JSTP/IFSTP (IATP)

"Offers exceptional combination of
KIT is your reliable partner in worldwide registration of cutting-edge
GLP systems, sound science andyour lead compounds including Asia, EU and U.S.A unmatched service to clients worldwide"
KIT Provides an Exlensive Array ol Tesls Including: General Toxicology, Carcinogenicity
Tesl, Non-Human Primate Toxicology, Reproductive Toxicology, Genelic Toxicology,
lmmunoloxicology, Pharmacology, Toxicokinelics, Toxicologic Palhology, Ecoloxicology,
and Environmental Chemistry

Korea Institute of Toxigycogy
P.O.Box 123 Yuseong Daejeon 305-600 Korea Tel:+82-42-610-8080 / Fax:+82-42-610-8085

Notice
The following corrections need to be made to the Abstract Book.
Please note the revisions below when referring to the corresponding parts.

JSTP/IFSTP (IATP) 2004 Secretariat

Chair's Name

p.29, p.95, p.286(correct) Gerd MORAWIETZ

Gerd MORAWITZ

Symposium 3 - Chair
Gerd MORAWJETZ

p.29, p.95-~

(The Fraunhofer Institute of Toxicology

Paul-Georg GERMANN(Preclinical Safety, Novartis Pharma AG)

and Aerosol Research, Drug Researchand Clinical Inhalation)

Special Presentation
Following presentation is scheduled to be held at the end of Symposium 3, February 18, 2004.

Title: Educational IT-Tools in Toxicological Pathology: The "Toxicopathology Case Collection"
Paul-Georg GERMANN

Preclinical Safety, Novartis Pharma AG

Gerd MORAWIETZ

The Fraunhofer Institute of Toxicology and Aerosol Research,
Drug Research and Clinical Inhalation

The above abstract will be inserted in the abstract book.

Symposium 3 Special Presentation

Abstract
Educational IT-Tools in Toxicological Pathology:"Toxicopathology Case Collection"

P.-G. Germann
Preclinical Safety, Novartis Pharma AG, Basel 303-0043, Switzerland
G. Morawietz
The Fraunhofer Institute of Toxicology and Aerosol Research, Drug Research andClinical Inhalation, Hannover 30625, Germany

Background - During his illustrious career as one of the founding fathers of toxicologic
pathology, Prof. Gerhard
MD, F.R.C. Path., (19241993) created a unique
histological slide collection (more than 1100 cases) from which about 1000 images have been
selected and stored on CD-ROM.

Scope - These images capture the essence of Prof. Zbinden's collection with emphasis on
toxicopathological changes produced by xenobiotics, although many images of normal tissues
and spontaneous lesions are included for reference. The collection is of considerable interest
to toxicologists and other preclinical safety specialists given the variety of case materialpresented, but is of particular value to the learning, teaching and practicing toxicologicalpathologist ( e.g. for the preparation of Pathology Board Examination).
What is different to- The important feature of the ESTP Toxicopathology
Case Collection is the availability of the original histological slide sets to purchasers of the
CD-ROM. The recipient can then study each case in more detail. Excluding
mailing/insurance costs, these slide sets can be ordered on short-term loan (up to 4 weeks)
free of charge. For teaching purposes, each case is available in 24 to 30 duplicate sets ofhistological slides.
Invited lectures - Special invited lectures from international recognized scientists areincluded as PDF files on this CD-ROM to cover additional important or rare aspects of
toxicological pathology. One special emphasis in this invited lectures is on the Toxicological
Pathology of Male
"Toxic Effects in the Testis"
Diane Creasy:
Danielle Roman:"Flow Cytometry evaluation of Testicular Toxicity""Sperm Motility"
Ulrich Hubel:"Evaluation of Leydig cell toxicity"
Jean-Loic Le Net
Gabriele Pohlmeyer: "Leydig cell tumors"
Additional special
Georg Krinke:
Heinrich Ernst:
Anne Provencher:on rare"Toxicologic Neuropathy- the PNS"
"Odontogenic Tumours in rodents""Hematopoesis & Hematotoxicity in Toxicological Pathology"

Costs - All editors & authors have contributed to this first CD-ROM edition without any
financial interest. Profit of the CD-ROM will be given to the ESTP, which is a non-profit
organization. The CD-ROM is available (03/2004) for 70.- €, please contact:
CaseCollection@Eurotoxpath.org (ISBN number: 3-9522681-1-9).

References - http://www.eurotoxpath.org/nomenclature/cdl index.htm

One Case of Ito Cell Tumor in Female F344/DuCrj Rat
Yumi UMEDA, Tetsuya TAKEUCHI, Hideki SENOR, Taku KATAGIRI, ShigetoshiAISO, Kasuke NAGANO
Japan Bioassay Research Center (JBRC)

ABSTRACTOne case of Ito cell tumor, spontaneously developing in the liver of a female rat, found from 5,000
females used for 25 studies of carcinogenicity bioassay, is presented. A mass, approximately 5 cm insize, was observed. This tumor was predominantly composed of clearly stained cells having a round,
ovoid or spindle-shaped nucleus and chromophobic cytoplasm. These clearly stained cells werearranged in a cord-like structure covered with the sinusoidal endothelium. This tumor metastasized
to the lung. The fluorescence microscopic examination of the liver mass revealed that fluorescence
was present in the cytoplasm of the clearly stained cell, suggesting the presence of a vitamin A being
characteristic of the Ito cell. Lipids were detected by Sudan-black Band Oil-red O stain.
Collagenous matrix was observed among the cord-like components of the tumor. From theabove-described findings, this tumor was diagnosed as a rare Ito cell tumor in the rat liver.
Table 1. Immunohistochemical characteristics of the liver tumor of 8DF1 mouse

Tumor cells

Sinusoidal
endothelial cells+

Factor VIII-related antigen
Cl:' -smooth muscle actin

Collagen I+

Collagen III++

DesminLaminin+Vimentin+

Sudan black B+*

Oil-red 0+*+*: Positive areas are only lipid in the cytoplasm.
CONCLUSION
Ito cells, which exist in Disse space, are the major vitamin A storage site of the body, and probably
play a major role in the extracellular matrix protein synthesis (collagen I, III-VI, laminin, fibronectin,
and tenascin) of the liver. The lesion with the presence of some phenotypes of the cell was observed
by detection of vaitamin A with fluorescent microscope and immunohistochemical staining with alight microscope.

In particular, the increased amounts of the extracellular matrix proteins, laminin

and collagen III, and the detection of vitamin A suggests that the present tumor originated from theIto cell.

Welcome
Dear Colleagues,
Welcome to Kobe. It is our great pleasure to welcome you in the Joint International
Meeting of the Japanese Society of Toxicologic Pathology (JSTP) and the International
Federation of Societies of Toxicologic Pathology (IFSTP), co-sponsered by theInternational Academy of Toxicologic Pathology (IATP); JSTP/IFSTP (IATP) 2004,
entitled Investigative Toxicologic Pathology-Present and Future.
The meeting includes one special lecture, one plenary lecture, 6 symposium sessions,
IATP educational session, and preferred-papers by posters. We also supply 9 luncheon
seminars. In addition, we provide commercial exhibitions in the meeting break areas.
We strongly hope that you will obtain important information by discussing andexchanging knowledge and experiences in the variety of fields in toxicologic pathology
during the meeting.
Kobe is a highly attractive city with an international atmosphere, located on the shore of
the Osaka Bay with a beautiful surrounding. Please enjoy not only the meeting, but alsosight-seeing and have a safety trip back with pleasant memories.
Sincerely yours,

Shoji Fukushima, M.D.

Yoichi Konishi, M.D.

20th JSTP Meeting Chair

5th IFSTP Meeting Chair

Organizing Committee

4

General Information

7

Instructions

11· · · ·

Social Events / Accommodation and Tour

13

AreaMap · · · ·14

Conference Venue

15

Acknowledgements

16

Schedule for Conference

18

Program

IATP · · · · · · · · · · · · · · · · · ·23

Memorial Keynote Lecture / Plenary Lecture24

Symposium

25

Posters31· · ·Luncheon Seminar

55

Abstracts
IATP · · · · · · · · · · · · · · · · · ·59

Memorial Keynote Lecture / Plenary Lecture69

Symposium

73

Posters · ·· 107Author Index· 281

(L--_ _JS_T_PI_IF_S_TP_(I_AT_1'_12_00_4_ _)(Organizing Committee
Meeting Chair
20th JSTP Meeting Chair

Shoji Fukushima (Osaka City University)
5th IFSTP Meeting Chair
Yoichi Konishi (Nara Medical University)

Secretary Generals
Dai Nakae (Sasaki Institute)Masahiro Tsutsumi (Nara Medical University)
Hideki Wanibuchi (Osaka City University)

Program Committee
Chair
Akihiko Maekawa (Sasaki Institute)Members
Ayumi Denda (Nara Medical University)
Kunio Doi (The University of Tokyo)
Kiyoshi Imai (Biosafety Research Center Foods, Drugs and Pesticides)
Katsumi Imaida (Kagawa University)
Satoru Mori (Shionogi & Co., Ltd.)
Keiichirou Morimura (Osaka City University)
Isao Narama (Setsunan University)
Masae Tatematsu (Aichi Cancer Center Research Institute)
Hiroyuki Tsuda (Nagoya City University)

Educational Committee
Chair
Kunitoshi Mitsumori (Tokyo University of Agriculture)Members
Tetsuo Nunoya (Nippon Institute for Biological Science)
Yasuyoshi Okuno (Sumitomo Chemical Co., Ltd.)Tomoyuki Shirai (Nagoya City University)
Seikou Tamano (Daiyukai Medical Research Institute)
Kazutoshi Tamura (Bozo Research Center)
Masahiro Tsutsumi (Nara Medical University))

Financial Committee
Chair
Shoji Fukushima (Osaka City University)
Members
Masao Hirose (National Institute of Health Sciences)
Yoichi Konishi (Nara Medical University)
Hiraki Kuniyasu (Nara Medical University)
Keizo Maita (The Institute of Environmental Toxicology)
Hiroaki Miyajima (Shin Nippon Biomedical Laboratories Ltd.)
Hideki Mori (Gifu University)
Takashi Nonoyama (Takeda Chemical Industries, Ltd.)
Michihito Takahashi (Pathology Peer Review Center)

International Advisory Committee
Robert A. Ettlin (IFSTP President)
Hugh E. Black (IFSTP Secretary General)
Members
Charles C. Capen (IATP Accreditation Committee, Past-Chairperson)
Patrizia Cristofori (IFSTP EC, Italian STP)
Eric Debruyne (IFSTP Treasurer)
Xavier Fouillet (IFSTP EC, French STP)
Jerry D. Frantz (IFSTP Past-President)
Paul-Georg Germann (IFSTP-councillor of the ESTP)
Chirukandath Gopinath (IFSTP Past-President)
Jerry F. Hardisty (IATP North American Director)
Michael J. Iatropoulos (IATP Secretary/Treasurer of the Board of Directors)
John M. Finch (IFSTP EC, British STP)
John Ishmael (IATP Accreditation Committee Member)
Eberhard Karbe (IATP Accreditation Committee Member)
D. Reid Patterson (IATP Board of Directors Chairperson)
Colin G. Rousseaux (IFSTP EC, North American STP)
Mikala Skydsgaard (IFSTP EC, Nordic STP)
Leander Tryphonas(IFSTP Past-Secretary General and IATP Past-Chairman of the Board of Directors)
Joseph G. Vos (IATP Accreditation Committee Member)
Piet W. Wester (IFSTP EC, Dutch STP)
Gary M. Williams (IATP Accreditation Committee Member)
Ki-Hwa Yang (IFSTP EC, Korean STP)

Subcommittee to Nominate Foreign Chairpersons of Symposia
Paul-Georg Germann (Novartis Pharma)
Dai Nakae (Sasaki Institute)Colin G. Rousseaux (University of Ottawa)

Honor Advisory Committee
Makoto Enomoto
Kousaku Fujiwara
Yuzo Hayashi
Eisei Ishikawa
Chitoshi Itakura
Nobuyuki Ito
Masayoshi Kanisawa
Sadashige Sakuma

6

-

..

.1

:

J

General:,;.

Information

•
?
.•

.,
~

•

r

'

~'

i

I

:
J

7

( ~ __
1S_T_'.P_1IF_S_T_1'(_IA_T_1')_20_0_4_ _)(General Information)

Date
Sunday, February 15 - Wednesday, February 18, 2004

Conference Venue
International Conference Center Kobe (ICCK)
6-9-1 Minatojima-nakamachi, Chuo-ku, Kobe 650-0046, Japan
Phone: +81-78-302-5200
Fax: +81-78-302-6485

Official Language
Official language is English.

Registration
Conference paiticipants can complete your registration during the following hours.
Sunday, February 15 ························ ll:30am-5:00pm
Monday, February 16· · · ·· · ·· · ·· · ·· · · · · · ·· 8:30am-6:00pm

Tuesday, February 17 · · · · · .. . · · ·· ·· · ·· · · ·· · · · 8:30am-6:00pm
Wednesday, February 18 ···· · · ··· · ······ · · 8:30am-5:00pm
1. Registration Desk is located in the Foyer (IF) at the International Conference Center Kobe (ICCK).

2. Advanced Registration: For those who have completed their registration in advance, please bring theconfirmation letter with you and present it at the registration desk.
3. On-site Registration: For those who have not completed their registration, please complete registration at theon-site registration desk. Registration fee is as follows:
Registration Type··········· ··· ········· ······· On-Site Registration
Member* 1 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · JPY50,000
Resident/Fellow/Student* 1*2 · · · · · · · · · · · · JPY20,000
Non-Member*l···················· ·········· JPY60,000

Accompanied person*3 ············ ···· ··· ·· JPYI0,000*1: The fee includes admission to all scientific sessions, exhibitions and Welcome and Gala reception.
*2: The registration form for Resident/ Fellow/ Student participants must be accompanied by a letter fromthe depaitment head or a photocopy of a current registration card. Applicants should be full-time emolled
at a degree-granting university or college through the academic yeai· of 2003 at time of registration.
*3: The fee includes admission to the meeting venue including the exhibition area, Welcome and Gala
receptions but not to the inside of the rooms where scientific sessions are being held.

9General Information Desk
General Information desk will be located next to the registration desk (IF). For inquiries, such as program
schedule, please inquire at the desk.

Messages
A message board will be located in the registration area. Participants are encouraged to check this board regularly.

Board Meeting
Board meeting will be held with the following schedule.
JSTP Board of Directors Meeting (Rijikai)
Sunday, February 15 · · · ·· · · · · ·· · · · · · · · .. · · · · 4:00pm-6:00pm SF (Room 505)
JSTP Council (Hyogi Iinkai)
Tuesday, February 17 · · · · · · · · · · · · · · · · · · · · · · · · 12:00pm-1:00pm SF (Room 505)
JSTP General Meeting (Soukai)
Wednesday, February 18 ·· ····· ··········· 11:30am-12:00pm IF (Main Hall)

Poster Awards
For those who have presented an outstanding work will be nominated to receive a poster award. Nomination will
be made by Program Committees. Recipients of the Poster Award will be announced on the last day of theconference.

Luncheon Seminar
There will be three (3) luncheon seminars held on each day, and total nine (9) seminars will be taken places
through the conference. List of sponsor company can be found in the program pages, and participants areencouraged to attend. Seminars will be held at Main Hall, Room 501, and 502.
Monday, February 16 ··· ····················· 12:00pm-1:00pm
Tuesday, February 17 · · · ·· · · · · · · · · · · · · · · · · ·· · 12:00pm-1 :OOpm
Wednesday, February 18 ···· ·· ············ 12:00pm-1:00pm

Exhibition
Exhibition will be located at the reception hall (3F). Participants are encouraged to visit the exhibition hall.
Monday, February 16 ······················ ·· 9:30am-5:30pm
Tuesday, February 17 · · · · · · · · · · · · · · · · · · · · · · · · 9:30am-5 :30pm
Wednesday, February 18 ············· ····· 9:30am-5:30pm

Cloak
Cloak is located in the basement (B IF).
Sunday, February 15 ···· ·················· ·· 11:30am-7:00pm
Monday, February 16 ········· ···· ········· ·· 8:30am-7:00pm
Tuesday, February 17··· ············ ··· 8:30am-7:00pm
Wednesday, February 18 ·················· 8:30am-7:00pm

10

('------_ _JS_T_~_1IF_S_T_1'(_IA_T_1')_20_0_4_ _)Instructions for /ATP Educational Session, Memorial Keynote/Plenary Lecture and Symposium
IATP Educational Session, Memorial Keynote Lecture, and Plenary Lecture and Symposium will be held at theMain Hall (B IF, IF).

Chairs are asked to be prepared at the Main Hall 30 minutes before the session begins.

Speakers are expected to check the connection of their laptop at least 30 minutes prior to the start of thepresentations at the preview room, Room 307 (3F).
Speakers are requested to ensure that they have a suitable connector for their laptop.
A 15 pin high density D plug connection for PCs and 15 pin D plug connection for Macs.
Please note no 35mm slides will be accepted at the conference.

(,____ _JS_T_P._
1IF_S_T_
1'(_IA_T_1')_20_0_
4 _ _)(

Instructions for Poster Presentation

1. Reception desk of the poster presentation is located at the entrance hall (3F). All the poster presenters are
expected to check in their names at the desk before posting the posters. This reception desk is only for theposter presentation, and all the conference participants are expected to come to the registration desk (lF) first
upon your arrival to the conference venue.
2. Every poster will be on display from Monday through Wednesday.
3. Poster should be set-up and removed during the following time.
Set-up: Monday, February 16 · · · · · · · · · · · · 8:30am-9:30am
Removal: Wednesday, February 18 · · · · · · 4:00pm-5:00pm· No individual presentation will be held for the poster presentation.
· Authors will be provided, by the Secretariat, with push pins to be used in attaching material to the poster
boards.
· Displays should be set up and removed strictly according to the times listed above.
· Posters not removed during the allocated times will be dismantled by the Secretariat.
4. Poster Presentation Halls will be Room 301 (3F) and Room 401, 402 (4F). Please stand next to your poster
during the appointed discussion time to prepare for discussion. Core discussion times of poster session are as
follow, and please refer to the program pages for your appointed date.
Monday, February 16 .................. ...... l:OOpm-2:00pm
Tuesday, February 17 · · · .. · · · · · · · · · · · .... · · · · 1:OOpm-2:00pm
Wednesday, February 18·· · · · · · · · · · .. ·....

1:00pm-2:00pm

5. Language: English
Poster Board Dimension

20cm

70cm
20cm

Poster Number-+(Prepared by, _ _ ~ - - --i Title, Name (s)+-Jthe Secretariat)(20cm x 20cm)

of author (s) ,----1

Affiliation (s)(20cm x 70cm)

Display Space(160cm x

160cm

90cm)

!

30cm

~-----~

12)

(~ _ _
1S_T_P._1IF_S_T_
1'(_IA_T_1')_20_0_4_ ~ )(Social Events/Accommodation and Tour)

Welcome Reception
Sunday, February 15 ·· ······ ................ 7:00pm-9:00pm
Welcome Reception will be held on Sunday, February 15 from 7:00pm at PORTOPIA Hotel Main Building,Grand Banquet Room "KAIRAKU" (B IF). Participation fee is included in the registration fee.

Gala Reception
Tuesday, February 17 · · · · · · ·· ·· ·· · · · ·· · · · · · · · 6:30pm-8:30pm
Gala Reception will be held on Tuesday, February 17 from 6:30pm at PORTOPIA Hotel South Wing Building,Grand Banquet Room "OWADA" (IF). Participation fee is included in the registration fee .

Official Travel AgentJTB Corp. Event & Convention Sales Dept, Western Japan Regional Headquarters has been appointed to be theofficial travel agent for JSTP/IFSTP (IATP) to handle all travel arrangements to and within Japan. For those whohave an inquiry about travel or sightseeing, please ask at the JTB travel desk.
JTB Corp. ,Event & Convention Sales Dept,
Western Japan Regional Headquarters
JSTP/IFSTP (IATP) 2004 Desk
Tel:+81-6-6260-5060
Fax:+81-6-6260-5090

Hotels for Participants
Hotel Name

Hotel Location

Phone

Portopia Hotel

Located next to the conference venue+81-78-302-1111

Hotel Pearl City Kobe

Walking distance from the conference venue+81-78-303-0100

Hotel Gaufres

Walking distance from the conference venue+81-78-303-5555

13

(,. ______ _J_ST_P._1IF_S_T_?(_IA_T_?)_2_00_4_ ____,)(

Area Map/Transportation)

Airplane
70min.
Haneda(TokyoInternational

Airport)ltami(Osaka
Internation

al Airport)
Limou sine Bus
Shinkansen

45min.

Shin-Kobe Stn.

§ Subway~----- =3min.

Hankyu Line
20-30min.

Umeda Stn.
Osaka Stn.

Sannomiya Stn.

JR Line

Airplane
75min.

JR" Haruka"
45min.
Limousine Bus
70min.

N

A

14

\\

Hotel Pearl City Kobe0-

(~ __
1S_T_P._VIF_S_T_1'(_IA_T_1')_20_0_4_ _)(Conjerenee Venue
f

503

5F

l

504

l

)

! 505

Luncheon
Seminar

4F

Poster Desk

3F

Poster Presentation l

2F

B 1F 1 F
Oral Presentation
Luncheon Seminar

Registration
DeskAcknowledgement

Supporting Society----------------------

Asian Pacific Organization for Cancer Prevention

The Japanese Cancer Association

International Life Sciences Institute (ILSI HESI/ILSI JAPAN)

The Japanese Society of lmmunotoxicology

Japanese Society for Cancer Prevention

The Japanese Society of Pathology

Japanese Society of Food Chemistry

The Japanese Society of Toxicology

The Japanese Environmental Mutagen Society

The Japanese Society of Veterinary Science

Subsidy
Kawano Masanori Memorial Foundation for Promotion of Pediatrics

The Portopia 81 Memorial Fund

The Japan Food Chemical Research Foundation

Tsutomu Nakauchi Foundation

SponsorDonation---------------------------Yuzo Hayashi

NPO International Life Sciences Institute of Japan

Adachi Co. ,Ltd .

(ILSI Japan)
Osaka Pharmaceutical Manufacturers Association
San-Ei Gen F.F.I., Inc.

Asahi Hospital
BASF Agro, Ltd.
Chemicals Evaluation and Research Institute, Japan
Daiichi Suntory Pharma Co. , Ltd.
Daiyu-kai Institute of Medical Science
JAPAN BIOASSAY RESEARCH CENTER
KANEKA CORPORATIONKIKO TECH CO., LTD.
Korean Society of Toxicologic Pathology
Mitsubishi Chemical Safety Institute Ltd.
MITSUI NORIN CO. , LTD.

SANWARIKEN Co., Ltd.
Shin Nippon Biomedical Laboratories, Ltd.
Sumitomo Chemical Co., Ltd.
Syngenta Japan K.K.
THE INSTITUTE OF ENVIRONMENTAL TOXICOLOGYThe Pharmaceutical Manufacturers' Association of Tokyo
Wakunaga Pharmaceutical Co., Ltd.
YASHIMA PURE CHEMICALS CO. , LTD.
ZERIA Pharmaceutical Co., Ltd.

NARD institute, ltd.

Advertisement-------------------------Nihon Bioresearch Inc.

CTBR (A Member of the lnveresk Research Group)
Daiyu-kai Institute of Medical Science

ORIENTAL YEAST CO., LTD.
Sakura Finetek Japan Co., Ltd.

Dako Cytomation Co. Ltd .

Shin Nippon Biomedical Laboratories, Ltd.
TAIHO PHARMACEUTICAL CO. , LTD.

lnvitrogen Japan K.K.
Korea Institute of Toxicology (KIT)KYORIN PHARMACEUTICAL CO., LTD.

16

KYOWA HAKKO KOGYO CO. , LTD.

Amersham Biosciences KK
Biopathology Institute, Co., Ltd .

TAKARA BIO INC.

Acknowledgement
Exhibition-------------------------Affymetrix Japan K.K.

Experimental Pathology Laboratories, Inc. (EPL)

AFIP, Department of Veterinary Pathology; RTPA

GENOSTAFF CO. , LTD.

ALOKACO ., LTD.

H & T Corporation

AZUMAYA CORPORATION

Huntingdon Life Sciences Co., Ltd.

BIOSAFETY RESEARCH CENTER, FOODS,DRUGS AND PESTICIDESKOGAKU Co. , Ltd.

BM EQUIPMENT CO., LTD.

MATSUNAMI GLASS IND., LTD.

Charles River Japan, Inc.

MEIWA SHOJI CO., LTD.

CLEA Japan, Inc.

Micro Edge Instruments Co., Ltd.

COVANCENICHIREI CORPORATION

CTC LABORATORY SYSTEMS Corporation

OLYMPUS CORPORATION

Daiyu-kai Institute of Medical Science

ORIENTAL YEAST CO. , LTD.

Dako Cytomation Co., Ltd.

Shin Nippon Biomedical Laboratories, Ltd.

Environmental Biological Life Science Research Center Inc.

SUMIKA TECHNOSERVICE CORPORATION

Maruzen Company, Limited

Luncheon S e m i n a r - - - - - - - - - - - - - - - - - - - - - - Affymetrix Japan K.K.

MEIWA SHOJI CO ., LTD.

Bozo Research Center Inc.

MORINAGA MILK INDUSTRY CO. , LTD.

Dako Cytomation Co., Ltd.
Fujisawa Pharmaceutical Co., Ltd.

NPO International Life Sciences Institute of Japan (ILSI Japan)/ ILSI Health and Environmental Sciences Institute (ILSI HESI)Japan SLC, Inc.

OLYMPUS CORPORATION

Drink---------------------------SUNTORY LIMITEDTAIHO PHARMACEUTICAL CO., LTD.

Coca-Cola (Japan) Company, Limited

Meiji Dairies Corporation

Schedule for Conference
Sunday February 15, 2004
Registration

Reception
Hall

Main Hall

Room 301

Room
401+402

Room 501

Room 502

Other

10:00am
11:00am-

12:00pm
11:30am
1: 00pm
JSTP

2:00pmI

3:00pmMicroexamination
Explanation
Opening Remarks

5:00pm
4:00pmIATP
Educational
Session

5:00pm--JSTP
Board ofDirectors
Meeting(Room 505)

6:00pm
7:00pmWelcome
Reception

8:00pm(Portopia Hotel)

9:00pmMonday, February 16, 2004
Registration

Main Hall-

8:00amReception
Hall

Room 301

Room
401+402

Room 501

Room 502--

Poster
Set-up9:00amPoster
Set-up

10:00am
11:00amSymposium
1-1
8:30am

12:00pm
Luncheon
Seminar I
1:00pmI

2:00pm
6:00pm
3:00pmExhibition
John Faccini
Memorial
Keynote Lecture

4:00pm
5:00pm
6:00pm18

Luncheon
Seminar 2

Symposium
1-2

Poster
Discussion

Poster
Discussion

Luncheon
Seminar 3

OtherSchedule for Conference
Tuesday, February 17, 2004
Registration

Main Hall

Reception
Hall

Room 301

Room
401+402

Room 501

Room 502

Other

Luncheon
Seminar S

Luncheon
Seminar6

JSTP Council

8:00am9:00amSymposium

10:00am2-1
11:00am

8:30am

12:00pm

Luncheon
Seminar4
1:00pmI

Exhibition

Poster
Discussion(Room SOS)

Poster
Discussion

2:00pm
6:00pm
3:00pmPlenary
Lecture4:00pmSymposium
2-2

5:00pm
6:00pm
7:00pmGala
Reception

8:00pm(Portopia Hoiel)Wednesday, February 18, 2004
Registration

Main Hall

Reception
Hall

Room 301

Room
401+402

Room 501

Room 502

Luncheon
Seminars

Luncheon
Seminar9

8:00am
9 :00amSymposium

10:00am3
11:00am

8:30amJSTP General Meeting

12:00pm
1:00pmI

Luncheon
Seminar?

Exhibition

Poster
Discussion

Poster
Discussion

PosterRemovalPosterRemoval2:00pm
5:00pm
3:00pmSymposium
4

4:00pm
5:00pmClosing Remarks

Other

.

Program'21

IATP PROGRAM

Main Hall
IATP

Sunday, February 15, 2004 I 3:00pm-6:30pm

CONTEMPORARY ISSUES IN TOXICOLOGIC PATHOLOGY
Chair : D. Reid PATTERSON
Reid Patterson Consulting, Inc., Illinois 60030, USA: Charles C. CAPEN
Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University,
Ohio 43210, USA

Enhancing the impact of toxicologic pathologists in global medical research
D. Reid PATTERSON
Reid Patterson Consulting, Inc., Illinois 60030, USA
Chemically-induced immunopathology and immune functional changes
Joseph G. VOSNational Institute for Public Health and the Environment (RIVM), Bilthoven 3720 BA,
The Netherlands
Thresholds in experimental chemical hepatocarcinogenesis
Gary M. WILLIAMS, A.M. JEFFREY, J. D. DUAN and M. J. IATROPOULOS
Department of Pathology, New York Medical College, New York 10595, USA
Endocrine disruption in wildlife: The role of toxicologic pathology in fish
Pieter W. WESTER
National Institute for Public Health and the Environment (RIVM), Bilthoven 3720 BA,
The Netherlands
Renal mesenchymal tumor vs. nephroblastoma: Revisited
John Curtis SEELY
Experimental Pathology Laboratories, Inc., Research Triangle Park, North Carolina 27709, USA
Demonstration of histogenesis and cytogenesis of mammary tumors from terminal
endbud, mammary duct, acinus and myoepithelium cells using human c-Ha-ras
proto- and mutated-oncogene transgenic rats
Hiroyuki TSUDA 1•2 , Shinobu UEDA 2 , Yoichiro MATSUOKA2, Tetsuya HAMAGUCHI2,
Akihiro NAIT0 2 and Nobuo TAKASUKA 2
Department of Molecular Toxicology, Nagoya City University Graduate School of Medical Sciences,
Aichi 467-8601, Japan 1
Experimental Pathology and Chemotherapy Division, National Cancer Center Research Institute2
The function and pathology of brown adipose tissue (BAT) in animals and humans
Michael IATROPOULOS and Gary WILLIAMSNew York Medical College, New York 10595, USA
Closing Comments
Charles C. CAPEN
Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University,
Ohio 43210, USA
23

MEMORIAL/PLENARY LECTURE PROGRAM

Main Hall
Second John Faccini Memorial Keynote Lecture

Monday, February 16, 2004 / 2:00pm-3:00pm

Molecular aspects of toxicologic pathology

This lecture is generously supported by
MOS Pharma Services.

~
Pharma Services

Chair: Yoichi KONISHINara Medical University

Science advancing health

Speaker : Okio HINO

www.mdsps.com

Department of Experimental Pathology, Cancer Institute, Japanese Foundation
for Cancer Research, Tokyo 170-8455, Japan

Main Hall
Plenary Lecture

Tuesday, February 17, 2004 / 2:00pm-3:00pm

Human relevance of carcinogenesis in rodents.
Chair : Shoji FUKUSHIMA
Osaka City University Medical School
Speaker : Samuel M. COHEN
Department of Pathology and Microbiology, University of Nebraska Medical Center,
Nebraska 68198, USA

24

JSTP/IFSTP IATP 200~
SYMPOSIUM PROGRAM
Main HallSYMPOSIUMl-1

Monday, February 16, 2004 I 9:30am-12:00pm

Carcinogenesis-Current understanding of mechanisms and risk assessments:
Non-genotoxic carcinogenesis/Low-dose carcinogenic potential of genotoxic andnon-genotoxic carcinogens
Chair : Colin G. ROUSSEAUX
Department of Cellular and Molecular Medicine, Faculty of Medicine, 451 Smyth Rd, University of Ottawa,
Ontario KlH 8M5, Canada: Masao HIROSE
Division of Pathology, National Institute of Health Sciences, Tokyo 15 8-8501, Japan

Sl-1-1
Risk assessment of low dose carcinogenesis by genotoxic carcinogens
Hideki WANIBUCHI, Keiichirou MORJMURA, Shoji FUKUSHIMA
Department of Pathology, Osaka City University Medical School, Osaka 545-8585, Japan

Sl-1-2Analysis of reporter genes for the molecular mechanisms underlying chemical
carcinogenesis
Akiyoshi NISHIKAWA and Masao HIROSE
Divisions of Pathology, National Institute of Health Sciences, Tokyo 158-8501 , Japan

Sl-1-3
Recent advances in tumor angiogenesis
Anne M. RYAN
Department of Pathology, Pfizer Global Research and Development, Connecticut 06340, USA

Sl-1-4
Gene inactivation in mammalian stem cells
Michael W. McBURNEY
Ottawa Regional Cancer Center and Department of Medicine, University of Ottawa,
Ottawa KlH 1C4, Canada

Main HallSYMPOSIUMl-2

Monday, February 16 2004 / 3:30pm-6:00pm

Carcinogenesis - Current understanding of mechanisms and risk assessments:
Genetically modified animal models
Chair : Jerrold M. WARD
National Institute of Allergy and Infectious Diseases, NIH, Maryland 20892, USA: Hiroyuki TSUDA
Department of Molecular Toxicology, Nagoya City University Graduate School of Medical Sciences,
Aichi 467-8601, Japan,
Experimental Pathology and Chemotherapy Division, National Cancer Center Research Institute,Tokyo 104-0045, Japan

Sl-2-1
Mouse models of human familial cancer syndromes as models for risk assessment
JeITold M. WARD
National Institute of Allergy and Infectious Diseases, NIH, Maryland 20892, USA

Sl-2-2
Mouse models of colon cancer and polyposis
Makoto Mark TAKETO
Department of Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501 , Japan

Sl-2-3Update on use of the Trp53 +!- mouse for short-term carcinogenicity testing ofpharmaceuticals
Eugenia FLOYD
Pfizer Groton Laboratories, Worldwide Safety Sciences, Connecticut 06498, USA

Sl -2-4
High susceptibility of human c-Ha-ras proto-oncogene transgenic rats to various
carcinogens - Possible application for a short term assay system for environmental
carcinogens
Hiroyuki TSUDA l,2, Takamasa ONISHI2, Shinobu UEDA2, Yoichiro MATSUOKA2, Katsumi FUKAMACHI2,
Akihiro NAITQ2, Cheol Beom PARK2, Beom Seok HAN2, Chuel Kyu KIM2 and Nobuo TAKASUKA2
Department of Molecular Toxicology, Nagoya City University Graduate School of Medical Sciences,
Aichi 467-8601, Japanl
Experimental Pathology and Chemotherapy Division, National Cancer Center Research Institute2

26

JSTP/IFSTP IATP 20.0~
Main Hall
SYMP0SIUM2-1

Tuesday February 17, 2004 / 9:00am-11:30am

Omics - A challenge for the toxicologic pathologists:Concerning general
toxicity and carcinogenicity
Chair : Michael L. CUNNINGHAM
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina27709, USA.
: Tomoyuki SHIRAI
Nagoya City University Graduate School of Medical Sciences, Aichi467-8601, Japan

S2-1-1
Toxicogenomic approach for detection of carcinogenic substances
Tomoyuki SHIRAII, Makoto ASAMOTOl, Kazunari TSUITMURAl ,2, Masaru SEKIJIMA3,
Nobuyoshi MIKAMI4 and Masanori OTUSKA2
Nagoya City University Graduate School of Medical Sciences, Aichi 467-8601, Japan 1
Chemical Evaluation and Research Institute2
Mitsubishi Chemical Safety Institute Ltd.3
Sumitomo Chemical Co. Ltd.4

S2-1-2
Proteomic expression profiling of liver toxicity in rat using 2D-DIGE technology
Hidenori YAMANAKA!, Kazunari TSUJIMURAI , Masanori OTSUKAI, Yoshikuni YAKABEI,
Satsuki HOSYUYAMAI, Koichi SAIT02, Kayo SUMIDA2, Masaru SEKIITMA3, Koji NAKAYAMA3,
Yukiko KAWAN03, Yasuro SHINOHARA4, Tomoyuki SHIRAI5
Chemical Assessment Center, Chemical Evaluation and Research Institute, Japan, Saitama 345-0043, Japan I
Sumitomo Chemical Co. , Ltd2
Mitsubishi Chemical Safety Institute Ltd3
Hokkaido University4
Graduate School of Medical Sciences, Nagoya City University5

S2-1-3
Gene expression profiling of rat liver fibrosis, cirrhosis, and cancer induced by cholinedeficient, L-amino acid-defined diet
Ivan RUSYNI , Oksana KOSYK I, Christine POWELL!, Ayumi DENDA2, Yoichi KONISHI2, Fumiyuki UEMATSU3
and Dai NAKAE3
Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill,
North Carolina 27599, USA I
Department of Oncological Pathology, Cancer Center, Nara Medical University2
Department of Pathology, Sasaki Institute, Sasaki Foundation3

S2-1-4
Gene expression changes in F344 rats following a pharmacological dose of acetaminophen
Michael L. CUNNINGHAM
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USAMain Hall
SYMP0SIUM2-2

Tuesday, February 17, 2004 I 3:30pm-6:00 m

Omics - A challenge for the toxicologic pathologists:
Concerning reproductive toxicity, immunotoxicity and toxicity of endocrine
disrupting chemicals
Chair : Paul-Georg GERMANN
Preclinical Safety, Novaitis Pharma AG, Basel 303-0043, Switzerland: Keizo MAITA
Study Planning and Consultation, The Institute of Environmental Toxicology, Ibaraki 303-0043, Japan

S2-2-1
Biological effects of diethylstilbestrol -Transgenerational effects of endocrine disrupting
chemicalsTetsuji NAGAO
Department of Life Science, Kinki University, Osaka 577-8502, Japan

S2-2-2Mechanisms of toxic effects and tumor development caused by DDT in F344 rats
Takanori HARADA, Ryoichi OHTSUKA, Makio TAKEDA, Naruto TOMIYAMA, Machiko SAKA,
Tadashi KOSAKA, Junya SASAKI, Sayuri KOJIMA, Naofumi TAKAHASHI, Yukiko TAKEUCHI,
Toshinori YOSHIDA, Maki KUWAHARA, Akiko ENOMOTO, Nobuaki NAKASHIMA and Keizo MAITA
The Institute of Environmental Toxicology, Mitsuka.ido-shi , Ibaraki 303-0043, Japan

S2-2-3lmmunotoxicology in drug safety evaluation: A medical toxicologist's view
Jacques DESCOTES
Poison Centre and Claude Bernard University, Lyon 69007, France

S2-2-4The sensitivity and predictability of current testing strategies to evaluate immunotoxicity
Dori GERMOLECI, Abraham NYSKAI , C Frieke KUPER2, Christopher PORTIER I, Michael KASHON3,
C KOMMINENP, Keith JOHNSON4 and Michael LUSTER3
National Institute of Environmental Health Sciences, North Carolina 27709, USAITNO Nutrition and Food Research2
National Institute of Occupational Safety and Health3
Dow Chemical Company4

JSTP/IFSTP IAT"i:, 200~
Main Hall
SYMP0SIUM3

Wednesday, February 18, 2004 / 9:00am-11:30am

Recent trend of information technology in toxicological pathology science
Chair: Gerd MORAWITZThe Fraunhofer Institute of Toxicology and Aerosol Research, Drug Research and Clinical Inhalation,
Hannover 30625, Germany: Ikuo HORII
Pfizer Global Research & Development, Worldwide Safety Sciences - Nagoya, Aichi 470-2393 , Japan

S3-1
Pathology information system
Nobuyuki OHASHI
Biosafety Research Center, Food, Drugs and Pesticides, Shizuoka 437-1 213 , Japan

S3-2
Linking worldwide pathology expertise with the telepathology system
Ingrid PRUIMBOOM-BREES, Mark TENGOWSKI and Ricardo OCHOA
Pfizer Research and Development, Department of Pathology - Groton Safety Sciences, Connecticut 06340, USA

S3-3
Image informatics: Current and future challenges
Judith A. NOLAN
Scimagix, Inc., California 94403 , USA

S3-4
AFIP online veterinary pathology training programs
Mark MENSE, Terrell BLANCHARD, Thelda ATKIN, Sophie BOUCHIHA-OLSON, Sean HAHN,
William INSKEEP and Dale DUNN
Department of Veterinary Pathology, Armed Forces Institute of Pathology, Washington DC 20306, USAMain Hall
SYMP0SIUM4

Wednesday February 18, 2004 / 2:00pm-4:30pm

Future challenges: Stem cell development, infectious agents and others
Chair : John FINCH
Inversk Research, Edinburgh EH33 2NE, UK: Kunio DOI
Veterinary Pathology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Tokyo
113-8657, Japan

S4-1
Establishment and manipulation of monkey and human ES cell lines for biomedical
research
Norio NAKATSUJI
Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan

S4-2
Clinical applications of stem cell research
David HARRISON
Department of Pathology, University of Edinburgh, Scotland EH8 9AG, UK

S4-3
DNA methylation pattern of CpG islands for epigenetics pathology and toxicology
Kunio SHIOTA
Cellular Biochemistry, Animal Resource Sciences/ Veterinary Medical Sciences, The University of Tokyo,
Tokyo 113-8657, Japan

S4-4
Emerging zoonotic diseases
Corrie BROWN
Department of Veterinary Pathology, College of Veterinary Medicine, University of Georgia, Georgia 30602, USA

