A n nu a lRepor t2 0 1 72017
Japan Chemical Industry Association

Annual Report 2017

Long-range Research InitiativeDevelopment of New Risk Assessment Methods with iPSCs

Title of Research:
13_S01-01-4

Development of the Novel Method with iPSCs to Assess ChemicalCompounds for the Risk of CSC Induction
Principal Investigator:
Masaharu Seno, PhD (Professor, Graduate School of Natural Science and Technology, Okayama
University) 3.1.1 Tsushima-Naka, Kita, Okayama 700-8530, Japan.
Collaborators:
Tomonari Kasai, PhD, Akifumi Mizutani, PhD, Jyunko Masuda, PhD, Akimasa Seno, PhD, Aun Ko
Ko Oo, PhD, Hideki Minematsu, MS, Eman Ahmed Taha, MS (Graduate School of Natural Scienceand Technology, Okayama University)
Yoshiaki Iwasaki, MD, PhD (Center of Healthcare, Okayama University)
Summary of Research:
The risk assessment for the cancer development caused by chemical compounds has long
been made by mutagenicity or repeated dose toxicity study, by which it generally takes long timeto get results. However, cancer is currently recognized consisting of heterogeneous population ofcells and cancer stem cells (CSCs). This is altering the idea that a cancer tissue consists of ahomogeneous population of clonal cells continuously proliferating due to the oncogenic mutations.
It appears necessary to develop a novel method to identify chemical compounds that may induce
cancer stem cells. In this study, we established a simple method to assess the risk of chemicalcompounds inducing CSCs employing mouse iPSCs, which have been reprogrammed from theembryonic fibroblast derived from a transgenic mouse carrying the Nanog promoter integrated
with GFP gene. This method allows the assessment in 8 days by judging the increase of spheres
and the fluorescence of GFP enhanced by Nanog gene expression. We evaluated 147 chemicalcompounds and 30 were judged as positive. Further, we tried to analyze the molecular mechanism
inducing CSCs from miPSCs by microarray, CpG methylation, lipid component, bioinformatics andso on. As the results, the relationship of some chemokine and its receptors, some specificcytoplasmic signal transduction pathway, inflammatory factors were implied to be importantly
involved. Collectively, it should be possible to find out the key relationships between the cancer
risks and the mechanism of action of chemical compounds in the future.
Timeline:
March 1, 2012 – February 28, 2018
Topics:Poster presentation entitled “Development of the Novel Method with iPSCs to Assess ChemicalCompounds for the Risk of CSC Induction” at 2017 Annual Meeting of LRI.
Publications:
Nair N, Calle AS, Zahra MH, Prieto-Vila M, Oo AKK, Hurley L, Vaidyanath A, Seno A, Masuda J,
Iwasaki Y, Tanaka H, Kasai T, Seno M. A cancer stem cell model as the point of origin ofcancer-associated fibroblasts in tumor microenvironment. Sci Rep. 2017;7(1):6838.
Ninomiya T, Ohara T, Noma K, Katsura Y, Katsube R, Kashima H, Kato T, Tomono Y, Tazawa H,
Kagawa S, Shirakawa Y, Kimura F, Chen L, Kasai T, Seno M, Matsukawa A, Fujiwara T. Iron
depletion is a novel therapeutic strategy to target cancer stem cells. Oncotarget.
2017;8(58):98405-98416.
OoA, Calle AS, Nair N, Mahmud H, Vaidyanath A, Yamauchi J, Khayrani AC, Du J, Alam MJ, Seno
A, Mizutani A, Murakami H, Iwasaki Y, Chen L, Kasai T, Seno M. Up-Regulation of PI 3-Kinases
and the Activation of PI3K-Akt Signaling Pathway in Cancer Stem-Like Cells Through DNA
Hypomethylation Mediated by the Cancer Microenvironment. Translational Oncology.
2018;11(3), 653-663.
Seno A, Seno M. Commonly expressed genes among cancer stem cells induced from hiPSCs and1

Development of New Risk Assessment Methods with iPSCs
obtained from cancer tissues or cell lines. Tumor and Microenvironment. 2018 in press.

2

Development and assessment of new risk assessment methods

Title of Research: 16_S01-01-2

Advanced system development for the hazard prediction and theenvironmental
risk
assessment/managementofchemical
substances
Principal Investigator: Bin-Le Lin, Chief Senior Researcher, Research Institute of Science for
Safety and Sustainability, National Institute of Advanced Industrial Science and Technology. 16-1
Onogawa Tsukuba City, Ibaraki, 305-8569 Japan. (Tel) +81-29861 8844, (e-mail) binlelin@aist.go.jp: Yoshimasa Takahashi, Professor of Toyohashi University of Technology. (Tel) +81532-446878, (e-mail) taka@cs.tut.ac.jp;
Summary of Research: In this research, collaboration between AIST and Toyohashi University ofTechnology will develop an advanced system to enable risk assessment management of
unconfirmed existing chemical substances and new substances without hazard information facedby Chemical Industry Association. Toward the goal of designated research, the following research
outputs were obtained. 1) MeRAM Ver.2.0 as the latest released version (Japanese version &English version) were open for public; (2) added a function to MeRAM that can automatically export
the built-in data of MeRAM according to various purposes of users; (3) In order to be used byenvironmental administration, a function that can automatically launch KATE from the interface of
MeRAM, and then can automatically acquire the predicted value of KATE and use it for risk
assessment; Although the release of the MeRAM latest version equipped with the functions of 2)
and 3) is not yet available, it was distributed to many participants at seminars of the Ministry of theEnvironment and workshops organized by AIST; (4) Started to develop our own methodology forpredicting the toxicity of chemical substances from October of last year. Based on various
fingerprint information (structure and physical information) of chemical substances and toxicity
information, toxicity estimation by machine learning but not using QSAR equations is underdeveloped.
Timeline: From March 2017 to February 2018
Topics: None
Publications:
1) (Paper Publication) Institution magazine "Toxicity Inquiry Box" of the Safety Assessment
Committee, 2017: "Ecological risk assessment of chemical substances in water environment".
2) (Presentation at Domestic Meeting) The 26th Environmental Chemistry Debate (7-9 June 2017),
Shizuoka Convention Center, Shizioka, “IT solution for Cumbersome Ecological Risk Assessment
and Management of Chemicals: AIST-MeRAM”.
3) (Invited Speech at Domestic Workshop) The 26th Environmental Chemistry Debate (7-9 June2017), Shizuoka Convention Center, Shizioka, "Open Science in Environmental Chemistry" Why
do not open your own data: Potential of using open data."
4) (Presentation at Domestic Meeting) The 20th Water Environment Symposium (26-27 September2017), Wakayama University, “Hierarchical ecological risk assessment and management ofchemical substances using MeRAM”.
5) (Presentation at Domestic Meeting) The Tougou-no-hi-no Symposium (4-5 October 2017), Tokyo
University, “Attempt to utilize integrated database for environmental risk assessment and risk
communication of chemical substances”.
6) (Presentation at Domestic Meeting) The 30th Annual Meeting of Japan Risk Research (27-29
October 2017), Shiga University, “Risk assessment of toxicity deficient substances by linking
MeRAM and KATE”.
7) (Invited Speech at Domestic Workshop) Environmental Science Seminar hosted by Ministry of
the Environment Seminar on chemical substance environment survey (15-16 January 2018),
Tokyo, " What MeRAM can do."8) (Invited Speech at Domestic Workshop) MeRAM Workshop hosted by AIST (23 January 2018),
3

Development and assessment of new risk assessment methods
Tokyo.

