22 results on '"Hisakazu Sanada"'
Search Results
2. Standard protocol for the total red blood cell Pig-a assay used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society
- Author
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Satsuki Chikura, Takafumi Kimoto, Satoru Itoh, Hisakazu Sanada, Shigeharu Muto, and Katsuyoshi Horibata
- Subjects
Pig-a assay ,Glycosylphosphatidylinositol ,Flow cytometry ,Red blood cells ,In vivo gene mutation ,CD59 ,Ecology ,QH540-549.5 ,Genetics ,QH426-470 - Abstract
Abstract The Pig-a assay, a promising tool for evaluating in vivo genotoxicity, is based on flow cytometric enumeration of red blood cells (RBCs) that are deficient in glycosylphosphatidylinositol anchor protein. Various approaches for measuring Pig-a mutant cells have been developed, particularly focusing on measuring mutants in peripheral RBCs and reticulocytes (RETs). The Pig-a assay on concentrated RETs—the PIGRET assay—has the potential to detect genotoxicity in the early stages of a study. To verify the potential and usefulness of the PIGRET assay for short-term testing, we conducted an interlaboratory trial involving 16 laboratories organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society (MMS/JEMS). The collaborating laboratories assessed the mutagenicity of a total of 24 chemicals in rats using a single-treatment design and standard protocols for conducting the Pig-a assay on total RBCs (the RBC Pig-a assay) and the PIGRET assay. Here, we describe the standard protocol for the RBC Pig-a assay in detail.
- Published
- 2019
- Full Text
- View/download PDF
3. Inhibitory Effects of Antiparkinsonian Drugs and Caspase Inhibitors in a Parkinsonian Flatworm Model
- Author
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Yoshihisa Kitamura, Masatoshi Inden, Hisakazu Sanada, Kazuyuki Takata, Takashi Taniguchi, Shun Shimohama, Hidehumi Orii, Makoto Mochii, Kiyokazu Agata, and Kenji Watanabe
- Subjects
Therapeutics. Pharmacology ,RM1-950 - Abstract
It has been known that rotenone and 1-methyl-4-phenylpyridinium ion (MPP+, a metabolite of MPTP), which inhibit mitochondrial complex I, are useful tools for parkinsonian models in vertebrates such as primates and rodents. Planarian, an invertebrate flatworm, has a high potential for regeneration, and dopamine plays a key role in its behavior. In the present study, we examined a cloned planarian, the GI strain from Dugesia japonica. Planarians that were treated with rotenone or MPTP underwent autolysis and individual death in a concentration- and time-dependent manner. In addition, these effects induced by rotenone or MPTP were inhibited by several antiparkinsonian drugs and caspase inhibitors. These results suggest that the degeneration of planarian dopaminergic system induced by rotenone or MPTP may be mediated through caspase-like activation.
- Published
- 2003
- Full Text
- View/download PDF
4. A bispecific antibody NXT007 exerts a hemostatic activity in hemophilia A monkeys enough to keep a non-hemophiliac state
- Author
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Yuri Teranishi-Ikawa, Tetsuhiro Soeda, Hikaru Koga, Kazuki Yamaguchi, Kazuki Kato, Keiko Esaki, Kentaro Asanuma, Miho Funaki, Mina Ichiki, Yuri Ikuta, Shunsuke Ito, Eri Joyashiki, Shun-Ichiro Komatsu, Atsushi Muto, Kei Nishimura, Momoko Okuda, Hisakazu Sanada, Motohiko Sato, Norihito Shibahara, Tetsuya Wakabayashi, Koji Yamaguchi, Akiko Matsusaki, Zenjiro Sampei, Hirotake Shiraiwa, Hiroko Konishi, Yoshiki Kawabe, Kunihiro Hattori, Takehisa Kitazawa, and Tomoyuki Igawa
- Abstract
Emicizumab, a factor (F)VIIIa-function mimetic bispecific antibody (BsAb) to FIXa and FX, has become an indispensable treatment for people with hemophilia A (PwHA). Although emicizumab is very potent, long-term outcomes from the clinical studies suggest that a small proportion of PwHA still experiences bleeds. Additionally, non-clinical studies indicate that the maximum cofactor activity of emicizumab is lower than international standard activity (100 IU/dL of FVIII). An increased cofactor activity BsAb would benefit such patients. Here, we report NXT007, a BsAb binding FIXa and FX developed through further engineering of emicizumab. Emicizumab has a common light chain, but through advances in antibody engineering, we were able to create a more potent BsAb with two new non-common light chains. After extensive optimization of the heavy and light chains, the resulting BsAb, NXT007, exerted in vitro thrombin generation (TG) activity in hemophilia A plasma equivalent to 100 IU/dL of FVIII when triggered by tissue factor. NXT007 demonstrated potent hemostatic activity in an acquired hemophilia A model in non-human primates at a much lower dosage than emicizumab, consistent with an around 30-fold dose shift in the in vitro TG activity between NXT007 and emicizumab. Moreover, together with Fc engineering that enhanced FcRn binding and reduced in vivo clearance, we demonstrate that NXT007 could be effective at a much lower dosage with a longer dosing interval compared to emicizumab. These non-clinical results suggest that NXT007 could maintain a non-hemophilic range of coagulation potential in PwHA and provides a rationale for its clinical testing.
