|Yoshimichi Nakatsu||Last modified date：2020.06.30|
Associate Professor / Bioregulation / Department of Basic Medicine / Faculty of Medical Sciences
|Yoshimichi Nakatsu||Last modified date：2020.06.30|
|1.||Mizuki OHNO, Kunihiko SAKUMI, Noriko TAKANO, Kosuke TESHIMA, Kyoko HIDAKA, Yoshimichi NAKATSU, Teruhisa TSUZUKI , Detection of de novo germline mutations in DNA repair-deficient mice lines, The Joint Meeting of The 6th Asian Congress on Environmental Mutagens(ACEM) and the 48th Annual Meeting of the Japanese Environmental Mutagen Society(JEMS), 2019.11, It is imperative to assess the trans generational effect of environmental mutagens such as radiation or chemicals. However, evaluation of the mutagenic effect, especially for low-dose mutagens, on de novo germline mutation (dGM) is still challenging because of low mutation rate in wild-type animals under natural conditions. Here, we use DNA repair-deficient mice lines for the detection of spontaneous dGMs in order to understand the molecular mechanisms behind germline genome integrity. We generated several mutant-mice family lines, deficient in 8-oxoguanine-related repair pathway, deficient in DNA mismatch repair (MMR) pathway or deficient in nucleotide excision repair (NER) pathway. Genomic DNA of parents and offspring were subjected to whole-exome or whole-genome sequencing for the detection of dGMs. Mth1/Ogg1/Mutyh-triple gene knocked out mice (8-oxoguaniene-induced mutations are usually well suppressed by MTH1, OGG1, MUTYH) exhibited 37-lold higher dGM rate than that in wild-type mice with a typical mutational pattern induced by 8-oxoguanine. In the MMR deficient mice, insertions/deletions at tandem repeat sequences were frequently observed in addition to high rate of G>A base substitution. On the other hand, dGM rate in NER deficient mice was almost same level as that in wild-type mice, suggesting that the repair targets of NER may not be frequently generated in the germ cells. Our results suggesting that dGM rate and spectra may fluctuate with the frequency of spontaneous DNA damages and DNA repair efficacy..|
|2.||Tsuzuki, T., Ohno, M., Takano, N., Taguchi, K., Nakatsu, Y., Oxidative stress-induced tumorigenesis: Lesson from the experiments with DNA repair-deficient mice., Beijing Symposium on “Genomic Stability and Accurate Gene Expression under Oxidative Stress”, 2018.11.|
|3.||Yoshimichi Nakatsu, Noriko Takano, Mizuki Ohno, Satoshi Kitazaki, Kazunori Koga, Akiyo Tanaka, Masaharu Shiratani, Application of transgenic mice to analyze genotoxic effects induced by non-thermal atmospheric air plasma
, 10th Anniversary International Symposium on Advanced Plasma Science and its Applications for Nitrides and Nanomaterials /11th International Conference on Plasma-Nano Technology & Science. (ISPlasma2018 / IC-PLANTS2018), 2018.03, Non-thermal atmospheric plasma has been used for biomedical applications such as blood coagulation, wound healing, and decontamination . In addition, the plasma has been proposed as a potential tool for cancer therapy because of its ability to induce apoptosis in cancer cells.
Non-thermal atmospheric plasma as well as ionizing radiation generates reactive oxygen species (ROS) in the liquid phase . ROS such as hydroxyl radical induce DNA double-strand break (DSB) and lead to cell death. The apoptotic induction of cancer cells by plasma treatment could be mediated by ROS. The cytotoxic activity was also observed in plasma-treated medium, so called plasma-activated medium (PAM) . PAM has been shown to have a selective cytotoxicity in several cancer cell lines, even chemotherapeutic agent resistant cell lines, providing a potential treatment for novel anti-cancer therapies . The cytotoxic effect of PAM is suggested to be mediated by ROS such as H2O2.
DSBs can be repaired by two different pathways: homologous recombination (HR) and non-homologous end joining (NHEJ) . HR is an error-free repair pathway, but it can be operated only in S and G2 phase of cell cycle because HR needs the sister chromatid as a repair template that is synthesized by DNA replication during the S phase. NHEJ is the predominant repair pathway during the G1 phase, and this is especially important for the cells in tissues/organs that is resting in G0/G1 phase or non-dividing status. NHEJ is characterized by rejoining of broken ends without the use of extensive homology, and is frequently associated with the presence of small insertions/deletions at DSB site. In addition to DSB, ROS induced various modified bases, and more than 20 different types of oxidatively altered purines and pyrimidines have been detected . These DNA damages also induce mutations and cell death, if not repaired.