4

Development and assessment of new risk assessment methods

Title of Research: 16_S01-01-2

Advanced system development for the hazard prediction and theenvironmental
risk
assessment/managementofchemical
substances
Subtitle: Development of the sophisticated basis tool environmental
hazard prediction
Principal Investigator:
Yoshimasa Takahashi, PhD (Professor, Dept. Comp. Sci. Eng., Toyohashi Univ. Tech.)1-1 Hibarigaoka, Tempaku-cho, Toyohashi 441-8580 JAPAN(tel)+81-0532-44-6878, (email)taka@cs.tut.ac.jp
Collaborators:
Tetsuo Katsuragi (Assit. Prof., Toyohashi Univ. Tech.)
Taku Izumihara (Graduate student, Toyohashi Univ. Tech.)
Mitsuru Sato (Graduate student, Toyohashi Univ. Tech.)
Tsubasa Ishikawa (Graduate student, Toyohashi Univ. Tech.)

Summary of Research:
In this fiscal year (Fiscal 2017) research, we designed external file specifications for data import to
PEACH(Prediction of Environmental Affect of Chemicals) with the aim of utilizing external data,and implemented the import function of external data utilizing the chemical structure dictionary. For
the chemical structure dictionary tool developed in the previous year, we expanded the data importfunction and revised the user-interface for strengthening cooperation with MeRAM. In addition, we
refined the estimation model of water solubility parameter logS by atomic fragment method. We
developed an estimation model of vapor pressure too. On the other hand, for the short-term
toxicity (96h-LC50) against fish, we developed alternative prediction models based on quantitative
activity-activity relationships (QAAR) for the toxicity tests with different fish species. The results
show that the QAAR models would be very useful for prediction of missing data.
Timeline:
1st Mar. 2016 – 28th Feb. 2017
Topics:Poster presentatikon at ICCA-LRI International Workshop 2016, Awaji, Jun., 2016
Publications:
1) Tsubasa Ishikawa, Tetsuo Katsuragi, Bin-Le Lin, Yoshimasa Takahashi, Studies oncomplementation of missing data of fish toxicity by Quantitative Activity-Activity Relationship, 26th
Symposium on Environmental Chemistry, Shizuoka, 2017.
2) Taku Izumihara, Tetsuo Katsuragi, Bin-Le Lin, Yoshimasa Takahashi, Construction of Chemical
Structure Dictionary for MeRAM, 26th Symposium on Environmental Chemistry, Shizuoka, 2017.
3) Mitsuru Sato, Dai Furukawa, Tetsuo Katsuragi, Yoshimasa Takahashi, Prediction of
Aqueous-Solubility Using an Atomic Group Contribution Method, 26th Symposium on
Environmental Chemistry, Shizuoka, 2017.
4) Yoshimasa TAKAHASHI and Ryota KIKUCHI, Algal toxicity prediction of chemicals using
TFS-PLS method in conjunction with active QSAR modeling, Abstracts of the 53rd Congress of
the European Societies of Toxicology (EUROTOX) Bratislava, Slovakia, 10th–13th September,2017. ; Toxicology Letters 280S (2017) S316.
5) Yoshitaka Inagaki, Tomoya Yamazaki, Yoshimasa Takahashi, “Development of a desk-top tool
ToxCalc for eco-toxicity prediction of chemicals”, The 43rd Symposium on Structure-Activity
Relationships, Tsuchiura, Nov., 2017.

5

Title of Research:16_S02-02-2

Prediction method (Amino acid Derivative Reactivity Assay: ADRA)
for skin sensitization using novel lysine and cysteine derivatives
Principal Investigator:
Toshihiko Kasahara, PhD (Engineering Manager, FUJIFILM Corporation, Safety Evaluation Center,
Ecology & Quality Management Division, CSR Division)
210, Nakanuma, Minamiashigara-shi, Kanagawa 250-0193, Japan(tel)+81-465-73-7440 (e-mail)ktoshihiko.kasahara@fujifilm.com
Collaborators:
Masaharu Fujita, PhD（FUJIFILM Corporation,Safety Evaluation Center）
210, Nakanuma, Minamiashigara-shi, Kanagawa 250-0193, Japan(tel)+81-465-73-7440 (e-mail)masaharu.fujita@fujifilm.com
Yusuke Yamamoto (FUJIFILM Corporation,Safety Evaluation Center）
210, Nakanuma, Minamiashigara-shi, Kanagawa 250-0193, Japan(tel)+81-465-73-7440 (e-mail)yusuke.yamamoto@fujifilm.com
Atsuishi Ono, PhD (Professor, Okayama University, Graduate school of Medicine, Dentistry andPharmaceutical sciences, Division of Pharmaceutical Sciences)1-1-1, Tsushimanaka, Kita-ku, Okayama-shi, Okanaya 700-8530, Japan(tel)086-251-7942 (e-mail) atsushiono3@okayama-u.ac.jp
Hajime Kojima, PhD (Director, National Institute of Health Sciences, Biological safety ResearchCenter, Division of Risk Assessment)
1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan(tel) 03-3700-9874 (e-mail)h-kojima@nihs.go.jp
Shinichi Wantanabe (LION CORPORATION, HUMAN & ENVIRONMENTAL SAFETYEVALUATION CENTER)
100, Tajima, Odawara-shi, Kanagawa 256-0811(tel)0465-49-4475 (e-mail)shin-w@lion.co.jp
Tsunetsugu Sugawara (LION CORPORATION, HUMAN & ENVIRONMENTAL SAFETYEVALUATION CENTER)
100, Tajima, Odawara-shi, Kanagawa 256-0811(tel)0465-49-4475 (e-mail) tsune-s@lion.co.jp
Koji Wakabayashi (Manager, MITSUI CHEMICALS, INC., Chemical Safety Department
1144, Togo, Mobara-shi, Chiba 297-0017, Japan(tel)0475-23-8251 (e-mail)Koji.Wakabayashi@mitsuichemicals.com
Yu Tahara (MITSUI CHEMICALS, INC., Chemical Safety Department
1144, Togo, Mobara-shi, Chiba 297-0017, Japan(tel)0475-23-8251 (e-mail)Yu.Tahara@mitsuichemicals.com
Nobuyuki Horie (Senior Research Scientist, SUMITOMO Chemical Co., Ltd., Environmental
Health Science Laboratory)
3-1-98, Kasugadenaka, Konohana-ku, Osaka-shi, Osaka 554-8558, Japan(tel)06-6466-5107 (e-mail)horien@sc.sumitomo-chem.co.jp
Keiichi Fujimoto (SUMITOMO CHEMICAL Co., Ltd., Environmental Health Science Laboratory)
3-1-98, Kasugadenaka, Konohana-ku, Osaka-shi, Osaka 554-8558, Japan(tel)06-6439-9753 (e-mail) fujimotok2@sc.sumitomo-chem.co.jp
Kei Kusakari (NISSAN CHEMICAL INDUSTRIES, LTD. Biological Research Laboratories)
1470, Shiraoka, Shiraoka-shi, Saitama 349-0294, Japan(tel)0480-92-2513 (e-mail)kusakarik@nissanchem.co.jp
Yoshihiko Kurokawa (NISSAN CHEMICAL INDUSTRIES, LTD. Biological Research Laboratories)
1470, Shiraoka, Shiraoka-shi, Saitama 349-0294, Japan(tel)0480-92-2513 (e-mail)kurokawayo@nissanchem.co.jp
Takashi Sozu, PhD (Assistant Professor, Department of Information and Computer Technology,
Faculty of Engineering, Tokyo University of Science)
6-3-1, Shinjuku, Katsushika-ku, Tokyo 125-8585, Japan(tel)03-5876-1688 (e-mail)sozu@rs.tus.ac.jp
Takuto Nakayama (Department of Management Science, Graduate School of Engineering, Tokyo