JSTP/IFSTP IATP -r.20021
POSTER PROGRAM

SESS10N1 : Environmental Toxicopathology
Room 301
Endocrine Disru ters

1:00pm-2:00pm

P-1 (Mon.)
Hershberger and uterotrophic assessments to evaluate potential endocrine disrupting
effects of diesel exhaust in rats
Tsuyoshi ITO', Hiraki OKUMURA' , Ryo OHTA2, Kiyoshi IMAP, Midori YOSHIDA4, Dai NAKAE4,
Akihiko MAEKAWAS
Health Effects Research Division, Japan Automobile Research Institute, Ibaraki 305-0822, Japan'
Safety Testing Laboratory, Hatano Research Institute, Food and Drug Safety Center2
Executive Director, Biosafety Research Center, Foods, Drugs and Pesticides3
Department of Pathology, Sasaki Institute, Sasaki Foundation4
Director, Sasaki Institute, Sasaki Foundations

P-2 (Tue.)
Application of hershberger assay protocol to the detection of thyroid function modulators
using castrated male rats
Masakuni SAWAKI', Shuji NODA' , Takako MUROI' , Hideo MITOMA', Saori TAKAKURA' ,Satoko SAKAMOTO', Masanori OTSUKA 1, Mineo TAKATSUK.1 1, Kanji YAMASAKI'
Chemicals Evaluation and Research Institute, Oita 877-0061 , Japan I

P-3 (Wed.)
Lack of modifying effects of 4-tert-octylphenol and benzyl butyl phthalate on
3,2'-dimethyl-4-aminobiphenyl-induced prostate carcinogenesis in rats
Hiroyuki KOHNO', Rikako SUZUK.Il ,2, Shigeyuki SUGIE' , Takuji TANAKA'
Department of Pathology, Kanazawa Medical University, Uchinada 920-0293, Japan 1
Research Fellow of the Japan Society for the Promotion of Science2

P-4 (Mon.)
Sex hormone responsiveness and ductal architecture after exposure to flutamide perinatallyin the rat ventral prostate
Kaori MIYATA 1, Setsuko YABUSHITA' , Masashi SAN02, Yasuyoshi OKUNO' , Masatoshi MATSU03
Environmental Health Science Lab. , Sumitomo Chemical Co. , Ltd., Osaka 554-8558, Japan'
Daiyu-Kai Institute of Medical Science2
Cooerative Research Center for Advanced Science and Technology, Osaka University3P-5 (Tue.)Is stem cell factor related to the testicular toxicity of thiamphenycol?
Maki KUWAHARA' , Yukiko TAKEUCHI' , Naofumi TAKAHASHI', Tadashi KOSAKA' , Nobua.ki NAKASHIMA' ,Toshinori YOSHIDA 1, Akiko ENOMOTO' , Toshiaki KITAZAWA3, Keizo MAITA2, Takanori HARADA'
Toxicology Division, The Institute of Environmental Toxicology, Ibaraki 303-0043, Japan I
Division of Study Plannning and Conslutation, The Institute of Environmental Toxicology2
Contract and Research Management Division, The Institute of Environmental Toxicology3

P-6 (Wed.)
No modifying effects of an estrogenic compound atrazine on 7, 12-dimethylbenz(a)anthracene-induced ovarian carcinogenesis in rats
Takuji TANAKA', Hiroyuki KOHNOI , Rikako SUZUK.Il ,2, Shigeyuki SUGIE'
Department of Pathology, Kanazawa Medical University, Ishikawa 920-0293 , Japan'
Research Fellow of the Japan Society for the Promotion of Science2

P-7 (Mon.)
The influence of pre- and post-natal exposure to methoxychlor on gene expression in theendometrium of rats after maturation
Ryoichi OHTSUKAI , Makio TAKEDA I, Satoru YAMAGUCHI !, Koichi HAYASHI!, Yukiko TAKEUCHJI ,Maki KUWAHARA!, Naofurni TAKAHASHI!, Tadashi KOSAKAI, Keizo MAITA!, Takanori HARADA!
Institute of Environmental Toxicology, Ibaraki 303-0043, Japan 1

P-8 (Tue.)
Effects of the fetal administration of diethylstilbestrol (DES) and 4-n-octylphenol (n-OP) on
7, 12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in SD rats
Hiroaki KAWAGUCHI! , Masakazu SOHDAl, Shuhei TAGUCHI! , Koichiro MIYAMOTO! , Hiroki YOSHIDA!
The Depar1ment of Tumor Pathology, Graduate School of Medical and Dental Sciences,
Kagoshima University, Kagoshima 890-8544, Japan t

P-9 (Wed.)
The influence of pre- and post-natal exposure to methoxychlor on the rat immune system
and renal function during aging process
Yukiko TAKEUCHI! , Naofurni TAKAHASHI 1, Tadashi KOSAKA 1, Koichi HAYASHI! , Yuko CHIBA 1,
Toshinori YOSHIDA! , Maki KUWAHARA! , Sayuri KOJIMA! , Ryoichi OHTSUKAI, Keizo MAITA2,
Takanori HARADA I
Toxicology Division II, Institute of Environmental Toxicology, Ibaraki 303-0043, Japan I
Division of Study Planning and Consultation, Institute of Environmental Toxicology2

P-10 (Mon.)
No effects of transplacental and lactational exposure to combination of bisphenol A and
4-nonylphenol, endocrine disruptors, on morphogenesis of male reproductive organs andspermatogenesis in F344 rats.
Kyoko NABAEI, Hiroko YOSHINO I, Norio IMAII , Takeshi HIROTA I, Mayumi KAWABEI , Syugo SUZUKI2,
Tomoyuki SHIRAI2
Daiyu-kai Institute of Medical Science, Aichi 491-0113 , Japan 1
Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of Medical
Sciences2

P-11 (Tue.)No effects of transplacental and lactational exposure to combination of bisphenol A and
4-nonylphenol, endocrine disruptors, on morphogenesis of male reproductive organs andspermatogenesis in /CR mice
Yuko DOil, Norio IMAJI, Kyoko NABAEI, Yousuke TODAI , Seiko TAMANOI, Hideki WANIBUCHI2,
Keiichirou MORIMURA2, Shoji FUKUSHIMA2
Daiyu-Kai Institute of Medical Science, Aichi 491-0113 , Japan I
Osaka City University Medical SchooI2

P-12 (Wed.)
Lack of carcinogenic risk in the prostate with transplacental and lactational exposure to
4-nonylphenol in F344 rats
Hiroko YOSHINO!, Kyoko NABAEI , Yuko DOJI, Takeshi HIROTA I, Kurniko OGAWA2, Tomoyuki SHIRAI2
Daiyu-Kai Institute of Medical Science, Aichi 491-0113 , Japan 1
Deptment of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of Medical
Sciences2

Risk Assessment

1:OOpm-2:00pm

P-13 (Mon.)
Using animal and human mode of action information in assessing human risk: a framework
for analysis of various pathologies
Penelope A FENNER-CRISP 1, Dorothy E PATTON 1, The ILSI RSI WORKGROUP 1
ILSI Risk Science Institute, Washington DC 20005, USA I

JSTP/IFSTP IATP 2001
P-14 (Tue.)
Carcinogenicity of low-dose MelQx, in relation to cancer risk assessment
Keiichirou MORIMURA I, Hideki WANIBUCHJI , Manabu HOSHJI, Anna KINOSHITAI, Shoji FUKUSHJMA I
Department of Pathology, Osaka City University Medical School, Osaka 545-8585 , Japan 1

P-15 (Wed.)
Presence of the threshold for carcinogenicity and in vivo mutagenicity induced bygenotoxic carcinogen, MelQx, at low doses
Saki NAITOI, Keiichirou MORIMURA' , Manabu HOSHJI , Nats uko MIYAZII , Shoji FUKUSHIMA I
Department of Pathology, Osaka City University Medical School, Osaka 545 -8585 , Japan 1

P-16 (Mon.)
Lack of modification of MelQx rat liver carcinogenesis by caffeine induction of CYP1A2
Hitoshi KANDORI1,2, Masanori KURIBAYASHJ l,3, Shingo INAGUMAI , Makoto ASAMOTOI ,Naorni HOKAIWADOI , Seishiro TAKAHASHJI , Tomoyuki SHIRAII
Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of Medical
Sciences, Aichi 467-8601, Japan 1
Takeda Chemical Industries Ltd. Drug Safety Research Center Hikari Branch2
Ono Pharmaceutical Co. Ltd. , Safety Research3

P-17 (Tue.)
Investigation of the complex carcinogenic risk by PhlP and Me/Qx in the initiation stage ofcarcinogenesis
Akihiro HIRATA 1, Tetsuya TSUKAMOTO!, Hiroki SAKAI2, Masarni YAMAMOTO! , Norimitsu SHIRAI1,
Takeshi IIDAKAI, Tokuma YANAF, Toshiaki MASEGF, Masae TATEMATSU I
Division of Oncological Pathology, Aichi Cancer Center Research Institute, Aichi 464-8681, Japan 1
Department of Veterinary Pathology, Faculty of Agriculture, Gifu University2

P-18 (Wed.)
Colon carcinogenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP) in rats:suggestion for carcinogenesis and practical threshold
Kenichiro DOIi , Elsayed I SALIMI , Anna KINOSHITA I, Rawiwan PUATANACHOKCHAII ,Hideki WANIBUCHJI, Shoji FUKUSHJMAI
Department of Pathology, Osaka City University Medical School, Osaka 545-8585, Japan 1

P-19 (Mon.)
Estimation of a no observed effect level for 2-acetylaminofluorene (2-AAF) and
2,4-diaminotoluene (2,4-DAT), genotoxic liver carcinogens, in 16-week feeding studies using
male F344 rats
Akihiro HAGIWARA' , Toshio ICHIHARAI , Hiroko YOSHINOI , Norio IMAII , Hideki WANIBUCHJ2,
Keiichirou MORIMURA2, Seiko TAMANOI
Daiyu-kai Institute of Medical Science, Aichi 491-0113 , Japan I
Department of Pathology, Osaka City University Medical SchooJ2

P-20 (Tue.)
Effect of phenobarbital on diethylnitrosamine-induced hepatocarcinogenesis in TGF-alphatransgenic mice-correlation with P450
Takayuki YUNOKI 1, Hideki WANIBUCHII , Rawiwan PUATANACHOKCHAII , Masakazu KAKUNII,
Kazuo HAKOII, Shoji FUKUSHIMA !
Department of Pathology, Osaka City University Medical School, Osaka 545-8585, Japan,1

P-21 (Wed.)
Possible mechanism of hormetic phenomenon of alpha benzene hexachloride ondiethylnitrosamine induced hepatocarcinogenesis
Rawiwan PUATANACHOKCHAII, Hideki WANIBUCHII , Mayuko OSADA2, Keiichirou MORIMURAI,
Yoshihiko FUNAE2, Shoji FUKUSHIMA!
Department of Pathology, Osaka City University Medical School, Osaka 545-8585, Japan 1
Department of Chemical Biology, Osaka City University Medical School2

JSTP/IFSTP IATP 20021
P-22 (Mon.)
Effect of low-dose DDT on liver carcinogenesis in rats
Masahiko KUSHIDAI , Tokuo SUKATA I, Keisuke OZAKII, Satoshi UWAGAWAI, Keiichirou MORIMURA2,
Hideki WANIBUCHI2, Yasuyoshi OKUNO!, Shoji FUKUSHIMA 2
Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., Osaka 554-8558, Japanl
Department of Pathology, Osaka City University Medical School 2

P-23 (Tue.)
Activation of the ras oncogene and its relationship to aflatoxins-DNA adduct formation in
rat liver treated with aflatoxins
Dae Joong KIMI, Tae Myoung KIMI, Jin Tae HONG2, Hwan Sao Y002, Chea! Beam PARK3, Beam Jun LEEI ,Jong-Koo KANGI , Young Won YUNI
College of Veterinary Medicine, Chungbuk National University! , Cheongju 361-763, Korea 1
College of Pharmacy, Chungbuk National University2
Biotoxtech3

P-24 (Wed.)
Lack of effects of 1439 MHz electromagnetic near field exposure on the blood-brain barrier
in immature and young rats
Masanori KURIBAYASHil .4, Jianqing WANG2, Osamu FUJIWARA2, Yuko DOP, Kyoko NABAE3,
Seiko TAMAN03, Tadashi OGISOI , Makoto ASAMOTOI , Tomoyuki SHIRAII
Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of Medical
Sciences, Aichi 467-8601, Japan 1
Department of Electrical and Computer Engineering, Nagoya Institute of Technology2
Daiy u- kai Institute of Medical Science3
Ono Pharmaceutical Co., Ltd. , Safety Research4

Genotoxic and Non-genotoxic Carcinogen

1:00pm-2:00pm

P-25 (Mon.)
Specific in vivo mutational spectra of genotoxic and non-genotoxic hepatocarcinogens in
gpt delta transgenic rats
Keita KANKII, Akiyoshi NISHIKAWA!, Ken-ichi MASUMURA2, Takashi UMEMURAI , Takayoshi IMAZAWAl ,Yasuki KITAMURA! , Takehiko NOHMF, Masao HIROSE 1
Division of Pathology, National Institute of Health Sciences, Tokyo 158-8501 , Japan I
Division of Genetics and Mutagenesis, National Institute of Health Sciences2P-26 (Tue.)
Mechanistic study using cDNA microarray on enhanced hepatocarcinogenesis in /CR mice
fed diet containing dicyclanil
Mitsuyoshi MOTOI , Miwa OKAMURA! , Tomoko MUTOH 1, Takao WATANABE! , Yoko KASHIDA 1,
Noboru MACHIDAI , Kunitoshi MITSUMORIILaboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan I

P-27 (Wed.)
Six-month carcinogenicity study of N-bis(2-hydroxypropyl)nitrosamine (DHPN) in rasH2
mice
Miwa OKAMURA I, Mitsuyoshi MOTOI , Yoko KASHIDAI, Noboru MACHIDA I, Kunitoshi MITSUMORil
Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan I

P-28 (Mon.)Genomic analysis of the difference between non-genotoxic carcinogen and genotocic
carcinogen in the urinary bladder
Junko HATAI , Azusa TAMURAI, Natsuki KITAJIMAI, Yoshinori KASAHARA!, Hiroshi UNOI,
Keiichirou MORIMURA2, Shoji FUKUSHIMA2
Pharmaceuticals Development Research Laboratories, Teijin Pharma Ltd. , Tokyo 191-8512, Japanl
Department of Pathology, Osaka City University Medical School2.

. . ~-~=-· ...,.

JSTP/IFSTP IATP 2004
Infectious Agents

1:00pm-2:00pm

P-29(Tue.)
Suppurative gastritis in BALB!c mice infected with Listeria monocytogenes via theintragastric route
Jong-Hwan PARKI , Yong-Ho PARK2, Seung-Hyeok SEOKl , Sun-A CHOI , Hui-Young LEEI, Dong-Jae KJMl,
So-Hyun KJM2, Jae-Hak PARKl, Young-Soon LEE3
Department of Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University, Seoul
151-742, Korea (South)I
Department of Veterinary Microbiology, College of Veterinary Medicine, Seoul National University 2
Deparment of Veterinary Public Health, College of Vete1inary Medicine, Seoul National University 3

P-30 (Wed.)
Analyses of immune response to candidate live oral Salmonella vaccine strain in mice
Sun A CHOI, In Soo LEE2, Jong Hwan Park PARKI , Seung Hyeok SEOKI, Hui Young LEE' , Dong Jae KJMI ,Jae Hak PARKI
Department of Laboratory Science, College of Veterinary Medicine, Seoul National University,
Seoul 151-742, Korea (South)lDepartment of Microbiology, Hannam University2

P-31 (Mon.)
Earlier Helicobacter pylori infection cause severer inflammation and more risk for stomach
carcinogenesis in N-methyl-N-nitrosourea treated Mongolian gerbils
Tetsuya TSUKAMOTO!, Xueyuan CAOI , Masami YAMAMOTO! , Harunari TANAKA I, Masae TATEMATSUIDivision of Oncological Pathology, Aichi Cancer Center Research Institute, Aichi 464-8681, Japan 1

P-32 (Tue.)
Increased oxidative stress associated with alterations in DNA damage and repair in urinary
bladder carcinomas associated and non-associated with Schistosomiasis
Elsayed I. SALIMI, Hideki WANIBUCHil, Amani ABDUL-HAMID2, Keiichirou MORIMURAI,
Shoji FUKUSHIMA I
Department of Pathology, Osaka City University Medica] School, Osaka 545-8585, Japan 1
Department of Pathology, Tanta Cancer Institute2

P-33 (Wed.)
Roles of the Thai liver fluke, Opisthorchis viverrini, in the development of cholangiocellular
carcinoma (CCC).
Witaya THAMAVIT1, Tomoyuki SHIRAI2, Malcolm A. MOORE2 , Nobuyuki IT0 2
Department of Pathobiology, Faculty of Science, Mahidol University 1
Department of Pathology, Nagoya City University, Medical SchooJ2

Heavy Metals and Natural Toxins

1 :OOpm-2:00pm

P-34 (Mon.)
In vitro and in vivo sensitivity of various cells to arsenic trioxide(As203)
Chang Suk KANGI , Yeon Sook MOONI , Yong Gu KIMI, Young Jin CHOII , Kyung Ja HANI, Sang In SHIM'
Department of Clinical Pathology, Catholic University of Korea, Seoul 150-713, Korea (South) 1

P-35 (Tue.)
Induction of glutathione 5-transferase placental form positive foci in liver and epithelial
hyperplasia in urinary bladder, but no tumor development in male Fischer 344 rats treatedwith monomethylarsonic acid for 104 weeks
Jun SHENI , Hideki WANIBUCHil, Elsayed SALIMI , Kenichiro DOI', Min WEI 1, Shoji FUKUSHIMA 1
Department of Pathology, Osaka City University Medical School, Osaka 545-8585, Japan 1

JSTP/IFSTP--tATP 200~
P-36 (Wed.)
Effects of sodium cacodylate and sodium nitrate on kidney tumor induced bydimethylnitrosamine in SD male rats.
Tsutomu HIBINOl, Takamasa YANAGIDA2, Junya MATUYAMAI, Ayumi KATil, Naoki YAMAMOT02
Department of Pathology, Fujita Health University College, Aichi 470-1192, Japan I
Joint Research Laboratory, Fujita Health University2

Food Safety

1:OOpm-2:00pm

P-37 (Mon.)
Involvement of choline and amino acid metabolism in different hepatocarcinogenicityof L-amino acid-defined semisynthetic and semipurified choline-deficient diets in rats
Maki IGARASHII , Masakazu TAKAHASHF, Hitoshi ASHIDA4, Mei-Heng MARS, Yutaka HATANAKA4,
Midori YOSHIDA2, Fumiyuki UEMATSU2, Naoto WATANABE! , Akihiko MAEKAWA3, Steven H ZEISEL5,
Dai NAKAE2
Laboratory of Protection of Body Function, Department of Food and Nutritional Science, Graduate school of
Agriculture, Tokyo University of Agriculture, Tokyo 101-0062, Japan!
Department of Pathology, Sasaki Institute, Sasaki Foundation2
Director, Sasaki Institute, Sasaki Foundation3
Division of Life Science, Graduate School of Science and Technology, Kobe University4
Department of Nutrition, School of Public Health and School of Medicine, University of North Carolina at Chapel
Hill5

P-38 (The.)
Induction of fatty liver by mebalonic acid in Fischer 344 rats
Naoto WATANABE! , Midori YOSHIDA2, Maki IGARASHil , Furniyuki UEMATSU2, Masakazu TAKAHASHF,
Akihiko MAEKAWA3, Dai NAKAE2
Department of Food and Nutritional Science, Laboratory of Protection of body function , Graduate School of Tokyo
University of Agriculture, Tokyo 101-0062, Japan!
Department of Pathology, Sasaki Institute, Sasaki Foundation2
Department Director, Sasaki Institute, Sasaki Foundation3

P-39 (Wed.)
Induction of multiple granulation in the liver with severe hepatocyte damage by montan
wax, a food additive, in a 90-day toxicity study in F344 rats
Mico IKEDA I, Kousuke SAOOI , Keiko YAMAKAWA I, Hijiri TAKEUCHII , Masanao YOKOHIRAI,
Yoko HOSOTANil, Yu ZENGt, Kyoko HOSOKAWAI, Shigemi KINOUCHI2, Katsumi IMAIDAl
Department of Onco-Pathology, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan I
Diagnostic Pathology and Cytology Institute, Shikoku Cytopathologic Institutes2

P-40 (Mon.)
Dietary intake of various lactic acid bacteria suppresses Type 2 helper T cell production in
antigen-primed mice splenocyte
Hui Young LEEl , Jong Hwan PARKI , Seung Hyeok SEOKl, Sun A CHOI, Min Won BAEKl, Dong Jae KIMI,
Yong Soon LEE2, Jae Hak PARKI
Department of Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University, Seoul 151742, Korea (South) 1
Department of Public Health, College of Veterinary Medicine, Seoul National University, Seoul, Korea2

Biomakers

1:00pm-2:00pm

P-41 (Tue.)
Examination of heat shock protein 70 as an early-stage injury-biomarker in rat testes after
stress-induced cell damage
Akiko IKEDA 1, Yoshihumi KANEKO!, Makiko TAKAHASHI!, Sumihisa SUEYOSHI!, Yoshihiro MASUMOTOl,
Kiyoyuki TSURUt
Kyorin Phamaceutical Co., Ltd, Research Center, Tochigi 329-0114, Japan!

JSTP/IFSTP IATP 2004
P-42 (Wed.)
Histopathological and endocrinological changes of cardiac natriuretic peptide in rats withdrug- induced cardiotoxicity
Hiroto MIYATA 1, Rie OHNO! , Rika SHIRANEI , Yutaka NAKANISHI! , Atsushi NAKAMURA I,
Masaaki KIMURA 1
Toxicology Laboratory, Medicinal Research Laboratories, Taisho Pharmaceutical Co. , Ltd. ,Saitama 33 1-9530, Japan I

P-43 (Mon.)
Histomorphometrical parameters for evaluation of osteocompatibility of metal implants withundecalcified bone section
Miki NISHIMORII, Shin-ichi KATSUDAL , Yoshimitsu OKAZAKI2, Emiko GOT03, Akihito SHIMOil,
Hidetaka SAT0 1
Japan Food Research Laboratories, Tokyo 206-0025 , Japanl
National Institute of Advanced Industrial Science and Technology2
National Institute of Technology and Evaluation3

P-44 (Tue.)
Genetic polymorphisms of alcohol-related metabolizing enzymes and the risk of esophageal
cancer in the Thai population
Pleumjit BOONYAPHIPHATI , Paramee THONGSUKSAII , Wanna SUDHIK.ARANI, Puttisak PUTTAWIBUL2
Department of Pathology, Faculty of Medicine, Prince of Songkla University, Songkla 90110, Thailand I
Department of Surgery, Facu lty of Medicine, Prince of Songkla University2

SESS10N2: Molecular Toxicopathology or Mechanism
Room 301
Genomics

1:00pm-2:00pm

P-45 (Wed.)
Toxicogenomics using "percellome" and "mille-feuille" data system
Jun KANNO!, Ken-ichi AISAKII, Atsushi ONOl, Katsuhide IGARASHI 1
Cellular & Molecular Toxicology Division, Biological Safety Research Center, National Institute of Health Sciences,
Tokyo 158-8501 , Japan!

P-46 (Mon.)
Microarray analysis of amplified RNA from laser- microdissected GST-P negative
hepatocellular preneoplastic lesion induced by peroxisome proliferators.
Tokuo SUKATAI, Satoshi UWAGAWAI , Keisuke OZAKII, Kayo SUMIDA1, Masahiko KUSHIDAI,
Kaoru KIKUCHI3, Koichi SAIT0 1, Kenji OEDA 1, Yasuyoshi OKUN0 1, Nobutoshi MIKAMI 1,
Shoji FUKUSHIMA2
Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd ., Osaka 554-8558, Japan 1
Department of Pathology, Osaka City University Medical School2
Sumitomo Pharmaceuticals Co. , Ltd.3

P-47 (Tue.)

Specific differences in gene expression profile revealed by cDNA microarray analysis ofglutathione S-transferase placental form (GST-P) immunohistochemically positive rat liverfoci and surrounding tissue
Shugo SUZUKI!, Makoto ASAMOTOI, Kazunaii TSUJIMURAl ,2, Kumiko OGAWA I, Mitsuru FUTAKUCHJL ,Tomoyuki SHIRAI 1
Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of Medical
Sciences, Aichi 467-8601 , Japan I
Chemicals Evaluation and Reseai·ch Institute, Chemical s Assessment Center2

P-48 (Wed.)
Potential for the prediction of carcinogenicity by gene expression profile in rat hepatoma
cells and comparison of expression patterns between chemically treated rat and humanhepatoma cells
Kazunai·i TSUJIMURAl ,2, Makoto ASAMOTOl , Shugo SUZUKI! , Shingo INAGUMAI, Kumiko OGAWA I,
Tomoyuki SHIRAI!
Depaitment of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of Medical
Sciences, Aichi 467-8601 , Japan 1
Chemicals Evaluation and Research Institute, Chemicals Assessment Center2

P-49 (Mon.)
Gene expression profiles in F344 rat livers treated with acetaminophen or cycloheximide
Takashi YAMOTOI, Naoki KIYOSAWAI, Kazumi ITOI , Takayuki SATOI , Kyoko SAKUMA 1, Miyuki KANBORII ,Noriyo NIINOI, Sunao MANABE2, Naochika MATSUNUMAI
Medicinal Safety Research Labs., Sankyo Co. , LTD, Shizuoka 437-0065 , Japan 1
Sankyo Pharma Inc.2

P-50 (Tue.)
Methacarn, a versatile fixation tool for quantitative mRNA expression analysis in
microdissected paraffin-embedded tissues using real-time RT-PCR and microarray systems
Makoto SHIBUTANII , Kyoung-Youl LEEI , Hironori TAKAGI! , Natsumi KATOI , Shu TAKIGAMII ,Masao HIROSE 1
Division of Pathology, National Institute of Health Sciences, Tokyo 158-8501, Japan 1

38

JSTP/IFSTP IATP 2004
P-51 (Wed.)
Transcription of rat amyloid precursor protein in rat livers is affected by the serum
cholesterol concentration
Naoki KIYOSAWAI, Kazumi ITOI, Kayoko ISHIKAWA I, Isao IGARASHII, Noriyo NIINOI , Kyoko SAKUMA! ,Miyuki KANBORJI, Takashi YAMOTOI , Sunao MANABE2, Naochika MATSUNUMAI
Medicinal Safety Research Labs., Sankyo Co., Ltd., Shizuoka 437-0065, Japan 1
Sankyo Pharma Inc.2

P-52 (Mon.)
Gene expression profiling of terminal end buds during rat mammary carcinogenesis
Koichiro MIYAMOTO!, Shuhei TAGUCHI!, Hiroaki KAWAGUCHI! , Yoshihisa UMEKITAI, Hiraki YOSHIDA I
Department of Tumor Pathology, Graduate School of Medical and Dental Sciences, Kagoshima University,
Kagoshima 890-8520, Japan 1

P-53 (Tue.)
Region-specific global gene expression analysis in the microdissected hypothalamic medialpreoptic area of rat neonates exposed perinatally to di(2-ethylhexyl)phthalate
Kyoung-Youl LEEI, Makoto SHIBUTANII , Hironori TAKAGI! , Natsurni KATOI , Shu TAKIGAMII,
Masao HIROSE 1
National Institute of Health Sciences, Tokyo 158-0098, Japan I

P-54 (Wed.)
Differential global gene expression in rat brown and white adipose tissues
Akira UNAMJI , Yasuo SHINOHARA2, Kazuaki KAJIMOT02, Kenjiro TSUBOTAI, Yoshimasa OKAZAKI!,
Shiro FUJIHIRAI , Masahiro MATSUMOTO!, Yuji OISHII, Yoshinobu BABA3
Toxicology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Osaka 532-8514, Japan!
Institute for Genome Research, The University of Tokushima2
Faculty of Pharmaceutical Sciences, The University of Tokushima3

P-55 (Mon.)
Microarray analysis of hepatocellular adenoma induced by di(2-ethylhexyl)phthalate in rasH
2mice
Kaoru TOYOSAWAI , Miwako HARADA2, Katsuji NAKAN02, Kohji TANAKA!, Nobuo MATSUOKA!
Safety Research Laboratories, Drug Research Division , Dainippon Pharmaceutical Co. , Ltd.,
Osaka 564-0053, Japan I
Pharmacology & Microbiology Research Laboratories, Drug Research Division, Dainippon Pharmaceutical Co.,
Ltd.2

P-56 (Tue.)
Mechanistic analyses using chip array analysis on inhibited uterine tumorigenesis in rasH2
mice initiated with N-ethyl-N-nitrosourea followed by dietary treatment of ethinylestradiol
Takao WATANABE!, Kayo SUMIDA2, Tomoko MUTOHI, Miwa OKAMURA!, Mitsuyoshi MOTOI,
Yoko KASHIDAI , Kunitoshi MITSUMORJI
Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan 1
Environmental Health Science Laboratory, Sumitomo Chemical Co. , Ltd.2

Carcinogenesis

1:00pm-2:00pm

P-57 (Wed.)
Tumor induction by colon carcinogen in rat gastric mucosa featuring intestinal metaplasia
caused by X-irradiation

Shoji KASHIWABARAI , Naoki KASHIMOTOI , Toshihiro UESAKAI , Osamu KATOH! , Keiji WAKABAYASHI2,
Hirornitsu WATANABE!
Department of Cellular Biology, Research Institute for Radiation Biology and Medicine,
Hiroshima University, Hiroshima 734-8553, Japan I
National Cancer Center Research Institute2

JSTP/IFSTP IATP 200~
P-58 (Mon.)
Expression of peroxisome proliferator-activated receptor gamma in helicobacter
pylori- associated mouse gastric cancer tissue and human gastric cancer cells.
Dae-Yong KIMI , Sang-Yeon OHi , Ki Taek NAM2, Beom Seok HAM2, Dong Deuk JANG2, Ki-Hwa YANG2,
Ki-Baik HAHM3
Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University I
National Institute of Toxicological Research2
Genome Research Center for Gastroenterology, Ajou University School of Medicine3

P-59 (Tue.)
Time-course and quantitative study of the relationship between transforming growth
factor-alpha and glutathione 5-transferase placental form expression during rat chemical
hepatocarcinogenesis
Mitsuaki KITANOI , Jutaro WADAl, Yutaka ARIKJI , Masanori KATOI , Hideki WANIBUCIIT2,
Keiichirou MORIMURA2, Kazunori HOSOEI , Shoji FUKUSHIMA2
Life Science Research Laboratories, Life Science RD Center, Kaneka Corporation, Hyogo 676-8688, Japan L
Department of Pathology, Osaka City University Medical School2

P-60 (Wed.)
Enhancing effects of combined treatment with IQ and sodium nitrite on colon and liver
carcinogenesis in rats
Yasuki KITAMURA I, Kazushi OKAZAKJL, Akiyoshi NISHIKAWA I, Keita KANK.11, Takashi UMEMURAI,
Takayoshi IMAZAWAI, Masao HIROSEIDivision of Pathology, National Institute of Health Sciences, Tokyo 158-8501, Japan I

P-61 (Mon.)
Involvement of the altered regulation of pre-mRNA splicing in the liver carcinogenic
processes
Masakazu TAKAHASHII, Fumi yuki UEMATSUI, Midori YOSHIDAI , Maki IGARASHII, Naoto WATANABEI,
Akihiko MAEKAWA2, Dai NAKAEI
Department of Pathology, Sasaki Institute, Sasaki Foundation, Tokyo 101-0062, Japan 1
Director, Sasaki Institute, Sasaki Foundatio n, Japan2

P-62 (Tue.)
Genetic alterations in the Catnb gene but not the H-ras gene in hepatocel/ular neoplasms
and hepatoblastomas of B6C3F1 mice following exposure to diethanolamine for 2 years
Shim-mo HAYASHII , Thai Vu TON2, Hue-Hua L. HONG2, Richard D. IRWIN2, Joseph K. HASEMAN2,
Theodora R. DEVEREUX2, Robert C. SILLS2
Pfizer Inc., Worldwide Safety Sciences, Taketoyo, Aichi 470-2393 , Japan I
National Institute of Environmental Health Sciences2P-63 (Wed.)
Carcinogenicity of phenobarbital in Mmh/OGG1 knockout mice
Anna KINOSHITAI , Hideki WANIBUCHJI , Keiichirou MORIMURA', Takayuki YUNOKI' , Shoji FUKUSHIMA!
Department of Pathology, Osaka City University Medical School, Osaka 545-8585, Japan 1

P-64 (Mon.)
Oxidative stress and aberrant DNA methylation in cancer
Yashige KOTAKEI , Kiyoshi ASADAI, Hong SANGI , Robert H. BROYLES', Robert A. FLOYD'
Oklahoma Medical Research Foundation, Oklahoma 73104, USA I

P-65 (Tue.)
Methylation-associated silencing of p53 responsive gene 2 and Fibril/in 2 in humanpancreatic cancers
Atsushi HAGIHARA', Kazuaki MIYAMOT02, Junichi FURUTA2, Shuichi SEKJ3, Shoji FUKUSHIMA',
Toshikazu USHIJIMA2
Department of Pathology, Osaka City University Medical School, Osaka 545-8585, Japan!
Carcinogenesis Division, National Cancer Center Research Institute2
Internal Medicine, Osaka City University Medical SchooP40

P-66 (Wed.)
Lack of influence of testicular castration or sialoadenectomy on sodium L-ascorbate
promotion of urinary bladder carcinogenesis in male F344 rats
Satoru MORii, Takashi MURAii , Keiichirou MORIMURAi , Hideki WANIBUCHli , Shoji FUKUSHIMAl
Deparment of Pathology, Osaka City Univercity Medical School, Osaka 545-8585, Japan i

P-67 (Mon.)
Effect of amiloride and ouabain on promotion by sodiium L-ascorbate in two-stage rat
urinary bladder carcinogenesis
Takashi MURAii, Hideyuki MIYAUCHii , Satoshi INOUEI , Takeki UEHARAi , Toshiyuki MARUYAMAI ,Satoru MORI2, Hideki WANIBUCHI2, Shoji FUKUSHIMA2
Developmental Research Laboratories, Shionogi & Co. , Ltd. , Osaka 561-0825, Japan 1
First Department of Pathology Osaka City University Medical SchooI2

P-68 (Tue.)
Susceptibility of p27kipt knockout mice on N-butyl-N-(4-hydroxybutyl) nitrosamine-induced
urinary bladder carcinogenesis
Toshiya MURASAKJl , Kumiko OGAWAi , Atsuya HIKOSAKAi, Satoshi SUGIURAi , Seishiro TAKAHASHii,
Tomoyuki SHIRAii
Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of Medical
Sciences, Aichi 467-860 1, Japanl

P-69 (Wed.)
Promoting effect of sodium L-ascorbate on N-butyl-N-(4-hydroxybutyl)nitrosamine-induced
renal pelvic carcinogenesis in SD/cShi rats of both sexes
Takashi MURAii , Hideyuki MIYAUCHii, Satoshi INOUEi , Takeki UEHARAi , Toshiyuki MARUYAMAi,
Aki hiro K0IDE2, Yukio MORF, Satoru MORI\ Hideki WANIBUCHP, Shoji FUKUSHIMA3
Developmental Research Laboratories, Shionogi & Co., Ltd., Osaka 561-0825, Japani
Laboratory of Radiochemistry, Gifu Pharmaceutical University2
Department of Pathology, Osaka City University Medical SchooP

P-70 (Mon.)
Renal cell carcinoma cell lines established from Nihon rat
Izumi MATSUMOTOi , Tadayoshi UEDAi , Kazu yasu KIJIMA i, Youko HIRAYAMA2, Hiroaki MITAN12,
Kazuo OKIMOTOi, Kohji TANAKAi , Okio HIN02, Nobuo MATSUOKAi
Drug Research Division , Dainippon Pharmaceutical Co. , Ltd. , Osaka 564-0053, Japan i
Department of Experimental Pathology, Cancer Institute2

P-71 (Tue.)

Strain-specific mammary proliferative lesion development following lifetime oral
administration of ochratoxin A in DA and Lewis rats
Yong-Soon LEEi, Woo-Chan SON2, Kyung-Sun KANG3
Department of Public Health, Seoul National University, Seoul 151-742, Korea (South) i
Huntingdon Life Sciences Pathology2
Department of Public Health, Seoul National University3

P-72 (Wed.)
Strain difference in spontaneous development of uterine adenocarcinoma between donryu
and Fischer344(F344)rats
Takaharu NAGAOKAi , Hiroaki MIYAJIMA i, Akihiko MAEKAWA2
Shin Nippon Biomedical Laboratori es, LTD. Kagoshima 891-1394, Japan i
Sasaki Institute2

P-73 (Mon.)
Expression profile of estrogen receptor- a -related signaling pathways in the development ofendometrial adenocarcinomas in Donryu rats
Midori YOSHIDAl, Takasumi SHIMOMOTOi , Yutaka HATANAKA3, Takuji MIHARA3, Akihiko MAEKAWA2,
Dai NAKAEi
Department of Pathology, Sasaki Institute, Sasaki Foundation, Tokyo 101-0062, Japan i
Director, Sasaki Institute, Sasaki Foundation2
BioMedical Science Department, Dako Cytomation Co., Ltd.3

JSTP/IFSTP IATP 2004
P-74 (Tue.)
Constitutive expression of cox-2 in thyroid follicular epithelial cells and its significant
reduction during DHPN-induced carcinogenesis in rats
Toshia IMAII, Mai HASUMURAI , Jun-ichi ONOSEl, Makoto UEDAI , Tamotsu TAKIZAWAI, Young-Man CHOI,
Masao HIROSE I
National Institute of Health Sciences, Tokyo 158-8501, Japan 1

P-75 (Wed.)
PCNA labeled index of spontaneous lung proliferating lesion in rats
Masayuki MITSUI!, Midori YOSHIDA2, Kasuke NAGAN03, Toshifumi TSUJIUCHII, Hiroshi MARUYAMA 4 ,Masahiro TSUTSUMI 1
Department of Oncological Pathology, Cancer Center, Nara Medical University, Osaka 556-0005, Japan 1
Department of Pathology, Sasaki Laboratory2
Division of Pathology, Japan Bioassay Research Center3
Department of Pathology, Hoshigaoka Koseinenkin Hospital 4

P-76 (Mon.)
The role of polymerase beta mediated DNA repair in skin tumorigenesis: using DNA
polymerase beta over-expressed transgenic mouse
Kazuo HAKOI', Raymond W TENNANT2, Ronald E CANNON2
Drug Safety Research Lab. , Taiho Pharmaceutical Co., Ltd ., Tokushima 771-0194, Japan 1
Cancer Biology Group, National Center for Toxicogenomics, National Institute of Environmental Health Sciences2P-77 (Tue.)
Possible relationships between immunosuppressive effects and carcinogenicity of thesynthetic gestagens with glucocorticoid-like effects in rodents
Shigeru HISADAI, Toshi HORIUCHI 1
Toxicology & Pharmacokinetics Research Department, Kawasaki 213-8522, Japan 1

Cancer Prevention

1:00pm-2:00pm

P-78 (Wed.)
Chemopreventive effects of plant derived compounds in a rat medium term liver bioassay
and on hepatocellular carcinoma cell lines and gene expression analysis by a microarray
Hiroyuki OHNISHII , Kazunari TSUJIMURAl ,2, Makoto ASAMOTO I, Masanori KURIBAYASHil ,3,Shuga SUZUKI 1, Tadashi OGIS0 1, Tornoyuki SHIRAl 1
Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of Medical
Sciences, Aichi 467-8601 , Japan I
Chemicals Evaluation and Research Institute, Chemicals Assessment Center2
Ono Pharmaceutical Co. Ltd ., Safety Research3

P-79 (Mon.)
The inhibitory effects of arctiin on F344 rat multiorgan carcinogenesis
Masaharu MOKUI , Hideki WANIBUCHII, Jin Seok KANGI , Saki NAITOI , Anna KINOSHITA!,
Shoji FUKUSHIMA I
Department of Pathology, Osaka City University Medical School, Osaka 545-8585, Japan 1

P-80 (Tue.)
Chemopreventive effect of PJJ-34 in rat multi-organ carcinogenesis model
Chiharu YAMAGUCHI!, Sakai KUNIYOSII , Takasi YAMAGUCHI I, Tomoko NAKATA I, Reiko TANAKA I,
Hideki WANIBUCHI2, Anna KINOSITA2, Keiichirou MORIMURA2, Shoji FUKUSIMA2
Department of Medicinal Chemistry, Osaka University of Parmaceutical Science, Osaka 569-1094, Japan 1
Department of Pathology, Osaka City University Medical School2

P-81 (Wed.)
Dietary protocatechuic acid inhibits progression of chemically induced rat tongue
carcinogenesis
Rikako SUZUKil ,2, Hiroyuki KOHNO I, Shigeyuki SUGIEI, Takuji TANAKA!
Department of Pathology, Kanazawa Medical University, Ishikawa 920-0293 , Japan I
Research Fellow of the Japan Society for the Promotion of Science2

JSTP/IFSTP IATP 200~
P-82 (Mon.)
Chemopreventive effect of FBRA on N-nitrosomethylbenzylamine-induced esophageal
tumorigenesis in rats
Toshiya KUNOI , Yoshinobu HIROSEI , Yasuhiro YAMADA I, Keiko SAKATA I, Nami KITAORII , Akira HARAI ,Hideki MORII
Department of Tumor Pathology, Gifu 500-8705, Japan I

P-83 (Tue.)
Acyclic retinoid inhibits the aberrant crypt foci formation induced by dimethylhydrazine in
the rat colon
Masumi SUZUJI , Takahiro SHIMIZUI , Takamitsu MORJOKAI , Viengvansay NABANDITHI , Morihiko INAMINEI ,Tatsuya KANESHIROI , Tatsuya KINJOI , Naoki YOSHIMII
Department of Pathology, University of the Ryukyus Faculty of Medicine, Okinawa 903-0215 , Japan I

P-84 (Wed.)
Time course observation on inhibitory effects of hepato-pro/iferative lesions in rats induced
by a liver-selective thyromimetic, KAT-681
Morimichi HAYASHII, Toru TAMURAI, Yuji OKUHARAI, Tatsuya NAGASAWAI , Tsuyoshi KITAMURA I,
Junji KURODAI , Nobuo SHIBATAI , Kunitoshi MITSUMORI2
Kissei Pharmaceutical Co. , Ltd ., Nagano 399-8305, Japanl
Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology 2

P-85 (Mon.)
Knockout of iNOS gene does not prevent hepatocarcinogenesis caused bya choline-deficient, I-amino acid-defined (CDAA) diet in mice
Ayumi DENDAI , Nao MURATA I, Toshifumi TSUJIUCHII , Masahiro TSUTSUMII , Dai NAKAE2,
Yoichi KONISHII , Hiroki KUNIYASUIDepartment of Oncological Pathology, Nara Medical University, Nara 634-8521 , Japan 1
Department of Pathology, Sasaki Institute2

P-86 (Tue.)
Modifying effects of Terminalia catappa on azoxymethane-induced colon carcinogenesis inmale F344 rats
Takamitsu MORIOKA I, Masumi SUZUII , Viengvansay NABANDITHI , Morihiko INAMINEI ,Tatsuya KANESHIROI , Tatsuya KINJOI , Takahiro SHIMIZUI , Miyuki AONAHATAI , Naoki YOSHIMII
Tumor Pathology, University of the Ryukyus Faculty of Medicine, Okinawa 903-0215 , Japan I

P-87 (Wed.)
Lovastatin inhibits tumor growth and metastasis in a mouse mammary carcinoma model by
p53-independent mitochondrial-mediated induction of apoptosis
Masa-aki SHIBATAI , Yuko ITOI , Junji MORIMOT02, Yoshinori OTSUKJI
Department of Anatomy & Biology, Osaka Medical College, Osaka 569-8686, Japan I
Laboratory Animal Center, Osaka Medical College2

P-88 (Mon.)
Prevention of H. pylori associated gastric carcinogenesis in N-methyl-N-nitrosourea-treatedMongolian gerbils using concentrate of Japanese apricot (Ume)Harunari TANAKAI , Ken-ichi INADA2, Toru NIWAI , Takafumi OTSUKAI, Mitsuo GOTOI ,Hirotoshi UTSUNOMIYA3, Tetsuya TSUKAMOTOI, Masae TATEMATSUIDivision of Oncological Pathology, Aichi Cancer Center Research Institute, Aichi 464-8681 , Japan 1
1st Department of Pathology, Fujita Health University2
Department of Pathology, Wakayama Medical University School of Medicine3

P-89 (Tue.)
The effect of N-acetylecysteine on low dose 2-amino-3,Bdimethylimidazof4,5-f]quinoxaline(MelQx)-induced rat hepatocarcinogenesis
Natsuko MIYAZII , Hideki WANIBUCHI1 , Masaharu MOKUI , Keiichirou MORIMURAI , Yoshiaki TAGAWA 1,
Shoji FUKUSHIMA I
Department of Pathology, Osaka City University Medical School, Osaka 545-8585 , Japan!

