ISSN 0910−0865

環境変異原研究
Environmental
MutagenResearch

Vol.24 No.1 2002

Coden：KHKEEN環境変異原研究
Environmental Mutagen Research

目
原

次

著
Genotoxicity of extracts of Japanese traditional herbal medicines（Kampo）
Makoto Katami, Haruo Kuboniwa, Shunichi Maemura and Toshihiko Yanagisawa ………………………… 1
Effect of a p53 codon 237 mutation on X ─ ray induced HPRT mutations
Shigeko Morimoto, Masamitsu Honma and Fumio Yatagai …………………………………………………… 17
A chromosomal aberration study of fibrillated PVA fiber in cultured mammalian cells
Takashi Hayashi and Fumiaki Arai ……………………………………………………………………………… 23
Antioxidative effects of fluvastatin and its metabolites in cultured human endothelial cells using single cell gel
electrophoresis
Tomonori Aoki, Atsumune Imaeda and Hisamitsu Nagase …………………………………………………… 29
Failure of stevioside to induce micronucleus formation in the rodent bone marrow cells
Temcharoen, P., S. Klongpanichpak, M. Suwannatrai, S. Apibal and C. Toskulkao ………………………… 37

特別企画
8th ICEM を終えて ………………………………………………………………………………………………… 43
書

評 …………………………………………………………………………………………………………………… 61

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Environ. Mutagen Res., 24: 1 - 15 (2002)

Genotoxicity of extracts of Japanese traditional herbal
medicines (Kampo)
Makoto Katami ＊, Haruo Kuboniwa, Shunichi Maemura and Toshihiko Yanagisawa
New Drug Discovery Laboratory, R ＆ D Division, TSUMURA ＆ CO.
3586, Yoshiwara, Ami-machi, Inashiki-gun, Ibaraki 300 -1192, Japan

Summar y
The possible genotoxicity potential of 128 Japanese traditional herbal medicines (Kampo) wasinvestigated using a bacterial reverse mutation test (the Ames test), an in vivo micronucleus test
(MN test) in mouse bone marrow cells and an unscheduled DNA synthesis test (UDS test) in rat
hepatocytes. Of 128 Kampo extracts examined, 98 did not induce mutations in bacteria while 30
induced mutations weakly in Salmonella typhimurium TA1537. Extracts of Scutellariae Radix, acommon herbal drug, and its major components, wogonin, baicalin, and baicalein showed positive
responses in the Ames test. Among 30 Ames-positive Kampo extracts, 27 contained these major
components of Scutellariae Radix extracts. It is possible that these components contribute to themutagenicity of the 27 Kampo extracts. Thirty Ames-positive Kampo extracts were also evaluatedfor their ability to induce chromosomal aberrations and DNA damage in in vivo micronucleus andunscheduled DNA synthesis assays. None of these extracts induced micronuclei and UDS.
Wogonin, baicalin and baicalein did not induce micronuclei in vivo. Based on the results in the present study, it is concluded that 98 Kampo extracts had no mutagenic potential in vitro, and that theother 30 Kampo extracts had no genotoxicity in vivo although they showed weak mutagenicity inbacteria.
Keywords: Japanese traditional herbal medicines (Kampo), genotoxicity, Ames test, micronucleus

test, unscheduled DNA synthesis test

Introduction
Japanese traditional herbal medicines (Kampo) are mixtures of crude extracts of herbal drugs. Each Kampo iswidely used to treat various types of disease in Japan. We
market 129 Kampo extract preparations for prescriptionuse. Based on their extensive history in clinical practice,Kampo are recognized as effective and safe biologicalagents. In order to verify the safety of Kampo extracts, wehave conducted some toxicological studies in animals(Iijima et al., 1995 ； Minematsu et al., 1995a-b ；Katsumata et al., 1997 ； Shimazu et al., 1997). Althoughgenotoxicity studies are useful for the prediction of carcinogenicity, relatively few Kampo extracts and herbal
drugs have been previously investigated for genotoxicity(Fujita et al., 1992, 1994 ； Fujita and Sasaki, 1993 ；Kikuchi and Oshio, 1983 ； Morimoto et al., 1982 ；＊katami_makoto@mail. tsumura.co.jp
Received: March 9, 2001, accepted: September 13, 2001
© Environmental Mutagen Society of Japan

Nozaka et al., 1987, 1991, 1994a-b ； Tadaki et al., 1995 ；Watanabe et al., 1983 ； Xue-jun et al., 1991 ； Yamamoto
et al., 1982). However, the data which do exist, show several inconsistencies.
In this study, the genotoxicity of 128 Kampo extracts
was examined first in vitro followed by in vivo tests forsamples that gave a positive in vitro response.

Materials and Methods
Test substances
Spray-dried extracts of 128 Kampo (Table 1), whichwere produced in Tsumura ＆ Co., Tokyo, Japan, consistof hot-water extracts from some herbal drugs mixed at theratios shown in Table 1. Freeze-dried water extracts ofScutellariae Radix and its components, wogonin, baicalin
and baicalein, were obtained from Tsumura ＆ Co.,Tokyo, Japan.
Bacterial reverse mutation test (Ames test)
The Ames test was carried out by the usual method(Ames et al., 1975 ； Maron and Ames, 1983). The
1

Table 1

Code No.

Summary of genotoxicity responses of 128 extracts of Japanese traditional herbal medicines (Kampo) in the bacterial reverse mutation test, the micronucleus test and the unscheduled DNA synthesis (UDS) test
Names of Kampo

TJ-1

Kakkon-to

TJ-2

Kakkon-to-ka-senkyushin’i

TJ-3

Otsuji-to

TJ-5

Anchu-sanTJ-6

Jumi-haidoku-to

TJ-7

a)

Hachimi-jio-gan

TJ-8a)

Dai-saiko-to

TJ-9a)

Sho-saiko-to

TJ-10a)Saiko-keishi-to

TJ-11

Saiko-keishi-kankyo-to

TJ-12a)Saiko-ka-ryukotsu-borei-to

TJ-14

Hange-shashin-to

TJ-15a)Oren-gedoku-to

TJ-16

Hange-koboku-to

TJ-17

Gorei-san

TJ-18

Keishi-ka-jutsu-bu-to

TJ-19a)Sho-seiryu-to

TJ-20

Boi-ogi-to

TJ-21

Sho-hange-ka-bukuryo-to2Constituents and combination ratio of herbal drugs
Puerariae Radix 4.0, Zizyphi Fructus 3.0, Ephedrae Herba 3.0,
Glycyrrhizae Radix 2.0, Cinnamomi Cortex 2.0, Paeoniae Radix 2.0,
Zingiberis Rhizoma 2.0
Puerariae Radix 4.0, Zizyphi Fructus 3.0, Ephedrae Herba 3.0,
Glycyrrhizae Radix 2.0, Cinnamomi Cortex 2.0, Paeoniae Radix 2.0,
Cnidii Rhizoma 2.0, Zingiberis Rhizoma 1.0, Magnoliae Flos 2.0
Angelicae Radix, 6.0, Bupleuri Radix 5.0, Scutellariae Radix 3.0,
Glycyrrhizae Radix 2.0, Cimicifugae Rhizoma 1.0, Rhei Rhizoma 0.5
Cinnamomi Cortex 4.0, Corydalis Tuber 3.0, Ostreae Testa 3.0,
Foeniculi Fructus 1.5, Glycyrrhizae Radix 1.0, Amomi Semen 1.0,
Alpiniae Officinarum Rhizoma 0.5
Platycodi Radix 3.0, Bupleuri Radix 3.0, Cnidii Rhizoma 3.0, Hoelen
3.0, Ledebouriellae Radix 1.5, Glycyrrhizae Radix 1.0,
Schizonepetae Spica 1.0, Zingiberis Rhizoma 1.0, Quercus Cortex
3.0, Araliae Rhizoma et Radix 1.5
Rehmanniae Radix 6.0, Corni Fructus 3.0, Dioscoreae Rhizoma 3.0,
Alismatis Rhizoma 3.0, Hoelen 3.0, Moutan Cortex 2.5, Cinnamomi
Cortex 1.0, Aconiti Tuber 0.5
Bupleuri Radix 6.0, Pinelliae Tuber 4.0, Scutellariae Radix 3.0,
Paeoniae Radix 3.0, Zizyphi Fructus 3.0, Aurantii Fructus
Immaturus 2.0, Zingiberis Rhizoma 1.0, Rhei Rhizoma 1.0
Bupleuri Radix 7.0, Pinelliae Tuber 5.0, Scutellariae Radix 3.0,
Zizyphi Fructus 3.0, Ginseng Radix 3.0, Glycyrrhizae Radix 2.0,
Zingiberis Rhizoma 1.0
Bupleuri Radix 5.0, Pinelliae Tuber 4.0, Scutellariae Radix 2.0,
Glycyrrhizae Radix 2.0, Cinnamomi Cortex 2.0, Paeoniae Radix 2.0,
Zizyphi Fructus 2.0, Ginseng Radix 2.0, Zingiberis Rhizoma 1.0
Bupleuri Radix 6.0, Scutellariae Radix 3.0, Trichosanthis Radix 3.0,
Cinnamomi Cortex 3.0, Ostreae Testa 3.0, Glycyrrhizae Radix 2.0,
Zingiberis Siccatum Rhizoma 2.0
Bupleuri Radix 5.0, Pinelliae Tuber 4.0, Cinnamomi Cortex 3.0,
Hoelen 3.0, Scutellariae Radix 2.5, Zizyphi Fructus 2.5, Ginseng
Radix 2.5, Ostreae Testa 2.5, Fossilia Ossis Mastodi 2.5, Zingiberis
Rhizoma 1.0
Pinelliae Tuber 5.0, Scutellariae Radix 2.5, Glycyrrhizae Radix 2.5,
Zizyphi Fructus 2.5, Ginseng Radix 2.5, Coptidis Rhizoma 1.0,
Zingiberis Siccatum Rhizoma 2.5
Scutellariae Radix 3.0, Coptidis Rhizoma 2.0, Gardeniae Fructus
2.0, Phellodendri Cortex 1.5
Pinelliae Tuber 6.0, Hoelen 5.0, Magnoliae Cortex 3.0, Perillae
Herba 2.0, Zingiberis Rhizoma 1.0
Alismatis Rhizoma 4.0, Atractylodis Lanceae Rhizoma 3.0,
Polyporus 3.0, Hoelen 3.0, Cinnamomi Cortex 1.5
Cinnamomi Cortex 4.0, Paeoniae Radix 4.0, Atractylodis Lanceae
Rhizoma 4.0, Zizyphi Fructus 4.0, Glycyrrhizae Radix 2.0,
Zingiberis Rhizoma 1.0, Aconiti Tuber 0.5
Pinelliae Tuber 6.0, Glycyrrhizae Radix 3.0, Cinnamomi Cortex 3.0,
Schisandrae Fructus 3.0, Asiasari Radix 3.0, Paeoniae Radix 3.0,
Ephedrae Herba 3.0, Zingiberis Siccatum Rhizoma 3.0
Astragali Radix 5.0, Sinomeni Caulis et Rhizoma 5.0, Atractylodis
Lanceae Rhizoma 3.0, Zizyphi Fructus 3.0, Glycyrrhizae Radix 1.5,
Zingiberis Rhizoma 1.0
Pinelliae Tuber 6.0, Hoelen 5.0, Zingiberis Rhizoma 1.5

Genotoxicity response
Ames
MN
UDS
N

−

−

N

−

−

P

N

N

N

−

−

N

−

−

N

N

−

P

N

N

P

N

N

P

N

N

P

N

N

P

N

N

P

N

N

P

N

N

N

−

−

N

−

−

N

−

−

N

N

−

N

−

−

N

−

−

(Table 1 continued）
Code No.
TJ-22

TJ-23a)TJ-24a)TJ-25a)
TJ-26

TJ-27
TJ-28

TJ-29
TJ-30
TJ-31
TJ-32
TJ-33
TJ-34
TJ-35
TJ-36
TJ-37

TJ-38

TJ-39
TJ-40
TJ-41

a)

TJ-43a)Names of Kampo
Shofu-sanConstituents and combination ratio of herbal drugs

Gypsum Fibrosum 3.0, Rehmanniae Radix 3.0, Angelicae Radix 3.0,
Atractylodis Lanceae Rhizoma 2.0, Ledebouriellae Radix 2.0,
Akebiae Caulis 2.0, Anemarrhenae Rhizoma 1.5, Glycyrrhizae
Radix 1.0, Sophorae Radix 1.0, Schizonepetae Spica 1.0, Arctii
Fructus 2.0, Sesami Semen 1.5, Cicadae Periostracum 1.0
Toki-shakuyaku-sanPaeoniae Radix 4.0, Atractylodis Lanceae Rhizoma 4.0, Alismatis
Rhizoma 4.0, Hoelen 4.0, Cnidii Rhizoma 3.0, Angelicae Radix 3.0
Kami-shoyo-san
Bupleuri Radix 3.0, Paeoniae Radix 3.0, Atractylodis Lanceae
Rhizoma 3.0, Angelicae Radix 3.0, Hoelen 3.0, Gardeniae Fructus
2.0, Moutan Cortex 2.0, Glycyrrhizae Radix 1.5, Zingiberis Rhizoma
1.0, Menthae Herba 1.0
Keishi-bukuryo-gan
Cinnamomi Cortex 3.0, Paeoniae Radix 3.0, Persicae Semen 3.0,
Hoelen 3.0, Moutan Cortex 3.0
Keishi-ka-ryukotsu-borei-to Cinnamomi Cortex 4.0, Paeoniae Radix 4.0, Zizyphi Fructus 4.0,
Ostreae Testa 3.0, Fossilia Ossis Mastodi 3.0, Glycyrrhizae Radix
2.0, Zingiberis Rhizoma 1.5
Mao-toArmeniacae Semen 5.0, Ephedrae Herba 5.0, Cinnamomi Cortex
4.0, Glycyrrhizae Radix 1.5
Eppi-ka-jutsu-to
Gypsum Fibrosum 8.0, Ephedrae Herba 6.0, Atractylodis Lanceae
Rhizoma 4.0, Zizyphi Fructus 3.0, Glycyrrhizae Radix 2.0,
Zingiberis Rhizoma 1.0
Bakumondo-to
Ophiopogonis Tuber 10.0, Pinelliae Tuber 5.0, Zizyphi Fructus 3.0,
Glycyrrhizae Radix 2.0, Ginseng Radix 2.0, Oryzae Fructus 5.0
Shimbu-to
Hoelen 4.0, Paeoniae Radix 3.0, Atractylodis Lanceae Rhizoma 3.0,
Zingiberis Rhizoma 1.5, Aconiti Tuber 0.5
Goshuyu-to
Zizyphi Fructus 4.0, Evodiae Fructus 3.0, Ginseng Radix 2.0,
Zingiberis Rhizoma 1.5
Ninjin-to
Glycyrrhizae Radix 3.0, Atractylodis Lanceae Rhizoma 3.0, Ginseng
Radix 3.0, Zingiberis Siccatum Rhizoma 3.0
Daio-botampi-to
Persicae Semen 4.0, Moutan Cortex 4.0, Rhei Rhizoma 2.0,
Benincasae Semen 6.0, Natrium Sulfuricum 1.8
Byakko-ka-ninjin-to
Gypsum Fibrosum 15.0, Anemarrhenae Rhizoma 5.0, Glycyrrhizae
Radix 2.0, Ginseng Radix 1.5, Oryzae Fructus 8.0
Shigyaku-san
Bupleuri Radix 5.0, Paeoniae Radix 4.0, Aurantii Fructus
Immaturus 2.0, Glycyrrhizae Radix 1.5
Moku-boi-toGypsum Fibrosum 10.0, Sinomeni Caulis et Rhizoma 4.0,
Cinnamomi Cortex 3.0, Ginseng Radix 3.0
Hange-byakujutsu-temma-to Aurantii Nobilis Pericarpium 3.0, Pinelliae Tuber 3.0, Atractylodis
Rhizoma 3.0, Hoelen 3.0, Astragali Radix 1.5, Alismatis Rhizoma
1.5, Ginseng Radix 1.5, Phellodendri Cortex 1.0, Zingiberis
Rhizoma 0.5, Gastrodiae Tuber 2.0, Hordei Fructus Germinatus 2.0,
Zingiberis Siccatum Rhizoma 1.0
Toki-shigyaku-ka-go
Zizyphi Fructus 5.0, Cinnamomi Cortex 3.0, Paeoniae Radix 3.0,
shuyu-shokyo-toAngelicae Radix 3.0, Akebiae Caulis 3.0, Glycyrrhizae Radix 2.0,
Evodiae Fructus 2.0, Asiasari Radix 2.0, Zingiberis Rhizoma 1.0
Ryo-kei-jutsu-kan-to
Hoelen 6.0, Cinnamomi Cortex 4.0, Atractylodis Lanceae Rhizoma
3.0, Glycyrrhizae Radix 2.0
Chorei-to
Alismatis Rhizoma 3.0, Polyporus 3.0, Hoelen 3.0, Asini Gelatinum
3.0, Kadinum 3.0
Hochu-ekki-to
Astragali Radix 4.0, Atractylodis Lanceae Rhizoma 4.0, Ginseng
Radix 4.0, Angelicae Radix 3.0, Bupleuri Radix 2.0, Zizyphi Fructus
2.0, Aurantii Nobilis Pericarpium 2.0, Glycyrrhizae Radix 1.5,
Cimicifugae Rhizoma 1.0, Zingiberis Rhizoma 0.5
Rikkunshi-to
Atractylodis Lanceae Rhizoma 4.0, Ginseng Radix 4.0, Pinelliae
Tuber 4.0, Hoelen 4.0, Zizyphi Fructus 2.0, Aurantii Nobilis
Pericarpium 2.0, Glycyrrhizae Radix 1.0, Zingiberis Rhizoma 0.5

Genotoxicity response
Ames
MN
UDS
N

−

−

N

N

−

N

N

−

N

N

−

N

−

−

N

−

−

N

−

−

N

−

−

N

−

−

N

−

−

N

N

−

N

−

−

N

N

−

N

N

−

N

−

−

N

−

−

N

−

−

N

−

−

N

−

−

N

N

−

N

N

−

3

(Table 1 continued）
Code No.

Names of Kampo

TJ-45

Keishi-to

TJ-46

Shichimotsu-koka-to

TJ-47a)Choto-sanTJ-48

Juzen-taiho-to

TJ-50

Keigai-rengyo-to

TJ-51

Juncho-to

TJ-52

Yokuinin-to

TJ-53

Sokei-kakketsu-to

TJ-54

Yoku-kan-san

TJ-55

Ma-kyo-kan-seki-to

TJ-56

Gorin-sanTJ-57

Unsei-in

TJ-58

Seijo-bofu-to

TJ-59

Ji-zuso-ippo

4

Constituents and combination ratio of herbal drugs
Cinnamomi Cortex 4.0, Paeoniae Radix 4.0, Zizyphi Fructus 4.0,
Glycyrrhizae Radix 2.0, Zingiberis Rhizoma 1.5
Paeoniae Radix 4.0, Angelicae Radix 4.0, Astragali Radix 3.0,
Rehmanniae Radix 3.0, Cnidii Rhizoma 3.0, Phellodendri Cortex
2.0, Uncariae Ramulus et Uncus 3.0
Gypsum Fibrosum 5.0, Aurantii Nobilis Pericarpium 3.0,
Ophiopogonis Tuber 3.0, Pinelliae Tuber 3.0, Hoelen 3.0, Ginseng
Radix 2.0, Ledebouriellae Radix 2.0, Glycyrrhizae Radix 1.0,
Zingiberis Rhizoma 1.0, Uncariae Ramulus et Uncus 3.0,
Chrysanthemi Flos 2.0
Astragali Radix 3.0, Cinnamomi Cortex 3.0, Rehmanniae Radix 3.0,
Paeoniae Radix 3.0, Cnidii Rhizoma 3.0, Atractylodis Lanceae
Rhizoma 3.0, Angelicae Radix 3.0, Ginseng Radix 3.0, Hoelen 3.0,
Glycyrrhizae Radix 1.5
Scutellariae Radix 1.5, Phellodendri Cortex 1.5, Coptidis Rhizoma
1.5, Platycodi Radix 1.5, Aurantii Fructus Immaturus 1.5,
Schizonepetae Spica 1.5, Bupleuri Radix 1.5, Gardeniae Fructus 1.5,
Rehmanniae Radix 1.5, Paeoniae Radix 1.5, Cnidii Rhizoma 1.5,
Angelicae Radix 1.5, Menthae Herba 1.5, Angelicae Dahuricae
Radix 1.5, Ledebouriellae Radix 1.5, Forsythiae Fructus 1.5,
Glycyrrhizae Radix 1.0
Rehmanniae Radix 6.0, Angelicae Radix 3.0, Scutellariae Radix 2.0,
Aurantii Fructus Immaturus 2.0, Armeniacae Semen 2.0, Magnoliae
Cortex 2.0, Rhei Rhizoma 2.0, Persicae Semen 2.0, Glycyrrhizae
Radix 1.5, Cannabidis Semen2.0
Coicis Semen 8.0, Atractylodis Lanceae Rhizoma 4.0, Angelicae
Radix 4.0, Ephedrae Herba 4.0, Cinnamomi Cortex 3.0, Paeoniae
Radix 3.0, Glycyrrhizae Radix 2.0
Paeoniae Radix 2.5, Rehmanniae Radix 2.0, Cnidii Rhizoma 2.0,
Atractylodis Lanceae Rhizoma 2.0, Angelicae Radix 2.0, Persicae
Semen 2.0, Hoelen 2.0, Achyranthis Radix 1.5, Aurantii Nobilis
Pericarpium 1.5, Sinomeni Caulis et Rhizoma 1.5, Ledebouriellae
Radix 1.5, Gentianae Scabrae Radix 1.5, Glycyrrhizae Radix 1.0,
Angelicae Dahuricae Radix 1.0, Zingiberis Rhizoma 0.5, Clematidis
Radix 1.5, Notopterygii Rhizoma 1.5
Atractylodis Lanceae Rhizoma 4.0, Hoelen 4.0, Cnidii Rhizoma 3.0,
Angelicae Radix 3.0, Bupleuri Radix 2.0, Glycyrrhizae Radix 1.5,
Uncariae Ramulus et Uncus 3.0
Gypsum Fibrosum 10.0, Armeniacae Semen 4.0, Ephedrae Herba
4.0, Glycyrrhizae Radix 2.0
Hoelen 6.0, Scutellariae Radix 3.0, Glycyrrhizae Radix 3.0,
Rehmanniae Radix 3.0, Plantaginis Semen 3.0, Alismatis Rhizoma
3.0, Angelicae Radix 3.0, Akebiae Caulis 3.0, Gardeniae Fructus 2.0,
Paeoniae Radix 2.0, Kadinum 3.0
Rehmanniae Radix 3.0, Paeoniae Radix 3.0, Cnidii Rhizoma 3.0,
Angelicae Radix 3.0, Scutellariae Radix 1.5, Phellodendri Cortex
1.5, Coptidis Rhizoma 1.5, Gardeniae Fructus 1.5
Scutellariae Radix 2.5, Platycodi Radix 2.5, Gardeniae Fructus 2.5,
Cnidii Rhizoma 2.5, Glehniae Radix cum Rhizoma 2.5, Angelicae
Dahuricae Radix 2.5, Forsythiae Fructus 2.5, Coptidis Rhizoma 1.0,
Glycyrrhizae Radix 1.0, Aurantii Fructus Immaturus 1.0,
Schizonepetae Spica 1.0, Menthae Herba 1.0
Cnidii Rhizoma 3.0, Atractylodis Lanceae Rhizoma 3.0, Forsythiae
Fructus 3.0, Ledebouriellae Radix 2.0, Glycyrrhizae Radix 1.0,
Schizonepetae Spica 1.0, Carthami Flos 1.0, Rhei Rhizoma 0.5,
Lonicerae Caulis et Folium 2.0

Genotoxicity response
Ames
MN
UDS
N

−

−

N

−

−

N

N

−

N

−

−

P

N

N

P

N

N

N

−

−

N

N

−

N

−

−

N

−

−

P

N

N

P

N

N

P

N

N

N

−

−

(Table 1 continued）
Code No.

Names of Kampo

Constituents and combination ratio of herbal drugs

TJ-60

Keishi-ka-shakuyaku-to

TJ-61

Tokaku-joki-to

TJ-62

Bofu-tsusho-san

TJ-63

Goshaku-san

TJ-64

Sha-kanzo-to

TJ-65

Kihi-to

TJ-66

Jinso-in

TJ-67

Nyoshin-sanPaeoniae Radix 6.0, Cinnamomi Cortex 4.0, Zizyphi Fructus 4.0,
Glycyrrhizae Radix 2.0, Zingiberis Rhizoma 1.0
Persicae Semen 5.0, Cinnamomi Cortex 4.0, Rhei Rhizoma 3.0,
Glycyrrhizae Radix 1.5, Natrium Sulfuricum 0.9
Scutellariae Radix 2.0, Glycyrrhizae Radix 2.0, Platycodi Radix 2.0,
Gypsum Fibrosum 2.0, Atractylodis Rhizoma 2.0, Rhei Rhizoma 1.5,
Schizonepetae Spica 1.2, Gardeniae Fructus 1.2, Paeoniae Radix
1.2, Cnidii Rhizoma 1.2, Angelicae Radix 1.2, Menthae Herba 1.2,
Ledebouriellae Radix 1.2, Ephedrae Herba 1.2, Forsythiae Fructus
1.2, Zingiberis Rhizoma 0.3, Kadinum 3.0, Natrium Sulfuricum 0.7
Atractylodis Lanceae Rhizoma 3.0, Aurantii Nobilis Pericarpium 2.0,
Angelicae Radix 2.0, Pinelliae Tuber 2.0, Hoelen 2.0, Glycyrrhizae
Radix 1.0, Platycodi Radix 1.0, Aurantii Fructus Immaturus 1.0,
Cinnamomi Cortex 1.0, Magnoliae Cortex 1.0, Paeoniae Radix 1.0,
Zingiberis Rhizoma 1.0, Cnidii Rhizoma 1.0, Zizyphi Fructus 1.0,
Angelicae Dahuricae Radix 1.0, Ephedrae Herba 1.0
Rehmanniae Radix 6.0, Ophiopogonis Tuber 6.0, Cinnamomi
Cortex 3.0, Zizyphi Fructus 3.0, Ginseng Radix 3.0, Zingiberis
Rhizoma 1.0, Cannabidis Semen 3.0, Glycyrrhizae Radix 3.0, Asini
Gelatinum 2.0
Astragali Radix 3.0, Ginseng Radix 3.0, Atractylodis Rhizoma 3.0,
Hoelen 3.0, Polygalae Radix 2.0, Zizyphi Fructus 2.0, Angelicae
Radix 2.0, Glycyrrhizae Radix 1.0, Zingiberis Rhizoma 1.0,
Saussureae Radix 1.0, Zizyphi Spinosi Semen 3.0, Longanae Arillus
3.0
Pinelliae Tuber 3.0, Hoelen 3.0, Puerariae Radix 2.0, Platycodi
Radix 2.0, Aurantii Nobilis Pericarpium 2.0, Zizyphi Fructus 1.5,
Ginseng Radix 1.5, Glycyrrhizae Radix 1.0, Aurantii Fructus
Immaturus 1.0, Perillae Herba 1.0, Zingiberis Rhizoma 0.5,
Peucedani Radix 2.0
Cyperi Rhizoma 3.0, Cnidii Rhizoma 3.0, Atractylodis Lanceae
Rhizoma 3.0, Angelicae Radix 3.0, Scutellariae Radix 2.0,
Cinnamomi Cortex 2.0, Ginseng Radix 2.0, Arecae Semen 2.0,
Coptidis Rhizoma 1.0, Glycyrrhizae Radix 1.0, Caryophylli Flos 1.0,
Saussureae Radix 1.0
Glycyrrhizae Radix 6.0, Paeoniae Radix 6.0
Hoelen 5.0, Atractylodis Lanceae Rhizoma 4.0, Aurantii Nobilis
Pericarpium 3.0, Ginseng Radix 3.0, Aurantii Fructus Immaturus1.5, Zingiberis Rhizoma 1.0
Cyperi Rhizoma 4.0, Perillae Herba 2.0, Aurantii Nobilis
Pericarpium 2.0, Glycyrrhizae Radix 1.5, Zingiberis Rhizoma 1.0
Rehmanniae Radix 3.0, Paeoniae Radix 3.0, Cnidii Rhizoma 3.0,
Angelicae Radix 3.0
Zizyphi Fructus 6.0, Glycyrrhizae Radix 5.0, Tritici Fructus 20.0
Bupleuri Radix 5.0, Pinelliae Tuber 5.0, Scutellariae Radix 3.0,
Zizyphi Fructus 3.0, Ginseng Radix 2.0, Coptidis Rhizoma 1.5,
Glycyrrhizae Radix 1.5, Zingiberis Rhizoma 1.0, Trichosanthis
Semen 3.0
Rhei Rhizoma 2.0, Glycyrrhizae Radix 1.0, Natrium Sulfuricum 0.5
Atractylodis Lanceae Rhizoma 4.0, Ginseng Radix 4.0, Hoelen 4.0,
Glycyrrhizae Radix 1.0, Zingiberis Rhizoma 1.0, Zizyphi Fructus 1.0
Rehmanniae Radix 5.0, Angelicae Radix 5.0, Akebiae Caulis 5.0,
Scutellariae Radix 3.0, Plantaginis Semen 3.0, Alismatis Rhizoma
3.0, Glycyrrhizae Radix 1.0, Gardeniae Fructus 1.0, Gentianae
Scabrae Radix 1.0

a)

TJ-68
TJ-69

Shakuyaku-kanzo-to
Bukuryo-in

TJ-70

Koso-sanTJ-71

Shimotsu-to

TJ-72
TJ-73

Kam-baku-taiso-to
Saikan-to

TJ-74
TJ-75

Choi-joki-to
Shikunshi-to

TJ-76

Ryutan-shakan-to

Genotoxicity response
Ames
MN
UDS
N

N

−

N

−

−

P

N

N

N

−

−

P

N

N

N

−

−

N

−

−

P

N

N

N
N

N
−

−
−

N

−

−

P

N

N

N
P

−
N

−
N

P
N

N
−

N
−

P

N

N

5

(Table 1 continued）
Code No.