6

University of Science)
6-3-1, Shinjuku, Katsushika-ku, Tokyo 125-8585, Japan(tel)03-5876-1688 (e-mail)4416624@ed.tus.ac.jp
Tsuyoshi Kawakami, PhD (Director, National Institute of Health Sciences, Biological safety
Research Center, Division of Risk Assessment)
1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan(tel) 03-3700-1141 (e-mail)tkawa@nihs.go.jp
Kohichi Kojima 、 PhD （ Director-General, Food and Drug Safety Center, Hatano ResearchInstitute）
729-5, Ochiai, Hadano-shi, Kanagawa 257-8523, Japan(tel)0463-82-4751 (e-mail)kojima.k@fdsc.or.jp
Nicol C. Kleinstreuer, PhD （NTP Interagency Center for the Evaluation of Alternative Toxicologicalmethods）(tel) (919) 541-7997 (e-mail) nicole.kleinstreuer@nih.gov
Jon Richmind, PhD （Ethical Biomedical Research and Testing Advice & Consultancy）
Doubledykes Carslogie Road, Cupar, Fife KY154HY, United Kingdom(tel)077-86-381-757 (e-mail)dr.richmond@gmail.com
Bae-Hwan Kim, PhD （Professor, Keimyung University, Department of Public Health, College of
Natural Science）
1095, Dalgubeol-daero, Dalseo-Gu, Daegu 42601, Republic of Korea(tel)+82-53-580-5933 (e-mail)kim9399@kmu.ac.kr
Summary of Research:
In this study, we performed the validation study so that the alternative method for skinsensitization (ADRA) is adopted as OECD test guideline.
Part1)For the four participating laboratories, the two training sessions were held, and the two
confirmation tests (Pre-training test and Training test) were performed because the lead laboratory
confirmed that the assay technique was correctly transferred for the participating laboratories.
Compared with cell-based in vitro test methods, DPRA is an easy-to-use test method and exhibits
excellent predictive capacity. Nevertheless, the DPRA test method has significant limitations: One of the nucleophilic reagents, cysteine peptide, is easily oxidized rendering some test
results invalid, unreliable or difficult to interpret.
 It requires relatively high concentrations of the test chemical, making it unsuitable for
evaluating poorly soluble chemicals.
 It requires a relatively large quantity of both test chemicals and nucleophilic reagents toperform.
 Hydrophobic test chemicals tend to precipitate easily in the reaction solution.
ADRA, an in chemico test which is also based on protein reactivity, does not have these limitations
thanks to the use of highly sensitive nucleophilic reagents, which allows reagent solutions to beprepared at test chemical concentrations just 1% of those required in DPRA.
Following a modular approach applying the ECVAM principles on test validity (OECD Series on
Testing and Assessment, Number 34, 2005, Hartung et al. 2004), the Validation Management
Team (VMT) empirically evaluated modules one through four (test definition, within-laboratory
reproducibility, transferability, and between-laboratory reproducibility), and used these results also
to evaluate modules five and six (predictive capacity and applicability domain).
During a preliminary training phase of the ADRA validation study using ten test chemicals, theresults obtained by the four participating laboratories were 100% concordant with the leadlaboratory. During the subsequent transferability phase of the study, three of the four participating
laboratories were 100% (10/10) concordant and the fourth laboratory was 90% (9/10) concordant
with the lead laboratory. The ADRA VMT considers these results to have confirmed that the ADRA
test method is easily transferred to naïve laboratories.
During Phase I of the Study Plan, each of the four participating laboratories performed three test
runs of identical sets of 10 coded test chemicals to evaluate within-laboratory reproducibility. The
results, based on concordance, were 100% (10/10), 100% (10/10), 100% (7/7), and 90% (9/10).
When the results from the 10 test chemicals of Phase I were combined with those of Phase II, in
which each participating laboratory performed one test run of identical sets of an additional 30 test
chemicals, between-laboratory reproducibility for 40 test chemicals was 92%. The VMT considers
7this to satisfy standards for both within- and between-laboratory reproducibility for this class of test
method.
Moreover, a review of predictive capacity relative to LLNA based on the results of Phases I and II
yielded a sensitivity of 81%, specificity of 98%, and accuracy of 86%. The VMT therefore
considers ADRA to be an in chemico method with sufficient specificity, sensitivity, and predictive
capacity for regulatory acceptance.
Part 2)
During a recent validation study conducted at multiple laboratories as part of the process toinclude ADRA in an existing OECD test guideline, one of the nucleophilic reagents used in
ADRA—N-(2-(1-naphthyl)acetyl)-L-cysteine (NAC)—was found to be susceptible to oxidation inmuch the same manner that the cysteine peptide used in DPRA was. Due to this, we undertook astudy to clarify the cause of the promotion of NAC oxidation.
In general, cysteine and other chemicals that have thiol groups are known to be oxidized in thepresence of minute quantities of metal ions. When Fe and Cu ions were added to the ADRA
reaction solution, the Cu ions facilitated NAC oxidation significantly. When 0.25 µM of EDTA wasadded in the presence of Cu ions, NAC oxidation was suppressed. Based on this, we predictedthat the addition of EDTA to the NAC stock solution would suppress NAC oxidation. Next, we
tested 82 chemicals used in developing ADRA to determine whether or not EDTA affects ADRA’s
ability to predict sensitization. The results showed that the addition of EDTA has virtually no effecton the reactivity of NAC with a test chemical and yielded an accuracy of 87% for predictions of skinsensitization, which was roughly the same as ADRA.
Timeline: March 1, 2017- (it continues from March 1, 2016)
Topics: none
Publications:
1. Presented at the 44th The Japanese Society of Toxicology (2017.7, Yokohama) 「Multicenter
Validation Study of Novel in chemico Skin Sensitization Assay (ADRA): 1st Report」
Atsushi Ono, Shin-ichi Watanabe, Tsunetsugu Sugawara, Koji Wakabayashi, Yu Tahara,
Nobuyuki Horie, Keiichi Fujimoto, Kei Kusakari, Yoshihiko Kurokawa, Takashi Sozu, Takuto
Nakayama, Takeru Kusao, Tsuyoshi Kawakami, Kohichi Kojima, Hajime Kojima, Jon
Richmond, Nicole Kleinstreuer, Bae-Hwa Kim, Yusuke Yamamoto, Masaharu Fujita, andToshihiko Kasahara
2. Presented at the 10th World Congress on Alternatives and Animals in the Life Sciences(WC10) (2017.8) (Seattle, WA, USA)「Phase-1 of the validation study of Amino acid Derivative
Reactivity Assay (ADRA) : a novel in chemico alternative test method of skin sensitization.」
Atsushi Ono, Shin-ichi Watanabe, Tsunetsugu Sugawara, Koji Wakabayashi, Yu Tahara,
Nobuyuki Horie, Keiichi Fujimoto, Kei Kusakari, Yoshihiko Kurokawa, Takashi Sozu, Takuto
Nakayama, Takeru Kusao, Tsuyoshi Kawakami, Kohichi Kojima, Hajime Kojima, Jon
Richmond, Nicole Kleinstreuer, Bae-Hwa Kim, Yusuke Yamamoto, Masaharu Fujita, andToshihiko Kasahara
3. Presented at the 30th Annual Meeting of the Japanese Society for Alternatives to Animal
Experiments」 (2017.11, Tokyo) 「Multi-laboratory validation study of ADRA as novel inchemico alternative test method for skin sensitization: 2nd report」Atsushi Ono, Shin-ichi
Watanabe, Tsunetsugu Sugawara, Koji Wakabayashi, Yu Tahara, Nobuyuki Horie, Keiichi
Fujimoto, Kei Kusakari, Yoshihiko Kurokawa, Takashi Sozu, Takuto Nakayama, Takeru Kusao,
Tsuyoshi Kawakami, Kohichi Kojima, Hajime Kojima, Jon Richmond, Nicole Kleinstreuer,
Bae-Hwa Kim, Yusuke Yamamoto, Masaharu Fujita, and Toshihiko Kasahara
4. The Cause of and Countermeasures for Oxidation of the Cysteine-Derived Reagent Used in
the Amino acid Derivative Reactivity Assay. (in submission to Journal ofAppliedToxicology) Masaharu Fujita, Yusuke Yamamoto, Shinichi Watanabe, Tsunetsugu Sugawara,
Koji Wakabayashi, Yu Tahara, Nobuyuki Horie, Keiichi Fujimoto, Kei Kusakari, Yoshihiko
Kurokawa, Tsuyoshi Kawakami, Kohichi Kojima, Hajime Kojima, Atsushi Ono, Yasuhiro
Katsuoka, Hideto Tanabe, Hiroshi Yokoyama, and Toshihiko Kasahara.