JSTP/IFSTP IATP-200~
Animal Models

1 :OOpm-2:00pm

P-90 (Wed.)
Glycogen granule, glycogen phosphorylase, and CYP1 after exposure to DMBA in rat liver
Shin WAKUI', Masakuni FURUSAT02
Department of Toxicologic Pathology, Azabu University School of Veterinary Medicine, Kanagawa 229-8501 , Japan'
Department of Pathology, Kyorin University School of Medicine2

P-91 (Mon.)
Comprehensive gene expression profiles of hepatocellular carcinomas induced in rats fed acholine-deficient, L-amino acid-defined dietFumiyuki UEMATSU', Naoto WATANABE2, Maki IGARASHJ.2, Masakazu TAKAHASHI', Midori YOSHIDAl,
Akihiko MAEKAWA3, Dai NAKAE 1
Department of Pathology, Sasaki Institute, Sasaki Foundation, Tokyo 101-0062, Japan'
Department of Food and Nutritional Sciences, Graduate School of Agriculture, Tokyo University of Agriculture2
Director, Sasaki Institute, Sasaki Foundation3

P-92 (Tue.)
Enhanced preneoplastic lesion induction in the livers of gap junction disrupted transgenicrats
Naomi HOKAIWADO', Makoto ASAMOTO', Kazunari TSUJIMURA l,2, Kumiko OGAWA' , Tadashi OGISO' ,Tomoyuki SHIRAI'
Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of Medical
Sciences, Aichi 467-8601, Japanl
Chemicals Evaluation and Research Institute, Chemicals Assessment center2

P-93 (Wed.)
In situ hybridization of interferon-gamma mRNA in concanavalin A-induced hepatitis in miceusing the polymerase chain reaction-derived cRNA probes
Hideki TANAKA' , Atsushi FUKUNARI', Tomomichi IWAKI', Shiro TAKAGI'
Mitsubishi Pharma Corporation, Kanagawa 227-0033, Japan I

P-94 (Mon.)
Rapid induction of type-1 diabetes mellitus by X-irradiation in Long-Evans Agouti (LEA) rats
Tokiko NAKAI 1, Akiko KUB0 1, Eiko TAKISHITA 1, Tamotsu TAKIZAWA 1, Takeshi TSUCHIGAUCHI',
Keiji KODAMA', Keisuke IZUMI'
Department of Molecular and Environmental Pathology, The University of Tokushima School of Medicine,
Tokushima 770-8503, Japan'

P-95 (Tue.)
Porcine-serum-induced hepatic fibrosis in Brown Norway and Wistar rats
Yasuko BABA', Koji UETSUKA', Hiroyuki NAKAYAMA' , Kunio DOI'
Department of Veterinary Pathology, Graduate School of Agricultural and Life Science, The University of Tokyo,
Tokyo 113-8657, Japan'

P-96 (Wed.)
Induction of mRNA related with hepatic injury response to oxidative stimuli in hepatocytes
and non-parenchymal cells
Toshinobu YAMAMOTO' , Hiroyuki UTSUMI' , Naoko SHIMADA' , Tetsu OGATAI, Naohisa TSUTSUil
Toxicology Laboratory, Mitsubishi Pharma Corporation, Chiba 292-0818, Japan'

P-97 (Mon.)
Persistence of liver cirrhosis is associated with altered location of a -smooth muscle actin( a -SMA) positive cells
Jin Seok KANG 1, Hideki WANIBUCHI', Keiichirou MORIMURA', Elsayed I. SALIM', Shoji FUKUSHIMA 'Department of Pathology, Osaka City University, Osaka 545-8585, Japan 1

JSTP/IFSTP IATP ·200~
P-98 (Tue.)
Spontaneous development of maxillary incisor lesions in aged poly(ADP-ribose)polymerase-1 (Parp-1) knockout mice
Osamu KUSUOKA 1, Masahiro TSUTSUMI2, Toshifumi TSUJIUCHJ2, Kazutoshi TAMURAI ,Kohsuke HORIGUCHJI, Kazumi SHIRAIWA3, Nobuo KAMADA4, Hitoshi NAKAGAMA5, Takashi SUGIMURA5,
Mitsuko MASUTANI5
Gotemba Laboratories, Bozo Research Center Inc., Shizuoka 4 12-0039, Japan I
Department of Oncological Pathology, Cancer Center, Nara Medical University2
Insti tute fo r Life Science Research, Asahi Kasei Corporation3
Chugai Research Insti tute for Medical Sciences, Inc.4
Biochemistry Division, National Cancer Center Research Institutes

P-99 (Wed.)
Pancreas anomaly and intestinal tumorsin the mouse small eye mutants, Pax6Sey3H and
Pax6Sey4H
Yumiko NITTA 1
Res. Inst. Radiat. Biol. Med., Hiroshima University, Hiroshima 734-8553 , Japan l

P-100 (Mon.)
Establishment and characterization of cancer cell lines from glandular stomach carcinoma
induced by MNU in p53-deficient mice
Masami YAMAMOTQI , Hayao NAKANISHJI , Tetsuya TSUKAMOTQI , Akihiro HIRATAI , Hiroki SAKAF,
Norimitsu SHIRAI I, Takeshi IIDAKAI , Tokuma YANAJ2, Toshiili MASEGJ2, Masae TATEMATSUILaboratory of Pathology, Aichi Cancer Center Research Institute, Aichi 464-8681 , Japan I
Department of Veterinary Pathology, Gifu University2

P-101 (Tue.)
Influence of p53 gene deficiency on spontaneous tumor development in TCRs ·1·/p53·1· mice
Kana HASHIMOTOI, Shoichi KADOI , Kazumi UCHIDA I, Shin IWATAl, Yuriko NAGATA I, Minoru AND Ql,
Hideyuki FUNABASHJ I, Masaharu ONQUE I
Yakult Central Institu te fo r Microbiological Research, Tokyo 186-8650, Japan 1

P-102 (Wed.)
Progression of glomerulonephritis in cynomolgus monkeys caused by autoimmunity to NC1
domain of type fl/ collagen [K35]
Masatoshi YAMAMOTQI, Iori ITAGAKII, Masami HIRUMAI , Yoshihiro TAKEII , Norio MUTQI,Flordeli za P. DE VILLA2, Tsukao YOKOYAMA3, Syu nji HATTQRJ4, Hidekazu SHIGEMATSU5
Ina Research Inc. , Ina-shi, Nagano, 399-4501 Japan, Nagano 399-450 1, Japanl
INA RESEARCH PHILIPPINES , INC.2
Collagen Research Center3
Nippi Reseach Institu te of Biomatrix4
Department of Pathology, Shinsyu University School of Medicines

P-103 (Mon.)
Histological changes of pancreatic islets in Zucker Diabetic Fatty rats, with progress in thetype 2 diabetes-like disease
Naoshi SHIMOJI I, Wakako TOGAI , Satoko TOMIQKA I, Haruyo SUGIYAMAI, Kaori SASAI ,Nobutaka DEMURA I, Haruko KAMEDAI, Yusuke NAGAE I
Preclinical Development Department, Novartis Pharma K. K., Ibarili 300-2611 , Japan I

P-104 (Tue.)
The mouse rasH2/BHT model as an in vivo rapid assay for lung carcinogens
Takashi UMEMURAI, Yukio KODAMA2, Keita KANKII, Yasuki KITAMURA !, Takayoshi IMAZAWAI ,Akiyoshi NISHIKAWA 1, Masao HIROSE lDivision of Pathology, National Institute of Health Sciences, Tokyo 158-8501, Japan I
Division of Toxicology, National Institute of Health Sciences2P-105 (Wed.)
The utility of EHBR for toxicity study of organic anion drugs
Chitose KUWAYAMAl, Hiroyas u NABA I, Chihaya KAKINUMA I, Takuo OGIHARAI , Shuhei OHNISHJ I,
Akihito SHIMOI I
Phamaceutical Research Center, Machida Phamacetical Co., Ltd., Tokyo 160-851 5, Japanl

JSTP/IFSTP-- IATP 200~
P-106 (Mon.)
Morphological evaluation of bone dynamics using in vivo micro-CT analysis andhistomorphometry in mice osteoporosis model
Anbo XIANGI, Masahiro KANEMATSU1, Mana MITAMURAI , Hideo KIKKAWAI , Kiyoshi KOBAYASHI! ,Hiroyuki HIGASHIYAMAI , Mine KINOSHITA I, Satoshi ASANOI
Pharmacology Department, GlaxoSmithKline KK., Ibaraki 300-4247, Japan 1

P-107 (Tue.)Expression of a system L amino acid transporter at testis of rats after prenatal exposure todi(n-butyl)phtalate
Tomoko MUTOI, Yoshikatsu KANAII, Hitoshi ENDOUIDepartment of Pharmacology and Toxicology, Kyorin University, Tokyo 181-8611 , Japan 1

P-108 (Wed.)
Infiltrating CD8+ T and NK cells, and IL-1 O and TGF- /J 1 cytokines expression in chemicallyinduced neoplasias in a medium-term alternative bioassay using male Wistar rats
Ana Lucia Tozzi SPINARDI-BARBISANt, Luis Fernando BARBISANI, Joao Lauro Viana de CAMARGO!,Maria Aparecida Marchesan RODRIGUES l
Dept. Pathology, UNESP Medical School, SP 18618-000, Brazi[IP-109 (Mon.)
Green fluorescent protein (GFP) as a marker of aryl hydrocarbon receptor (AHR) function intransgenic zebrafish (Danio rerio)
Seung H. SEOK1 , Jong-H. PARKI, Sun A. CHOI , Min W. BAEK I, Hui Y. LEEI , Dong J. KIMI, Yong S. LEE2,
Jae H. PARKI
Department of Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University, Seoul 151 742, Korea (South)I
Department of Public Health, College of Veterinary Medicine, Seoul National Un iversity2

JSTP/IFSTP IATP 200~
SESS10N3 : Organ Toxicopathology
Room 401+402
Nervous System

1:00pm-2:00pm

P-110 (Tue.)
Pathology of Minamata disease (methylmercury poisoning) - special reference to theperipheral nerves
Komyo ETOI , Akira YASUTAKE' , Motohiro TAKEYA2, Michio AKIMA3
National Institute for Minamata Disease, Kumamoto 867-0008 , Japan'
Graduate School for Medicial and Pharmaceutical Science, Kumamoto University2
Toho University, School of Medicine3

P-111 (Wed.)
Mechanisms and disease control of N-methyl-N-nitrosourea-induced retinal degeneration in
animals
Katsuhiko YOSHIZAWA 1, Katsuji KIUCHP, Kaei MORIGUCHP, Yuji OISHI', Nobuaki SHIKATA2,
Airo TSUBURA2
Department of Toxicologic Pathology, Toxicology Research Laboratories, Fujisawa Pharmaceutical Co. , Ltd. , Osaka
532-8514, Japan I
Department of Pathology II, Kansai Medical University2
Department of Ophthalmology, Kansai Medical University3

P-112 (Mon.)
Neuronal cell death in the fetal rat spinal cord following NMDA-treatment to dams
Hirotake TAKAI 1, Tsuneo IT0 1, Kiyoka KATSUYAMA' , Masami SUZUKI' , Kei-ich KATAYAMA2,
Kunio DOI2
Safety Assessment Department, Chugai Pharmaceutical Co. , Ltd. , Shizuoka 412-8513 , Japan'
Department of Veterynary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo2P-113 (Tue.)
Analysis of cell cycle, migration and apoptosis of neural stem cells in the 5-azacytidine(5AzC)-treated rat fetal brain
Masaki UEN0 1, Kei-ichi KATAYAMA', Hiroyuki NAKAYAMA' , Kunio DOI'
Department of Veterinary Pathology, The University of Tokyo, Tokyo I 13-8657, Japan 1

P-114 (Wed.)
T-2 toxin-induced changes in the rat fetal brain
Shinya SEHATA' , Naoki KIYOSAWA2, Toshihiko MAKIN02, Fusako ATSUMI2, Kazurni IT02,
Takashi YAMOT02, Munihiro TERANISHI2, Sunao MANABE2, Koji UETSUKA', Hiroyuki NAKAYAMA',
Kunio DOI'
Department of Veterinary Pathology, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Tokyo 113-8657, Japan 1
Medicinal Safety Research Laboratories, Sankyo Co. , Ltd.2

P-115 (Mon.)
Examination of discriminative diagnosis of brain tumors difficult to be stained withhematoxylin-eosin
Yoshihurni KANEK0 1, Akiko IKEDA 1, Makiko TAKAHASHI 1, Surnihisa SUEYOSHI', Yasukazu SATO' ,Yoshihiro MASUMOTO', Kiyoyuki TSURU'
Kyorin Pharmaceutical Co. , Ltd. Research Center, Tochigi 329-0114, Japan'

P-116 (Tue.)A spontaneous cholesterol granuloma of choroid plexus in a beagle dog
Shuji HAYASHI 1, Satoshi SUZUKI', Fumiko NINOMIYA' , Kazuo HAKOI' , Shuji YAMAGUCHI' ,Kenji IRIMURA' , Shoji FUKUSHIMA2
Drug Safety Research Laboratory, Taiho Pharmaceutical Co., LTD. , Tokushima 771 -0194, Japan'
Department of Pathology, Osaka City University Medical School2

JSTP/IFSTP IATP·- 2oogi
P-117 (Wed.)
A study on the developmental process of distal axonopathy caused by acrylamide
Naofumi TAKAHASHI 1, Maki KUWAHARA 1, Yukiko TAKEUCHJI , Toshinori YOSHIDA I, Akiko ENOMOTO],
Nobuaki NAKASHIMAI, Yasufurni SHUTOHt , Sayaka ISHIMINEl , Toshiaki KITAZAWA3, Keizo MAITA2,
Takanori HARADA t
Toxicology Division II, The Institute of Environmental Toxicology, lbaraki 303-0043, Japan!
Division of Study Planning and Consultation, The Institute of Environmental Toxicology2
Contract and Research Management Division, The Institute of Environmental Toxicology3

P-118 (Mon.)
Screening test for central neurotoxicity by a newly developed human hybrid neuron
Min-Cheol LEEl, JK RYU2, SU KIM2, KH YANG3
Department of Pathology, Chonnam National University Medical School, Chonnam 501-190, Korea (South)l
Brain Disease Research Center, Ajou University2
Department of General Toxicology National Toxicology Institute3

P-119 (Tue.)Developmental neurotoxicity of domoic acid in rat
Min-Cheol LEEI, YS KIMI , JY WOOi , J KIMI , KH YANG2
Department of Pathology, Chonnam National University Medical School, Chonnam 501-190, Korea (South)lDepartment of General Toxicology National Toxicology Institute2

P-120 (Wed.)
Newly-established tumor lines from a spontaneous malignant schwannoma in F344 rats
Jyoji YAMATEI, Hisae YASUI 1, Mitsuru KUWAMURAI, Takao KOTANII , Sadashige SAKUMA I,
Jonathan LAMARRE2
Laboratory of Veterinary Pathology, Osaka Prefecture University, Osaka 599-8531, Japan I
Department of Biomedical Sciences, University of Guelph2

P-121 (Mon.)
Spontaneous malignant schwannoma in F344 rat
Bang Hyun KIM 1, Dong Deuk JANGI , Mina CHOII , Beom Seok HANI , Ki Taek NAMI, Chu i Kyu KIM1 ,Kook Kyoung LEE2, Ki Dae PARKI , Wan Seob CHOI , Ki Hwa YANGI
Department of General Toxicology, National Institute of Toxicological Research, Seoul 122-704, Korea (South) l
Department of Veterinary Medicine, Cheju National University2

Res iratory System

1:00pm-2:00pm

P-122 (Tue.)Histopathological characterization of nasal airway lesions induced by intranasal challengewith ovalbumin in sensitized guinea pigs
Emiko KUWASAKil , Koshi.mu KATOKUI, Takafumi OSHIKATAl, Yutaka NAKAHARA! , Hideyuki WATANABE! ,Masao HAMAMURA 1, Kosuke MORIZUMI2
Department of Pathology, Panapharm Laboratories Co., Ltd. , Kumamoto 869-0425, Japan I
Department of Parmacology, Panapharm Laboratories Co., Ltd.2

P-123 (Wed.)
Hydroxyurea(HU)-induced apoptosis and changes in apoptosis-related genes expression in
the mouse fetal lung
Gye-Hyeong WOOi , Eun-jung BAK2, Koji UETSUKAI , Hiroyuki NAKAYAMA I, Kunio DOIi
Department of Veterinary Pathology, The University of Tokyo, Tokyo 113-8657, Japan!
Department of Biomedical Science, The University of Tokyo2P-124 (Mon.)
In vivo detection of lung tumors in mice by high-resolution X-ray microtomography
Horst WEILER I, Kris MEURRENS2, Nora M. DE CLERCK3, Andrei A. POSTNOV3, Piter M. TERPSTRA2
PHILIP MORRIS Research Laboratories, GmbH, Cologne 51149, Germany!
PHILIP MORRIS Research Laboratories Bvba2
University of Antwerp3

JSTP/IFSTP IATP~l004
P-125 (Tue.)
Eosinophilic change of the lining cells of the nasal cavity in rasH2 mice administereddi(2-ethylhexyl)phthalate
Takatoshi KOUJITANII , Kaoru TOYOSAWAI, Izumi MATSUMOTO! , Kazuo OKIMOTOI , Marni KOUCHII ,Koji KUROKII , Kohji TANAKA!, Nobuo MATSUOKA!
Safety Research Laboratories, Dainippon Pharmaceutical Co. , Ltd. , Osaka 564-0053 , Japanl

P-126 (Wed.)
Biological persistence and pathological changes of potassium octatitanate of two different
shapes.
Akira OGAMII , Takako OYABUI , Yasuo MORIMOTO! , Hiroshi YAMATO! , Izumi AKIYAMA I, Isamu TANAKA I
Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health,Fukuoka 807-8555, Japan!

P-127 (Mon.)
Sequential change in acute lung damage due to intratracheal instillation of Quartz in F344
male rats establishment of a biological bioassay for detection of lung toxicity by fineparticles
Masanao YOKOHIRAI , Hijiri TAKEUCHI! , Keiko YAMAKAWA I, Kousuke SAOOI, Mico IKEDA I,
Yoko HOSOTANII , Zeng YUi, Kyoko HOSOKAWAI , Katsumi IMAIDAI , Makoto SHIRAISHI2
Department of Onco-Pathology, Kagawa University, Kagawa 761-0793 , Japan I
Diagnostic Pathology and Cytology Institute, Shikoku Cytopathologic Institutes2

P-128 (Tue.)lmmunohistochemical demonstration of IL-13 protein in rat late athmatic response model
Kiyoshi KOBAYASHI!, Osamu KURUSUI, Hiroyuki HIGASHIYAMAI, Anbo XIANGI, Hideo KIKKAWAI ,Satoshi ASANOI , Mine KINOSHITA I
Pharmacology Department, High Throughput Biology, TRL, GlaxoSmithKline K.K., Tsukuba 300-4247, Japan 1

P-129 (Wed.)
One case of pleural mesothelioma in male F3441DuCrj rat
Hideki SENOHI , Tetsuya TAKEUCHI!, Yumi UMEDAI , Taku KATAGIRII , Shigetoshi AISOI , Kasuke NAGANOIJapan Bioassay Research Center, Kanagawa 257-0015 , Japan!

Cardiovascular System

1:00pm-2:00pm

P-130 (Mon.)
Incidence of cardiomyopathy in rats treated with PhlP varies with the rat strain
Shoji KASHIWABARAI, Naoki KASHIMOTOI, Toshihiro UESAKAI , Osamu KATOHI , Keiji WAKABAYASHI2,
Hiromitsu WATANABE!
Department of Cellular Biology, Research Institute for Radiation Biology and Medicine,
Hiroshima University, Hiroshima 734-8553 , Japanl
National Cancer Center Research Institute2

P-131 (Tue.)Histopathological effect of Enalapril maleate on fetal heart development in rat.
Arash KHAKII, Iraj SOHRABI HAGHDOST I, Amir Afshin KHAKI2, Mahnaz HAYDARI3
Department of Veterinary Pathology, Islarnic Azad University, Tabriz/Iran, Tabriz 51388, Iran 1
Department of Histology in Medical College of Tabriz University/Iran2
Department of Pharmacology, Shahid Beheshty Medical College Tehran/Iran3

P-132 (Wed.)
Human heart glutamate receptors (GluRs) - possible effector sites: An opportunity for drug
discovery.
Olga PULIDOI , John VEINOT2, Ruedi MUELLERI , Meghan KAVANAGH! , Colin ROUSSEAUX3,
Santokh GILLI.
Pathology Section, Toxicology Research Division, Food Directorate Bureau of Chemical Safety, HPFB , Health
Canada, Ontario KlA OL2, Canada !
Department of Pathology and Laboratory Medicine, University of Ottawa, Civic Hospital Campus 2
Department of Cellular and Molecular Medicine, Faculty of medicine, University of Ottawa3

JSTP/IFSTP IATP 2~00~
Digestive System

1:00pm-2:00pm

P-133 (Mon.)
Enzyme- and immuno-histochemical changes in rat salivary glands induced by theophylline
Satoru KAITKAWAI, Kenji NAKANOI , Aisuke NII1, Hiroyuki NAKAYAMA2, Kunio DOJ2
Safety Research Labs., Yamanouchi Pharmaceutical Co. , Ltd., Tokyo 174-8511 , Japanl
Laboratory of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University of Tokyo2

P-134 (Tue.)Histopathological study of the time course changes in the PTHrP-induced incisor lesions ofrats.
Atsuhiko KAT0 1, Masami SUZUKII , Yayoi KARASAWAI, Tetsuro SUGIMOTO!, Kunio DOJ2
Safety Assessment Dept. Chugai Pharmaceutical Co. , Ltd. , Shizuoka 412-8513, Japan I
Department of Veterinary Pathology, Faculty of Agriculture, University of Tokyo2P-135 (Wed.)
Spontaneous ectopic sebaceous glands (Fordyce's granules) in the oral mucosa ofSprague-Dawley rats
Masako IMAOKAI, Hiroshi SATOHI, Kiyonori KAIi , Tetsuyo KAJIMURA2, Kazuhisa FURUHAMAI
Drug Safety Research Laboratory, Daiichi Pharmaceutical Co., Ltd., Tokyo 134-8630, Japan!
Research Planning & Administration Department, Daiichi Pharmaceutical Co., Ltd.2

P-136 (Mon.)
Acquisition of a gastric or duodenal phenotype on heterotropic transplantation ofesophagus diaphragm, trachea and bladder tissues in F344 rats
Hiromitsu WATANABE 1, Takashi OCHIYA2, Seiichi KAWAMATA3, Tomoyuki KUROSE3, Yoko KOMINAMII,
Masayo NISHIKII , Atsushi SASAKII, Miho SHIRAISHI! , Rina GONI , Masaomi HAYASHJI ,Shoji KASHIWABARAI, Naoki KASHIMOTOI , Toshihiro UESAKAI, Osamu KATOHI
Department of Cellular Biology, Research Institute for Radiation Biology & Medicine, Hiroshima University,
Hiroshima 734-8553, Japan 1
Department of Cancer Metastesis, National Institute of Cancer2
Institute of Health Science, Faculty of Medicine, Hiroshima University3

P-137 (Tue.)
Proliferative changes in upper digestive duct of rats treated with a single dose of Alloxan
Kiyokazu OZAKII , Yasushi KODAMAI , Tetsuro MATSUURA!, Isao NARAMAI
Research Institute of Drug Safety, Setsunan University, Osaka 573-0101, Japan!

P-138 (Wed.)
Gastric carcinoma in mustard gas factory workers in the Ohkuno island
Takamitsu SASAKI! , Tomonori SASAHJRAI, Wataru YASUI2, Hiroki KUNIYASUIDepartment of Oncological Pathology, Nara Medical University Cancer Center, Nara 634-8521, Japan I
Department of Molecular Pathology, Hiroshima University Graduate School of Biomedical Sciences2P-139 (Mon.)
Gastric carcinoma in a Japanese macaque (Macaca fuscata)
Tokuma YANAII , Masato KOBAYASHI!, Shunji GOTOU2, Akino KATOU2, Hiroki SAKAII , Akihiro HIRATA! ,Toshiaki MASEGII
Department of Veterinary Pathology, Gifu University, Gifu 501-1193, Japan I
Primate Research Institute, Kyoto University2

P-140 (Tue.)One case of Ito cell tumor in female F3441DuCrj rat
Yumi UMEDAI , Tetsuya TAKEUCHI! , Hideki SENOHI, Taku KATAGIRil, Shigetoshi AISOI , Kasuke NAGANOIJapan Bioassay Research Center, Kanagawa 257-0015, Japanl

'-a---·---

JSTP/IFSTP IATP 2004
P-141 (Wed.)
Effects of bandage of the torso to hepatomegaly induced by phenobarbital and clofibrate inrats
Kazuhiro HAYAKAWA I, Satoru HOSOKAWAI, Toyohiko AOKJI, Atsushi INAGAMII, Akira INOMATAI,
Jiro SONODA I, Satoru MOTOOKAI, George LOSOS2, Kazuo TSUKIDATE 1
Drug Safety Research Laboratories, Eisai Co., Ltd., Japan, Gifu 501-6195, Japan 1
Asia Pacific Consulting Company2

P-142 (Mon.)
Different susceptibilities of rat liver lobes in carbon tetrachloride-induced hepatotoxicity
Takeki UEHARAI, Takashi MURAII, Satoshi INOUEI , Akira TOUCHII, Satoru MORII, Toshiyuki MARUYAMA I
Developmental Research Laboratories, Shionogi & Co., Ltd., Osaka 561-0825, Japan 1

P-143 (Tue.)
Characterization of drug metabolic reaction involved in the acquired resistance tobromobenzene-induced hepatotoxicity
Kohji TANAKA I, Satoko SATOI, Isao IGARASHII , Naoki KIYOSAWAI, Toshiyuki WATANABE!,
Munehiro TERANISHI!, Sunao MANABEI
Medicinal Safety Research Laboratories, Sankyo Co., Ltd., Shizuoka 437-0065, Japan 1

P-144 (Wed.)
Depletion of tumor-infiltrating macrophages is associated with amphoterin expression incolon cancer
Tomonori SASAHIRAI, Takarnitsu SASAKJI, Hiraki KUNIYASU 1
Department of Oncological Pathology, Nara Medical University Cancer Center, Nara 634-8521, Japan 1

P-145 (Mon.)
A new medium-term bioassay system for the detection of colon carcinogenesis modifiers
Young-Man CHOI, Jun-ichi ONOSEI, Toshio IMAII, Mai HASUMURAI, Makoto UEDAI, Masao HIROSE!
Division of Pathology, National Institute of Health Sciences, Tokyo 158-8501, Japan 1

U rinar S stem

1:00pm-2:00pm

P-146 (Tue.)
Renal damage in newborn rats treated with p-Cumylpheno1 ·Yuko YAMAGUCHI!, Nobuo NISHIMURA2, Megurni YAHATA2, Hiroshi EDAMOT0 1, Shinichiro IKEZAKI 1,
Kazutoshi TAMURA! , Eiichi KAMATA4, Makoto EMA4, Ryuichi HASEGAWA3
Division of Pathology, Gotemba Laboratory, Bozo Research Center Inc., Shizuoka 412-0039, Japan 1
Division of The Second Laboratory for Safety Evaluation, Gotemba Laboratory, Bozo Reseach Center Inc. 2 Divisionof Medicinal Safety Sciences, National Institute of Health Sciences3
Divi sion of Risk Assessment, Biological Safety Research Center, National Institute of Health Sciences4P-147 (Wed.)
A photographic spectrum of non-neoplastic renal tubule and transitional epithelial
structures within selected renal neoplasm of the rat
John Curtis SEELY! , Gordon C HARD2
Experimental Pathology Laboratories, Inc., Research Triangle Park, North Carolina 27709, USA 1
Consultant in Toxicology, Pathology, Carcinogenesis.2

P-148 (Mon.)
Effect of water deprivation or furosemide on cefalotin and glycerol-induced proximal tubular
toxicity in rats
Miyoko OKADAl, Naoki OHYAMA I, Shun-ichiro ISHI11, Naoya MASUTOMI 1, Fumiko SAN0 1,
Jira SUGIMOTOI, Shiro TAKAGI!
Toxicology Laboratory, Mitsubishi Pharma Corporation, Chiba 292-0818, Japan 1

P-149 (Tue.)
Pathological observation of karyomegalic cells in the kidney on male rats treated with
Paecilomyces japonica
Yong-Bum KIMI, Chang-Su HA I, Hwa-Young SON2, Sung-Whan CH02, Boo-Hyon KANG !
Korea Institute of Toxicology, KRICT, Yuseong, Daejeon 305-600, Korea (South) I
College of Veterinary Medicine, Chungnarn National University2

P-150 (Wed.)
Long-term observation of anti-Thy-1 glomerulonephritis in uninephrectomized rats:
Relationship between morphological changes and renal function
Shunji NAKATSUTII, Kenjiro TSUBOTAl, Yoshimasa OKAZAKI!, Masahiro MATSUMOTO!, Shiro FUJIHIRAl,
Yuji OISHI!, Masayuki TOMITA2, Hajime SOGABE2, Shoko NAKAZAT02,
Hiroshi KAWACHP , Fujio SHIMIZU3
Toxicology Research Laboratories, Fujisawa Pharmaceutical Co. ,LTD. , Osaka 532-8514, Japan I
Medicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co. , Ltd.2
Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences3P-151 (Mon.)
Spontaneous glomerulonephritis in cynomolgus monkeys
Xiuying YANG! , Shigeru SATAKE 1, Yasuhiro KAMIMURA I, Kimiaki HIRAKAWAI , Hiroshi MAEDA! ,Hiroaki MIYAJIMA I
Shin Nippon Biomedical Laboratories, Ltd. , Kagoshima 891-1394, Japan 1

P-152 (Tue.)
Spontaneous collagen glomerulopathy in a cynomolgus monkey
Kae FUJISAWA !, Nobuo TAKASUI , Noriko TSUCHIYA I, Shuuichi MATSUSHIMA I, Haruhisa INAGAKI2,
Mikinori TORIII
Developmental Research Laboratories, SHIONOGI & CO. , LTD. , Osaka 561-0825, Japan!
Discovery Research Laboratories, SHIONOGI & CO. , LTD.2

Endocrine System

1 :OOpm-2:00pm

P-153 (Wed.)
Expression of somatostatin mRNA and peptides in C-cell tumours of the thyroid gland in
Han Wistar rats
Andrew PILLING!, Stewart JONES2, John TURTON3
Huntingdon Life Sciences, Huntingdon PE28 4HS , UKI
GlaxoSmithKline2
School of Pharmacy, University of London3

P-154 (Mon.)
A spontaneous dwarf mutation in BrlHan:WIST@Jcl (GALAS) rat Histopathological characteristics of the endocrine system
Takuya DOIi , Masato NAMIKII , Michiko ASHINAI, Naoto TOYOTA I, Hiroko KOKOSHIMAI ,Yuki TOMONARII , Junko SATO! , Takeshi KANNO! , Yumi WAKOI , Minoru TSUCHITANII
Mitsubishi Chemical Safety Institute Ltd., Ibaraki 314-0255, Japanl

P-155 (Tue.)
Association of adrenal pheochromocytoma and lung pathology in inhalation studies withparticulate compounds in the male F344 rat--the National Toxicology Program experience.
Keisuke OZAKil, Joseph K. HASEMAN2, James R. HAILEY3, Robert R. MARONPOT3, Abraham NYSKA3
Environmental Health Science Laboratory, Sumitomo Chemical Co. , Ltd. , Osaka 554-8558, Japan!
Biostatistics Branch, National Institute of Environmental Health Sciences (NIEHS)2
Laboratory of Experiment Patholgy, National Institute of Environmental Health Sciences (NIEHS)352

JSTP/IFSTP IATP 2ooi
Re roductive System

1:00pm-2:00pm

P-156 (Wed.)
Renal toxicity induced by folic acid is directly responsible for the enhancement of male
reproductive toxicity of di (n-butyl) phthalate in male rats
Toshio ICHIHARA I, Tomoko TSUTSUMil , Mayumi KAWABEI , Hiroko YOSHINOI , Makoto ASAMOT02,
Syugo SUZUKI2, Tomoyuki SHIRAI2
Daiyu-kai Institute of Medical Science, Aichi 49 1-0 11 3, Japan I
Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of Medical
Sciences 2

P-157 (Mon.)
Motional and pathological analysis for rat testicular toxicity induced by boric acid
Masakazu KA KUNI I, Naoya KIMOTO I, Tsuyos hi TAKEDA I, Masato MIZUTANI I, Kazuo SUZUKi l,
Hitoshi SATOI , Katsumi TAKABA I, Takuji BARAi
Toxicological Research Laboratories, KYOWA BAKKO KOGYO CO. , LTD. , Yamaguchi 755-850 1, Japanl

P-158 (Tue.)
Early ultrastructural changes of the Sertoli cells with vacuole formations
Yoshiaki SAITOI , Kenj i USUMII , Noriko OSAWA I, Tomoko NAGATA I
Food and Drug Safety Center, Hatano Research Institute, Kanagawa 257-8523, Japan I

Others

1:00pm-2:00pm

P-159 (Wed.)
International advancement in education in veterinary & comparative pathology
D. Reid PATTERSONI
Reid Patterson Consul ting, Inc. , Grayslake 60030, USA 1

P-160 (Mon.)
Histopathology, immunocytochemistry and electron microscopy of spontaneous uveal
melanoma in two HAN Wistar rats
Alexandra MORANI, Christopher BARTON I
Pathology Department, Covance Laboratories, Harrogate HG3 l PY, UK I

P-161 (Tue.)
Case report: naturally occuring proliferative vitreoretinopathy with multiple ocularalterations in a rabbit
Osamu KATSUTA I, Takes hi YAMAGUCHI I, Kenj i TAKASEIDrug Safety/Drug Metabolism Gro up, Research and Development Center, Santen Pharmaceutical Co., Ltd., N ara
630-0 101 , Japanl

P-162 (Wed.)
Histopathological findings of cleft palate in rat embryos induced by triamcinolone acetonide
Satoshi FURUKAWAI, Koji USUDA I, Masayoshi ABE i, Izumi OGAWA I
Biological Research Laboratories, Nissan Chernical Industries, LTD ., Saitama 349-0294, Japanl

P-163 (Mon.)
Spontaneous neural crest tumors of pinna in mice
Hiroshi EDAMOTOI , Mariko NAGATANII, Rie ANDO I, Sachiko TAMAII, Kenichiro KASAHARA I,
Shuzo OKAZAKII , Kazutoshi TAMURAIDivision of Pathology, Gotemba Laboratory, Bozo Research Center Inc. Shizuoka, 4 12-0039, Japan I
Division of the Second Laboratory fo r Safety Evaluation, Gotemba Laboratory, Bozo Research Center Inc.2

53

JSTP/IFSTP -lATP 2·00~
P-164 (Tue.)Histological changes in the dorsal skin of YPC mice irradiated with UVB for 4weeks
Taro OKADA! , Akira YASOSHIMAI , Kaoru TAKAN02, Junichiro MATSUDA2, Koji UETSUKAI,
Hiroyuki NAKAYAMA!, Kunio DOIi
Department of Veterinary Pathology, Graduate School of Agricultural and Life Sciences,
the University of Tokyo, Tokyo 113-8657, Japanl
Department of Veterinary Science, National Institute of Infectious Diseases 2

P-165 (Wed.)
Effects of scratching on the onset and exacerbation of dermatitis in NC/Nga mice
Yutaka NAKANISHI!, Atsushi NAKAMURA I, Yuki HASHIMOT02, Iwao ARAI2, Hirota MIYATA 1,
Masaaki KIMURA 1
Toxicology Laboratory, Medical Research Laboratories, Taisho Pharmaceutical Co., Ltd., Saitama 331 -9530, Japan 1
Phamacology Laboratory, Medical Research Laboratories, Taisho Pharmaceutical Co., Ltd. 2

P-166 (Mon.)
Morphological changes of auricular tissue in the mouse TNCB-repeated application model.
Chie TAKADAI , Daisuke HARADA I, Yukihito TSUKUMOl, Toyoko KASHIWAGI 1, Haruhiko MANABE 1,
Satoshi NISHIKAWA! , Kazuo SUZUKII , Katsumi TAKABAI
Kyowa Hakko Kogyo Co .,Ltd. , Shizuoka 411-8731 , Japan 1

P-167 (Tue.)
Application of special staining to decalcified skeletal tissues in routine toxicity studies
Aisuke NII1 , Kenji NAKANOI , Takanori HANADAI
Safety Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., Tokyo 174-8511, Japan 1

P-168 (Wed.)
Histopathological characterization of the skeletal myopathy in rasH2 mice
Takayuki TSUCHIYA! , Fumiko SANOl , Kazuhiro GOTOI , Mamoru MUTAII , Toshimi USUI2, Jiro SUGIMITOI ,Shiro TAKAGI 1
Toxicology Laboratory, Mitsubishi Pharma Corporation , Chiba 292-0818, Japan 1
Central Institute of Experimental Animals2

P-169 (Mon.)
Spontaneous degenerative lesions of the femoral growth plate in rats
Shinichiro IKEZAKII , Osamu KUSUOKAI, Mizuho TAKAGII , Katsuhiko WARITAI, Maiko TSURUKAMEl,
Kayoko KUDOI, Sachiko TAMAI I, Atsushi NAKAMURA I
Division of Pathology, Bozo Research Center Inc., Shizuoka 419-0101, Japan 1

P-170 (Tue.)
Early pathophysiological feature of arthropathy in juvenile dogs induced by ofloxacin,a quinolone antimicrobial agent
Koichi YABEl , Hiroshi SATORI, Toshirnasa JINDOI, Tadaki SUGAWARAl , Kazuhisa FURUHAMAI ,Masanobu GORY02, Kosuke OKADA2
Drug Safety Research Lab., Daiichi Pharmaceutical Co. , Ltd ., Tokyo 134-8630, Japanl
Veterinary Pathology, Iwate University2P-171 (Wed.)
Organ calcification in monkeys treated intradermally with rhPTH1-34 for 6 months followed
by a 1-month recovery period
Jihong YANG! , Peng WANGI , Xiangting XUI , Guoping XUI , Ling ZHANG! , Liuyi ZHANG I
Department of Pathology, Yunnan Pharmacological Laboratories of Natural Products, Kunming Medical College,
Kunming City P.R. 650031, China (PRC)lP-172 (Mon.)
Physiological parameters and background histopathology findings from chronic (2 year)
carcinogenicity studies in the HAN wistar rat
Christopher BARTON 1
Department of Pathology, Covance Laboratories, North Yorkshire HG31PY, UKl

54

JSTP/IFSTP IATP 200_~
LUNCHEON SEMINAR PROGRAM
Main HallLUNCHEON SEMINAR 1

Monday, February 16, 2004 I 12:00pm-1:00pm
Co-sponsored by: Fujisawa Pharmaceutical Co.,Ltd.