Biomedical applications of plasma in either direct or indirect way are always associated with the generation of ROS that cause oxidation of cellular DNA in the treated tissues. If the plasma-induced DNA damages are not correctly repaired in the surviving cells, the accumulation of mutations occurs in the surviving cells, leading to the increase of a risk for carcinogenesis in the treated tissues. Therefore, biomedical applications of plasma need to be strictly evaluated in terms of safety implications. Here, we performed mutation analysis using transgenic mice exposed by non-thermal atmospheric air plasma. Direct exposure of air plasma jet induced the mutations, mainly deletions, in the in vitro cultured splenocytes from rpsL-transgenic mice in a dose-dependent manner. Direct exposures of air plasma jet induced DNA damages in the epidermis of mouse skin. These results suggested that further investigations are needed to establish safety protocols for the long-term applications of plasma such as wound-healing and cancer treatment.
 G. Fridman, G. Friedman, A. Gutsol, A. B. Shekhter, V. N. Vasilets and A. Fridman: Plasma Processes and Polymers. 5, 503 (2008).
 M. U. Rehman, P. Jawaid, H. Uchiyama and TKondo: Arch Biochem Biophys. 605,19 (2016).
 H. Tanaka, M. Mizuno, K. Ishikawa, K. Nakamura, H. Kajiyama, H. Kano, F. Kikkawa and M. Hori: Plasma Medicine. 1, 265 (2011).
 F. Utsumi, H. Kajiyama, K. Nakamura, H. Tanaka, M. Mizuno, K. Ishikawa, H. Kondo H. Kano, M. Hori and F. Kikkawa: PLoS One. 8, e81576 (2013).
 K. Rodgers and M. McVey: J Cell Physiol. 231, 15 (2016).
 E. Gajewski, G. Rao, Z. Nackerdien and M. Dizdaroglu: Biochemistry. 29, 7876 (1990)..
|4.||Tsuzuki Teruhisa, Ohno Mizuki, Takano Noriko, Taguchi Ken-ichi, Nakabeppu Yusaku, Nakatsu Yoshimichi, DNA repair system as a constituent of mechanism underlying practical threshold of oxidative stress-induced tumorigenesis, The 12th International Conference and the 5th Asian Congress on Environmental Mutagens, 2017.11.|
|5.||Tsuzuki Teruhisa, Ohno Mizuki, Takano Noriko, Taguchi Ken-ichi, Nakabeppu Yusaku, Nakatsu Yoshimichi, Oxidative stress-induced tumorigenesis: Lesson from the experiments with DNA repair-deficient mice, 4th Transgenic Technology Meeting, 2017.10.|
|6.||Tsuzuki Teruhisa, Ohno Mizuki, Takano Noriko, Taguchi Ken-ichi, Nakabeppu Yusaku, Aoki Yasunobu, Nohmi Takehiko, Nakatsu Yoshimichi, Oxidative stress-induced intestinal tumors in Mutyh-deficient mice treated with low doses of potassium bromate, 6th US-Japan DNA Repair Meeting, 2017.05.|
|7.||Yoshimichi Nakatsu, Noriko Takano, Mizuki Ohno, Satoshi Kitazaki, Kazunori Koga, Akiyo Tanaka, Masaharu Shiratani, Teruhisa Tsuzuki, Analyses of oxidative mutagenesis and carcinogenesis using genetically modified mice: application to plasma medicine, The 4th International Workshop on Plasma for Cancer Treatment (IWPCT-2017), 2017.03, Oxygen radicals attack DNA, and induce various lesions into the DNA. Among such lesions, 8-oxo-7, 8-dihydroguanine (8-oxoG) is highly mutagenic because of its ambiguous pairing property. Three enzymes, MTH1, OGG1, and MUTYH, play important roles in avoiding the 8-oxoG-related mutagenesis in mammalian cells. We have established an experimental system for oxidative DNA damage-induced mutagenesis and tumorigenesis in the intestine of mice. Oral administration of oxidizing reagent, potassium bromate (KBrO3), effectively induced G:C to T:A mutations and epithelial tumors in the small intestines of Mutyh-deficient mice, implying the significance of Mutyh in the suppression of mutagenesis and tumorigenesis induced by oxidative stress. To elucidate the roles of other DNA repair genes in the suppression of oxidative stress-induced tumorigenesis, we performed KBrO3-induced tumorigenesis experiments using various types of DNA repair-deficient mice. We observed an enhanced tumor-formation in the small intestines of Msh2-deficient mice, as compared with the wild type. No such enhancement was observed in Xpa-deficient mice. These results indicate that mismatch repair, but not nucleotide excision repair, is involved in the suppression of oxidative stress-induced intestinal tumorigenesis in mice. The number of tumors was marginally increased in Ogg1- and Mth1-deficient mice, in comparison to the wild-type mice, suggesting that in contrast to Mutyh, Ogg1 and Mth1 may play a limited role in the suppression of intestinal tumorigenesis caused by oxidative stress. Our data indicate that among the repair factors examined, only Mutyh and Msh2 play a significant role in the suppression of oxidative stress-induced intestinal tumorigenesis in mice.