Names of Kampo

TJ-77

Kyuki-kyogai-to

TJ-78

Ma-kyo-yoku-kan-to

TJ-79

Heii-sanTJ-80

Saiko-seikan-to

TJ-81

Nichin-to

TJ-82

Keishi-ninjin-to

TJ-83

Yoku-kan-san-ka-chimpihange

TJ-84
TJ-85

Daio-kanzo-to
Shimpi-to

TJ-86

Toki-inshi

TJ-87

Rokumi-gan

TJ-88

Nijutsu-to

TJ-89

Ji-daboku-ippo

TJ-90

Seihai-to

TJ-91

Chikujo-untan-to

TJ-92

Jiin-shiho-to

6Constituents and combination ratio of herbal drugs
Rehmanniae Radix 5.0, Paeoniae Radix 4.0, Angelicae Radix 4.0,
Glycyrrhizae Radix 3.0, Cnidii Rhizoma 3.0, Artemisiae Folium 3.0,
Asini Gelatinum 3.0
Coicis Semen 10.0, Ephedrae Herba 4.0, Armeniacae Semen 3.0,
Glycyrrhizae Radix 2.0
Atractylodis Lanceae Rhizoma 4.0, Magnoliae Cortex 3.0, Aurantii
Nobilis Pericarpium 3.0, Zizyphi Fructus 2.0, Glycyrrhizae Radix
1.0, Zingiberis Rhizoma 0.5
Bupleuri Radix 2.0, Scutellariae Radix 1.5, Phellodendri Cortex 1.5,
Coptidis Rhizoma 1.5, Trichosanthis Radix 1.5, Glycyrrhizae Radix
1.5, Platycodi Radix 1.5, Gardeniae Fructus 1.5, Rehmanniae Radix
1.5, Paeoniae Radix 1.5, Cnidii Rhizoma 1.5, Angelicae Radix 1.5,
Menthae Herba 1.5, Forsythiae Fructus 1.5, Arctii Fructus 1.5
Pinelliae Tuber 5.0, Hoelen 5.0, Aurantii Nobilis Pericarpium 4.0,
Glycyrrhizae Radix 1.0, Zingiberis Rhizoma 1.0
Cinnamomi Cortex 4.0, Glycyrrhizae Radix 3.0, Atractylodis
Lanceae Rhizoma 3.0, Ginseng Radix 3.0, Zingiberis Siccatum
Rhizoma 2.0
Pinelliae Tuber 5.0, Atractylodis Lanceae Rhizoma 4.0, Hoelen 4.0,
Cnidii Rhizoma 3.0, Aurantii Nobilis Pericarpium 3.0, Angelicae
Radix 3.0, Bupleuri Radix 2.0, Glycyrrhizae Radix 1.5, Uncariae
Ramulus et Uncus 3.0
Rhei Rhizoma 4.0, Glycyrrhizae Radix 2.0
Ephedrae Herba 5.0, Armeniacae Semen 4.0, Magnoliae Cortex 3.0,
Aurantii Nobilis Pericarpium 2.5, Glycyrrhizae Radix 2.0, Bupleuri
Radix 2.0, Perillae Herba 1.5
Angelicae Radix 5.0, Rehmanniae Radix 4.0, Paeoniae Radix 3.0,
Cnidii Rhizoma 3.0, Ledebouriellae Radix 3.0, Astragali Radix 1.5,
Schizonepetae Spica 1.5, Glycyrrhizae Radix 1.0, Tribuli Fructus
3.0, Polygoni Multiflori Radix 2.0
Rehmanniae Radix 5.0, Corni Fructus 3.0, Dioscoreae Rhizoma 3.0,
Alismatis Rhizoma 3.0, Hoelen 3.0, Moutan Cortex 3.0
Pinelliae Tuber 4.0, Atractylodis Lanceae Rhizoma 3.0, Scutellariae
Radix 2.5, Cyperi Rhizoma 2.5, Aurantii Nobilis Pericarpium 2.5,
Atractylodis Rhizoma 2.5, Hoelen 2.5, Glycyrrhizae Radix 1.0,
Zingiberis Rhizoma 1.0, Clematidis Radix 2.5, Arisaematis Tuber
2.5, Araliae Radix 2.5
Cinnamomi Cortex 3.0, Cnidii Rhizoma 3.0, Nupharis Rhizoma 3.0,
Glycyrrhizae Radix 1.5, Rhei Rhizoma 1.0, Caryophylli Flos 1.0,
Quercus Cortex 3.0
Angelicae Radix 3.0, Ophiopogonis Tuber 3.0, Hoelen 3.0,
Scutellariae Radix 2.0, Platycodi Radix 2.0, Armeniacae Semen 2.0,
Gardeniae Fructus 2.0, Mori Cortex 2.0, Zizyphi Fructus 2.0,
Aurantii Nobilis Pericarpium 2.0, Glycyrrhizae Radix 1.0,
Schisandrae Fructus 1.0, Zingiberis Rhizoma 1.0, Phyllostachysis
Caulis in Taeniam 2.0, Asparagi Radix 2.0, Fritillariae Bulbus 2.0
Pinelliae Tuber 5.0, Bupleuri Radix 3.0, Ophiopogonis Tuber 3.0,
Hoelen 3.0, Platycodi Radix 2.0, Aurantii Fructus Immaturus 2.0,
Cyperi Rhizoma 2.0, Aurantii Nobilis Pericarpium 2.0, Coptidis
Rhizoma 1.0, Glycyrrhizae Radix 1.0, Zingiberis Rhizoma 1.0,
Ginseng Radix 1.0, Phyllostachysis Caulis in Taeniam 3.0
Cyperi Rhizoma 3.0, Bupleuri Radix 3.0, Paeoniae Radix 3.0
Anemarrhenae Rhizoma 3.0, Aurantii Nobilis Pericarpium 3.0,
Angelicae Radix 3.0, Ophiopogonis Tuber 3.0, Atractylodis Rhizoma
3.0, Hoelen 3.0, Glycyrrhizae Radix 1.0, Menthae Herba 1.0, Lycii
Radicis Cortex 3.0, Fritillariae Bulbus 2.0

Genotoxicity response
Ames
MN
UDS
N

−

−

N

−

−

N

−

−

P

N

N

N

−

−

N

−

−

N

N

−

N
N

−
−

−
−

N

−

−

N

−

−

P

N

N

N

−

−

P

N

N

N

−

−

N

−

−

(Table 1 continued）
Code No.
TJ-93

Jiin-koka-to

TJ-95
TJ-96a)

TJ-97

TJ-98
TJ-99
TJ-100
TJ-101
TJ-102

TJ-103
TJ-104

TJ-105

TJ-106

TJ-107

Names of Kampoa)

TJ-108

TJ-109

TJ-110Constituents and combination ratio of herbal drugs

Atractylodis Lanceae Rhizoma 3.0, Rehmanniae Radix 2.5, Paeoniae
Radix 2.5, Aurantii Nobilis Pericarpium 2.5, Angelicae Radix 2.5,
Ophiopogonis Tuber 2.5, Phellodendri Cortex 1.5, Glycyrrhizae
Radix 1.5, Anemarrhenae Rhizoma 1.5, Asparagi Radix 2.5
Goko-toGypsum Fibrosum 10.0, Armeniacae Semen 4.0, Ephedrae Herba
4.0, Mori Cortex 3.0, Glycyrrhizae Radix 2.0
Saiboku-to
Bupleuri Radix 7.0, Pinelliae Tuber 5.0, Hoelen 5.0, Scutellariae
Radix 3.0, Magnoliae Cortex 3.0, Zizyphi Fructus 3.0, Ginseng
Radix 3.0, Glycyrrhizae Radix 2.0, Perillae Herba 2.0, Zingiberis
Rhizoma 1.0
Dai-bofu-to
Astragali Radix 3.0, Rehmanniae Radix 3.0, Paeoniae Radix 3.0,
Atractylodis Lanceae Rhizoma 3.0, Angelicae Radix 3.0,
Ledebouriellae Radix 3.0, Cnidii Rhizoma 2.0, Glycyrrhizae Radix
1.5, Achyranthis Radix 1.5, Zizyphi Fructus 1.5, Ginseng Radix 1.5,
Notopterygii Rhizoma 1.5, Eucommiae Cortex 3.0, Zingiberis
Siccatum Rhizoma 1.0, Aconiti Tuber 1.0
Ogi-kenchu-to
Paeoniae Radix 6.0, Astragali Radix 4.0, Cinnamomi Cortex 4.0,
Zizyphi Fructus 4.0, Glycyrrhizae Radix 2.0, Zingiberis Rhizoma 1.0
Sho-kenchu-to
Paeoniae Radix 6.0, Cinnamomi Cortex 4.0, Zizyphi Fructus 4.0,
Glycyrrhizae Radix 2.0, Zingiberis Rhizoma 1.0
Dai-kenchu-toGinseng Radix 3.0, Zanthoxyli Fructus 2.0, Zingiberis Siccatum
Rhizoma 5.0
Shoma-kakkon-to
Puerariae Radix 5.0, Paeoniae Radix 3.0, Cimicifugae Rhizoma 2.0,
Glycyrrhizae Radix 1.5, Zingiberis Rhizoma 0.5
Toki-toAngelicae Radix 5.0, Pinelliae Tuber 5.0, Cinnamomi Cortex 3.0,
Magnoliae Cortex 3.0, Paeoniae Radix 3.0, Ginseng Radix 3.0,
Astragali Radix 1.5, Zanthoxyli Fructus 1.5, Glycyrrhizae Radix 1.0,
Zingiberis Siccatum Rhizoma 1.5
Sansonin-to
Hoelen 5.0, Cnidii Rhizoma 3.0, Anemarrhenae Rhizoma 3.0,
Glycyrrhizae Radix 1.0, Zizyphi Spinosi Semen 10.0
Shin’i-seihai-to
Gypsum Fibrosum 5.0, Ophiopogonis Tuber 5.0, Scutellariae Radix
3.0, Gardeniae Fructus 3.0, Anemarrhenae Rhizoma 3.0,
Cimicifugae Rhizoma 1.0, Lilii Bulbus 3.0, Magnoliae Flos 2.0,
Eriobotryae Folium 2.0
Tsu-do-sanAurantii Fructus Immaturus 3.0, Rhei Rhizoma 3.0, Angelicae Radix
3.0, Glycyrrhizae Radix 2.0, Carthami Flos 2.0, Magnoliae Cortex
2.0, Aurantii Nobilis Pericarpium 2.0, Akebiae Caulis 2.0, Sappan
Lignum 2.0, Natrium Sulfuricum 1.8
Unkei-toOphiopogonis Tuber 4.0, Pinelliae Tuber 4.0, Angelicae Radix 3.0,
Glycyrrhizae Radix 2.0, Cinnamomi Cortex 2.0, Paeoniae Radix 2.0,
Cnidii Rhizoma 2.0, Ginseng Radix 2.0, Moutan Cortex 2.0, Evodiae
Fructus 1.0, Zingiberis Rhizoma 1.0, Asini Gelatinum 2.0
Gosha-jinki-gan
Rehmanniae Radix 5.0, Achyranthis Radix 3.0, Corni Fructus 3.0,
Dioscoreae Rhizoma 3.0, Plantaginis Semen 3.0, Alismatis Rhizoma
3.0, Hoelen 3.0, Moutan Cortex 3.0, Cinnamomi Cortex 1.0, Aconiti
Tuber 1.0
Ninjin-yoei-toRehmanniae Radix 4.0, Angelicae Radix 4.0, Atractylodis Rhizoma
4.0, Hoelen 4.0, Ginseng Radix 3.0, Cinnamomi Cortex 2.5,
Polygalae Radix 2.0, Paeoniae Radix 2.0, Aurantii Nobilis
Pericarpium 2.0, Astragali Radix 1.5, Glycyrrhizae Radix 1.0,
Schisandrae Fructus 1.0
Sho-saiko-to-ka-kikyo-sekko Gypsum Fibrosum 10.0, Bupleuri Radix 7.0, Pinelliae Tuber 5.0,
Scutellariae Radix 3.0, Platycodi Radix 3.0, Zizyphi Fructus 3.0,
Ginseng Radix 3.0, Glycyrrhizae Radix 2.0, Zingiberis Rhizoma 1.0
Rikko-sanGardeniae Fructus 2.0, Cimicifugae Rhizoma 2.0, Ledebouriellae
Radix 2.0, Glycyrrhizae Radix 1.5, Gentianae Scabrae Radix 1.0

Genotoxicity response
Ames
MN
UDS
N

−

−

N

−

−

P

N

N

N

−

−

N

−

−

N

−

−

N

−

−

N

−

−

N

−

−

N

−

−

P

N

N

N

−

−

N

N

−

N

N

−

N

−

−

P

N

N

N

−

−

7

(Table 1 continued）
Code No.
TJ-111

TJ-112

TJ-113
TJ-114a)

TJ-115

TJ-116

TJ-117

TJ-118
TJ-119TJ-120

TJ-121
TJ-122

TJ-123
TJ-124

TJ-125
TJ-126

TJ-127
TJ-128

TJ-133
TJ-1348

Names of Kampo
Seishin-renshi-in

Constituents and combination ratio of herbal drugs

Ophiopogonis Tuber 4.0, Hoelen 4.0, Scutellariae Radix 3.0,
Plantaginis Semen 3.0, Ginseng Radix 3.0, Astragali Radix 2.0,
Glycyrrhizae Radix 1.5, Nelumbinis Semen 4.0, Lycii Radicis Cortex
2.0
Chorei-to-go-shimotsu-to
Rehmanniae Radix 3.0, Paeoniae Radix 3.0, Cnidii Rhizoma 3.0,
Alismatis Rhizoma 3.0, Polyporus 3.0, Angelicae Radix 3.0, Hoelen
3.0, Asini Gelatinum 3.0, Kadinum 3.0
San’o-shashin-to
Scutellariae Radix 3.0, Coptidis Rhizoma 3.0, Rhei Rhizoma 3.0
Sairei-to
Bupleuri Radix 7.0, Alismatis Rhizoma 5.0, Pinelliae Tuber 5.0,
Scutellariae Radix 3.0, Atractylodis Lanceae Rhizoma 3.0, Zizyphi
Fructus 3.0, Polyporus 3.0, Ginseng Radix 3.0, Hoelen 3.0,
Glycyrrhizae Radix 2.0, Cinnamomi Cortex 2.0, Zingiberis Rhizoma
1.0
Irei-toMagnoliae Cortex 2.5, Atractylodis Lanceae Rhizoma 2.5, Alismatis
Rhizoma 2.5, Polyporus 2.5, Aurantii Nobilis Pericarpium 2.5,
Atractylodis Rhizoma 2.5, Hoelen 2.5, Cinnamomi Cortex 2.0,
Zingiberis Rhizoma 1.5, Zizyphi Fructus 1.5, Glycyrrhizae Radix 1.0
Bukuryo-in-go-hangePinelliae Tuber 6.0, Hoelen 5.0, Atractylodis Lanceae Rhizoma 4.0,
koboku-to
Magnoliae Cortex 3.0, Aurantii Nobilis Pericarpium 3.0, Ginseng
Radix 3.0, Perillae Herba 2.0, Aurantii Fructus Immaturus 1.5,
Zingiberis Rhizoma 1.0
Inchin-gorei-sanAlismatis Rhizoma 6.0, Atractylodis Lanceae Rhizoma 4.5,
Polyporus 4.5, Hoelen 4.5, Cinnamomi Cortex 2.5, Artemisiae
Capillaris Spica 4.0
Ryo-kyo-jutsu-kan-to
Hoelen 6.0, Atractylodis Rhizoma 3.0, Glycyrrhizae Radix 2.0,
Zingiberis Siccatum Rhizoma 3.0
Ryo-kan-kyo-mi-shinArmeniacae Semen 4.0, Pinelliae Tuber 4.0, Hoelen 4.0,
ge-nin-to
Schisandrae Fructus 3.0, Glycyrrhizae Radix 2.0, Asiasari Radix 2.0,
Zingiberis Siccatum Rhizoma 2.0
Oren-toPinelliae Tuber 6.0, Coptidis Rhizoma 3.0, Glycyrrhizae Radix 3.0,
Cinnamomi Cortex 3.0, Zizyphi Fructus 3.0, Ginseng Radix 3.0,
Zingiberis Siccatum Rhizoma 3.0
Sammotsu-ogon-to
Rehmanniae Radix 6.0, Scutellariae Radix 3.0, Sophorae Radix 3.0
Haino-san-kyu-to
Platycodi Radix 4.0, Glycyrrhizae Radix 3.0, Aurantii Fructus
Immaturus 3.0, Paeoniae Radix 3.0, Zizyphi Fructus 3.0, Zingiberis
Rhizoma 1.0
Toki-kenchu-to
Paeoniae Radix 5.0, Cinnamomi Cortex 4.0, Zizyphi Fructus 4.0,
Angelicae Radix 4.0, Glycyrrhizae Radix 2.0, Zingiberis Rhizoma 1.0
Senkyu-chacho-san
Cyperi Rhizoma 4.0, Cnidii Rhizoma 3.0, Schizonepetae Spica 2.0,
Menthae Herba 2.0, Angelicae Dahuricae Radix 2.0, Ledebouriellae
Radix 2.0, Glycyrrhizae Radix 1.5, Notopterygii Rhizoma 2.0, Theae
Folium 1.5
Keishi-bukuryo-gan-kaCoicis Semen 10.0, Cinnamomi Cortex 4.0, Paeoniae Radix 4.0,
yokuinin
Persicae Semen 4.0, Hoelen 4.0, Moutan Cortex 4.0
Mashinin-gan
Rhei Rhizoma 4.0, Aurantii Fructus Immaturus 2.0, Armeniacae
Semen 2.0, Magnoliae Cortex 2.0, Paeoniae Radix 2.0, Cannabidis
Semen 5.0
Mao-bushi-saishin-to
Ephedrae Herba 4.0, Asiasari Radix 3.0, Aconiti Tuber 1.0
Keihi-to
Atractylodis Lanceae Rhizoma 4.0, Hoelen 4.0, Dioscoreae Rhizoma
3.0, Ginseng Radix 3.0, Alismatis Rhizoma 2.0, Aurantii Nobilis
Pericarpium 2.0, Glycyrrhizae Radix 1.0, Nelumbinis Semen 3.0,
Crataegi Fructus 2.0
Dai-joki-to
Magnoliae Cortex 5.0, Aurantii Fructus Immaturus 3.0, Rhei
Rhizoma 2.0, Natrium Sulfuricum 1.3
Keishi-ka-shakuyaku-daio-to Paeoniae Radix 6.0, Cinnamomi Cortex 4.0, Zizyphi Fructus 4.0,
Glycyrrhizae Radix 2.0, Rhei Rhizoma 2.0, Zingiberis Rhizoma 1.0

Genotoxicity response
Ames
MN
UDS
P

N

N

N

−

−

P
P

N
N

N
N

N

−

−

N

−

−

N

−

−

N

−

−

N

−

−

N

−

−

P
N

N
−

N
−

N

−

−

N

−

−

N

−

−

N

−

−

N
N

−
−

−
−

N

−

−

N

−

−

(Table 1 continued）
Code No.

Names of Kampo

TJ-135

Inchinko-to

TJ-136

Seisho-ekki-to

TJ-137Kami-kihi-to

TJ-138

Kikyo-toConstituents and combination ratio of herbal drugs
Gardeniae Fructus 3.0, Rhei Rhizoma 1.0, Artemisiae Capillaris
Spica 4.0
Atractylodis Lanceae Rhizoma 3.5, Ginseng Radix 3.5,
Ophiopogonis Tuber 3.5, Astragali Radix 3.0, Aurantii Nobilis
Pericarpium 3.0, Angelicae Radix 3.0, Phellodendri Cortex 1.0,
Glycyrrhizae Radix 1.0, Schisandrae Fructus 1.0
Astragali Radix 3.0, Bupleuri Radix 3.0, Atractylodis Lanceae
Rhizoma 3.0, Ginseng Radix 3.0, Hoelen 3.0, Polygalae Radix 2.0,
Gardeniae Fructus 2.0, Zizyphi Fructus 2.0, Angelicae Radix 2.0,
Glycyrrhizae Radix 1.0, Zingiberis Rhizoma 1.0, Saussureae Radix
1.0, Zizyphi Spinosi Semen 3.0, Longanae Arillus 3.0
Glycyrrhizae Radix 3.0, Platycodi Radix 2.0

Genotoxicity response
Ames
MN
UDS
N

−

−

N

−

−

N

−

−

N

−

−

Ames: Bacterial reverse mutation test (Ames test), MN: Micronucleus test using mouse bone marrow cells,
UDS: Unscheduled DNA synthesis (UDS) tests using rat hepatocytes, P: Positive responses,a)
N: Negative responses, −: Not tested, Already published (Kuboniwa et al., 1999a-q).

Scutellariae Radix extracts and their major components
were also submitted to the Ames test. Salmonella
typhimurium strains (TA100, TA1535, TA98 and TA1537)
and an Escherichia coli strain (WP2uvrA) were obtained
from the Institute for Fermentation, Osaka, Japan. Eachassay mix of 1.0 mL included 0.1 mL of S9 as an exogenous metabolic activation system, 8 µmol of MgCl2, 33µmol of KCl, 5 µmol of glucose-6-phosphate, 4 µmol of
NADPH, 4 µmol of NADH and 100 µmol of sodium phosphate buffer. The maximum dose levels of test substances were determined by dose-finding studies. The
dose at which growth was inhibited was used as the maximum dose level. In the case of no growth inhibition, 5000
µg/plate was selected as the maximum dose level as recommended in regulatory guidelines. All test substanceswere suspended in distilled water. As positive controls, 2(2-furyl) -3- (5-nitro-2-furyl) acrylamide (CASRN 3688-537), 2-aminoanthracene (CASRN 613-13-8), 9-aminoacridinehydrochloride hydrate (CASRN 52417-22-8), sodium azide(CASRN 26628-22-8) and N-ethyl-N’-nitro-N-nitrosoguanidine (CASRN 63885-23-4) were used.
The tests were carried out by the plate incorporation
method in the presence or absence of the S9mix. Afterincubation for 48 h, the number of revertant colonies wascounted. If the number of revertant colonies in a test substance treatment group increased dose-dependently morethan 2-fold over the number of revertant colonies in thenegative controls, it was declared positive. Mutagenic
potencies were calculated as the number of induced revertant colonies per µg substance.
Micronucleus test
The micronucleus test was conducted to assess chromosomal damage in vivo according to the Japanese guideline for nonclinical studies (Japanese Ministry of Healthand Welfare, 1994). Thirty Ames-positive and 17 Amesnegative Kampo extracts (Table 1) and wogonin, baicalin
and baicalein were tested.
Male Crj ： CD-1 (ICR) mice, purchased from Charles
River Japan, Inc., Yokohama, Japan, were used at 8 weeks
old. Dose levels of the test substances were determined
experimentally, or from technical limits for administration. All test substances were suspended in distilledwater. Mice were treated with the test substances by asingle, or repeated oral administration for 2 or 4 days.
Bone marrow cells were flushed out from femora withfetal bovine serum at 24 - 72 h after the final administration, smeared on glass slides, and stained with Giemsa or
Acridine orange for microscopy. The incidence of
micronucleated polychromatic erythrocytes (MNPCE)
was evaluated based on observation of 1000 polychromatic erythrocytes (PCE) per mouse. Statistical analysis wasdone by the method of Kastenbaum and Bowman(Kastenbaum and Bowman, 1970). Mitomycin C (CASRN
50-07-7, 2 mg/kg, i.p. injection) was used as a positive
control. If the incidence of MNPCE in a treated groupincreased significantly and dose-dependently in comparison with negative control groups, it was declared positive.
UDS test
UDS test was carried out by the method of Furihata etal. (Furihata and Mori, 1994 ； Sawada et al., 1993) to evaluate the induction of DNA damage in vivo. Thirty Amespositive Kampo extracts (Table 1) were tested. Male
F344/du Crj rats, purchased from Charles River Japan,
Inc., Yokohama Japan, were used at 8 weeks old. Doselevels of the test substances were determined by the technical limits of administration. Test substances were suspended in distilled water and administered to rats by gavage. Hepatocytes were isolated by perfusion with collage9

Table 2 Positive responses on 30 extracts of Japanese traditional herbal medicines (Kampo) in the S. typhimurium TA1537
reverse mutation test
Code No.

Names of Kampo

TJ-3
TJ-8d)
TJ-9d)
TJ-10d)
TJ-11
TJ-12d)
TJ-14
TJ-15d)
TJ-50
TJ-51
TJ-56
TJ-57

Otsuji-to
Dai-saiko-to
Sho-saiko-to
Saiko-keishi-to
Saiko-keishi-kankyo-to
Saiko-ka-ryukotsu-borei-to
Hange-shashin-to
Oren-gedoku-to
Keigai-rengyo-toJuncho-toGorin-sanUnsei-in

TJ-58
TJ-62
TJ-64
TJ-67
TJ-71
TJ-73
TJ-74
TJ-76
TJ-80
TJ-88
TJ-90
d)
TJ-96
TJ-104
TJ-109
TJ-111
TJ-113
TJ-114d)
TJ-121

Seijo-bofu-to
Bofu-tsusho-san
Sha-kanzo-to
Nyoshin-san
Shimotsu-to
Saikan-to
Choi-joki-to
Ryutan-shakan-to
Saiko-seikan-to
Nijutsu-to
Seihai-to
Saiboku-to
Shin’i-seihai-to
Sho-saiko-to-ka-kikyo-sekko
Seishin-renshi-in
San’o-shashin-to
Sairei-to
Sammotsu-ogon-to

Metabolicactivation

Mutagenicitya)

Negative control
valueb)Mutagenic
potencyc)

＋
＋
＋
＋
＋
＋
＋
＋
＋
＋
＋
−
＋
＋
＋
−
＋
−
＋
−
＋
＋
＋
＋
＋
＋
＋
＋
＋
＋
−
＋

20/15029/1500
16/15016/15038/40030/1500
26/40017/50
54/3000
26/50047/60012/31322/62535/60024/31331/3000
37/60026/4000
44/1000
31/2500
27/50022/31
21/50
18/62535/1250
48/80016/15035/60025/132
20/313
14/15038/400

8
11
8
8
13
12
13
8
17
12
17
5
10
17
10
7
18
9
18
10
12
11
9
8
10
19
7
12
9
10
7
12

0.080
0.012
0.053
0.053
0.063
0.012
0.033
0.180
0.012
0.028
0.050
0.022
0.019
0.030
0.045
0.008
0.032
0.004
0.026
0.008
0.030
0.355
0.240
0.016
0.020
0.036
0.060
0.038
0.121
0.032
0.047
0.065

a)

No. of revertant colonies/dose (in µg) of Kampo incorporated in the plate, b) No. of revertant colonies in negative control group,
Net number of revertant colonies per µg, d) Already published (Kuboniwa et al., 1999a-d; Kuboniwa et al., 1999o-q),
TA1537: S. typhimurium TA1537, ＋: Presence of metabolic activation (S9mix), −: Absence of metabolic activation (S9mix).

c)nase at 2 and 12 h after administration, and were cultured5(1.0 × 10 cells/mL) in William’s E (WE) medium for 2 h.
The medium was then replaced with fresh WE medium
containing 370 KBq/mL (10 µCi/mL) of thymidine
3[methyl- H] for labeling DNA of hepatocytes. For autoradiography processing, cultured hepatocytes were harvested and dipped in emulsion for X-ray. After storage at −20℃ for 10 to 11 days, the exposed DNA of hepatocytes wasdeveloped for observation as grains. The number ofgrains over the nucleus (Nuclear grains) and in threeadjacent nuclear-size areas over the cytoplasm(Cytoplasmic grains) were counted. Net nuclear grains
were calculated as Nuclear grains minus the mean of
Cytoplasmic grains over the three respective areas.
Dimethylnitrosamine (CASRN 62-75-9) was used at 5
mg/kg as a positive control. If the mean of Net nuclear
10

grains in the treated groups increased more than 5, thetest substance was declared positive.

Results
Ames test
Of 128 Kampo extracts, 98 were negative in the Ames
test either in the presence or absence of the exogenous
metabolic activation system (data not shown). In the positive control groups, the number of revertant colonies wasmarkedly increased (data not shown). Thirty Kampo
extracts (TJ-3, TJ-8, TJ-9, TJ-10, TJ-11, TJ-12, TJ-14, TJ-15,
TJ-50, TJ-51, TJ-56, TJ-57, TJ-58, TJ-62, TJ-64, TJ-67, TJ-71,
TJ-73, TJ-74, TJ-76, TJ-80, TJ-88, TJ-90, TJ-96, TJ-104, TJ109, TJ-111, TJ-113, TJ-114 and TJ-121), exhibited a weak
but reproducible positive effect in S. typhimurium TA1537(Tables 1 and 2).