8Research on the effects of chemical substances on children, elderly
people, and those with gene disorders
Title of Research:
15_S03-01-3

Establishment of in vitro evaluation system for the prediction of
respiratory sensitizing potential of chemicals
Principal Investigator:
Takayuki Yoshimoto, PhD (Professor, Tokyo Medical University, Institute of Medical Science)
6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan(tel)+81-3-3351-6141 Ext. 431, (e-mail) yoshimot@tokyo-med.ac.jp
Collaborators:
Izuru Mizoguchi, PhD (Research Associate, Tokyo Medical University, Institute of Medical
Science) 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan(tel) +81-3-3351-6141 Ext. 448, (e-mail) imizo@tokyo-med.ac.jp
Summary of Research:
Recently, several in vitro assays to predict the respiratory sensitizing potential of chemicals
have been developed, because the use of animal models in safety testing of chemicals issignificantly limited. However, these alternative methods cannot distinguish chemical respiratory
sensitizers and skin sensitizers, although the risk management systems for them are quite
different. Therefore, in the present study, we aim at developing a novel in vitro assay, which candiscriminate chemical respiratory sensitizers from skin sensitizers by taking advantage of thefundamental differences between their modes of function; development of helper T (Th) 2 immune
responses, which is critically important for respiratory sensitization.
In this year, we have established an immunocompetent 3D co-culture system of human upper
airway, which consists of human airway epithelial cells, immature dendritic cells (DCs) derived
from human peripheral blood CD14+ monocytes, and human fibroblast cells cultured in individual
scaffolds. Then, these scaffolds were assembled into a 3D multi-cell tissue model, which moreclosely mimics the in vivo situation. Then, three typical chemicals for respiratory and skinsensitizers were added on the 3D co-culture system. The present results indicate that this 3D
co-culture system can predict the respiratory sensitizing potential of chemicals by means ofenhanced expression of key costimulatory molecule OX40L, which is important for Th2
differentiation, as markers in DCs (1). Moreover, to improve the versatility, we have next utilized
iPS cell-derived DCs based on published protocols, although this efficacy is not good. Therefore,
we are currently trying to establish a novel method to generate infinitely growing myeloid
progenitors by stimulation with various cytokines and incorporation of genes related to cell cycle
and survival. In addition, we are also trying to develop a novel 3DC/T system, which consists ofsensitized DCs and allogenic naive CD4+ T cells. In this 3DC/T system, IL-4 up-regulation could be
the best marker for the prediction of respiratory sensitizing potential of chemicals.
Timeline:
March 1, 2017-Feberary 28, 2018
Topics:The 5th Annual Meeting of The Japan Chemical Industry Association LRI, poster presentation“Establishment of a novel in vitro evaluation system for the prediction of respiratory sensitizing
potential of chemicals” (The Tokyo Shoken Building, August 25, 2017)
Publications:
1. Mizoguchi I, Ohashi M, Chiba Y, Hasegawa H, Xu M, Owaki T, Yoshimoto T. Prediction ofchemical respiratory and contact sensitizers by OX40L expression in dendritic cellsusing anovel 3D coculture system. Front Immunol. 8, 929, 2017 (IF=6.429).
2. Ohashi M, Mizoguchi I, Chiba Y, Hasegawa H, Xu M, Yoshimoto T. Prediction of chemical
respiratory and contact sensitizers by OX40L expression in dendritic cells using a novel 3D
co-culture system. The 46th Annual Meeting of The Japanese Society for Immunology, oral
poster presentation in English (Sendai, December 12-14, 2017)9

Development and assessment of new risk assessment methods

Title of Research:
17_S05-01

Development of Novel Analytical System of Biological Responses to
Chemicals by Deep Phenotyping Method
Principal Investigator:
Hiroyuki Kusuhara (Professor, University of Tokyo, Graduate School of Pharmaceutical Sciences,
Department of Molecular Pharmacokinetics)
7-3-1 Hongo, Bunkyo, Tokyo, 113-0033, Japan(Tel) +81-3-5841-4770, (E-mail) kusuhara@mol.f.u-tokyo.ac.jp
Collaborators:
Tadahaya Mizuno (Assistant Professor, University of Tokyo, Graduate School of Pharmaceutical
Sciences, Department of Molecular Pharmacokinetics)
7-3-1 Hongo, Bunkyo, Tokyo, 113-0033, Japan(Tel) +81-3-5841-4771, (E-mail) tadahaya@mol.f.u-tokyo.ac.jp
Setsuo Kinoshita (Contract Research Student, University of Tokyo, Graduate School of
Pharmaceutical Sciences, Department of Molecular Pharmacokinetics)
7-3-1 Hongo, Bunkyo, Tokyo, 113-0033, Japan
Summary of Research:
Today, organisms are always exposed to chemicals in every situation to some extent. Widevariety of chemicals exist around us and the chemicals affect organisms not one by one but
simultaneously like a complex. The fact prompts us to care “multiple exposure”. To achieve theappropriate and correct prediction of multiple exposure of chemicals, it is necessary tocomprehend the effects of chemicals even if those are hidden and difficult to be detected. In thisstudy, we tackle with the theme by omic approaches with innovative techniques in both dataacquisition and data analysis method.
This study consists of two pillars: (1) a novel acquisition method of proteome profile data by
2-dimensional electrophoresis (2DE) and (2) a new analysis method of profile data. In the 5th term,
we carried out fine-tuning of these two. As for the novel data analysis method, aiming theestablishment and the operation, performance tests were conducted by employing data obtained
from public database (GEO dataset, CMap, etc). The data were mainly transcriptome data andwere subjected to the novel method, “Orthogonal Liner Separation Analysis”, OLSA, followed byliterary survey, gene ontology analysis, gathering information from compounds database(PubChem, Drugbank, etc.) and so on. The results indicate the following: (a) OLSA enables theseparation and the extraction of cellular responses as vectors, (b) a vector generated by OLSA
may be possible to be added or subtracted the others, and (c) the prediction by OLSA wasconfirmed experimentally to some extent. Of note, (b) is quite curious since it supports our
hypothesis that cellular responses (, at least a part of them,) can be linearly separated anddescribed with the separated vectors, which is essential in the prediction and evaluation of multiple
exposure. Although, as for 2DE, a problem existing in fluorescent dye due to contaminantsdelayed the progress, the problem was already solved. Therefore, we believe our progress in theestablishment of the prediction and the evaluation method of multiple exposure.
Timeline:
1/4/2017Topics:
2017 LRI annual meeting, “Development of Novel Analytical System of Biological Responses to
Chemicals by Deep Phenotyping Method”
Publications:
Nothing10

Development and assessment of new risk assessment methods
Title of Research:
13_PT01-01