To establish the application of plasma to humans, it is important to evaluate the potential risk of plasma to organisms. Plasma is a gas composed of electrons, various ions, and reactive oxygen/nitrogen species. The plasma exposure produced RONS not only in external environment but also in cells, thus anticipated the inductions of DNA damages and mutations. Therefore, we performed the mutation analyses using mouse cells exposed by non-thermal atmospheric air plasma. Direct exposure of air plasma jet induced the mutations, mainly deletions, in the in vitro cultured splenocytes from rpsL-transgenic mice in a dose-dependent manner. On the other hand, direct exposures of air plasma jet induced DNA damages in the epidermis of mouse skin. However, the increases of mutation frequencies were not detected in the air plasma-exposed mouse skins. These results suggest that genetically modified mice are useful tool for the risk assessment of plasma..
|8.||Yoshimichi Nakatsu, EFFECTS OF NON-THERMAL ATMOSPHERIC AIR PLASMA IRRADIATION ON MOUSE SKIN
, International Conference on Plasma Medical Science Innovation (ICPMSI) 2017, 2017.02.
|9.||Yoko Yamanishi, Ryotaro Tanaka, Yuta Arakawa, Yoshimichi Nakatsu, Gene transfer by circulating plasma bubble flow, 30th IEEE International Conference on Micro Electro Mechanical Systems, MEMS 2017, 2017.02, We have succeeded in injection of plasmid to adherent cells which are suspended in the plasma-bubbles laden circulation flow in a chamber. High-speed plasma-bubbles are generated by glass electrode and the air-liquid interface has a stiction force which draws the gene (plasmid) and stick to the air-liquid interface. The circulating flow increased the chance for cells to contact air-liquid interface of bubbles which enclosed plasma or reactive gas. Finally, the high reactive interface enables gene transfer to cells efficiently. This technology of two-dimensional microfluidic chip contributes an option to the high-throughput gene transfer..|
|10.||Yoshimichi Nakatsu, Noriko Takano, Mizuki Ohno, Satoshi Kitazaki, Kazunori Koga, Akiyo Tanaka, Masaharu Shiratani, Teruhisa Tsuzuki, Analyses of oxidative mutagenesis and carcinogenesis using genetically modified mice: application to plasma medicine, 第26回日本MRS年次大会/Internatinal Symposium C-4, 2016.12.|
|11.||Hayashida Genki, Nakatsu Yoshimichi, Hidaka Kyoko, Fujikane Ryosuke, Hidaka Masumi, Tsurimoto Toshiki, Tsuzuki Teruhisa, Development of assay systems to characterize the variants of mismatch repair factor MSH2 found in Lynch syndrome, The 10th International 3R (Replication, Recombination and Repair) Symposium, 2016.11.|
|12.||Yoshimichi Nakatsu, Noriko Takano, Mizuki Ohno, Satoshi Kitazaki, Kazunori Koga, Takaaki Amano, Akiyo Tanaka, Miyuki Hirata, Masaharu Shiratani, Teruhisa Tsuzuki
, Mutagenesis in Human and Mouse Cells Irradiated by Non-thermal Atmospheric Air Plasma
, 6th International Conference on Plasma Medicine (ICPM-6), 2016.09.