Table 3 Positive responses on extracts of Scutellariae Radix and its main components in the bacterial reverse mutation tests
Names ofherbal drugs or components

Tester strain for
detection of
positive responses

Scutellariae Radix

TA1537

Wogonind)

TA100
TA1537
TA1537
TA1537

Baicalind)
Baicaleind)Metabolicactivation

Mutagenicitya)

Negative
controlvalueb)Mutagenic
potencyc)

−
＋
＋
＋
＋
−
＋

11/50
28/50
468/50127/50
31/50
15/50
41/50

5
10
92
13
13
5
13

0.120
0.360
7.520
2.800
0.360
0.200
0.560

a)

No. of revertant colonies/dose (in µg) of herbal drugs or components incorporated in the plate,
No. of revertant colonies in negative control group, c) Net number of revertant colonies per µg,d)Component in Scutellariae Radix,
TA1537: S. typhimurium TA1537, TA100: S. typhimurium TA100,＋: Presence of metabolic activation (S9mix), −: Absence of metabolic activation (S9mix).
b)

Extracts of Scutellariae Radix, which is a common
herbal drug used in 27 of the 30 Ames-positive Kampo
extracts, and its major components, wogonin, baicalin andbaicalein, induced positive responses in TA1537 andwogonin was also positive in TA100 (Table 3).
Mouse bone marrow MN test
All 30 Ames-positive and 17 Ames-negative Kampo
extracts did not increase the MNPCE frequencies. The
incidence of MNPCE ranged from 0.0 to 0.4% per mousein all the treatment groups as shown in Table 4 for theAmes-positive extracts ； this range was comparable tothat of the negative controls. The major components ofScutellariae Radix, wogonin, baicalin, and baicalein werealso negative in the MN test ； their MNPCE frequencies
ranged from 0.0 to 0.5% per mouse (Table 5). In the positive control groups, the incidence of MNPCE was significantly increased as expected (data not shown).
The ratio of PCE to total erythrocytes in the treatmentgroups did not decrease indicating an absence of cytotoxicity (data not shown).
Rat liver UDS test
None of the Ames-positive Kampo extracts elicited UDS
induction at 2 and 12 h after administration (Table 6).
The range of Net nuclear grains was ─ 0.5 to ─ 3.4 in all
the treatment groups (Table 6), and was comparable tothe negative controls. The positive control groups
showed the expected responses (data not shown).

Discussion
In this present study, the genotoxicity of Kampo
extracts in vitro and in vivo has been examined. Out of
128 Kampo extracts evaluated by the Ames bacterial
mutation test, 98 were negative and the remaining 30
extracts showed a positive response. Previously, Rokumigan and Juzen-taiho-to were reported with contradictory

Ames test results (Nozaka et al., 1994a ； Fujita et al.,
1992, 1994 ； Fujita and Sasaki, 1993). In the present
study, they were judged negative in the Ames test. In
contrast, Otsuji-to, Dai-saiko-to, Sho-saiko-to, Saiko-karyukotsu-borei-to, Hange-shashin-to, Oren-gedoku-to,
Keigai-rengyo-to, Unsei-in and Saiboku-to were positive inthe present study although they were reported negativeelsewhere (Nozaka et al., 1994a). Such contradictory findings might be due to variations in the origin of the herbal
drugs, with some herbs having many subspecies the components of which are likely to be altered by climatic andgeographical conditions. Their variations can easily affectthe results of in vitro assays such as the Ames test.
The extracts of Scutellariae Radix are used quite widely
as a component of Kampo. In the present study, it wascompounded in 27 of the 30 Ames-positive Kampo
extracts. These Scutellariae Radix extracts and components wogonin, baicalin and baicalein were positive in theAmes test, therefore these compounds may have contributed to the positive responses of these 27 Kampo
extracts in the Ames test. However, more extensive analyses are needed to further probe the components of theseAmes-positive Kampo extracts, as well as the componentsof the 3 Ames-positive extracts that did not contain
Scutellariae Radix.
Several extracts of herbal drugs or their components,
such as flavonoids, anthraquinones, alkaloids and theirderivatives, are known to be mutagenic (Brown andDietrich, 1979 ； Brown, 1980 ； Czeczot et al., 1990 ；Fujita et al., 1993, 1994 ； Kikuchi and Oshio, 1983 ；Morimoto et al., 1982, 1983 ； Nagao et al., 1981 ； Nozaka
et al., 1987, 1990, 1991, 1994b ； Sugimura et al., 1977 ；Tadaki et al., 1995 ； Watanabe et al., 1983 ； Xue-jun etal., 1991 ； Yamamoto et al., 1982). These components
may have contributed to the positive responses of the
Kampo extracts studied here.
All 30 Ames-positive and 17 Ames-negative Kampo
11

Table 4 Micronucleus test for 30 extracts of Japanese traditional herbal medicines (Kampo) showing positive responses in the bacterial
reverse mutation test
Code No.

Names of Kampo

Dose (mg/kg)

Sampling time (h)Range of
MNPCE/PCE(%)a)

TJ-3
TJ-8c)
TJ-9c)
TJ-10c)
TJ-11
TJ-12c)
TJ-14
TJ-15c)
TJ-50
TJ-51
TJ-56
TJ-57
TJ-58
TJ-62
TJ-64
TJ-67
TJ-71
TJ-73
TJ-74
TJ-76
TJ-80
TJ-88
TJ-90
TJ-96c)
TJ-104
TJ-109
TJ-111
TJ-113
TJ-114c)
TJ-121

Otsuji-to
Dai-saiko-to
Sho-saiko-to
Saiko-keishi-to
Saiko-keishi-kankyo-to
Saiko-ka-ryukotsu-borei-to
Hange-shashin-to
Oren-gedoku-to
Keigai-rengyo-toJuncho-toGorin-sanUnsei-inSeijo-bofu-to
Bofu-tsusho-san
Sha-kanzo-to
Nyoshin-san
Shimotsu-to
Saikan-to
Choi-joki-to
Ryutan-shakan-to
Saiko-seikan-to
Nijutsu-to
Seihai-to
Saiboku-to
Shin’i-seihai-to
Sho-saiko-to-ka-kikyo-sekko
Seishin-renshi-in
San’o-shashin-to
Sairei-to
Sammotsu-ogon-to

2500, 5000, 10000
500, 1000, 2000
2500, 5000, 10000
500, 1000, 2000
2500, 5000, 10000
250, 500, 1000, 2000
2000, 4000, 8000
500, 1000, 2000
2500, 5000, 10000
2500, 5000, 10000
2500, 5000, 10000
2500, 5000, 10000
2500, 5000, 10000
2500, 5000, 10000
2500, 5000, 10000
2500, 5000, 10000
2500, 5000, 10000
2500, 5000, 10000
1250, 2500, 5000
2500, 5000, 10000
2500, 5000, 10000
2500, 5000, 10000
2500, 5000, 10000
500, 1000, 2000
2500, 5000, 10000
2500, 5000, 10000
2000, 4000, 8000
2500, 5000, 10000
500, 1000, 2000
2500, 5000, 1000024b)
24, 48, 72
24
24, 48, 72
24b)
24, 4824b)
24, 48, 72
24b)
24b)
24b)
24b)
24b)
24b)
24b)
24b)
24b)
24b)
24b)
24b)
24b)
24b)
24b)
24, 48, 72
24b)
24b)
24b)
24b)
24, 48, 72
24b)

0.0 〜 0.3
0.0 〜 0.3
0.0 〜 0.3
0.0 〜 0.4
0.1 〜 0.3
0.0 〜 0.2
0.0 〜 0.4
0.0 〜 0.4
0.0 〜 0.4
0.0 〜 0.4
0.0 〜 0.3
0.0 〜 0.3
0.0 〜 0.4
0.0 〜 0.4
0.0 〜 0.3
0.0 〜 0.4
0.0 〜 0.3
0.0 〜 0.3
0.0 〜 0.3
0.0 〜 0.4
0.0 〜 0.4
0.0 〜 0.4
0.0 〜 0.4
0.0 〜 0.4
0.0 〜 0.3
0.0 〜 0.2
0.0 〜 0.2
0.0 〜 0.4
0.0 〜 0.4
0.0 〜 0.3a)Minimum and maximum incidences (%) of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes in 5 mice,Time after final administration,c)
Already published (Kuboniwa et al., 1999a-d; Kuboniwa et al., 1999o-q).
b)Table 5 Micronucleus test for main components of Scutellariae Radix showing positive responses in the bacterial reverse mutation testa)b)Names of the components

Dose (mg/kg)

Sampling time (h)a)

Range of MNPCE/PCE (%)b)

WogoninBaicalin
Baicalein

500, 1000, 2000
500, 1000, 2000
500, 1000, 2000

24, 4824, 4824, 480.0 〜 0.4
0.0 〜 0.5
0.0 〜 0.4

Time after final administration,
Minimum and maximum incidences (%) of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes in 5 mice.

extracts and major components of Scutellariae Radix were
subjected to the mouse bone marrow MN test. None
induced micronuclei even at doses as high as 2000 mg/kg
or more. These 30 Ames-positive Kampo extracts are usually recommended for human consumption at doses of1250 - 7000 mg/body/day (ca. 21-117 mg/kg/day). Thus
the human ex p osure levels of Kampo extracts are at least
one order of magnitude below those shown to be negativein the MN test. The 30 Ames-positive Kampo extracts
were also evaluated by the rat liver UDS test. None
12

showed any DNA damaging potential under the test conditions.
In conclusion, the majority (98 of 128) of Kampo
extracts tested were not mutagenic in the Ames test. On
further testing of the 30 Ames-positive extracts in a mousebone marrow MN test and a rat liver UDS test, none were
found to elicit an effect. Overall, these results suggestthat all Kampo extracts evaluated here pose little or norisk for human health from the viewpoint of genotoxicity.

Table 6

Unscheduled DNA synthesis (UDS) test for 30 extracts of Japanese traditional herbal medicines (Kampo) showing positive responses in the bacterial reverse mutation test
Dose (mg/kg)

Time after treatment (h)No. of Net nuclear grainsa)

Otsuji-to

4000

TJ-8b)

Dai-saiko-to

2000

TJ-9b)

Sho-saiko-to

2000

TJ-10b)

Saiko-keishi-to

2000

TJ-11

Saiko-keishi-kankyo-to

2500

TJ-12b)

Saiko-ka-ryukotsu-borei-to

2000

TJ-14

Hange-shashin-to

2000

TJ-15b)Oren-gedoku-to

2000

TJ-50

Keigai-rengyo-to

4000

TJ-51

Juncho-to

4000

TJ-56

Gorin-san

2500

TJ-57

Unsei-in

4000

TJ-58

Seijo-bofu-to

4000

TJ-62

Bofu-tsusho-san

4000

TJ-64

Sha-kanzo-to

4000

TJ-67

Nyoshin-san

4000

TJ-71

Shimotsu-to

4000

TJ-73

Saikan-to

2500

TJ-74

Choi-joki-to

3000

TJ-76

Ryutan-shakan-to

3000

TJ-80

Saiko-seikan-to

4000

TJ-88

Nijutsu-to

2500

TJ-90

Seihai-to

4000

TJ-96b)

Saiboku-to

2000

TJ-104Shin’i-seihai-to

4000

TJ-109Sho-saiko-to-ka-kikyo-sekko

2500

TJ-111Seishin-renshi-in

2000

TJ-113San’o-shashin-to

3000

TJ-114b)

Sairei-to

2000

TJ-121

Sammotsu-ogon-to

4000

2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12
2
12

− 1.2 ± 1.5− 1.0 ± 1.7− 2.3 ± 0.6− 2.6 ± 1.0− 2.5 ± 0.9− 3.3 ± 0.3− 2.8 ± 0.3− 2.4 ± 1.1− 2.2 ± 0.2− 3.1 ± 0.9− 2.4 ± 0.5− 2.7 ± 0.3− 2.3 ± 0.3− 2.8 ± 1.3− 2.2 ± 0.2− 2.3 ± 0.5− 1.7 ± 1.1− 2.2 ± 0.7− 1.3 ± 1.8− 0.8 ± 1.8− 1.3 ± 0.4− 2.6 ± 0.7− 1.8 ± 0.8− 3.4 ± 0.2− 0.5 ± 1.6− 1.0 ± 1.9− 2.7 ± 0.1− 2.2 ± 1.0− 2.8 ± 0.9− 3.3 ± 0.2− 1.2 ± 0.4− 2.2 ± 0.4− 0.7 ± 1.6− 1.2 ± 1.8− 0.6 ± 1.7− 0.9 ± 1.8− 0.8 ± 1.7− 1.2 ± 1.8− 0.8 ± 1.6− 1.6 ± 2.3− 1.7 ± 0.1− 1.5 ± 0.6− 2.0 ± 0.4− 2.4 ± 0.7− 0.7 ± 1.5− 1.2 ± 2.0− 3.1 ± 0.6− 2.3 ± 0.7− 0.5 ± 1.2− 1.8 ± 2.1− 2.3 ± 0.6− 3.0 ± 0.9− 3.0 ± 0.4− 2.2 ± 0.9− 2.8 ± 0.3− 2.3 ± 0.9− 1.0 ± 0.4− 2.3 ± 0.2− 0.6 ± 1.7− 0.9 ± 1.6

Code No.

Names of Kampo

TJ-3

a)

Mean ± S.D. of 3 rats, b) Already published (Kuboniwa et al., 1999a-d; Kuboniwa et al., 1999o-q).
13

Acknowledgment
We thank Dr. Makoto Hayashi, National Institute of
Health Sciences, for giving counsel to the contents of ourmanuscript.
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15Original Article

Environ. Mutagen Res., 24 ： 17 - 21 (2002)

Effect of a p53 codon 237 mutation on X-ray induced
HPRT mutations
Shigeko Morimoto1, Masamitsu Honma3 and Fumio Yatagai1,2＊
1

2

Division of Radioisotope Technology and Cellular Physiology Laboratory, The Institute of Physical3and Chemical Research, Saitama 351-0198, Japan; Division of Genetics and Mutagenesis, National
Institute of Health Sciences, Tokyo, 158 -8501, Japan

Summar y
To determine the influence of mutant p53 on HPRT mutations induced by ionizing radiation, we
irradiated human lymphoblastoid cell lines TK6 (wild-type p53) and WI-L2-NS (mutant p53 at codon
237) with X-rays. We applied multiplex PCR analysis for independent mutants arising spontaneously
or induced by X-ray exposure. X-rays enhanced the HPRT mutation frequency, yielding values thatwere 6 times the spontaneous level in TK6 and 6.8 times that level in WI-L2-NS. Point mutations (allexon regions amplified by PCR), which comprised the major portion of spontaneous mutations (60〜 80 ％) in both cell lines, were decreased by X-ray irradiation. In contrast, the proportion of complete deletions (no exon region amplified) was increased. These tendencies of decrease andincrease after irradiation were the same as those reported by Phillips et al. (1995) but not statistically proved by chi-2 test. Although such test also can not show the statistic significance in the following differences, the proportion of partial deletions (some exon regions amplified) increased in TK6
but decreased in WI-L2-NS, reflecting the relatively high spontaneous level (29 ％) of such mutations in WI-L2-NS relative to the level (8 ％) in TK6. The high level of spontaneous partial deletions
in p53 mutant cells was primarily attributable to a frequent (6/8) specific event-deletion of exons 2
to 5. X-ray reduced that frequency to a more ordinary level (1/8). As reported by Phillips et al.
(1995), we also did not detect any effect of the codon 237 of p53 mutation on the distribution of point
mutations, partial deletions, or complete deletions in X-ray-induced HPRT mutants.
Keywords: human lymphoblastoid cell,

HPRT (hypoxanthine phosphoribosyltransferase) mutation, multiplex PCR (polymerase chain reaction) analysis, X-ray exposure

Introduction
Since p53 protein is critical in the maintenance ofgenomic stability, we set out to study the influence of p53
gene status on the specificity of spontaneous and radiation-induced mutations in cultured human cells. Suchinformation might provide insight into the mechanisms ofmutation induction in relation to DNA replication, transcription, and repair. The closely related human lymphoblastoid cell lines TK6 and WI-L2-NS are derived fromWI-L2 cells originally isolated from human spleen ( Levy
et al., 1968). WI-L2-NS contains an ATG (Methionine) to
ATA (Isoleucine) homozygous change in codon 237 of the＊

yatagai@postman.riken.go.jp
received: November 5, 2001, accepted: January 29, 2002
© Environmental Mutagen Society of Japanp53 gene, while TK6 contains the wild-type (Xia et al.,1995). WI-L2-NS is more resistant than TK6 to X-rays, andthis resistance is accompanied by higher mutability at the
hypoxanthine phosphoribosyltransferase (HPRT) locus(Amundson et al., 1993; Zhen et al., 1995). These differences in radiation resistance may reflect differences in anerror-prone (Amundson et al., 1993), or recombinational
repair (Xia et al., 1994) system.
Inactivation of wild-type p53 leads to loss of G1/S
checkpoint control and to genomic instability, including
an increased spontaneous level of homologous recombination (Mekeel et al., 1997; Willers et al., 2000). Enhanced
spontaneous and radiation-induced intrachromosomal
homologous recombination was observed in mouse L
cells with p53 mutations at codons 175, 248, or 273(Bertrand et al., 1997; Saintigny et al., 1999). WTK1 cells,
derived from WI-L2-N2, which carry the same p53 muta17

Table 1 Primers used in multiplex PCR analysis of HPRT mutations
Band size

PrimerPosition

Sequence

393bp

ex2pF
ex2pR
ex3pF
ex3pR
ex4pF
ex4pR
ex5pF
ex5pR
ex6pF
ex6pR
ex78pFex78pR
ex9pF
ex9pR

S 14674 to 14700
A 15066 to 15044
S 16519 to 16540
A 16977 to 16954
S 27754 to 27776
A 28085 to 28063
S 31500 to 31522
A 31735 to 31712
S 34817 to 34841
A 35074 to 35052
S 39596 to 39619
A 40495 to 40471
S 41261 to 41281
A 42054 to 42032

CACCTAAATTTCTCTGATAGACTAAGGGATACTAAGTAATTAGTAAGGCCGTGGAAGTTTAATGACTAAGAGGTATATATCCTCCAAGGTGACTAGGCTATGGATATTAGCTAGCTAAC
GCTTCCAAGGGTTAAATAACCCA
AGCATCTAAAAACAAGAGTTTGG
AACTGATGCAGAGGAATTTCTCTCGATTTTGGTGAGAATTACTGTGCTG
CACTTAATCCCCCTTCAAATGAGACCAAGTGCCTTGTCTGTAGTGTCTCCTAAATCTTCCTCAACCATGTTCACTAATGTGATAGACTACTGC
GAACTGCTGACAAAGATTCACTG

459bp
332bp
236bp
258bp
900bp
794bp

tions at codon 237 as WI-L2-NS, show higher spontaneous
mutation frequencies at the TK locus as well as at the
HPRT locus (Honma et al., 1997a). WTK1 cells also
demonstrate a high frequency of TK allelic loss and unbalanced translocation (Honma et al., 1997b). This mutant
p53 gene is constitutively expressed (Xia et al., 1995) andresults in loss of p21 transactivation following genotoxic
stress (Wenz et al., 1998). In human gliomas overexpression of p21 have been implicated in radio-resistance(Kokunai and Tamaki 1999). That observation, however,is difficult to reconcile with other reports showing radioresistance in p21-uninduced cells. A lower frequency
and/or delayed appearance of apoptosis in WI-L2-NS cells
relative to wild-type cells after X-ray exposure may berelated to such resistance (Xia et al., 1995, Zhen et al.,1995).
After X-ray irradiation, TK6 and WTK1 cells show no
significant difference in HPRT mutation spectrum (distribution of point mutations, partial deletions, and complete
deletions) (Phillips et al., 1995). If WTK1 loci other than
p53 also mutated during establishment of the cell line,
those mutations might mask the influence of mutant p53
on mutational specificity. Thus, in this study, we aimed toconfirm the reported characteristic in WI-L2-NS, a related
cell line carrying the same p53 mutation.

Materials and Methods
Cell culture and exposure to X-rays
Human TK6 and WI-L2-NS lymphoblastoid cells were
cultured at 37 ℃ in RPMI 1640 medium (Gibco-BRL) supplemented with 1 mM α-ketoglutaric acid and 10 ％ fetal
bovine serum (REHATUIN) in a humidified atmosphere
containing 5 ％ CO2. They were cultured for two days in
RPMI medium containing CHAT (10 µM cytidine, 200 µM
hypoxanthine, 0.2 µM aminopterin, and 17.5 µM thymidine) and one more day in CHT medium (CHAT excluding aminopterin). Cells were then resuspended in fresh
18

5

RPMI at 8 × 10 cells/ml and exposed to X-rays at 1
Gy/min (Softex-Rigaku, Japan, 250 kVp).
Determination of cell survival and selection ofmutant clones
Determination of cell survival and selection of HPRT
mutants were based upon a limiting dilution methodology(Kagawa et al., 1999). Briefly, following X-ray irradiation,
the cells were incubated for 4 days in normal medium toallow phenotypic expression and then treated with 6thioguanine (6-TG; 5 µg/ml final concentration). The
treated cells were incubated in eighty 96-well dishes of
4(4 × 10 cells/ml per well) for 2 weeks for selection of 6TG resistant mutants. Each 96-well dish was preparedfrom a single phenotypic expression dish. One HPRT
mutant clone was picked from each 96-well dish and characterized by PCR analysis to make a mutational spectrum
consisting of independent mutant clones. Spontaneous
mutants were collected by the same procedure, but withno phenotypic expression period. Putative HPRT mutant
clones were diluted 20-fold in the selection medium andincubated for another 5 days to confirm their HPRT status. Cell survival was also measured after 2 weeks incubation in 96-well dishes (inoculation of approximately 1.6
cells per well).
Multiplex PCR analysis of genomic DNA
TM
ISOGEN (Nippon Gene, Japan) was used for the
extraction of genomic DNA from HPRT mutant clones. All
exon regions of the HPRT locus, except the exon 1 region,
were amplified by multiplex PCR (Gibbs et al., 1990). The
PCR reaction conditions, including primers (Table 1),
were essentially the same as in our previous study(Kagawa et al., 1999). All the PCR products were analyzed in a 2 ％ agarose gel.

Fig. 1 Western blot analysis of p53 and p21 protein expression by
TK6 and WI-L2-NS cells 2h after 3 Gy of X-ray, C-ion, andFe-ion irradiation.

Western blot analysis
Total protein was extracted from the cells 2h after they
were irradiated, and 60 µg of the extract was loaded into
each lane in a 15 ％ SDS-PAGE mini-gel. The protein waselectrophoresed, blotted onto a PVDF membrane(Millipore), and probed with monoclonal antibodies
against p53 (DO-1, Santa Cruz Biotech) and p21 (EA10,
Oncogene Sciences).

Results
HPRT mutation induction
Western blot analysis showed that p53 protein was
induced in TK6 2 hours after exposure to 3 Gy X-ray radiation, while mutant p53 was constitutively produced in WIL2-NS (Fig. 1). Induction of p21 protein was accompanied
by an increase in p53 protein only in TK6 but not in WI-L2NS. We know from survival curves that WI-L2-NS is moreresistant than TK6 to X-ray irradiation (P ＝ 0.025 at D10 byStudent T-test) (data not shown). That is in accordancewith a previous study (Amundson et al., 1993), as is ourprevious observation of higher mutability in WI-L2-NS
than in TK6 after X-ray exposure (Amundson et al., 1993,
Zhen et al., 1995).
In this study, we compared the HPRT mutation spectrum between both cell lines after X-ray irradiation
reduced cell survival to approximately 10 ％. Exposure of
TK6 cells to 1.7 Gy of X-rays reduced cell survival to 12 ％and increased the HPRT mutation frequency from the−6−6
spontaneous level of 2.0 ± 0.7 × 10 to 12.0 × 10 .
Exposure of WI-L2-NS cells to 2.5 Gy of X-rays reduced
survival to 14 ％ and increased HPRT mutation frequency−6−6from 3.4 ± 0.5 × 10 to 23.0 × 10 .

Comparison of HPRT mutation spectra
Table 2 shows the results of multiplex PCR analysis forindependently arising HPRT mutants. In both cell lines,
the majority of spontaneous HPRT mutations were point
mutations (PCR amplification was normal). The proportion of spontaneous partial deletions (only some exon
regions were amplified) was higher in WI-L2-NS (29 ％)than in TK6 (8 ％). Interestingly, 6 of the 8 partial deletions observed in WI-L2-NS were of type b (exon regionsb)
2-5 were not amplified (Table 2 ). This “hot-spot” existed
only in WI-L2-NS and after X-ray exposure, when the frequency of that deletion was reduced to 1/7. The frequency of spontaneous complete deletions (no exon region was
amplified) was similar for the two cell lines, 〜 10 ％ of thetotal mutations.
X-ray irradiation reduced the proportion of point mutations from the spontaneous level in both strains. It
increased the proportion of partial deletions in TK6, but
decreased it in WI-L2-NS. This result suggests that theinduction of partial deletions is more efficient in TK6 thanin WI-L2-NS. X-ray irradiation increased the proportion ofcomplete deletions in both strains, which suggests thatthe DNA double-strand breaks caused by X-ray exposure
were involved in induction of deletion mutations.
Statistical analysis of the data revealed no significant difference between the two cell lines in the distribution ofpoint mutations, partial deletions, and complete deletions.
Among the 6 and 7 partial deletions observed in TK6 andWI-L2-NS, respectively, we found only one that reflectedb)the same deletion pattern (f) (Table 2 ). A single com＊b)plex deletion mutation (k in Table 2 ) involving noncontiguous exons was recovered in WI-L2-NS cells
exposed to X-rays. The same complex deletion has also
been identified in human lymphoblastoid cells exposed toaccelerated carbon- and neon-ion beams (Kagawa et al.,1999).

Discussion
Increase of mutation frequency in TK6 and WI-L2-NS
by X-ray irradiation is consistent with those reported by
Nelson et al. (1994). The relatively high proportion of partial deletions among the HPRT mutations occurring spontaneously in p53 mutant cells compared to 53 wild-type
cells suggests the possible involvement of mutant p53
function in the generation of spontaneous mutations. The
distribution of HPRT mutational class (point mutation,
partial deletion, and complete deletion) obtained after Xray irradiation was not influenced by this p53 mutation.
This tendency is consistent with a similar finding for TK6
and WTK1 cells reported by Phillips et al. (1995). They,
however, noted that a particular type of partial deletion - adeletion inside the HPRT locus - was about twice as frequent in WTK1 as in TK6 cells both before and after X-ray
exposure. Similarly we found a distinct difference in the19Table 2 Analysis of independent HPRT mutants by multiplex PCRa) Distribution of HPRT mutation classes
TK6

Point mutation
Partial deletion
Complete deletion
Total number of mutants

WI-L2-NS

Spont.

X-rays(1.7 Gy)

Spont.

X-rays(2.5 Gy)

20(80)2(8)3(12)

32(67)
6(13)
10(20)

17(60)8(29)
3(11)

25(52)
7(15)
16(33)

25

48

28

48

The numbers in parenthesis represent relative proportion of eachmutation class as ％.
b) Detailed characteristics of partial deletion mutantsa 〜 o: PCR amplification pattern (deletion profile) of exon regions: indicates exon regions of HPRT that were amplified by multiplex PCR.
＊
: Complex deletion mutation (see text)

pattern of partial deletions recovered after X-ray exposure.
The p53 protein is believed to contribute to genetic stability through DNA homologous recombination in TK6(Honma et al., 2000). The function of mutant p53 has beensuggested to be involved in this homologous recombination resulting in the enhancement of recombinogenic
activity associated with higher mutability (Xia et al., 1994,
Bertrand et al., 1997, Mekeel et al., 1997, Saintigny et al.,1999). Such high recombinogenic activity in WTK1
(which has the same p53 mutation as WI-L2-NS) is alsosuggested by spontaneously and radiation-induced
enhancement of LOH at the TK locus (Honma et al.,1997a & b, Wiese et al., 2001). A novel integrated plasmidbased recombination assay performed to elucidate theeffect of p53 status on homologous and nonhomologous
recombination resulting in deletions could not account for
how those deletions arose (Gebow et al., 2000).
Acknowledgments
This research was supported in part by a Grant-in-Aid
from the Science and Technology Agency of Japan and inpart by the “Ground Research for Space Utilization” program promoted by NASDA and the Japan Space Forum.

20

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Wenz, F., E. I. Azzam and J. B. Little (1998) The response of proliferating cell nuclear antigen to ionizing radiation in human lymphoblastoid cell lines is dependent on p53, Radiat. Res., 149(1): 3240.
Wiese, C., S. S. Gauny, W. C. Liu, C. L. Cherbonnel-Lasserre and A.
Kronenberg (2001) Different mechanisms of radiation-induced
loss of heterozygosity in two human lymphoid cell lines from a single donor, Cancer Res., 61(3): 1129-1137.
Xia, F., S. A. Amundson, J. A. Nickoloff and H. L. Liber (1994)
Different capacities for recombination in closely related human
lymphoblastoid cell lines with different mutational responses to Xirradiation, Mol. Cell. Biol., 14(9): 5850-5857.
Xia, F., X. Wang, Y. H. Wang, N. M. Tsang, D. W. Yandell, K. T.
Kelsey and H. L. Liber (1995) Altered p53 status correlates withdifferences in sensitivity to radiation-induced mutation and apoptosis in two closely related human lymphoblast lines, Cancer Res.,
55(1): 12-15.
Zhen, W., C. M. Denault, K. Loviscek, S. Walter, L. Geng and A. T.
Vaughan (1995) The relative radiosensitivity of TK6 and WI-L2-NS
lymphoblastoid cells derived from a common source is primarily
determined by their p53 mutational status, Mutat. Res., 346(2): 8592.