- Published
- 2022
5. Standard protocol for the PIGRET assay, a high-throughput reticulocyte Pig-a assay with an immunomagnetic separation, used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen and Genome Society
- Author
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Hisakazu Sanada, Katsuyoshi Horibata, Satsuki Chikura, Takafumi Kimoto, Shigeharu Muto, and Satoru Itoh
- Subjects
0301 basic medicine ,Reticulocytes ,Social Psychology ,lcsh:QH426-470 ,Method ,HIS49 ,Mutagen ,Environmental Science (miscellaneous) ,Biology ,medicine.disease_cause ,Immunomagnetic separation ,Genome ,Flow cytometry ,03 medical and health sciences ,0302 clinical medicine ,Reticulocyte ,lcsh:QH540-549.5 ,Genetics ,medicine ,Immunomagnetics ,medicine.diagnostic_test ,Mutant cell ,Glycosylphosphatidylinositol ,lcsh:Genetics ,030104 developmental biology ,medicine.anatomical_structure ,Biochemistry ,CD71 ,030220 oncology & carcinogenesis ,Standard protocol ,lcsh:Ecology ,Pig-a assay ,In vivo gene mutation ,Genotoxicity ,CD59 - Abstract
The PIGRET assay is one of the Pig-a assays targeting reticulocytes (RETs), an in vivo genotoxicity evaluation method using flow cytometry with endogenous reporter glycosylphosphatidylinositol anchor protein. The PIGRET assay with RETs selectively enriched with anti-CD71 antibodies has several desirable features: high-throughput assay system, low background frequency of mutant cells, and early detection of mutation. To verify the potential and usefulness of the PIGRET assay for short-term testing, an interlaboratory trial involving 16 laboratories organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen and Genome Society was conducted. The collaborating laboratories assessed the mutagenicities of a total of 24 chemicals in rats using a single-treatment design and standard protocols for conducting the Pig-a assay on the total red blood cell assay and the PIGRET assay. Here the standard protocol for the PIGRET assay was described in detail.
- Published
- 2021
6. Standard protocol for the total red blood cell Pig-a assay used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society
- Author
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Hisakazu Sanada, Satsuki Chikura, Shigeharu Muto, Satoru Itoh, Takafumi Kimoto, and Katsuyoshi Horibata
- Subjects
0301 basic medicine ,Social Psychology ,lcsh:QH426-470 ,Mutagen ,Environmental Science (miscellaneous) ,Biology ,medicine.disease_cause ,Red blood cells ,Flow cytometry ,03 medical and health sciences ,0302 clinical medicine ,In vivo ,lcsh:QH540-549.5 ,Genetics ,medicine ,Glycosylphosphatidylinositol anchor ,medicine.diagnostic_test ,Mutant cell ,Red blood cell ,Glycosylphosphatidylinositol ,lcsh:Genetics ,030104 developmental biology ,medicine.anatomical_structure ,Biochemistry ,030220 oncology & carcinogenesis ,Standard protocol ,lcsh:Ecology ,Pig-a assay ,In vivo gene mutation ,Genotoxicity ,CD59 - Abstract
The Pig-a assay, a promising tool for evaluating in vivo genotoxicity, is based on flow cytometric enumeration of red blood cells (RBCs) that are deficient in glycosylphosphatidylinositol anchor protein. Various approaches for measuring Pig-a mutant cells have been developed, particularly focusing on measuring mutants in peripheral RBCs and reticulocytes (RETs). The Pig-a assay on concentrated RETs—the PIGRET assay—has the potential to detect genotoxicity in the early stages of a study. To verify the potential and usefulness of the PIGRET assay for short-term testing, we conducted an interlaboratory trial involving 16 laboratories organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society (MMS/JEMS). The collaborating laboratories assessed the mutagenicity of a total of 24 chemicals in rats using a single-treatment design and standard protocols for conducting the Pig-a assay on total RBCs (the RBC Pig-a assay) and the PIGRET assay. Here, we describe the standard protocol for the RBC Pig-a assay in detail.
- Published
- 2019
7. The PIGRET assay, a method for measuring Pig-a gene mutation in reticulocytes, is reliable as a short-term in vivo genotoxicity test: Summary of the MMS/JEMS-collaborative study across 16 laboratories using 24 chemicals
- Author
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Hideki Adachi, Yoshifumi Uno, Yuki Okada, Kunio Wada, Yosuke Ogiwara, Shigeharu Muto, Katsuyoshi Horibata, Shuichi Hamada, Mika Yamamoto, Akihisa Maeda, Eri Tsutsumi, Masami Yamada, Yasuaki Uematsu, Hironao Takasawa, Masamitsu Honma, Takeshi Morita, Ikuma Yoshida, Satoru Itoh, Hisako Hori, Shiho Nakayama, Hisakazu Sanada, Akiko Ukai, Miyuki Shigano, Kazunori Narumi, Naomi Koyama, Takafumi Kimoto, Yuta Suzuki, Daishiro Miura, Takayuki Fukuda, Yohei Fujiishi, Ken Goto, Satsuki Chikura, Ryuta Kikuzuki, and Takahiro Kyoya
- Subjects
0301 basic medicine ,Erythrocytes ,Reticulocytes ,Health, Toxicology and Mutagenesis ,Transferability ,Mutant ,Pig a gene ,Biology ,medicine.disease_cause ,03 medical and health sciences ,In vivo ,medicine ,Genetics ,Humans ,Whole blood ,Mutation ,Mutagenicity Tests ,Membrane Proteins ,Reproducibility of Results ,Molecular biology ,Interinstitutional Relations ,030104 developmental biology ,Ethylnitrosourea ,Erythropoiesis ,Laboratories ,Genotoxicity - Abstract
The in vivo mutation assay using the X-linked phosphatidylinositol glycan class A gene (Pig-a in rodents, PIG-A in humans) is a promising tool for evaluating the mutagenicity of chemicals. Approaches for measuring Pig-a mutant cells have focused on peripheral red blood cells (RBCs) and reticulocytes (RETs) from rodents. The recently developed PIGRET assay is capable of screening >1×106 RETs for Pig-a mutants by concentrating RETs in whole blood prior to flow cytometric analysis. Additionally, due to the characteristics of erythropoiesis, the PIGRET assay can potentially detect increases in Pig-a mutant frequency (MF) sooner after exposure compared with a Pig-a assay targeting total RBCs (RBC Pig-a assay). In order to test the merits and limitations of the PIGRET assay as a short-term genotoxicity test, an interlaboratory trial involving 16 laboratories was organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagenicity Society (MMS/JEMS). First, the technical proficiency of the laboratories and transferability of the assay were confirmed by performing both the PIGRET and RBC Pig-a assays on rats treated with single doses of N-nitroso-N-ethylurea. Next, the collaborating laboratories used the PIGRET and RBC Pig-a assays to assess the mutagenicity of a total of 24 chemicals in rats, using a single treatment design and mutant analysis at 1, 2, and 4 weeks after the treatment. Thirteen chemicals produced positive responses in the PIGRET assay; three of these chemicals were not detected in the RBC Pig-a assay. Twelve chemicals induced an increase in RET Pig-a MF beginning 1 week after dosing, while only 3 chemicals positive for RBC Pig-a MF produced positive responses 1 week after dosing. Based on these results, we conclude that the PIGRET assay is useful as a short-term test for in vivo mutation using a single-dose protocol.
