Vol. 23-2 Contents
光遺伝毒性特集（特別編集委員 田中憲穂，島田弘康）
特集号によせて
Preface
田中憲穂……………………………………………………………………………………………………………45
Noriho Tanaka
総 説
Review
Photochemical genotoxicity testing：experience with the Ames test and the in vitro chromosomal aberration assay
Gocke E and Chetelat AA……………………………………………………………………………………47
Acute phototoxicity testing
Spielmann H ……………………………………………………………………………………………………53
光増感剤によるDNA損傷の化学
中西郁夫，宮田直樹………………………………………………………………………………………………65
In vitro 光毒性評価における活性酸素種の影響
Effects of reactive oxygen species in in vitro phototoxicity assays
岡本裕子……………………………………………………………………………………………………………73
Yuuko Okamoto
フルオロキノロン系合成抗菌剤の光遺伝毒性
Photogenotoxicity of fluoroquinolone antibacterial agents
島田弘康……………………………………………………………………………………………………………83
Hiroyasu Shimada
原 著
Original Article
Photogenotoxicity and apoptosis in human HaCaT keratinocytes induced by 8-methoxypsoralen and lomefloxacin
Zhang J, Kersten B, Kasper P and Muler L ………………………………………………………………89
Effects of visible light absorbing chemicals in the photo-micronucleus test in Chinese hamster V79 cells
Kersten B, Kasper P, Brendler-Schwaab SY and Muler L ……………………………………………97
A note on artificial induction of mutation upon testing 7,12-dimethylbenz[a]anthracene mutagenicity under
fluorescent light in the absence of microsomal enzymes
Takahashi K, Asanoma M, Miyabe M and Watanabe-Akanuma M …………………………………103
The rapid screening of photogenotoxic compounds using photo plasmid-relaxation assay
Nakagawa Y, Takigawa Y and Tanaka N …………………………………………………………………107
資料・情報
医薬品における光遺伝毒性試験
Photogenotoxicity testing for pharmaceuticals
森田 健，若田明裕………………………………………………………………………………………………119
Takeshi Morita＊ and Akihiro Wakata
________________________________________Vol. 23-2 Summary
Preface
Noriho Tanaka
Cell Toxicology, Hatano Research Institute, Food and Drug Safety Center
特集号によせて
田中 憲穂
特別編集委員 （財）食品薬品安全センター秦野研究所
近年，化粧品，医薬品，工業原料，環境汚染物質の中には，光照射下の光化学反応によって遺伝毒性が発現す
る事が注目されるようになり，そのメカニズムの研究，試験法の開発とその評価試験などが行われ，それにとも
なって，業界や行政による対応等もみられるようになってきました．今回の特集号では，この分野で第一線の研
究を進めておられる内外の先生方に，総説，原著論文を含めて多数の寄稿をいただき，極めて内容の濃い特集号
を組む事ができました．
海外から寄稿いただきました先生方のうち，Horst Spielmann 先生（Germany）は，光毒性試験の分野で，早
くからヨーロッパを中心に試験方法の提案とその評価試験を実施され，現在Spielmann 先生を中心にOECDの光
毒性試験のガイドラインが提案されています．Spielmann 先生には光毒性試験法についての総説を寄稿いただき
ました．Elmar Gocke 先生（Switzerland）は，光遺伝毒性を検出するためのAmes 試験の条件設定に早くから取
り組まれ，先年ワシントンで開催されたIWGT（International Workshop on Genotoxicity Test Procedures）の会
議でも，光遺伝毒性試験法の問題点を中心になってまとめられました．Elmar Gocke 先生には光Ames 試験と光
染色体異常試験についての総説をいただきました．また，ドイツにおいて行政の立場で早くから光遺伝毒性の問
題に取り組まれているLutz Muler 先生（現Switzerland）には，2 編の原著論文を寄稿いただきました．
国内からは，製薬企業の立場でフルオロキノロン系抗菌剤の光遺伝毒性について先進的に取り組んでおられる
島田弘康先生に，また化粧品の分野で光毒性の検出等に取り組まれている岡本裕子先生には活性酸素種の光毒性
発現における役割について，それぞれ寄稿いただきました．一方，中西郁夫先生には光化学的側面から化学物質
による光DNA損傷についての総説をいただきました．また原著論文として，高橋和彦先生にはDMBAの光遺伝
毒性の発現について，中川ゆづき先生にはplasmid-relaxation assay を用いた光（遺伝）毒性の検出法についての
論文をいただきました．さらに，資料・情報として，日本製薬工業協会による日本における医薬品の光遺伝毒性
に関する調査結果を掲載いたしました．
光遺伝毒性の検出は，太陽光に暴露された化学物質のphotodynamic action による相乗的な生物への反応によ
って生じると考えられる光発がん物質の検出に極めて重要であります．地球環境の面からもオゾン層破壊による
紫外線の増大が懸念されている時期でもあり，特に，環境変異原の面からは，環境中に放出されている多環芳香
族炭化水素をはじめとする光遺伝毒性物質の生物への影響が危惧されるところでもあります．本特集号を契機と
して，この研究分野のより一層の進展のきっかけにもなれば，企画者の1 人として大変幸いです．重ねて，寄稿
いただきました諸先生に厚く御礼を申し上げます．
2001 年8 月
________________________________________Review
Photochemical genotoxicity testing: experience with the Ames