21

Original Article

Environ. Mutagen Res., 24 ： 23 - 27 (2002)

A chromosomal aberration study of fibrillated PVA fiberin cultured mammalian cells
Takashi Hayashi ＊ and Fumiaki Arai
Ina Research Inc. 8047 Nishiminowa, Ina-shi, Nagano-ken 399 - 4501, Japan

Summar y
We studied the effect of a modified form of polyvinyl alcohol fiber (43.4, 86.8, and 173.5 µg/mL),an asbestos substitute, with and without a metabolic activation system on chromosomal aberrations
in a Chinese hamster cell line (CHL/IU cells). The fiber did not increase the frequency of polyploidy or of cells with structural aberrations under various exposure conditions.


Keywords: chromosomal aberrations, PVA fiber, Kuralon RM182, asbestos, asbestos substitute

Introduction

Materials and Methods

Asbestos was extensively used in the manufacture of
insulating materials, abrasives, flame retardants, andcement until the 1970s, when inorganic polymer asbestos
fibers were found to cause mesotheliomas following along latent period and were classified as a direct-acting
carcinogen (Borow et al., 1973; Stanton, 1977;
Chamberlain and Brown, 1978; McDonald and McDonald,
1978, 1980). As a result, worldwide regulatory authorities
prohibited its manufacture and use (Asbestos
International Association, 1983, 1984), and in the 1980s,
several types of polyvinyl alcohol (PVA) fibers were developed as substitute reinforcement materials (Mizobe, 1985;
Hikasa, 1986, 1994; Rosenbruch et al., 1992; Koshi, 1997).

The most popular of those, Kuralon RM182 andRMH182, cannot be subjected to routine in vitro tests
because the size of the fibers ─ approximately 14 µm in
width (diameter) and 4 to 6 mm in length ─ makes them
unsuitable for such tests. Following several unsuccessfulattempts, however, we were able to modify a special typeof PVA fiber, Kuralon RMU182, which is not of cement
reinforcing grade, into a testable form.
Here we describe a chromosomal aberration study offibrillated PVA fiber in cultured mammalian cells.

Test article
A sample of Kuralon was cut and disintegrated in adisk refiner to accomplish fibrillation and was placed in a
tube, sealed and irradiated with γ-rays (25 kGy) by
Kuraray Co., Ltd. The actual test article was provided tous as a white suspension in distilled water (1.735
mg/mL). The distribution of the dimensions of the fibril
lated Kuralon , referred to as “A-3”; Fig. 1, is shown in
Table 1. The sealed tubes were placed in an airtight container and stored at approximately 4 ℃. On the day oftreatment, the stock suspension was sonicated andapplied to each treatment level as it is while changing its
volume.

＊haya-ta@ina-research.co.jp
Received: August 21, 2000, accepted: February 20, 2002
© Environmental Mutagen Society of Japan

Positive control articles
Mitomycin C (MMC: CAS 50-07-7), 1000 µg/mg potency, or 99.5 ％ pure N-nitrosodimethylamine (DMN: CAS
62-75-9), both from Wako Pure Chemical Industries, Ltd.,was used as the positive control in the absence (continuous treatment) or presence (short treatment), respectively, of the metabolic activation system. The compounds
were dissolved in physiological saline J.P. (Otsuka
Pharmaceutical Factory, Inc.) and filtered through a 0.20

µm membrane (Corning , Iwaki Glass Company Ltd.).
Negative control article
Japanese Pharmacopeia water for injection (Otsuka
Pharmaceutical Factory, Inc.) was used as the negative
control and was stored at room temperature.
23

Table 1 Distribution of dimensions of fibrillated PVA fiber Kuralon
(A-3)
Distribution ofdimensions
Length(µm)

Width (diameter)(µm)

Aspect ratio(L/W)
Fig. 1 An electron micrograph of fibrillated PVA fiberStandard control article
The standard control article, zimbabwe chrysotile, waspurchased from Wako Pure Chemical Industries, Ltd. To
make a 50 µg/mL suspension, we added an appropriate
amount to water for injection and pulverized/stirred it in a
mixer (Bio-mixer , Nihonseiki Kaisha Ltd.) for 3 minutes.
The suspension was autoclaved and stored at approximately 4 ℃.
Cell line and culture conditions
We purchased CHL/IU cells from Dainippon
Pharmaceutical Co., Ltd. Frozen aliquots (approximately 196 ℃) of the preserved cell suspension were thawed andserially subcultured in Eagle’s minimum essential medium (Kohjin Bio Co., Ltd., 87 - 603) supplemented with
10 ％ (v/v) heat-inactivated calf bovine serum (Cansera
International Inc., Lot No. 2608312) every 2 - 5 days. The
cells were checked during this acclimation period andjudged to be normal if they had a satisfactory proliferation
rate, were free of mycoplasma, and had a modal chromosomal number of 25. The passage numbers of the cells
used for continuous treatment (24 hrs or 48 hrs) andshort treatment (6 hrs) were 17 and 19, respectively.
S9 mixWe prepared the S9 mix from a purchased S9 fraction(Oriental Yeast Co., Ltd., Lot No. 97112809) and CofactorC (Oriental Yeast Co., Ltd). The S9 fraction was derived
24

No. offibersFiber content
(％)

L＜1
1≦L＜3
3≦L＜5
5 ≦ L ＜ 1010 ≦ L ＜ 30
30 ≦ L ＜ 500
500 ≦ L

3
87
25
15
6
0
0

2.2
64.0
18.4
11.0
4.4
0.0
0.0

Total

136

100

W ＜ 0.1
0.1 ≦ W ＜ 0.2
0.2 ≦ W ＜ 0.3
0.3 ≦ W ＜ 0.4
0.4 ≦ W ＜ 0.5
0.5 ≦ W ＜ 1
1 ≦ W ＜ 30
30 ≦ W

1
66
51
13
4
1
0
0

0.7
48.5
37.5
9.6
2.9
0.7
0.0
0.0

Total

136

100

L/W ＜ 33 ≦ L/W ＜ 55 ≦ L/W ＜ 1010 ≦ L/W ＜ 2020 ≦ L/W ＜ 30
30 ≦ L/W ＜ 4040 ≦ L/W ＜ 50
50 ≦ L/W

0
9
44
50
21
6
2
4

0.0
6.6
32.4
36.8
15.4
4.4
1.5
2.9

Total

136

100

from the livers of 7-week-old male Sprague-Dawley rats
pre-treated with phenobarbital and 5,6 - benzoflavone. The
S9 mix was prepared just prior to use and preserved in an
ice water bath until completion of the tests. The components of 1 mL S9 mix were as follows: 0.3 mL S9 fraction,
0.1 mL 50 mmol/L MgCl2, 0.1 mL 330 mmol/L KCl, 0.1
mL 50 mmol/L G-6-P, 0.1 mL 40 mmol/L NADP, 0.1 mL
distilled water, and 0.2 mL 20 mmol/L HEPES buffer solution (pH 7.2).
Treatment
We based treatment levels on the results of a preliminary study in which the A-3 did not inhibit cell growth at
up to 173.5 µg/mL, the concentration of the stock suspension. We applied that concentration and 86.8 and 43.4µg/mL for both continuous and short treatments. We
used zimbabwe chrysotile at 50 µg/mL, which produced
chromosomal aberrations in a preliminary study.
4
CHL/IU cell suspension was seeded at 3 × 10 /5 mL
onto a plastic plate (diam. 60 mm) and incubated for 3
days. For continuous treatment, 0.125 to 0.5 mL of themedium was removed from each culture and thenreplaced with an equal volume of test or control article,

Table 2 Chromosomal aberration study of fibrillated PVA fiber in cultured CHL/IU cells ─ Continuous treatment ─

Compound

Dose

Trt-Reca)(µg/mL)(hr)

Number of cells with structural aberrationsb)

Polyploid cellsb)(Pccc) in polyploid cells)

Total
gap

ctb

d)

e)

f)

cte

csb

cse

g)

oth

h)

+ gap(％) - gap(％) No. of cells％

Water for injection
Fibrilated PVA Fiber
Fibrilated PVA Fiber
Fibrilated PVA Fiber
Mitomycin C
Untreated
Zimbabwe Chrysotile

24-0
43.4
86.8
173.5
0.2
50

0
24-0
24-0
24-0
24-0
24-0
24-0

0
0
0
0
6
1
1

0
0
0
0
67
0
4

0
0
0
0
100
0
3

0
0
0
0
0
0
1

0
0
0
0
2
0
0

0.0
0
0
0
0
0
0

0.0
0.0
0.0
0.0
58.5
0.5
4.0

3
0.0
0.0
0.0
58.5
0.0
3.5

1.5
0
1
2
3
0
34 (8)

0.0
0.5
1.0
1.5
0.0
17.0 (23.5)

Water for injection
Fibrilated PVA Fiber
Fibrilated PVA Fiber
Fibrilated PVA Fiber
Mitomycin C
Untreated
Zimbabwe Chrysotile

43.4
86.8
173.5
0.2
50

48- 0
48-0
48-0
48-0
48-0
48-0
48-0

0
1
0
0
14
3
3

0
0
0
0
126
0
7

0
0
0
0
176
0
6

0
0
0
0
1
0
4

0
0
0
0
3
0
1

0
0
0
0
0
0
0

0.0
0.5
0.0
0.0
92.5
1.5
8.0

0.0
0.0
0.0
0.0
91.0
0.0
7.0

1
1
1
2
4
1
95 (11)

0.5
0.5
0.5
1.0
2.0
0.5
47.5 (11.6)

a)Treatment time - Recovery time
Results are based on counts of 200 cellsc)Premature chromosome condensation (=PVZ: pulverization)d)
Chromatid breaks e) Chromatid exchanges f) Chromosome breaksb)and the plates were incubated an additional 24 or 48
hours. For short treatment, 2.075 to 2.8 mL of the medium
was removed and replaced with 0.075 to 0.3 mL test or
control article plus 0.5 mL S9 mix (final S9 concentration,
5 ％ v/v), and the plates were then incubated 6 hours
more. When treatment was complete in short treatment,
the treatment medium was removed and replaced withfresh medium and the cultures were incubated for an
additional 18 hours. For both treatments, we added a colcemid (final colcemid concentration, 0.2 µg/mL) to each
culture at approximately 2 hours before cell harvesting.
After that, the cells were trypsinized and incubated in
0.075 mol/L KCl hypotonic solution for 15 min at 37 ℃
and fixed several times with freshly prepared Carnoy fixative (methanol : acetic acid = 3 : 1). Two drops of the cellsuspension were spread onto degreased glass slides andstained with Giemsa solution. We examined 100
metaphase cells per plate by a blind method and scoredstructural chromosome aberrations and polyploidy(Ishidate, 1987; Japan Environmental Mutagen Society,1988). We further classified structural aberrations into thefollowing categories: gaps, chromatid breaks (ctb), chromatid exchanges (cte), chromosome breaks (csb), chromosome exchanges (cse), and others. A gap was defined
as a short achromatic region (length equal to or slightly
longer than the width of a chromatid) existing along the
major axis of a chromatid or chromosome. If the achromatic region was considerably larger, it was classified as abreak.
The results were judged according to the following criteria: negative (─) if less than 5 ％ of cells were aberrant,g)

Chromosome exchangesh)Othersequivocal (±) if 5.0-9.0 ％ cells were aberrant, and positive (+) if 10 ％ or more cells were aberrant and the incidence of cells with chromosomal aberrations, excluding
cells with gap only, increased in a dose-dependent manner.

Results
For 24- and 48-hour continuous treatment, Table 2,
results were negative in the PVA fiber-treated groups. In
the standard control group, results were negative forstructural aberrations but polyploidy was frequently andpositively observed and the incidence increased overtime. Among the polyploid cells, premature chromosome
condensation (pulverization) was evident, but the incidence did not change over time.
The same results were obtained for short treatments inboth the presence and absence of S-9 (Table 3).

Discussion
The harmful action of asbestos is attributed to its sizeand shape, which enables it to invade cells (Pott, 1980;
Stanton, 1977; Stanton et al., 1981). More than 90 ％ ofasbestos used until recently was chrysotile, which is lessthan 1 µm in width (diameter) and more than 5 mm inlength, and most chrysotile was used as a reinforcing
ingredient in cement material. Following the prohibitionof the manufacture and use of asbestos (Asbestos
International Association, 1983, 1984), substitutes weredeveloped (Mizobe, 1985; Hikasa, 1986, 1994). The mostpopular substitutes for cement reinforcing material are
the PVA fibers Kuralon RM182 and RMH182, whose per25

Table 3 Chromosomal aberration study of fibrillated PVA fiber in cultured CHL/IU cells ─ Short treatment ─
Dose Trt-Reca)
Compound（µg/mL） (hr)Number of cells with structural aberrationb)
S9 mixPolyploid cellsb)(Pccc) in polyploid cells)

Total
gap

ctb

d)

e)

f)

cte

csb

cse

g)

oth

h)

+ gap(％) - gap(％) No. of cells％

Water for injection
Fibrilated PVA Fiber
43.4
Fibrilated PVA Fiber
86.8
Fibrilated PVA Fiber 173.5
N-Nitrosodimethylamine 1000
Untreated
Zimbabwe Chrysotile
50

6-18
6-18
6-18
6-18
6-18
6-18
6-18

+
+
+
+
+
+
+

0
1
0
2
5
1
4

0
0
0
0
28
1
6

0
0
0
0
31
0
4

0
0
0
0
0
0
1

0
0
0
0
0
0
0

0
0
0
0
0
0
0

0.0
0.5
0.0
1.0
22.0
1.0
4.5

0.0
0.0
0.0
0.0
20.5
0.5
4.0

2
0
1
1
3
2
45 (9)

1.0
0.0
0.5
0.5
1.5
1.0
22.5 (20.0)

Water for injection
Fibrilated PVA Fiber
43.4
Fibrilated PVA Fiber
86.8
Fibrilated PVA Fiber 173.5
N-Nitrosodimethylamine 1000
Untreated
Zimbabwe Chrysotile
50

6-18
6-18
6-18
6-18
6-18
6-18
6-18

-

0
0
0
2
0
0
2

0
0
0
1
0
0
2

0
1
0
0
1
0
1

0
0
1
0
0
0
0

0
0
0
0
0
0
0

0
0
0
0
0
0
0

0.0
0.5
0.5
1.0
0.5
0.0
2.0

0.0
0.5
0.5
0.5
0.5
0.0
1.0

0
3
2
2
1
4
60 (7)

0.0
1.5
1.0
1.0
0.5
2.0
30.0 (11.7)

a)Treatment time - Recovery time
Results are based on counts of 200 cellsc)Premature chromosome condensation (=PVZ: pulverization)d)
Chromatid breaks e) Chromatid exchanges f) Chromosome breaksb)formance is comparable to that of chrysotile (Mizobe,
1985; Hikasa, 1986, 1994). Although those materials are
not fibrillated under ordinary conditions (Saito et al.,1999), a special type of PVA fiber, Kuralon RMU182,
which is not of cement reinforcing grade, can be fibrillated because this fiber was produced with PVA resin of
higher degree of polymerization and was highly drawn on
the fiber production stage more than the case of conven
tional fibers. We therefore fibrillated a sample of Kuralon
RMU182 and investigated its clastogenicity.
Although asbestos and the majority of its substitutes,
such as a glass fiber, mineral wool, and ceramic fiber, generally induce cytotoxicity and/or clastogenicity in in vitro
assay systems, the fibrillated PVA fiber (A-3) tested in thisstudy induced neither. Zimbabwe chrysotile that is known
its in vitro clastogenicities and in vivo toxicities (Daniel,
1983; Koshi et al., 1986, 1991; Smith and Wright, 1996) did
induce polyploidy and structural chromosome aberrations
under the same test conditions. We believe that the geo
metric characteristics of the PVA fiber Kuralon RM
series may be responsible this result and that the materials may be safer than asbestos and other asbestos substitutes even if it is fibrillated.
References
Asbestos International Association (1983) Asbestos International
Association Reports: Asbestos banning situation in the world - asof August 15, 1983, Japan Asbestos Association, Sekimen, No. 455,
6-10. (in Japanese)
Asbestos International Association (1984) Asbestos International
Association Reports: Asbestos banning situation in the world - asof July 1, 1984, Japan Asbestos Association, Asbestos, No. 465,6-8.
26g)Chromosome exchangesh)Others(in Japanese)
Asbestos International Association (1984) Asbestos International
Association Reports; Asbestos banning situation in the world (continued) - as of July 1, 1984, Japan Asbestos Association, Sekimen,
No. 466, 6-8. (in Japanese)
Borow, M., A. Conston, L. Livornese and N. Schalet (1973)
Mesothelioma following exposure to Sekimen, A review of 72
cases, Chest, 64, 641-646.
Chamberlain, M. and R.C. Brown (1978) The cytotoxic effects ofasbestos and other mineral dust in tissue culture cell lines, Brit. J.
Exptl. Pathol., 59, 183-189.
Daniel, F. B. (1983) In vitro assessment of asbestos genotoxicity,
Environ. Health Perspect, 53, 163-167.
Hikasa, J. (1986) Substitute fibre for asbestos, Sen-i Kikai Gakkaishi(Sen-i Kogaku), 39 (4), 22-28. (in Japanese)
Hikasa, J. (1994) Synthetic fibers - Vinylon, Sen-i Gakkaishi (Sen-i to
Kogyo), 50 (6), 136-138. (in Japanese)
Ishidate, M. Jr. (Ed) (1987) Data book of chromosomal aberration
test in vitro (revised edition), L.I.C. Inc., Tokyo. (in Japanese)
Japan Environmental Mutagen Society, Section on MMS. (Ed)(1988) Atlas of chemicals-induced chromosomal aberrations,
Asakura Shoten, Tokyo. (in Japanese)
Koshi, K., N. Kohyama, T. Myojo and K. Fukuda (1986) Biologicaleffects of asbestos and its substitutes: Cell toxicity, hemolytic
action and clastogenic activity in vitro (Fiscal Year 1985 - 1987),
Kankyo Hozen Kenkyu, 94-1 - 94-19. (in Japanese)
Koshi, K. (1991) Biological effects of man-made mineral fibers by invitro tests, Aerosol Res., 6 (1), 25-33. (in Japanese)
Koshi, K. (1997) Biological Effects of asbestos substitute fibers,
Roudou no Kagaku, 52 (2), 26-31. (in Japanese)
McDonald, A.D. and J.C. McDonald (1978) Mesothelioma after crocidolite exposure during gas mask manufacture, Environ. Res., 17,340- 346.
McDonald, A.D. and J.C. McDonald (1980) Malignant mesothelioma
in North America, Cancer, 46, 1650-1656.
Mizobe, A. (1985) Fibers for reinforcing cement and concrete, Sen-i

Gakkaishi (Sen-i to Kogyo), 41 (6), 14-19. (in Japanese)Pott, F. (Ed) (1980) Animal experiments on biological effects of mineral fibers. In: Biological Effects of Mineral Fibers, Wagner JC.
IARC Sci. Publ. No. 30, Lyon, IARC. pp. 261- 272.
Rosenbruch, M., K.H. Friedrichs and H.-W. Schlipköter (1992) Zur
gesundheitlichen Bedeutung der zur Herstellung vonFaserzement verwendeten Asbestersatzfasern, Zbl Arbeitsmed,
42, 355-362.
Saito, T., I. Sone, J. Hikasa, J. Morisawa, Y. Hayashi and K. Miyazaki(1999) Fibrillation of synthetic fibers as a reinforcement, The
Report on Works of Kochi Prefectural Paper Technology Center,

4, 76-88. (in Japanese)
Smith, A.H. and C.C. Wright (1996) Chrysotile asbestos is the maincause of pleural mesothelioma, Amer. J. of Industrial Medicine, 30,
252- 266.
Stanton, M.F. (1977) Carcinogenicity of fibrous glass: Pleural
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27

Original Article

Environ. Mutagen Res., 24 ： 29 - 35 (2002)

Antioxidative effects of fluvastatin and its metabolites incultured human endothelial cells using single cell gel
electrophoresis
Tomonori Aoki1, Atsumune Imaeda1＊ and Hisamitsu Nagase2
1

Safety Research Laboratory, Tanabe Seiyaku Co., Ltd.,
3-16-89 Kashima, Yodogawa-ku, Osaka 532 -8505, Japan2
Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502-8585, Japan

Summar y
In the present study, we investigated the antioxidative effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, and its metabolites (M2, M3, and M4) on oxidative DNA
damage in cultured human umbilical vein endothelial cells (HUVEC), as well as the effects of otherinhibitors of this enzyme, pravastatin and simvastatin. Single cell gel electrophoresis was used toevaluate the protective effects of fluvastatin on reactive oxygen species (ROS)-induced DNA damage in HUVEC exposed to either t-butylhydroperoxide or hydrogen peroxide. Fluvastatin and its
metabolites showed protective effects on DNA damage as potent as the reference antioxidants,
ascorbic acid, trolox, and probucol. It was suggested that fluvastatin has the potential to protect
cells against ROS -induced DNA damage, although pravastatin and simvastatin did not exhibit clear
protective effects. Fluvastatin may contribute to reduce ROS in humans and also reduce ROS related diseases.
Keywords: fluvastatin,
trophoresis assay

HUVEC,

oxidative DNA damage,

Introduction
Oxidative stress may be an important factor in thedevelopment of various diseases. In atherosclerosis, forexample, an increased plasma concentration of low-density lipoprotein (LDL) has been considered to be a majorrisk factor. The impaired removal of plasma LDL in suchpatients results in abnormal persistence of LDL in theplasma, allowing more time for oxidative modification to
occur (Hussein et al., 1997). Oxidative modification of
LDL has been reported to be one of the important steps inthe progression of atherogenesis (Goldstein et al., 1979;
Leonhardt et al., 1997). In addition, it has been indicatedthat oxygen-derived free radicals may play a major role asinitiators and promoters of DNA damage and in mutations
that may be related to cancer, diabetes, heart disease, andother age-related diseases (Ames, 1983; Wiseman andHalliwell, 1996; Anderson et al., 1997; Araki, 1997;＊

a-ima@tanabe.co.jp
Received: February 20, 2002, accepted: March 30, 2002
© Environmental Mutagen Society of Japan

antioxidant,

single cell gel elec-

Leinonen et al., 1998).
Fluvastatin (FV) is a highly potent inhibitor of 3hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which is used as a hypolipidemic drug (Plosker andWagstaff, 1996). FV and its metabolites (Fig. 1) (Dain etal., 1993) have been proved to have antioxidative activities(Suzumura et al., 1999a; Suzumura et al., 1999b). FV wasconfirmed to scavenge hydroxyl radical (HO•) generated
by the Fenton’s reaction using an electron spin resonance(ESR) spectrometer (Yamamoto et al., 1998). The antioxidative activities are related to their unique chemical
structures (Nakashima et al., 1999; Nakamura et al.,2000).
In a previous study, FV showed protective effects
against the oxidation of LDL in vitro (Hussein et al.,1997), and a reduction in the level of serum thiobarbituric
acid-reactive substances in cholesterol-fed rabbits (Mitani
et al., 1996; Hussein et al., 1997). Thus FV may help toreduce the risk of atherosclerosis by protecting LDL fromoxidative modification as well as reducing the plasma LDLlevels (Suzumura et al., 1999c).
In the present study, we investigated the protective
29

Fig. 1 Structures of fluvastatin and its major metabolites (M2, M3, and M4), pravastatin, and simvastatin

Table 1

Test materials used in this study

Test material
Fluvastatin
Fluvastatin metabolites
Pravastatin
Simvastatin
Ascorbic acid
Trolox
ProbucolHydroxyl radical
Hydrogen peroxide
tert-Butylhydroperoxide

Abbreviation or formula
FV
M2, M3, M4
PV
SV
AsA
TX
PB
HO•
H2O2
t-BuOOH

effects of FV and its human metabolites (M2, M3, and
M4) against oxidative DNA damage using normal humanumbilical vein endothelial cells (HUVEC) in vitro. We also
compared the protective effects of FV on oxidative DNA
damage with those of two other HMG-CoA reductase
inhibitors, pravastatin (PV) and simvastatin (SV) (Fig. 1).

Materials and Methods
Reagents
Fluvastatin (FV) was donated from the Department ofPharmacology, Novartis Pharma (Ibaraki, Japan). FV
metabolites (M2, M3, and M4) and Probucol (PB) weresynthesized by the Discovery Research Laboratory,
Tanabe Seiyaku Co., Ltd. (Saitama, Japan) (Fig. 1). tButylhydroperoxide (t-BuOOH), dimethyl sulfoxide(DMSO), and hydrogen peroxide (H2O2) were purchasedfrom Katayama Chemical Industries (Osaka, Japan).
Trolox (TX) and ascorbic acid (AsA) were purchasedfrom Wako Pure Chemical Industries (Osaka, Japan)(Table 1).
30

Cell culture and treatments
Frozen normal human umbilical vein endothelial cells(HUVEC), purchased from Kurabo Industries Ltd., wereused as the indicator cell line. Prior to exposure, thefrozen cells were thawed and maintained by subculture
for 5 - 7 days in a CO 2 incubator at 37 ℃ in a 5 ％ CO2
atmosphere. HuMedia-EG2 purchased from Kurabo
Industries Ltd. was used as the culture medium.
5
The cells (7 × 10 ) were seeded on a tissue culture dish
(60 mm in diameter containing 5 mL of medium) andincubated for 4 days. After replacement with 2.7 mL offresh medium, 0.15 mL of test solution and 0.15 mL of
ROS generation solution were added to the culture for 1 h.
The 0.15mL aliquot of culture medium and vehicle wereadded to the untreated and solvent control culturesinstead of the test solution, respectively. After exposure,the cells were washed twice with saline and analyzed
immediately for DNA damage.
Single cell gel electrophoresis assay
DNA damage in individual cells was detected using thesingle cell gel electrophoresis assay (SCG assay) as DNA
single strand breakage. The slides were prepared by thethree-layer procedure. After exposure, the cells were4
resuspended at the concentration of 2.0 × 10 cells / 100µL in 1.0 ％ low-melting agarose in saline and immediately
pipetted onto a slide precoated with a layer of regularagarose, and then a top layer of regular agarose wasadded. After the agarose had solidified, the slides wereplaced in ice-cold lysing solution (2.5 M NaCl, 100 mM
EDTA, 10 mM Tris, 1 ％ sarkosyl, 5 ％ dimethyl sulfoxide,and 1 ％ Triton X100 [pH 10.0] ) for 60 min. Prior to elec-

Fig. 2 Tail moment, tail length, and tail DNA of t-BuOOH-treated
HUVEC using SCG assay.
The cells were treated with 0.0313-0.5 mM t-BuOOH for 60
min and immediately assayed for DNA damage with SCG
assay. t-BuOOH, t-butylhydroperoxide. The data represent
mean ± S.E. ＊＊ p<0.01 as compared with control (0 µM tBuOOH).

trophoresis, the slides were incubated in an alkaline
buffer (300 mM NaOH and 1 mM EDTA; pH > 13) for 10
min to allow the DNA to unwind. After alkali unwinding,the single-stranded DNA in the gel was electrophoresedat 300 mA and 1.0 V/cm for 15 min in the same alkaline
buffer maintained below 4 ℃. Following electrophoresis,
the alkali in the gels was neutralized with Tris-HCl buffer(pH 7.4) for 10 min and rinsed with saline. The DNA wasvisualized by staining with ethidium bromide andobserved using a Nikon Labophot fluorescence microscope. A specialised SCG analysis system (Komet 3.0,
Kinetic Imaging Ltd., Liverpool, UK) was used to analyze
the data. Tail length (µm), tail DNA (％), and tail moment
were recorded for 50 cells per slide with 2 slides per variable investigated. The parameter used to describe theextent of DNA damage was the tail moment, which isdefined as the product of the tail length and the fraction of
DNA in the tail in this software (tail length × tail DNA /100). The measurements from 100 randomly selected
cells per treatment condition were averaged.
Effects on DNA Damage Induced by ROS in vitro
The protective effects against oxidative DNA damage
induced by ROS were measured by the SCG assay. tBuOOH and H2O2 were each used as the ROS generating
systems. Well-known antioxidants AsA and TX were used.
PB has been reported to have a highly lipophilic property
and phenolic moieties, which show antioxidant properties
partly due to their free radical scavenging effect (Valoti etal., 1989; Hiramatsu et al., 1994). Each reagent was dissolved in distilled water, except for TX and PB. TX wasdissolved in 1M NaHCO3 and PB in DMSO. FV was diluted to adjust the final concentrations to 0.4, 0.8, 4, and 8

Fig. 3 The effect of FV on t-BuOOH-induced DNA damage.
The cells were treated with 0.0625-1 mM t-BuOOH
for 60 min in the absence or presence of 0.4-8 µM
FV, and immediately assayed for DNA damage
with SCG assay. Results are expressed as the tailmoment, and represent the mean of at least two
separate experiments, each performed in duplicate. t-BuOOH, t-butylhydroperoxide; FV, fluvastatin. The data represent mean ± S.E. ＊ p<0.05,＊＊
p<0.01 as compared with control (t-BuOOH
only).

µM, and the metabolites (M2, M3, and M4), AsA, TX, andPB were 0.8 and 8 µM in the reaction mixture. t-BuOOH
was diluted to adjust the final concentrations to 0.0313,
0.0625, 0.125, 0.25, 0.5, and 1 mM in the reaction mixtureand H2O2 was diluted to adjust the final concentrations to
0.5, 1, 2, 3, and 4 µM.
Duplicate plates were prepared and observed for eachcondition. The protocol was performed at least twice forreliability, and satisfactory results were obtained between
repeated tests.
Statistical analysis
Statistical significance among the groups was analyzed
by the Dunnett’s multiple comparison test. The significance of differences was evaluated by the Dunnett’s parametric test when variances were equal (p ≧ 0.05), by the
Bartlett’s test for homogeneity of variance, and theDunnett’s non-parametric test when variances were
unequal (p < 0.05). Results were considered to be significant at a probability level of p < 0.05 or 0.01.