Chair : Yoichi KONISHINara Medical University

Aery/amide in foods: A global dilemma
Speaker : James R. COUGHLIN
Coughlin & Associates

Prevention of Aery/amide induced toxicity in rat
Speaker : Masao HIROSE
National Institute of Health Sciences

Room 501
LUNCHEON SEMINAR 2

Monday, February 16, 2004 / 12:00pm-1:00pm
Co-sponsored by: MORINAGA MILK INDUSTRY CO., LTD.

Recent advances in lactoferrin research
Chair : Hiroyuki TSUDA
Department of Molecular Toxicology, Nagoya City University Graduate School of Medical Sciences

Biological and physico-chemical features of /actoferrin
Speaker : Kei-ichi SHIMAZAKI
Dairy Science Laboratory, Graduate School of Agriculture, Hokkaido University

Recent advances of clinical application of lactoferrin for the treatment of chronic hepatitis C
Speaker: Katsuaki TANAKA
Gastroenterological Center, Yokohama City University Medical Center

Room 502
LUNCHEON SEMINAR 3

Monday, February 16, 2004 I 12:00pm-1:00pm
Co-sponsored by: MEIWA SHOJI CO., LTD.

Morphological Genomics - Gene analysis of the disease using Laser microdissection
Chair : Hideki WANIBUCHI
Osaka City University Medical School
Speaker: Tetsuhiko TACHIKAWA
Department of Oral Pathology, School of Dentistry, Showa University

Main HallLUNCHEON SEMINAR 4

Tuesday, February 17, 2004 / 12:00pm-1:00pm
Co-sponsored by: Affymetrix Japan K.K.

GeneChip® technology in the identification of mechanisms of toxicity
Chair: Sadashi SUZUKI
Affymetrix Japan K.K.
Speaker : Michael PAUMEN
Affymetrix Japan K.K.

Room 501
LUNCHEON SEMINAR 5

Tuesday, February 17, 2004 / 12:00pm-1:00pm
Co-sponsored by: OLYMPUS CORPORATIONOLYMPUS advanced technology for toxicogenomics
Chair : Kiyotsugu KOTIMAOLYMPUS CORPORATION
Speaker : Keiichirou MORIMURA
Osaka City University Medical School

Room 502
LUNCHEON SEMINAR 6

Tuesday, February 17, 2004 / 12:00pm-1:00pm
Co-sponsored by: Bozo Research Center Inc.

Chair : Kunitoshi MITSUMORITokyo University of Agriculture & Technology

In situ mRNA hybridization method with formalin-fixed and paraffin-embedded tissue section
Speaker : Yuichi SATO
Department of Molecular Diagnostics, Kitasato University School of Allied Health Sciences

Histopathology background data in laboratory beagles over ten years.
Speaker : Kazutoshi TAMURA
Division of Pathology, Gotemba Laboratories, Bozo Research Center Inc.

Inhalation toxicology-21st century equipment and techniques for precise animal treatment Speaker : Cristopher N. CROUCH
Division oflnhalation Toxicology, ITR Laboratories Canada

56

JSTP/IFSTP IATP 2004
Main HallLUNCHEON SEMINAR 7

Wednesday, February 18, 2004 I 12:00pm-1:00pm

Co-sponsored by: NPO International Life Sciences Institute of Japan (ILSI Japan)
ILSI Health and Environmental Sciences Institute (ILSI HESI)

Concordance of animal toxicity data with human clinical data
Chair : Samuel M. COHEN
University of Nebraska Medical Center
Speaker : James E. SANDERS
Aventis Phannaceuticals

Room 501
LUNCHEON SEMINAR 8

Wednesday, February 18, 2004 I 12:00pm-1:00pm
Co-sponsored by :Japan SL(, Inc.

lnterbreeder's comparison of SD and F344 rats from the standpoint of pathological examinations
Chair : Kiyoshi IMAI
Biosafety Research Center Foods, Drugs and Pesticides
Speaker : Kunitoshi MITSUMORITokyo University of Agriculture & Technology

Room 502
LUNCHEON SEMINAR 9

Wednesday, February 18, 2004 I 12:00pm-1:00pm
Co-sponsored by: Dako Cytomation Co. Ltd.

lmmunohistochemical detection of the altered expression of proteins involved in rat
carcinogenesis and their digital image analysis by automated cellular scanning system(Ario/ SL-50)
Chair : Katsumi !MAIDA
Faculty of Medicine, Kagawa University
Speaker : Dai NAKAE
Sasaki Institute, Sasaki Foundation

/ATPJSTP/IFSTP IATP 200~

/ATPEnhancing the impact of toxicologic pathologists in global medical research
D. Reid PATTERSON
Reid Patterson Consulting, Inc., Illinois 60030, USA
In the late 1990s, the International Federation of Societies of Toxicologic Pathology, the organization
bridging the numerous independent organizations of toxicologic pathologists around the world, had a vision
for establishing a mechanism for recognizing or accrediting leaders and innovators in the field. These
accomplished medical and veterinary pathologists would represent the field by continuing to contributeintellectually in timely scientific symposia, by challenging outdated scientific dogma, by assisting in theorganization of new societies of toxicologic pathology, and by advising governmental bodies andharmonization conferees on scientific policy. The innovators envisaged this academy to establish aninternationally acceptable standard of knowledge, contributions and continued competency within the field
for the benefit of the global society. Thus, the International Academy of Toxicologic Pathology (IATP) wasestablished in 1999. While still in its formative years, the Academy has attracted noted medical andveterinary pathologists from academia, government, and the pharmaceutical industry from Canada, Europe(Germany, Italy, The Netherlands), Japan, Korea, Taiwan, the United Kingdom and the United States.
Additional members are needed to realize the vision of the founders of the IATP.

JSTP/IFSTP IATP 200~

IATP

Chemically-induced immunopathology and immune functional changes
Joseph G. VOSNational Institute for Public Health and the Environment (RIVM), Bilthoven 3720 BA, The Netherlands
Toxicological pathology plays a most important role in the risk assessment process by identifying anddefining the health effects following exposure to xenobiotics. This includes the field of immunotoxicology, as
identification of chemicals that have the potential to cause injury to the immune system is of considerable
public health significance in view of the role of the immune system in infectious diseases, hypersensitivity
reactions and autoimmune diseases. In assessing immunotoxicity, a two-tier testing system is usuallyemployed in rodents in which the first tier is a general screen for (immuno) toxicity including enhanced
histopathology of lymphoid organs and the second tier consists of more specific immune function studies
including host resistance tests or mechanistic studies. Studies with TCDD, TBTO, HCB, azathioprine andcyclosporin A will be discussed that provided data correlating histopathology with induced immune functionchanges. This will be followed by a discussion of the outcomes of enhanced histomorphology in theinterlaboratory validation studies of azathioprine, cyclosporin A and HCB in the rat, as well as the results ofa recent evaluation of extended histopathology in the mouse as an indicator of immunotoxicity. From thesestudies, that have been the basis for a number of regulatory activities, the following conclusions can be
drawn: i) the consistency between histopathology and functional tests, ii) the complimentary information ofpathology and immunology observations, and iii) the dependence on standardised protocols and trained
toxicologic pathologists to accurately identify and grade microscopical changes in lymphoid organs andtissues.

62

IATP

Thresholds in experimental chemical hepatocarcinogenesis
Gary M. WILLIAMS, A.M. JEFFREY, J. D. DUAN and M. J. IATROPOULOS
Department of Pathology, New York Medical College, New York 10595, USA
In order to explore for potential thresholds in liver carcinogenicity, we conducted a series of studies utilizing
an initiation/promotion (I/P) model in male F344 rats with the hepatocarcinogens 2-acetylaminofluorene(AAF) and diethylnitrosamine (DEN) (Toxicol. Pathol. 28, 388, 2000), which, after bioactivation, react with
DNA in target tissues, particularly liver. The I/P protocol results in hepatocarcinogenicity comparable to thatreported for chronic exposure to these carcinogens. In these studies, AAF or DEN was administered in exact
intragastric doses during the initiation segment (IS) of up to 12 weeks followed in some experiments byphenobarbital (PB) during the promotion segment (PS) for 24 weeks to enhance manifestation of initiation.
We have documented in the IS that there are no-observed-effect levels (NOELs) for formation of DNA
adducts, cytotoxicity, compensatory hepatocellular proliferation, formation of preneoplastic hepatocellularaltered foci (HAF), and in the PS, for tumor development. The earliest parameter to be affected with very
low exposure is the formation of DNA adducts, followed at higher exposures by increased cell proliferation.
The finding of NOELs for critical events of initiation of hepatocarcinogenesis supports the finding of a
NOEL for PB-promotable liver tumors. We concluded that there is a practical threshold for the
carcinogenicity of DNA-reactive carcinogens. Moreover, these findings provide a basis for understanding
why low level environmental exposures of humans to DNA-reactive carcinogens may convey no cancer risk.
Accordingly, a safe exposure level can be calculated for DNA-reactive carcinogens using DNA adducts as
the most sensitive NOEL, and application of safety factors.

JSTP/IFSTP IATP.2'00~/ATP

Endocrine disruption in wildlife: The role of toxicologic pathology in fish
Pieter W. WESTER
National Institute for Public Health and the Environment (RIVM), Bilthoven 3720 BA, The Netherlands
An emerging issue in toxicological pathology is pathology of laboratory fish. Laboratory fish are
increasingly used in environmental toxicology, in particular in the context of the issue of endocrine
disruption. Classical tests using fish are mainly focussed at general endpoints in ecotoxicity, i.e. withsustainability of biomass as principal goal. However, endocrine disruption as an ecotoxicological concern
requires more refined endpoints, especially for shorter studies (i.e. not being multi-generation tests).
Histopathology is therefore the screening tool of choice, in analogy with rodent toxicology. Efforts are
currently undertaken by various organisations to develop databases, knowledge and harmonization in order tovalidate this emerging field where toxicologic pathologist are bound to play a principal role. We have studied
some small fish species used in the laboratory (medaka, guppy and zebrafish) with emphasis on endocrine
disrupting effects. The small size of these fish allows total body embedding of multiple specimen which, instep serial sectioning, enables quick screening for all relevant organs . In comparison with rodents, most
histopathological reactions are rather similar, taking into account the usual typical interspecies differences. In
addition, comparable data in rodents and fish enable integrated (human and environmental) risk assessment.
This paper will address a series of fish- and species-specific features and effects with emphasis on endocrine
disruption.

64

...

-~--

JSTP/IFSTP IATP 2004

/ATP

Renal mesenchymal tumor vs. nephroblastoma: Revisited
John Curtis SEELY
Experimental Pathology Laboratories, Inc., Research Triangle Park, North Carolina 27709, USA
Among the non-epithelial (tubular cell) neoplasms recognized in the rat kidney, lipoma/liposarcoma;
nephroblastoma (NB); renal mesenchymal tumor (RMT); and renal sarcoma are the most commonly
diagnosed. Even though excellent guideline criteria for each of these neoplasms have been published thedifferential diagnosis between NB and RMT may remain problematic in some cases . In the rat, the diagnosis
of NB is dependent upon the identification of the highly basophilic blastemal cells which are pathognomonic
for the diagnosis of NB. A biphasic cellular pattern of blastemal and epithelial elements may form clusters,
nests, alveolar, papillary or tubular patterns. Organoid epithelial differentiation into glomeruli, ducts and/or
tubules may also be recognized. The stromal component of the rat NB may contain stellate cells or moremature appearing cells resembling fibroblasts. On the other hand, the most important feature of RMT is thepresence of a small fibroblastic-like cell which proliferates between tubules and glomeruli. These cells often
form whirling patterns around encircled, pre-existing renal tubules. Occasionally, fibrous tissue and/or
smooth muscle may be identified. More rarely, striated muscle, vascular, cartilaginous or osteoid tissue may
be observed. RMT often contains hyperplastic tubular epithelium and urothelium. The distinction between
NB and RMT may be complicated by several factors:some cases of induced RMT have been en-oneously reported as NB;
difficulty in comparing the rat NB and RMT to human NB;
overlapping of similar diagnostic features;the presence of hyperplastic tubules and urothelium, particularly in RMT;
the presence of neoplasms which do not easily fit into patterns typical of the rat RMT or NB.

JSTP/IFSTP IATP 200~

/ATP

Demonstration of histogenesis and cytogenesis of mammary tumors fromterminal endbud, mammary duct, acinus and myoepithelium cells using humanc-Ha-ras proto- and mutated-oncogene transgenic rats
Hi royuki TSUDA 1•2 , Shinobu UEDA2, Yoichiro MATSUOKA2 , Tetsuya HAMAGUCHI2,
Akihiro NAIT0 2 and Nobuo TAKASUKA 2
Department of Molecular Toxicology, Nagoya City University Graduate School of Medical Sciences,
Aichi 467-8601, Japan 1
Experimental Pathology and Chemotherapy Division, National Cancer Center Research Institute2
We have established a rat line carrying human c-Ha-ras proto-oncogene (Hras128), including its own
promoter region. Both sexes were found to be highly susceptible to chemically-induced mammary, skin,bladder and esophageal carcinogenesis. Palpable multiple mammary carcinomas developed in almost all
female animals in 8-12 weeks and microscopic early carcinomas in 25 days of a single i.v. dose of N-methyl-N-nitrosourea (MNU) at 50 days after birth. Preferential mutation of the transgene was found in all tumors.
Furthermore, similar mutation was detected in laser captured normal looking terminal endbuds, a target tissueof MNU , at day 5 after MNU application, indicating the importance of transgene mutation.
To investigate the role of the mutated ras gene in mammary carcinogenesis, we used transgenic rats carrying
a human c-Ha-ras mutated oncogene (codon 12) with Cre/loxP. construct (Hras250), thus enabling activationof the transgene by infecting the adenovirus can·ying cre-recombinase (Adex/Cre). Five days after injectionof th e Adex/Cre into the main mammary duct, the ras oncogene activated ductal, acinar and myoepithelial
cells were clearly identified by Cre-recombinase immunostaining. Myoepithelial cells were identified bypositive reaction for a-smooth muscle antigen (a -SMA). Subsequently, development of atypical cell
hyperplasia of the duct and acinar epithelium and myoepithelial cells was observed at day 10. Carcinoma insitu lesions of these 3 cell types developed at day 15 20 and was followed by invasive progression by week
4. Development of the myoepithelial lesions was found to take 2 patterns, one was an epithelia-like growthoccurring within the ductual or acinar cell hyperplasia lesions, and the other was a sacoma-like proliferationin the stromal tissue, the former being positive for vimentin and the latter negative. Thus, we were able tocharacterize the histogenesis of MNU-induced mammary carcinomas from 4 different tissure/cell types bydemonstration of in vivo activation of endbud tissue, mammary duct, acinus and myoepithelial cells usingrats carrying ras proto- and mutated-oncogene.

/ATPThe function and pathology of brown adipose tissue (BAT) in animals andhumansMichael IATROPOULOS and Gary WILLIAMSNew York Medical College, New York 10595, USA
Brown adipose tissue (BAT), an endocrine tissue, is an important regulator of nonshivering thermogenesis,
energy metabolism and mitochondrial biogenesis. There is a paucity of data about BAT response inlaboratory animals because BAT has not been routinely included in regulated safety assessment study
protocols. We will present information on the structure, function and pathology of BAT, focusing mainly on
younger and older rats. The energy supply process is stimulated in mammals mainly by hypothermia, by
hypoxia and in primates by hypoglycemia. Of these, hypothermia is the main stimulus of brown adipocyte(BA) proliferation. Thermogenesis is achieved in BA in the mitochondria (mt), which are 50 to 100 times
more abundant in BA than in any other cell type. The mt response involves a membranous protein in the
inner membrane, uncoupling protein (UCP) or thermogenin. Triodothyronine (T3) is implicated in theregulation of the UCP gene. UCP allows mt to oxidize substrates rapidly without ADP phosphorylation, thus
uncoupling oxidation from phosphorylation and promoting instead the dissipation of oxidation energy as
heat. Norepinephrine (NE) binds to the s3-adrenoreceptor in BA leading to activation of lipoprotein lipase(LPL), which stimulates lipolysis and liberates FFA. Both NE and insulin (I) stimulate glucose uptake by
BA, and NE increases UCP in BA. Nitric oxide (NO) stimulates the biogenesis of mt, redistribution of heatgenerated by the mt and inhibition of BA proliferation. NO also regulates the binding and releasing ofoxygen from hemoglobin (Hgb) and the mt pathway of apoptosis. These effects of NO are mediated byperoxisome proliferator-activated receptor coactivator 1 alpha (PPARgamrnaCAlapha) . NO is produced byendothelial NO synthase (eNOS) mainly in BA, but also in the hepatocytes and cells of the zona
glomerulosa. PPARgarnrnaCAlalpha in tum increases the expression of nuclear respiratory factor-I (NRF-1)
and mitochondrial transcription factor A (mtTFA). Thus, UCP, T3 , I, NE, NO, PPARgamrnaCAlalpha,cGMP, eNOS , NRF-1 and mtTFA are all parts of an interactive cascade connecting the BA functions throughthe extracellular matrix (EMA) of all tissues to controlling the processes of EMA homeostasis and signaling,tissue normoxia and normothermia, apoptosis, proliferation, and differentiation of all cells.

Second John Faccini Memorial Keynote Lecture
Plenary Lecture

Memorial
Second John Faccini Memorial Keynote Lecture
Molecular aspects of toxicologic pathology
Okio HINO
Department of Experimental Pathology, Cancer Institute, Japanese Foundation for Cancer Research,
Tokyo 170-8455, Japan
Carcinogenesis can be compared to an opened Japanese fan, because initiated cells grow in several directions
and clinical tumors suggest that the edge of the fan has many gene abnormalities.
Cancer is a heritable disorder of somatic cells. Environment and heredity both operate in the otigin of humancancer. The Eker (Tse 2 gene mutant) rat model of hereditary renal carcinoma (RC) is an example ofMendelian dominantly inherited predisposition to a specific cancer in an experimental animal. To the best of
our knowledge, this was the first isolation of a Mendelian dominantly predisposing cancer gene in anaturally occurring animal model.
Recently, we discovered a new hereditary renal carcinoma in the rat in Japan, and the rat was named the"Nihon" rat and its predisposing (Nihon) gene could be a novel renal tumor suppressor gene.
We will present these unique models, comparing these two predisposing genes, for the study of problems in
carcinogenesis d"eg., species-specific difference in tumorigenesis, cell stage and tissue/cell-type specifictumorigenesis, multistep carcinogenesis, modifier gene(s) in renal carcinogenesis, cancer prevention and thedevelopment of the therapeutic treatments which can be translated into human patients, as well as how
environmental factors interact with cancer susceptibility gene(s).

JSTP/IFSTP IATP 200~

Plenary

Plenary Lecture
Human relevance of carcinogenesis in rodents.
Samuel M. COHEN
Department of Pathology and Microbiology, University of Nebraska Medical Center, Nebraska 68198, USA
Over the past five decades, there has been an increasing reliance on the two-year rodent bioassay to screen
chemicals for possible carcinogenic activity in humans. Considerable effort has been invested in thedevelopment of a variety of in vitro screening assays to assist in this judgment, but most of these have reliedon one or more aspects of genotoxicity. In vitro cell transformation assays have also been developed, andmore recently, bioassays in specialized transgenic organisms or other in vivo screening assays have beendeveloped in attempts to shorten this process and to raise the specificity for determination of humancarcinogenicity. Any time an animal model is used to evaluate any aspect of human disease, there are two
fundamental assumptions: 1) observations in the species studied are pertinent to the human situation(interspecies extrapolation); and 2) effects at doses used in animal studies are relevant to the exposure levelsof humans (dose extrapolation). Although these assumptions may be appropriate for many chemicals,particularly those that are DNA-reactive, increasingly, mechanisms of action have been determined in animals
that either are not relevant to humans or not pertinent at the doses to which humans are exposed. ·Considerable insight has been gained into the various modes of action that occur in animal models, andevaluation of their relevance to humans has been ascertained. Based on this knowledge, I am proposing aconsiderably shortened evaluation process for screening for potential carcinogenicity in humans thateliminates the need for the two-year bioassay in rodents, both mice and rats. The process will involve
screening for DNA-reactivity, in contrast to a generalized screening for genotoxicity, reliance on structure
activity relationships, and an evaluation of 4-week and 13-week in vivo studies in mice and rats extensively
evaluating the histopathologic effects of treatment, toxicokinetics, and cell kinetics (proliferation, necrosis,
and apoptosis). Although these sho1t-term studies require intensive investigation, the results would provide
adequate information for reasonable judgments concerning potential risks to humans. Since shorter time
periods are involved, a larger number of doses could be evaluated to more precisely evaluate the dose
response. In addition, mechanistic evaluations could be pursued evaluating the relevance of species and doseto the human situation. The advent of newer technologies offer the possibility of further refining andshortening this process, with greater specificity regarding effects in humans.

72

SymposiumSymposium
Sl-1 Chair
Carcinogenesis-Current understanding of mechanisms and risk assessments:
Non-genotoxic carcinogenesis/Low-dose carcinogenic potential of genotoxic
and non-genotoxic carcinogens
Colin G. ROUSSEAUX
Department of Cellular and Molecular Medicine, Faculty of Medicine, 451 Smyth Rd, University of Ottawa,
Ontario KlH 8M5, Canada
Masao HIROSE
Division of Pathology, National Institute of Health Sciences, Tokyo 158-8501, Japan
Dr. Wanibuchi will talk about the risk assessment of low dose carcinogenesis in rats. It has long been
believed that there is no threshold level in genotoxic carcinogens, and thus they are not allowed to include as
food additives and pesticides. He will present the existence of threshold levels for several endpoints such as
tumor incidence, DNA damage and mutation even in genotoxic carcinogens. The results may give us
important information for the future risk assessment of envirnnmental genotoxic carcinogens.
Dr. Nishikawa will present the recent results of reporter gene analysis of several genotoxic and nongenenotoxic carcinogens using transgenic animal models such as lac! and gpt delta. In this assay,
carcinogenicity, and mutation frequency and spectra of target organs can be evaluate in a same animal.
Therefore risk assessment of some possible genotoxic carcinogens have recently been conducted using thisassay. However, there may be problems as well as merits in this assay. He also will discuss the problems asa tool for assessing the risk of unknown environmental carcinogens.
In the second part of the session,two speakers will address conference attendees regarding the latest
developments in gene inactivation and its role in carcinogenicity and current developments in angiogenesis as
it relates to the development and progression of neoplasia.
Dr. Ryan will discuss the critical role that angiogenesis plays early in the multistage process ofcarcinogenesis. Following an overview of angiogenesis as a normal process embryogenesis (implantation,organ development and tissue differentiation) and also a key role in postnatal physiologic processes such as
wound healing, longitudinal bone growth and reproduction, Dr. Ryan will brief outline the role ofangiogenesis in pathologic processes, including rheumatoid arthritis, psoriasis, and intraocular
neovascularization. The majority of the presentation will focus on the positive and, particularly, negative
regulators of angiogenesis, which many of these negative factors are currently being evaluated in clinical
trials as potential anti-angiogenic therapeutics for oncology indications. Dr. Ryan's presentation will highlightthe various experimental methods and models used in elucidating the mechanisms of induction and inhibitionof angioneogensis.
Gene silencing is necessary for growth, differentiation, repair and maintenance of healthy tissue by limiting
the expression of some genes while permitting the expression of others. Invasion, a characteristic of many
malignancies, may seem to contradict this concept and appear to be a "new event." However, the recognition
that none of us would be here today if, as a trophoblast, we had no method of invading our mothers
endometrium through the expression of certain genes. These genes then normally become and remain silent.
The converse also holds true. Human cancers arise from cells that have inactivated tumor suppressor genes
either by mutation or transcriptional inactivation. The means by which genes become transcriptionallysilenced is poorly understood in mammalian cells. Dr. McBurney will present his resent research in murine
embryonal carcinoma stem cells growing in cell culture. His research suggests that the silencing oftransgenes in stem cells is triggered by chromatin assembly or reassembly following DNA recombination andthat the active splicing of transcripts maintains chromatin in the open configuration that supports continued
transcription.

JSTP/IFSTP IATP -~2~00~

Symposium

Sl-1-1
Risk assessment of low dose carcinogenesis by genotoxic carcinogens
Hideki WANIBUCHI, Keiichirou MORIMURA, Shoji FUKUSHIMA
Department of Pathology, Osaka City University Medical School, Osaka 545-8585, JapanFor a long period, it has been generally considered that genotoxic carcinogens have no threshould in exerting
carcinogenic potential. This is because genotoxic carcinogens are mutagenic, and seem to act throughinteraction with DNA to produce irreversible genetic changes in target organ cells. This theory is based on
acceptance of a linear relationship which approaches zero at low doses for risk assessment of exposure to
man with chemicals found to be carcinogenic in animal studies. However, there are limited data available for
estimation of cancer risk assessment in man exposed to genotoxic carcinogens, and the validity of the nothreshold theory can be challenged in this case.
In these situation, low dose hepatocarcinogenicity of genotoxic, environmental carcinogens were examined as
an aid to cancer risk assessment in humans. Male F344 rats, 21-day-old at the start of the experiment, wereadministered 2-arnino-3,8-dimethylimidazo[4,5j]quinoxaline (MeIQx), a food-derived hepatocarcinogen, inthe diet at various doses for a maximum of 32 weeks. Quantitative values of glutathione S -transferase
placental form (GST-P) positive foci, a preneoplastic marker for tumors in the liver, were similar among the
0 to 1 ppm MeIQx groups, while MeIQx at a dose of 10 ppm showed a tendency for increase and 100 ppm
significantly elevated their numbers. MeIQx-DNA adduct formation in the liver demonstated a linear
relationship with all the doses tested. Levels of 8-hydroxy-2' -deoxyguanosine (8-0HdG) in the liver werelinearly elevated from 1 ppm MeIQx at week 4, and from 0.01 ppm MeIQx at week 16. Interestingly, there
were no differences in H-ras mutation frequencies with 0.01 ppm MeIQx or less, while they weresignificantly increased at 0.1 ppm MeIQx and above. In a Big Blue transgenic rat mutagenesis assay, MeIQx
at low doses was found not to induce lac! gene mutations in the liver, this tending to correlate with noninduction of GST-P positive foci. However, the dose of MeIQx at which in vivo mutagenicity wassignificantly increased was lower than that for GST-P positive foci. Data for GST-P positive focus
development with MeIQx followed by phenobarbital treatment were similar to those with MeIQx-alone. Coadministration of carbon tetrachloride or ethanol enhanced the induction of liver GST-P positive foci by
MeIQx in each group, numbers being significantly increased with 10 ppm and higher doses of MeIQx. Thus,
these results indicate that MeIQx have no-observed effect levels for induction of carcinogenesis-relatingchanges in the rat lives, implying a threshold for carcinogenicity of genotoxic carcinogens.

JSTP/IFSTP IATP 20021

Symposium

Sl-1-2Analysis of reporter genes for the molecular mechanisms underlying chemical
carcinogenesis
Akiyoshi NISHIKAWA and Masao HIROSE
Divisions of Pathology, National Institute of Health Sciences, Tokyo 158-8501 , Japan
Recent progress in transgenic animal models with reporter genes such as lacl, lacZ and gpt has enabled us
to assess in vivo genotoxicity and carcinogenicity simultaneously. In order to analyze the molecularmechanisms underlying chemical carcinogenesis, we have investigated genotoxic as well as non-genotoxic
carcinogens using lacl transgenic and gpt delta rodents. As genotoxic carcinogens, the heterocyclic amines
MelQx and IQ, the nitroso compounds dimethylnjtrosamine (DMN) and N -nitrosopyrrolidine (NPYR), andthe <linking water contaminants potassium bromate (PB) and MX were given to these mice or rats . Dietary
12- or 13-week MelQx or IQ treatment consistently increased mutant frequencies (MFs) of reporter genes in
the liver of mice or rats, of wruch predominant type of mutations was GC to TA transversion. Interestingly,
although the MFs were significantly increased even at low doses such as 30 ppm, 78-week feeding of 300
ppm MeIQx failed to induce murine liver tumors. It was thus suggested that post-mutational epigenetic
processes might also play a key role in chemical carcinogenesis. Intraperitoneal DMN treatment significantly
increased MFs in the liver, kidneys and lungs, of which predominant type of mutations was deletions as well
as GC to AT transition, but not in other non-target organs. In fact, the mice receiving DMN for 78 weeks
proved to have liver cell adenomas. It was also suggested that both induced and spontaneous MFs areaccumulated with aging. Although no significant increase in the MFs was detected in the kidney of mice
given PB their drinking water at 800 pm for 12 weeks, a significant increase in the MFs was detected in rats
given at 500 ppm, in line with 8-oxoG formation. However, molecular analysis of mutations indicated basesubstitutions other than GC to TA transversion in addition to short deletions. Surprisingly, a strong in vitro
mutagen MX did not increase the MFs in mice at all. Meanwhile, as a non-genotoxic carcinogen, di(2ethylhexyl)phthalate (DEHP) was given to gpt delta rats for 13 weeks. However, no increase in gpt MFs wasfound in rats given DEHP. In addition,. DEHP failed to increase deletions detected by Spi- assay. Our results
thus strongly suggest that these reporter gene transgenic models could offer powerful tools for analyzing
molecular mechanisms underlying chemical carcinogenesis and for assessing the risk of unknown
environmental carcinogens.

JSTP/IFSTP IATP 200ZI

Symposium

Sl-1-3
Recent advances in tumor angiogenesis
Anne M . RYAN
Department of Pathology, Pfizer Global Research and Development, Connecticut 06340, USA
Angiogenesis plays a critical role in normal embryogenesis (implantation, organ development and tissue
differentiation) and also a key role in postnatal physiologic processes such as wound healing, longitudinalbone growth and reproduction. Angiogenesis has also been implicated in a variety of pathologic processes,
including rheumatoid arthritis, psoriasis, intraocular neovascularization and carcinogenesis. Several positive(aFGF, bFGF, TGF-a, TGF-~, HGF, IL-8, VEGF, angiopoietin and av~3) and negative (TSP, SPARC,
angiostatin and endostatin) regulators of angiogenesis have recently been identified. Many of the latter are
currently being evaluated in clinical trials as potential anti-angiogenic therapeutics for oncology indications.
Recent data from several rodent models implicates angiogenesis as a key early event in the multistage
process of carcinogenesis. VEGF up-regulation (and subsequent increases in rnicrovessel density) has beenreported in the early stages of BEN-induced bladder cancer and MNU-induced prostate cancer in rats. COX2, which may play a role in carcinogenesis by stimulating tumor angiogenesis via VEGF, is upregulated in 3methylcholanthrene + diethylnitrosarnine-induced lung tumors in rats and correlated with tumor progression.
COX-2 inhibitors have been shown to decrease tumor progression and rnicrovessel density and to enhance
the anti-cancer effects of radiotherapy and chemotherapy in experimental animals. In addition toexperimental rat models of carcinogenesis, several genetically modified mouse models (H-ras Tg, TSP-1 Tg,
K14-HPV16 Tg, RIP1-TAG2, APC min/+) have also recently demonstrated the pivotal role of angiogenic
factors in the pathogenesis of skin, intestine and pancreatic islet neoplasia. Finally, several new knockout
mouse models (TSP-1 , TSP-2, IFNR) have been reported with increased susceptibility to carcinogenesis due
to alterations in endogenous angiogenic signaling pathways.

Symposium
Sl-1-4
Gene inactivation in mammalian stem cells
Michael W. McBURNEY
Ottawa Regional Cancer Center and Department of Medicine, University of Ottawa,
Ottawa KlH 1C4, Canada
Human cancers arise from cells that have inactivated tumor suppressor genes either by mutation ortranscriptional inactivation. The means by which genes become transcriptionally silenced is poorly
understood in mammalian cells. We have studied this process in murine embryonal carcinoma stem cells
growing in cell culture. Following their stable integration into the genome, transfected reporter genes
spontaneously inactivate at high frequency. This gene silencing can be forestalled by treating cells with
inhibitors of histone deacetylases such as trichostatin A but once inactivation has occurred, trichostatin A
does not reactive expression. DNA methylation of transfected genes occurs but does not seem involved in theinitiation of gene silencing although DNA methylation may be important for maintaining the silent state andfor suppressing recombination between repeated sequences in the transgene. Direct repeats of identical genesequences do predispose to more efficient silencing of the repeated genes suggesting that mammalian cells
carry out repeat-induced gene silencing (RIGS), a phenomenon well desc1ibed in simpler eukaryotic systems.
The promoters driving expression of our reporter genes appear to promote transcription in both directions
creating the potential for repeated genes to give rise to double stranded RNA that could trigger gene
inactivation by a process similar to that described in yeast that is dependent on the enzymology of the RNAinterference pathway. Finally, we find that inactivation of transgenes can be forestalled by creating transgenes
comprised of introns and exons. Our evidence is that the active splicing of primary transcripts feeds back on
the local chromatin structure to prevent the invasion of heterochromatinization. Our data suggests that thesilencing of transgenes in stem cells is triggered by chromatin assembly or reassembly following DNA
recombination and that the active splicing of transcripts maintains chromatin in the open configuration that
supports continued transcription .

JSTP/IFSTP IATP. 20·0~

Symposium

Sl-2 Chair
Carcinogenesis - Current understanding of mechanisms and risk assessments:
Genetically modified animal models
Jerrold M. WARD
National Institute of Allergy and Infectious Diseases, NIH, Maryland 20892, USA
Hiroyuki TSUDA
Department of Molecular Toxicology, Nagoya City University Graduate School of Medical Sciences,
Aichi 467-8601 , Japan,
Experimental Pathology and Chemotherapy Division, National Cancer Center Research Institute,Tokyo 104-0045, Japan
Over the past 30 years, carcinogenesis assessment of chemicals for human risk assessment has utilized the 2year rat and mouse biossay as a standard test. Recently, the costs of these tests has risen to 1-2 million
dollars per GLP study. Also, modem day human risk assessment requires considerations of possible
mechanisms of toxicity and carcinogenesis in rodents and humans. In the search for a more cost effective
and perhaps more relevant assays, genetically engineered mice (GEM) and rats (GER) have been developed.
These new rodent models have genetic changes in genes for which similar changes have been found in
human tumors or in human familial cancer syndromes . Use of these new rodent assays require less animals,
time and costs. In addition, relevance to the human situation may be more applicable. Studies to date haveshown that genotoxic and nongenotoxic carcinogens usually induce tumors in these rodents faster and withearlier and more multiple preneoplastic and neoplastic lesions than in conventional rodents. Some exceptions
to these usual findings have also been seen. The new rodent GEM and GER models may offer faster, __ more
efficient and more relevant models for carcinogenicity assays and human risk assessment. Although further
work is needed to develop these models for routine use, they presently have offered initial benefit for drug
and chemical screening. The presentations will review concepts and models with the eye to the future.

Symposium
Sl-2-1
Mouse models of human familial cancer syndromes as models for risk
assessment
Jerrold M. WARD
National Institute of Allergy and Infectious Diseases, NIH, Maryland 20892, USA
Of the more than 50 characterized familial cancer syndromes, most involve diseases affecting multiple organs
and many can be traced to one or more abnormalities in specific genes. These syndromes may be responsible
for up to 5% of all human cancers. The genes are often tumor suppressor genes and the syndromes are
inherited as a dominant trait. Mutations in the same genes are also often found in sporadic cancers.
Genetically engineered mouse models of these syndromes provide a direct application of a mouse model to ahuman disease including human risk of exposure to carcinogenic substances. Genetically engineered mice(null, heterozygous, conditional knockout) as models for human familial cancer syndromes include thosewith mutations in Ape, Atm, Elm, Breal, Brea2, Lkbl, Menl, Mlh, Msh, Nfl, Tp53, Pten, Rbl , Tscl , Tse2,
Vhl, and Xpa. These mouse lines provide models not only for clinical disease and pathology, but also
provide avenues to investigate carcinogenesis of chemicals and other substances, chemoprevention, molecularpathology, gene and protein functions and therapeutic intervention. This presentation reviews these mousemodels and examines their usefulness in toxicology assessment and other aspects of medical research. The
mice are available in mouse repositories (http://ernice.nci.nih.gov/emice/resources/repositories).

~\:~ St:,· 17., if 2.6-u SC
HRc&oriajcf_

JSTP/IFSTP IATP . 200~

Symposium

Sl-2-2
Mouse models of colon cancer and polyposis
Makoto Mark TAKETO
Department of Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, JapanAs a model for human familial adenomatous polyposis (PAP), we earlier constructed a knockout mousestrain Apc 11716 • Using this mouse model, we found that cyclooxygenase 2 (COX-2) is markedly induced in
the polyp stromal cells, and that inhibition of COX-2 can suppress intestinal polyposis, either by introductionof a COX-2 gene knockout mutation, or by dosing COX-2 inhibitors to the Apc 6716 mice (Cell 87:803-809,
1996). Although the direct metabolite of arachidonic acid by COX-2 is PGH2, it is then converted to varisous
PGs . Among them, PGfa appears to be the major player in the polyposis and carcinogenesis, and PGfa
signals through four different cell surface receptors, EPl, EP2, EP3 and EP4. We recently found that themRNA for the EP2 receptor is strongly induced in the polyps . We then crossed the ApcMl 6 knockout micewith knockout mice for the EPl, EP2, and EP3 genes, respectively. Only in the EP2 compound mutants, thepolyp number and size were reduced dramatically, showing very similar phenotypes to those in the COX-2
gene compound mutants. These results indicate that the PGfa secreted from the stromal cells activates cellsurface receptor EP2 in an autocrine manner, and increases the cellular cAMP level, which in tum induces
more COX-2; a positive feedback mechanism. At the same time, the increased cAMP level induces
biosynthesis of angiogenic factors such as VEGF and Ang2 (Nat. Med. 7:1048-1051, 2001) . In fact, themicrovessel densities (MVD) increased when polyps reached beyond 1 mm in diameter, and this increase
was inhibited in the compound Apc 6 716 mutants with the COX-2 or EP2 gene mutation, indicating thattumorigenic PGE2 signal is mediated through EP2 to cause angiogenesis (Cancer Res. 62:506-511 , 2002).
While COX-2 plays a key role in tumorigenesis, our recent data suggest that COX-1 is equally important(Cancer Res. 62:in press, 2003) . These results will be presented and discussed.