test and the in vitro chromosomal aberration assay
Elmar Gocke*, Andr仔-Anne Ch師elat
F. Hoffmann-La Roche Ltd, Basle, Preclinical development
PRNS, bldg 73/215 CH 4020 Basel, Switzerland
Summary
Photochemical genotoxicity can be detected using appropriately adapted versions of the bacterial
mutagenicty test and the in vitro chromosomal aberration assay. For screening programmes it has
been debated whether a photo-clastogenicity assay will be sufficient or whether the photo-Ames test
will be necessary for complementation. In order to facilitate this discussion, we present data from in
house investigations and refer to pertinent literature data showing that there is no clear precedent
of a photochemical genotoxin which is exclusively positive in the bacterial assay. Furthermore,
many of the photogenotoxins show only weak activity in the bacterial assay but are potently genotoxic
in the clastogenicity assay. On the other hand, it might be prudent to include the bacterial
assay because at present there is still a rather limited experience in photochemical mutagenesis
testing.
Keywords: photoactivation, phototoxicity, genotoxicity test battery, reactive oxygen species, radicals
* elmar.gocke@roche.com
Received: April 26, 2001, accepted: May 7, 2001
Environmental Mutagen Society of Japan
Acute phototoxicity testing
Horst Spielmann
National Centre for Documentation and Evaluation of Alternative Methods to Animal Experiments
(ZEBET), Federal Institute for Health Protection of Consumers and Veerinary Medicine (BgVV)
Diedersdorfer Weg 1, D-12277 Berlin, Germany
Summary
Phototoxicity (=chemical phototoxicity) is an acute reaction which can be induced by a single
treatment with a chemical and UV or visible radiation. In vivo, the reaction can be evoked in all subjects,
provided that concentration of chemical and dose of light are appropriate. This acute phototoxic
reactions in skin can be induced by substances applied topically to the skin or via the systemic
route and exposure to UV or visible light. This reaction has to be distinguished from other photosensitized
reactions, e.g. photoallergy, photogenotoxicity and photocarcinogenicity. Acute phototoxic
reactions can be measured in vivo in humans and laboratory animals and in vitro in a wide spectrum
of cell and tissue models. Due to differences in species specificity the acute phototoxicity data
obtained in animal models showed a poor correlation to results from human patch testing.
Therefore, in 1992 the European Commission represented by the EU validation centre ECVAM
decided to develop and validate the most promising in vitro assays for acute phototoxicity in close
co-operation with COLIPA, the European Cosmetic, Fragrance and Perfumery Association. From
1992-1998 a series of joint ECVAM/COLIPA prevalidation, formal validation and special studies
were conducted in order to achieve regulatory acceptance of the most promising in vitro phototoxicity
test both in Europe and at a world wide level. The studies were managed by ZEBET, the national
German validation centre, and showed that a photocytotoxicity assay employing the established
mouse fibroblast cell line 3T3 provided an almost perfect prediction of the phototoxic potential of
chemicals in humans even when testing was conducted under blind conditions in several laboratories.