Development of in vivo fluorescent imaging of neuronaldifferentiation in zebrafish for developmental neurotoxicity testing
Principal Investigator:
Yuhei Nishimura (Department of Integrative Pharmacology, Mie University Graduate School of
Medicine)
Collaborators:
Toshio Tanaka (Department of Systems Pharmacology, Mie University Graduate School of
Medicine)
Summary of Research:
The developing brain is extremely sensitive to many chemicals. Exposure to neurotoxicantsduring development has been implicated in various neuropsychiatric and neurological disorders,
including autism spectrum disorder, attention deficit hyperactive disorder, schizophrenia,
Parkinson’s disease, and Alzheimer’s disease. Although rodents have been widely used fordevelopmental neurotoxicity testing, experiments using large numbers of rodents are timeconsuming, expensive, and raise ethical concerns. Using alternative non-mammalian animalmodels may relieve some of these pressures by allowing testing of large numbers of subjects while
reducing expenses and minimizing the use of mammalian subjects. Recently, zebrafish has
emerged as an alternative non-mammalian animal model that allows testing of large numbers ofsubjects while reducing expenses and minimizing the use of mammalian subjects.
In this study, we have developed a transgenic zebrafish line whose neurons, astrocytes, andoligodendrocytes can be visualized using fluorescence stereomicroscope. Using the zebrafish, weevaluated the developmental neurotoxicity of 10 positive and 3 negative control chemicals. The
neuronal differentiation of the transgenic zebrafish was significantly affected by 9 out of the 10
positive control chemicals. The 3 negative control chemicals did not show any significant effects on
the neuronal differentiation. Hierarchical clustering of the 13 chemicals based on the fluorescentsignals revealed the similarity among valproic acid, trichostatin A, and carbamazepine and thesimilarity between nicotine and chlorpyrifos. Valproic acid, trichostatin A, and carbamazepine are
histone deacetylase inhibitors. Nicotine and chlorpyrifos can disrupt acetylcholine signaling. These
results suggest that in vivo fluorescent imaging of neuronal differentiation in the transgenic
zebrafish developed in this study can be a useful tool not only to detect the developmental
neurotoxicity of various chemicals but also to reveal the adverse outcome pathways.
Timeline:
Nov 2013 ~ Feb 2018
Topics:Presented at TEST SMART DNT4 (2014) and ICCA-LRI and NIHS workshop（2016）
Publications:
1)

2)

3)

Nishimura Y, Murakami S, Ashikawa Y, Sasagawa S, Umemoto N, Shimada Y, Tanaka T:
Zebrafish as a systems toxicology model for developmental neurotoxicity testing.
Congenital anomalies 2015; 55(1):1-16.
Nishimura Y, Okabe S, Sasagawa S, Murakami S, Ashikawa Y, Yuge M, Kawaguchi K, Kawase
R, Tanaka T: Pharmacological profiling of zebrafish behavior using chemical and geneticclassification of sleep-wake modifiers. Frontiers in pharmacology 2015; 6:257.
Nishimura Y, Inoue A, Sasagawa S, Koiwa J, Kawaguchi K, Kawase R, Maruyama T, Kim S,
Tanaka T: Using zebrafish in systems toxicology for developmental toxicity testing.
Congenital anomalies 2016; 56(1):18-27.

11Development and assessment of new risk assessment methods

Title of Research:
15_PT01-01

Development of new in vivo screening method to validate thelow-dose effects induced by estrogenic chemicals using estrogen
reporter micePrincipal Investigator:
Tsuyoshi Nakanishi (Laboratory of Hygienic Chemistry and Molecular Toxicology, Gifu
Pharmaceutical University)
Collaborators:
Hisamitsu Nagase (Laboratory of Hygienic Chemistry and Molecular Toxicology, Gifu
Pharmaceutical University)

Summary of Research:
Background: The uterotrophic bioassay has been proposed as a short-term in vivo screening test
to detect the estrogenic properties of potentially endocrine disrupting chemicals by theOrganisation for Economic Co-operation and Development (OECD). Although the test is alreadystandardized as a OECD test guideline (Test No.440), there is a possibility that the test mightoverlook a certain estrogenic properties which has no effect to the uterus. On the other hand, we
recently generated a new transgenic mouse carrying “E-Rep” reporter gene which consisted of theluciferase (Luc) under the control of estrogen-responsive elements coupled to a minimal promoter.
E-Rep mouse is a potential powerful tool for assessing the kinetics of gene activation by
estrogenic properties in vivo. In the 4th term, we have established a provisional protocol for thenovel in vivo screening test, which employs bioluminescence imaging in the whole body as theend point for estrogenic action of test chemicals.
Objective: In the current term, we verified the validity of the provisional protocol by confirming
whether the estrogen-induced bioluminescence imaging in the whole body reflects the estrogenic
action in each organ. In addition, using the bone tissue-specific selective estrogen receptor
modulators (SERM), which have little effect on the uterus, we confirmed whether our novel in vivo
screening test have an advantage over the uterotrophic bioassay.
Methods: The experimental protocols of the new assay using E-Rep mice are based on theuterotrophic bioassay with some modifications. Briefly, E-Rep female mice (8 weeks of age) were
ovariectomized and then fed phytoestrogen-free purified diet during the experimental period. After
7 days of ovariectomy, the mice were orally gaveged with 17α-ethinylestradiol (EE) or SERMs for
7 consecutive days. The Luc activity was monitored in living animals with IVIS imaging at Day 1, 2,
4 and 7. After last detection of in vivo bioluminescence imaging, mice were dissected andmeasured uterine weight in accordance with the uterotrophic bioassay. For measurement of daily
Luc activity induced by EE in each organ, EE-gaveged mice were dissected and removed eachorgan at Day 1, 2, 4 and 7.
Results: According to measurement of daily Luc activity in each organ, it was confirmed that theEE-induced bioluminescence imaging in the whole body reflects its estrogenic action in eachorgan. Furthermore In the experiment using the bone tissue-specific SERMs, our novel in vivo
screening test could determine the SERMs as positive estrogens, whereas the uterotrophic
bioassay could not determine a SERM even at a dose which show sufficient estrogen activity inthe bone tissue.
Conclusion: Our novel in vivo screening test using E-Rep mice with in vivo bioluminescenceimaging is an excellent method that can detect SERMs that cannot be detected by the uterotrophic
bioassay.
Timeline: April, 2017 – March, 201812

Development and assessment of new risk assessment methods
Topics:1) Dr. Tsuyoshi Nakanishi was awarded the 4th JCIA-LRI award for the research theme"Comprehensive research on reproductive toxicity of nuclear receptor-activated hazards andestablishment of its evaluation system".
Publications:
1) Nishioka Y, Tamai K, Onda M, Hiromori Y, Kimura T, Hu J, Nagase H, Nakanishi T, Potential
interference of oil vehicles on genital tubercle development during the fetal period in ICR mice,
Biol. Pharm. Bull., 41:266-271 (2018)13