Results
Effects of FV on DNA Damage Induced by t BuOOH in vitro
We assessed the sensitivity of the conventional SCG
assay to detect the DNA damage of HUVEC. The cells
were exposed to t-BuOOH at 37 ℃ at various concentra31

(A) Control(B) 1 mM t-BuOOH(C) 1 mM t-BuOOH + 0.8 µM FV(D) 1 mM t-BuOOH + 8 µM FVFig. 4

Fluorescence photographs of HUVEC after exposure to 1 mM t-BuOOH in the SCG assay.
(A) (Control), nuclei of undamaged negative control cells, 200× magnification. (B) (1 mM t-BuOOH), severely
damaged cell. The fragmented DNA has migrated out of the nucleus (comet head) and streamed out in a longtail towards the anode. Highly fragmented DNA has migrated away from the nuclear cage, leaving a small
comet head. (C) (1 mM t-BuOOH + 0.8 µM FV) and (D) (1 mM t-BuOOH + 8 µM FV), FV inhibited the tBuOOH-induced DNA damage in a dose-dependent manner. t-BuOOH, t-butylhydroperoxide; FV, fluvastatin.

tions. Fig. 2 shows a representative SCG assay of DNA
damage as a function of t-BuOOH concentration exposedfor 60 min. Significant increases in tail length, tail DNA,and tail moment were observed in HUVEC exposed to tBuOOH showing a dose-dependent manner. The effect of
FV on DNA damage produced by t-BuOOH was investigated. In these experiments, the cells were exposed tovarious concentrations of t-BuOOH for 60 min, in theabsence or presence of FV, and the level of DNA damage
was measured. The addition of FV significantly reduced
the levels of tail moment induced by t-BuOOH in a dosedependent manner (Fig. 3). Fig. 4 shows representative
photomicrographs of the ethidium bromide-stained nuclei
indicating that 0.8 and 8 µM FV virtually inhibited theextensive DNA cleavage caused by 1 mM t-BuOOH in adose-dependent manner.
As shown in Fig. 5, the addition of 8 µM FV significantly reduced t-BuOOH-induced DNA damage; the results
were comparable to those with AsA, TX, and PB. FV
reduced the DNA damage to an extent similar to TX and
PB. In AsA, the DNA damage was reduced significantly
32

even at 0.8 µM. M2, M3, and M4 also inhibited t-BuOOHinduced DNA strand scission significantly even at 0.8 µM,
and these effects showed dose-dependency. Meanwhile
no protective effects of PV and SV were shown in this system.
Effects of FV on DNA Damage Induced by H2O2 invitro
The cells were exposed to H2O2 at 37 ℃ in various concentrations. Fig. 6 shows a representative SCG assay of
DNA damage as a function of H2O2 concentration exposedfor 60 min. Significant increases in tail length, tail DNA,and tail moment were observed in HUVEC exposed to
H2O2 in a dose-dependent manner.
The effect of FV on DNA damage produced by H2O2
was investigated. The cells were exposed to various concentrations of H2O2 for 60 min, with or without of FV, andthe level of DNA damage was measured immediately after
the exposure using the SCG assay. The concentrations of
0.8, 4, and 8 µM FV significantly inhibited the H 2O 2induced tail moment (Fig. 7).

Fig. 5 The effect of FV, its metabolites (M2, M3, and M4), andantioxidants on 1 mM t-BuOOH-induced DNA damage.
The cells were treated with 1 mM t-BuOOH for 60 min in
the absence or presence of 0.8 - 8 µM FV, its metabolites(M2, M3, and M4), and various antioxidants, and immediately assayed for DNA damage with SCG assay. Results are
expressed as the tail moment, and represent the mean of atleast two separate experiments. M2, M3, and M4 designate principal FV metabolites in humans. t-BuOOH, tbutylhydroperoxide; FV, fluvastatin; AsA, ascorbic acid;
TX, trolox; PB, probucol; PV, pravastatin; SV, simvastatin.
The data represent mean ± S.E. ＊ p<0.05, ＊＊ p<0.01 ascompared with control (t-BuOOH only).

Fig. 6 Tail moment, tail length, and tail DNA of H 2O 2-treated
HUVEC using SCG assay.
The cells were treated with 0.5 - 4 µM H2O2 for 60 min and
immediately assayed for DNA damage with SCG assay.
The tail moment is the product of the length of the tail andthe amount of DNA present within the tail. The data represent mean ± S.E. ＊＊ p<0.01 as compared with control (0µM H2O2).

Fig. 7 The effect of FV on H2O2-induced DNA damage.
The cells were treated with 0.5 - 4 µM H2O2 for 60
min in the absence or presence of 0.4 - 8 µM FV,
and immediately assayed for DNA damage with
SCG assay. Results are expressed as the tailmoment, and represents the mean of at least two
separate experiments, each performed in duplicate. FV, fluvastatin. The data represent mean ±S.E. ＊＊ p<0.01 as compared with control (H 2O 2only).

Fig. 8 The effect of FV, its metabolites (M2, M3, and
M4) and antioxidants on 3 µM H2O2-induced DNA
damage.
The cells were treated with 3 µM H2O2 for 60 min
in the absence or presence of 0.8 - 8 µM FV, its
metabolites (M2, M3, and M4), and various antioxidants, and immediately assayed for DNA damage
with SCG assay. Results are expressed as the tailmoment, and represent the mean of at least two
separate experiments. M2, M3, and M4 designate
principal FV metabolites in humans. FV, fluvastatin; AsA, ascorbic acid; TX, trolox; PB, probucol;
PV, pravastatin; SV, simvastatin. The data repre＊＊p<0.01 as compared with consent mean ± S.E.
trol (H2O2 only).

33

As shown in Fig. 8, FV effectively reduced DNA damage caused by H2O2, and this effect was dose-dependent.
The addition of 0.8 and 8 µM FV significantly reduced the
DNA damage. AsA, TX, and PB reduced H2O2-induced
DNA damage to a similar extent. M2, M3, and M4,
metabolites of FV, also reduced the DNA damage to asimilar extent, in a dose-dependent manner. The protective effects of FV against DNA damage were comparedwith those of PV or SV. FV exhibited protective effects
against H2O2 -induced breakage, but PV and SV did not.

Discussion
ROS may play a major role as endogenous initiators andpromoters of DNA damage and in mutations that contribute to cancer, diabetes, heart disease, and other agerelated diseases (Ames, 1989; Wiseman and Halliwell,1996; Anderson et al., 1997). On the other hand, recent
studies have suggested that antioxidant treatment
reduced the risk of various diseases. Antioxidants may
represent an important defense against cellular injury
mediated by ROS, and many antioxidants (Smith, 1998;
Blask et al., 1999; Ishino et al., 1999; Zhao et al., 1999;
Owen et al., 2000).
We investigated the protective effects of FV and its
human metabolites against oxidative DNA damage using
HUVEC in vitro, and compared the effects with two other
HMG-CoA reductase inhibitors. The protective effects onoxidative DNA damage induced by H2O2 and t-BuOOH
were examined using the SCG assay. The alkaline (pH >13) version of the SCG assay is known to be capable ofdetecting DNA single-strand breaks, alkali-labile sites,
DNA-DNA/DNA-protein cross-linking, and DNA singlestrand breaks associated with incomplete excision repair
sites (Singh et al., 1988; McKelvey-Martin et al., 1993;
Fairbairn et al., 1995; Hartmann and Spier, 1995; Tice etal., 2000).
FV showed protective effects against oxidative DNA
damage of HUVEC induced by both H2O2 and t-BuOOH.
FV and its metabolites caused a reduction in DNA damage induced by 1 mM t-BuOOH, and the protective potency was equal to that of AsA, TX, and PB, which are wellknown antioxidants. FV and its metabolites also reduced 3µM H2O2-induced DNA damage to the same extent as
AsA, TX, and PB. In addition, only FV consistently
showed the protective effect on the oxidative DNA damage in comparison with two other HMG-CoA reductase
inhibitors, PV and SV. The previous study indicated thatthe chemical structure, including the fluorophenyl indole
moiety, differs from that of PV and SV (Suzumura et al.,1999b), and the active site for the antioxidative ability of
FV is the allylic carbon conjugated with the indole ring(Nakamura et al., 2000).
FV have been confirmed to chemically scavengehydroxyl radical (HO•) using an ESR spectrometer34(Yamamoto et al., 1998). FV exhibited protective effects
against the oxidation of LDL (Mitani et al., 1996; Hussein
et al., 1997; Suzumura et al., 1999c). It has been suggested
that FV may help to reduce the risk of atherosclerosis byprotecting LDL from oxidative modification (Suzumura etal., 1999c). We have reported on the protective effects of
FV and its metabolites on oxidative DNA damage from
ROS in vitro. The protective effects have been proved interms of the DNA breakage rate using a single-stranded
phage DNA system, and in the Ames test with Salmonella
typhimurium TA102 and TA104 test strains (Imaeda et al.,
2000; Imaeda et al., 2001). The antioxidative activities indicated in previous studies are consistent with the protective effects against oxidative DNA damage of HUVEC.
These results suggested that FV and its metabolites protect against oxidative DNA damage. Metabolites provided
even stronger protection against DNA damage of HUVEC
than FV. The metabolites M2 and M3 having a phenolichydroxyl group on the indole moiety showed more potent
effects than FV itself, the fluorophenyl indole moiety wassuggested to be important for the manifestation of thehydroxyl radical scavenging activity, and the phenolichydroxyl group was considered to enhance this activity(Suzumura et al., 1999b).
In conclusion, FV and, to an even greater effect, itsmajor human metabolites, may reduce the risk of various
diseases associated with oxidative stress, such as
atherosclerosis, cancer, diabetes, and other age-relateddiseases, by inhibiting the oxidation of LDL, the oxidative
modification in human vascular endothelial cells, andother risk factors.
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35

Original Article

Environ. Mutagen Res., 24 : 37 - 41 (2002)

Failure of stevioside to induce micronucleus formation in therodent bone marrow cells
Temcharoen, P.1＊, S. Klongpanichpak2, M. Suwannatrai2,3, S. Apibal4 andC. Toskulkao2
1

2

3

Departments of Pathobiology, Physiology and Graduate Program in Toxicology, Faculty of4
Science, Mahidol University, Department of Pathology, Faculty of Medicine, Ramathibodi Hospital,
Mahidol University, Rama Ⅵ Road, Bangkok 10400, Thailand

Summar y
Stevioside, a major constituent of Stevia rebaudiana Bertoni which is commonly used as a noncaloric sugar substitute in many countries, was evaluated for its chromosome damaging potential by
the bone marrow micronucleus test in both sexes of hamsters, rats and mice. Experimental animals
received an oral administration of 10 g stevioside/kg body weight (BW). Animals were killed 24, 30,
48 and 72 hours after the treatment. Bone marrow cells were collected and 2000 polychromatic erythrocytes (PCEs) per animal were analyzed for the presence of micronuclei. Four hundred erythrocytes were also scored for the PCEs: normochromatic erythrocytes (NCEs) ratio. Stevioside at thedose of 10 g/kg BW had no effect on the frequencies of micronucleus formation in the bone marrow
erythrocytes of either sex of hamsters, rats and mice at any time point when compared to the negative control. Moreover, there were no apparent changes in the PCE:NCE ratio in either sexes of thetested species with an exception of male hamsters which exhibited significantly lower PCE:NCE
ratio at 48 h after the treatment. These results suggest that stevioside at the given dosage had nochromosome damaging effect on the bone marrow cells of the tested rodent species. However the
genotoxic risk of stevioside to humans is an issue that needs further investigation.
Keywords: stevioside, micronucleus test, rodent bone marrow cell

Introduction
Stevioside, a diterpene glycoside, is a major sweet component isolated from the leaves of the herbal plant Stevia
rebaudiana Bertoni (Crammer and Ikan, 1986). It is one of
the most important natural sweeteners and has been widely used to treat hypoglycemia in Paraguay for a number ofyears (Soejarto et al., 1983). Because stevioside is a white
crystalline, odorless substance, approximately 300 times
sweeter than sucrose (Kakayama and Isima, 1976), it ispresently used as a non-nutritive sweetening agent in various kinds of food and food products in countries such as
Japan, Brazil and People’s Republic of China (Sasaki,
1983; Kinghorn and Soejarto, 1991). Stevioside has been＊

scptm@mahidol.ac.th
Received: February 18, 2002, accepted: April 12, 2002
© Environmental Mutagen Society of Japansubjected to various assessments for its toxic effect andno serious toxic effects have been reported (Yamada etal., 1985; Yodyingyuad and Bunyawong, 1991; Xili et al.,1992). For its genotoxic effect, stevioside and crude
extracts of Stevia rebaudiana have been evaluated by several test systems including bacterial reversion test
(Okumura et al., 1978; Matsui et al., 1996), Ames test
(Suttajit et al., 1993; Klongpanichpak et al., 1997), Recassay (Okumura et al., 1978; Matsui et al., 1996), forward
mutation assay in bacteria (Pezzuto et al., 1985; Matsui etal., 1996; Temcharoen et al., 1998), chromosomal aberration test (Ishidate et al., 1984; Suttajit et al., 1993; Matsui
et al., 1996), host mediated assay (Tama Biochemical Co.
Ltd., 1981) and dominant lethal test (Tama Biochemical
Co. Ltd., 1981). In all of the tested systems steviosideshowed negative results, while crude extracts (50% purityof stevioside) from the Stevia leaf showed a slight positive37

result only in the in vitro chromosomal aberration test
(Ishidate et al., 1984; Matsui et al., 1996). However, all ofthese tested systems were performed in vitro.
Therefore, there was a need for the genotoxicity of stevioside to be further assessed in animal models. In thepresent study, the micronucleus test was performed inboth sexes of hamsters, rats and mice. The results
showed that stevioside did not induce micronuclei ineither sex of all tested animal species at various time
points of treatment.

Materials and Methods
Adult male and female Syrian golden hamsters (80 -100g) were supplied by the Animal Production Center,
Faculty of Science, Mahidol University, Bangkok,
Thailand. Adult male and female Wistar rats (100 -120 g)
as well as Swiss albino mice (30- 35 g) were obtained fromthe National Animal Center, Mahidol University, Salaya,
Thailand. They were housed in a controlled condition at aroom temperature of 25 ± 2˚C, relative humidity of 60%,
with an automatic light cycle period of 12 hours light/12
hours dark. The animals were kept in stainless steel cageswith wire mesh cover and fed with CP rat diet (Pokapan
Animal Food Co. Ltd.) and water ad libitum. Prior to eachexperiment, the animals were fasted overnight but
allowed free access to water. Stevioside (approx. 96% purity) was extracted and purified from dried Stevia rebaudiana leaves as previously described by Adduci et al.
(1987).
Groups of 20 male and female animals of each species
were administered by gavage with 10 g of stevioside/kg
BW (dissolved in distilled water to a concentration of 250
mg/ml). Positive control animals received an intraperitoneal administration of 80 mg cyclophosphamide/kg
BW, while the negative control were administered by gavage with distilled water. Five animals of each sex were
killed by cervical dislocation at 24, 30, 48 and 72 h after
the treatment. Femoral marrow cells were flushed out
with fetal bovine serum, smeared on clean glass slides,
and fixed with methanol for 5 minutes before being air
dried and double stained with May-Grunwald and Giemsa(Schmid, 1975). The slides were labeled and examined
without any reference to the treatment. The frequenciesof micronucleated polychromatic erythrocytes (MNPCEs)
in 2000 PCEs per animal and the ratio of PCE to NCE in
400 erythrocytes were determined under light microscope.
The data for micronucleus induction were analyzed statistically by the Kastenbaum-Bowman method(Kastenbaum and Bowman, 1970). Data for PCE/NCE
ratio were analyzed statistically by using Student’s t test.
38

Results
The effects of stevioside on micronucleus formation inboth male and female hamsters, rats and mice are shownin Tables 1-3, respectively. Stevioside at the dose of 10
g/kg BW did not significantly alter the frequencies of
MNPCEs at any time point. Furthermore, there were noapparent changes in the ratio of PCE to NCE in either sexof all those species examined except the PCE:NCE ratio
in the male hamsters at 48 h after the treatment, which
was significantly lower than that of the negative control
group. In the positive control animals, cyclophosphamide(80 mg/kg BW, i.p.), induced a significant increase in thefrequency of MNPCEs in every treated animal species.
These results suggested that stevioside did not induce
micronucleus formation in bone marrow polychromaticerythrocytes of hamsters, rats and mice of either sex.

Discussion
The mutagenicity and clastogenicity of crude products,
crude extracts of the leaves of Stevia rebaudiana and stevioside have been determined in a number test systems.
Crude extracts of Stevia (18% stevioside) and stevioside (>80%) gave negative results in bacterial reverse mutation
test with Escherichia coli WP2 uvrA/pKM 101 andSalmonella typhimurium G46 (Okumura et al., 1978;
Matsui et al., 1996), and Rec-assay with Bacillus subtilis+
H17 (Rec ) and M45 (Rec ) with or without rat liver S9
fractions (Okumura et al., 1978; Matsui et al., 1996). In
Ames test, the results were negative for all components
against Salmonella typhimurium TA1535, TA1537,
TA1538, TA97, TA98, TA100, TA102 and TA104, evenwhen the dose was as high as 50 mg/plate with or without
liver S9 fractions from rat, mouse, hamster, guinea pig
and rabbit (Suttajit et al., 1993; Klongpanichpak et al.,1997), with an exception of the crude product whichshowed a slight positive result in S. typhimurium TA100
with rat liver S9 fraction (Tama Biochemical Co. Ltd.,
1981). Furthermore, the test with stevioside also yielded
negative results in the forward mutation assay using S.
typhimurium TM677, with or without liver S9 fraction
from rat (Pezzuto et al., 1985; Matsui et al., 1996;
Temcharoen et al., 1998), umu test in S. typhimurium
TA1535/pSK1002 with or without liver S9 fraction (Matsui
et al.,1996), host mediated assay via mouse (TamaBiochemical Co. Ltd., 1981), and mutation test using germinal cells of silk worm (oocyte) system (TamaBiochemical Co. Ltd., 1981). In the clastogenic study, stevioside showed negative results in chromosomal aberration test in cultured human lymphocyte (Suttajit et al.,1993), human fetal fibroblast (Tama Biochemical Co. Ltd.,
1981), Chinese hamster lung fibroblast cell line (Ishidate
et al., 1984; Matsui et al., 1996), rat medullary cells (TamaBiochemical Co. Ltd., 1981), and sister chromatid

Table 1 Micronucleus test in hamster bone marrow after oral administration of stevioside.
Compound

Dose

Time
after

(g/kg BW) admini
stration(hours)No. of MNPCEs / 1,000
PCEsa

PCE / NCE ratiob

male

femalemale

femaleStevioside0(water)
10

0
24
30
48
72

2.20 ± 0.58
0.80 ± 0.37
0.40 ± 0.24
2.00 ± 0.52
1.25 ± 0.75

2.00 ± 0.55
1.60 ± 0.40
0.60 ± 0.24
1.40 ± 0.93
1.60 ± 0.51

0.77 ± 0.04
0.68 ± 0.03
0.66 ± 0.03
0.54 ± 0.01c
0.74 ± 0.04

0.71 ± 0.02
0.72 ± 0.04
0.67 ± 0.02
0.65 ± 0.06
0.73 ± 0.03

Cyclophosphamide( i.p. )0.08

30

32.6 ± 4.25d

34.0 ± 4.30d

0.65 ± 0.02c

0.56 ± 0.03d

aMean ± SEM, n = 5 , 2000 PCEs scored per animal , bMean ± SEM, n = 5, 400 erythrocytes( PCE + NCE ) scored per animal.
Significant difference from control : p < 0.05c, p < 0.01d.

Table 2 Micronucleus test in rat bone marrow after oral administration of stevioside.
Compound

Dose

Time
after

(g/kg BW) admini
stration(hours)No. of MNPCEs / 1,000
PCEsa

PCE / NCE ratiob

male

femalemale

femaleStevioside0(water)
10

0
24
30
48
72

1.14 ± 0.14
2.00 ± 0.52
0.80 ± 0.31
1.80 ± 0.58
1.60 ± 0.75

1.45 ± 0.33
1.20 ± 0.58
2.80 ± 1.36
2.00 ± 1.31
0.80 ± 0.58

0.79 ± 0.01
0.76 ± 0.04
0.80 ± 0.03
0.80 ± 0.05
0.75 ± 0.03

0.69 ± 0.03
0.70 ± 0.04
0.81 ± 0.03
0.73 ± 0.02
0.79 ± 0.01

Cyclophosphamide( i.p. )0.08

30

17.4 ± 2.04d

16.0 ± 2.43d

0.36 ± 0.04d

0.41 ± 0.03caMean ± SEM, n = 5 , 2000 PCEs scored per animal , bMean ± SEM, n = 5, 400 erythrocytes( PCE + NCE ) scored per animal.
Significant difference from control : p < 0.05c, p < 0.01d.

Table 3 Micronucleus test in mouse bone marrow after oral administration of stevioside.
Compound

Dose

Time
after

(g/kg BW) admini
stration(hours)No. of MNPCEs / 1,000
PCEsa

PCE / NCE ratiob

male

femalemale

femaleStevioside0(water)
10

0
24
30
48
72

0.50 ± 0.27
1.40 ± 0.40
1.00 ± 0.50
0.40 ± 0.39
0.40 ± 0.30

1.00 ± 0.57
1.60 ± 0.49
2.40 ± 1.16
1.20 ± 0.65
0.80 ± 0.20

0.55 ± 0.04
0.60 ± 0.04
0.52 ± 0.01
0.56 ± 0.03
0.53 ± 0.03

0.73 ± 0.05
0.76 ± 0.05
0.80 ± 0.03
0.77 ± 0.07
0.75 ± 0.07

Cyclophosphamide( i.p. )0.08

30

31.4 ± 7.07d

28.2 ± 5.88d

0.50 ± 0.03

0.39 ± 0.03d

aMean ± SEM, n = 5 , 2000 PCEs scored per animal , bMean ± SEM, n = 5, 400 erythrocytes( PCE + NCE ) scored per animal.
Significant difference from control : p < 0.05c, p < 0.01d.

39

exchange in human fetal fibroblasts with or without rat
liver S9 fraction (Tama Biochemical Co. Ltd., 1981).
However, the crude product (50% purity of stevioside)
yielded a slightly positive result when tested by the chromosomal aberration test in Chinese hamster lung fibroblast (Tama Biochemical Co. Ltd., 1981; Ishidate et al.,1984). In the dominant lethal test, crude product wasadministered orally at the doses of 10, 15 and 20 g/kg BW
to 9-week-old male mice and only at the dose of 15 g/kg
BW that it induced a slight but significant difference inthe number of implantations. However, with no doseresponse relationship, it was regarded as a negative result(Tama Biochemical Co. Ltd., 1981). The observed positive
results of crude products and crude extracts in some test
systems were probably caused by the impurities of the
products. Previous studies of stevioside, all of whichshowed negative results, were performed only in the invitro test systems. The present study was the first toassess the clastogenic effect of stevioside in the in vivo
test system using micronucleus test in rodents including
hamster, mouse and rat. It was found that stevioside at thedose as high as 10 g/kg BW had no clastogenic potential
in either sex of all treated animals at various time intervals
after an oral administration. These results were consistent
with the previous in vitro studies confirming an absenceof mutagenic and clastogenic potential of stevioside. The
present results also showed no changed in the PCE:NCE
ratio in most animals. The slight but significant decreasein PCE:NCE ratio observed in male hamsters after 48 htreatment (Table 1) was, however, in the acceptable normal range (Adler, 1984). Nevertheless, it indicated thatstevioside at a certain dose could somehow reach thebone marrow to cause a slight cytotoxicity but did notexert clastogenic effect on the bone marrow cells.
Stevioside, given orally, is known to be converted bycolon microflora bacteria to steviol which is subsequently
completely absorbed (Wingard et al., 1980; Nakayama etal., 1986). Hamster was found to be more susceptible tothe toxicities of stevioside and steviol when compared to
the rat and mouse (Toskulkao et al., 1997). The liver S9
fraction from Aroclor pretreated hamsters exhibited higher efficiency in activating both the mutagenic and cytotoxic effects of steviol, when compared to other rodents(Temcharoen et al., 1998). In addition, steviol at the doseof 4 g/kg BW for hamsters, and at the higher dose of 8
g/kg BW for rats and mice exhibited a slight cytotoxicity
to the bone marrow cells (Temcharoen et al., 2000). Baseon the total conversion of stevioside to steviol in the colonand their molecular weights, 10 g stevioside/kg BW usedin this study was equivalent to 3.96 g steviol/kg BW incase of completely converted by colon microflora bacteria.
Therefore, it was possible that stevioside itself was not
cytotoxic to the bone marrow cells, but after being con40

verted to steviol and reabsorbed into the circulation.
Steviol could reach the bone marrow and caused theobserved slight cytotoxicity to the bone marrow cells.
In summary, the present results indicated that theobserved slight cytotoxic effect on bone marrow cells may
have resulted directly from stevioside or its metabolites.
As for the assessment of the genotoxic risk of stevioside,
it could be concluded that stevioside had no genotoxic
potential. Moreover the average human consumption of 2
mg of stevioside/kg BW/day or the acceptable daily
intake (ADI) of 7.938 mg of stevioside/kg BW/ day (Xiliet al., 1992) was much lower than the tested dose in thisstudy, stevioside should be not harmful for use as a sweetener for human. However, the genotoxic risk of steviosideneed to be fully studied in human.
Acknowledgement
The authors are thankful to Assoc. Prof. Dr. Monthip
Tiensuwan, Department of Mathematics, for her statisticalassistance and also thanks to Mrs. Y. Sengsai for her typing. Stevioside was obtained from the Thai ResearchInstitute of Pharmacognosy, Chiang Mai. Financial support was provided by the National Science andTechnology Development Agency (NSTDA), the National
Research Council and Mahidol University, Thailand.
References
Adduci, J., D. Buddhasukh and B. Ternai (1987) Improved isolationand purification of stevioside, J. Sci. Soc. Thailand, 13, 179-183.
Adler, I.D. (1984) Cytogenetic tests in mammals, In : S. Venitt and
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Crammer, B. and R. Ikan (1986) Sweet glycosides from the Stevia
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Ishidate, M. Jr., T. Sofuni, K. Yoshikawa, M. Hayashi, T. Nohmi, M.
Sawada and A. Matsuoka (1984) Primary mutagenicity screeningof food additives currently used in Japan, Fd. Chem. Toxicol., 22,623- 636.
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Kinghorn, A.D. and D.D. Soejarto (1991) Stevioside. In L. O’Brien
Nabors and R.C. Gelardi (Eds), “ Alternative sweeteners,” Marcel
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Klongpanichpak, S., P. Temcharoen, C. Toskulkao, S. Apibal and T.
Glinsukon (1997) Lack of mutagenicity of stevioside and steviol in
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Nakayama, K., D. Kasahara and F. Yamamoto (1986) Absorption,
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Pezzuto, J.M., C.M. Compadre, S. M., Swanson, N.P.D. Nanayakkara
and A.D. Kinghorn (1985) Metabolically activated steviol, the aglycone of stevioside, is mutagenic, Proc. Natl. Acad. Sci., 82, 24782482.
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Yamada, A., S. Ohgaki, T. Noda and M. Shimizu (1985) Chronic toxicity study of dietary Stevia extracts in F 344 rats, J. Food Hyg. Soc.
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41

特別企画

Environ. Mutagen Res., 24 ： 43 - 60（2002）

8th ICEM を終えて8th ICEM と「9 月 11 日」

8th ICEM 実行委員長

木苗直秀（静岡県立大学食品栄養科学部）
8th ICEM 組織委員会委員長

黒田会員撮影早津彦哉

第 8 回国際環境変異原学会（8th ICEM）

誰しもあの 9 月 11 日の事件にはショッ

は，平成 13 年 10 月 21 日（日）より 26 日

クを受けたにちがいない．私もたまたま（金）まで静岡県コンベンションアーツセ

テレビでこの実況を見てしまった．8th

ンター「グランシップ」で日本学術会議と共同主催の形

ICEM が間近になって，その準備に忙殺されていた中で，で開催されました．9 月 11 日に米国で発生したテロ事件

この惨事が起こり，これは大変だ，どうしようと思ったのため，およそ 90 名のキャンセルがありましたが，最一人である．これで科学が中心となると思っていた 21

終的には 44 ヵ国 1 地域より外国人が 412 名，日本人が世紀はいったいどうなっていくのか？科学の一分野とし409 名の計 821 名が参加されました．4 年毎に世界各国ての環境変異現研究の位置づけは？など，心に浮かんでで開催されている本学会を 21 世紀初頭に日本（静岡）は消え，また浮かんでくるといった揺れ動く心境となっで開催できましたことは，大変意義のあることと考えてた．

おります．今大会の特徴としては，20 世紀に問題とな

以後 8th ICEM が開催された 10 月下旬までが組織委員った生活環境中の変異原・発がん物質について遺伝子へ会の最大に苦しい日々であったといえる．外国，特にアの修飾様式の解析など基礎的研究とともにそれらのリス

メリカ，カナダからは，主要な参加者達の苦悩や失意のク評価，リスク管理，リスクコミュニケーションなど応

言葉や，逆に，なにくそ負けるか頑張ろうといった決意

用面にまでアカデミックな討論ができたこと，またそれ表明などが寄せられた．われわれは，いつ戦争が始まる

ら化学物質の作用を封じ込む抗変異原・抗発がん物質にか，世界中の主要空港が閉鎖になる可能性まで考えなが関しても新しい知見が数多く発表され，参加者の関心がら，かたずをのんで成り行きを見守るしかなかった．こ

非常に高まったことなどが挙げられます．大会を通しての間の危機的状況を示すものとして，私が受信した e-

基調講演 3 題，特別講演 5 題のほか，シンポジウム 25，

mail の数がある．9 月 11 日以降 9 月末日まで合計 230 通，

ワークショップ 2，スペシャルセッション 2，一般口頭

10 月には入ってからさらに増えて 1 日当たり平均 25 通

発表 160 題，ポスター発表 360 題などとても実り多い学となり，最大の受信数は 10 月 15 日の 1 日 71 通であった．

会となりました．さらに静岡県，静岡市の御協力を頂き，8th ICEM の初日 10 月 21 日の夜のレセプションが会の21 世紀にこの分野を担う若き科学者達 60 名がホームス成否を占う鍵であると思っていたが，そこで見た会場にテイできましたことは，国際交流にも貢献できたことをあふれんばかりの大勢の参加者達とその笑顔で，私はこ表すもので，国際学会の新しいモデルを提案できたもの

れは大丈夫だとの確信を得た．用意されたスナック類はと考えております．

あっという間になくなってしまい，私自身殆ど何も口に入らなかったのも喜び以外の何物でもなかった．

本大会の開催に際しまして，日本学術会議，国際環境変異原連盟，日本環境変異原学会をはじめ，地方自治体会議の最中および終了後に参加された皆さんから寄せや企業の皆様の温かい御支援を賜りまして誠にありがとられた沢山の嬉しいメッセージからも，今回の ICEM はうございました．お陰をもちまして，本大会を無事且つ成功であったといってよいと思う．学問的にも 80 ％く

成功裡に終えることができました．ここに，本大会終了

らいは期待通りの成果があったと思うし，ソーシャルイの御報告とともに，重ねて厚く御礼申し上げます．

ベントは，ホームステイの成功ともあいまって，素晴ら
産総研分子細胞しかったと皆からほめられた．

須藤鎮世

会が終って 3 ヵ月を経た現在も，世界各国から 8th

個人的なことですが，City of Hope に留学する前に故ICEM は良かったとの言葉が寄せられ続けている．この大野乾先生から Jeffreys の論文は重要だから読んでこい会を盛り上げるのに大きな役目を果たしていただいたと指示された．親子鑑定など DNA 診断の基礎となった

JEMS 会員の皆様に深く感謝するとともに，8th ICEM

マイクロサテライト多型に関する最初の論文である．今が「9 月 11 日」の悪夢を振り払って，環境変異原研究の回，聴講の機会を得て嬉しく思う．大野先生は 1928 年 2

21 世紀のスタートダッシュの勢いをつけることに成功

月 1 日生まれ，2000 年 1 月 13 日に亡くなられた． Heしたことを皆さんとともに喜びたいと思う．

was a great manと早すぎる逝去を惜しまれた．
43

Jeffreys さんも great man で愛妻家，great men の条件はていたように思う．その後，第 2 回（98 年 11 月名古屋），

My wife and I both fell

第 3 回準備委員会（99 年 2 月，静岡）を経て，99 年 11 月

愛妻家のようだ．初来日ですがin love with your country!