- Published
- 2016
- Full Text
- View/download PDF
8. Standard protocol for the total red blood cell
- Author
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Satsuki, Chikura, Takafumi, Kimoto, Satoru, Itoh, Hisakazu, Sanada, Shigeharu, Muto, and Katsuyoshi, Horibata
- Subjects
Glycosylphosphatidylinositol ,Method ,HIS49 ,Flow cytometry ,Red blood cells ,Pig-a assay ,In vivo gene mutation ,CD59 - Abstract
The Pig-a assay, a promising tool for evaluating in vivo genotoxicity, is based on flow cytometric enumeration of red blood cells (RBCs) that are deficient in glycosylphosphatidylinositol anchor protein. Various approaches for measuring Pig-a mutant cells have been developed, particularly focusing on measuring mutants in peripheral RBCs and reticulocytes (RETs). The Pig-a assay on concentrated RETs—the PIGRET assay—has the potential to detect genotoxicity in the early stages of a study. To verify the potential and usefulness of the PIGRET assay for short-term testing, we conducted an interlaboratory trial involving 16 laboratories organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society (MMS/JEMS). The collaborating laboratories assessed the mutagenicity of a total of 24 chemicals in rats using a single-treatment design and standard protocols for conducting the Pig-a assay on total RBCs (the RBC Pig-a assay) and the PIGRET assay. Here, we describe the standard protocol for the RBC Pig-a assay in detail.
- Published
- 2018
9. Nonclinical safety assessment of Efinaconazole Solution (10%) for onychomycosis treatment
- Author
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Radhakrishnan Pillai, Marian Glynn, William J. Jo, Kenji Minowa, Hisakazu Sanada, Linda Mutter, Barry Calvarese, Hiroaki Nejishima, and Hisato Senda
- Subjects
Male ,Antifungal Agents ,Time Factors ,Erythema ,Swine ,Administration, Topical ,Injections, Subcutaneous ,Urinary system ,Hyperkeratosis ,Inflammation ,Pharmacology ,Administration, Cutaneous ,Toxicology ,Rats, Sprague-Dawley ,Mice ,Subcutaneous injection ,Onychomycosis ,medicine ,Animals ,Efinaconazole ,Skin ,chemistry.chemical_classification ,Mice, Inbred ICR ,No-Observed-Adverse-Effect Level ,Dose-Response Relationship, Drug ,business.industry ,General Medicine ,Triazoles ,medicine.disease ,Rats ,Pharmaceutical Solutions ,chemistry ,Toxicity ,Swine, Miniature ,Azole ,Female ,medicine.symptom ,business ,medicine.drug - Abstract
Efinaconazole is a triazole developed as a 10% solution for topical treatment of onychomycosis, a common fungal nail infection. Efinaconazole solution and topical formulation vehicle administered dermally to mice (13weeks), rats (6months) and minipigs (9months) produced transient erythema, minimal to modest hyperkeratosis, and mild microscopic skin inflammation. The liver was the target organ of systemic toxicity; reversible, minimal to moderate vacuolated changes were noted in the rat dermal study at 15 and 50mg/kg/day. No systemic toxicity was observed in mice and minipigs, at approximate high dermal doses of 930 and 170mg/kg/day, respectively. Daily subcutaneous injection of propylene glycol vehicle or efinaconazole to rats for 6months produced severe local inflammation and systemic spread, evidenced by peritoneal adhesions, spinal cord necrosis and urinary tract disease. Mortalities occurred in all groups but were increased at the high dose (30 or 40mg/kg/day), suggesting that vehicle effects were exacerbated by efinaconazole. Efinaconazole was not carcinogenic in a 2-year mouse dermal study and was not genotoxic. Exposure-based safety margins at the NOAEL were 70-698 relative to onychomycosis patients. In conclusion, efinaconazole demonstrated low/moderate toxicity, consistent with other azole antifungals, and high safety margins for topical onychomycosis therapy.
