidation of the enediol, rather than the enediol itself, was responsible for the rapid phase reaction which followed addition of cyanide. This was confirmed by exploring the cyanide-catalyzed oxidation of the α-ketoaldehyde, phenylglyoxal. The inhibitory effect of the manganese-containing superoxide dismutase indicated that O₂^- was a kinetically important intermediate of the rapid phase reaction. A reaction mechanism is proposed which is consistent with the results presented. © 1987.

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α,β-Dihydroxyisovalerate dehydratase. A superoxide-sensitive enzyme

Kuo C. F., Mashino T., Fridovich I.

Journal of Biological Chemistry 262 ( 10 ) 4724 - 4727 1987

Research paper (scientific journal), Joint Work, ISSN 0021-9258

Increasing the intracellular flux of O₂^- by incubating aerobic Escherichia coli with paraquat or plumbagin markedly lowered the α,β-dihydroxyisovalerate dehydratase activity detectable in extracts from these cells. This effect was not seen in the absence of dioxygen and was exacerbated by inhibiting protein biosynthesis with chloramphenicol. These effects of paraquat and of plumbagin were both time- and concentration-dependent. Transfer of E. coli from aerobic to anaerobic conditions caused a rebound of the dehydratase activity, in the continued presence of paraquat and of chloramphenicol, indicating the presence of a mechanism for reactivating this enzyme. The instability of the dehydratase activity in cell extracts was exacerbated by selective removal of superoxide dismutase, but not of catalase, by immunoprecipitation. Addition of exogenous superoxide dismutase reversed the effect of immunoprecipitation; whereas catalase or inactive superoxide dismutase were ineffective. We conclude that the dehydratase is inactivated by O₂^-. This could account for the bacteriostatic effects of dioxygen and of paraquat.

Epoxidations of olefins by peroxy intermediate generated in situ from CCl₄ and superoxide

Yamamoto Hiroshi, Mashino Tadahiko, Nagano Tetsuo, Hirobe Masaaki

Journal of the American Chemical Society 108 ( 3 ) 539 - 541 1986

Research paper (scientific journal), Joint Work, ISSN 0002-7863

Reduction of imines by cluster [Fe₄S₄(SR)₄]^3-

Mashino Tadahiko, Nagano Tetsuo, Hirobe Masaaki

Tetrahedron Letters 24 ( 46 ) 5113 - 5116 1983

Research paper (scientific journal), Joint Work, ISSN 0040-4039

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