Development and assessment of new risk assessment methods

Title of Research:
16_PT01-01

Establishment of a comprehends test for identifying mutagenic
chemical compounds and molecular mechanisms underlying
mutagenesis
Principal Investigator:
Shunichi Takeda, MD, PhD (Professor, Kyoto University, Graduate School of Medicine,
Department of Radiation Genetics)
Yoshida-Konoecho, Sakyo-ku, Kyoto 606-8501, Japan(tel)+81-(0)75-753-4410, (email) stakeda@rg.med.kyoto-u.ac.jp
Collaborators:
Masamitsu Honma (National Institute of Health Sciences, Division of Genetics and Mutagenesis)
3-25-26, Tonomachi, Kawasaki-ku, Kasasaki, Kanagawa 210-9501, Japan(tel)+81-(0)44-270-6600, (email) honma@nihs.go.jp
Manabu Yasui (National Institute of Health Sciences, Division of Genetics and Mutagenesis)
3-25-26, Tonomachi, Kawasaki-ku, Kasasaki, Kanagawa 210-9501, Japan(tel)+81-(0)44-270-6600, (email) m-yasui@nihs.go.jp
Summary of Research:(1) Creation of TK6 cells deficient in individual DNA repair proteins
The OECD countries have employed the human TK6 B lymphoid cells as a standard cell line for
evaluating the genotoxicity of industrial chemical compounds. However, quick accurate repair of
DNA damage induced by chemicals in DNA-repair proficient TK6 cells significantly limits thesensitive identification of mutagenic chemicals. To overcome this problem, we have disrupted
genes encoding various DNA repair factors and shown the list of 119 DNA-repair mutants
generated from TK6 cells in the web site (http://www.nihs.go.jp/dgm/tk6.html). The DNA-sensitivity
profile of various DNA-repair mutants to given chemicals would allow for assessing the type of
DNA lesion induced by them.
(2) Analysis of molecular mechanisms for mutagenesis induced by nucleoside analogs
Nucleoside analogs have been used for anti-viral and cancer therapies. We created a bioassay forcomprehensively analyzing various mechanisms for mutagenesis caused by nucleoside analogs.
This analysis will be published in OncoTarget.
(3) Establishment of micro nucleus (MN) assay using DNA repair deficient TK6 cells
We established XRCC1-deficient (XRCC1-/-) TK6 cells, and conducted the MN assay using typicalDNA damaging agents, such as X-rays and mitomycin-C. MN analyses with XRCC1-/- cells
showed several times higher sensitivity than conventional MN analyses with wild-type TK6 cells.
(4) Discovery of an alternative excision repair of DNA damage induced by UV
We created XPA-/-/XRCC1-/- TK6 cells, and uncover the functional overlap between nucleotide
excision repair (NER) and base excision repair (BER). It has been believed that BER is unable toremove bulky DNA lesions induced by UV and crosslinking agents such as cisplatin. We found that
XRCC1-dependent BER contributes to removal of the bulky DNA lesions in NER-deficient XPA-/TK6 cells. Moreover, XRCC1-dependent BER is capable of removing the bulky DNA lesions on the
DNA of mitochondria, where no functional NER is present.
Timeline:
March 1, 2016Topics:
2017 LRI Annual Meeting, “Improvement of MN test sensitivity in the detection of mutagenic
chemicals by employing DNA damage repair mutant TK6 cells”
Publications:Tsuda, M et al., The dominant role of proofreading exonuclease activity of replicative polymerase εin cellular tolerance to cytarabine (Ara-C). Oncotarget. 8(20):33457-33474, 2017.
14

Development and assessment of new risk assessment methods

Title of Research:
16_PT01-02-2

Construction of prediction models for toxicities induced by drugs
and chemicals based on chemical structures and AOP
Principal Investigator:
Yoshihiro Uesawa
Summary of Research:
Quantitative structure–activity relationship (QSAR) analysis uses structural, quantum chemical,
and physicochemical features calculated from molecular geometry as explanatory variables
predicting physiological activity. Recently, deep learning based on advanced artificial neuralnetworks has demonstrated excellent performance in the discipline of QSAR research. While it
has properties of feature representation learning that directly calculate feature values frommolecular structure, the use of this potential function is limited in QSAR modeling. The present
study applied this function of feature representation learning to QSAR analysis by incorporating
360° images of molecular conformations into deep learning. Accordingly, we successfully
constructed a highly versatile identification model for chemical compounds that induce
mitochondrial membrane potential disruption with the external validation area under the receiver
operating characteristic curve of ≥0.9.
Timeline:
March, 2017 – February, 2018
Topics:Uesawa, Y., inventor; Meiji Pharmaceutical University, assignee. Prediction systems, prediction
methods, prediction programs, learning model input data generation systems, and learning model
input data generation programs. Pending Japanese patent application 2017-129823. 2017.
Publications:
1. Yuki Asako, Yoshihiro Uesawa, “High-Performance Prediction of Agonists on Human Estrogen
Receptor Based on Chemical Structures”, Molecules, 23;22(4). pii: E675, 2017
2. Adverse effect predictions based on computational toxicology techniques and large-scale
databases, 137th annual meeting of the pharmaceutical society of Japan, symposium, Sendai
Japan (invited), 2017/3
3. “Application of JADER in palliative pharmacy care”, The 11th Annual Meeting of JapaneseSociety for Pharmaceutical Palliative Care and Sciences, symposium, Sapporo, Japan(invited),
2017/6
4. “Drug toxicity predictions based on databases for adverse effects and chemical structures”, The
24th HAB annual meeting, Tokyo, Japan(invited), 2017/6
5. “Analyses of adverse outcome pathways based on artificial intelligence”, The 44th Annual
Meeting of the Japanese Society of Toxicology, Yokohama, Japan(invited)2017/7
6. “Construction of prediction models for toxicity reactions based on AOP and chemical structures”,
Japan Chemical Industry Association LRI symposium, Tokyo, Japan(invited) 2017/8
7. “Application of AI technology in liver toxicity prediction via adverse outcome pathways”,
Informatics In Biology, Medicine and Pharmacology2017, Sapporo, Japan(invited) 2017/9/
8. “The future of toxicity prediction developed by artificial intelligence”, 196th R&D Conference,
Tokyo, Japan (invited)2017/12
9. Yurie Yoshida, Ryota Higuchi, Jun Teraoka, Yuhei Mashiyama, Hajime Kagaya, Yoshihiro
Uesawa, “Comprehensive analysis of drug-induced thrombocytopenia in Japanese Adverse Drug
Event Report database with chemical-structural information”, 137th annual meeting of thepharmaceutical society of Japan, , Sendai Japan, 2017/3
10. Yuhei Mashiyama, Yurie Yoshida, Ryota Higuchi, Jun Teraoka, Hajime Kagaya, Yoshihiro
Uesawa, “Comprehensive analysis of drug-induced erythema exudativum multiforme major inJapanese Adverse Drug Event Report database with chemical-structural information”, 137th
15

Development and assessment of new risk assessment methods
annual meeting of the pharmaceutical society of Japan, , Sendai Japan, 2017/3

16

Development and assessment of new risk assessment methods

Title of Research:
17_PT01-01

Chip device for in vitro systemic toxicology
Principal Investigator:
Junji Fukuda, PhD (Professor, Yokohama National University, Engineering)79-5 Tokiwadai, Hodogaya, Yokohama, Kanagawa, 240-8501, Japan(tel) +81-045-339-4008, (e-mail) fukuda@ynu.jp

Collaborators:
Hiroshi Itagaki, PhD (Professor, Yokohama National University, Engineering)79-5 Tokiwadai, Hodogaya, Yokohama, Kanagawa, 240-8501, Japan
Masatoshi Watanabe, MD, PhD (Professor, Mie University, Medicine)1577 Kurimamachiya, Tsu, Mie, 514-8507, Japan