- Published
- 2014
10. Evaluation for a Mutagenicity of 4,4^|^prime;-Methylenedianiline on Hematopoietic Cells by a Pig-a Gene Mutation Assay in Rats
- Author
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Toshiyuki Nakamura, Tomoka Ohsumi, Hisakazu Sanada, and Minako Okamoto
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Genetics ,Haematopoiesis ,chemistry.chemical_compound ,4,4'-Methylenedianiline ,Social Psychology ,chemistry ,Mutation (genetic algorithm) ,Pig a gene ,Environmental Science (miscellaneous) ,Biology ,Molecular biology - Published
- 2014
11. Interlaboratory trial of the rat Pig-a mutation assay using an erythroid marker HIS49 antibody
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Katsuyoshi Horibata, Masami Yamada, Satoru Itoh, Hisakazu Sanada, Masamitsu Honma, Kazuyuki Hashimoto, Yoshifumi Uno, Takafumi Kimoto, Shigeharu Muto, and Satsuki Chikura
- Subjects
Male ,Erythrocytes ,Reticulocytes ,Glycosylphosphatidylinositols ,9,10-Dimethyl-1,2-benzanthracene ,Health, Toxicology and Mutagenesis ,DMBA ,CD59 Antigens ,Gene mutation ,medicine.disease_cause ,Sensitivity and Specificity ,Flow cytometry ,Japan ,Antigen ,In vivo ,Genetics ,medicine ,Animals ,Erythroid Precursor Cells ,Mutation ,biology ,medicine.diagnostic_test ,Mutagenicity Tests ,Erythrocyte Membrane ,Antibodies, Monoclonal ,Membrane Proteins ,Reproducibility of Results ,Flow Cytometry ,Molecular biology ,4-Nitroquinoline-1-oxide ,Peripheral blood ,Rats ,Ethylnitrosourea ,biology.protein ,Antibody ,Laboratories - Abstract
The peripheral blood Pig-a assay has shown promise as a tool for evaluating in vivo mutagenicity. In this study five laboratories participated in a collaborative trial that evaluated the transferability and reproducibility of a rat Pig-a assay that uses a HIS49 antibody reacts with an antigen found on erythrocytes and erythroid progenitors. In preliminary work, flow cytometry methods were established that enabled all laboratories to detect CD59-negative erythrocyte frequencies (Pig-a mutant frequencies) of
- Published
- 2013
12. Evaluation of the PIGRET assay in rats by single oral dosing with azidothymidine
- Author
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Kazuto Hashimoto, Tomoka Ohsumi, Hisakazu Sanada, Naomi Koyama, and Taishi Miyashita
- Subjects
0301 basic medicine ,Reticulocytes ,Anti-HIV Agents ,Health, Toxicology and Mutagenesis ,Mutant ,Administration, Oral ,Transferrin receptor ,Mutagen ,Gene mutation ,Biology ,medicine.disease_cause ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,In vivo ,Genetics ,medicine ,Animals ,Dosing ,Nucleoside analogue ,Mutagenicity Tests ,Membrane Proteins ,Molecular biology ,Rats ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,Zidovudine ,DNA ,medicine.drug ,Mutagens - Abstract
In vivo phosphatidylinositol glycan, class A (Pig-a) gene mutation assay using peripheral blood is known to be a novel and useful tool to evaluate the mutagenicity of compounds. Recently, the rat PIGRET assay which is an improved method for measuring Pig-a mutant cells in reticulocytes with magnetic enrichment of CD71 positive cells has been developed. Several reports showed that the PIGRET assay could detect the increase of Pig-a mutant frequency earlier than the Pig-a assay in total red blood cells (RBC Pig-a assay). Therefore, as part of a collaborative study by the Mammalian Mutagenicity Study (MMS) Group of the Japanese Environmental Mutagen Society, the usefulness of the PIGRET assay in comparison to the RBC Pig-a assay has been assessed for 24 compounds with various mechanisms of action. In the present study, we performed the PIGRET assay and RBC Pig-a assay with a nucleoside analogue, azidothymidine (AZT), and compared the results in these assays. We administered a single dose of AZT to rats by oral gavage up to 2000mg/kg and examined Pig-a mutant frequencies at days 7, 14 and 28 by PIGRET and RBC Pig-a assays. No significant increases in mutant frequency were observed after administration of AZT in both the RBC Pig-a and PIGRET assays and comparable to the previous results of the International Workshop on Genotoxicity Testing (IWGT) workgroup. AZT has been thought to induce not only DNA chain termination as a pharmacological effect but also a large deletion on the genome DNA. The Pig-a assays may be less sensitive to compounds such as AZT which induce large deletions on the genome DNA.