とのことであった．晩餐会でには岐阜で第 1 回の組織委員会が開かれた．この時点で，は英人たちがノーベル賞をもらうかも知れないと噂，ノ長尾プログラム委員長から 8 ICEM でのプログラム編

ーベル委員会の Gustafsson さん，頼みます．私が担当

成の概要が示された．最終的に 8th ICEM では，plenary

した DNA Technology シンポジウムでは 7 演者中 2 名がsession が 8，シンポジウムが 23，特別セッションが 3，欠席した．その 1 人はサテライト会議 Functional

ワークショップが 2，それぞれ開催され幅広い内容が深Genomics を要約するはずの Aaron さんで，これに参加く論議されたように思う．特に plenary lecture は，いずされた Lohman さんが急遽，Aaron さんからデータを入れも素晴らしい講演ばかりであった．

th手，代役を果たされた．有難う．Aaron さんは座長でも私自身も Translesion DNA synthesis，Transgenic

あり，英国の Powell さんに代役をお願いした．が，なrodents，Structural biology という 3 つのシンポジウムのかなか返事が来ない．座長代役は会期中に確認した．後

モデレーターを引き受けさせていただいた．

で知ったが，彼女は 9 月 11 日，San Diego での SAGE

「Translesion DNA synthesis」は，主に DNA 損傷を乗り（serial analysis of gene expression）の学会に参加中で，越えて複製を行う新しいタイプの DNA ポリメラーゼ（Y空港閉鎖中は予定どおり親戚のところで過ごしたので問ファミリー DNA ポリメラーゼ）に関するセッションであ題なかったという．あの時期，幼い子供達を留守に 2 度り，この 2 年ほどの間にその重要性が認識されてきた課

海外に出してくれた家族の皆さんに謝意を表したい．彼

題である．前回の ICEM では，あまり論議されなかった

女の職場は 11 月までの契約で，次の課題として蛋白構内容であり，シンポジウムの当日（10 月 22 日）は，はた造の勉強を開始したという．成功を祈る．同じ座長の林

してどれくらいの人が聞きに来てくれるかと心配した崎さんはエジンバラでのマウスゲノム学会（10/21 〜 24）

が，200 人近く入る C 会場が満員になる盛況ぶりであっからのとんぼ返りであった．行きのジャンボ機は乗客がた．「Transgenic rodents」は，遺伝子改変を行ったマウ2 人とのことであった．困難な時に多数の方々の協力をス，ラットを用いて変異と発がんの研究を行うセッショ

得て成功裡に終わったことを嬉しく，誇りに思う．私はンであり，米国，カナダ，オランダ，日本から講演があプログラム，行事接遇，展示会に参画したが，晩餐会のった．この研究は主に環境変異原分野で発展してきた

司会は良い経験になった．多々至らぬところはお詫びし，

made in EMS の研究であり，これからも新しいマウス，

皆様のご協力に感謝します．特に静岡部会の皆様ご苦労

ラットが作られ変異原，がん原研究に新風を吹き込むこさまでした．とてもよい学会であったという数多くの声とが期待された．「Structural biology」は，やはり前回を代表して Sir Alec Jeffreys の言葉で締める．

の ICEM では論議されなかった課題であるが，今回の演The meeting was excellent.

者の顔ぶれ（T.A. Kunkel, J.A. Tainer, W. Yang, D.B.
Wigley, T. Shibata, K. Morikawa）はどこの学会にも負け

国立医薬品食品衛生研究所変異遺伝部

ない豪華なものであり，「Translesion DNA synthesis」

能美健彦に続いて多くの人が聞きに来てくれるかと期待したが，2001 年 10 月に静岡のグランシップで開開催日が最終日に近い木曜の午前であったせいか聴衆がth

少なく，その点は残念であった．セッションの終了後にICEM）は盛会のうちに幕を閉じ，準備に座長の一人であった T.A. Kunkel 博士（NIEHS）から「自関わった者の一人としてたいへんうれしく思っている．

分が以前に Gordon Conference で Structural biology のセ

か れ た 第 8 回 国 際 環 境 変 異 原 学 会（ 8th

思い起こせば 8 ICEM への道は，その 4 年前フランス
thッションを開いた時にも，その意義が理解されずあまり（ツールーズ）の 7 ICEM において，次の国際環境変異

人が集まらなかったが，今では Structural biology がいろ

原学会が日本で開催されると決まったところから始まっ

いろな分野で主要な研究方法となっている．今にこの環た．日本地図を配して次の ICEM が日本で開かれること

境変異原研究の分野でもその重要性が理解されるだろを知らせるビラが，ツールーズの会場のそこここに貼らう」と励まされた．

れていたことを今でも思い出す．それから 3 ヵ月，1997

会場のグランシップは建物が大きくて新しく，議論を年 12 月に開かれた日本環境変異原学会（JEMS）の評議員

深め交流を図るには絶好の場所であった．会場の内外でth会において，8 ICEM は早津委員長を中心に静岡，奈良，は，黄色いウインドブレーカーに身を包んだ担当者が，ソウル，上海で行うことが合意され，翌年 4 月には国立

朝早くから夜遅くまで会の円滑な運営のために努力さ

がんセンターで第 1 回目の準備会が開かれた．準備会でれ，頭の下がる思いがした．木苗 JEMS 会長を中心にしは「質素で効率よく，かつ at home な雰囲気で会を運営

た静岡の組織委員の先生方の熱意と善意が会場全体を包する」というモットーが早津委員長から提案され，結局んでいるような気がした．

このモットーが学会運営全体の原則として最後まで生き44th2004 年にサンフランシスコで開かれる 9 ICEM では，

はたしてどのようなセッションが新しく設けられ変異原

major challenges at every juncture including financial

研究を引っ張ってゆくのであろうか．それはこれから 4

ones and even the terrorist events of September 11 in the

年間の私たちの研究の進展にかかっている．

US that threatened international travel. In spite of thesechallenges, the meeting and its satellites were held essentially as planned. The hard work for the meeting was per-thth

Reflections on the 8 International Conference on

formed by the 8 ICEM Organizing Committee under theEnvironmental Mutagens（ICEM）and Satellite Meetings

capable leadership of its President, Hikoya Hayatsu; Vice
President, Treasurer and Local Chairperson, Naohide

1＊

2

and Paul H.M. Lohman

Kinae; Vice President for Finance, Yoshinari Ohnishi, and（ U.S. Environmental Protection Agency and National

Scientific Program Chairperson, Minako Nagao. Makoto

Institute of Environmental Health Sciences Research

Hayashi organized the IWGT
（that unfortunately had to beMichael D. Waters
1

2

Triangle Park, NC USA, Laboratory of Radiation Genetics

cancelled）, the IAEMS Internet course and the 5th ISCA.

and Chemical Mutagenesis Leiden University Medical

The organizers of the 8th ICEM and all satellite meetings

Center Wassenaarseweg 72, 2333 AL Leiden The

truly deserve our recognition for staying the course andNetherlands）

presenting an outstanding scientific conference.
th

The IAEMS was very pleased to join with the

The 8 ICEM also turned out beautifully in a social con-Environmental Mutagen Society of Japan（JEMS）and thetext. The people of Shizuoka Prefecture, the University ofthScience Council of Japan in sponsoring the 8 ICEM in

Shizuoka, and the city itself were extremely kind and hos-

Shizuoka, Japan, on October 21-26, 2001. Satellite meet-

pitable. The new GranShip proved to be an exciting

ings organized in conjunction with the 8th ICEM included

venue for the meeting. Professor Kinae together with his

Seattle, USA, and Seoul, Korea, before the ICEM andassociates and student assistants were constantly avail-Nara, Japan, and Shanghai, China, afterwards. In addi-

able and helpful to all attendees. We were certainly treat-thtion, the 5 International Symposium on ChromosomethAberrations（5 ISCA）was held on Awaji Island, Japan,
immediately after the ICEM.

ed as honored guests in Japan.
With the ICEM behind us we look forward to a new millennium of international research collaboration. The

This was the second time in twenty years that thepleasant memories and new knowledge from Shizuoka,

ICEM has been held in this beautiful country of Japan.

Nara, and Awaji Island will strengthen our continuingrd

The 3 ICEM was organized in Tokyo, and its satellite

friendships and encourage us in our scientific pursuits inmeetings were held in Kyoto and Mishima. It has been an

the future.
thastounding twenty years for environmental mutagenesis

The 8 ICEM represented the completion our tenures

research! In 1981, we were discussing fundamental mech-as President and Secretary-General of the IAEMS. We

anisms of mutagenesis and debating the merits of thewant to sincerely thank all of the members of the JEMS

Ames test. In 2001, we were exploring the implications offor their constant support of our efforts over the past four

sequencing the human genome and applying toxicoge-

years and for their lasting gifts to the IAEMS Member

nomics in groundbreaking research on global gene/pro-Societies-the 8th ICEM, the Nara satellite meeting, andtein expression.

the 5 ISCA.

th（20 December 2001）

In concert with its theme, Research for the New
Millenniumth, the 8 ICEM truly crossed the thresholdinto what is already being called theGolden Age of

8th ICEM に IAEMS - Fellow として参加してthBiology . The 8 ICEM was indeed a great scientific success. This important international meeting enabledexchange of views on current scientific research and静岡県立大学大学院生活健康科学研究科
食品栄養科学専攻

藤田理英子

served to transfer important new information worldwide.

今回の 8th ICEM は私にとって初めて

It undoubtedly catalyzed the establishment of many new

参加した国際学会でした．発表だけでなinternational scientific collaborations. And, through theくスタッフとしても参加し，学会の表舞

novel home-stay program , it served to educate many

台と裏舞台を体験したわけですが，感想としては「とにinternational students and to introduce them to the won-

かく忙しかった」「追われていた」，でも「やり遂げた」

derful Japanese culture.

という満足感に浸ることができたと思います．始めは，thThe organization the 8 ICEM was impeccable, despite

慣れない英会語，接し方，習慣の違い等，戸惑うことば45かりで，日本にいながら，どこか違う国にでも来たような感じでした．それでも後半はなんとか雰囲気に馴染むことができ，身振り手振りも板につき，言葉を交わすことができるようになりました．ポスター発表では，演題数の多さに圧倒されました．私のところにも多くの方が見に来て下さり，自分とは異なった視点から鋭い指摘，

4. SNP Analysis Leads to New Inferences about
Disease, Therapy and Risk
5. Functional Genomic Approaches Using Whole
Genomes and Comparative Genomics
6. Assessments of Genome Function throughProteonics

質問を頂き，とても有意義な時間を過ごすことができま

7. Emerging Technologies

した．特に，変異原の分野の最先端で活躍されている，

講演はベンチャー企業からの最新の Technology と論文上でしかお目にかかれない先生方と直接お話できた

Academia からの basic な Lecture それに製薬企業の開発ことは，とても貴重な経験でした．このような機会にめ

段階での Toxicogenoimics の実例などを織り交ぜたもの

ぐり会えたのも，国際学会ならではのできごとだったと

で，アメリカでのこれに対する取り組みの一端をうかが思います．8th ICEM は，研究面だけでなく，学会運営

うことが出来た．また，ポスターが 30 題程度発表されの大切さを知る，本当に良い機会になりました．一生忘ていたが，その内容は玉石混交といった感じであった．

れない素晴らしい学会だったと思います．

全体の印象は，テロ直後にもかかわらず，アメリカの最後に，8th ICEM に参加する機会を与えて下さいまAdventure Sprit が充分に感じられるものであった．
JEMS にとって，今後 Toxicogenoimics にどのように取した木苗直秀教授に深く感謝致します．

り組むかが大きな課題であると感じられた．
この会の正式な報告は近く
Molecular Mutagenesis

Environmental andに Chair を勤めた Sid Aaron（会社の命令で日本の ICEM に参加出来ずぼやいていた）サテライトミーティングを終えてによってまとめられるとのことですから，興味のある方はこちらを見ていただきたい．また，Abstract に興味のFunctional Genomics 印象記

ある方は小生までお問い合わせ下さい．

（財）食品薬品安全センター秦野研究所
澁谷
8

th

徹

ICEM ソウルサテライトミーティング参加報告

ICEM の Satellite Meeting である

Functional Genomics に参加した．

岐阜大学医学部

森秀樹

会場はアメリカ西海岸の Seattle

第 8 回 ICEM のソウルサテライトミー

Tacoma 空港のすぐ横の Wyndham Hotel で，会期は 10

ティングは 10 月 17 日〜 19 日に Yonsei 大

月 16 日から 18 日であった．直前におきたテロの影響が学の Centennial Hall にて開催された．

あって，アメリカ国内からの speaker 数名のキャンセル

テーマはDietary and Medical Anti-があった．日本からの事前登録者もすべて参加を断念さ

mutagens and Anticarcinogens: Molecular Mecha-nisms

れたようで，現地参加の私と阪大の三宅先生（JEMS 非

and Chemoprevention Potential

会員）の 2 名が参加したのみであった．私は Washington

ーターはソウル大学の Yong-Joon Surth 教授と Yonsei 大州立大学に行く用があり，同じ Seattle でこの会が開催

学の Kwang Kyun Park 教授である．11 ヵ国から 29 名のされていたので参加した．折からイチローのいる

招待演者が 6 の sessions（1. Antioxidative, Antigenotoxicであった．コーディネMariners が World Series 出場を争っていたので，ホテルand Antitumorigenic Compounds in the Diet, 2.

のテレビを横目で見ながらの出席でもあった．会の全体

Chemopreventive and Chemoprotective Potential ofは以下のようでした．

Dietary and Medicinal Plants: New Paradigm for

ま ず S.H. Taylor（ UCSD）の Keynote Address

Functional Foods, 3. Biochemical and Molecular Markers

Structure, Function and Dynamics of PKA Signaling がfor Chemoprevention Studies, 4. Molecular Mechanisms

あり，以下の 5 つの Sessions があった．

of Chemopreventive or Chemoprotective Phytochemicals:

1. Gene Function Impacts Human Populations Exposed
to Xenobiotics
2. Determining Gene Function Depends on DNA
Sequencing and Annotation
3. Expression of DNA Sequence Information is Integral
to Function
46

Modulation of Intracellular Signaling Cascades, 5. Dietary
Modulation of Formation, Metabolism, and Action ofCarcinogens, 6. Effects of Edible Phytochemicals andTheir Synthetic Derivatives on Carcinogenesis andMutagenesis: From Basic Science to ClinicalApplications）に分かれて講演した．この中には埼玉がん

ICEM 奈良サテライトミーティング

センターの藤木先生と近畿大学の村上先生と私が含まれている．招待講演者でキャンセルしたのは米国の 1 名だけであった．別に 91 のポスター発表があったがこちら

産業医科大学葛西宏の殆どは韓国の研究者からのものであった．講演に際し去る 10 月 27，28 日（土，日）奈良県新公会堂においてての討論もかなり活発であったが，多くは招待演者間で「突然変異と発癌における活性酸素と活性窒素の役割」

あった．Yonsei 大学の会場は申し分無かった．Yonsei

に関する 8th ICEM 奈良サテライト会議が開かれた．同大学には羨ましい程の Guest House があり，Welcome

時多発テロ事件後という悪条件にもかかわらず，海外かReception が行われた大学内のホールも立派なものであらも多数の参加者があり，主催者の一人としてホッとしった．会期中に学生食堂や購買部も覗いてみたが，日本

た（招待者，海外 14 名，国内 10 名，ポスター発表 49 演の大学生協とそっくりであった．Reception も at home

題（うち海外から 20），参加者総計 146 名）．Tannenbaum

で学生による音楽の演奏などがあり，結構楽しかった．

の基調講演を皮切りに，国内外の招待演者による，最先いずれにせよ，この 3 日間の抗変異，抗発がん関係のサ端の話が聞け，分野も DNA 損傷，修復，シグナルトラ

テライトミーティングは充分成果があったものと思われンスダクション，遺伝子発現など多岐に渡り，有意義なる．この会議が行われた前の週に別の会議で那覇へ行っ会となった．静岡から到着した人達からは，奈良というた．近場であるにも関わらず，私自身沖縄へ行くのも韓独特な雰囲気のため疲れを忘れ新たな気持ちで参加でき国へ行くのも初めてである．不思議なことになぜか那覇た，という声が多く聞かれた．また早朝にもかかわらずよりもソウルの街の方により日本的な雰囲気を感じた． 「鹿寄せ」には多数の参加者があり笑いを誘っていた．
所変われば品変わると思ったのはあちこちで白い紋様をこの会が成功したのは，奈良医大に在籍されていた小西

有する小型のカラスの様な鳥を見た時ぐらいである．

先生，中江先生のご尽力の賜であり，また多くの企業，
組織からのご支援によるものである．この場を借りて深謝したい．

「第 5 回国際染色体シンポジウム」開催報告
大会長

京都教育大学

生島隆治

ICEM 上海サテライトミーティング参加報告

第 8 回 ICEM のサテライトミーティン
グとして，昨年 10 月 26 〜 28 日に標記シ

国立医薬品食品衛生研究所

本間正充

ンポジウムを兵庫県淡路島国際会議場で

上海サテライトミーティングと第 9 回開催した．ドイツのエッセン大学で 4 年

アレクサンダーホランダーコースは 10 月

毎に開催されてきたもので，今回が初の日本開催となっ

30，31 日に上海市にある復旦大学で開催

た．「21 世紀における染色体異常研究の展望」という主された．中国環境変異原学会（CEMS）が題の下に，染色体の構造的・数的異常の基礎と応用の両主催し，翌日から始まる年次総会に先駆けて行われたも面に関し，2 題の key lecture，16 題の招待講演，20 題ののである．全体で約 120 名の参加のうち，招待者を含む

ポスター発表が行われた．折からの国際テロ事件の影響

外国人が 10 名程度で，ここでもやはりテロの影響で数を受け，2 名の招待講演者が出席できないというハプニ

名の外国人発表者のキャンセルがあり，プログラム内容

ングもあったが，15 ヵ国より 100 名近くの参加者を迎え，の変更を余儀なくされた．テーマはHuman Population

連日夜遅くまで熱気に満ちた討論で瞬く間に会期が終わMonitoring for Cancer Prevention

で，2 日目の午後にってしまった．生命科学分野の目覚ましい発展を反映しは遺伝毒性モニタリングとしての小核試験と，コメットて，染色体異常の研究にも顕著な進展が多々見られ，そ試験のワークショップが行われた．ワークショップではのすべてを国際的に共有するとともに近未来における更この分野の世界的権威である Dr. Kirsh-Volder，Dr.

なる飛躍を展望できたことは最大の成果であった．日本

Fenech，Dr. Tice 等の熱心な教育講演と技術指導に，質丸による大阪湾クルージング・ディナーパーティーは外問が相次ぎ，予定時間を大幅にオーバーし，夕方から行国人には殊の外好評で絶賛を博した．ともあれ，日本国

われる予定の懇親会が 1 時間以上も遅れる事態となっ発祥の地，淡路島の「夢舞台」（安藤忠雄設計）で大いた．中国での環境変異原研究はあまり知られていないが，に「夢」を見ようという当初の目論見は十二分に叶えら

CEMS は今年で 10 年目を迎え，総会での演題数も 100れたと自己評価している．これも，偏に組織委員会メンを越える規模となっている．発表内容も国際的に十分通バーをはじめ関係各位の温かい御援助の賜物と心より感用するものも少なくない．今後の発展と，同じアジアの謝申し上げる次第である．なお，本シンポジウムで発表隣人であることを考えると，これまで欧米一辺倒であっされた研究成果は，近く Mutation Research の特別号とた我々の研究の方向を見直してもいいのではないかと思して出版されることになっている．

わせた．
47

3rd International Workshop on Genotoxicity Testing

IWGT の目的が国際的な合意を得ることにあり，その（8th ICEM Satellite Meeting）の開催延期の経緯について

ため各グループメンバーに不参加者がでると，国際的な合意を得たことにはならないことから，どれだけ不参加

祖父尼俊雄，林真

者がでるかは深刻な問題である．そのため，各グループ

International Workshop on Genotoxicity Testing

メンバーについて参加の確認を行ったところ，時間の経（IWGT）は，1993 年にメルボルンで，1999 年にワシント

緯に伴い不参加者が増加した．10 月に入った時点で，ンで開催され，第 3 回 IWGT は 8th ICEM のサテライト

71 名の討論者のうち 22 名が不参加であった．また，開ミーティングとして開催することが計画された

催か否かの決断のための時間的余裕もなくなってきた．

（Organizers: D. Kirkland（UK）, L. Müller（Switzer-land）,

サテライトミーティングの中止は ICEM 本会議に影響し

M. Hayashi（Japan）, T. Sofuni（Japan）, J. MacGregor

かねないことから，組織委員，グループリーダーのみの（USA）, L. Schechtman（USA））．IWGT の目的は国際的議論でなく，IAEMS の Drs. M. Waters, P. Lohman や 8thに受け入れられる試験手法の確立にあり，今回は 1）

ICEM の早津，木苗先生らとの意見交換など，最終判断

Mouse lymphoma assay，2）In vitro micronucleus assay，

は至難なものであった．IWGT の目的からみて開催する

3）Transgenic genotoxicity models，4）p53 and Hras2

意義がなくなることと，本会議への影響が少ないことがtransgenic tumor models，5）Strategy and classification

予測されたことから，最終的に 10 月 4 日に 3rd IWGT のを取り上げた．会場は静岡県立大学で，受け入れ態勢な

延期を公式に発表した．

どの具体的な準備が着々と進められた最中に 9 月 11 日

IWGT の延期によりこれに関連した本会議のシンポジの事件が発生した．当初はこの事件が IWGT 開催へどの

ウムが 1 つ中止になり，また IWGT の延期に伴い本会議ように影響するかは掴みかねたが，しばらくすると海外への参加を取り止めた人もおり，全く本会議に影響がな

旅行に対する懸念のメールが交換されるようになり，9かったとはいえない．しかし，本会議は盛会のうちに終月 25 日頃からは米国行政，企業などでの海外旅行の一了したことから，実質的な影響はきわめて少なかったと

時的禁止の情報が流れるようになった．一方，欧州側かいえる．現在，延期された 3rd IWGT の開催計画が日本

らは比較的冷静な反応が多く，予定通りの参加の意志を

および英国で検討されている．静岡での 3rd IWGT の開示した人々もいた．日本側から予定通りの開催のために催に協力された皆様方に改めて深謝するとともに，引続参加を依頼したが，事態を深刻に受け止める流れは止め

きのご協力をお願いしたい．

られるものではなかった．

48

8thICEM プログラム掲載にあたり
昨年 9 月 11 日に起きた同時多発テロの余波は遠い日本で行われた国際学会にも及びました．講演，発表のキャンセルもあり，プログラムや参加者リストの変更を余儀なくされ，不自由な中での開催となりました．そこで，環境変異原研究第 24 巻 1 号を 8th ICEM の記念号とするにあたり，写真，回顧記事などのほかに，実際に行われた発表を記録として残すことは意義あることと考えました．演題番号と演者名，会場などを実際の発表順に並べただけの簡単なものですが，お手元のプログラムの演題番号と照らし合わせると，演題名はわかるようになっています．ご活用いただければ幸いです．
編集委員一同

21 October, 2001
Opening ceremony

17:00 ─ 18:30

Room A

Opening address
H Hayatsu（Japan）
Message
The Science Council of Japan
Welcome message
N Kinae（Japan）
Message
MD Waters（USA）
Message from the Prime Minister of Japan（to be read on behalf of the minister）
President’s lecture
PR-1

H Hayatsu（Japan）

18:30 ─ 20:30

Welcome Cocktail

22 October, 2001
Plenary session

Room C+D

8:30 ─ 12:00
Keynote lecture
KL ─ 1T Sugimura（Japan）

Plenary lecture
PL ─ 1

L Loeb（USA）

Plenary lecture
PL ─ 2C Barrett（USA）

Plenary lecture
PL ─ 3

D MacPhee（Japan）

RoomA

49

Lunch, Poster discussion
Lunch
Poster discussion P1 ─ 1 〜 P27 ─ 51（odd number posters）

Plenary session

12:00 ─ 14:30 6F, Ocean（1F）

14:30 ─ 15:15
Plenary lecture
PL ─ 4J ─ A Gustafsson（Sweden）

Symposia 1 and Special session 2
Symposium 1B: Dietary Mutagens and Carcinogens
1B ─ 2
M Nagao（Japan）
R Sinha（USA）
1B ─ 3
1B ─ 4
LR Ferguson（NewZealand）
H Bartsch（Germany）
1B ─ 5
JE Trosko（USA）
1B ─ 7

15:30 ─ 19:20

Symposium 1C: Translesion DNA Synthesis as a Mechanism of Mutagenesis
GC Walker（USA）
1C ─ 1
1C ─ 2
RPP Fuchs（France）
1C ─ 3CW Lawrence（USA）
F Hanaoka（Japan）
1C ─ 4
R Woodgate（USA）
1C ─ 5
1C ─ 6
M Moriya（USA）
Symposium 1D: Molecular Cytogenetic Approach to Gene Mapping and Function
1D ─ 1AT Natarajan（Netherlands）
J Inazawa（Japan）
1D ─ 2
1D ─ 4
HG Weier（USA）
1D ─ 5
M Ohimura（Japan）
U Wintersberger（Austria）
1D ─ 650

Room A

Room B 〜 F
Room B

Room CRoom D

Symposium 1E: Ecosustem and Human Health
WF Grant（Canada）
1E ─ 1HNB Gopalan（Kenya）
1E ─ 2
GM Bohm（Brazil）
1E ─ 3
1E ─ 4
G Cabrera（Mexico）
S Knasmueller（Austria）
1E ─ 6
TH Ma（USA）
1E ─ 7

Room E

Special session 2: The Future of the IAEMS
MD Waters（USA）
1F ─ 1
1F ─ 3
PHM Lohman（Netherlands）
DM DeMarini（USA）
1F ─ 5
WW Au（USA）
1F ─ 6, 7
1F ─ 8LR Ribeiro（Brazil）
JM Gentile（USA）
1F ─ 9, 10
open discussion

