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 forevaluating the genotoxicity of industrial chemical compounds. However, quick accurate repair ofDNA 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 ofDNA 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 thatXRCC1-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 theDNA 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.