- Published
- 2016
13. Discrimination of genotoxic and non-genotoxic hepatocarcinogens by statistical analysis based on gene expression profiling in the mouse liver as determined by quantitative real-time PCR
- Author
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Emiko Okada, Akihisa Maeda, Shuichi Hamada, Yohei Miyamoto, Yasuyoshi Ishikawa, Ayami Tadakuma, Masakatsu Natsume, Shizuyo Sutou, Hiroshi Honda, Kazunori Narumi, Tomohiro Sakuma, Akiko Koeda, Izumi Hanahara, Takashi Watanabe, Madoka Nakajima, Yohei Fujiishi, Michiasa Hirayama, Hisakazu Sanada, Keiyu Oshida, Takayoshi Suzuki, Mahoko Kido, Wakako Ohyama, Rina Minamiguchi, and Chie Furihata
- Subjects
Male ,DNA repair ,DNA damage ,Gene Expression Profiling ,Health, Toxicology and Mutagenesis ,Double Effect Principle ,Cell cycle ,Biology ,Real-Time Polymerase Chain Reaction ,Molecular biology ,Gene expression profiling ,Mice ,Liver Neoplasms, Experimental ,Real-time polymerase chain reaction ,Liver ,Gene expression ,Carcinogens ,Genetics ,Animals ,DNA microarray ,Gene ,Injections, Intraperitoneal ,Mutagens - Abstract
The general aim of the present study is to discriminate between mouse genotoxic and non-genotoxic hepatocarcinogens via selected gene expression patterns in the liver as analyzed by quantitative real-time PCR (qPCR) and statistical analysis. qPCR was conducted on liver samples from groups of 5 male, 9-week-old B6C3F(1) mice, at 4 and 48h following a single intraperitoneal administration of chemicals. We quantified 35 genes selected from our previous DNA microarray studies using 12 different chemicals: 8 genotoxic hepatocarcinogens (2-acetylaminofluorene, 2,4-diaminotoluene, diisopropanolnitrosamine, 4-dimethylaminoazobenzene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, N-nitrosomorpholine, quinoline and urethane) and 4 non-genotoxic hepatocarcinogens (1,4-dichlorobenzene, dichlorodiphenyltrichloroethane, di(2-ethylhexyl)phthalate and furan). A considerable number of genes exhibited significant changes in their gene expression ratios (experimental group/control group) analyzed statistically by the Dunnett's test and Welch's t-test. Finally, we distinguished between the genotoxic and non-genotoxic hepatocarcinogens by statistical analysis using principal component analysis (PCA) of the gene expression profiles for 7 genes (Btg2, Ccnf, Ccng1, Lpr1, Mbd1, Phlda3 and Tubb2c) at 4h and for 12 genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Gdf15, Lrp1, Mbd1, Phlda3, Plk2 and Tubb2c) at 48h. Seven major biological processes were extracted from the gene ontology analysis: apoptosis, the cell cycle, cell proliferation, DNA damage, DNA repair, oncogenes and tumor suppression. The major, biologically relevant gene pathway suggested was the DNA damage response pathway, resulting from signal transduction by a p53-class mediator leading to the induction of apoptosis. Eight genes (Aen, Bax, Btg2, Ccng1, Cdkn1a, Gdf15, Phlda3 and Plk2) that are directly associated with Trp53 contributed to the PCA. The current findings demonstrate a successful discrimination between genotoxic and non-genotoxic hepatocarcinogens, using qPCR and PCA, on 12 genes associated with a Trp53-mediated signaling pathway for DNA damage response at 4 and 48 h after a single administration of chemicals.
- Published
- 2012
14. Evaluation of the in vivo Mutagenicity of Nickel Subsulfide in the Lung of F344 gpt delta Transgenic Rats Exposed by Intratracheal Instillation: A Collaborative Study for the gpt delta Transgenic Rat Mutation Assay
- Author
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Hisakazu Sanada, Takehiko Nohmi, Shuichi Hamada, Tomoyuki Kamigaito, Kazunori Narumi, Masayuki Hasuko, Tadashi Noguchi, Kenichi Masumura, Rie Takashima, and Hiroyuki Hayashi
- Subjects
Mutation ,animal structures ,Lung ,Social Psychology ,Chemistry ,Transgene ,Mutant ,Inflammation ,Environmental Science (miscellaneous) ,medicine.disease_cause ,Molecular biology ,Nickel Subsulfide ,Toxicology ,medicine.anatomical_structure ,In vivo ,Genetics ,medicine ,medicine.symptom ,Carcinogen - Abstract
This study was conducted to evaluate the effectiveness of a transgenic rat mutation assay using F344 gpt delta rats. We investigated the mutagenic potential in the lung of nickel subsulfide (Ni3S2), an insoluble fine-crystalline-metallic compound and a carcinogen in the rodent and human lung. Ni3S2 carcinogenicity has been proposed to act via both genotoxic and non-genotoxic mechanisms. Ni3S2 was intratracheally instilled into male gpt delta rats at doses of 0.5 and 1 mg/animal once a week for four weeks; these doses of Ni3S2 are high enough to induce inflammation in the lung. Following a period of 28 and 90 days after the first administration, the gpt mutant frequencies (MFs) in lung were determined in four independent laboratories, and Spi− selection for larger deletion mutations was done in one laboratory. The gpt MFs of the rats treated with Ni3S2 were not increased: all four laboratories obtained similar results with no statistical differences. The Spi− MFs were also not increased by exposure to Ni3S2. These results indicate that intratracheally instilled Ni3S2 is non-mutagenic in the lung of gpt delta transgenic rats; however, whether Ni3S2 is non-mutagenic in the lung or it induces mutations which are not detectable by transgenic rodent mutation assays requires further investigation.
- Published
- 2012
15. Evaluation for a mutagenicity of aristolochic acid by Pig-a and PIGRET assays in rats
- Author
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Naomi Koyama, Michi Nakamura, Hisakazu Sanada, and Yutaka Yonezawa
- Subjects
0301 basic medicine ,Male ,Erythrocytes ,Reticulocytes ,Health, Toxicology and Mutagenesis ,Mutant ,Aristolochic acid ,Mutagen ,Gene mutation ,Biology ,medicine.disease_cause ,Rats, Sprague-Dawley ,03 medical and health sciences ,chemistry.chemical_compound ,In vivo ,Genetics ,medicine ,Animals ,Carcinogen ,Mutagenicity Tests ,Body Weight ,food and beverages ,Membrane Proteins ,Molecular biology ,In vitro ,Rats ,030104 developmental biology ,chemistry ,Micronucleus test ,Aristolochic Acids ,Mutagens - Abstract
The Pig-a assay, which uses the endogenous phosphatidylinositol glycan, class A gene (Pig-a) as a reporter of mutation, has been developed as a method for evaluating in vivo mutagenicity. Pig-a gene mutation can be detected by identifying the presence of CD59, the glycosylphosphatidylinositol anchor protein, on the surface of erythrocytes (RBC Pig-a assay) and reticulocytes (PIGRET assay). The International Workshop on Genotoxicity Testing (IWGT) showed the usefulness of the RBC Pig-a assay through the evaluation of several compounds. Aristolochic acid (AA), one of the evaluated compounds in the IWGT workgroup, is a carcinogenic plant toxin that is a relatively strong gene mutagen both in vitro and in vivo, but a weak inducer of micronuclei in vivo. In the present study, we examined the mutagenicity of AA in the peripheral blood of rats treated orally with a single dose of AA using Pig-a assays. Furthermore, we evaluated the advantages of the PIGRET assay compared with the RBC Pig-a assay. The results showed that a statistically significant increase in mutant frequency of the Pig-a gene was detected at day 28 by the RBC Pig-a assay, and at days 7, 14 and 28 by the PIGRET assay. In addition, the mutant frequency by the PIGRET assay was higher than that by the RBC Pig-a assay. These results indicate that the mutagenicity of AA can be detected using the Pig-a assays, as reported by the IWGT, and the PIGRET assay can detect Pig-a mutants at an early time point compared with the RBC Pig-a assay.