Summary of Research:
The purpose of this study is to fabricate microdevices for in vitro drug testing to evaluate
systemic toxicity of chemicals, so-called Body on a chip. Unlike previous typical animal testingalternatives, our device may possibly enable to replicate in vivo kinetics and interactions between
several tissues and organs including the skin, the small intestine, and the lung for drug absorption,
liver and myocardial tissues for drug metabolism and exposure.
In the first year of the project, we engineered skin and small intestine tissues as cellular layers
and liver tissues as spherical aggregates. Furthermore, a microdevice where these tissues were
integrated and connected with micro fluidic channels was fabricated. To circulate culture medium
in the microdevice, a seesaw-like bioreactor was designed, with which culture medium were
perfused in the microdevice without any external pump and air pressure supply. We demonstrated
that multiple tissues were formed and cultured in the microdevice on the bioreactor. Our next stepis to demonstrate that our approach can be used to evaluate absorption dynamics of chemical
substances passing through the skin and small intestine tissues and metabolic dynamics with theliver tissues.
Timeline:
May 1, 2017Topics:
Junji Fukuda, “Chip device for in vitro systemic toxicology”, JCIA LRI Research Conference, Tokyo,
Japan, Aug 2017
Publications:
Keito Iseki and Junji Fukuda, “Development of drug screening device using seesaw typebioreactor”, 30th The Japanese Society for Alternative to Animal Experiences, Tokyo, Japan, Nov
2017

17

Development and assessment of new risk assessment methods

Title of Research:
17_PT01-02

Development of novel screening system to predict the effects ofenvironmental factors in each organ by using cell-free circulating
DNA in serum
Subtitle: Advanced system for prediction of hazards fromenvironmental factors in fetus using cell free fetal DAN in mother’s
blood
Principal Investigator:
Wataru Miyazaki, PhD (Associate Professor, Department of Integrative Physiology, Graduate
School of Medicine, Gunma University)
3-39-22 Showa-machi, Maebashi, 371-8511, Japan(tel) +81-27-220-7923, (e-mail) miya@gunma-u.ac.jp
Collaborators:
Noriyuki Koibuchi (Professor, Department of Integrative Physiology, Graduate School of Medicine,Gunma University)
Asahi Haijima (Assistant Professor, Department of Integrative Physiology, Graduate School of
Medicine, Gunma University)
Yusuke Takatsuru (Assistant Professor, Department of Integrative Physiology, Graduate School of
Medicine, Gunma University)
Takahiko Katoh (Professor, Department of Public health, Graduate School of Medicine, Kumamoto
University))
Summary of Research:
To prevent the several adverse effects induced by environmental factors including chemicals, it isvery important to develop a novel screening system for each organ. Moreover, the system should
be low invasiveness and simple method to obtain the results immediately. In this study, to develop
the system, we have focused on the differences of epigenetic status in each organ-derived
genome DNA and utilized cell free circulating DNA(cfDNA) / cell free fetal circulating DNA(cffDNA)
to predict and evaluate the effects of environmental factors in whole organs. This advanced
system also have possibilities to predict the effects of the factors in fetus using mother’s serum.
Through this term, first, we performed whole genome bisulfite sequence (WGBS) to explore theorgan specific markers in brain, liver, and heart from embryonic day14.5 (E14.5) fetuses. These
data were combined with the others of dams and E12.5 fetuses from Gene Expression Omnibus(GEO), and we picked some candidates out for each organ. To investigate whether thesecandidates are useful for the system or not, we have evaluated these using the dummy of cfDNA
derived from fetal tissues. In addition, we have produced organ specific injured-model mice forbrain, liver, kidney, and pancreas to examine the specificities of the candidates for these organs.
Timeline:
March 1, 2017 Topics:
Poster presentation at 2017th JCIA LRI Annual Workshop, title: Development of novel screening
system to predict the effects of environmental factors in each organ by using cell-free circulating
DNA in serum
Publications:18Title of Research:
17_PT02-01

Establishment of mechanism based assay protocol for hasard andcarcinogenic risk of carbon based nanomaterials
Principal Investigator:
Hiroyuki Tsuda, MD, PhD (Professor, Nagoya City University, Nanotoxicology Project Lab)
3-1, Tanabedohri, Mizuho-ku, Nagoya, 467-8603, Japan(tel)+81-52-836-3496, (e-mail) htsuda@phar.nagoya-cu.ac.jp
Collaborators:
Susumu Tomono, PhD(Assistant Professor, Aichi Medical University School of Medicine,
Department of Microbiology and Immunology)
1-1 Yazakokarimata, Nagakute, Aichi, 480-1195 Japan(tel) +81-561-62-3311 (e-mail)tomono@aichi-med-u.ac.jp
Summary of Research:
For the purpose of evaluating the toxicity and carcinogenicity of nanomaterials, particularly carbon
nanotubes, we are establishing a method that is suitable for risk assessment and is far cheaper thanwhole body inhalation exposure. Whole body inhalation exposure requires specialized equipment andis exceedingly expensive, making it impossible to use except for a very small number of research
institutes. Consequently, whole body inhalation testing can not be used to test the large number of
inhalable carcinogens being produced. The method we are establishing for toxicity and carcinogenicity
testing uses Trans-tracheal Intra-pulmonary Spraying (TIPS). In contrast to whole body inhalation
exposure, TIPS can be used by many hundreds of researchers, making it possible to test all of the
carcinogens workers and users are being exposed to. To examine the usefulness of the TIPS methods
for practical use, we are currently testing MWCNT-7 (a known carcinogen composed of 40 layers ofgraphene) and Double-Walled Carbon Nanotubes (DWCNT) (2 layers). Method: Based on preliminary
results using another DWCNT available from Sigma-Aldrich, 10 week old male F344 rats were treatedwith the test DWCNT or MWCNT-7 suspended in a vehicle containing 0.5% PF68 (apolyoxyethylene-polyoxypropylene block copolymer) on alternate days for 15 days (8 times in total) atfinal doses of 0.25 and 0.5mg/rat of the CNTs. Rats were sacrificed at 3 and 8 weeks after the start ofadministration, and the lung, tracheal lung lavage fluid (BALF), and supernatant and cell pellet of thethoracic lavage fluid (PLF) were analyzed.
Results: An inflammatory reaction was induced by DWCNT but the reaction was markedly less in rats
administered DWCNT compared to rats administered MWCNT-7.

Timeline:
March, 2017 – February, 2018
Topics:The 5th Research Achievement Presentation Meeting
Date:Augst 25th, 2017
Title: Establishment of mechanism based assay protocol for hasard and carcinogenic risk ofcarbon based nanomaterials
Publications:
None

19

Research on the effects of chemical substances on children, elderly
people,and those with gene disorders
Title of Research:
12_PT03-01-5

Development of high sensitivity in vitro assessment system ofchemical-mediated hypersensitivity by using serine proteaseinhibitor-deficient cells
Subtitle: Analysis of in vitro assessment for respiratory allergy