Therefore, in the year 2000 the 3T3 NRU PT test has officially been accepted by the European
Commission and all EU member states for classification and labelling of chemicals to assess their
acute phototoxic potential. In the present review the potential of the current in vitro and in vivo phototoxicity
tests will be described with particular reference to the underlying mechanisms and the
results obtained in the European validation study of in vitro phototoxicity tests.
Keywords: chemical phototoxicity, in vitro test, 3T3 NRU PT test, phototoxic potential, acute photocytotoxicity
zebet@bgvv.de
Received: June 6, 2001, accepted: July 17, 2001
Environmental Mutagen Society of Japan
In vitro 光毒性評価における活性酸素種の影響
岡本 裕子
（株）コーセー研究本部・基盤技術研究所 〒174-0051 東京都板橋区小豆沢1-18-4
Effects of reactive oxygen species in in vitro phototoxicity assays
Yuuko Okamoto
Fundamental Research Laboratory, KOS Corporation Research ＆ Development Division
1-18-4 Azusawa, Itabashi-ku, Tokyo 174-0051, Japan
Summary
Three in vitro phototoxicity assays - the photohaemolysis assay, the haemoglobin photo-oxidation assay,
and the neutral red uptake assay - were evaluated for use as screening methods in predicting the in vivo phototoxicity
of test substances. The photohaemolysis assay evaluates oxygen-dependent membrane damage.
The haemoglobin photo-oxidation assay detects haemoglobin oxidation from oxyhaemoglobin to
methaemoglobin with UVA irradiation. The neutral red uptake assay evaluates cell survival by assessing the
ability of viable cells to take up neutral red dye. Thirty-three test substances were assessed in this study.
The phototoxicity predicted in each assay was compared with that in guinea pigs and in human. The phototoxicity
predictions made by the in vitro methods were comparatively good. These results suggest that the
three in vitro phototoxicity assays could be used to effectively screen chemicals for phototoxicity.
The effects of reactive oxygen species in two in vitro phototoxicity methods were assessed. Sixteen test
substances that indicated positive reaction in the photohaemolysis assay or the 3T3 neutral red uptake assay
were evaluated by using scavengers. Of the 16 test substances, 12 indicated the production of singlet oxygen.
Nine of these reacted to histidine used as a scavenger of singlet oxygen. In this study we have confirmed
that photodynamic mechanisms play a major role in in vitro phototoxicity reactions. These results
suggest that the photohaemolysis assay and the 3T3 neutral red uptake assay could be used to evaluate the
photodynamic mechanisms of photosensitizing chemicals.
Thus, from the genetic point of view, the production of reactive oxygen species and singlet oxygen responsible
for phototoxicity is closely related to the induction of photochemical genotoxicity.
Keywords ： in vitro photohaemolysis, 3T3 neutral red uptake phototoxicity assay, reactive oxygen species
yu-okamoto@kose.co.jp
受付： 2001 年7 月9 日 受理： 2001 年7 月27 日
･日本環境変異原学会
フルオロキノロン系合成抗菌剤の光遺伝毒性
島田 弘康
第一製薬株式会社 〒104-8369 東京都中央区京橋2-16-1
Photogenotoxicity of fluoroquinolone antibacterial agents
Hiroyasu Shimada
Daiichi Pharmaceutical Co., Ltd., 16-1, Kyobashi 2-Chome, Chuo-ku, Tokyo 104-8369, Japan
Summary
Fluoroquinolone antibacterial agents are widely used for their broad and strong antimicrobial spectrum.
Recent reports on their photogenotoxicity indicated that some fluoroquinolones have potent genotoxicity
under UV or visible light irradiation. This review paper describes the characteristics of photogenotoxicity of
fluoroquinolones, mechanisms, structure-activity relationship and a predictivity of photocarcinogenicity.