- Published
- 2015
16. Changes in expression of hepatic cytochrome P450 subfamily enzymes during development of adjuvant-induced arthritis in rats
- Author
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Masashi Sekimoto, Hisakazu Sanada, Ayaka Kamoshita, and Masakuni Degawa
- Subjects
Male ,medicine.medical_specialty ,CYP3A ,Liquid paraffin ,Arthritis ,Biology ,Toxicology ,Gene Expression Regulation, Enzymologic ,Proinflammatory cytokine ,Internal medicine ,Gene expression ,medicine ,Animals ,Cytochrome P-450 CYP3A ,RNA, Messenger ,Intradermal injection ,Interleukin ,medicine.disease ,Arthritis, Experimental ,Rats ,Endocrinology ,Liver ,Rats, Inbred Lew ,Cytochrome P-450 CYP2B1 ,Microsomes, Liver ,Cytokines ,Tumor necrosis factor alpha - Abstract
An animal model of rheumatoid arthritis can be elicited in male Lewis rats by a single intradermal injection of liquid paraffin containing dead Mycrobacterium tuberculosis (MT adjuvant) into the planar surface of the right hind-foot. In the present study, we used this animal model to examine the changes in expression of hepatic cytochorme P450 (CYP) enzymes during the development of the arthritis. Swellings of the MT adjuvant-injected hind-foot initially occurred at 1-8 days after the injection. Thereafter, the swelling gradually become more severe up to 13 days later and was maintained for up to 25 days. Swellings of the other hind-foot was also observed after 12 days and gradually become more severe up to 15 days with maintenance of the severe swelling for up to 25 days. The gene expression levels and enzyme activities of hepatic CYP 3A and CYP2B subfamily enzymes at 1, 12, and 25 days after the MT adjuvant injection were significantly decreased, compared with the corresponding time-matched controls. The decreases in the gene expression levels and activities of all the enzymes examined were closely correlated with increases in the expression levels of the inflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-1α, interleukin-1β and interleukin-6, which were produced in the liver. All of the present findings demonstrate that hepatic CYP3A and CYP2B subfamily enzymes are decreased during the development of MT adjuvant-induced arthritis and further suggest that the decreases are dependent on the production of inflammatory cytokines in the liver.
- Published
- 2011
17. Repeated-dose liver micronucleus test of 4,4'-methylenedianiline using young adult rats
- Author
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Naomi Koyama, Shuichi Hamada, Kazufumi Kawasako, Yumi Wako, and Hisakazu Sanada
- Subjects
Male ,medicine.medical_specialty ,Societies, Pharmaceutical ,4,4'-Methylenedianiline ,Health, Toxicology and Mutagenesis ,Administration, Oral ,Biology ,Drug Administration Schedule ,Toxicology ,Rats, Sprague-Dawley ,chemistry.chemical_compound ,Japan ,Bone Marrow ,Internal medicine ,Genetics ,medicine ,Animals ,Humans ,Young adult ,Cooperative Behavior ,Carcinogen ,Chromosome Aberrations ,Aniline Compounds ,Micronucleus Tests ,Dose-Response Relationship, Drug ,Age Factors ,Rats ,Endocrinology ,medicine.anatomical_structure ,chemistry ,Liver ,Organ Specificity ,Toxicity ,Micronucleus test ,Day treatment ,Carcinogens ,Hepatocytes ,Bone marrow ,Micronucleus - Abstract
Liver micronucleus (MN) tests using partial hepatectomized rats or juvenile rats have been shown to be useful for the detection of hepatic carcinogens. Moreover, Narumi et al. established the repeated-dose liver MN test using young adult rats for integration into general toxicity. In the present study, in order to examine the usefulness of the repeated-dose liver MN test, we investigated MN induction with a 14 or 28 day treatment protocol using young adult rats treated with 4,4′-methylenedianiline (MDA), a known hepatic carcinogen. MDA dose-dependently induced micronuclei in hepatocytes in 14- and 28-day repeated-dose tests. However, although statistically significant increases in micronuclei were observed in bone marrow cells at two dose levels in the 14-day study, there was no dose response and no increases in micronuclei in the 28-day study. These results indicate that the evaluation of genotoxic effects using hepatocytes is effective in cases where chromosomal aberrations are not clearly detectable in bone marrow cells. Moreover, the repeated-dose liver MN test allows evaluation at a dose below the maximum tolerable dose, which is required for the conventional MN test because micronucleated hepatocytes accumulate. The repeated-dose liver MN test employed in the present study can be integrated into the spectrum of general toxicity tests without further procedural modifications.