|13.||Mizuki Ohno, Noriko Takano, SAKUMI Kunihiko, Ryutaro Fukumura, Yuki Iwasaki, Toshimichi Ikemura, Yoichi Gondo, Yusaku Nakabeppu, Yoshimichi Nakatsu, Teruhisa Tsuzuki, Role of the oxidative DNA damage repair system in somatic and germline mutations in mice, Zing conference "Genome Integrity" , 2015.08.|
|14.||Mizuki Ohno, Noriko Takano, Kunihiko Sakumi, Ryutaro Fukumura, Yuki Iwasaki, Toshimichi Ikemura, Yoichi Gondo, Yusaku Nakabeppu, Yoshimichi Nakatsu, Teruhisa Tsuzuki, Influence of Oxidative DNA Damage on the Rate of Somatic and Germiline Mutation, 15th International Congress of Radiation Research, 2015.05.|
|15.||Teruhisa Tsuzuki, Mizuki Ohno, Noriko Takano, Kenichi Taguchi, Yusaku Nakabeppu, Yoshimichi Nakatsu, Oxidative Stress-induced Tumorigenesis: Lesson from the Experiments with DNA Repair-deficient Mice, Advances in Understanding the Biological Consequences by Environmental Stressors, 15th International Congress of Radiation Research, 2015.05.|
|16.||Yoshimichi Nakatsu, Jingshu Piao, Takuya Hashizume, Mizuki Ohno, Kenichi Taguchi, Teruhisa Tsuzuki, Mismatch Repair Deficient Mice Show Susceptibility to Oxidative Stress-induced Intestinal Carcinogenesis, 15th International Congress of Radiation Research, 2015.05.|
|17.||Teruhisa Tsuzuki, Mizuki Ohno, Noriko Takano, Kenichi Taguchi,, Yusaku Nakabeppu, Yasunobu Aoki, Takehiko Nohmi, Yoshimichi Nakatsu, Oxidative stress-induced intestinal tumors in Mutyh-deficient mice treated with low doses of potassium bromate, 4th Asian Conference on Environmental Mutagen, 2014.12.|
|18.||Teruhisa Tsuzuki, Mizuki Ohno, Noriko Takano, Kenichi Taguchi, Yusaku Nakabeppu, Yasunobu Aoki, Takehiko Nohmi, Yoshimichi Nakatsu, Oxidative stress-induced intestinal tumors in Mutyh-deficient mice treated with low doses of potassium bromate, 5th US-Japan DNA Repair Meeting, 2014.10.|
|19.||Charatda Punvittayagul, Yoshimichi Nakatsu, Rawiwan Wongpoomchai, Mizuki Ohno, Teruhisa Tsuzuki, In vitro study for mutagenicity of purple rice hull extract using fibroblasts derived from rpsL-transgenic mouse, 日本癌学会, 2013.10.|
|20.||Teruhisa Tsuzuki, Jing Shu Piao, Noritaka Matsumoto, Yoshimichi Nakatsu, The roles of mismatch repair system and p53 in the suppression of oxidative stress-induced intestinal tumor-formation in mice., 4th US-Japan DNA Repair Meeting, 2012.04, In living cells, reactive oxygen species (ROS) is constantly generated by cellular metabolic reactions such as mitochondrial respiration, and also by external cause such as exposure to radiation and chemicals. DNA and its substrate, nucleotides, are continuously oxidized by ROS, and resulting oxidative DNA damages appear to cause mutagenesis and carcinogenesis in mammal. To counteract this hazardous effects of ROS, mammalian cells are equipped with several enzymatic systems. Following KBrO3 treatment in all mouse types, tumor-formation in the small intestines of Msh2-/- mice significantly increased as compared with Msh2+/+ and Msh2+/- mice. These results suggest that mismatch repair (MMR) is involved in the suppression of oxidative stress-induced intestinal tumorigenesis in mice. Following KBrO3-treatment, tumor-formation in the small intestine of Trp53-/- mice significantly increased as compared with Trp53+/+ and Trp53+/- mice and tumor-formation in the small intestine of Trp53+/- mice moderately increased as compared with Trp53+/+ mice. In addition to previous observation with Mutyh-deficient mice, our data suggest that MMR system as well as Trp53 is also involved in the suppression of oxidative stress-induced intestinal tumorigenesis in mice..|
|21.||Oxidartive stress-induced intestinal tumorigensis in Trp53-deficient mice.|
|22.||Oxidartive stress-induced intestinal tumorigensis in Trp53-deficient mice.|
|23.||The roles of tumor-associated genes, Mutyh, Msh2 and Trp53, in the suppression of oxidative stress-induced tumorigenesis in mice.|
|24.||A MAPO1 protein complex that involved in the induction of apoptosis triggered by O6-methylguanine.|
|25.||Oxidative stress and intestinal carcinogenesis.|
|26.||Mutagenesis caused by oxidized nucleotides and its preventive mechanisms in mammal.|
|27.||Mutation analysis of the Apc gene in the small intestinal tumors induced by oxidative stress in Mutyh-deficient mice.|
|28.||Oxidative stress-induced intestinal tumors in Mutyh-deficient mice.|
|29.||Mutagenesis and tumorigenesis in mice with a targeted disruption of the Mutyh gene.|