___
JSTP/IFSTP IATP 200~
- -·_ ..,,.

Symposium

Sl-2-3Update on use of the Trp53 +!- mouse for short-term carcinogenicity testing ofpharmaceuticals
Eugenia FLOYD
Pfizer Groton Laboratories, Worldwide Safety Sciences, Connecticut 06498, USA
Over two years have passed since publication of the results from the ILSI-HESI international consortium forthe evaluation of alternative models for short-term carcinogenicity testing of pharmaceuticals . The Trp53 +/mouse is now generally accepted to be the alternative model of choice for compounds, or compounds withmetabolites, that are clearly or equivocally positive in one or more tests of the standard genetic toxicology
battery. Regulatory agencies have used negative Trp53 +/- assay results in weight-of-evidence arguments toallow the continued development of a number of compounds having positive Ames and/or in vitro
chromosomal aberration assays. As their experience with the Trp53 +/- assay has grown, regulators haveupdated their informal guidance on protocol design and study interpretation. In conjunction, they haveidentified areas of regulatory concern regarding assays with the Trp53 +/- mouse. The major recommended
protocol change is the increase in group size from 15 to 25 mice per sex per dose to increase the statisticalpower. A positive control group remains an essential element of current protocol design, although discussions
continue about the possibility of substituting genotype confumation and periodic colony testing for use of
concurrent positive controls. Regulatory agencies have expressed concern about several documented failuresof response to positive control compounds, the most commonly used being p-cresidine. In response, somelaboratories are evaluating methyl nit:rosourea (MNU) for use as a standard positive control. Routine
inclusion of a wild-type high-dose arm also remains highly recommended by regulatory agencies. Twostrains of the TSG-p53® mouse of are now commercially available, the NS and N12 backcrosses, creating a
new challenge for sponsors performing short-term bioassays. The N12 is more closely congenic to the C57
BL/6 (B6) background strain, making it scientifically preferable to the NS. However, the ILSI studies used
the NS strain, and use of the N12 has lagged because of concerns about the extended applicability of the
ILSI historical database. Use of Trp53 +/- mice in dose range finding studies is the safest way to selectdoses for a bioassay, but wild-type mice (NS or B6) still can be substituted for genetically altered strains (NSor N12, respectively). Of most importance, some regulators ' concerns about the sensitivity of the current Trp
53 +/- bioassay have lead to discussions about increasing its duration from 6 to 9 months, a protocol changewith potential to greatly impact future use of this model by pharmaceutical companies.

w~lo\ ~ I~ Tt> ~ J1 bij~
1,bl'l --6;)JSTP/IFSTP IATP 200~

Symposium

Sl-2-4High susceptibility of human c-Ha-ras proto-oncogene transgenic rats tovarious carcinogens - Possible application for a short term assay system forenvironmental carcinogens
Hiroyuki TSUDA 1•2 , Takamasa ONISHF, Shinobu UEDA2, Yoichiro MATSUOKA 2 , Katsumi FUKAMACHF,
Akihiro NAIT0 2 , Cheol Beom PARK2 , Beom Seok HAN2 , Chuel Kyu KIM 2 and Nobuo TAKASUKA2
Department of Molecular Toxicology, Nagoya City University Graduate School of Medical Sciences,
Aichi 467-8601, Japan 1
Experimental Pathology and Chemotherapy Division, National Cancer Center Research Institute2
A rat line carrying three copies of the human c-Ha-ras proto-oncogene, including its own promoter region
was established ( Hras128) and both sexes were found to be highly susceptible to chemically-induced
mammary, skin, bladder and esophageal carcinogenesis. Multiple mammary carcinomas developed in almost
all animals in as short as 8-12 weeks after oral doses of MNU, DMBA or PhIP on day 49 after birth.
Furthermore, skin tumors were induced in males with a DMPA-TPA protocol. Preferential mutation of thetransgene but not the endogenous c-Ha-ras gene and relative increase in the activated form of ras protein inthese tumors indicated an importance of transgene mutation. An obvious increase in the number of endbuds,
the targets of carcinogens, and enhanced proliferation of the component epithelial cells, associated with anincrease in the pMAPK/MAPK ratio and c-myc expression, might explain the elevated susceptibility tocarcinogens. Furthermore, the observation that mammary carcinomas can be induced by various carcinogens
including mammary and various other non-mammary carcinogens in 12 weeks and induction of early stage
carcinomas in the abdominal-inguinal mammary glands in all rats within as short as 20 days after MNU
application may indicate possible utilization for medium to short term screening of environmental
carcinogens irrespective of their organotropism.

Symposium
S2-1 Chair
Omics - A challenge for the toxicologic pathologists:concerning general
toxicity and carcinogenicity
Michael L. CUNNINGHAM
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina27709, USA.
Tomoyuki SHIRAI
Nagoya City University Graduate School of Medical Sciences, Aichi467-8601, Japan
Toxicogenomics is a new scientific field that studies how the genome is involved in responses toenvironmental stressors or toxicants. It includes studies of genetics, genomic-scale mRNA expression celland tissue-wide protein expression(poteomics),metabolite profiling(metabonomics)and bioinformatics forunderstanding the role of gene-environment interactions in disease. New technologies, such as DNA
microarray analysis, can measure the expression of thousands of genes at a time providing the potential toaccelerate discovery of toxicant pathways and specific gene targets. Studies of gene and protein expression
can provide mechanistic insight into toxic responses and disease pathologies. It is expected that the power
and potential of these new toxicogenomics methods are capable of revolutionizing the field of toxicology.
Changes in gene expression as a result of chemical exposure can indicate activation of a signal transduction
pathway that is responsible for disorder of cell function. Such information can be combined with otherbiological and chemical information to explain the mechanism(s)underlying a toxic response. Global gene
expression profiles for chemicals from different mode-of-action classes can provide gene expression"
signatures"of chemical. Gene expression profiling may be consistently and mechanistically linked to specifictoxic effects and disease. In other words, if gene profiling specific to certain class or category of chemicals
was established, these new technologies can be applied to predict outcome of a certain chemical (predictive
toxicology). Here we have4speakers focusing liver disorders such as liver cell degeneration/necrosis and liver
tumors. Two of them will present current data on gene expression profilings or protein profilings as
predictive tool for hepatocarcinogenic potential of chemicals, and the other two will talk about gene
expression profiles in the liver of rats exposed to a hepatotoxin or stress induced by a specific diet.

Symposium
S2-1-1
Toxicogenomic approach for detection of carcinogenic substances
Tomoyuki SHIRAI1, Makoto ASAMOT0 1, Kazunari TSUJIMURA' ,2 , Masaru SEKIJIMA3,
Nobuyoshi MIKAMI4 and Masanori OTSUKA2
Nagoya City University Graduate School of Medical Sciences, Aichi 467-8601, Japan 1
Chemical Evaluation and Research Institute2
Mitsubishi Chemical Safety Institute Ltd. 3
Sumitomo Chemical Co. Ltd. 4
A long-term rodent bioassay for carcinogenicity is a standard method to predict carcinogenic hazard ofchemicals to human beings. However, this assay needs a long term and a lot of costs. To overtake theseissues, short or medium-term bioassays have been developed, but there are still limitations in these assays
especially for detection of non-genotoxic carcinogens. To address these, the Ministry of Economy, Trade andIndustry, Japan has started a toxicogenomics project in 2001 to develop a novel bioassay based on
toxicology studies and gene expression profiles.
Five-week old male Fisher F344 rats were treated by daily oral gavages with known carcinogenic and noncarcinogenic compounds at two doses for up to 28 days. Reference compounds are classified tocategorizations; mutagenic hepatocarcinogen, non-mutagenic hepatocarcinogen, mutagenic nonhepatocarcinogen, non-mutagenic non-hepatocarcinogen and non-carcinogen. The liver and colon were
sampled at 1, 3, 7, 14 and 28 days. Besides of classical toxicological studies, gene expression profiles wereobtained using in-house cDNA microarrays containing approximately 9,000 genes. Genes for prediction ofcarcinogenicity were selected by statistical methods and analyzed by cross validation probability tests,
clustering analysis and principal components analysis. Correct predictive ratio for carcinogenicity was over
95% when rats were treated with chemicals over 14 days.
These results indicate short-term bioassay systems of rats for carcinogenicity using gene expression profiles
are prolTilsmg.

86

JSTP/IFSTP IATP 2004

Symposium

S2-1-2
Proteomic expression profiling of liver toxicity in rat using 2D-DIGE technology
Hidenori YAMANAKA' , Kazunari TSUJIMURA 1, Masanori OTSUKA', Yoshikuni YAKABE 1,
Satsuki HOSYUYAMA 1, Koichi SAIT02 , Kayo SUMIDA2, Masaru SEKIJIMA3, Koji NAKAYAMA3,
Yukiko KAWAN03, Yasuro SHIN0HARA4, Tomoyuki SHIRAI5
Chemical Assessment Center, Chemical Evaluation and Research Institute, Japan, Saitama 345-0043 , Japan 1
Sumitomo Chemical Co., Ltd 2
Mitsubishi Chemical Safety Institute Ltd 3
Hokkaido University 4
Graduate School of Medical Sciences, Nagoya City University 5
Hepatic responses to 28-day repeated dose of 24 chemicals were monitored using two-dimensional
differential gel electrophoresis (2D-DIGE) technology (Amersham Biosciences, UK Limited). The method,which has ability to measure expression change of hundreds of proteins at once, provided new insights into
the effects of chemicals on biological systems. For example, the genotoxic hepatocarcinogen
diethylnitrosoamine (DEN), peroxisome proliferator Clofibrate and non-carcinogen 4-nitro-ophenylenediamine gave rise to greater than 1.5-fold quantitative changes compared to control in 236 and 108
and 60 liver proteins after 28 days of 20, 250 and 250 mg/kg/day repeated dose, respectively. Especially,
glutathione S-transferase, pi, that known to express in a wide variety of tumors, showed 95-fold increase byadministration of DEN. The proteomic expression signatures of 24 chemicals including genotoxic
carcinogen, non-genotoxic carcinogen and non-carcinogen were compared and hierarchical clustering wasapplied to them to find the characteristic pattern of liver protein to the type of toxicity of chemicals.

JSTP/IFSTP IATP "2DO~

Symposium

S2-1-3
Gene expression profiling of rat liver fibrosis, cirrhosis, and cancer induced bycholine-deficient, L-amino acid-defined diet
Ivan RUSYNt, Oksana KOSYK 1, Christine POWELU, Ayumi DENDA2 , Yoichi KONISHF,
Fumiyuki UEMATSU 3 and Dai NAKAE 3
Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill,
North Carolina 27599, USA t
Department of Oncological Pathology, Cancer Center, Nara Medical University 2
Department of Pathology, Sasaki Institute, Sasaki Foundation3
The consecutive emergence of the fatty liver, necrosis, compensatory proliferation, accumulation ofpreneoplastic foci , development of fibrosis , cirrhosis, adenomas and carcinomas are the hallmark pathologicalchanges induced in rat liver by choline-deficient, L-arnino acid-defined (CDAA) diet. This model ofhepatocarcinogenesis has been extensively studied and the mechanisms that are thought to underline therapid progression of the disease include, among other things, altered fatty acid metabolism, hypomethylationof various genes, changes in gene expression, activation of hepatic stellate cells, oxidative stress to DNA,and accumulation of mutations. The goals of this study were to investigate the mechanisms of oxidative
DNA damage and repair induce@by CDAA diet, and to determine what genes are regulated at different
stages of liver injury in this model. Male Fisher 344 rats were given CDAA diet, or matching cholinesufficient (CSAA) control diet for up to 80 weeks and liver samples were collected. A significant increase inoxidized purines in genomic DNA was detected as early as 4 weeks of CDAA treatment. The number ofmutagenic apurinic/apyrirnidinic sites was increased at 12 and 30 weeks on a choline-deficient diet.
Furthermore, the expression of base excision DNA repair genes for 8-0H-dG DNA glycosylase, Nmethylpurine DNA glycosylase, apurinic/apyrimidinic endonuclease, polymerase beta, methylguanine
methyltransferase and proliferating cell nuclear antigen was significantly elevated in CDAA-treated animals
beginning at 4 weeks. Gene expression profiling was conducted on liver samples from 4, 12 and 80-weekgroups, as well as on liver tumors from the 80 week CDAA-fed animals. The selection of tissue samples was
performed to reflect various stages of liver pathology in this model. In general, three biological replicateswere analyzed in each group . Affymetrix® rnicroarrays (RAE230A, ~16,000 genes) were used. Several
normalization and image intensity calculation procedures were used to analyze the results. Clustering
analysis of the individual samples on all of the genes represented on the array revealed a strong segregationof CSAA and CDAA groups with a further separation of replicate samples into individual treatment groups.
The analysis of individual clusters of genes specific for each time point and treatment showed that CDAAinduced liver pathology could be correlated to specific changes in gene expression. Collectively, this study
demonstrated that global changes in gene expression can be phenotypicaly anchored to liver pathology, andthis new information may be used to uncover the complexity of the molecular changes associated withvarious stages of liver disease.

JSTP/IFSTP IATP 2004

Symposium

S2-1-4
Gene expression changes in F344 rats following a pharmacological dose of
acetaminophen
Michael L. CUNNINGHAM
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA
Acetaminophen (APAP) is a commonly used over-the-counter analgesic and antipyretic that is hepatotoxic inhigh doses. Centrilobular necrosis occurs in the liver following APAP exposure after glutathione stores are
depleted, allowing its toxic metabolite N-acetyl-p-benzoquinone irnine to react with cellular macromolecules
causing mitochondrial dysfunction and hepatotoxicity. We treated ad libitum fed male Fisher 344 rats orallywith APAP at doses that may be experienced during normal therapeutic regimes, 50 and 150 mg/Kg andexamined gene expression changes in the liver after 6 hours. Although no clinical signs of toxicity wereobserved by histopathologic examination or clinical chemistry measurements, gene expression changes wereobserved consistent with energy metabolism disruption. The adaptive response by the liver included down
regulation of fatty acid synthesis and cholesterol metabolism, pathways that are energy intensive.
Upregulation of stress related genes such as heme oxygenase and metallothionein 1 were also observed.
Several of the changes were consistent with previous reports but additional novel gene changes wereobserved. By 48 hours after dosing only a small number of genes are differentially expressed and theseappear to be associated with a return to energy homeostasis. These early changes in gene expression indicate
a disruption of energy metabolism and of normal metabolic processes at pharmacologic doses of APAP.
These data suggest that differential gene expression analysis may provide a more sensitive indicator of
toxicity than routine histology and clinical chemistry evaluations.

JSTP/IFSTP IATP ·200~

Symposium

S2-2 Chair
Omics - A challenge for the toxicologic pathologists:
Concerning reproductive toxicity, immunotoxicity and toxicity of endocrine
disrupting chemicals
Paul-Georg GERMANN
Preclinical Safety, Novaitis Pharma AG, Basel 303-0043, Switzerland
Keizo MAITA
Study Planning and Consultation, The Institute of Environmental Toxicology, Ibaraki 303-0043, Japan
There has been an increasing concern among people if their substantial system maintaining healthy life andreproductive activity might be affected by environmental toxicants while they are not aware of it. To develop
proper methods evaluating the toxicity and to study the mechanism of the mode of action with those
toxicants should be an urgent task for the toxicologic pathologists.
This first part of this session will present sophisticated interactions between environmental endocrine active
contaminants and sensitive tissues in the talks of Drs. Nagao and Harada. Dr. Nagao will give his
presentation on "Biological effects of diethylstilbestrol in rats and mice" and Dr. Harada on "Mechanism of
toxic changes and tumor development by DDT in F344 rats."
In the second part of this session the current understanding of immunological responses against chemical
exposure will be presented by Drs. Descotes and Germolec. During the last years, accumulating evidence
occurred that potential immunotoxicity might be associated with significant increase in incidence andseverity of clinical side effects. The scope of the second part of this IFSTP/JSTP workshop is dedicated toimmunotoxicology & immunotoxicopathology. There we would like to present different aspects, views andissues of the current state of the art of this important field, also by giving specific examples. The awarenessof immunotoxicology has led to an intensified and controversial discussion within the different Guideline
Committees to address this impo1tant field adequately in preclinical studies. Specialised animal models andmodified study designs in preclinical immunotoxicology studies are of increasing importance. Theirdifficulties especially their limitations in interpretation are the topic of Dr. Descotes presentation: "Importanceof Immunotoxicology on Safety Assessment: A Toxicologists View." Dr. Germolec is contributing thetoxicological pathologists' view in her talk about "Improving the Sensitivity and Predictability of Current
Testing Strategies for Immunotoxicology." Pathologists are playing an increasing role with their interpretationof immunotoxic effects. An international pathology working group report with its results is pointing to theusefulness of extended pathological contributions. Finally, in the discussion we will try to figure out future
development trends in the assessment of new drugs as well as open and emerging issues in
immunotoxicology.
The result of the discussions will be also included in the full summary of the workshop.

JSTP/IFSTP IATP -200·4

Symposium

S2-2-1
Biological effects of diethylstilbestrol -Transgenerational effects of endocrine
disrupting chemicalsTetsuji NAGAO
Department of Life Science, Kinki University, Osaka 577-8502, Japan
In parallel with increasing concern about the reproductive effects of endocrine disrupting chemicals in theenvironment, evidence has been accumulating that developmental exposure to estrogenic hormones can cause
a variety of abnormalities in the male reproductive tract (reviewed by Nagao et al., 1998, 1999, 2003).
However, little is known about transgenerational toxicity of estrogens, except for the potential of atransgenerational carcinogenic effect of diethylstilbestrol (DES) (Newbold et al., 1998, 2000). We carried out
the present study as an attempt to fill this gap in our knowledge. Male mice prenatally treated with DES
were mated with untreated females, and the resulting fetal offspring were inspected for externalmalformations. A significant increase in the incidence of malformed fetuses over the control level was
noticed in the offspring whose sires were treated with a high DES dose, but it was not seen in the offspringof males treated with a low dose. The high dosing, but not the low dosing, caused partial atrophy andfeminization in the genital tracts. Ethinyl est.radio! and est.radial benzoate also showed transgenerational
teratogenicity when applied prenatally at a dose which caused histopathological changes in the testes. Germcell series in normal testis have mechanisms to select against spontaneously arising mutations. However,these selection mechanisms may not function efficiently in chemically damaged testis. Based on these results
and considerations, I propose as a working hypothesis that male-mediated developmental toxicity(teratogenicity) may occur as a consequence of testicular toxicity.
(supported by a grant from The Science and Technology Agency: Special Coordination Funds for Promoting
Science and Technology)
Nagao T., Fujikawa K. , 1998, Cong Anom 38:1-8.
Nagao T., 1999, Environ Mutagen Res 21:267-72
Nagao T., 2003, Advances in Male Mediated Developmental Toxicity. , Kluwer Academic/Plenum Publishers
Newbold RR. , Hanson RB., Jefferson WJ. et al ., 1998, Carcinogenesis 19: 1655-63.
Newbold RR., Hanson RB., Jefferson WJ. et al., 2000, Carcinogenesis 21:1355-63.

JSTP/IFSTP IATP 200fl

Symposium

S2-2-2Mechanisms of toxic effects and tumor development caused by DDT in F344
rats
Takanori HARADA, Ryoichi OHTSUKA, Makio TAKEDA, Naruto TOMIYAMA, Machiko SAKA,
Tadashi KOSAKA, Junya SASAKI, Sayuri KOJIMA, Naofumi TAKAHASHI, Yukiko TAKEUCHI,
Toshinori YOSHIDA, Maki KUWAHARA, Akiko ENOMOTO, Nobuaki NAKASHIMA and Keizo MAITA
The Institute of Environmental Toxicology, Mitsukaido-shi, Ibaraki 303-0043, Japan

In order to clarify the mechanisms of toxic effects and tumor induction by DDT, we conducted a 2-year
feeding study of p,p' -DDT in F344 rats of both sexes at dose levels of 0, 5, 50, and 500 ppm. These
animals were sacrificed after 26, 52, 78, and 104 weeks of treatment and subjected to toxicokinetics,
hematology, biochemistry, necropsy, and histopathology. In addition, potential factors involved inhepatocarcinogenesis including microsomal enzymes, oxidative stress, cell proliferation, gap junctional
intercellular communication (GJIC), and gene expression in target tissues were examined. Hormone assay
was also performed on control and high-dose animals at termination of treatment.
Males and females in the high-dose group showed tremor and depression of body weight gain which weremore evident in females. A marked hair loss was also noted in females. The concentrations of DDT and its
metabolites in the plasma and brain tended to be higher in females, while those in the liver were higher inmales. This difference might be one of the factors contributing to sex differences in toxic effects and tumor
induction. Analysis of hepatic microsomal enzymes demonstrated dose-dependent increases in P450 isozyme
contents of CYP2B 1 and CYP3A2 which were more evident in females. Since CYP3A2 is androgendependent, the induction of this isozyme in females indicates that DDT is able to modulate sexual metabolic
dimorphism. As to serum hormone levels in the high-dose group, a decrease in testosterone and an elevationin progesterone were noted in males and a decline in estrogen in females. Histopathologically, centrilobular
hepatocellular hypertrophy was observed in all dose groups. Eosinophilic hepatocellula.r foci increased in
number and size with time in a dose-dependent manner and the incidence of hepatocellular tumors increasedin males in the mid-dose group and both sexes in the high-dose group. These groups exhibited increases inhepatic lipid peroxide and 8-hydroxydeoxyguanosine and an inhibition of GJIC. The oxidative stress may
play an important role in the hepatocarcinogenesis by DDT. In other organs/tissues, the occurrence ofchronic nephropathy was accelerated in the mid- and high-dose groups of both sexes . In the high-dose group,
the incidences of pituitary adenomas and testicular Leydig cell tumors increased in males and hypertrophy/
hyperplasia of ovarian interstitial gland were frequently observed in females. These findings may reflect an
endocrine disrupting effect of DDT. Microarray analysis of gene expression in target tissues is now
underway.

JSTP/IFSTP IATP ·20~04

Symposium

S2-2-3lmmunotoxicology in drug safety evaluation: A medical toxicologist's view
Jacques DESCOTES
Poison Centre and Claude Bernard University, Lyon 69007, France
Over the past decades, the clinical experience unambiguously demonstrated that drug-induced immunotoxic
effects can result in significant morbidity and even mortality. Immunotoxic effects are divided into four
broad categories: immunosuppression, immunostimulation, hypersensitivity and autoimmunity. Each category
is associated with a specific pattern of possible adverse health consequences, the nonclinical prediction ofwhich should follow different strategies. Concern was initially on immunosuppression, but hypersensitivity
and immunostimulation are increasingly key issues. Although absolutely essential, the histologicalexamination of the lymphoid organs is not considered sufficient to assure the reliable detection of
unexpectedly immunosuppressive drugs. At least one immune function assay, primarily a T-dependent
antibody response assay, is recommended. Additional testing is decided case by case depending on early
toxicity findings, possible histology changes, and the structure and pharmacological properties of the drug. It
is not known whether function assays typically used to predict immunosuppression are also applicable todetect unexpectedly immunostimulatory drugs. Specific assays, e.g. cytokine release assays, can be helpful insome circumstances. For the purpose of immunotoxicity risk assessment, host resistance assays, e.g.
experimental infections (immunosuppression) or autoimmunity models (immunostimulation) may be needed.
Current animal models and assays are seldom valid to predict the risk of drug-induced hypersensitivity.
Guinea pig models, but not the LLNA can be used for the prediction of contact sensitization induced bydrugs. Immunogenicity of therapeutic proteins is sometimes evaluated in monkeys. Finally, drug-induced
autoimmune reactions cannot be predicted in most instances. The timing and design of nonclinicalimmunotoxicity studies are critical issues. Although no guidelines require the evaluation of drug
immunotoxicity prior to clinical trials, it would be logical to perform some immunotoxicity studies early innonclinical safety evaluation. Inclusion of a T-dependent antibody response assay in safety pharmacology is
an interesting alternative. Other critical factors to be considered include particular dose-response
relationships, the redundancy of immune responses, the role of genetic factors and inter-species differences.
Finally, as more and more studies will be pe1formed routinely to meet recent regulatory requirements, awealth of unexpected immune changes is likely to be seen in nonclinical studies . It is therefore essential that
selected endpoints of nonclinical studies can also be measured in clinical trials.

JSTP/IFSTP IATP 200~

Symposium

S2-2-4The sensitivity and predictability of current testing strategies to evaluate
immunotoxicity
Dori GERMOLEC 1, Abraham NYSKA 1, C Frieke KUPER2 , Christopher PORTIER 1, Michael KASHON3,
C KOMMINENP, Keith JOHNSON4 and Michael LUSTER3
National Institute of Environmental Health Sciences, North Carolina 27709, USA1TNO Nutrition and Food Research 2
National Institute of Occupational Safety and Health3
Dow Chemical Company 4
The identification of chemicals that have the potential to cause injury to the immune system is of
considerable public health significance, as alterations in immune function can lead to increased incidence ofhypersensitivity and autoimmune disorders, infectious diseases or neoplasias. Experimental animal datacollected using standardized testing panels have provided a database from which the sensitivity andpredictability of a variety of tests commonly used for the screening of chemicals for immunotoxicity has
been evaluated. A working group was established to design and implement a study to evaluate the sensitivity
and predictability of extended histopathology as an indicator of immunotoxicity, as compared with the
National Toxicology Program's functional testing battery. Standardized slide sets from thymus; spleen andmesenteric lymph nodes were generated for 10 chemicals, which had previously been evaluated for theirimmunotoxicity using functional tests. A semiquantitative assessment was adopted in order to estimate thehistopathological changes within different anatomical compartments of the lymphoid organs using thediagnostic terms for identifying and semiquantitating the histopathologic changes recommended by Kuper etal. (2000) . Findings from each pathologist were entered into a common database and evaluated foragreement between pathologists, sensitivity of individual parameters and co1Telation with the "immunotoxic"
call. A direct comparison of the ratings for each pathologist indicated that certain individuals tended to be
more conservative while others were more able to discern subtle changes. Additional analyses examined theconsistency of a pathologist's ratings in a single tissue by investigating the correlation among all themeasures in the same tissue type. When data were examined for each pathologist independently, it was notedthat specific histopathological changes such as cortical cellularity in the thymus were more easily evaluatedand demonstrated the best correlation. For several spleen and lymph node measures, the outcomes for eachpathologist were less well correlated. When comparing histopathology to functional tests, the antibody
formin g cell (AFC) assay detected immune suppression in two instances where no changes in pathology
were indicated. In contrast, the AFC assay failed to detect oxymetholone as immunotoxicant, although otherimmune parameters, as well as extended histopathology indicated immunologic changes . These studies
suggest that there is not a single most sensitive parameter for assessing damage to the immune system, butrather that one or two functional tests combined with pathology examinations would be needed to accurately
flag a compound as immunotoxic and provide data into its mechanism of action.

JSTP/IFSTP IATP 200A

Symposium

S3 Chair
Recent trend of information technology in toxicological pathology science
Gerd MORAWITZThe Fraunhofer Institute of Toxicology and Aerosol Research, Drug Research and Clinical Inhalation,
Hannover 30625, Germany
Ikuo HORII
Pfizer Global Research & Development, Worldwide Safety Sciences - Nagoya, Aichi 470-2393, Japan
Recently, introduction and integration of novel information technologies (IT) has become the dominant trend
in various scientific domains, and yielded considerable results in data management with synergistic effects onscientific data evaluation. Application of IT has been spreading deeply and widely also into the field of
toxicological pathology (TP) science.
This symposium has been organized to study the necessity of the effective use of IT and its applicability inthe process of data gathe1ing, data retrieval, report preparation and evaluation relevant to TP. It is essential tosmartly introduce / develop the IT system to establish a significant database for effective use of dataobtained, and also indispensable in terms of TP safety evaluation to develop a synergistic workstation
towards the goal of establishing a mutually accessible database between TP science and the other fields ofscience. In other words, sufficient database establishment using a warehousing approach is expected in orderto improve the difficulties in data management due to the diversity and complexity in TP safety evaluations.
Related to these, we will touch upon the valid database management, telepathology system, imaging
informatics system and pathology training program.
Presentations and discussions will be held mainly on the following themes.
(1) Pathological data gathering / retrieval / evaluation and its usability as a database(2) Strategy and prospect of telepathology system(3) TP imaging technology as a new approach(4) Pathological evaluation / training program under the information technology
We hope that information / outcome at this symposium, basically on IT, will offer insights that will lead tofuture development in the field of TP science.

JSTP/IFSTP IATP 200~

Symposium

S3-1
Pathology information system
Nobuyuki OHASHI
Biosafety Research Center, Food, Drugs and Pesticides, Shizuoka 437-1213, Japan
An-Pyo Center has been using the PATHOTOX, an Integrated Pathology and Toxicology System Network,
since 1991 with the successional improvement by user's requirement and for the increased reliability. Thissystem has been used to evaluate many toxicological examinations including over one hundred
carcinogenetic studies up to now. This computer system offers high user interface with a network multiwindows system (X-windows) and prompt response by using UNIX server (Solaris 8) and windows 2000
client. Simultaneous recording of microscopic findings can be done on a parallel with evaluating observationof histopathological slides . Entry of histopathological findings is done by selecting words or phrases frommultiple windows comprising organ and tissue specific finding list, a general histological dictionary, an organ
dictionary, a comment list, and cause of death section. The management of these dictionaries is important to
use this system more efficiently because pathologists need to confirm every word in the dictionary for every
histopathological lesion according to the universally accepted criteria. Currently, the combined image systemwith histopathological findings is available to share criteria for each pathological finding by pathologists.
Pathologists are able to discuss on the histopathological features with dictionary images on the computer
screen. Furthermore, necropsy findings and clinical data, including hematology, clinical chemistry, andurinalysis data of individual animals can be displayed in screens. The system produces a variety of reporting
and retrieval functions combined with statistical analyses. Peto's trend test can be executed for some oftumor findings to reveal a significant occurrence of tumors. For the authorized user, senior pathologist, thissystem has a peer review function, being divided into the macro findings input, the mjcro findings input, thefindings data reference, the master management, etc. For security, each user has the account and must change
the password every three months. Network security system is equipped with number of firewalls andoperating system functions. Users living in the remote area from the An-Pyo Center can safely use thissystem from another site (The Saitama branch) through the VPN (Virtual Private Network).

96

JSTP/IFSTP IATP 2004

Symposium

S3-2
Linking worldwide pathology expertise with the telepathology system
Ingrid PRUIMBOOM-BREES, Mark TENGOWSKI and Ricardo OCHOA
Pfizer Research and Development, Department of Pathology - Groton Safety Sciences, Connecticut 06340,
USA
The telepathology system consists of a suite of integrated internet-enabled, computer-driven microscopes
installed at each user site. Each system is equipped with a standard computer and internet server, a robotic
optical microscope; a video or digital camera and an automatic multi slide holder. By automating the slideloading and unloading process, up to 50 slides can be scanned and digitized unattended, and overnight.
Because the computer connects the microscopes to the Internet, the users can view and navigate themicroscopic slides from across the globe or just down the hall using their standard PC and internet
connection. The microscopic images appear on the users computer screen in real time, and the software
allows selection and full navigation of the slide, including control of the objective (up o lOOX dry objective),
focus, illumination and digital photography. Thanks to the automatic loading and the remote internet access,pathologists can collaborate asynchronously or concurrently with colleagues across the globe, and this makethe telepathology system ideal for applications such as consultation around individual microscopic finding s
or to peer review an entire toxicology study. In conclusion, the telepathology system allows industries tobreakdown distance barriers and to leverage worldwide pathology expertise while reducing travel expenses
and the costs of duplicating areas of expertise at multiple sites.

JSTP/IFSTP IATP 200~

Symposium

S3-3
Image informatics: Current and future challenges
Judith A. NOLAN
Scimagix, Inc., California 94403, USA
Digital imaging techniques have provided researchers with new capabilities but with an equal number ofdifficulties. With the growing acceptance of digital imaging technology in medical research, there is a rapidly
expanding need for systems to aid in the management and mining of this type of data. One of the main
challenges is to develop a system that can read and display a large number of different image formats,including proprietary formats from instrument manufacturers. These images may be created with a variety oftechniques, including histochemical staining of tissues, confocal and video output and multi-channel
fluorescent detection. Users require the ability to search both the images and their associated metadata, andto be able to easily compare them on any workstation. Centralized management enables these capabilities,
along with providing a mechanism to consistently annotate and share the data among colleagues. Once sucha system is put in place, users will be able to access images readily and may concentrate on gaining morequantitative data from the images using image analysis techniques . Most imaging scientists will combine dataobtained from commercial and their own proprietary algorithms. The main difficulty with the analyses iscomparing results from different analyses when data is in very different formats. Technology producing large
volumes of images, such as High Content Screening, further magnifies the problem. Researchers in preclinical and clinical environments have additional validation considerations for images and associated data,
since these electronic records may invoke the 21 CPR Part 11 ruling. This presentation will discuss theseissues and review methodologies that will assist imaging scientists to best control and extract valuable
info rmation from their data.

98Symposium
S3-4
AFIP online veterinary pathology training programs
Mark MENSE, Terrell BLANCHARD, Thelda ATKIN, Sophie BOUCHIHA-OLSON, Sean HAHN,
William INSKEEP and Dale DUNN
Depa11ment of Veterinary Pathology, Armed Forces Institute of Pathology, Washington DC 20306, USA
The Department of Veterinary Pathology, Armed Forces Institute of Pathology, has implemented three online
resources in veterinary pathology. 1) The Veterinary Systemic Pathology Resource, funded from a
Department of Education, Fund for the Improvement of Post-secondary Education grant, utilizes the same
eleven-organ system training archive as the AFIP residency program. Each organ system is divided into
bacterial, fungal, metabolic/miscellaneous, neoplastic, parasitic, toxic, and viral entities. The database can beviewed as unknowns or sorted by organ system, animal species, and disease category for over 675 entities innumerous species. Linked sequential images with legends are provided to simulate examination of the tissue
at the microscope. Lower magnification images contain hyperlinked "hotspots" which delineate the viewablearea contained in the next higher magnification. Each case manuscript is reviewed and updated every three
years. Future projections include the addition of virtual slides, gross pathology images, cytology/clinicalpathology data, electron micrographs, immunohistochemistry micrographs, links from references to online
publications, and development of online testing modules. 2) The Registry of Toxicologic Pathology web
conference provides toxicologic pathologists with an anonymous forum for the exchange of ideas andinformation concerning toxicologic pathology research and related issues. There are nine conferences
annually, with four cases per conference. Registered participants in the conference include more than 45
international corporate and government agencies, greater than 675 individual users, and nine US veterinary
schools. Four hours of continuing education credit is offered per conference under the AAVSB of RACE
approval program. 3) The Department of Veterinary Pathology provides the Wednesday Slide Conference on
the World-Wide-Web to veterinary pathologists worldwide in an effort to enable everyone to participate inthis valuable forum. The Wednesday Slide Conference is in its 51 st year, has 135 international participants
and is considered an integral part of many international veterinary training programs. This yearly conferenceis held on each Wednesday for 25 weeks from September through May for residents and board-certified
veterinary pathologists. Each conference presents four unknown cases of classic or newly discovered disease
entities, encompassing modern concepts and the most recent literature references. The conference focuses onmorphologic and etiologic diagnoses, board quality descriptions, differential diagnoses, and classic gross andhistologic lesions, with comparative aspects among the common species affected.

JSTP/IFSTP IATP,,· 2004

Symposium

S4 Chair
Future challenges: Stem cell development, infectious agents and others-Introduction
Kunio DOI
Veterinary Pathology, Graduate School of Agriculture and Life Sciences, The University of Tokyo,
Tokyo 113-8657, JapanThe last symposium of JSTP/IFSTP (IATP) 2004 deals with some possibilities of future challenges intoxicologic pathology. Recently stem cells have been attracting a great attention in the field of biomedical
science. During the last decade, a good deal of information about stem cells has been newly added every
year, and it is no doubt that such information will be necessary for the development of toxicologic pathology
in near future. In this symposium, at first, three researchers show us the current of stem cell research fromvarious viewpoints. Namely, Dr. Nakatsuji gives lecture on establishment and manipulation of monkey andhuman ES cell lines as well as reprogrammrning of somatic cells into pluripotent stem cells, and further
mentions ethical problems of usage of human ES cells. Dr. Shiota emphasizes an importance of DNA
methylation pattern of CpG islands, and proposes a need of evaluation of mutagens as well as epimutagens
in pathological and toxicological researches. Dr. Hanison introduces many examples of clinical applicationsof stem cell research. Next, Dr. Brown gives us a general view of emerging zoonotic diseases such as bovine
spongiform encephalopathy, and refers to how emerging zoonotic diseases reflect upon toxicologic
pathology.

100

JSTP/IFSTP IATP 2004

Symposium

S4-1
Establishment and manipulation of monkey and human ES cell lines forbiomedical research
Norio NAKATSUJI
Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
We have established several ES cell lines from blastocysts of the cynomolgus monkey. After various
improvements of culture methods, they can be maintained in culture stably for long periods. They maintainthe normal karyotype and produce several differentiated cell types in culture. When such ES cells weretransplanted into SCID mice, they produced teratomas containing many differentiated tissues. Unlimited
proliferation capacity of ES cells in culture and subsequent differentiation into various types of functional
cells could be used for cell transplantation therapy. For example, we have succeeded to produce
dopaminergic and other neurons and pigmented epithelia from our cynomolgus ES cells.
For further progress in basic research and the medical application of primate and human ES cells, we needto improve many aspects of the manipulation of stem cells. For example, we like to produce geneticallymodified monkey and human ES cells for reduction of antigenicity in cell therapy or efficient selection ofparticularly useful cell types. After improvement of the transfection and selection methods, we can produce
ES cell clones with integrated transgenes at efficient and reliable rates.
Nonhuman primate ES cell lines provide important research tools for basic and applicative research. In mostcountries, usage of human ES cells is strictly regulated because of ethical problems . In such situations,monkey ES cells provide invaluable materials for research to advance various aspects of regenerative
medicine. Also, they are indispensable for pre-clinical research using primate models of allogenic celltransplantation therapy to evaluate effectiveness, safety and immunological reaction in physiologicalconditions similar to the treatment of human diseases. Our cynomolgus ES cells have been already
distributed and utilized by many laboratories.
We are now carrying out a project to establish human ES cell lines from frozen surplus human embryos. The
Japanese government guidelines, issued in September 2001, require strict procedures for establishment andresearch usage of human ES cells. We have obtained the approval and national grants to produce human ES
cell lines in April 2002. So far, we have established and characterized one human ES cell lines. We expect toproduce additional few lines. We have obligation to distribute ES cells to other institutes in Japan withapproved research plans, and we plan to start the distribution by the end of this year.
We are also investigating reprogramming of somatic cells into pluripotent stem cells by cell fusion with ES
cells. Such reprogramming could be used to produce pluripotent stem cells that have the same genome witha patient to avoid immunological rejection without production of the nuclear-transfer-cloned embryos, which
would raise various ethical problems in many countries including Japan.

JSTP/IFSTP IATP 200fl

Symposium

S4-2
Clinical applications of stem cell research
David HARRISON
Department of Pathology, University of Edinburgh, Scotland EH8 9AG, UK
One of the challenges is knowing which stem cells are going to be useful for therapeutic purposes. Should
we be relying on embryonal stem cells or can adult derived stem cells, haematopoietic or otherwise, be used.
In this overview we will explore possible uses as well as discussing, from a pathology perspective, some of
the key issues still to be resolved.
The first key issue is plasticity: the ability of a stem cell to adopt the characteristics of the differentiated cellintended. Whilst it is clear that embryonal cells are totipotent there is considerable debate about the plasticity
of other stem cells, for example bone marrow derived.
There are a number of overlapping possible uses for stem cell therapeutics some of which, for example bonemarrow transplantation, are already widely used. They are (i) replacement (ii) substitution (iii) regeneration(iv) repair of gene defects (v) reproductive cloning.
Replacement therapy may offer hope in Parkinson's disease or Alzheimer's by directly adding back cells thathave been damaged or destroyed. The combination of gene therapy with stem cell therapy offers thepossibility of treating old diseases in new ways, for example glucagons-like peptide 1 in treatment of type 1
diabetes. Regeneration is widely thought to offer possibilities in diseases like liver failure, renal disease andemphysema. Yet, as pathologists we are aware that chronic disease is not always a deficit of cells but rather aproblem of order, architecture and remodelling. Specific repair of gene defect, for example sickle cellanemia, is an attractive target with many millions of potential patients. Reproductive cloning and theassociated technology of nuclear transfer raise moral and ethical problems, as well as considerable technicalproblems.
Stem cell research is expanding rapidly. It is swallowing up gene therapy and there is a need to pause andreflect what we are trying to achieve, and to place therapy in the context of an understanding of the disease
we are trying to treat.

102

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JSTP/IFSTP IATP 2004

Symposium

S4-3
DNA methylation pattern of CpG islands for epigenetics pathology andtoxicology
Kunio SHIOTA
Cellular Biochemistry, Animal Resource Sciences / Veterinary Medical Sciences, The University of Tokyo,
Tokyo 113-8657, Japan"Epigenetics" means the study of heritable changes in gene-activity without changes in DNA sequences. In
vertebrates, methylation of DNA mainly occurs at the 5' -position of cytosine in a CpG dinucleotide forming
5-methylcytosine. Methylation of DNA plays a profound role in transcriptional repression of gene expression
through several mechanisms. Sequences of CpGs are not evenly distributed in the mammalian genome. They
appear at a 10 to 20 times higher density in selected regions than in other regions, and regions enriched with
CpGs are known as CpG islands. These CpG islands are used as landmarks to find genomic regions in bulk
DNA sequences, because CpG islands are generally found in transcription units. Generally, it has beenrecognized that CpG islands are unmethylated in normal tissues, except the CpG islands involved in X
inactivation and genomic imprinting. However, most data on DNA methylation mediated gene repression
concerns TATA-less and CG-rich promoters that are associated with CpG islands . Scanning of a few
thousands of islands from various cells and tissues of mouse, including ES cells and embryo revealed thatCpG islands having tissue-dependently and differentially methylated region (T-DMR) were numerous andwidespread in the genome. The T-DMR panel clearly indicates that DNA methylation is cell type specific.
The human genome project identified 30,000-40,000 protein coding genes, and there are approximately
29,000 CpG islands. There are 30,000 genes and 15,000 CpG islands in the mouse genome. Tissue-specificpromoters revealed that 50% of CpG islands are linked to tissue-specific genes. The remaining tissue-specificpromoters do not associate with CpG islands. A single fertilized egg gives rise to a complex multi-cellularorganism consisting of at least 200 differentiated cell types. Most cells differentiate without changes in DNA
sequence through activation of a particular set of genes and inactivation of others. The molecular basis forthe memory for activated or inactivated gene sets, which is inherited to the cells' next generations, is criticalfor differentiation and development of multicellular organisms. Thus, formation of DNA methylation patternunderlies mammalian development, and epigenetic errors cause other diseases. Changes in heritable DNA
methylation which alter phenotype are referred to as epimutagen. Now, evaluation of mutagens as well as
epimutagens are needed in the pathology and toxicology research.

Ref
Shiota K. et al., Genes Cells 7:961-969 (2002).
Shiota K, Yanagimachi R: Differentiation 69:162-166 (2002).