- Published
- 2015
18. Evaluation of the repeated-dose liver and gastrointestinal tract micronucleus assays with 22 chemicals using young adult rats: summary of the collaborative study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/The Japanese Environmental Mutagen Society (JEMS) - Mammalian Mutagenicity Study Group (MMS)
- Author
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Shoji Matsumura, Hironao Takasawa, Hajime Kojima, Makoto Hayashi, Hajime Sui, Yumi Wako, Yasushi Shimada, Nobuyuki Ohashi, Masamitsu Honma, Emiko Okada, Takeshi Morita, Satoru Kawakami, Wakako Ohyama, Tomomi Takayanagi, Yosuke Ogiwara, Izumi Ogawa, Shigeharu Muto, Masaki Sano, Kenji Inoue, Hisakazu Sanada, Kazunori Narumi, Keisuke Shimada, Aya Hayashi, Kazufumi Kawasako, Kazumi Matsumoto, Fuyumi Uno, Akihisa Maeda, Rie Takashima, Shuichi Hamada, Tadashi Imamura, and Yukari Terashima
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Male ,Societies, Pharmaceutical ,Reticulocytes ,Health, Toxicology and Mutagenesis ,Mutagen ,Pharmacology ,Biology ,medicine.disease_cause ,Sensitivity and Specificity ,Drug Administration Schedule ,Hazardous Substances ,Rats, Sprague-Dawley ,Japan ,In vivo ,Bone Marrow ,medicine ,Genetics ,Animals ,Humans ,Young adult ,Cooperative Behavior ,Carcinogen ,Chromosome Aberrations ,Gastrointestinal tract ,Micronucleus Tests ,Age Factors ,Reproducibility of Results ,Rats ,Gastrointestinal Tract ,medicine.anatomical_structure ,Liver ,Organ Specificity ,Micronucleus test ,Carcinogens ,Hepatocytes ,Female ,Bone marrow ,Micronucleus ,DNA Damage - Abstract
The repeated-dose liver micronucleus (RDLMN) assay using young adult rats has the potential to detect hepatocarcinogens. We conducted a collaborative study to assess the performance of this assay and to evaluate the possibility of integrating it into general toxicological studies. Twenty-four testing laboratories belonging to the Mammalian Mutagenicity Study Group, a subgroup of the Japanese Environmental Mutagen Society, participated in this trial. Twenty-two model chemicals, including some hepatocarcinogens, were tested in 14- and/or 28-day RDLMN assays. As a result, 14 out of the 16 hepatocarcinogens were positive, including 9 genotoxic hepatocarcinogens, which were reported negative in the bone marrow/peripheral blood micronucleus (MN) assay by a single treatment. These outcomes show the high sensitivity of the RDLMN assay to hepatocarcinogens. Regarding the specificity, 4 out of the 6 non-liver targeted genotoxic carcinogens gave negative responses. This shows the high organ specificity of the RDLMN assay. In addition to the RDLMN assay, we simultaneously conducted gastrointestinal tract MN assays using 6 of the above carcinogens as an optional trial of the collaborative study. The MN assay using the glandular stomach, which is the first contact site of the test chemical when administered by oral gavage, was able to detect chromosomal aberrations with 3 test chemicals including a stomach-targeted carcinogen. The treatment regime was the 14- and/or 28-day repeated-dose, and the regime is sufficiently promising to incorporate these methods into repeated-dose toxicological studies. The outcomes of our collaborative study indicated that the new techniques to detect chromosomal aberrations in vivo in several tissues worked successfully.
- Published
- 2014
19. Integration of in vivo genotoxicity and short-term carcinogenicity assays using F344 gpt delta transgenic rats: in vivo mutagenicity of 2,4-diaminotoluene and 2,6-diaminotoluene structural isomers
- Author
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Akiyoshi Nishikawa, Tomoki Inoue, Hiroyuki Hayashi, Kenichi Masumura, Naomi Toyoda-Hokaiwado, Yuji Kawamura, Yasushi Kurata, Masami Yamada, Takashi Umemura, Makiko Takamune, Hisakazu Sanada, and Takehiko Nohmi
- Subjects
Male ,3R principle ,Carcinogenicity Tests ,Genetic Toxicology ,2,4-Diaminotoluene ,Biology ,Phenylenediamines ,Toxicology ,medicine.disease_cause ,gpt delta transgenic rat ,chemistry.chemical_compound ,Shuttle vector ,In vivo ,medicine ,carcinogenicity ,Animals ,Pentosyltransferases ,Carcinogen ,Genetics ,Reporter gene ,Mutation ,Mutagenicity Tests ,Escherichia coli Proteins ,genotoxicity ,Glutathione ,Molecular biology ,Rats, Inbred F344 ,Rats ,chemistry ,Liver ,Rats, Transgenic ,diaminotoluenes ,Genotoxicity ,Mutagens - Abstract
An important trend in current toxicology is the replacement, reduction, and refinement of the use of experimental animals (the 3R principle). We propose a model in which in vivo genotoxicity and short-term carcinogenicity assays are integrated with F344 gpt delta transgenic rats. Using this model, the genotoxicity of chemicals can be identified in target organs using a shuttle vector lambda EG10 that carries reporter genes for mutations; short-term carcinogenicity is determined by the formation of glutathione S-transferase placenta form (GST-P) foci in the liver. To begin validating this system, we examined the genotoxicity and hepatotoxicity of structural isomers of 2,4-diaminotoluene (2,4-DAT) and 2,6-diaminotoluene (2,6-DAT). Although both compounds are genotoxic in the Ames/Salmonella assay, only 2,4-DAT induces tumors in rat livers. Male F344 gpt delta rats were fed diet containing 2,4-DAT at doses of 125, 250, or 500 ppm for 13 weeks or 2,6-DAT at a dose of 500 ppm for the same period. The mutation frequencies of base substitutions, mainly at G:C base pairs, were significantly increased in the livers of 2,4-DAT-treated rats at all three doses. In contrast, virtually no induction of genotoxicity was identified in the kidneys of 2,4-DAT-treated rats or in the livers of 2,6-DAT-treated rats. GST-P-positive foci were detected in the livers of rats treated with 2,4-DAT at a dose of 500 ppm but not in those treated with 2,6-DAT. Integrated genotoxicity and short-term carcinogenicity assays may be useful for early identifying genotoxic and nongenotoxic carcinogens in a reduced number of experimental animals.