Keywords ： photogenotoxicity, fluoroquinolone
shima4dx@daiichipharm.co.jp
受付： 2001 年8 月17 日 受理： 2001 年9 月3 日
･日本環境変異原学会
________________________________________Original Article
Photogenotoxicity and apoptosis in human HaCaT
keratinocytes induced by 8-methoxypsoralen and
lomefloxacin
Zhang, J.a, B. Kerstenb, P. Kasper and L. Mulerc*
Federal Institute for Drugs and Medical Devices (BfArM), Bonn, Germany
Summary
The HaCaT cell line is a spontaneously transformed human epithelial cell line from adult skin,
which retain some differentiation characteristics of human skin cells. These cells are considered to
more closely resemble the in vivo skin condition than other cell lines which are usually employed in
genetic toxicity testing and may therefore be particularly appropriate for in vitro photogenotoxicity
testing. We investigated chromosomal damage as evidenced by micronucleus (MN) formation in
cytokinesis-blocked HaCaT keratinocytes after treatment with 8-methoxypsoralen (8-MOP) or lomefloxacin
plus UV irradiation with a UVB/UVA relationship of 1:30. Our results show that 8-MOP
induced MN in a dose-dependent manner over a concentration range from 1×10-7 to 1×10-4 M using
low and intermediate UVA/UVB irradiation dose levels. Lomefloxacin (LOM) induced MN in
HaCaT cells in the range from 1×10-5 to 1×10-4 M only in conjunction with high UVA/UVB irradiation
doses at which 8-MOP was too toxic to the cells. Both, photoactivated 8-MOP and LOM were
also found to be strong inducers of apoptosis in HaCaT cells, probably as a consequence of their
DNA damaging activities. Apoptosis was analysed at different days of cell culture, i.e., day 3, 7 or 11,
at which HaCaT cells proceed from a proliferation state to differentiation. The highest sensitivity
towards apoptosis was found in day-11 HaCaT cells suggesting that keratinocytes become increasingly
prone to apoptosis during the differentiation process. In summary, our studies indicate that
HaCaT cells are suitable target cells to assess multiple cellular effects, such as clastogenicity, cytotoxicity
and apoptosis in response to UV-irradiated photosensitizers.
Keywords: photogenotoxicity, apoptosis, phototoxicity, psoralen, lomefloxacin
* lutz.mueller@pharma.novartis.com
Present addresses: a U.S. Environmental Protection Agency,
Research Triangle Park, North Carolina
b Max Planck Institute of Molecular Genetics, Berlin, Germany
c Novartis Pharma AG, WSH 2881.2.28, CH-4002 Basel, Switzerland
Received: June 14, 2001, accepted: July 6, 2001
Environmental Mutagen Society of Japan
Effects of visible light absorbing chemicals in the photomicronucleus
test in Chinese hamster V79 cells
Kersten, B.1a*, P. Kasper1, S.Y. Brendler-Schwaab2 and L. M殕ler1b
1Federal Institute for Drugs and Medical Devices, Friedrich-Ebert-Allee 38, D-53113 Bonn, Germany
2Toxicology, Bayer-AG, D-42096 Wuppertal, Germany
Summary
The photon energy of visible light is not sufficient to cause direct damage of DNA. However,
endogenous or exogenous photosensitizers which absorb in the visible light range may have the
potential to damage DNA indirectly. Energy transfer from triplet excited sensitizers to molecular
oxygen seems to be the most important mechanism (type II-photoreactions). Excited singlet oxygen
may directly damage DNA or other cellular structures and/or give rise to other damaging reactive
oxygen species (ROS). The lifetimes of ROS are very short, so that the effects (phototoxic/photogenotoxic)
are influenced by the type of molecular targets which are in the immediate vicinity of
their site of generation. Photogenotoxic effects induced by visible light absorbing compounds in
mammalian cells have been studied only for a few chemicals.
In this study we used the photo-micronucleus assay in V79 cells to investigate photogenotoxic
effects induced following treatment of the cells with 5 visible light absorbing chemicals as well as
following induction of endogenous porphyrin synthesis by 5-aminolevulinic acid. Irradiation was
performed using solar light conditions. Proflavine, neutral red, methylene blue and protoporphyrin
IX induced clear photogenotoxic effects in our hands, whereas acridine was negative for this endpoint.
All compounds reduced the proliferation index following irradiation, which indicates photocytotoxic
effects. Prolonged incubation of V79 cells with 5-aminolevulinic acid (2-6 h) led to clear photogenotoxic
effects in the photo-micronucleus assay probably caused by the time-dependent induction
of intracellular porphyrin synthesis.