JSTP/IFSTP IATP 200~

Symposium

S4-4
Emerging zoonotic diseases
Corrie BROWN
Department of Veterinary Pathology, College of Veterinary Medicine, University of Georgia, Georgia 30602,
USA
The interface between animal and human health is becoming increasingly blurred as new diseases crosscontinents and oceans and from one species to another. The last few years has witnessed a dismaying arrayof novel human/animal disease events. Bovine spongiform encephalopathy traveled to many new parts of theworld, with each appearance creating considerable consternation about health of both agriculture and the
public. Avian influenza continues to emerge and re-emerge, often with movement into human populations.
Particularly disquieting is the possibility of a reassortment event occurring between human and avian
influenza viruses within a human host. A newly recognized human disease, severe acute respiratory
syndrome, galvanized the international public health community into concerted efforts to contain the spreadof a newly recognized coronavirus, which presumably moved into the human population from an animalreservoir. An outbreak of monkeypox in prairie dogs and humans in the Midwestern United States
underscores the potential for any disease to show up anywhere and unexpectedly affect new species.
Basically, given the interconnectedness of today's world, trying to separate diseases into the categories of"human" and "non-human" has become increasingly artificial. In fact, as we continue to change theenvironment, modify habitats, move species around, and alter the way we live and raise animals, pathogens
from the animal and human worlds will not only continue to proliferate and mutate but also collide in a
multitude of unpredictable ways. It is imperative that medical and veterinary fields interact and communicate
in a fluid manner, so that we can respond effectively to each new disease as it arises, and continue to protect
our global populations of all species.

104Symposium
S4 Chair
Future challenges: Stem cell development, infectious agents and others-Summary
John FINCH
Inveresk Research, Edinburgh EH33 2NE, UK
As Dr Doi said in his introduction to our symposium, we have been trying this afternoon to describe futurechallenges to toxicological pathology. Whilst some of what we have heard today might seem remote fromthe day to day concerns of the toxicological pathologist the principles that are applied to these newchallenges are those that we are already applying in the more traditional aspects of our profession. Dr
Nakatsuji described the establishment of embryonal stem cell lines, and how monkey ES cells provide a
valuable alternative to human cells in research. The establishment of human ES cell lines was also described.
Dr Harrison told us about the uses such cells can have in the armamentarium of the clinician. Exciting
possibilities include treatments for Parkinson's disease, Alzheimer's disease, diabetes, liver failure and manyothers. Here the challenge is one of recognising opportunity and ensuring that as guardians of public safety
we share the enthusiasm of our colleagues in drug discovery and pure research.
Other challenges involve the recognition of new threats to our well being. Genotoxicity is well known to us,
but in Dr Shiota's talk were introduced to epigenetics as a real possibility of extending our safetyassessments of medicines and environmental chemicals. Changes in heritable DNA methylation can affectthe phenotype and evaluation of the epimutagenic of substances may one day be as routinely performed as
current genotoxicity tests. A central theme of our symposium has been the introduction of foreign cells as
therapeutic agents. These do of course carry a risk of carrying unwanted pathogens and it is there fore
appropriate that finally we turned our attention to the world of infectious disease to get Dr Brown 's excellent
update on what's new in human and animal disease from SARS to BSE.
I hope that this glimpse of what the future may hold has been as stimulating for you as it has for me. In
any event our goal remains what it has always been - better and safer medicines for mankind.

Posters

Posters
P-1
Hershberger and uterotrophic assessments to evaluate potential endocrine
disrupting effects of diesel exhaust in rats
Tsuyoshi IT0 1, Hiroki OKUMURA 1, Ryo OHTA2, Kiyoshi IMAP, Midori YOSHIDA4, Dai NAKAE4,
Akihiko MAEKAWA 5
Health Effects Research Division, Japan Automobile Research Institute, Ibaraki 305-0822, Japan 1
Safety Testing Laboratory, Hatano Research Institute, Food and Drug Safety Center2
Executive Director, Biosafety Research Center, Foods, Drugs and Pesticides3
Department of Pathology, Sasaki Institute, Sasaki Foundation 4
Director, Sasaki Institute, Sasaki Foundation 5
In recent years, potential endocrine disrupting effects of diesel exhaust, in particular those on reproductive
organs have received much attention. Diesel exhaust is demonstrated to inhibit the spermatogenesis in rats
and mice. It is also indicated that diesel exhaust particle extracts exert an antagonistic effect on androgen
receptors and an agonistic effect on estrogen receptors in vitro. Such effects is attributed mainly topolycyclic aromatic hydrocarbons represented by benzo[a]pyrene (B[a]P), major component of diesel
exhaust particles, because B[a]P is antiandrogenic, its hydroxylated metabolites being estrogenic in culture
cells. In this context, the present study was conducted to clarify whether diesel exhaust modulates overall
androgen- and estrogen-mediated biological mechanisms in vivo by using Hershberger and uterotrophic
assays in Fischer 344 rats. In the Hershberger assay, castrated male rats were inhaled diluted diesel exhaustat the concentrations of particles of 0, 0.1, 0.5 or 2.0 mg/m 3 or filtered exhaust with particles of 2.0 mg/m 3
for 16 hours/day for 10 days. To examine the antiandrogenic activity, subcutaneous doses of 0.2 mg/kg ofteststerone propionate was additionally administrated to half of the animals in every group on each day. Afterthe final diesel exhaust exposure, weights of the seminal vesicle and coagulating gland, ventral prostate,levator ani, bulvocavemous muscle and cowper's glands were measured. In the uterotrophic assay,
ovariectomized female rats were inhaled diluted diesel exhaust with conditions identical to those used in the
Hershberger assay for 16 hours/day for 14 days. To examine the antiestorogenic activity, subcutaneous dosesof 0.1 µg/kg of ethynylestradiol was additionally administered to half of the animals in every group on eachday. After the final diesel exhaust exposure, weight of the uterus was measured. As a result, diesel exhaust
did not change any organ weights in either assay. These results indicate that diesel exhaust does notmodulate the overall androgen- and estrogen-mediated biological mechanisms in vivo.

JSTP/IFSTP IATP '200~

Posters

P-2
Application of hershberger assay protocol to the detection of thyroid function
modulators using castrated male rats
Masakuni SAWAKI 1, Shuji NODA1, Takako MUROI 1, Hideo MITOMA1, Saori TAKAKURA1,
Satoko SAKAMOT0 1, Masanori OTSUKA 1, Mineo TAKATSUKI 1, Kanji YAMASAKI 1
Chemicals Evaluation and Research Institute, Oita 877-0061, Japan 1
In vivo screening methods for detection of thyroid function modulators are now under development in many
research laboratories. We examined the applicability of Hershberger assay protocol to the screening of
thyroid function modulators. Hershberger assay is a screening method for (anti-) androgenic compounds
using castrated male rats. Castrated male BrlHan WIST@Jcl (GALAS) rats at 8 weeks of age wereadministered by gavage with a potent thyroid peroxidase inhibitor 3-amino-1,2,4-triazole (AT) 0, 40, and 200
mg/kg/day for 10 consecutive days (n = 5 or 6/group). Castration was performed at the age of 6 weeks. To
examine the effect of castration, intact male rats were also subjected to the same protocol. All animals weresacrificed approximately 24 h after the final dosing. The thyroid glands and hypophysis of each rat werecollected and weighed approximately 24 h after fixation with 10% neutral-buffered formalin to avoid
crushing during manipulation. In addition, the thyroid glands of all animals were prepared for hematoxylin
and eosin staining for histopathological examination and morphometry of the follicular epithelial height. As aresult, significant increase of the thyroid weight was observed at the 200 mg/kg group in the castrated andintact rats . The hypophysis weight was not changed by AT treatment, however, comparing the vehicle-treatedgroups, the hypophysis weight of the castrated rats was higher than that of the intact rats. Enlargement of the
thyroid glands in the AT-treated rats was observed at necropsy. Histopathlogically, the thyroid glands of all
the AT-treated animals showed hypertrophy and hyperplasia of the follicular epithelial cells and the height offollicular epithelium of the thyroid glands increased in a dose-dependent manner in both of the castrated andintact rats . These results suggest that the effect of AT could be detected using Hershberger assay protocol
regardless of castration and this protocol may be useful for screening of thyroid function modulators,
although other compounds with mechanisms different from AT, such as deionase inhibitors and thyroid
hormone metabolism enhancers should be tested.

I JO

.

~

. ,, ---~u-i,,-:.._....--JS TP/IFS TP IATP 2004

Posters

P-3
Lack of modifying effects of 4-tert-octylphenol and benzyl butyl phthalate on
3,2'-dimethyl-4-aminobiphenyl-induced prostate carcinogenesis in rats
Hiroyuki KOHN0 1, Rikako SUZUKI 1•2 , Shigeyuki SUGIE 1, Tak:uji TANAKA1
Department of Pathology, Kanazawa Medical University, Uchinada 920-0293, Japan 1
Research Fellow of the Japan Society for the Promotion of Science2
Certain types of cancer development is related to environmental factors including diet and environmental
chemicals. In the current study, the effects of dietary feeding of estrogenic compounds 4-tert-octylphenol (4-t-OP) and benzyl butyl phthalate (BBP) on 3,2' -dimethyl-4-aminobiphenl (DMAB)-induced prostatic
carcinogenesis were investigated in male F344 rats to know whether these compounds exert modifying
effects on prostatic carcinogenesis. We also assessed the effects of test compound on proliferating cell
nuclear antigen (PCNA)-index in induced neoplasms, hyperplastic lesions, and non-lesional glands in theprostate. Rats were given intraperitoneal injections of DMAB (25 mg/kg body weight) every other week for
10 times to induce prostatic neoplasms. They also received the experimental diet containing 10 or 100 ppm
OP and BBP for 40 weeks, starting one week after the last dosing of DMAB. DMAB exposure produced
prostatic adenocarcinoma with an incidence of 41 % (7 /17 rats) at the end of the study (week 60). Dietaryadministration of 4-t-OP and BBP did not affect the incidence of prostatic adenocarcinoma: 44% (7/16 rats)
in the DMAB -+ 10 ppm 4-t-OP group; 25 % (4/16 rats) in the DMAB -+ 100 ppm 4-t-OP group; 44% (7/16 rats) in the DMAB -+ 10 ppm BBP group; and 44% (7/16 rats) in the DMAB -+ 100 ppm BBP group.
The incidences of prostatic adenocarcinoma in other groups were 0% (0/8 rats) in the 100 ppm 4-t-OP alonegroup, 13% (1/8 rats) in the 100 ppm BBP alone group, and 0% (0/8 rats) in the untreated group. The
PCNA-indices in adenocarcinomas, hyperplastic lesions, and non-lesional glands in rats treated with DMAB
and 4-t-OP or BBP were slightly lower than that of the DMAB alone group, and the differences among thegroups were not statistically significant. These results might suggest dietary feeding of two estrogenic
compounds 4-t-OP and BBP did not have modulating effects on DMAB-induced rat prostatic carcinogenesis.

JSTP/IFSTP IATP 21)0~

PostersP-4
Sex hormone responsiveness and ductal architecture after exposure to
flutamide perinatally in the rat ventral prostate
Kaori MIYATA1, Setsuko YABUSHITA1, Masashi SAN02 , Yasuyoshi OKUN0 1, Masatoshi MATSU0 3
Environmental Health Science Lab., Sumitomo Chemical Co ., Ltd., Osaka 554-8558, Japan 1
Daiyu-Kai Institute of Medical Science2
Cooerative Research Center for Advanced Science and Technology, Osaka University 3
Effects on ventral prostate in male rats exposed to flutamide perinatally were examined to clarify thehormone responsiveness and the main factor with respect to the irreversible effects.
Material & Methods: [Study 1] Pregnant rats were administered flutarnide at a daily dose of lOmg/kg fromgestation day 14 (GD14) to post-parturition day 3. Male pups were castrated on post-natal day 36 (PND36)
and treated with vehicle, testosterone propionate (TP; 2 mg/kg/day, s.c.) or dihydrotestosterone (DHT; 1.25
mg/kg/day, s.c.) for 10 days, beginning at PND46. On PND56, serum testosterone, LH and FSH levels,
ventral prostate weight were measured, and RT-PCR (for AR, C3, VEGF, TGF-betal, beta2, KGF and CK8
mRNAs) was carried out. [Study 2] Pregnant rats were administered flutamide at doses of lOmg/kg from
GD12 to GD21, or 30mg/kg on 2 successive days in the period from GD14 to 21 (GD14-15, GD16-17, GD
18-19, and GD20-21). RT-PCR at PNDl for the lOmg/kg group, and morphological analysis at PND7, 14
and 21, and organ weight measurement and receptor binding assay at PND76 or 78 for all groups wereconducted.
Results: [Study 1] The degree of increase in the weight after TP or DHT treatment was smaller in the 10mg
/kg group. Serum hormone levels and the results of RT-PCR revealed no significant differences. [Study 2]
RT-PCR at PNDl demonstrated increase in the expression of AR and KGF mRNAs. Morphological analysis
showed a consistent reduction in the number of main ducts and ductal branchpoints, as well as thecomplexity of the terminal ductal network in the lOmg/kg group, and the 30mg/kg GD 16-17 and GD 18-19
groups . The organ weights at PND76 or 78 were reduced in all treated groups, and the severity of effect wasmatched with those on ductal architecture. Receptor binding assays exhibited no significantly difference
regarding maximum binding capacity and dissociation constant.
Conclusion: In flutamide exposure group, the increased degree of organ weight after androgen treatment was
small, and an irreversible effect on the prostatic ductal architecture caused. The morphological effect was
severest when the prostatic buds are developing, and accompanied an increase in KGF mRNA expression
just after exposure. The effect on organ weight after androgens treatment was likely related to the ductal
architecture, there were not any effects on serum hormone levels, expression of mRNAs, nor androgen
receptor function later in life.

112

JSTP/IFSTP IATP 2004:

PostersP-5
Is stem cell factor related to the testicular toxicity of thiamphenycol?
Maki KUWAHARA1, Yukiko TAKEUCHI1, Naofumi TAKAHASHI', Tadashi KOSAKA 1,
Nobuaki NAKASHIMA', Toshinori YOSHIDA' , Akiko ENOMOTO' , Toshiaki KITAZAWA3, Keizo MAITA2,
Takanori HARADA 1
Toxicology Division, The Institute of Environmental Toxicology, Ibaraki 303-0043, Japan 1
Division of Study Plannning and Conslutation, The Institute of Environmental Toxicology2
Contract and Research Management Division, The Institute of Environmental Toxicology 3
Previously, we demonstrated the testicular toxicity of thiamphenicol (TAP), derivate of chroramphenicol, in
Spraque-Dawley rats. In the testis of rats treated with 200 mg/kg/day TAP, spermatogonia in the stage V andpreleptoten spermatocyte in the stage VII were significantly decreased from week 2. At week 4, degeneration
and apoptosis of germ cells were frequently seen in most of the seminiferous tubules together with retentionof sperm, dissociation of germ cell arrangement, and frequent presence of giant cells. There were no obvious
changes in the Leidig cells. These changes incompletely disappeared after a week-10 of withdrawal period
following 4 weeks of treatment, in spite of the presence of BrdU positive spermatogonia in seminiferous
tubules which appeared to be "Sertoli only syndrome". These data have suggested that Sertoli cells are most
likely to be the primary target in TAP testicular toxicity. As it was recently repo11ed that irreversible
testicular toxicity of hexandione was promoted by deficient of stem cell factor (SCF), which is secreted by
Sertoli cells and plays key roles in the development of the testis in the neonatal stage, we examined therelationship between the SCF and thiamphenicol-induced testicular toxicity. Testes of rats orally treated with
200 mg/kg/day TAP for 7, 14, and 28days were fixed with FSA fixatives , routinely sectioned andimmunostained with anti-SCF by ABC method. In the control testis, a strong positive reaction was observedin elongated spermatid in stage XI to XIII and spermatogonia in stage I to II. These types of germ cells arereported to express c-kit, the receptor protein of the SCF, and SCF binding to c-kit is necessary todifferentiate these cells. No Sertoli cells showed positive reaction against the SCF antibody. Testes treatedwith 200 mg/kg/day TAP for 7 and 14 days showed almost same staining manner with the controls. After 28
days of treatment, although positive reaction observed in elongated spermatid and spermatogonia weremaintained, some sprematocytes in disarranged seminiferous tubules exhibited a positive reaction. It is
concluded that Sertoli cells still produce SCF in the TAP-treated testis, however, some functional alteration
might occur.

JSTP/IFSTP IATP 200~

PostersP-6
No modifying effects of an estrogenic compound atrazine on 7, 12dimethylbenz(a)anthracene-induced ovarian carcinogenesis in rats
Takuji TANAKA', Hiroyuki KOHN0 1, Rikako SUZUKI 1•2, Shigeyuki SUGIE 1
Department of Pathology, Kanazawa Medical University, Ishikawa 920-0293, Japan'
Research Fellow of the Japan Society for the Promotion of Science2
Atrazine with an estrogenic action is one of the widely used pre-emergence and post-emergence triazine
herbicides that have made their way into the potable water supply of many agricultural communities.
Atrazine was reported to induce mammary tumors in experimental animals. In the current study, themodifying effect of dietary feeding of a synthetic estrogenic compound atrazine, on 7,12-dimethylbenz(a)anthracene (DMBA)-induced ovarian carcinogenesis was studied in female Sprague-Dawley rats. We also
assessed the effect of atrazine on the growth of ovarian adenocarcinoma by measuring proliferating cell
nuclear antigen (PCNA)-index and by assessing the immunohistochemical expression of estrogen receptor(ER)-aand -~and androgen receptor (AR) in induced neoplasms. Rats were given a single injection of
DMBA (0.01 ml of 0.5% DMBA suspended in olive oil) into their left ovary to induce ovarian neoplasms.
They also received the experimental diet containing 5, 50 or 500 ppm atrazine for 50 weeks, starting one
week after the dosing of DMBA. DMBA exposure produced ovarian adenocarcinoma with an incidence of
45 % (9/20 rats) at the end of the study (week 51). Dietary feeding with atrazine reduced the incidence ofovarian adenocarcinoma without statistically significance: 22% (4/18 rats), 28% (5/28 rats), and 26% (5/19
rats) incidences in rats fed 5 ppm, 50 ppm, and 500 ppm atrazine containing diets after DMBA exposure,respectively. The PCNA-index in adenocarcinomas was greater than that of surface ovarian epithelium. ER-a,~and AR were expressed in a variable percentage of moderately and poorly differentiated adenocarcinoma
cell nuclei, but their reactivity was extremely weak or negative in well differentiated adenocarcinoma cells.
In additionm atrazine treatment did not affect tumor development in other organs (mammary gland andsubcutaneous tissue). These results might suggest dietary feeding of a synthetic estrogenic compoundatrazine did not affect DMBA-induced rat ovarian carcinogenesis.

114

··-----

JSTP/IFSTP IATP 2004

PostersP-7
The influence of pre- and post-natal exposure to methoxychlor on gene
expression in the endometrium of rats after maturation
Ryoichi OHTSUK.A1, Makio TAKEDA1, Satoru YAMAGUCHI 1, Koichi HAYASHI 1, Yukiko TAKEUCHI 1,
Maki KUWAHARA1, Naofumi TAKAHASHI1, Tadashi KOSAKA 1, Keizo MAITA1, Takanori HARADA1
Institute of Environmental Toxicology, lbaraki 303-0043 , Japan 1
In order to determine whether pre- and post-natal exposure to methoxychlor (MXC), an endocrine disrupting
chemical, influences the gene expression in the rat uterus after maturation, we conducted microan·ay analysis
for mRNA expression in endometrial cells captured by Laser Microdissection (LCM) system.
For the present study, 32 female pups were obtained from SD rat dams receiving MXC at a dietary level of
0 or 1000 ppm during the gestation/lactation period. The pups were fed a normal diet after weaning and kept
up to 52 weeks of age. At 10 and 52 weeks of age, 8 animals from each group were euthanized and theiruteri were sampled immediately after necropsy. The uterine tissue samples were embedded in tissue
compound for microarray analysis and also fixed in 10% neutral buffered formalin for conventional
histopathology.
For microarray analysis, endometrial cells were captured from the frozen uterine sections stained with
HistoGene LCM Frozen Section Staining Kit using LCM system, and the total RNA samples were extracted
by Absolutely RNA Microprep Kit. The antisense RNA (aRNA, reflecting mRNA expression) samples weresynthesized from extracted total RNA samples by MessageAmp aRNA Kit for 2 rounds according to the
manufacture's protocol. Before starting microarray analysis, a pooled control aRNA sample was preparedfrom 8 control samples and was used as reference of each sample by labeling with Cy3. The samples fromeach animal (including controls) were labeled with Cy5, and incubated with Cy3-labeled pooled control on
Microarrays (24 arrays were used) and scanned with array scanner.
In MXC-treated rats at 10 weeks of age, about 300 genes expressed higher (>150%) and 5 genes expressed
lower (<66.7%) signals than those of Cy3-labeled pooled control. In control rats, only 6 genes expressedhigher signals than that of pooled control. These results may indicate the possibility of the fetal imprinting
effect of MXC on the rat endometrial cells . Analysis for animals at 52 weeks of age is now underway.

JSTP/IFSTP IAT.P . ,200~

Posters

P-8
Effects of the fetal administration of diethylstilbestrol (DES) and 4-noctylphenol (n-OP) on 7, 12-dimethylbenz(a)anthracene (DMBA)-induced
mammary carcinoma in SD rats
Hiroaki KAWAGUCHI1, Masakazu SOHDA 1, Shuhei TAGUCHI', Koichiro MIYAMOTO',
Hiroki YOSHIDA 1
The Department of Tumor Pathology, Graduate School of Medical and Dental Sciences,
Kagoshima University, Kagoshima 890-8544, Japan 'Introduction Recently, breast cancer in humans has been increasing both in Japan and the rest of the world.
Endocrine disrupting chemicals are thought to be one of the factors involved in this phenomenon. We have,therefore, studied the effects of the fetal administration of diethylstilbestrol (DES) and 4-n-octylphenol (nOP) on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in SD rats.
Materials and Methods Pregnant rats were divided into 3 groups. The rats in group I were intact during the
gestation period. The other rats were given DES (0.1, 1, 10, 100 ppm) and/or n-OP (10, 100, 1000 ppm) intheir feed on gestation days 0-21 (group II) and on gestation days 13-21 (group III). All offspring were givenl O mg DMBA by gastric intubation at the age of 50 days, and palpated for mammary masses.
Result Early abortion (Group II) and abnormal delivery (Group III) were seen in pregnant rats given DES,
and growth disorders were seen in their offspring. However, no abnormal changes in pregnancy or delivery
were seen in pregnant rats given n-OP or their offspring. The induction of mammary tumors induced by
DMBA was suppressed in the female fetal rats given DES in Group II, but was promoted in the female
fetuses given DES in Group III and n-OP in Groups II and III.
Discussion We suggest that the fetal administration of DES (strong estrogen) induced disorders of theendocrine mechanism and induction of mammary tumors, whereas, the fetal administration of n-OP (veryweak estrogen) promoted the induction of mammary tumors.

116

Posters
P-9
The influence of pre- and post-natal exposure to methoxychlor on the rat
immune system and renal function during aging process
Yukiko TAKEUCHI1, Naofumi TAKAHASHI 1, Tadashi KOSAKA 1, Koichi HAYASHI1, Yuko CHIBA 1,
Toshinori YOSHIDA 1, Maki KUWAHARA1, Sayuri KOJIMA1, Ryoichi OHTSUKA1, Keizo MAITA2,
Takanori HARADA 1
Toxicology Division II, Institute of Environmental Toxicology, Ibaraki 303-0043, Japan 1
Division of Study Planning and Consultation, Institute of Environmental Toxicology 2
We previously demonstrated that apoptotic cell death occutTed in the thymus of rat pups of both sexes
exposed to a high dose of methoxychlor (MXC) during the pre- and post-natal period. Based on the result,
we have undertaken a further investigation to determine whether the pre- and post-natal exposure to MXC
persistently influence the rat immune system until a mature age. For the present study, male and female rat
pups were obtained from dams receiving MXC at dietary levels of 0, 30, 100, 300, and 1000 ppm during the
gestation and lactation period. They were fed a normal diet after weaning and kept up to 52 weeks of age.
These animals were examined for immune response at 10 and 52 weeks and also for renal function at 52
weeks. At 10 weeks of age, there were no abnormalities in the thymus of either sex, but splenic T-cell
subsets were significantly decreased in females at 300 and 1000 ppm. At 52 weeks of age, significant
increases in the relative weights (ratio to body weight) of the spleen and kidneys were observed in females at
I 000 ppm although there were no significant changes in the thymus and splenic lymphocyte subsets in either
sex . These female rats showed an abnormal excretion of urinary protein and increases in plasma creatinineand chloride which indicate renal dysfunction. However, such changes were not observed in male rats.
Histopathologically, the incidence of nephropathy increased in females at 1000 ppm and their kidneys had
significantly large-sized glomeruli. In addition, increases in glomerular IgG and IgM deposits and plasma
IgM levels were observed in these female rats. These results indicate that the immune system damage causedby the pre- and post-natal exposure to MXC could be repaired during aging process but not completely infemales even at a mature age. The increased incidence of nephropathy characterized by enlarged glomeruli
associated with increased IgG and IgM deposits may reflect an imbalance of population among lymphocyte
subsets which persistently remains after exposure to MXC.

JSTP/IFSTP IATP·'t200~

PostersP-10
No effects of transplacental and lactational exposure to combination ofbisphenol A and 4-nonylphenol, endocrine disruptors, on morphogenesis ofmale reproductive organs and spermatogenesis in F344 rats.
Kyoko NABAE 1, Hiroko YOSHIN0 1, Norio IMAI' , Takeshi HIROTA1 , Mayumi KAWABE 1,
Syugo SUZUKI2, Tomoyuki SHIRAI2
Daiyu-kai Institute of Medical Science, Aichi 491-0113, Japan 1
Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of
Medical Sciences2
Bisphenol A (4,4' -isopropylidene-2-diphenol, BPA) is widely used in the polycarbonate plastic industry. BPA
has been reported to be weakly estrogenic, both in vitro and in vivo. BPA has been found in umbilical cord
blood and mother's milk, and both fetus and newborn are suspected to be much more sensitive to estrogenic
compounds than adult. 4-Nonylphenol (4-NP), a surfactant used in the production of laboratory plasticware,and was also reported to exert estrogenic activity. Previously, we also reported that transplacental andlactational exposure to BPA and 4-NP did not each exert any adverse effects on morphogenesis of accessory
sex organs or spermatogenesis in F344 male rats. This study was conducted to evaluate the combination riskof endocrine disruptors on the accessory sex organs and spermatogenesis in F344 male rats offspring
exposed them preneonatally and neonatally. Non-treated female and male F344 rats were mated at 11 weeks
of age, and the pregnant rats were administered by gavage O (control), BPA 0.05 mg/kg, 4-NP 0.1 mg/kg
and mixture of BPA 0.05 mg/kg and 4-NP 0.1 mg/kg during gestation and lactation period. When male Flrats reached 13 weeks old, they were euthanized, and final body and organs weights were recorded. And
spermatogenesis (account of sperm, sperm mobility and morphological abnormality) and distribution of stageof seminiferous epithelium were investigated, and serum levels of testosterone were measured. No effects on
the body weights of treated dams, the gestational period and the numbers of stillborn and newborn wereapparent. No differences in the sex ratios of live newborn at birth was observed between the treated andcontrol groups. Furthermore, no effects on male Fl rats in body weights and food consumption wereapparent. No differences in organ to final body weight ratios of testes, prostate, epididymides, seminal
vesicles and LA-BC muscle were noted between the treated and control groups. No differences inspermatogenesis in the testis and epididymis were noted between the treated and control groups. No effects
on distributions of stage of seminiferous epithelium were also observed. And no observable histopathologicalabnormalities were present in the accessory sex organs. We concluded that transplacental and lactational
exposure to combination of BPA and 4-NP did not exert any adverse effects on the spermatogenesis and themorphogenesis of rat accessory sex organs.

ll8

JSTP/IFSTP IATP 2004

PostersP-11
No effects of transplacental and lactational exposure to combination ofbisphenol A and 4-nonylphenol, endocrine disruptors, on morphogenesis ofmale reproductive organs and spermatogenesis in /CR mice
Yuko DOI', Norio IMAI', Kyoko NABAE1, Yousuke TODA', Seiko TAMAN0 1, Hideki WANIBUCHI2,
Keiichirou MORIMURA 2 , Shoji FUKUSHIMA2
Daiyu-Kai Institute of Medical Science, Aichi 491 -0113, Japan 1
Osaka City University Medical School2
Recently, a number of chemicals, which disrupt normal endocrine functions and misgivings in worldwide
have been identified. Bisphenol A (BPA) and 4-nonylphenol (4-NP) are widely used and both compounds
were reported to have weak estrogenic effects. The important endocrine-disrupting effects might bemodification of development in hormone-sensitive organs such as the prostate, uterus, breast and testis.
Previously, we exposed transplacentally and lactationally to female mice BPA or 4-NP alone at 0.05 mg/kg/
day by gavage and concluded that transplacental and lactational exposure to BPA or 4-NP was no effect inmale offspring mice. Since we are exposed by many chemicals, we investigated the combination effect of
two chemicals, BPA and 4-NP. We designed to examine the effect of transplacental and lactational exposureof BPA and 4-NP in combination on morphogenesis of male reproductive organs and spermatogenesis.
Female and male ICR mice were mated, and the pregnant female mice were given vehicle (corn oil), BPA at
0.05 mg/kg/day, 4-NP at 0.05 mg/kg/day or mixture of BPA and 4-NP intragastrically during the gestationand lactation period. Male Fl body weights and food consumptions were measured once a week until 13
weeks old. At week 13, all Fl male mice were killed and reproductive organ weights were recorded .
Thereafter, they were observed histopathologically. The numbers of sperm in the testis and epididymis werecounted, and the motility and malformation of sperm were also observed. In maternal mice, implantation
marks were decreased in combination group. No. of newborn and live newborn, and sex ratio of newborn
were also decreased. Body weights and food consumptions were no different during experimental period.
Relative testis, prostate (ventral lobe) and epididymis weights were not affected and no histopathologicalabnonnality was observed compared with control group. In each group treated with chemicals, there were nochanges on morphological abnormalities in the reproductive organs of male offsprings. For Fl male mice,
number of sperms in testis and epididymis were not affected in combination group. Furthermore, data forother parameters (movement and morphological abnormalities) also did not significantly differ between
control and treatment group. These data indicate that transplacental and lactational exposure to combinationof BPA and 4-NP does not affect on morphogenesis and spermatogenesis in male offspring mice.

JSTP/IFSTP IATP ·2:oofl

Posters

P-12
Lack of carcinogenic risk in the prostate with transplacental and lactational
exposure to 4-nonylphenol in F344 rats
Hiroko YOSHIN0 1, Kyoko NABAE 1, Yuko D0I1, Takeshi HIROTA!, Kumiko OGAWA2, Tomoyuki SHIRAF
Daiyu-Kai Institute of Medical Science, Aichi 491 -0113, Japan 1
Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of
Medical Sciences24-Nonylphenol (4-NP), a surfactant used in the production of laboratory plasticware, and has been reported
to have estrogenic activity. The compound has been concerned as an estrogenic xenobiotic. One of theendocrine-disrupting effects might be modification of carcinogenicity in hormone-sensitive organs such as theprostate, uterus, breast and testis. We previously reported that estrogen may play an important role inprostate carcinogenesis. Therefore, the cun-ent study was designed to examine the modulating effects of 4-NP
on prostate cancer risk in male offspring exposed transplacentally and lactationally. Non-treated female andmale F344 rats were mated at about 11 weeks of age, and females which were confirmed to be pregnant wasgiven intragastrically by gavage O (control), 0.1, 10 or 100 mg/kg/day of 4-NP during the gestation andlactation periods. The body weight of each dam was monitored during gestation and lactation period. WhenF1 males reached 5 weeks old, all groups were given 2-amino-1-methyl-6-phenylimidazo[4,5 -b]pyridine
intragastrically by gavage at a dose of 100 mg/kg b.w. weekly for 19 weeks. All surviving rats were killed atexperimental week 60 and subjected to complete autopsy. The body weight of F1 male rats was monitored
weekly for experimental period, at autopsy, F1 rats were euthanized, and final body and organ weights
recorded. The prostates, seminal vesicles and all other organs were examined for gross abnormalities andfixed in 10% buffered formalin, processed routinely, and examined histopathologically. The body weights ofdams were significantly suppressed by 4-NP lOOmg/kg/day administration during gestation and lactation
period. And gestation period of rats treated 100 mg/kg/day were elongated compared to control group.
However, the numbers of stiilborn and newborn were not affected. There were no differences in the sex
ratios at PND 4 between the treated and control groups. There were no observable external anormalies atbirth and no significant differences in offspring body weights at any point before weaning.The final bodyand organ weights were not significantly affected. The preneoplastic and neoplastic lesions of the accessory
sex organs were histopathologically classified into prostatic intraepithelial neoplasia (PIN) and carcinomas of
prostate, as well as atypical hyperplasia of the seminal vesicle. None of the incidences in the experimentalgroups were significantly different from those in the control group. Our data suggest that maternal exposure
to 4-NP during gestation and lactation periods does not affect the risk of prostate carcinogenesis in maleoffspring.

120

Posters
P-13
Using animal and human mode of action information in assessing human risk:a framework for analysis of various pathologies
Penelope A FENNER-CRISP 1, Dorothy E PATTON 1, The ILSI RSI WORKGROUP 1
ILSI Risk Science Institute, Washjngton DC 20005, USA1
In late 2003, the International Life Sciences Institute Risk Science Institute (ILSI RSI) published the productof a two-year effort on using animal and human mode of action (MOA) information to evaluate the humanrelevance of tumor induction by non-genotoxic carcinogens. This report featured case study analyses of six
carcinogens, which then led to the development of a four-part Human Relevance Framework (HRF) for
systematic and transparent analysis of MOA data and related information. With such information increasingly
available for various pathologies in addition to neoplasia, the sponsors of this project, the U.S.
Environmental Protection Agency and Health Canada, requested ILSI RSI to evaluate the utility andapplicability of thls new HRF for other endpoints. The current RSI Workgroup, including some participantsfrom the original project, has applied the HRF to case studies involving developmental effects such as
skeletal and other abnormalities, male reproductive effects such as the androgen insufficiency syndrome(AIS), neurotoxic effects as a result of premature onset of cell replication in the developing brain and renal
tubular damage. The Workgroup also examined the value of application of the Framework to genotoxk
carcinogens, including those with and without mutagenic properties. Thls poster will exhibit the Frameworkand several of the cases used to exarrune its utility for analysis of a broad variety of pathologies.

JSTP/IFSTP IATP.~200~

Posters

P-14
Carcinogenicity of low-dose MelQx, in relation to cancer risk assessment
Keiichirou MORIMURA1, Hideki WANIBUCHI 1, Manabu HOSHI 1, Anna KINOSHITA1,
Shoji FUKUSHIMA 1
Department of Pathology, Osaka City University Medical School, Osaka 545-8585, Japan'
More than 80% of human cancers may be related to carcinogenic environmental chemicals, indicating animportance for managing the hazards of environmental carcinogens. For managing these hazards, anassessment based on some scientific data should be necessary. As for the genotoxic carcinogens, it has been
generally accepted that there have no threshold in exerting carcinogenic potential, because genotoxic
carcinogens are mutagenic. In the present study, for clarification of human risk assessment of genotoxic
carcinogens, we examined the low-dose carcinogenicity of 2-amino-3,8-dimethylimidazo[4,5-f]quinoline(MelQx) which is one of food-derived heterocyclic amine mutagens. A total of 885 male 21-day-old F344
rats were received MeIQx at doses of 0, 0.001 (equivalent to human daily intake), 0.01, 0.1, 1, 10, 100 ppm
in powered diet for 4 or 16 weeks. After 16 weeks treatment, the numbers of GST-P positive foci of rat liverin groups receiving 0.001 to 1 ppm of the carcinogen did not differ from the control value, in contrast to theincrease observed with 10 ppm and the clear, significant elevation with 100 ppm. We also examined theformatio n of MeIQx-DNA adducts and 8-Hydroxy-2' -deoxyguanosine (8-0HdG) levels in the liver. There
was linear relationship between the various doses of MeIQx and the levels of MeIQx-DNA adducts.
Concerning the 8-0HdG levels, no significant differences among groups receiving MeIQx from 0.001 ppm
to 0.1 ppm and the control group were apparent, although values were linearly elevated from 1 ppm andabove, with statistical significance. cDNA microarray using 3D an·ay system was applied for liver tissues of
16 weeks treatment groups. No over-expression was detected in metabolizing enzymes for O to 1 ppm but
slight increase was seen in high dose treatment. Some genes related with signaling pathways were downregulated. Moreover, the in vivo mutagenicity assay using Big Blue rats treated for 16 weeks with same low
doses of MeIQx was examined. The mutation frequency from 0.001 ppm to 1 ppm were same level atcontrol group and the values for 10 ppm and 100 ppm showed significant increase compared with thecontrol group. The present results indicate that the dose-response curve of genotoxic carcinogen, MeIQx, at
low-dose is different from that of high-dose. Threshold may exist for the hepatocarcinogenic potential of
MeIQx. These results are impo1tant regarding how we should view the impact of genotoxic carcinogens, inthe human environment in relation to cancer risk assessment and management.

122

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JSTP/IFSTP IATP 200~

Posters

P-15
Presence of the threshold for carcinogenicity and in vivo mutagenicity induced
by genotoxic carcinogen, Me/Ox, at low doses
Saki NAIT0 1, Keiichirou MORIMURA1, Manabu HOSHI1, Natsuko MIYAZI1 , Shoji FUKUSHIMA 1
Department of Pathology, Osaka City University Medical School, Osaka 545-8585 , Japan 1
More than 80% of human cancers may be related to carcinogenic environmental chemicals. Carcinogenicity
is generally detected by long-term carcinogenicity testing in rodents using doses considerably in excess ofhuman exposure levels. The situation is complicated by the fact that carcinogens can be classified as either
genotoxic or non-genotoxic according to results of in vivo genotoxity tests. The currently accepted view isthat no threshold exists for the cartcinogenic potential of genotoxic carcinogens, with the response curve
approaching zero in a linear fashion with extrapolation from the doses used in carcinogenicity testing. Actual
human cancer risk of genotoxic carcinogens is very difficult to assess, because few directly obtained data are
available from the carcinogenecity testing at low doses such as human daily exposure levels . Therefore,
practical information concerning cancer risk remains inadequate, and the need for investigation in this field isurgent.To elucidate the relationship between in vivo carcinogenic and mutagenic potentials of genotoxic
carcinogens, low doses were tested in the Big Blue transgenic rats with 2-arnino-3,8-dimethylirnidazo[4,5-f]quinoxaline (MeIQx). In disagreement with a general belief that genotoxic carcinogens show no threshold for
carcinogenic potential, a threshold was demonstrated for both carcinogenicity in terms of preneoplastic lesion
induction and in vivo mutagenicity of MeIQx.

12

JSTP/IFSTP IATP 200~

Posters

P-16
Lack of modification of Me/Qx rat liver carcinogenesis by caffeine induction of
CYP1A2
Hitoshi KANDORI 1•2 , Masanori KURIBAYASHJl ·3, Shingo INAGUMAI, Makoto ASAMOT0 1,
Naomi HOKAIWADOt, Seishiro TAKAHASHI 1, Tomoyuki SHIRAI'
Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of
Medical Sciences, Aichi 467-8601 , Japan 1
Takeda Chemical Industries Ltd. Drug Safety Research Center Hikari Branch2
Ono Pharmaceutical Co. Ltd., Safety Research 3
2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), one of the most abundant carcinogenic
heterocyclic amine in cooked meat and fish, is speculated to be associated with human carcinogenesis.
MeIQx is known to be metabolically activated by several enzymes, especially CYPIA2, to form DNA
adducts. Therefore analysis of interactions between MeIQx with various chemicals which affect MeIQx
metabolism is important for risk assessment. In order to evaluate modifying effects of CYPIA2 inducers on
MeIQx hepatocarcinogenesis, caffeine or diethylnitrosamine (DEN) was given together with MeIQx to rats.
Modification was assessed using a medium-term liver bioassay system. Six-week-old male F344 rats were
given a single dose of DEN, 200 mg/kg b.w., i.p. as an initiator. Starting 2 weeks later, MeIQx was given
together with or without 0.1 % caffeine or 0.01 % DEN in the diet or drinking water, respectively for 6
weeks. At week 3, all rats were subjected to a two-thirds partial hepatectomy. At week 8, the experiment was
terminated and the livers were taken for analysis. First, we analyzed the numbers and areas of glutathione Stransferase (GST-P) positive liver foci, putative preneoplastic lesions, after immunohistochemical staining.
Then, mRNA expression levels of several metabolic enzymes were compared between GST-P positive fociand the surrounding liver tissue. As a result, DEN given together with MeIQx showed a synergistic
promoting effect on development of GST-P positive foci. However, no modification was found with caffeine,
although it up-regulated CYPIA2 mRNA expression. DEN is reported to also be a CYPIA2 inducer but wewere not able to confirm this. In the GST-P positive foci, phase II detoxifying enzymes including GSTs and
UDPGTs were up-regulated. In the background tissue of rat livers after exposure to DEN as a CYP inducer,
specific expression of extracellular matrix-related genes including spare and laminin receptor I was detected.
These results suggest that modifying effects of caffeine on MeIQx through CYPIA2 are limited because
caffeine also up-regulates various phase II detoxifying enzymes. Moreover, our findings indicate that changes
in extracellular matrix-related genes in the surrounding tissue are important for progression of GST-P
positive foci .