- Published
- 2009
20. Evaluation for a Mutagenicity of 4,4-Methylenedianiline on Hematopoietic Cells by a Pig-a Gene Mutation Assay in Rats.
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Hisakazu Sanada, Minako Okamoto, Tomoka Ohsumi, and Toshiyuki Nakamura
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- *
ERYTHROCYTES , *DIAMINODIPHENYLMETHANE , *MUTAGENESIS , *GLYCOSYLPHOSPHATIDYLINOSITOL , *LABORATORY rats - Abstract
The Pig-a gene is involved in the synthesis of glycosyl-phosphatidylinositol (GPI) anchors. Pig-a gene mutations can be detected by identifying the presence of CD59, the GPI anchor protein, on the surface of erythrocytes (RBC Pig-a assay) and reticulocytes (PIGRET assay) and can be identified using flow cytometry. The usefulness of these Pig-a gene mutation assays has been confirmed in multi-laboratory trials with referenced mutagens. Although 4, 4-methylenedianiline (MDA) is an aromatic amine and has been identified as a potent hepatic carcinogen, in vivo micronucleus tests for MDA in hematopoietic cells determined that it was negative to weakly positive for genotoxicity. In the present study, we examined the mutagenicity of MDA in the peripheral blood of rats after 1- and 28-day MDA dosing using the Pig-a gene mutation assays. We also examined the utility of the RBC Pig-a and PIGRET as-says. No changes in mutation frequency were observed after one-day MDA administration. Repeated dosing caused a moderate increase in mutation frequency compared to vehicle control at days 14 and 28, as measured by the RBC Pig-a assay and at day 14 by the PIGRET assay. The highest mutation frequency was found on days 7 and 14 by the PIGRET and RBC Pig-a assays, respectively. In this study, we detected the mutagenicity of MDA in peripheral blood samples using gene mutation assays and judged to be positive for the MDA mutagenicity since a significant increase in mutation frequency was observed at high dose. These assays are expected to be easily integrated into general toxicity tests and to be combined with existing genotoxicity studies. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
- View/download PDF
21. Changes in expression of hepatic cytochrome P450 subfamily enzymes during development of adjuvant-induced arthritis in rats.
- Author
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Hisakazu Sanada, Masashi Sekimoto, Ayaka Kamoshita, and Masakuni Degawa
- Abstract
An animal model of rheumatoid arthritis can be elicited in male Lewis rats by a single intradermal injection of liquid paraffin containing dead Mycrobacterium tuberculosis (MT adjuvant) into the planar surface of the right hind-foot. In the present study, we used this animal model to examine the changes in expression of hepatic cytochorme P450 (CYP) enzymes during the development of the arthritis. Swellings of the MT adjuvant-injected hind-foot initially occurred at 1-8 days after the injection. Thereafter, the swelling gradually become more severe up to 13 days later and was maintained for up to 25 days. Swellings of the other hind-foot was also observed after 12 days and gradually become more severe up to 15 days with maintenance of the severe swelling for up to 25 days. The gene expression levels and enzyme activities of hepatic CYP 3A and CYP2B subfamily enzymes at 1, 12, and 25 days after the MT adjuvant injection were significantly decreased, compared with the corresponding time-matched controls. The decreases in the gene expression levels and activities of all the enzymes examined were closely correlated with increases in the expression levels of the inflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-1α, interleukin-1β and interleukin-6, which were produced in the liver. All of the present findings demonstrate that hepatic CYP3A and CYP2B subfamily enzymes are decreased during the development of MT adjuvant-induced arthritis and further suggest that the decreases are dependent on the production of inflammatory cytokines in the liver. [ABSTRACT FROM AUTHOR]
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- 2011
- Full Text
- View/download PDF
22. Inhibitory effects of antiparkinsonian drugs and caspase inhibitors in a parkinsoninan flatworm model
- Author
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Shun Shimohama, Kiyokazu Agata, Kazuyuki Takata, Kenji Watanabe, Hisakazu Sanada, Makoto Mochii, Takashi Taniguchi, Yoshihisa Kitamura, Hidehumi Orii, and Masatoshi Inden
- Subjects
Metabolite ,Pharmacology ,Inhibitory postsynaptic potential ,Antiparkinson Agents ,chemistry.chemical_compound ,Parkinsonian Disorders ,Dopamine ,medicine ,Animals ,Enzyme Inhibitors ,biology ,MPTP ,Dopaminergic ,lcsh:RM1-950 ,Rotenone ,Planarians ,biology.organism_classification ,Caspase Inhibitors ,nervous system diseases ,Disease Models, Animal ,lcsh:Therapeutics. Pharmacology ,chemistry ,nervous system ,Planarian ,Platyhelminths ,Caspases ,Molecular Medicine ,Dugesia japonica ,medicine.drug - Abstract
It has been known that rotenone and 1-methyl-4-phenylpyridinium ion (MPP(+), a metabolite of MPTP), which inhibit mitochondrial complex I, are useful tools for parkinsonian models in vertebrates such as primates and rodents. Planarian, an invertebrate flatworm, has a high potential for regeneration, and dopamine plays a key role in its behavior. In the present study, we examined a cloned planarian, the GI strain from Dugesia japonica. Planarians that were treated with rotenone or MPTP underwent autolysis and individual death in a concentration- and time-dependent manner. In addition, these effects induced by rotenone or MPTP were inhibited by several antiparkinsonian drugs and caspase inhibitors. These results suggest that the degeneration of planarian dopaminergic system induced by rotenone or MPTP may be mediated through caspase-like activation.
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