Principal Investigator:
Akira Nakamura, Department of Immunology, Tohoku Medical and Pharmaceutical University
1-15-1 Fukumuro, Wakabayashi-ku, Sendai, Japan 981-8536 (tel) +81-22-290-8725(e-mail) aki-n@tohoku-mpu.ac.jp
Summary of Research:
The allergic disorders triggered by various chemicals are separated into two types of diseases,
IgE-dependent immediate allergy and IgE-independent chronic allergy. Mast cells, basophils andeosinophils act as the effector cells in both allergic reactions. In particular, basophils andeosinophils rapidly infiltrate into respiratory tissue, and cause airway hyper-responsiveness andairway obstruction. These cells secrete abundant serine proteases as well as chemical mediatorsand cytokines. Serine proteases, such as elastases, disrupt the basement membrane, leading to
the infiltration of inflammatory cells. The serine proteases are repressed by the serine proteaseinhibitors. Because we found that basophils and eosinophils lacking a secretory leukoproteaseinhibitor SLPI produced more cytokines than wild type cells after stimulation with IgE or LPS.
Therefore, we have planed to establish the highly sensitive cell line by the deletion of serine
protease inhibitors using a human basophilic cell line, KU812-F. Because KU812-F expresses highlevels of Serpin b1 and b6, and we have generated stable Serpin b1 knockdown KU812-F celllines by using lentiviral-delivered sh (short hairpin) RNA. The knockdown KU812-F cells (KD cells)
produced IL-6 about 2 times higher than KU812-F after administration with toluene diisocyanate(TDI). KD cells also secreted IL-13 upon stimulation with TDI. In contrast, scramble and wild type
KU812-F cells did not produce IL-13. we investigated gene alteration between KD cells andscramble KU812-F cells in responses to TDI by mRNA sequencing analysis. We found significant
elevation of three genes as follows: CD69 (T cell activation marker), CXCR4 (chemokine receptor),
sphingosine-1-phosphate receptor 4 (S1PR4: cell migration). We evaluated the relative
fluorescence intensities (RFI) in accordance with human Cell Line Activation Test (H-CRAT).
KU812-F and KD cells showed higher RFI of CD69 than scramble cells upon TDI andformaldehyde stimulation, whereas all cell lines did not display any alteration of CD69 expression
after DNCB and Methyl Salicilate. In the present LRI research, we investigated the numbers of
optimal cells and the expression of CD69 mRNA. We confirmed that KD cells displayed high levelsof CD69 expression, and also defined that the numbers of 3 x 104 is adequate for the evaluation.
In addition, we found that the levels of CD69 mRNA are significantly augmented in KD cells after
TDI stimulation. Collectively, our data suggested that analysis of CD69 expression and IL-13
ELISA is a suitable application for the detection of respiratory sensitizes by using Serpin b1
knockdown KU812-F cells.
Timeline:
2017/3/1-2018/2/28
Topics:Poster presentation entitled “Development of high sensitivity in vitro assessment system ofchemical-mediated hypersensitivity by using serine protease inhibitor-deficient cells.” at the annual
conference of 2017 LRI, Tokyo, Japan, August 201720

Assessment on the effects on ecosystems and the environment

Title of Research:
16_PT04-01

Development of the platform to manage the monitoring data ofchemicals in environment, ChemTHEATRE
Principal Investigator:
Kei Nakayama, PhD (Assistant Professor, Ehime University, Center for Marine Environmental
Studies)
2-5 Bunkyo-cho, Matsuyama 790-8577, Japan(tel) +81-89-927-8132, (e-mail) kei_n@ehime-u.ac.jp

Collaborators:
Tatsuya Kunisue (Ehime University, Center for Marine Environmental Studies)
Itsuki C Handoh (Niigata University, Educational Strategy Coordination Office)Seiichi Uno (Kagoshima University, Faculty of Fisheries)
Tomohiko Isobe (National Institute for Environmental Studies, Center for Health andEnvironmental Risk Research)
Nobuaki Ohno (University of Hyogo, Graduate School of Simulation Studies)
Daisuke Ueno (Saga University, Faculty of Agriculture)
Summary of Research:
We have launched a platform to register and visualize the monitoring data of environmental
contaminants, named ‘ChemTHEATRE’ (Chemicals in the THEATRE: Tractable and Heuristic
E-Archive for Traceability and Responsible-care Engagement). So far, the data of 58 publications
have been registered to the database. Users can find the chemical concentration data inenvironmental and biological specimens with their metadata, such as sampling date and location,
species, biometrics, and so on, in addition to experimental methods including extraction andcleanup processes, used standards, instruments, etc. ChemTHEATRE is now available at
http://chem-theatre.com/.
Updated information is provided from Facebook and Twitter.
Facebook: https://www.facebook.com/ChemTHEATRE/
Twitter: https://twitter.com/Chem_THEATRE/
Timeline:
March 1, 2017 – February 28, 2018
Topics:Our research was presented at ICCA-LRI and JRC Workshop in June 21–22, 2017.
“Introduction to ChemTHEATRE: Open data leads to a new era for integrated exposure and effects
analysis.”
Publications:21

Other issues to be urgently addressed
Title of Research:
13_PT05-01-4

Development and practical verification of a comprehensive system
to monitor multiple instances of environmental pollution in the
Mekong River basin
Principal Investigator:
Kazumasa Hirata, PhD (Professor, Osaka University, Pharmaceutical Sciences; 1-6 Yamadaoka,
Suita, Osaka 565-0871, Japan)(tel) +81-06-6879-8236, (e-mail) hirata@phs.osaka-u.ac.jp
Collaborators:
Kazuo Harada, PhD (Osaka University, Pharmaceutical Sciences); Hideyuki Matsuura, PhD(Osaka University, Pharmaceutical Sciences); Yoshinori Sumimura (Osaka University, Center for
Global Initiatives); Nguyen Quang Trung (Institute of Environmental Technology, Department ofEnvironmental Toxic Analysis); Misa Muraoka (Osaka University, Pharmaceutical Sciences); Tran
Thi My Duyen (Can Tho University, College of Aquaculture and Fisheries); Jun-ichi Nishikawa,
PhD (Mukogawa Women’s University, School of Pharmacy and Pharmaceutical Sciences); Taku
Yamashita (Mukogawa Women’s University, School of Pharmacy and Pharmaceutical Sciences)
Summary of Research:Multiple instances of serious environmental pollution in Southeast Asia have been caused bycontamination from agricultural chemicals, endocrine disruptors, heavy metals, and antibiotics. It istherefore important to monitor food and environmental samples from polluted areas to ensure
public safety. However, it is difficult to introduce monitoring systems based on expensive,high-quality analytical instruments in areas that often lack other resources. The aim of our projectis to develop a novel comprehensive system to monitor contamination in such localities. In thisstudy, we focused mainly on constructing low-cost, easy-to-use, and easy-to-transport
paper-based bioassays for antibiotics and endocrine disruptors, in which the biomolecular
reactions constructed to detect target chemicals were embedded on paper through the process offreeze-drying. The response signal of this bioassay can be detected by a digital camera and thus
does not require expensive conventional laboratory instruments, making it especially suitable in
low-resource localities.
Timeline:
November, 2013 Topics:
JCIA-LRI 5th Research Report Meeting (Tokyo，August, 2017; Poster presentation）

Publications:
1.
2.

3.

4.

5.

氏家和紀、松浦秀幸、Tran Thi My Duyen、原田和生、平田收正: 「無細胞反応系を用いた抗菌剤検出のための紙基板バイオセンサーの開発」、第 69 回日本生物工学会大会、東京、2017 年 9 月
松浦秀幸、氏家和紀、Tran Thi My Duyen、原田和生、平田收正: 「Development of paper-based
biosensors using cell-free system for detection of antibiotics」、2017 年度生命科学系学会合同年次
大会、神戸、2017 年 12 月
氏家和紀、松浦秀幸、Tran Thi My Duyen、原田和生、平田收正: 「組換え葉酸合成酵素を用いた
紙基板サルファ剤センサーの開発」、日本農芸化学会 関西・中四国・西日本支部 2017 年度合同
大阪大会、大阪、2017 年 9 月
Kazuki Ujiie, Hideyuki Matsuura, Tran Thi My Duyen, Koki Izutsuu, Kazuo Harada, Hideyuki
Matsuura, Kazumasa Hirata: “Paper-based luminescence bioassay method embedding asequence of enzymatic reactions to detect sulfonamide groups” submitted
Kazuki Ujiie, Hideyuki Matsuura, Tran Thi My Duyen, Koki Izutsuu, Kazuhito Fujiyama, Shinichiro
Maeda, Kazuo Harada, Hideyuki Matsuura, Kazumasa Hirata: “Development of a paper-based
luminescence bioassay for therapeutic monitoring of aminoglycosides” in preparation

22

A n n u a l R e p o r t 2 017
Japan Chemical Industry Association
1-4-1 Shinkawa, Chuo-ku Tokyo, 104-0033, Japan
TEL. +81-3-3297-2575

FAX. +81-3-3297-2612

URL：http://www.nikkakyo.org/
URL for LRI : http://www.j-lri.org/