JSTP/IFSTP IATP 2004

Posters

P-17
Investigation of the complex carcinogenic risk by PhlP and MelQx in theinitiation stage of carcinogenesis
Akihiro HIRATA 1, Tetsuya TSUKAMOT0 1, Hirok:i SAKAF, Masami YAMAMOT0 1, Norimitsu SHIRAI 1,
Takeshi IIDAKA 1, Tokuma YANAF, Toshiak:i MASEGF, Masae TATEMATSU 1
Division of Oncological Pathology, Aichi Cancer Center Research Institute, Aichi 464-8681, Japan 1
Department of Veterinary Pathology, Faculty of Agriculture, Gifu University 2[Purpose] In order to examine the complex carcinogenic risk by different heterocyclic amines (HCAs), theinitiation activities of 2-amino-1-methyl-6-phenylimidazo[4,5-b ]pyridine (PhIP), 2-amino-3,8dimethylimidazo[ 4,5-b ]quinoxaline (MeIQx) and their combination were investigated with an in vivo 5-week
initiation assay model.
[Method] Seven-week-old male F344 rats were undergone two-thirds partial hepatectomy (PH) and thecompounds were administered intragastrically at 12 and 30 hours after PH. Group 1 animals received
simultaneous administration of PhIP (200 mg/kg body weight (b.w.)) and MeIQx (25 mg/kg b.w.) at 12hours and then vehicle (com oil) alone at 30 hours. Rats in groups 2 were given both compounds separately(PhIP at 12 hours and then MeIQx at 30 hours). Group 3 received both of them in a reverse order(MeIQx -> PhIP). Single compounds were given at 12 or 30 hours to group 4 (PhIP -> vehicle), group 5(vehicle-> PhIP), group 6 (MeIQx -> vehicle), and group 7 (vehicle -> MeIQx). Control group rats were
given only vehicle (group 8). Subsequently, the rats were fed on basal diet for 2 weeks, followed by dietcontaining 0.015% 2-acetylaminofluorene for the next 2 weeks. Three weeks after PH, all rats received
carbon tetrachloride as a stimulus for proliferation. At the end of week 5, all the survivors were sacrificed.
Initiation activities of each group were assessed with the induction of glutathione S-transferase placental
form (GST-P) positive foci in the liver.
[Results] The value of GST-P positive foci in the group taking MeIQx prior to PhIP (group 3) was
significantly higher than those in the groups having PhIP or MeIQx alone at the comparable time points(P<0.05, vs. group 5 and 6), indicating the summation effect of the two chemicals. Nonetheless, there wasno significant difference in the number of GST-P positive foci between the groups with serial treatment of
PhIP then MeIQx (group 2) and with PhIP (group 4) or MeIQx (group 7) alone. Furthermore, the number of
GST-P positive foci induced with MeIQx alone (group 6) was significantly suppressed with the simultaneousadministration of PhIP (group 1) (P<0.05, vs. group 6).
[Conclusion] PhIP, when administered beforehand or simultaneously, could inhibit the carcinogenic potentialof MeIQx in the initiation stage of carcinogenesis.

JSTP/IFSTP IATP,,~200~

Posters ·P-18
Colon carcinogenicity of 2-amino-1-methyl-6-phenylimidazof4,5-b}pyridine(PhlP) in rats: suggestion for carcinogenesis and practical threshold
Kenichiro DOI', Elsayed I SALIM', Anna KINOSHITA', Rawiwan PUATANACHOKCHAI',
Hideki WANIBUCHI 1, Shoji FUKUSHIMA'
Department of Pathology, Osaka City University Medical School, Osaka 545-8585, Japan'
Colon carcinogenesis is thought to be a multi-step process caused by plural genetic alterations. Genotoxic
carcinogens, such as food-derived heterocyclic amines (HCAs), have been implicated as initiating agents toexert its mutagenicity through binding DNA and forming DNA adduct. Most abundant HCA, 2-amino-1methyl-6-phenylimidazo[4,5 -b]pyridine (PhIP), is revealed to be rodent's colon carcinogen and constantly
taken from our usual environment, resulted in suspicion of an etiology for human colon cancer. However,
PhIP could induce quite low levels of tumor yield when administered to rats. Therefore, we presently hoped
to examine an actual potential of PhIP on rat colon carcinogenesis at lower dose levels that mimic to humanpractical exposure. A total of 192, 6-week-old, male F344 rats were divided into 8 groups (24 rats in each)and all animals were subcutaneously injected two times of 15 mg/kg body weight, azoxymethane (AOM),
followed by continuous feeding of PhIP in basal diet. At weeks 16 and 36, animals were sacrificed andhistopathologically examined. As a result, PhIP exerted so strong enhancing effects on colon tumorigenesis at
more than 50 ppm, oppositely which clearly revealed no-observed effect levels (0.001-10 ppm) including
human daily exposure. Furthermore, several modifying effects on tumor distribution or histological types
were observed at higher-doses. These results may postulate the strong promotion effect of PhIP in the latter
carcinogenic process, also may suggest a 'practical threshold' for rats, providing further possibility forhuman risk assessment.

126

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•~--,

JSTP/IFSTP IATP 2004

Posters

P-19
Estimation of a no observed effect level for 2-acetylaminofluorene (2-AAF) and
2,4-diaminotoluene (2,4-DAT), genotoxic liver carcinogens, in 16-week feedingstudies using male F344 rats
Akihiro HAGIWARA 1, Toshio ICHIHARA 1, Hiroko YOSHINO', Norio IMAI', Hideki WANIBUCHI2,
Keiichirou MORIMURA 2 , Seiko TAMANO'
Daiyu-kai Institute of Medical Science, Aichi 491 -0113, Japan'
Department of Pathology, Osaka City University Medical School2
The carcinogenic potential at very low doses of 2-AAF and 2,4-DAT was evaluated in 16-week feedingstudies, which applied quantitative analysis of glutathione S-transferase placental form (GST-P) positive
hepatocytic foci, using male F344 rats. In experiment 1, groups of 100 or 50 animals (21-day old atcommencement) were given O (as control), 0.001, 0.01, 0.1, 1, 10 and 100 ppm 2-AAF in their diet for 16
weeks. Marked body weight retardation was found in 100 ppm group, but no influence was evident ingroups fed a diet containing less than 10 ppm. Administration of a dietary concentration of 100 ppm 2-AAF
to rats caused prominent elevation of liver weight, serum g-glutamyltranspeptidase (GGT),
bromodeoxyuridine (BrdU) labeling indices (LI) in the hepatocyte, and quantitative values for small sizes of
GST-P positive foci (comprised from 2-4 cells, 5-10 cells, and 11 cells). Significant increase in liver weight
was also noted in rats fed 10 ppm 2-AAF, but not in rats receiving less than 1 ppm 2-AAF. Quantitativevalues for GST-P positive foci were statistically increased in the 1 and 10 ppm groups, but not at 0.1 ppm orlower. No significant elevation of the 8-hydroxyguanine (8-0HdG) level in the liver DNA was found at thehighest dietary level. In experiment 2, groups of 90 or 30 animals were also given dietary levels of 0, 0.001,
0.01, 0.1, 1, 10, 100 and 300 ppm 2,4-DAT for same period. Marked retardation of body weight increase
was found in 300 ppm groups, but not in animals fed a diet containing less than 100 ppm. Administration ofa dietary concentration of 300 ppm 2,4-DAT to rats caused prominent elevation of liver weight, andquantitative values for small GST-P positive foci. Significant increased liver weight was also noted in rats fed
100 ppm 2,4-DAT, but not with doses of 10 ppm 2,4-DAT or less. Quantitative values for GST-P positivefoci were significantly increased in 100 ppm groups, but at 10 ppm and lower there were no differencesfrom controls. BrdU LI in the hepatocyte and 8-0HdG level in the liver DNA were not affected at 300 ppm
2,4-DAT group. Thus, practical threshold may exist for carcinogenicity of 2-AAF and 2,4-DAT, and a noobserved effect level of 2-AAF and 2,4-DAT, as assessed with reference to GST-P positive foci, wasestimated to be 0.1 ppm (6.81 mg/kg/day) and 10 ppm (0.654 mg/kg/day), respectively, under the present
experimental conditions.

JSTP/IFSTP IATP 200~

Posters

P-20
Effect of phenobarbital on diethylnitrosamine-induced hepatocarcinogenesis in
TGF-alpha transgenic mice-correlation with P450
Takayuk:i YUNOKI 1, Hideki WANIBUCHI1, Rawiwan PUATANACHOKCHAI 1, Masakazu KAKUNI1,
Kazuo HAKOI1, Shoji FUKUSHIMA 1
Department of Pathology, Osaka City University Medical School, Osaka 545-8585, Japan, 1[Objective] To study the effect of various concentrations of phenobarbital on DEN induced
Hepatocarcinogenesis with TGF-alpha transgenic mice.
[Methods] All mice was administerd DEN intraperitoneal dose of 5mg/kg body weight at 15 days old,and
given PB at dietary concentration of 0,2 15,500ppm from the end of week2.
[Result] All mice were sacrificed at the of 22 weeks .The incidence of hepatocellular carcinoma was 80% at
dietary concentration of 500ppm.The incidence was 20% and 24 % at DEN alone group and DEN+PB2%
grup,respectively.But hepatocellular carcinoma was not obserbed at DEN+PB15%grup.8-0HdG in liverslightly decreased at PB 2ppm group,but there was no significant difference among all groups.The lever of
The level of TGF-alpha slightly decreased at PB 2ppm group and 15ppm group.
[Conclusion] Phenobarbital enhanced DEN-induced liver hepatocarcinogenesis at 500 ppm, on the other
hand, tend to suppressed hepatocarcinogenesis at 15 ppm in TGF-alpha transgenic mice. One possible
mechanism of the suppression of liver carcinogenicity by medium dose of phenobarbital might be influence
on TGF-a expression.The result suggest that the mechanism correlate with p-450.

128

JSTP/IFSTP IATP 2004

Posters

P-21
Possible mechanism of hormetic phenomenon of alpha benzene hexachloride
on diethylnitrosamine induced hepatocarcinogenesis
Rawi wan PUATANACBOKCBAI 1, Bideki WANIBUCBI 1, Mayuko OSADA2, Keiichirou MORIMURA1,
Yoshihiko FUNAE2, Shoji FUKUSBIMA1
Department of Pathology, Osaka City University Medical School, Osaka 545-8585, Japan 1
Department of Chemical Biology, Osaka City University Medical School2
Alpha benzene hexachloride (alpha-BBC), an organochlorine insecticide, was banned about 25 years ago
from Japan but some countries still employed. Male F344 rats were injected by 100 mg/kg body weight ofdiethylnitrosamine (DEN) once a week for 3 times via intraperitoneum. After the last injection 1 week, dietcontaining various concentrations of alpha-BBC, range from 0.01 to 500 ppm, was fed for 10 weeks. It was
found that high concentration of alpha -BBC, 500 ppm, treated rats significantly increased the number andarea of glutathione S-transferase placental form-positive foci (GST-P), a marker of preneoplastic lesion of theliver. On the other hand, low doses of alpha-BBC, 0.05 ppm, significantly reduced both number and area of
GST-P positive foci. Thus, in plotting concentration of alpha-BBC versus number and area of GST-P positivefoci results in a U-shape dose-response curve. Furthermore, biphasic effect of alpha-BBC on cytochrome P
450 contents was revealed that significantly reduced at low dose, 0.05 ppm, but significantly raised at highdose. 8-hydroxydeoxyguanosine, a marker of oxidative DNA damage, production was significantly increasedin high dose treated groups but significantly decreased in low dose treated groups. In addition, the levels of
CYP2Bl , a source of free radicals production, mRNA and protein using competitive RT-PCR and western
blotting, respectively, were significantly induced in 50 and 500 ppm of alpha-BBC treated group. There,
however, is no significantly difference among low dose treated group. From these results suggested thathormetic phenomenon of alpha-BBC might be involved in xenobiotic metabolism, cytochrome P450
oxidoreductase system, that produce free radicals followed by oxidative DNA damage and consequently
pathological change in the liver.

JSTP/IFSTP IATP ,'2 ·00~

Posters

P-22
Effect of low-dose DDT on liver carcinogenesis in rats
Masahiko KUSHIDA 1, Tokuo SUKATA1, Keisuke OZAKJl , Satoshi UWAGAWA1, Keiichirou MORIMURA2 ,Hideki WANIBUCHF, Yasuyoshi OKUN01, Shoji FUKUSHIMA2
Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., Osaka 554-8558, Japan 1
Department of Pathology, Osaka City University Medical SchooF
Inhibitory effects of DDT on rat hepatocarcinogenesis have been reported, when given to rodents at a doseof 0.005, 0.01 ppm, while higher doses exhibited promoting activity. The mechanism of DDT promotion as
well as its inhibitory activity remain unclear and raise the question of whether the treatment with low dosesof DDT will reduce cancer risk. We investigated its effect in male 6-week-old F344 rats injected by Ndiethylmitrosoamine (DEN) once a week for 2 times, ip. After 1 week of the last injection, diet containing
various concentrations of DDT, 0.005, 0.5, 500 ppm, was fed for 11 and 43 weeks. The inhibitory effect of
DDT treatment at a dose of 0.005 ppm on formation of GST-P positive foci and tumors has been observed
after 11 and 43 weeks of treatment, respectively. CYP3A2 and 8-hydroxy-2' -deoxyguanosin (8-0HdG),
which is the marker of oxidative DNA damage, levels in rat livers were correlated to induction of GST-P
positive foci. On the contrary, when DDT was applied at a high dose, a formation of GST-P positive fociand tumors, CYP3A2 protein expression and 8-0HdG levels in rat livers highly elevated in the rat liver atweeks 11 and 43 , respectively. Thus these results suggested that hormetic phenomenon of DDT might beinvolved in CYP450 system that produce free radicals followed by oxidative DNA damage and resulting topathological change in the liver.

130

JSTP/IFSTP IATP 2004

Posters

P-23
Activation of the ras oncogene and its relationship to aflatoxins-DNA adductformation in rat liver treated with aflatoxins
Dae Joong KIM 1, Tae Myoung KIM 1, Jin Tae HONG2, Hwan Soo Y00 2 , Cheol Beom PARK3,
Beom Jun LEE 1, Jong-Koo KANG 1, Young Won YUN 1
College of Veterinary Medicine, Chungbuk National University1, Cheongju 361-763, Korea 1
College of Pharmacy, Chungbuk National University 2
Biotoxtech 3
Aflatoxins are produced by Aspergillus flavus, parasiticus and their related fungi that grow in improperly
stored foods such as corn, rice, peanuts and other cereals. In addition to its high mutagenicity andcytotoxicity, aflatoxin B1(AFB1) is a potent hepatocarcinogen in experimental animals and an importantfactor for the human liver cancer. In spite of a high attention to the hepatocarcinogenicity of aflatoxins, therelative toxicity, for the risk assessment, of other types (AFB2, AFG1 and AFG2) of the toxin was not fully
studied. In the present study, the relative potency for the hepatotoxicity, mutagenicity, and DNA-adductformation by aflatoxins B1 , B2, G1 and G2 were investigated. Sprague-Dawley male rats were orally
administered with AFB1, AFB2, AFG1 or AFG2 at doses of 0.25, 1.25 or 2.5 mg/kg. Animals were killed at
12, 24 or 48 hrs following alflatoxin exposure, the histopathological examination, expression of ras oncogene
and 8-0xodG production as the biomarkers of hepatotoxicity, mutagenicity and DNA-adduct formation,
respectively, were examined and analyzed for the relative toxicity of the types of aflatoxins.

JSTP/IFSTP IATP -21)0;

Posters

P-24
Lack of effects of 1439 MHz electromagnetic near field exposure on the bloodbrain barrier in immature and young rats
Masanori KURIBAYASHI 1A, Jianqing WANG 2, Osamu FUJIWARA 2, Yuko D013, Kyoko NABAE3,
Seiko TAMAN0 3, Tadashi OGIS0 1, Makoto ASAMOT0 1, Tomoyuki SHIRAI 1
Depm1ment of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of
Medical Sciences, Aichi 467-8601, Japan 1
Depm1ment of Electrical and Computer Engineering, Nagoya Institute of Technology 2
Daiyu-kai Institute of Medical Science3
Ono Pharmaceutical Co., Ltd. , Safety Research4
The use of cellular phones has been spreading rapidly all over the world, especially in the developed
countries. They are being used not only by adults but also by children and the potential risk ofelectromagnetic nem·-fields (EMFs) from their antennae to human health, especially with respect to brain
tumor development or disturbance of the blood-brain barrier (BBB), is a source of concern. In the present
study, we performed experiments to explore whether EMF exposure causes any damage to the BBB in rats
with ages equivalent to the time of BBB genesis (4-weeks-old) and the teenage (IO-weeks-old) period. For
this purpose, alteration of BBB -related genes, such as those encoding p-glycoprotein, aquaporin-4 andclaudin-5, was assessed by immunohistochemistry and quantitative RT-PCR in the brain. First, we verified
that expression of the three BBB-related genes is influenced by 1,3-dinitrobenzene (DNB), a chemicalreported to destroy the BBB, and then we performed an EMF exposure experiment with 0, 2.0 and 6.0 W/kg
brain-average specific absorption rates (SARs) at 1.5 GHz for 90 min/day for 1 or 2 weeks. Vascularpermeability was also investigated using dye-transfer technique by FITC-dextran and albumin
immunohistochemistry. As a result, expression of the three BBB-related genes was found to be clearlydecreased after administration of DNB as a positive control , when compared with the control values.
However, there were no biologically meaningful differences with the EMF at any exposure level at either
age. Vascular permeability, monitored with reference to transfer of FITC-dextran, was not affected by EMF
exposure. Furthermore, albumin irnrnunohistochemistry clearly demonstrated albumin leakage from capillary
blood vessels to brain parenchymal tissue in the brains of rats receiving DNB , although there was no changewith EMF exposure. Thus, these findings suggest that local exposure to 1.5 GHz EMF exerts no adverse
effects on the BBB in immature and young rats.

132

Posters
P-25
Specific in vivo mutational spectra of genotoxic and non-genotoxic
hepatocarcinogens in gpt delta transgenic rats
Keita KANKI 1, Akiyoshi NISHIKAWA 1, Ken-ichi MASUMURA2, Takashi UMEMURA1,
Takayoshi IMAZAWA1, Yasuki KITAMURA 1, Takehiko NOHMF, Masao HIROSE 1
Division of Pathology, National Institute of Health Sciences, Tokyo 158-8501, Japan 1
Division of Genetics and Mutagenesis, National Institute of Health Sciences 2
Environmental carcinogens are classified into genotoxic and non-genotoxic carcinogens based on in vitro
bacterial mutagenicity. However, data from transgenic rodents with reporter genes including lac! and lacZ
have suggested some discrepancy between in vitro and in vivo mutagenicity of carcinogens. gpt delta
Transgenic rodents have advantages to efficiently detect different types of mutations. For example, point
mutations and deletions can be detected by 6-thioguanine selection using the gpt gene of E. coli and Spi·
selection using the red/gam genes of lambda phage, respectively. In the present experiment, the in vivo
mutant frequency (MF) and mutation spectrum of the known genotoxic rodent hepatocarcinogens 2-amino-3methylimidazo[ 4,5-f]quinoline (IQ) and N -nitrosopyrrolidine (NPYR), and the non-genotoxic
hepatocarcinogen di(2-ethylhexyl)phthalate (DEHP) were investigated in gpt delta transgenic rats. Although
there was no growth inhibition in any group after the 13-week treatment, liver/body weight ratio wassignificantly increased in all groups. GST-P positive liver cell foci were significantly increased in the 300
ppm IQ and 200 ppm NPYR groups, but little was induced in 12,000 ppm DEHP group. 6-Thioguanine
selection revealed significant increases of gpt MF in the liver of IQ group (18.8XI0- 5) and NPYR group(5.7XI0- 5 ), being 35- and 10-fold higher than that of control (0.55X 10·5 ), respectively. Predominant type ofbase substitutions observed in gpt genes were G:C to T:A transversion in IQ group, and A:T to G:Ctransition in NPYR group. There was no difference of gpt MF and mutation spectrum between the control
and DEHP groups. Similarly, there was no difference of Spi· MF and mutation spectrum in DEHP groups.
Thus, IQ and NPYR showed potent mutagenicity with specific mutation spectrum in the liver of gpt delta
rats. G:C to T:A base substitution induced by IQ has been also reported in other transgenic rodents such as
MutaMouse and BigBlue. Although NPYR has been reported to form deoxyguanosine adducts by simple
alkylation, adduction to deoxyadenosine by its metabolites such as crotonaldehyde may be responsible to invivo mutation of NPYR because A:T to G:C transition was a major type of mutation in the present study.
Since no mutagenicity was detected with DEHP, hepatocarcinogenicity of DEHP in rodents was confirmed to
be non-genotoxic pathways. In conclusion, our data indicates that analysis for specific in vivo mutational
spectrum can reversely provide useful information on the mechanism underlying chemical carcinogenesis.

JSTP/IFSTP IATP ·21,0~

Posters

P-26
Mechanistic study using cDNA microarray on enhanced hepatocarcinogenesis
in /CR mice fed diet containing dicyclanil
Mitsuyoshi MOT0 1, Miwa OKAMURA1, Tomoko MUTOH 1, Takao WATANABE 1, Yoko KASHIDA 1,
Noboru MACHIDA' , Kunitoshi MITSUMORI 1
Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan 'Dicyclanil, a pyrimidiamine-derived insect growth regulator used as a veterinary medicine, is known as ahepatocarcinogen in mice. In our previous study, negative results were obtained in an in vivo comet assay inmice and a liver initiation assay in rats using dicyclanil. These results suggested that dicyclanil was a nongenotoxic carcinogen having a potent tumor promoting effect. However, detailed mechanism of thepromoting effects for hepatocarcinogenesis of dicyclanil is still unclear. In the present study, to investigate
the mechanism of hepatocarcinogenesis in mice induced by dicyclanil, cDNA microarray and RT-PCR were
performed in mice fed diet containing dicyclanil. At first, to obtain the gene expression profile by theadministration of dicyclanil, male ICR mice were given O or 1500 ppm dicyclanil for 2 weeks. Although anincrease in relative liver weights and slight swelling of hepatocytes were observed in the mice treated withdicyclanil, no other histopathological changes were observed in the liver. In microarray analyses,
upregulations of gene expression of oxidation and/or redox-related metabolic enzyme, such as alcohol
dehydrogenase, cholesterol 24-hydroxylase, coproporphyrinogen oxidase, CYP lAl, CYP 1A2, Glu-6
phosphate dehydrogenase, glutathione peroxidase and thioredoxin reductase 1 (TrxR 1), were observed in theliver of dicyclanil-treated mice. Especially, the mRNA expressions of CYP lAl, CYP 1A2 ,md TrxR 1 wereremarkably increased. The gene expression of thioredoxin, known as an oxidative stress marker, was also
increased in the liver of mice given dicyclanil. Based on these results, using the two stage
hepatocarcinogenesis model, male ICR mice received an i.p. injection of 5 mg/kg of N-nitrosodimethylamine
and were then given diet containing O or 1500 ppm dicyclanil for 7 weeks with two thirds partial
hepatectomy, to examine liver promoting effects and alteration of gene expressions induced by dicyclanil. In
histopathological investigations, the centrilobular swelling of hepatocytes were observed in the dicyclanilgroup, and the numbers of gamma glutamyltransferase positive micro foci were increased in the liver of thisgroup, indicating enhanced tumor promoting effects. In RT-PCR and real-time PCR analyses, upregulationsof mRNAs that were similar to those in 2-week study were also observed in the liver of the dicyclanil group.
These results suggest that oxidative stress partly plays a role in the enhancement of liver tumor promoting
effects by dicyclanil. Additional molecular pathological investigations are now in progress in our laboratory.

134

JSTP/IFSTP IATP 2004

PostersP-27
Six-month carcinogenicity study of N-bis(2-hydroxypropyl)nitrosamine (DHPN)
in rasH2 mice
Miwa OKAMURA1, Mitsuyoshi MOT0 1, Yoko KASHIDA 1, Noboru MACHIDA1, Kunitoshi MITSUMORI 1
Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, Tokyo 183-8509, Japan 1[Purpose] The rasH2 mice carrying the human prototype c-Ha-ras gene have been used in many 6-monthshort-term carcinogenicity tests. In the present study, we evaluated the carcinogenic susceptibility of rasH2
mice to N-bis(2-hydroxypropyl)nitrosamine (DHPN).
[Methods] Total of 73 male rasH2 mice and 35 their wild-type littermates (non-Tg mice), 6-week old, were
given DHPN in drinking water at 0, 20 or 200 ppm, and O or 200 ppm, respectively, for 26 weeks . All
animals were subjected to a complete necropsy for histopathological examinations. The unilateral lobes ofthe lung, including normal lung tissue (0 ppm) and the whole lung tissue containing a neoplastic massinduced by DHPN (200 ppm), were frozen for RT-PCR analyses of transgene and endogenous mouse ras
gene.
[Results and Discussion] The experiment of rasH2 and non-Tg mice given 200 ppm DHPN was completed at
20 weeks, since the survival rates of these animals were remarkably decreased as compared with that in thecontrol group. Histopathologically, adenomas and adenocarcinomas of the lung and squamous cell
hyperplasias and papillomas of the trachea were frequently observed in rasH2 and non-Tg mice given 20 or
200 ppm DHPN. Hemangiosarcomas of the liver were induced with a high incidence in both mice of the
200 ppm group, but there was no induction of hemangiosarcomas of the liver in rasH2 mice of the 20 ppm
group. In addition, squamous cell hyperplasias and papillomas of the forestomach, transitional cell
hyperplasias and papillomas of the urethra, squamous cell papillomas and carcinomas of the excretory ductof salivary glands were induced in rasH2 mice in DHPN treated group. No induction of neoplastic lesions of
the thyroids and kidneys was found in rasH2 and non-Tg mice. RT-PCR analysis showed that there was nomarked difference in the expression of mRNA of the t:ransgene and endogenous mouse ras gene between thewhole lung tissue of rasH2 mice with a neoplasm and normal lung tissue in the control rasH2 mice. These
results suggest that rasH2 mice are highly susceptible to DHPN and have many tumor target organs such as
the forestomach, salivary gland and urethra, which have not been found in long-term carcinogenicity studiesof rats and mice.

JSTP/IFSTP IATP .200~

Posters

P-28
Genomic analysis of the difference between non-genotoxic carcinogen andgenotocic carcinogen in the urinary bladder
Junko HATA 1, Azusa TAMURA' , Natsuki KITAJIMA 1, Yoshinori KASAHARA1, Hiroshi UNO',
Keiichirou MORIMURA2 , Shoji FUKUSHIMA2
Pharmaceuticals Development Research Laboratories, Teijin Pharma Ltd., Tokyo 191-8512, Japan 1
Department of Pathology, Osaka City University Medical SchooF
Uracil is known to be non-genotoxic carcinogen, calculus is induced by the uracil-administration and longterm stimulation of the calculus produces the neoplastic lesion in the urinary bladder. However, N-butyl-N(4-hydroxybutyl) nitrosamine (BBN) and p-Cresidine is known to be genotoxic carcinogen. We examined thedifference of the expression levels of various mRNA in the minary bladder treated with these substances. For
the purpose of this study, we used the glass array system (Atlas Glass Array, Rat Toxicology 1.0) and realtime PCR system method. The urinary bladder was prepared from twelve F344 rats 4-week treated with 3%
uraci l in the feeding, 0.05 % BBN in the drinking water or 0.5 % p-Cresidine in the feeding, respectively.
Histopathologicaly, the papillomatosis was observed in all Uracil-treated urinary bladder, and simple
hyperplasia was observed in all of the BEN-treated. But no change was observed in the control andp-Cresidine-treated. The mRNA levels were compared and the differences in mRNA expressions between
these three compounds were detected by glass array. Although extreme differences were not detected, theincreasing mo.re than two-times or decreaasing more than half were detected. We selected several genes, andreco nfi rmed the expression level by real time PCR. The number of the gene detected the difference fromcontrol was the largest in the BBN treated group and the smallest in the p-Cresidine treated group . Someselected data of mRNA level was reconfirmed by real time PCR, and we confirmed 70% data of the Glass
Array was the same data of the real time PCR. Moreover, by using the in situ RT-PCR method, we examined
the localization of the mRNA expression of the Glutation S-transferase (GST), which detected down
regul ation in the Uracil-treated urinary bladder only in the glass array analysis. The expression was observedin the cytoplasm of the transitional cell. Especially, the signal of the normal transitional cell in control andp-Cresidine-treated was stronger than that of the papillomatosis (Uracil) and simple hyperplasia (BBN). In
conclusion, we detected the difference of the mRNA level of three compounds by using glass array and realtime PCR for the trigger of this study, and we aimed at the GST mRNA expression. Though the differenceof the gene expression between the carcinogen and non-carcinogen was not clarified regarding the GST
mRNA expression, we detected the GST mRNA in the normal transitional cell stronger than that ofpapillomatosis treated with Urasil and simple hyperplasia treated with BBN.

136

JSTP/IFSTP IATP 2004/):~

Posters

P-29
Suppurative gastritis in BALB/c mice infected with Listeria monocytogenes via
the intragastric route
Jong-Hwan PARK', Yong-Ho PARK2, Seung-Hyeok SEOK 1, Sun-A CHO', Hui-Young LEE ',Dong-Jae KIM', So-Hyun KIM 2 , Jae-Hak PARK' , Young-Soon LEE3
Department of Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University,
Seoul 151-742, Korea (South)'
Department of Veterinary Microbiology, College of Vete1inary Medicine, Seoul National University 2
Deparment of Veterinary Public Health, College of Veterinary Medicine, Seoul National University 3
Suppurative gastritis was demonstrated in BALB/c mice 3 days after intragastric inoculation with 10 9organisms of Listeria monocytogenes strain ATCC19113 (serotype 3). Also tested were four other strains ofmice (C3H, C57BL/6, FVB and ICR). After inoculation with ATCC19113 the numbers of bacteria found in
the stomach wall were greater in C57BL/6 and ICR mice than in C3H and FVB mice; moreover, the gastritis
produced in BALB/c and C57BL/6 mice was more severe than that produced in the other mouse strains The
inflammatory response occurred in the lamina muscularis and mucosa of the fundus. Massive necrosis of thegastric epithelium was observed, and oedema in a large part of the mucosa! layer of the fundus. In addition,
the submucosal layer was apparently expanded due to oedema, and in the cardia, the mucosal layer had
become thin and flattened. Immunohistochemically, a polyclonal antibody against Listeria spp. produced
labelling was observed in areas of the gastric mucosa in which there was an inflammatory response andgastric epithelial necrosis.

JSTP/IFSTP IATP ·200~

Posters

P-30
Analyses of immune response to candidate live oral Salmonella vaccine strain
in mice
Sun A CHO ', In Soo LEE2 , Jong Hwan Park PARK', Seung Hyeok SEOK 1, Hui Young LEE 1,
Dong Jae KIM 1, Jae Hak PARK 1
Department of Laboratory Science, College of Veterinary Medicine, Seoul National University,
Seoul 151-742, Korea (South)'
Department of Microbiology, Hannam University 2This study was performed to provide the guideline for evaluating the efficacy of a recombinant Salmonella
live vaccine. We examined the immunologic efficacy of a candidate typhoid vaccine strain (recombinantsalmonella typhimurium LF 878). Ten each mouse was orally administered with 105 and 106 CFU of LF878,
107and 109of S. typhi Ty2, respectively, and control with sterile PBS. Five mice of each group were
necropsied at 5 and 12 days after infection. On ELISA and RT-PCR using serum and spleen tissue,
expression of immunoglobulins and cytokines was higher in mice immunized with LF878 and S. typhi Ty2
th an in control. When treated with T- and B-cell mitogens, spleen cells of mice immunized with LF878 andS. typhi Ty2 was more proliferative than those of control. And expression of CD4+ T lymphocyte in spleensof mice with LF878 and S. typhi Ty2 also increased more than control.

138

•

•

,.s...~r

JSTP/IFSTP IATP 2004

Posters

P-31
Earlier Helicobacter pylori infection cause severer inflammation and more risk
for stomach carcinogenesis in N-methyl-N-nitrosourea treated Mongolian
gerbils
Tetsuya TSUKAMOT0 1, Xueyuan CA0 1, Masami YAMAMOT0 1, Harunari TANAKA 1,
Masae TATEMATSU 1
Division of Oncological Pathology, Aichi Cancer Center Research Institute, Aichi 464-8681, Japan 1
Purpose: Helicobacter pylori (H. pylori) is now well known to be associated with stomach carcinogenesis.
Infection during childhood rather than in adults is considered to be more important.
Methods: [Experiment l] To evaluate the difference in susceptibility to stomach carcinogenesis among
various ages to acquire H. pylori infection, we designed an experiment with inoculation of H. pylori (ATCC
43504) followed by N-methyl-N-nitrosourea (MNU) treatment at different ages. Four-week-old male
Mongolian gerbils were divided into twelve groups. H. pylori were inoculated at 4, 18 and 32 weeks old, as
representative of early, middle and late infection, respectively. Two weeks later, the animals were treated with
MNU. Groups without H. pylori and/or MNU were included as controls. [Experiment 2] To compare theseverity of inflammation in association with stomach carcinogenesis, duration of H. pylori infection was set
to long (20 weeks), intermediate (8 weeks), and short (2 weeks) terms as a model of severe, moderate, andmild inflammation before the administration of MNU at 27 weeks old. Gerbils were sacrificed at 77 weeks
old. Controls were designed as above.
Results: [Experiment l] The incidences of adenocarcinomas at 52 weeks after the inoculation in the early(H. pylori +MNU), middle (H. pylori +MNU), and late (H. pylori +MNU) groups were 60% (12/20), 18.4%(2/11), and 10% (2/20), respectively. The corresponding figures were 14.8% (4/27), 0% (0/11), and 0%(0/21) in the MNU-alone groups. A higher titer of serum IgG for H. pylori and gastrin level was seen in theearly-infected compared to the middle and the late groups (P<0.01). [Experiment 2] Anti-H. pylori antibody
and serum gastrin level were the highest in the long-term group, indicating the severest inflammation.
Incidence of adenocarcinomas were 45 % (9/20), 20% (2/10), and 23.1 % (3/13) in the long, intermediate, andshort Hp+MNU groups, respectively. The tumor incidence in the long-term group was significantly higher(P<0.05) than that in the corresponding MNU alone group (0%=0/16) .
Conclusion: The results clearly demonstrated that early acquisition of H. pylori with MNU significantly
increases gastric chemical carcinogenesis, as compared to the case with later infection, possibly because ofdifferences in host gastric mucosal factors, immunologic responses, and subsequent inflammation.

139

JSTP/IFSTP IATP 200~

PostersP-32
Increased oxidative stress associated with alterations in DNA damage andrepair in urinary bladder carcinomas associated and non-associated with
Schistosomiasis
Elsayed I. SALIM 1, Hideki WANIBUCHI 1, Amani ABDUL-HAMID 2, Keiichirou MORIMURA 1,
Shoji FUKUSHIMA 1
Department of Pathology, Osaka City University Medical School, Osaka 545-8585, Japan'
Department of Pathology, Tanta Cancer Institute2
To cast light on the underlying mechanisms predisposing Egyptian urinary bladder carcinomas associated andnon-associated with Schistosomiasis to high grades of aggressiveness, we analyzed immuno-histochemicallythe relationship between oxidative stress markers, DNA single strand breaks (ssDNA) and DNA repair genes
in function with nitric oxide synthases expression levels in bladder cancers of 36 Egyptian cases with orwithout Schistosoma infection. Marked elevated levels of 8-hydroxy-2'-deoxyguanosine (8-0HdG) were
found in SCCs and TCCs associated with Schistosomiasis as compared with non-Schistosomal TCCs. This
was accompanied by strong overexpression of inducible nitric oxide synthase (iNOS), ssDNA marker, andDNA damage repair genes; the 8-oxoguanine-DNA-glycosylase (OGGl) and apirinic/apirimidinic
endonuclease (APE-1) in Schistosomal carcinomas. However, expression of the endothelial nitric oxide
synthase (eNOS) was slightly stronger in non-Schistosoma-associated than the Schistosoma-associatedcancers. In conclusion, these findings suggest a strong correlation between Schistosoma haematobium
infection in Egyptian patients, and explains mechanisms of induction by increased levels of oxidative stressaccompanied by a continuous DNA damage in urothelium followed by a subsequent overexpression of DNA
repair genes, all together may function in a possible correlation with increased levels of iNOS in
Schistosomal bladder cancers.

JSTP/IFSTP IATP 2004~1~

Posters

P-33
Roles of the Thai liver fluke, Opisthorchis viverrini, in the development ofcholangiocellular carcinoma (CCC).
Witaya THAMAVIT 1, Tomoyuki SHIRAI2, Malcolm A. MOORE 2 , Nobuyuki IT0 2
Department of Pathobiology, Faculty of Science, Mahidol University 1
Department of Pathology, Nagoya City University, Medical School2
Association of CCC development and 0.viverrini infection has been well known in the Thai Northeast. In
experimental animals that received combination of the liver fluke and dimethylnitrosamine (DMN) developed
100% CCC. Animals with single DMN-initiated dose and subsequent infection by the liver fluke also
developed CCC 44% of animals with other bile duct precancerous lesions ie. cholangiofibrosis, mucinous
cystadenoma etc. in almost all the animals. Whereas, no CCC development were detected in animals monthly
treated for 10 times with 50, 25 and 12 metacercariae in both sexes and left for one year. The results
indicates promoting roles of the parasite in causing chronic cell death and cell profiferation of bile ductepithelium which persist as long as the presence of the parasite of which its lifespan is not less than 25
years. The histopathological lesions that imply cell death and cell proliferation of bile ducts are: egg andparasite granulomas, hyperplasia of main ducts, cholangiectatic cyst of bile duct resulting in partial
obstruction of larger duct and bile ductule proliferation as a consequence, and mechanical damage of bile
duct due to movement of the parasite etc. In the Thai Northeastern people, children received infection via
mother feeding of infected raw fishes at the age of two month old. Continuous induction of bile duct cell
death and cell proliferation creates fertile soil for initiation and subsequent promotion which perpetuates forlife reflecting coinitiator and promoter roles of the parasite. In Thailand, initiators of carcinogens are
believed to contaminate widely in foods and foodstuffs and everywhere. These reasons might explain world
highest incidence of CCC in that region".

JSTP/IFSTP IATP-. 2"()0~

Posters

P-34
In vitro and in vivo sensitivity of various cells to arsenic trioxide(As203)
Chang Suk KANG 1, Yeon Sook MOON 1, Yong Gu KIM 1, Young Jin CHOI1, Kyung Ja HAN 1,
Sang In SHIM 1
Department of Clinical Pathology, Catholic University of Korea, Seoul 150-713, Korea (South) 1
Recently, inorganic arsenic trioxide (As203) was reported to induce complete remission in many patientswith refractory acute promyelocytic leukemia (APL) and its growth inhibitory effects on several lymphoidneoplasms and solid tumors, in vitro, has been studied. However, the effect of As203 on cancer cells frommalignant patients is not established. especially, in vivo. We assayed arsenic sensitivities on Lo Vo, OVCAR3 and PA-1 cell lines and cancer cells obtained from patients with peritoneal carcinomatosis. And weevaluated the therapeutic effects of As203 on mice implanted cancer cell line. We cultured the Lo Vo,
OVCAR-3 and PA-1 cell lines in media with variable As203 concentration for 24 and 48 hours. The
detection methods for apoptotic or damaged cells were flow cytometry using annexin V-FITC/PI, terminal
deoxynucleotide transferase (Tdt) -mediated dUTP- digoxigenin nick-end labeling (TUNEL), trypan blue dye
exclusion test, acridine orange/ethidium bromide (AO/EtBr) uptake test, morphologic examination after
Wright staining, and electrophoresis using 1% agarose gel for DNA ladder pattern identification. The cancer
cells from patients are incubated by same process for 24 hours and 48 hours and measured percentage ofdamaged cells by trypan blue dye exclusion method. To find out in vivo therapeutic effect of As203, we
introduced the PA-1 cells into the peritoneal cavity of mice and injected As203 into peritoneal cavity of thetumor implanted mice. Then, we weighed the mice (group I; peritoneal carcinomatosis group without As203
injection, group II; As203 injected group after peritoneal carcinomatosis developed, group III; PA-1 cells
and As203 injected group at first time) and analyzed macro- and microscopic findings of tumor masses andsurvival rate between group II and group III. Among the above mentioned methods, Trypan blue dye
exclusion was the most useful and reproducible method for As203 susceptibility test, and PA-1 cell line wasmost susceptible. Ladder pattern was not observed in DNA electrophoresis of As203 exposed cell lineds. Of
eight peritoneal carcinomatosis cases, two in Sµmol/L , four in lOµmol/L, seven in SOµmol/L weresusceptible. In vivo study, group III developed peritoneal carcinomatosis more lately, and lived longer thangroup I and IL Conclusively, the As203 sensitivity was variable in each cancer cell lines and cancer cells
from patients. The As203 injection could delay development of peritoneal carcinomatosis in the early stageof peritoneal seeding. The cell death effect of As203 exposure might be result from not apoptosis induction
but other mechanism.