Keywords: photo-micronucleus test, photogenotoxicity, phototoxicity, photosensitizer, 5-aminolevulinic acid
* kersten@molgen.mpg.de
Present address: a Max Planck Institute of Molecular Genetics,
Ihnestrasse 73, D-14195 Berlin, Germany
b Novartis Pharma AG, Toxicology/Pathology, WSH 2881.2.28,
CH-4002 Basel, Switzerland
Received: June 21, 2001, accepted: July 4, 2001
Environmental Mutagen Society of Japan
A note on artificial induction of mutation upon testing 7,12-
dimethylbenz[a]anthracene mutagenicity under fluorescent
light in the absence of microsomal enzymes
Kazuhiko Takahashi1＊, Masaharu Asanoma2, Masaki Miyabe2
and Mie Watanabe-Akanuma3
1Faculty of Pharmaceutical Sciences, Nagoya City University
3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan
2Nagoya City Public Health Research Institute, 1-11 Hagiyama-cho, Mizuho-ku, Nagoya 467-8615, Japan
3Institute of Environmental Toxicology, 2-772 Suzuki-cho, Kodaira, Tokyo 187-0011, Japan
Summary
When mutagenicity assay was done under fluorescent lamps commonly used for room lighting,
7,12-dimethylbenz[a]anthracene (DMBA) showed mutagenicity without any enzymatic activation.
The light between wavelength of 370 and 450 nm is responsible for the photoactivation of DMBA,
which is corresponding to the absorption peak of DMBA. The formation of the direct-acting mutagen
was inhibited by addition of cofactor for S9mix. These findings have relevance to the routine
testing of chemicals for mutagenic activity.
Keywords ： photoactivation, photogenotoxicity, DMBA, visible light, mutagenicity
* takahasi@phar.nagoya-cu.ac.jp
Received : February 21, 2001, accepted : April 6, 2001
Environmental Mutagen Society of Japanﾊ
The rapid screening of photogenotoxic compounds using
photo plasmid-relaxation assay
Yuzuki Nakagawa＊, Yuko Takigawa and Noriho Tanaka
Cell Toxicology, Hatano Research Institute, Food and Drug Safety Center
729-5 Ochiai, Hadano, Kanagawa 257-8523, Japan
Summary
To develop a simple and rapid screening system for detection of photogenotoxic chemicals, we
applied the plasmid-relaxation assay to the in vitro photochemical genotoxicity assay system. Using
54 chemicals as model compounds, including known phototoxic and non-phototoxic chemicals, we
evaluated the performance of the plasmid-relaxation assay. The results obtained here were comparable
with published data that were assessed from in vivo phototoxicity systems. We concluded that
the plasmid-relaxation assay is useful as a primary screening method to detect chemicals with photochemical
genotoxicity.
Keywords ： plasmid-relaxation assay, phototoxicity, photochemical genotoxicity
* nakagawa.y@fdsc.or.jp
Received ： August 23, 2001, accepted ： September 5, 2001
Environmental Mutagen Society of Japan
________________________________________資料・情報
医薬品における光遺伝毒性試験
森田 健＊，若田 明裕
日本製薬工業協会 〒103-0023 東京都中央区日本橋本町3-4-1 トリイ日本橋ビル
Photogenotoxicity testing for pharmaceuticals
Takeshi Morita＊ and Akihiro Wakata
Japan Pharmaceutical Manufactures Association
3-4-1 Hon-cho, Nihonbashi, Chuo-ku, Tokyo 103-0023, Japan
Summary
Interest in photosafety testing, especially for pharmaceuticals, has grown over recent years in Europe and
the US. Therefore, the Genotoxicity Working Group of the Japanese Pharmaceutical Manufactures
Association（JPMA）surveyed the area of photogenotoxicity to provide information to members. Current
status, literature surveys and questionnaires on photogenotoxicity were conducted. Compounds suspected
to be photomutagenic and/or photocarcinogenic（M殕ler and Kasper, 1998）are shown in Table 1.
Questionnaires in JPMA are shown in Table 2. Decision trees in several draft guidance are given in Figs. 1-
3. Furthermore, the results of literature surveys are given in Appendix.
Keywords ： photogenotoxicity, JPMA, questionnaires
* takeshi.morita@gsk.com
受付： 2001 年5 月11 日 受理： 2001 年5 月15 日
･日本環境変異原学会