Room F23 October, 2001
Plenary session

8:30 ─ 9:15
Keynote lecture
KL ─ 2RoomA

AJ Jeffreys（UK）

Symposia 2

9:30 ─ 12:30

Room A 〜 F

Symposium 2A: Tea and Health
N Kinae（Japan）
2A ─ 1
2A ─ 2
C Ioannides（UK）
Y Kuroda（Japan）
2A ─ 3
Z Dong（USA）
2A ─ 42A ─ 5
VE Steele（USA）
2A ─ 6
H Mukhtar（USA）
discussion
Symposium 2B: Mutagens and Carcinogens in Water, Air and Soil:
Significance to Human Health
K Wakabayashi（Japan）
2B ─ 1
2B ─ 2PA White（Canada）
T Watanabe（Japan）
2B ─ 32B ─ 4
J Lewtas（USA）
2B ─ 7
DM DeMarini（USA）
Symposium 2C: Molecular Epidemiology with DNA Damage as Markers
2C ─ 2
D Turner（Australia）
T Kamataki（Japan）
2C ─ 4
SA Kyrtopoulos（Greece）
2C ─ 5
2C ─ 6RJ Sram（Czech）
Symposium 2D: Individual Difference in Human Metabolizing Capacity:
Genetic and Environmental Influences
2D ─ 2
H Autrup（Denmark）
Y Fujii ─ Kuriyama（Japan）
2D ─ 3
Y Yamazoe（Japan）
2D ─ 5
P27 ─ 48
M Waters（USA）

Room B

Room CRoom D

Symposium 2E: Health and Aging
2E ─ 1Y Furuichi（Japan）
V Bohr（USA）
2E ─ 2
2E ─ 3SC Park（Korea）
J Vijg（USA）
2E ─ 4
T Ono（Japan）
2E ─ 5
SV Vasilieve（Russia）
O8 ─ 10

Room E

Symposium 2F: Radiation and DNA Repair ─ Protein Interactions
DJ Chan（USA）
2F ─ 1
K Komatsu（Japan）
2F ─ 2
2F ─ 3
A Pastink（Netherlands）
K Miyagawa（Japan）
2F ─ 4

Room F51

2F ─ 52F ─ 6

J Thacker（UK）
PC Hanawalt（USA）

Lunch, Poster discussion

12:00 ─ 14:30 6F,
Ocean（1F）

Lunch
Poster discussion P1 ─ 2 〜 P27 ─ 52（even number posters）

12:30 ─ 14:30

Plenary session
Plenary lecture
PL ─ 5

R Holliday（Australia）

Symposia 3, Workshop 1, and Special session 1a
15:30 ─ 19:20
Workshop 1: Validation of the in vitro micronucleus assay for safety
evaluation of clastogenic ／ aneugenic compounds
T Sofuni（Japan）
3A ─ 2
D Marzin（France）
3A ─ 33A ─ 4
H Norppa（Finland）
Workshop 2: A SFTG International Collaborteive Study on in vitro
Micronucleus Test, using human lymphocytes and
CHO, CHL and L5178Y cell lines
3A ─ 5E Lorge（France）
A Wakata（Japan）
3A ─ 6
Symposium 3B: Mechanisms of Antimutagenesis and Anticarcinogenesis
RH Rdashwood（USA）
3B ─ 1
3B ─ 2
MJ Plewa（USA）
3B ─ 3
YJ Surh（Korea）
S De Flora（Italy）
3B ─ 4
TG Rossman（USA）
3B ─ 5
3B ─ 6
H Nishino（Japan）
Symposium 3C: Transgenic Rodents as a Tool for Modern Risk Assessment
JA Healdle（Canada）
3C ─ 1
3C ─ 2JG de Boer（Canada）
H Van Steeg（Netherlands）
3C ─ 3
T Nohmi（Japan）
3C ─ 4
3C ─ 6
H Tsuda（Japan）
JE French（USA）
3C ─ 7
Symposium 3D: Genomic Instability
H Ohmori（Japan）
3D ─ 1J ─ S Hoffman（France）
O19 ─ 3
3D ─ 3
H Nakagama（Japan）
RH Schiestl（USA）
3D ─ 4
MA Kadhim（UK）
3D ─ 5
3D ─ 6C Streffer（Germany）

52

Room A

Room A 〜 F

Room A

Room B

Room CRoom DSymposium 3E: Endogenous Mutagens and DNA Damage
3E ─ 1J Cadet（France ）S Loft（Denmark）
3E ─ 2B Demple（USA）
3E ─ 3J Laval（France ）3E ─ 4
I Rusyn（USA）
3E ─ 5

Room E

Special session 1a: Science in Countries with Developing Environmental
Mutagenesis Programs
JO Akerele（Nigeria）
3F ─ 1
3F ─ 2OA Badary（Egypt）
K Fahmy（Egypt）
3F ─ 3
RC Chaubey（India）
3F ─ 4
3F ─ 5G Murin（Slovakia）
5F ─ 5
A Cebulska ─ Wasilewska（Poland）
KSA Mossanda（South Africa）
3F ─ 7
3F ─ 8LS Hoyos G（Colombia）
M Abdel ─ Hamid（Egypt）
3F ─ 9A Tagne（Cameroon）
3F ─ 1024 October, 2001
General oral sessions

8:30 ─ 11:45
Session 7: Antimutagens ─ Anticarcinogens
U Vinitket ─ Kumnuen（Thailand）
O7 ─ 14O7 ─ 1E Elmore（USA）
CAM Krul（Netherlands）
O7 ─ 2
J ─ K Lin（Taiwan）
O7 ─ 3O7 ─ 4JL Marnewidk（South Africa）
DM Simic（Yugoslavia）
O7 ─ 6
S Arimoto ─ Kobayashi（Japan）
O7 ─ 7O7 ─ 8EO Farombi（Nigeria）
J ─ H Jang（Korea）
O7 ─ 9OJ Park（Korea）
O7 ─ 10IM Villaseor（Philippines）
O7 ─ 11O7 ─ 12
U Rannug（Sweden）
Session 2: Mutagens and Carcinogens in Water, Air and soil
O2 ─ 1
M Dybdahl（Denmark）
GM Bohm（Brazil）
O2 ─ 3O2 ─ 4
D Pra（Brazil）
AC Basilio（Brazil）
O2 ─ 5
N Sera（Japan）
O2 ─ 6
G Bronzetti（Italy）
O2 ─ 7
O2 ─ 8K ─ T Chung（USA）
S Wakuri（Japan）
O2 ─ 9M Machala（Czech）
O2 ─ 10BJ Majer（Austria）
O2 ─ 11
O2 ─ 12
P Joseph（USA）

Room F

Room B 〜 H
Room B

Room C

53

Session 6: Mehcanisms of Mutations
P19 ─ 5
I Kobayashi（Japan）
K Negishi（Japan）
O6 ─ 1E Sage（France）
O6 ─ 2
C Otsuka（Japan）
O6 ─ 3J O ─ Wang（Japan）
O6 ─ 4Y ─ N Yu（China）
O6 ─ 5O6 ─ 6
S Liu（China）
K Felix（USA）
O6 ─ 7

Room D

Session 18: Transgenerational Effects of Environmental Factors
T Shibuya（Japan）
O18 ─ 1
Session 14: Molecular Epidemiology
O14 ─ 1
C Bolognesi（Italy）
G Speit（Germany）
O14 ─ 2O14 ─ 4
P Schmezer（Germany）
A Horska（Slovakia）
O14 ─ 5
S Loft（Denmark）
O14 ─ 6
H Ohshima（France）
O14 ─ 7O14 ─ 8
K Nakachi（Japan）
K Savela（Finland）
O14 ─ 9

Room E

Session 9: Genetic Disease in DNA Repair
P Mosesso（Italy）
O9 ─ 1J Surralles（Spain）
O9 ─ 2
Session 1: Mutagens and Carcinogens in Diet
Y Totsuka（Japan）
O1 ─ 1O1 ─ 2P ─ M Leong ─ Morgenthaler（Switzerland）
P Moller（Denmark）
O1 ─ 3K Hiramoto（Japan）
O1 ─ 4O1 ─ 5JE Lampinen（Finland）
R Hussein（Egypt）
O1 ─ 6UB Vogel（Denmark）
O1 ─ 7
Session 13: DNA Technology
O13 ─ 1O13 ─ 2O13 ─ 3O13 ─ 4

R Gealy（USA）
C Furihata（Japan）
MHL Green（UK）
PB Veronique（Cameroon）

Session 3: Solar and Ionizing Radiation Mutagenesis
K Kikugawa（Japan）
O3 ─ 1
A Czich（Germany）
O3 ─ 2O3 ─ 3
K Fujikawa（Japan）
LA Vasilyeva（Russia）
O3 ─ 4
AN Osipov（Russia）
O3 ─ 5A Masaoka（Japan）
O3 ─ 6

54

Room G

Session 21: Comet Assay
O21 ─ 1
O21 ─ 2
O21 ─ 3
O21 ─ 4
O21 ─ 5

RC Chaubey（India）
VJ McKelvey ─ Martin（UK）
R Saran（India）
A ─ H Tu（China）
J ─ R Meunier（France）

Session 19: Genomic and Chromosomal Instability
N Shima（USA）
O19 ─ 1E Wintersberger（Austria）
O19 ─ 2
1D ─ 6U Wintersberger（Austria）
S Ban（Japan）
O19 ─ 4SR Moore（USA）
O19 ─ 5
O19 ─ 6
S Sutou（Japan）
Session 5: DNA AdductsO5 ─ 2O5 ─ 3O5 ─ 4O5 ─ 5

Room H

E Eder（Germany）
U Harju（Finland）
PT Henderson（USA）
A Umemoto（Japan）

Session 25: Genomics and Proteomics in Genetic Toxicology
O25 ─ 1AJ Van der Ed（Netherlands）
Lunch, Excursion

12:00 ─25 October, 2001
Plenary session

8:30 ─ 9:15
Keynote lecture
KL ─ 3

Room AEC Friedberg（USA）

Symposia 4 and Workshop 2
Workshop 2: The Comet Assay ─ Recent Advances and New Applications
G Speit（Germany）
4A ─ 1
D Anderson（UK）
4A ─ 2
4A ─ 3
YF Sasaki（Japan）
AR Collins（UK）
4A ─ 44A ─ 5BL Pool ─ Zobel（Germany）
RR Tice（USA）
4A ─ 6
Symposium 4B: Mechanistic Dynamics and Genetic Diseases in DNA Repair
4B ─ 4J Jiricny（Switzerland）
T Tsuzuki（Japan）
4B ─ 2PK Cooper（USA）
P8 ─ 2
K Tanaka（Japan）
4B ─ 5
4B ─ 6JHJ Hoeijmakers（Netherlands）

Room A

Room B

55

Symposium 4C: Transgenetational Effects of Environmental Agents
4C ─ 1
T Nomura（Japan）
LM Anderson（USA）
4C ─ 2K Mabuchi（USA）
4C ─ 4
WW Au（USA）
4C ─ 5
K Sankaranarayanan（Netherlands）
4C ─ 6
Symposium 4D: Protective Mechanisms and the Concept of Genotoxic Activity
J Rueff（Portugal）
4D ─ 1PD Josephy（Canada）
4D ─ 2
4D ─ 3
MJM Nivard（Netherlands）
I ─ D Adler（Germany）
4D ─ 4
S Fukushima（Japan）
4D ─ 5
4D ─ 6JM Parry（UK）

Room CRoom D

Symposium 4E: Epidemiology and Environmental Mutagens ─ Relevance andLimitations ─ Epidemiology is the touchstone of our science. How reliable is it ?
Introduction
BA Bridges（UK）
P Vineis（Italy）
4E ─ 2BA Bridges（UK）
4E ─ 1
4E ─ 3O Hino（Japan）
Introduction to Discussion
DG MacPhee（Japan）
Discussion
Symposium 4F: Structural Biology on DNA Replication and Its Relevance to
Mutation Research
TA Kunkel（USA）
4F ─ 1
W Yang（USA）
4F ─ 2
DB Wigley（UK）
4F ─ 3
4F ─ 4T Shibata（Japan）
JA Tainer（USA）
4F ─ 5K Morikawa（Japan）
4F ─ 6
Lunch, Lunch time seminar
Symposia 5 and Special session 1b
Symposium 5A: Micronucleus Assay in Human Monitoring andthe HUMN Project
5A ─ 1E Zeiger（USA）
5A ─ 2S Bonassi（Italy）
M Fenech（Australia）
5A ─ 3
5A ─ 4
NT Holland（USA）
M Kirsch ─ Volders（Belgium）
5A ─ 5WP Chang（Taiwan）
5A ─ 6
D Scott（UK）
5A ─ 7
Symposium 5B: Chemical and Biological Properties of DNA Adducts,
Relevance to Mutagenesis and Carcinogenesis
PB Farmer（UK）
5B ─ 2
5B ─ 3
DH Phillips（UK）

56

12:30 ─ 14:30

Room F

6F ／ Room C

Room A

Room B5B ─ 4
5B ─ 5

S Shibutani（USA）
E Ohtsuka（Japan）

Symposium 5C: Epigenetic Changes Related to Human Health
5C ─ 2
M Costa（USA）
GP Pfeifer（USA）
5C ─ 4
T Sekiya（Japan）
5C ─ 5
5C ─ 6
T Ushijima（Japan）

Room C

Symposium 5D: Solar Radiation and Mutagenesis
RM Schaaper（USA）
5D ─ 1
5D ─ 2
L Mullenders（Netherlands）
T Ishikawa（Japan）
5D ─ 3
A Sarasin（France）
5D ─ 4
5D ─ 5T Negishi（Japan）
RC von Borstel（Canada）
5D ─ 6
MHL Green（UK）
5D ─ 7

Room D

Special session 1b: Science in Countries with Developing Environmental
Mutagenesis Programs
R Aroutiounian（Armenia）
5F ─ 1
5F ─ 3
RI Bersimbaev（Kazakhstan）
AR Muotri（Brazil）
P9 ─ 1
H Groot ─ Restrepo（Colombia）
5F ─ 6
5F ─ 7E Mirkova（Bulgaria）
RD Montero（Mexico）
5F ─ 85F ─ 9AK Giri（India）
5F ─ 10
S Kietthubthew（Thailand）
Banquet26 October, 2001
Symposium 6

Room F

18:00 ─ 21:30

Ocean

8:30 ─ 12:00

Room A

Symposium 6A: DNA Techonology
M Miwa（Japan）
6A ─ 1
6A ─ 2J Powell（UK）
TS Tanaka（USA）
6A ─ 3Y Suh（Korea）
6A ─ 4
6A ─ 6
Y Hayashizaki（Japan）
PH Lohman（Netherlands）
6A ─ 7

General oral sessions

8:30 ─ 12:00
Session 7: Antimutagens ─ Anticarcinogens
H Steinkellner（Austria）
O7 ─ 13
L Ao（China）
O7 ─ 16
O7 ─ 17
AM Aboul ─ Enein（Egypt）
E Lansky（Israel）
O7 ─ 18

Room B 〜 H
Room B

57

Session 11: Transgenic Animals in Genetic Toxicology
O11 ─ 1
K Amanuma（Japan）
T Chen（USA）
O11 ─ 4H ─ J Martus（Switzerland）
O11 ─ 5
Session 20: Micronuclei in vitro and in vivo
L Abramsson ─ Zetterberg（Sweden）
O20 ─ 1
O20 ─ 2ME Gonsebatt（Mexico）
S ─ H Kim（Japan）
O20 ─ 3YG Izyumov（Russia）
O20 ─ 4

Room C

Session 22: Germline Mutations
CL Yauk（UK）
O22 ─ 2
Session 2: Mutagens and Carcinogens in Water, Air and Soil
T Grummt（Germany）
O2 ─ 13
O2 ─ 14
Y Yamazaki（Japan）
J ─ H Kwon（Korea）
O2 ─ 15
S Kira（Japan）
O2 ─ 16
T Takamura ─ Enya（Japan）
O2 ─ 17
Session 8: DNA Repair and its Mechanisms
O8 ─ 1
CS Griffin（UK）
O8 ─ 3
M Frankenberg ─ Schwager（Germany）
TP Nouspikel（USA）
O8 ─ 4
M Satoh（Canada）
O8 ─ 5O8 ─ 7
I Rusyn（USA）
H Ide（Japan）
O8 ─ 8B Tudek（Poland）
O8 ─ 9O8 ─ 12B Kaina（Germany）
Session 16: Individual Differnces in Mutagenesis and Carcinogenesis
O16 ─ 1RK Elespuru（USA）
S ─ M Hou（Sweden）
O16 ─ 3
A Rannug（Sweden）
O16 ─ 4
H Tokiwa（Japan）
O16 ─ 5
O16 ─ 6
AJ Van der Eb（Netherlands）
C Keshava（USA）
O16 ─ 8

Room D

Room E

Session 4: Endogenous Mutagens
M Kruszewski（Poland）
O4 ─ 2O4 ─ 3
T Nunoshiba（Japan）
T Tsutsui（Japan）
O4 ─ 4
Session 17: Molecular Cytogenetics, Chromosomal Aberraions
O17 ─ 1
J Boei（Netherlands）
ET Sakamoto ─ Hojo（Brazil）
O17 ─ 2
F Darroudi（Netherlands）
O17 ─ 3IP Aranha（Brazil）
O17 ─ 4
O17 ─ 5
Y Ishii（Japan）
M Dusinska（Slovakia）
O17 ─ 6

58

Room FO17 ─ 7
O17 ─ 8J Nath（USA）
VA Ratner（Russia）

Session 12: Epigenetic Changes
O12 ─ 1
M Suganuma（Japan）
BM Thomson（New Zealand）
O12 ─ 2
Session 10: Metabolisms of Genotoxic Agents
K Fujita（Japan）
O10 ─ 1
M Miyazaki（Japan）
O10 ─ 2
WP Watson（UK）
O10 ─ 3O10 ─ 4
A Koide（Japan）
C Fred（Sweden）
O10 ─ 7
Session 15: Health and Aging
O15 ─ 1P15 ─ 6

Room GC Dellacroce（Italy）
S Kawanishi（Japan）

Session 23: Thresholds in Genetic Toxicology
AM Lynch（UK）
O23 ─ 1
Session 25: Ecosystem and Human Health
RI Bersimbaev（Kazakhstan）
O26 ─ 1
Session 27: Others
O27 ─ 1
O27 ─ 2
O27 ─ 3
O27 ─ 4
O27 ─ 5
O27 ─ 6
O27 ─ 7
O27 ─ 8O27 ─ 9O27 ─ 10
O27 ─ 11
O27 ─ 12
Closing session

H Hayatsu
N Kinae
PC Hanawalt

Room HM Honma（Japan）
J ─ R Meunier（France）
P Rettberg（Germany）
RA Sidorov（Russia）
JEE Haglund（Sweden）
H Lu（China）
L Chen（China）
A Matsuoka（Japan）
MA Ghoneim（Egypt）
M Tornqvist（Sweden）
S Tokudome（Japan）
RWL Godschalk（Netherlands）
12:00 ─ 12:15

Room A

59

60

Environ. Mutagen Res., 24 ： 61（2002）

書 評
Food Borne Carcinogens ─ Heterocyclic
AminesM. Nagao and T. Sugimura（Eds.）
John Wiley ＆ Sons, Ltd（2000）
373 pages, Price ￡90, ISBN 0─471─98399─3

人間の健康に最も関係の深い「環境」といえば，まず，反応について解説があったのちに，Nagao による変異原

日常の食べ物がある．わが国における環境変異原研究は，性の詳細なレビューと考察がなされ，次いで発がんのデ

その歴史の初めから食べ物に関係していた．すなわち，ータが動物別，標的臓器別に紹介されている．また，が食品保存料として使われていた AF2 に強い変異原性が発ん以外の病変についての一章があり，さらに抗変異原性，見され，次いでそれに発がん性があることがわかって使

抗がん性物質についての詳しい解説と考察が Dashwood

用禁止になったことが，日本環境変異原学会のスタート

によってなされている．各論の最後は，疫学者によるヘのきっかけになったといえよう．1977 年に国立がんセテロサイクリックアミンのヒト発がん物質としての位置

ンター研究所の杉村隆，長尾美奈子両博士のグループがづけがなされ，半肯定的な議論が展開されている．本書

魚や肉の焦げに強い変異原性があることを見出し，そのの締めくくりは，Sugimura グループによる 2 つの章で，本体がヘテロサイクリックアミンであることを突きとめ「結論と展望」が述べられている．

て以来，日本の変異原研究は，その成果を中心に多くの私が通読して感心したのは，資料として有用な章の中研究者が集まり，今日の隆盛をもたらしたといってよい．

にあって，読み物としても面白く，同じ分野の研究者と世界的にみても，この発見のインパクトは大きく，このしてスリルをも味わえるような章がいくつもあることで20 年間で，アメリカ，ヨーロッパをはじめ各国の優秀ある．ところどころに眼を開かせるようなヒントが散りな科学者が競って「食べ物による発がんとヘテロサイク

ばめられたこれらのエッセイを読むことは，単にヘテロ

リックアミンの因果関係」を究明しようと，猛烈な勢い

サイクリックアミン研究者のみでなく，変異原−発がんで研究を進めてきた．2000 年という区切りの年度を迎研究者の誰でもが感銘するところであろう．特に力作は，えて，今回この分野の創始者であるお二人が編集して，序章と結論の 3 つの章であり，ここに著者らの研究の哲

本書を刊行されたことはまことに時宜を得たものといえ学が込められている．「肉や魚の焼けこげが，人間の発よう．

がんの原因であることをきちんと立証するにはまだ時間

本書は，12 章 30 名の著者（日本 10 名，外国 20 名）のがかかるであろうが，これまでの研究結果からこの可能分担執筆により，ヘテロサイクリックアミン化合物群の性が高いこと，従って焼けこげの摂取は控えよう」といすべてについて，それらの多岐にわたる生物への作用をう著者らの結論は，本書全体を読んで十分納得できる．

「発がん性」に焦点をしぼって網羅的に解説したもので引用文献は延べ 1300 を越え，1999 年までカバーしてある．まず Sugimura の"Preface"によって発見の端緒がいる（2000 年の分もわずかにある）．ヘテロサイクリッ

語られ，第 1 章でさらに拡大して発がんの現象全体をカクアミンについてのほぼ全貌を網羅したこの本は，20バーしつつ論じられた後，「各論」がスペシャリスト達

世紀終期の科学の新発展を記録したものとして，大きなによって詳細に記述されている．すなわち，合成法，食価値がある．

べ物，飲料，タバコ煙の中の含量；代謝；細胞成分との（早津彦哉・就実学園

岡山市西川原 1 ─ 6 ─ 1）61

日本環境変異原学会会則

研究に必要な知識と経験を有し，定められた会
第1章

名

称

費を納入した者とする．
第8条

学生会員は，大学または大学院等に在籍し，毎第1条

本会は日本環境変異原学会と称する．

年所定の手続きを経て，定められた会費を納入

第2条

本会の英語の名称は The Japanese Environ-

した者とする．

mental Mutagen Society と称し，JEMS と略称
第3条

第9条

賛助会員は，本会の目的に賛同し，本会の事業する．

を後援するために，定められた会費を納入した

本会は事務所を〒 170-0003 東京都豊島区駒込

個人または法人とする．

1−43−9

駒込 TS ビル

財団法人口腔保健協第10条

購読会員は，学会誌「環境変異原研究」および
会報「Jems News」の購読のみを行う個人また会内に置く．

は法人とする．
第2章

目

的

第11条

名誉会員は，変異原の研究または本会の発展に特に功績のあった者で，理事会が推薦し，評議

第4条

本会は人間・生物・地球環境における変異原，

員会の承認を得た者とする．また，名誉会員はとくに公衆の健康に重大な関係を有する変異原とこれに関連する基礎研究の推進，ならびに関会費の納入を免除される．
第12条

連情報・技術の伝達を目的とする．

本会に入会を希望するものは，正会員 1 名の推薦書付きの所定の申込書に記入の上，年会費の納入とともに，本会事務所に申込むものとする．

第3章

事

業

正式の入会の可否は，理事会および評議員会において決定する．

第5条

本会は前章の目的を達成するために，次の事業

第13条

会員は毎年年会費を納入しなければならない．

を行う．

次年度の年会費の額は理事会において審議し，

1.

評議員会において定める．

年 1 回大会を開催し，総会，ならびに学術上の研究成果の発表および知識・情報の交

2.
3.

4.

第14条

会員は次の事由によって会員，役員および評議換を行う．

員の資格を喪失する．

学会誌「環境変異原研究」および会報「Jems1.

退会の届け出をしたとき．

News」を発行し，会員に配布する．

2.

会費を滞納し，かつ催促に応じないとき．

学会賞等（学会賞，研究奨励賞，功労賞）

3.

死亡，または法人が解散したとき．

を設け，環境変異原の分野ですぐれた研究

4.

本会の名誉及び信用を甚だしく傷つけ，あを行った会員および将来の成果が期待され

るいは本会則に違反し，評議員会で除名のる会員（原則として個人）に授与する．

決議がなされたとき．

国際環境変異原学会連合に加入し，国際協第5章

力に必要な活動を行う．
5.

て購入配付する．
6.

第15条

本会には次の役員（理事および監事）および評

その他公開シンポジウムの開催，研究会の議員を置く．

支援など本会の目的を達成するために必要

1.

第4章

会

員
第16条

第7条

理事 11 名（うち，会長 1 名，会長指名理事
2 名）

な活動を行う．

第6条

役員および評議員

Mutation Research 等の英文誌をまとめ2.

監事 2 名3.

評議員 40 名以上

評議員のうち 30 名は正会員の選挙により，正会本会の会員は，正会員，学生会員，賛助会員，

員から選出する．それ以外の評議員（推薦評議

購読会員および名誉会員とする．

員と称す）は，正会員 3 名以上，評議員，また正会員は，本会の目的に賛同し，環境変異原のは理事の推薦により，理事会で正会員歴ならび

に学会活動歴を審査の上，評議員会で承認を得
第6章て選出される．
第17条

第29条

事）2 名を指名する．

第21条

総会は，正会員をもって構成し，大会開催時に
年 1 回開催される．

第31条

総会において会則の改廃制定，予算・決算の承会長は本会を代表し，会務を掌握し，理事会，認，その他評議員会において審議した重要事項

評議員会および総会を招集する．また，評議員の承認を行う．

会および総会において主たる会務について報告
第20条

第30条

会長は選出された理事 9 名の互選によって定める．

本会の会議は，総会，評議員会，および理事会とする．

正会員から選出する．会長は理事（会長指名理

第19条

議

理事のうち 9 名および監事 2 名は選挙で選出された評議員（推薦評議員は除く）の選挙により，

第18条

会

第32条

評議員会は原則として年 2 回開催する．評議員をしなければならない．

会（臨時評議員会を含む）は評議員総数の過半

会長および理事は理事会を構成し，会務を執行

数（委任状を含む）をもって成立し，出席者のする．会務執行のために理事会には，総務，会過半数の賛否をもって議決する．評議員会の議

計，広報，国際協力，企画，編集，表彰人事，

長は，会長または会長が指名した者が務める．

および書記担当理事を置く．

理事は評議員会に出席できるが，議決には参加

監事は本会の財産の状況，および理事の業務執できない．

行の状況を監査し，不整の廉あることを発見し第33条報告する．また，監事は理事会，および評議員

会長は総数の 1/3 以上の評議員の要請があるときは臨時評議員会を開催しなければならない．

たときにはこれを評議員会および総会において第34条

理事会は理事の過半数の出席をもって成立し，出席者の過半数の賛否をもって議決する．

会に出席して意見を述べることができる．ただし，理事，各種委員会委員を兼ねることはでき

第7章

ない．
第22条

評議員は評議員会を構成し，会務を審議する．

第23条

役員および評議員の任期は選出された年の翌年

第24条

総務担当理事の担当事項は次の通りとする．
1.

会員の入退会に関すること．

たは増員により選任された役員および評議員の2.

会則等制度，規則に関すること．

任期は，補欠の場合は前任者の残任期間とし，

3.

総会，評議員会，および理事会に関すること．

増員の場合は現任者の残任期間とする．

4.

役員および評議員の選挙に関すること．

役員および評議員は，再任されることができる．

5.

事務所との連絡．

ただし，会長は原則として 1 期をもって限度と6.

研究会等関連事業全般にわたること．

する．また，選挙で，得票数上位 9 名に入った

7.

関係委員会に関すること．

前理事は 5 名以内まで，第 25 条の限度内で再任

8.

その他，他の理事担当事項に入らない事項．

第36条

会計担当理事の担当事項は次の通りとする．

理事および監事は連続 2 期，生涯 4 期をもって

1.

予算，決算に関すること．

限度とする．ただし，会長指名理事は生涯 1 期

2.

旅費の算出．

しか指名されず，この 1 期は生涯 4 期に含まれ3.

Mutation Research 等英文誌購入，配布に関すること．

る．
第26条

4.

会長は必要に応じ，理事会の承認を得て，会長
または担当理事を含む委員会を設けることがで

第37条

る．委員の任期は会長の任期に合わせて 2 年とインターネット「ホームページ」の開設等
広報に関すること．

し，再任は妨げない．委員会委員長には，会長

2.

各種団体との連絡調整に関すること．

が就任するか，または会長が担当理事か適当な

3.

学会誌の広告に関すること．

委員に委嘱する．

4.

名簿の作成，配布に関すること．

大会会長は理事会の推薦に基づき評議員会の承5.

会員数の増強に関すること．

6.

関係委員会に関すること．

認を得て選出される．
第28条

関係委員会に関すること．

広報担当理事の担当事項は次の通りとする．
1.

きる．委員は理事会の承認を得て会長が委嘱す

第27条

務

の 1 月 1 日から 2 年間とする．ただし，補欠ますることができる．
第25条

第35条

会

大会会長は大会を主宰し，総会の議長となる．

第38条

国際協力担当理事の担当事項は次の通りとす
る．

1.

3.

国際環境変異原学会連合および国際会議事関係委員会に関すること．

務局との連絡に関すること．
2.
第39条

第40条

第8章

関係委員会に関すること．

1.

公開シンポジウムの企画，開催に関すること．

2.

本会の事業全般の企画に関すること．

3.

関係委員会に関すること．

第43条
第44条

著作権に関すること．

3.

関係委員会に関すること．

本会の経費は，本会会費，各種補助金，寄付金，
て充てる．

第45条

2.

本会の会計年度は 1 月 1 日に始まり，12 月 31 日

事業に伴う収入，財産から生ずる収入等をもっ学会誌等の企画，編集，出版および配布に関すること．

収支の予算および決算は，評議員会および総会の承認を得なければならない．

表彰人事担当理事の担当事項は次の通りとする．
1.

付

則

学会賞等および学会の推薦を必要とする研1.

本会則は平成 12 年 1 月 1 日より施行した．

究助成金または賞等の候補者の公募，審査，

2.

第 3 章第５条の 3，第 41 条の 1 については，平成
13 年 10 月 21 日に改定した．

選考，推薦に関すること．

第42条

計

に終わる．

編集担当理事の担当事項は次の通りとする．
1.

第41条

会

企画担当理事の担当事項は次の通りとする．

2.

名誉会員の選考，その他表彰に関すること．

3.

関係委員会に関すること．

3.

正会員，学生会員，賛助会員，および購読会員の会費は，それぞれ年額 7,000 円，5,000 円，50,000

書記担当理事の担当事項は次の通りとする．

円，および 10,000 円とする．ただし，Mutation

1.

理事会の議事を記録し，会長および理事のResearch 等の英文誌の配布を希望するものは，会

承認後，公表すること．

費の他に別途定める購読料を本会へ前納するもの

評議員会および総会の議事を記録し，公表

とする．

2.

すること．

日本環境変異原学会細則担当理事を含む）4 名が行う．開票には当該委
第1章

総員 3 名以上と監事 1 名以上が立ち会う．

則
第5条

第1条

日本環境変異原学会細則（以下細則という）は議員によって原則として先ず理事を選出し，次日本環境変異原学会会則（以下会則という）の目的を遂行するために必要な細目を定める．
第2条

まず評議員の選挙を行い，30 名を選出し，新評に監事を選出する．

第6条

会長，理事，監事には会則第 24，25 条により細則の改廃制定は理事会で審議・議決し，評議就任任期数に制限があるが，任期途中に，やむ

員会の承認を得るものとする．

をえず辞任したり，補充就任した場合，その期間は 1 期と計算しない．

第2章

会員資格の喪失
第4章

第3条

評議員の選出

会則第 14 条第 2 項は下記のように適用する．
会費を 2 年間滞納した会員は学会誌等を受け取第7条

る資格を失い，3 年間未納の場合は会員資格を

行う．
第8条

喪失する．

評議員の選挙に関する事務は選挙管理委員会が
選挙管理委員会は選挙人（正会員）名簿および被選挙人（正会員）名簿を作成し，公表しなけ

第3章

選

挙

ればならない．
第9条

第4条

投票は，被選挙人の中から 6 名，または 6 名以

会則に基づく選挙に関する事務は，会則第 26 条

下を連記し，無記名小封筒に入れ，さらに記名によって決められた選挙管理委員会委員（総務

した封筒で郵送することによって行う．

第10条
第11条

当選は得票数順に 30 名とし，得票同数のときは返し，過半数得票者が出れば決定とするが，過選挙管理委員による抽選により決定する．

半数を得たものがいない場合には上位 2 名の決

当選者はやむをえない理由のある場合，選挙管

選投票を行う．

理委員会宛にその旨を書面に付して提出し，辞第8章

退することができる．辞退の申し出は告示を受

学会賞等の規定

けてから 1 週間以内にしなければならない．
第12条

当選者が辞退した場合は次点者を繰り上げて当

第25条

第13条

日本環境変異原学会学会賞
1.

選とする．

日本環境変異原学会学会賞(以下，学会賞選挙で選出された評議員の定数に欠員が生じたと略)は，学会員のうち，環境変異原研究

場合には任期途中に補充はしない．ただし，推分野における業績がきわめて顕著であり，薦評議員を加えて合計 40 名未満になったときかつ本学会の進歩発展に多大な寄与をしたには，推薦評議員を追加することにより 40 名以者(原則として個人)に対して授与される．
2.

上にする．

学会賞受賞候補者推薦募集要項は，会報誌（Jems News）に掲載するものとし，学会

第5章

理事の選出

正会員によって推薦された受賞候補者は，学会所定の推薦申請書用紙を使用し，必要第14条

理事の選挙に関する事務は選挙管理委員会が行事項を記入の上，推薦者を経て指定期日以内に学会長宛申請するものとする．

う．
第15条

投票は，選挙で選出された 30 名の評議員が，正第16条

当選は得票数順に 9 名とし，得票同数のときは

3.

会員の中から 3 名連記無記名で行う．

第17条

持つ学会員に限られる．
4.

学会賞の授賞件数は，毎年 1 件以内とし，選挙管理委員による抽選により決定する．

選考に関しては表彰人事委員会がこれにあ当選者が辞退した場合は次点者を繰り上げて当たり，学会賞の選考の結果に関しては，理選とする．次点者が 2 名以上のときは選挙管理事会と評議員会の承認を得るものとする．
5.

委員による抽選により決定する．
第18条

学会賞受賞候補者は，5 年以上の会員歴を名により補充する．

第26条

日本環境変異原学会研究奨励賞
1.

第6章

受賞者には賞状ならびに副賞を贈呈し，総会の席上で表彰するものとする．

理事の定数に欠員が生じた場合には，会長の指日本環境変異原学会研究奨励賞(以下，研究奨励賞と略)は，学会員のうち，環境変監事の選出

異原研究分野において顕著な寄与をする発
第19条
第20条

監事の選挙に関する事務は選挙管理委員会が行表を行い，かつ将来の研究の発展を期待しう．

得る者(原則として個人)に対して授与され投票は，選挙で選出された 30 名の評議員が，正る．

会員の中から単記無記名で行う．
第21条
第22条
第23条

2.

研究奨励賞受賞候補者推薦募集要項は，会

当選は得票数順に 2 名とし，得票同数のときは報誌（Jems News）に掲載するものとし，

選挙管理委員による抽選により決定する．

学会正会員によって推薦された候補者は，当選者が辞退した場合は次点者を繰り上げて当学会所定の申請用紙(推薦書ならびに申請

選とする．

書)を使用し，必要事項を記入のうえ，推監事の定数に欠員が生じた場合には，評議員に薦者を経て指定期日以内に学会長宛申請すよる投票により補充する．

るものとする．
3.

第7章

研究奨励賞受賞候補者は，3 年以上の会員歴を持ち，かつ募集締め切り日において，会長の選出

満 45 才以下の学会員に限られる．
第24条

会長は選出された理事 9 名の互選によって決定

4.

研究奨励賞の授賞件数は，毎年 2 件程度とするが，原則として，単記無記名の投票によっし，選考に関しては表彰人事委員会がこれて過半数を得たものが就任する．過半数を得たにあたり，研究奨励賞の選考の結果に関しものがいない場合には上位 2 名の決選投票を行ては，理事会と評議員会の承認を得るもの

い，最高得票者が就任する．ただし，上位 3 名とする．

以上が同数の場合は，同数得票者の投票を繰り5.

受賞者には賞状ならびに副賞を贈呈し，総4.

会の席上で表彰するものとする．
第27条

日本環境変異原学会功労賞
1.

を得る．

日本環境変異原学会功労賞（以下，功労賞と略）は，学会員のうち，環境変異原研究

第32条

表彰人事委員会委員長は原則として表彰人事担分野における応用研究，変異原研究を通じ当理事が就任し，表彰人事委員会を召集，主催

た社会貢献および学会の運営への寄与などする．表彰人事担当理事を除いた委員は 6 名とを通じ学会の進歩発展に対する総合的な貢献が顕著なもの（原則として個人）に対し

する．
第33条

て授与される．
2.

功労賞受賞候補者推薦募集要項は，会報誌

委員会は委員長を含む委員 5 名以上の出席によって成立する．

第34条（Jems News）に掲載するものとし，学会委員会における議決
1.

正会員によって推薦された候補者は，学会賛成により可決される．但し，この際白票経て指定期日以内に学会長宛申請するもの

は投票総数に参入しない．
3.

功労賞受賞候補者は，10 年以上の会員歴をが，投票に参加することはできない．

功労賞の授賞件数は，毎年 1 件程度とし，

4.

選考に関しては表彰人事委員会がこれにあたり，功労賞の選考の結果に関しては，理委員会に出席できない委員は，書面を以て審議事項に対し意見を述べることができる

持つ学会員に限られる．

5.

投票による審議事項は，出席員の過半数の使用し，必要事項を記入のうえ，推薦者を
とする．

4.

重要とみなされる審議事項については，無記名投票により賛否を問うものとする．

2.

所定の申請用紙(推薦書ならびに申請書)を

3.

選考結果を評議員会・理事会へ報告し承認

会長は本委員会に出席し，意見を述べることができるが，議決には参加できない．

第35条

学会賞等受賞者の選考

事会と評議員会の承認を得るものとする．

本委員会委員は学会賞等の推薦者となることは受賞者には賞状ならびに副賞を贈呈し，総できず，受賞候補者として推薦された場合は，会の席上で表彰するものとする．

当該賞の選考に参加することはできない．学会
賞，研究奨励賞，功労賞候補者の会員歴の適合性を確認した後に，投票による最終選考に先立

第9章

委員会の運営って，各候補者につき，予め配布された資料に基づき，下記に従い研究内容等に関する討議を

第28条

第29条
第30条

委員および委員長は会則第 26 条によって会長

行う．

が委嘱する．委員会委員長は委員会を招集，主1.

学会賞受賞候補者について催する．

研究の独創性委員長は委員会開催通知を委員全員，会長，総研究の高度性務担当理事，および会計担当理事に送付する．

研究の普遍性委員長は委員会開催に必要な最小の経費を会計

学会の進歩発展に対する貢献
2.

担当理事に要求することができるが，その採否

研究奨励賞受賞候補者については会計担当理事により，本学会の予算の範囲内

研究の独創性とする．

研究の高度性研究の将来性第 10 章

表彰人事委員会の運営

学会における活動状況
3.

第31条

功労賞受賞候補者について委員会は下記の事項につき担当する．

研究の応用性1.

学会賞等，他団体の助成金または賞等，名学会における研究発表などの活動状況

誉会員の推薦に関する書類の提出日を定

変異原研究を通じた社会貢献

め，編集委員会委員長に Jems News への掲

学会に対する貢献（評議員・理事・年会

載を依頼する．
2.
3.

表彰人事委員会委員に関係書類を配布す

長などとしての貢献）
第36条

学会賞等受賞者の選出

る．

学会賞等の受賞候補者について，「信任投票」

表彰人事委員会を開催し受賞者を選考す(可または否を記入)を行い，有効投票数の過半る．

数を獲得したものを信任された候補者とみなし，これらの者についてのみ以下の手続きに従

第39条

編集委員会は委員長および 6 名の委員より構成って受賞者を決定する．

される．委員は委員長の意見を参考にし，理事

1.

会の承認を得て会長が委嘱する．委員の任期は学会賞，功労賞（1）信任された候補者が 1 名の場合，この者を

1 期 2 年とし，原則として連続 2 期とする．

受賞者とする．
（2）信任された候補者が 2 名の場合，「単記名方式投票」による上位得票者を受賞者とす付
1.

（3）候補者が 3 名以上の場合

2.

「単記名方式投票」の併用により受賞者を下記の方法に従い選出する．
「序列記入方式投票」においては，候補
者名を連記した投票用紙の各候補者に対して序列を記入する．但し，複数の候補者に対して同一序列を記入してはならない．序列は点数化し，例えば候補者が 3 名のとき，最高序列点を 3，次いで 2，1 点とし，有効票について，各候補者の得点を集計する．
「序列記入方式投票」において，最多序列得点者が 1 名の場合，この者を受賞者とする．
最多序列得点者が 2 名の場合，これらの者について「単記名方式投票」を行い，上位得点者を受賞者とする．
研究奨励賞（1）信任された候補者が 2 名以下の場合，この者またはこれらの者を受賞者とする．
（2）信任された候補者が 3 名以上の場合「序列記入方式投票」を基本とする下記の方法に従い選出する．
最多序列得点者が 1 名，および 2 位の得点者が 1 名の場合，または最多序列得点者
が 2 名の場合，これらの者を受賞者とする．
最多序列得点者が 1 名で，2 位の者が 2名の場合，最多序列得点者を受賞者とし，
これら 2 位の者に対しては，「単記名方式
投票」を行い，上位者を受賞者とする．
学会賞等の英文名は学会賞を JEMS Award，研究奨励賞を JEMS Achievement Award，功労賞
JEMS Service Award とする．必要に応じ，
JEMS のあとに（Japanese Environmental Mutagen Society），また Award のあとに（西暦年号）を付すことができる．
第 11 章
第38条

編集委員会の運営

編集委員会委員長は原則として編集担当理事が就任し，編集委員会を召集，主催する．

第 1 章，第 3 章，第 9 章，第 11 章は平成 12 年 1 月
1 日より施行した．

「序列記入方式投票」または，これと第37条

本細則の第 4 ─ 7 章は新会則のもとでの選挙のため
平成 11 年 6 月 1 日より施行した．

る．

2.

則

3.

第 2 章，第 8 章，第 10 章は平成 14 年 1 月 1 日より施行した．

日本環境変異原学会
役員名簿（平成 14 年度）
会
理

監

長

林

真

事

評議員名簿（平成 14 〜 15 年度）
赤

沼

三

恵

荒

木

明

宏

宇

野

芳

文

敏

博

大

塚

雅

則

宏

鎌

滝

哲

也

大

澤

浩

一

太

田

総務担当

田

中

憲

穂

大

西

克

成

葛

西

会計担当

若

林

敬

二

川

西

正

祐

木

苗

直

秀

後

藤

純

雄

広報担当

布

柴

達

男

佐々木

有

島

田

弘

康

清

水

英

佑

国際協力担当

長

尾

美奈子

下

位

香代子

祖父尼

俊

雄

高

橋

和

彦

企画担当

木

苗

直

秀

田

中

憲

穂

出

川

雅

邦

寺

尾

良

保

葛

西

宏

長

尾

美奈子

中

島

圓

糠

谷

東

雄

編集担当

森

田

健

布

柴

達

男

根

岸

和

雄

根

岸

友

恵

表彰人事担当

川

西

正

祐

能

美

健

彦

林

真

早

津

彦

哉

書記担当

赤

沼

三

恵

藤

川

和

男

降

旗

千

恵

本

間

正

充

後

藤

純

雄

松

元

郷

六

宮

川

誠

望

月

正

隆

菊

川

清

見

森

秀

樹

森

雄

森

田

烈

矢

嶋

信

浩

山

添

康

吉

川

若

田

明

裕

若

林

事

企画委員

編集委員

表彰人事委員

広報委員

渡

部

木

苗

直

秀（委員長）

宇

野

芳

文

葛

西

降

旗

千

恵

本

間

正

充

森

田

赤

沼

三

恵

高

橋

和

彦

矢

嶋

信

浩

山

田

雅

巳

若

田

明

裕

若

栗

川

西

正

祐（委員長）

太

田

敏

博

澁

谷

徹

下

位

香代子

長

尾

美奈子

能

美

健

彦

早

津

彦

哉

布

柴

達

男（委員長）

赤

沼

三

恵

荒

木

明

宏

宇

野

芳

文

田

中

憲

穂

宏

健（委員長）

忍

林

真

兵

庫

森

田

淳

志

若

林

敬

田

辺

健一郎

健
二

幸
敬

健
邦

衛

二
（五十音順）

入会手続きのご案内
入会申込書に必要事項をご記入の上，年会費とともに現金書留にて事務局宛てお送りください．
年 会 費

7,000 円（正会員）
5,000 円（学生会員）

事業年度 （1 月 1 日〜 12 月 31 日）（お申し込みについて）
1. 入会申込書に必要事項を楷書でご記入の上，年会費とともに現金書留にて事務局までお送りください．
2. 正会員は評議員の推薦を必要とします．推薦者がおられない場合は，事務局にご相談ください．
3. 学生会員は指導教官の推薦と在学証明書を必要とします．翌年の 3 月 31 日まで会員として登録されます．
4. 年度末に入会申し込みをされる場合で，翌年度から入会希望の場合は，その旨お知らせください．
5. 学会誌は入会後に発行した号からお送りしますが，当該年度で入会前の学会誌をご希望の場合は事務局までご連絡ください．
6. 住所変更（学会誌送付先の変更）の際は，会員番号（学会誌等送付する際，宛名ラベルに印刷されています），氏名，新・旧住所をご記入の上，事務局宛，書面にてご連絡ください．

日本環境変異原学会〒 170 ― 0003

東京都豊島区駒込 1 ― 43 ― 9

駒込 TS ビル 3F
電話

財団法人

口腔保健協会内

03 ― 3947 ― 8891（代） FAX

03 ― 3947 ― 8341

環境変異原研究 投稿規定
1． 掲載論文

4． 論文原稿の送付

環境変異原研究に関する未発表の「総説」，「一般論

論文原稿は 5 部と本誌に綴じ込みの「投稿論文チェ

文」
，「短報」
，および「特別企画（受賞講演）」，「論ックリスト」を，下記に（簡易）書留便または宅配説」，「資料・情報」などを掲載する．なお，投稿論便で送付する．

文の採否は編集委員会の審査により決定する．

〒 170 − 0003

「総説」 は，一つのテーマに関連する多くの研究論

東京都豊島区駒込 1 − 43 − 9

文の総括，評価，解説などである．原則として編集委員会より寄稿を依頼する．

日本環境変異原学会誌編集係「一般論文」 は，変異原に関する独創的研究の原著

Tel．03 − 3944 − 2591

論文で，それ自身独立して価値ある結論あFax．03 − 3947 − 8073

るいは事実を含むものとする．陰性データも受け付ける．

駒込 TS ビル

インテルナ出版

5． 著作権
本誌に掲載された記事，論文などの著作権は日本環「短報」 は，新しい技術の紹介や価値あるデータを

境変異原学会に帰属するものとする．従って，本会含む短い報告とする．陰性データも受け付が必要と認めた場合は転載し，また外部から引用のける．

申請があった場合には，編集委員会において検討の「論説」 は，一つのテーマに関連する多くの研究論上許可することがある．ただし，著作者自身が自分文の総括，評価，解説などで，会員からのの記事，論文などの一部の複製，翻訳などの形で利投稿によるものとする．

用することを妨げるものではない．しかし，著作者「資料・情報」は，環境変異原に関する調査の結果自身であっても，全文を複製の形で他の著作物に利などをまとめたもの，および公開シンポジ

用する場合には，事前に文書にて学会長に申し出をウム，研究会の要旨などとする．
（Letter to Editor も受け付ける．）
2． 投稿資格
共著者のうちの 1 人は日本環境変異原学会会員でなければならない．ただし，招待寄稿の場合にはこの限りではない．
3． 論文原稿の書き方
論文原稿の用語は英語または日本語とし，最新の執筆規定に従い簡潔にわかりやすく書く．総説，一般
論文，論説は，写真・図表を含めて刷り上がり 8 頁

行い，許諾を求めなければならない．
6． 校正
著者校正は原則として原稿に対する誤植の訂正に限る．原稿にない加筆・変更はしないこと．
7． 著者負担金
1） 投稿料は無料とする．ただし頁数の超過分や多額の経費を要する図表の実費は著者負担とする場合がある．
2） カラー印刷等の特殊印刷のため付加的に発生する費用は著者負担とする．

以内．短報は 4 頁以内とする．ただし規定の頁数を3） 別刷りはすべて著者負担とする．別刷り希望者

超えることが明らかな場合は，事前に編集委員に相は著者校正時に添付する申込書に 50 部単位で

談する．

申し込む．

編集委員会からのお願い「環境変異原研究 Environmental Mutagen Research」への投稿論文の，投稿から印刷までの時間の短縮を目指し，投稿論文の査読を評議員にお願いすることになりました．それに伴い，投稿規定の見直しも行い，今後は年大会での発表と同様に，投稿論文の分野を著者に申告していただき，同一分野を登録していただいている評議員に査読していただくことになります．今回投稿時のチェックリストをお届けしますので，投稿論文と一緒に必ずお送り下さい．
現在 7 名の編集委員が分担して学会誌，Jems News の編集作業を行っていますが，それぞれ自分の仕事を抱え
ボランティアとして活動しています．そこで，会員の皆様の積極的な雑誌作りへの協力として，年大会で発表されたものを一報でも多く論文にまとめ，本学会誌に投稿していただきますようお願い致します．また，Jems Newsへの投稿，情報，意見，公募等もお願いします．
本誌の将来像に関する議論を進めております．英文誌化の話も出ており，編集委員を中心に検討を始めたとこ
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1．用語は日本語または英語とする．
2．原稿は原則としてワープロを用いて作成する．
日本文の原稿：

10．図と写真は原図またはキャビネ大の光沢写真版とし，裏面に Fig. 1, 2, …および上下を鉛筆書きする．
11．表は上部に一連の番号 Table 1, 2, …と英文の表題を原稿は A4 判用紙に 1 行約 40 字，1 頁 30 〜 31 行

記入する（文頭のみ大文字）．脚注を要するときに表で印字する（刷り上がりの約 1/2 頁に相当する）．

示の語句の右肩にａ, ｂ, ｃ, …を付記し，表の下欄

ただし，要約は英文（300 語以内）とする．また，別に題名，著者名（フルネーム），所属機関名ならびに所在地を英文で付ける．
英文の原稿：

外にそれぞれの説明を記す．
12．本文中の文献引用は著者名および年号をもってする．
13．参考文献は筆頭著者名のアルファベット順に配列し，原稿は A4 判用紙にダブルスペースで印字（12 ポ雑誌の省略名は Chemical Abstracts の記載方法に従イント）する．1 頁 25 〜 27 行を標準とする．原う．記載順序は著者氏名，（年），題名，雑誌名，巻，稿は著者の責任において英語の添削訂正を受けた頁（単行本の一部引用の場合は著者氏名，（年），題名，ものに限る．

編者名，書名，発行所，発行地，頁）の順とする．

3．論文の記述は，第 1 頁は表題，著者名，所属および単行本そのものを引用する場合は，編者名あるいは所在地，第 2 頁は英文の要約（Summary）およびキ

著者名，（年），書名，発行所，発行地の順とする．

ーワード（英文 5 語以内，固有名詞や遺伝子名など

文献の記載方法は下記の例に従う．

で大文字の使用が必要な場合を除き，原則として小Ames, B.N., J. MaCann and E. Yamasaki（1975）

文字表記とする），第 3 頁以下，緒言（Introduction），

Methods for detecting carcinogens and muta-

実験材料および方法（Materials and Methods），結gens with the Salmonella/mammalian-micro-

果（Results），考察（Discussion）または結果および考some mutagenicity test, Mutat. Res., 31, 347-

察，結語（Conclusion），謝辞（Acknowledgments），
参考文献（References），表，図の説明および図の順

364．
Ashby, J., F.J. de Serres, M. Draper, M. Ishidate

序とする．ただし，総説の記述は，第 3 頁以下，緒Jr., B.H. Margolin, B. Matter and M.D. Shelby

言，1. ……，2. ……，結語，謝辞，参考文献，表，（1985）Overview and conclusion of the IPCS

図の説明および図の順序とする．なお図と表の説明

collaborative study on in vitro assay systems,

文はすべて英文とする．
4．学名，遺伝子記号などはイタリックとし，その他まぎらわしい記号については原稿に適宜指示を与える．
5．化学物質名は原則として英語とし，一般名を用いる．
また，化学物質の CAS 登録番号を記載する．文中に用いる英語の単語あるいは句は固有名詞を除いて小文字で書きはじめる（文頭の場合は大文字）．
6．数字は算用数字を用い，単位は慣用による省略記号を用いる．
7．略語を使用するときは，論文中にはじめて使用する
ときに完全な語とその略語を括弧内に示す．
8．句読点はカンマ（，）
およびピリオド（．）とする．
9．表，図は本文と別にし，それらの挿入箇所を本文のIn ： J. Ashby, F.J. de Serres et al.（Eds）,
Evaluation of Short-Term Tests for Carcinogens, Elsevier, Amsterdam, pp. 117-174．
Friedberg, E.C., G.C. Walker and W. Siede（1995）
DNA Repair and Mutagenesis, ASM Press,
Washington, D.C.
藤川和男，梁治子，近藤宗平（1984）
ハエの翅毛

スポットテスト―近ごろ注目されている短期試
験法，環境変異原研究，6，107-113．
佐々木正夫（1983）環境変異原と染色体異常，外村晶（編），染色体異常，朝倉書店，東京，pp．
107-113．
松影昭夫（編）（1996）DNA 複製，修復と発癌，羊土社，東京．

右余白に明示する．グラフ，写真，線画等はすべて図とし，一連の番号 Fig. 1, 2, …を付し，説明文（英文）
を別紙に添える．

（改訂

2000 年 12 月）

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送付前のチェック項目□ 投稿規定，執筆規定に沿っていますか（特に引用文献）□ 英文校閲はお済みですか□ 投稿論文は 5 部ありますか編集後記
今さらのようですが，環境変異原学会の編集委員は専属ではありません．本業の傍ら，イ
ンテルナ出版の方々に気を揉んでいただきながらの作業です．いつも感心するのは，インテルナの方が連絡を下さるタイミングです．ぎりぎりまで待って，かつ，言われてから手をつけても間に合う…そんな時期です．今号に「特別企画」と銘打って登場した 8thICEM の記
事は Jems News から移ってきたものです．どうぞお楽しみください．各編集委員のノルマ
は Jems News と環境変異原研究各 2 号です．今号は私の記念すべき卒業作品となりました．
雑誌編集に興味のある方，私の任期満了の折にはぜひ！交代してください．
（M.Y.）
担当編集委員

編

集

委

山田

雅巳

員

森田

健（委員長）（2002 ─）〒 151 ─ 8566 東京都渋谷区千駄ヶ谷 4 ─ 6 ─ 15 GSK ビル
グラクソ・スミスクライン株式会社
Tel ： 03 ─ 5786 ─ 5067 Fax ： 03 ─ 5786 ─ 5223
E-mail ： takeshi.5.morita@gsk.com

矢嶋

信浩（1999 ─）〒 485 ─ 0059 愛知県小牧市小木東 3 ─ 45
雪印ラビオ株式会社・開発研究所
Tel ： 0568 ─ 77 ─ 3235 Fax ： 0568 ─ 76 ─ 1046
E-mail ： n-yajima@snowlabio.com

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山之内製薬株式会社・安全性研究所
Tel ： 03 ─ 5916 ─ 2177 Fax ： 03 ─ 3960 ─ 5141
E-mail ： wakata@yamanouchi.co.jp

山田

雅巳（1999 ─）〒 158 ─ 8501 東京都世田谷区上用賀 1 ─ 18 ─ 1
国立医薬品食品衛生研究所・変異遺伝部
Tel ： 03 ─ 3700 ─ 1141
（ext 282）
Fax ： 03 ─ 3707 ─ 6950
E-mail ： myamada@nihs.go.jp

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E-mail ： mie.a@viola.ocn.ne.jp

和彦（2002 ─）〒 467 ─ 8603 愛知県名古屋市瑞穂区田辺通 3 ─ 1
名古屋市立大学大学院薬学研究科
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Tel ＆ Fax ： 052 ─ 836 ─ 3780
E-mail ： takahasi@phar.nagoya ─ cu.ac.jp

評
赤沼 三恵
大塚 雅則
木苗 直秀
下位香代子
寺尾 良保
根岸 和雄
藤川 和男
望月 正隆
山添
康

荒木 明宏
大西 克成
後藤 純雄
祖父尼俊雄
長尾美奈子
根岸 友恵
降旗 千恵
森
秀樹
吉川 邦衛

議

員

宇野 芳文
葛西
宏
佐々木 有
高橋 和彦
中嶋
圓
能美 健彦
本間 正充
森
幸雄
若田 明裕

環境変異原研究第 24 巻平成 14 年 5 月 24 日

印

刷

平成 14 年 5 月 31 日

発

行

発

行

者

大澤
鎌滝
島田
田中
糠谷
林
松元
森田
若林

浩一
太田 敏博
哲也
川西 正祐
弘康
清水 英佑
憲穂
出川 雅邦
東雄
布柴 達男
真
早津 彦哉
郷六
宮川
誠
健
矢嶋 信浩
敬二（五十音順）

第1号

2002 年

日本環境変異原学会

発行責任者

林

製

インテルナ出版作真環境変異原研究

Environmental Mutagen Research目原Vol. 24 No. 1 2002

次著
Genotoxicity of extracts of Japanese traditional herbal medicines（Kampo）
Makoto Katami, Haruo Kuboniwa, Shunichi Maemura and Toshihiko Yanagisawa ………………………… 1
Effect of a p53 codon 237 mutation on X─ray induced HPRT mutations
Shigeko Morimoto, Masamitsu Honma and Fumio Yatagai …………………………………………………… 17
A chromosomal aberration study of fibrillated PVA fiber in cultured mammalian cells
Takashi Hayashi and Fumiaki Arai ……………………………………………………………………………… 23
Antioxidative effects of fluvastatin and its metabolites in cultured human endothelial cells using single cell gel
electrophoresis
Tomonori Aoki, Atsumune Imaeda and Hisamitsu Nagase …………………………………………………… 29
Failure of stevioside to induce micronucleus formation in the rodent bone marrow cells
Temcharoen, P., S. Klongpanichpak, M. Suwannatrai, S. Apibal and C. Toskulkao ………………………… 37

特別企画
8th ICEMを終えて…………………………………………………………………………………………………… 43
書

評 …………………………………………………………………………………………………………………… 61

付 記
日本環境変異原学会

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