142

JSTP/IFSTP IATP-2004

Posters

P-35
Induction of glutathione S-transferase placental form positive foci in liver andepithelial hyperplasia in urinary bladder, but no tumor development in maleFischer 344 rats treated with monomethylarsonic acid for 104 weeks
Jun SHEN' , Hideki WANIBUCHI 1, Elsayed SALIM', Kenichiro DOI', Min WEI', Shoji FUKUSHIMA'
Department of Pathology, Osaka City University Medical School, Osaka 545-8585, Japan'
The carcinogenicity of monomethylarsonic acid (MMA(V)), a major metabolite of inorganic arsenics inhuman and experimental animals, was investigated in male Fischer 344 rats. A total of 129 rats at 10 weeks
of age were randomly divided into three groups and received drinking water containing MMA(V) at doses of
0 (Control), 50 and 200 ppm ad libitum for 104 weeks. No significant differences were found between thecontrol and MMA(V)-treated groups regarding clinical signs, mortality, hematological and serum
biochemistry findings. Quantitative analysis of glutathione S-transferase placental form (GST-P) positive fociin liver revealed a significant increase of numbers and areas in the 200 ppm MMA(V)-treated group. In theurinary bladder MMA(V) induced simple hyperplasia, and significantly elevated the proliferating cell nuclear
antigen (PCNA) positive index in the urothelium. A variety of tumors developed in rats of all groups,
including the controls, but all were histologically similar to those known to occur spontaneously in F344 rats
and there were no significant differences among the groups. Thus, it could be concluded that, under thepresent experimental conditions, MMA(V) induced lesions in the liver and urinary bladder, but did not cause
tumor development in male F344 rats even after two years exposure.

JSTP/IFSTP IATP 2004

Posters

P-36
Effects of sodium cacodylate and sodium nitrate on kidney tumor induced bydimethylnitrosamine in SD male rats.
Tsutomu HIBIN0 1, Takamasa YANAGIDA2, Junya MATUYAMA 1, Ayumi KATI 1, Naoki YAMAMOT0 2
Department of Pathology, Fujita Health University College, Aichi 470-1192, Japan 1
Joint Research Laboratory, Fujita Health University 2This study examined the effects of sodium cacodylate as an arsenic compound and sodium nitrate as a foodadditative on kidney tuomorigenesis induced by dimethylnitrosamine (DMN) in rats. Male Sprague-Dawley
rats, 6 weeks olds at the start of the experiment, were divided into experiment 1 and 2. A single dose of 40
mg/kg DMN were dissolved in 0.5 ml of saline, were intragastrically intubated. One gram/liter Sodium
cacodylate (SC) and ten grams/liter sodium Nitrate (SN) were mixed in drinking water respectively and wereadministered ad libitum. Experiment 1: Group 1 (16 rats) was gavaged DMN and 1 week later, wasadministrated SC. Group 2 (10 rats) was gavaged DMN alone. Group 3 (14 rats) was gavaged saline and SC.
Group 4 (10 rats) was control. At 40 weeks, the incidence of renal cell carcinoma (RCC) was in 13 of 16
rats (8 1.3%) of group 1, and in 5 of 9 rats (55.6%) of group 2. Experiment 2: Group 1 (15 rats) was treatedwith DMN and SN. Group 2 (15 rats) was gavaged DMN alone. Group 3 (10 rats) was administered SN
alone. Group 4 (10 rats) was controls. At 40 weeks, the incidence of RCC was in 15 of 15 rats (100%) of
gro up 1, and 14 of 15 rats (93.3%) in Group 2. We concluded that sodium cacodylate were promoted theincidence of renal cell carcinoma induced by a single dose of DMN in male Sprague-Dawley rats, but
sodium nitrate was not promoted.

144

JSTP/IFSTP IATP 20021

Posters

P-37
Involvement of choline and amino acid metabolism in different
hepatocarcinogenicity of L-amino acid-defined semisynthetic and semipurified
choline-deficient diets in rats
Maki IGARASHI1 , Masakazu TAKAHASHI2, Hitoshi ASHIDA4, Mei-Heng MAR5, Yutaka HATANAKA4,
Midori YOSHIDA2, Fumiyuki UEMATSU2, Naoto WATANABE', Akihiko MAEKAWA3, Steven H ZEISEL5,
Dai NAKAE2
Laboratory of Protection of Body Function, Department of Food and Nutritional Science, Graduate school of
Agriculture, Tokyo University of Agriculture, Tokyo 101-0062, Japan 'Department of Pathology, Sasaki Institute, Sasaki Foundation2
Director, Sasaki Institute, Sasaki Foundation 3
Division of Life Science, Graduate School of Science and Technology, Kobe University 4
Department of Nutrition, School of Public Health and School of Medicine, University of North Carolina at
Chapel Hill5

Introduction : Choline is a vitamin that is involved in a wide variety of physiological regulations. Alteredbioavailability of choline thus causes various pathological conditions. For instance, its dietary deficiency ishepatocarcinogenic in rats. In order to clarify its underlying mechanisms, we developed a L-amino aciddefined semisynthetic choline-deficient (CDAA) diet and found its much stronger carcinogenicity than aconventional, semipurified choline-deficient (CD) diet containing proteins with an equivalent amino acidprofile to the CDAA diet. To assess factors involved in this phenomenon, the present study compared plasmaand hepatic levels of choline derivatives and amino acids in male Fisher 344 rats given a short-term feedingof the CDAA or CD diets.
Methods : A total of 30 male Fisher 344 rats were used and equally divided into 5 groups at their age of 7
weeks old. Group 1 received a basal laboratory diet. Groups 2 and 3 received a semipurified, cholinesupplemented (CS) diet and the CD diet, respectively. Groups 4 and 5 received a L-amino acid-defined
semisynthetic, choline-supplemented (CSAA) diet and the CDAA diet, respectively. At the end of a 4-week
feeding, all rat were killed, and the and heparinized plasma and liver were collected. Plasma and liver levelsof choline derivatives; the choline, phosphatidylcholine, betaine, sphingomyeline, glycerophophocholine andphosphocholine; were analyzed by a liquid chromatography-mass spectrometry. Plasma and liver levels ofamino acids were measured by a liquid chromatography.
Results and discussion : Both the CDAA and CD diets decreased levels of all determined choline
derivatives, reflecting insufficient choline availability, and increased levels of almost all amino acids,suggesting enhanced protein degradation and diminished amino acid utility, either in plasma or livers. In
particular, the increase of an essential amino acid cysteine, urea cycle members urea and ornithine, andfibrosis-related praline may correspond to hepatotoxicity induced by the diets. While the changes of choline
derivative levels occmTed in a similar extent for the by CDAA and CD diets, the changes of amino acidlevels were substantially more prominent in the CDAA case than in the CD case . These results suggest thatthe different amino acid metabolism, but not the similar choline metabolism, may be involved in the different
hepatocarcinogenicity in between rats fed the CDAA diet and these feed the CD diets.

JSTP/IFSTP IAT"i1~200"1

Posters

P-38
Induction of fatty liver by mebalonic acid in Fischer 344 rats
Naoto WATANABE', Midori YOSHIDA 2 , Maki IGARASHI 1, Fumiyuki UEMATSU2,
Masakazu TAKAHASHF, Akihiko MAEKAWA3, Dai NAKAE2
Department of Food and Nutritional Science, Laboratory of Protection of body function, Graduate School ofTokyo University of Agriculture, Tokyo 101-0062, Japan 1
Department of Pathology, Sasaki Institute, Sasaki Foundation2
Department Director, Sasaki Institute, Sasaki Foundation 3
Purpose Mebalonic acid (MA) is used as a food additive or an integrant of cosmetics, but its safety
evaluation including a repeated toxicity remains undetermined. In the present study, therefore, we conducteda 13-week oral toxicity study to evaluate subchronic oral toxicity and its reversibility for MA in rats.
Materials and methods Groups each consisting of 15 male or female Fisher 344 rats were administered MA
at concentrations of O (a basal diet only), 400, 2,000 and 10,000 ppm in diet from their 6 weeks of age for
13 weeks. At this time-point, ten each of animals were sacrificed, and the remaining 5 each of animals were
switched to the basal diet for additional 4 weeks. After terminations of the treatment or recovery periods, theblood was collected from all animals for the hematological and blood biochemical analyses. After a complete
necropsy, all organs and/or tissues were fixed in a 10% neutrally buffered formaldehyde solution androutinely processed for the histopathological examination.
Results No clinical abnormalities or no death attributed to the MA treatment were observed in any animals.
The body weight gain of the highest dose groups in either sex was slightly but significantly depressed at thelast half of the MA-treated period, whereas the depression was abolished during the recovery period. Foodconsumptions in all treated groups were comparable to those in the control groups. At the necropsy, thelivers of the highest dose group in either sex showed fatty-liver appearance, and the changes
histopathologically correlated to fatty changes of the hepatocytes in the centrilobular zone with a slightaggregation of inflammatory cells. Also in the highest dose groups, the ALT activity was increased withslight anemia for both sexes, while the total cholesterol and triglyceride levels were increased for only
females. After the recovery period, all of such histopathological, hematological and blood biochemicalchanges were almost reversed. There were no treatment-related changes observed in the other dose groups ofeither sex.
Discussion and conclusion These results indicate that a 13-week treatment of 10,000 ppm of MA induces
fatty change of the liver with its related blood biochemical and hematological changes in male and female
rats. The depression of the body weight at the highest dose is also considered treatment-related. All thesechanges are, however, reversible after a 4-week recovery period.

146

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..

-~-

JSTP/IFSTP IATP 2004

Posters

P-39
Induction of multiple granulation in the liver with severe hepatocyte damage
by montan wax, a food additive, in a 90-day toxicity study in F344 rats
Mico IKEDA', Kousuke SA00 1, Keiko YAMAKAWA', Hijiri TAKEUCHI', Masanao YOKOHIRA 1,
Yoko HOSOTANI1, Yu ZENG', Kyoko HOSOKAWA 1, Shigemi KINOUCHF, Katsumi !MAIDA'
Department of Onco-Pathology, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan'
Diagnostic Pathology and Cytology Institute, Shikoku Cytopathologic Institutes 2
Montan wax is a mineral wax which is extracted from lignite formed by the decomposition of vegetable
substances. As it is hard and resistant to decomposition, when used as an ingredient in polishes and waxes, it
imparts scuff resistance, water repellency and a high gloss. It has many other applications, including its use
in emulsions, as an asphalt additive, in casting and leather finishing and as an additive in paper making andlubricant manufacture. Montan wax has also found employment as a food additive. As a part of the safetyassessment of montan wax, we have performed a 90-day toxicity study in Fischer 344 rats.
Groups of 10 males and 10 females were given the material at dose levels of 0, 0.56, 1.67 or 5 % in the dietfor 90 days. Clipical signs were noted once a day and body weights and food intakes were measured once a
week. During the experiment, there were no remarkable changes in general appearance and no deaths
occurred in any group. All animals were sacrificed after 90 days treatment. Hematological and serum
biochemical examinations as well as necropsies were performed at the end of the experiment, when blood
samples were collected after 16 hours of starvation. On hematological examination, Hb, Ht, MCV and MCH
were significantly decreased and WBC was significantly increased in all treated rats. On serum biochemical
examination, AST and ALT were found to be elevated more than four fold in all treated groups as comparedto the control group in both sexes. Furthermore, significant increases of total cholesterol, platelets andpotassium were also observed in all treated rats in all treated rats. Relative organ weights of liver, spleen,lung and kidneys in all treated groups of both sexes were increased. Histopathological observation revealed
diffuse multiple microgranulation in the liver with severe hepatocyte damage and lymphocytic infiltration.
Granulomatous lesion were also apparent in the mesenteric lymph nodes in all treated males and females .
These findings clearly demonstrate that montan wax, at doses of more than 0.56 % in the diet can induce
severe granulomatous inflammation in the liver. Because of pathological, hematological and serum
biochemical changes were observed in the lowest dose group, a no-observed-adverse-effect level (NOAEL)
could not be determined in a present study.

JSTP/IFSTP IAT:r·· :mo~Posters

P-40
Dietary intake of various lactic acid bacteria suppresses Type 2 helper T cell
production in antigen-primed mice splenocyte
Hui Young LEE1, Jong Hwan PARK 1, Seung Hyeok SEOK 1, Sun A CH0 1, Min Won BAEK 1,
Dong Jae KIM 1, Yong Soon LEE 2 , Jae Hak PARK 1
Department of Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University,
Seoul 151-742, Korea (South) 1
Department of Public Health, College of Veterinary Medicine, Seoul National University, Seoul, Korea2
Lactic acid bacteria (LABs) have been proposed as a potential oral allergy-therapeutic mean of modulating
immune phenotype expression in vivo, via promoting or reducing cytokine production. This study
investigated the ability of LABs to suppress allergic response via modulating cytokine production in mice
splenocytes. BALB/c mice were intraperitoneally primed to ovalbumin in alum adjuvant to invoke antigenspecific Thlffh2 cytokine-secreting cell populations in splenocytes. Spleen cells from mice fed with
Lactobacillus confusus PL9001(KCCM-10245), L. fermentum PL9005(KCCM-10250), L. plantarum PL9011(KCCM-10358) and Bifidobacterium infantis PL9506(KCCM-10406) during antigen sensitization suppressed
the levels of Th2 cell cytokines such as IL-4 and IL-5. And all mice fed with LABs induced Thl cell
cytokines such as IL-2 secretion by splenocyte. Results suggested that these LABs are anti-allergic agents, inthe view of the Thl/anti-Th2 immunoregulation.

148

JSTP/IFSTP IATP 2002i

PostersP-41
Examination of heat shock protein 70 as an early-stage injury-biomarker in rat
testes after stress-induced cell damage
Akiko IKEDA 1, Yoshihumi KANEK0 1, Maldko TAKAHASHI 1, Sumihisa SUEYOSHI 1,
Yoshihiro MASUMOT0 1, Kiyoyuki TSURU 1
Kyo1in Phamaceutical Co. , Ltd, Research Center, Tochigi 329-0114, Japan 1
Heat shock proteins (HSPs) are increased at an early stage of injury associated with stress conditions such as
hyperthermia, ischemia, UV radiation, oxidative stress, and chemicals. Heat shock protein 70 (HSP70), oneof such proteins, may serve as a sensitive biomarker for cell damage. Thus, this study was aimed to examine
whether HSP70 would be used as an injury-biomarker for testes by determining the time-courses for HSP70
and apoptosis after cell damaging. Cell damages were induced by surgical crytorchidism or administration ofhuman chorionic gonadtrophin (hCG). Surgical cryptorchidism has been frequently used to study germ cell
apoptosis due to heat stress. Administration of hCG is known to induce precapillary vasoconstriction
resulting in cell damage.
In cryptorchidism with Wistar rats, the day of surgery was designed as day 0, and the testes were removed 4
h, 8 h, 24 h, and 3 days, 5 days, 7 days and 14 days after surgery. In administration of hCG, Wistar rats
were injected s.c. with 100 i.u. hCG, and then sacrificed to remove the testes 4 h, 8 h, 24 h and 2 days after
injection. The levels of HSP70 expression and apoptosis in spermatocytes were detected byimmunoperoxidase staining and by TUNEL, respectively.
In cryptorchidism, HSP70 was rarely observed in pachytene spermatocytes and round spermatids at O h after
surgery. HSP70 increased in pachytene spermatocytes from 4 h after surgery, and peaked 8 h after surgery.
The increase in HSP70 preceded germ cell apoptosis, which tended to increase from 24 h after surgery.
Germ cell apoptosis was noted in the majority of tubules from 3 days after surgery, and the apoptotic cells
peaked 5 days after surgery. Staining with periodic acid shiff showed the presence of degenerating germ cells
in almost all tubules from 3 days after surgery. Similarly, administration of hCG increased the HSP70
expression, which preceded germ cell apoptosis in pachytene spe1matocytes. HSP70 increased at 8 h, andpeaked at 24 h after administration. In the both types of cell damage induction, HSP70 was temporally
correlated with germ cell apopstosis despite some time lag each other. In conclusion, HSP70 increased
earlier than germ cell apoptosis in pachytene spermatocytes under stress conditions both in surgicalcryptorchidism and administration of hCG. These results suggest that HSP70 may serve as an early injurybiomarker for rat testes.

JSTP/IFSTP IATP ,_200fl

Posters

P-42
Histopathological and endocrinological changes of cardiac natriuretic peptide
in rats with drug- induced cardiotoxicity
Hiroto MIYATA1, Rie OHN0 1, Rika SHIRANE 1, Yutaka NAKANISHP, Atsushi NAKAMURA1,
Masaaki KIMURA 1
Toxicology Laboratory, Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd.,
Saitama 331 -9530, Japan 1
Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are circulating hormones secreted fromheart predominantly in cases of hypertension or congestive heart failure. We have previously reported on therelationship between these hormones in plasma and cardiac injury in rats. In the present study, in order toconfirm whether ANP and BNP levels can be appropriate indicators in evaluation of cardiac injury, weinvestigated the relationship between the plasma or heart levels of these hormones and heart injury in rats
given isoproterenol (ISO) to induce cardiac injury. Eighty eight male Crj; CD (SD) IGS rats (10 weeks old)
were given a single intravenous injection of ISO. Blood and heart samples were collected 0.5, 2, 4, 8, 48
hrs, 7 and 14 days after the dosing. Six hearts per group were used for histopathological investigations. Eachspecimen was fixed in 10% neutral phosphate-buffered formalin, dehydrated in absolute alcohol, andembedded in paraffin wax. 4µm-thick sections processed from these paraffin blocks, were stained withhematoxylin and eosin (H&E) or used for immunohistochemical studies using anti-ANP or anti-BNP
antibody. Measurements of plasma and heati concentration were perfomed by radioimmunoassay. Myocardial
degeneration or necrosis with inflammatory cell infiltration was observed in the cardiac ventricle 4 hrs after
the dosing. At 48 hrs there was myocarditis with neutrophil and mononuclear cell infiltration in the ventricle,
more extensively than at earlier points. In some specimens, fibrosis had formed in the myocarditis 4 days
after the dosing. BNP positive cells were recognized in myocardial degenaration part or its circumference 4days after the dosing. ANP levels in plasma and heart concentration did not change after ISO dosing.
However, BNP levels in plasma significantly increased 0.5-48 hrs after the dosing, peaking 4 hrs after thedosing. BNP levels in cardiac atrial measurement increased 7 days after the dosing while those in the cardiac
ventricle significantly increased 2-8 hrs after the dosing. These data indicate that peak BNP levels in plasma
parallel those of the cardiac ventricle, revealing that they reflect histopathological alterations of the heart.
These results suggest that plasma ANP and BNP levels can be useful indicators of the location of injuries in
the heart.

- .

··-~--JS TP/IFS TP IATP 2004

Posters

P-43
Histomorphometrical parameters for evaluation of osteocompatibility of metal
implants with undecalcified bone section
Miki NISHIMORI 1, Shin-ichi KATSUDA1, Yoshimitsu OKAZAKI2, Emiko GOT03, Akihito SHIMOI 1,
Hidetaka SAT0 1
Japan Food Research Laboratories, Tokyo 206-0025, Japan 1
National Institute of Advanced Industrial Science and Technology 2
National Institute of Technology and Evaluation 3
We evaluated histocompatibility of alloys such as Co-CrF-75, Ti6-4ELI and Ti-15Zr by comparing
osteogenesis histomorphometrically on undecalcified bone section. Alloys were implanted into femoral andtibial bones of male Wistar/ST rats for 1, 3, 6, and 12 months. At the end of each implantation period, bones
including implants were excised and polyester-resin-sections were ground to approximately 70 to 80
micrometers thick. They were stained with Villanueva bone stain, Toluijine blue stain or Giemsa stain, andobserved with light and/or fluorescent microscopes. For histomorphometry, (1) thickness of new bonesurrounding the surface of the implant, (2) rate of bone contact with the implant, (3) rate of perimeter of
new bone, (4) rate of osteoid and calcified bone in new bone were measured with a quantitative television
microscope. (1) Thickness, (2) rate of bone contact, and (3) rate of perimeter were increased from 1 to 6
months in all alloys. However, these factors were constant after 6 months. The calcified new bonesurrounded all alloys, (4) the rate of calcified bone being more than 80 % of the new bone in 1 month. At Ti-15Zr implanted sites, rates of osteoid were approximately 10, 6, 6, 3 % in 1, 3, 6 and 12 months,
respectively. At Co-CrF-75 and Ti6-4ELI implants, rates of osteoid were approximately 5 % in 3 months.
However, the rates of osteoid were increased up to approximately 10 % after 6 months. From these finding,Ti- l 5Zr appeared to show good osteocompatibility and osteoconductivity, and histomorphometricalmeasurements of the relation between new bones and implants were suggested to be good parameters forosteogenesis with undecalcified bone section.

JSTP/IFSTP IATP·, :too~

Posters

P-44
Genetic polymorphisms of alcohol-related metabolizing enzymes and the riskof esophageal cancer in the Thai population
Pleumjit BOONYAPHIPHAT 1, Pa.ramee THONGSUKSAI 1, Wanna SUDHIKARAN 1,
Puttisak PUTTAWIBUL2
Department of Pathology, Faculty of Medicine, Prince of Songkla University, Songkla 90110, Thailand 1
Department of Surgery, Faculty of Medicine, Prince of Songkla University 2
The association of life style habits and polymorphism of ADH2 , ALDH2 and CYP2El genes with the risk ofesophageal squamous cell carcinoma in the Thai population was investigated in a hospital-base case-control
study: l 63cases and22 lcontrols. The results of multivariate logistic analysis revealed that alcohol
consumption>40g/day;tobacco smoking> lOcigarettes/d and betel chewing>Squids/day increased the risk
significantly(odds ratio(OR)7 .75,95 %confidence interval(CI)3.65-16.44;2.72,95%CI1 .10-6.68and2.3695%CI
l.25-4.44respectively).The associations between individual gene and the interaction of genes andenvironment on the risk of cancer were not found. Interestingly, a significant gene-gene interaction wasobserved. Individual with CYP2E l(c 1/c2)and ALDH2(2*/2*)had the risk6.27folds(95%CI1.96-20.01).In
conclusion, these results demonstrate that alcohol is a strong risk factor for esophageal cancer. Furthermore,
the combined genotype of mutant CYP2E land ALDH2is also a significant risk factor for esophageal cancer
in our population.
This work was supported by a grant from Prince of Songkla University

Posters
P-45
Toxicogenomics using "percellome" and "mille-feuille" data system
Jun KANNO 1, Ken-ichi AISAK.1 1, Atsushi ON0 1, Katsuhide IGARASHI 1
Cellular & Molecular Toxicology Division, Biological Safety Research Center, National Institute of Health
Sciences, Tokyo 158-8501, Japan'
The whole genome information for humans and rodents, and technology for monitoring whole genome
expression will enable us to perform the comprehensive profiling of transcriptome. The major characteristicsof this profiling are that the overt phenotypes are not the primary factors for the construction of toxicology
database/informatics. This "gene first, phenotype second" approach named "Phenotype-Independent
Toxicology" will compile the future predictive toxicogenomics database in combination with the current
toxicology based on "phenotype first, gene second" strategy.
To build up such database, multiple experiments must be conducted and data collected over a certain periodof time. Commonly, DNA microarray data are normalized relatively against mRNA concentration of certain
housekeeping gene(s) or total rnRNA quantity, or other empirical baselines that are considered to beuniversal among samples. Although such relative measurements are satisfactory for smaller numbers of genesthat are changing drastically, smaller changes in many genes are not well analyzed. To overcome these
problems, here we introduce a system that will absolutize the gene expression values in a "per one cell"basis ("percellome") . Once absolutized, the data from each samples and studies can be compared without
further normalization. This system is made primarily for Affymetrix GeneChips, but can be expanded toother platforms and RT-PCR, as long as they fulfill the demands of this system. This "percellome"
absolutization will enable us to express data in a linear scale from zero, thus generating "mille-feuille" datausing virtually all gene data generated by the microarray.
The "percellome" will facilitate the analysis of low expression genes including those of knocked out genes ingene knock out mice. Unsupervised clustering is much easier for this type of data than commonly used ratio
data against concurrent controls. As a whole, this system will contribute to the predictive toxicology by thedevelopment of a high-precision large-scale database/informatics.

JSTP/IFSTP IATP~200~

Posters

P-46
Microarray analysis of amplified RNA from laser- microdissected GST-P
negative hepatocellular preneoplastic lesion induced by peroxisome
pro/iterators.
Tokuo SUKATA1, Satoshi UWAGAWA 1, Keisuke OZAKI 1, Kayo SUMIDA1, Masahiko KUSHIDA1,
Kaoru KIKUCHI3, Koichi SAITO 1, Kenji OEDA1, Yasuyoshi OKUNO 1, Nobutoshi MIKAMI 1,
Shoji FUKUSHIMA2
Environmental Health Science Laboratory, Sumitomo Chemical Co. , Ltd., Osaka 554-8558, Japan'
Department of Pathology, Osaka City University Medical School2
Sumitomo Pharmaceuticals Co., Ltd. 3
In the present study, we tried to obtain a gene expression profile, which is specific for glutathione-Stransferase placenatal form (GST-P) negative foci induced by peroxisome proliferator (PP), by microarray
analysis of micro-lesions. GST-P negative foci were induced using the initiation-promotion model. Study 1: 6week-old, F344 male rats were given a single i.p. injection of diethylnitrosamine (DEN, 200mg/kg) at thestart of the experiment and subjected to two-thirds partial hepatectomy at week 3. Two weeks after the
initiation by DEN, Wy-14,643 and clofibrate were fed to rats for 10 weeks at doses of 0, lOOOppm (Wy14,643) and 3000ppm (clofibrate), respectively. Study 2: one week after the i.p. injection of DEN (lOOmg/kg) once a week for 2 weeks, clofibrate was fed to 6-week-old, F344 male rats for 26 weeks at dose of 3000
ppm. GST-P positive (induced by DEN alone and DEN+clofibrate in Study 1) and GST-P negative foci(induced by DEN+ Wy-14,643 in Study 1 and DEN+clofibrate in Study 2), and the adjacent normal tissues
were microdissected using laser microdissection system. Subseq uently, a slight amount of total RNA was
isolated, linear- amplified, and analyzed using gene tips. The genes were filtered with a minimum standardof at least a 1.5-fold change in expression level compared to the adjacent normal tissue. As the results, thenumbers of up-regulated genes were 175 in Wy-14,643 and 260 in clofibrate, and the numbers of downregulated genes were 30 in Wy-14,643 and 190 in clofibrate. In the up- regulated genes, 18 genes werecommon in the GST-P negative foci induced by the PPs (EST, 7; cell growth, 2; protease, 2; metabolism, 1;
signal transduction, 1; receptor, 1; membrane protein, 1; the others, 3), and only one gene was specific for
GST-P negative foci (not expressed in GST-P positive foci) among them. On the other hand, in the downregulated genes, 8 genes were common in the GST-P negative foci induced by the PPs (metabolism, 4;
genetic stability, 1; steroid hormone synthesis, 1; EST, 1; the other, 1), and none of them were specific for
GST-P negative foci. Furthermore, fluctuation of these GST-P negative foci susceptibility genes generally got
to be more distinct in hepatocellular adenoma. These lesion-specific analysis will be useful to elucidate therelation between morphological change and fluctuation of gene expressions.

154

.

~

...

..:.-~

JSTP/IFSTP IATP 200~

Posters

P-47
Specific differences in gene expression profile revealed by cDNA microarray
analysis of glutathione S-transferase placental form (GST-P)
immunohistochemically positive rat liver foci and surrounding tissue
Shugo SUZUKI1, Makoto ASAMOT01, Kazunari TSUJIMURA 1•2, Kumiko OGAWA',
Mitsuru FUTAKUCHI 1, Tomoyuki SHIRAI'
Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of
Medical Sciences, Aichi 467-8601, Japan'
Chemicals Evaluation and Research Institute, Chemicals Assessment Center2
The glutathione S-transferase placental form (GST-P), one of the glutathione S-transferase family of
detoxification enzyme, is a very useful marker of rat liver preneoplastic lesions. We here investigated thegene expression profile in GST-P positive foci as compared with sun-ounding GST-P negative areas in thesame livers of rats . For induction of the GST-P positive foci, seven-week-old male rats received an
intraperitoneal injection of diethylnitrosamine at a dose of 200 mg/kg b.w. for initiation, and two weeks later,
they were administered basal diet containing 100 ppm 2-acetylaminofluorene for 4 weeks. The rats were
subjected to two-thirds partial hepatectomy at the end of experimental week 3. All surviving animals were
killed under ether anesthesia at week 6. For the purpose of extracting RNA from prepared GST-P
immunostained tissue, liver frozen sections were fixed in ice-cold acetone, and treated with rabbit anti-rat
GST-P antibody and secondary antibody at 4 °C with RNase inhibitor. The sites of peroxidase binding were
demonstrated with diarninobenzidine at room temperature. After the immunostaining of frozen tissue,
microdissection was perfonned using the AS LMD system. Total RNAs were extracted to allow gene
expression profiles to be assessed by cDNA microru.Tay technology. As a result we found 37 genes to be upregulated and 10 genes to be down-regulated in the GST-P positive foci. All these genes were found to be
associated with metabolism in the biological process category, and catalytic activity in the molecular function
category. The results provide for an important role of metabolism in cai·cinogenesis included GSTs, GCS ,aldo-keto reductases, glutathione synthetase and P450s. Transaldolase, rat aflatoxin Bl aldehyde reductase(AFAR) and gamma-glutamylcysteine synthetase (GCS) were found as up-regulated genes and regucalcin as
a down-regulated gene, in line with findings for hepatocellular carcinomas. The results indicate that theapproach adopted is useful for understanding mechanisms of hepatocarcinogenesis and identification of newmarkers for rat liver preneoplastic foci.

JSTP/IFSTP IATP 200~

Posters

P-48
Potential for the prediction of carcinogenicity by gene expression profile in rat
hepatoma cells and comparison of expression patterns between chemicallytreated rat and human hepatoma cells
Kazunari TSUJIMURA1 ·2, Makoto ASAMOT0 1, Shugo SUZUKI 1, Shingo INAGUMA1, Kumiko OGAWA',
Tomoyuki SHIRAI'
Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of
Medical Sciences, Aichi 467-8601, Japan 1
Chemicals Evaluation and Research Institute, Chemicals Assessment Center2[Object] The long-te1m rodent bioassay is the standard method to predict carcinogenic hazard of chemicals
for human beings. However, this assay takes a long time and high costs. Recently, gene expression profiling,with the use of microarray technology, has be increasingly contributing to an improved understanding of
global, coordinated cellular events under a variety of conditions. In the field of toxicology, this approach has
been also applied to assess the toxicity of unknown compounds, in the field of "Toxicogenomics".
Assessment using cell lines is simple and reproducible and our aim has been to develop a rapid and reliableprediction method for carcinogenicity based on microarray technology in cultured cells.
[Materials and Methods] We selected 39 chemicals that have been well characterized for carcinogenicity. Rat
hepatoma cells (MHlCl) and human hepatoma cells (HepG2) were treated with the chemicals for three days
at a non-toxic dose. Gene expression changes were analyzed using our in-house microarray for MHlCl cells
and the AceGene microarray (Hitachi soft ltd.) for HepG2. Genes for prediction of carcinogenicity wereselected with statistical methods and analyzed by clustering analysis, with a support vector machine (SVM),
that is supervised machine learning classifiers. As a test set, additional 6 chemicals underwent prediction ofcarcinogenicity based on the results of the SVM analysis and comparison with actual findings . Twenty-fourof 39 chemicals were compared for expression responses in MHlCl and HepG2 regarding ortholog
significant genes using the Homologene database.
[Result] A significant gene set for the prediction of carcinogenicity was selected. As the result of crossvalidation, the predictive ratio for carcinogenicity was approximately 80%. With the six test set chemicals,
the results predicted by SVM were reasonable. In contrast to MHlCl, when human HepG2 cells were used,significant genes could not be selected for the prediction of carcinogenicity, and the pattern of gene
expression induced by chemicals was very different from the human (HepG2) case.
[Discussion] These results indicate that short-term bioassay systems of hepatoma cells for carcinogenicity
using gene expression are promising. With interspecies analysis, careful consideration is needed for theapplication of rat data to the human situation.
[Acknowledgement] This study was financially supported by the New Energy and Industrial Technology
Development Organization (NEDO) for basic research on "Development of a High Precision Summary
Toxicity (Hazard) Assessment System".

156

JSTP/IFSTP IATP 2ooir: 1

Posters

P-49
Gene expression profiles in F344 rat livers treated with acetaminophen or
cycloheximide
Takashi YAMOT0 1, Naoki KIYOSAWA 1, Kazumi IT0 1, Takayuki SAT01, Kyoko SAKUMA',
Miyuki KANBORI 1, Noriyo NIIN0 1, Sunao MANABE 2, Naochika MATSUNUMA 1
Medicinal Safety Research Labs., Sankyo Co., LTD, Shizuoka 437-0065, Japan 1
Sankyo Pharma Inc. 2
Acetaminophen (APAP) and cycloheximide (CHX) are known as chemicals that induce the cell death inhepatocytes by different mechanisms. The aim of this study was to evaluate the feasibility of gene expression
data for the analysis of the cytotoxic mechanisms of APAP and CHX. F344/DuC1j male rats (9 weeks ofage) were administrated 1000 mg/kg of APAP or 6 mg/kg of CHX. The liver of treated animals werecollected at 1, 2, 6 and 24 hours after dosing of APAP, or at 1, 2 and 6 hours after dosing of CHX. Rat
Genome U34A Array (Affymetrix) was used for the collection and analysis of comprehensive gene
expression data in rats liver. Among the 8799 probes in the Rat Genome U34 Array, 1049 and 1315 probes
were selected as the probes that represent the remarkable gene expression changes according to the APAP
and CHX treatment, respectively. The expression of the genes associated with inflammation detected
relatively soon after APAP treatment and the gene expression profile was changed in the course of time. On
the other hand, specific changes were observed at first and then the inflammation related gene expression
was detected in the CHX toxicity study. The gene expression changes in rat livers caused by the treatmentswith APAP or CHX could be classified as different clusters. In conclusion, the difference between thecytotoxic mechanisms of APAP (necrosis) and CHX (apoptosis) in rat livers was distinguished by thedifference in their gene expression profiles. The accumulation of data that associates between pathologicalobservations and the changes in gene expression profiles will provide vast useful information for theprediction of the toxicity of chemicals.

JSTP/IFSTP IATP~-2·00~

Posters

P-50
Methacarn, a versatile fixation tool for quantitative mRNA expression analysis
in microdissected paraffin-embedded tissues using real-time RT-PCR andmicroarray systems
Makoto SHIBUTANI1, Kyoung-Youl LEE 1, Hironori TAKAGI1, Natsumi KAT0 1, Shu TAKIGAMI1,
Masao HIROSE 1
Division of Pathology, National Institute of Health Sciences, Tokyo 158-8501 , Japan 1
We have previously demonstrated methacarn to be a versatile fixation tool for molecular analysis of proteins,
DNAs and RNAs in paraffin-embedded tissues (PETs). This time, we wish to clarify the performance of
methacarn for quantitative measurement of rnRNA expression in microdissected PET specimens by real-time
RT-PCR and microarray systems. RNA-yield from methacarn-fixed PET section was equivalent to theunfixed cryosection and hematoxylin staining also did not affect the yield. By real-time RT-PCR, relative
abundance of measurable rnRNAs in methacarn-fixed PET section was found to be 80-100 % of unfixedcryosection. Hematoxylin staining resulted in only 0-20 % reduction of amplifiable rnRNAs from theunstained case. Correlation between the expression levels of target genes and input amount of extracted RNA
in the range of 1-1000 pg was very high (correlation coefficient:>0.98) with regression curves equivalent tothe unfixed cryosection case. Although the cell number within the dynamic range of PCR amplification for
cytochrome P450 2B 1 and glyceraldehyde 3-phosphate dehydrogenase was found to be 52 and 13,
respectively in the 10 µm-thick section of the phenobarbital (PB)-treated rat liver, measured values werevaried among samples when the size of microdissected areas was decreased, and ~ 300 cells would be
required for accurate measurement if normalization of expression values with the input amount of total RNA
was intended. As for microarray analysis, up to 0.5 million-fold of antisense RNAs (aRNAs) could be
amplified by two-round (2x) in vitro transcription with 50 ng of total RNA from PB-treated rat liver PET
section. With the amplified aRNAs, expression profile was compared with those in lx or 2x-amplified
aRNAs from unfixed tissue, and high fidelity was confirmed by a correlation efficient of 0.91 with Ixamplified aRNAs from unfixed tissue. For practical measurement, region-specific gene expression profiles in
the microdissected medial preoptic area (MPOA) were analyzed in rat neonates injected s.c. with estradiol
benzoate (EB; 10 µg/pup) or flutamide (FA; 250 µg/pup), to identify genes linked to disruption of brain
sexual differentiation. MPOA-specific gene expression profile by each treatment was examined 24 hour later,and genes showing altered expression by EB or FA with sex-dependent manner could be identified as
candidates to play a role in central endocrine disruption. These results demonstrate the high performance of
methacarn close to that of unfixed tissues in the quantitative expression analysis of rnRNAs in microdissected
PET-specimens by both real-time RT-PCR and microarray systems.

158

•

,,-

.. .,.

'"C..

::.._~. v,,,,,v-r

JSTP/IFSTP IATP 2004

Posters

P-51
Transcription of rat amyloid precursor protein in rat livers is affected by theserum cholesterol concentration
Naoki KIYOSAWA1, Kazumi ITO', Kayoko ISHIKAWA', Isao IGARASHI1, No1iyo NIIN0 1,
Kyoko SAKUMA 1, Miyuki KANBORI 1, Takashi YAMOT0 1, Sunao MANABE2, Naochika MATSUNUMA1
Medicinal Safety Research Labs., Sankyo Co., Ltd., Shizuoka 437-0065, Japan'
Sankyo Pharma Inc. 2
Microarray analysis is a useful tool for detecting and investigating unknown behaviors of gene expression.
Analyzing a gene expression database constructed with Rat Genome U34 Array (Affymetrix Inc.), we foundthat transcription of amyloid precursor protein (APP) in male F344 rat liver is progressively increased by therepetitive phenobarbital (PB) treatments. APP transcriptional induction by the PB treatment did not showdose dependency, suggesting it is not an extension of direct pharmacological effect of PB, but caused fromsecondary effect by repetitive PB treatments. On the other hand, APP transcription decreased in the clofibrate(CPIB)-treated rat livers . The objective of this study is to investigate the molecular mechanism of hepatic
APP induction and repression in rat livers after repetitive PB or CPIB treatments.
The hepatic APP transcription profiles correlated with serum total cholesterol (T.CHO) concentration profiles
in both chemical-treated rats. Therefore we explored the genes whose time-course transcription profiles arecorrelated highly with serum T.CHO concentration profile, using microaiTay database on rat livers treatedwith PB or CPIB. Of the 8799 probes in the Rat Genome U34 Array, transcription of GST alpha, UDPGT-1,
Apolipoprotein A-I or cMOAT, all of which are involved in bilirubin or bile acid metabolism and efflux,
were highly affected by the serum T.CHO concentration, and APP transcription exhibited the 6th mostcorrelated transcription profiles to that of serum T.CHO concentration. These data suggest that hepatic APPtranscription is strongly affected by the serum T.CHO concentration, supposedly involved in cholesterol andbile acid metabolism. To confirm the notion, we fed a high-cholesterol (1 %) diet for 33 days, and both
serum T.CHO and hepatic APP transcription were measured. Serum T.CHO concentration and hepatic APPtranscription in treated rat livers increased by 4.6-fold andl.9-fold compared to those in the control,
respectively. These data suggest that the APP transcription in rat liver is positively regulated by the serum T.
CHO, and that increase in APP transcription in the PB-treated rat liver was brought about via an elevation ofserum T.CHO concentrations.

JSTP/IFSTP IATP 2004

Posters

P-52
Gene expression profiling of terminal end buds during rat mammary
carcinogenesis
Koichiro MIYAMOTO', Shuhei TAGUCHI', Hiroaki KAWAGUCHI ' , Yoshihisa UMEKITA 1,
Hiroki YOSHIDA 1
Department of Tumor Pathology, Graduate School of Medical and Dental Sciences, Kagoshima University,
Kagoshima 890-8520, Japan 1
We have reported that a rat mammary carcinoma is induced in rats by the administration of 7,12dimethylbenz[a]anthracene (DMBA). In the present study, we tried to clarify molecular paths involved inprogression of normal tissue to malignancy using cDNA microarray and laser microdissection methodology.
Inbred female Sprague-Dawley rats were divided into two groups. At the age of 50 days, rats of Group I and
II were given 20 mg of 7, 12-dimethylbenz[a]anthracene (DMBA) and 1 ml of sesame oil, respectively. Two
weeks after the administration of DMBA and sesame oil, rats of both groups were killed for removal of
mammary glands . Removed mammary glands were frozen on dry ice and embedded in OCT. Frozen sections
were stained with HistoGene (Arcturus Engineering) . Terminal end buds were transferred onto caps using the
PixCell Ile LCM System (Arcturus Engineering). Total RNA was extracted from caps using PicoPure(Arcturus Engineering). cDNA was synthesized from total RNA and amplified by Atlas SMART Probe
Amplification Kit (CLONTECH). 32P-labeled cDNA probes were hybridized to Atlas Rat Toxicology 1.2
Array membranes. The hybridization pattern was analyzed by autoradiography and quantified byphosphorimaging. Results were normalized by global normalization and compared between two groups using
Atlaslmage (CLONTECH) . Fifteen genes that demonstrated more than 1.69-fold change values between
Group I and Group II were evaluated as significant. Especially transcripts for ribosomal proteins, receptors of
the ex tracellular matrix and folding-related proteins were up-regulated in DMBA-treated rats. Study for
abnormal gene expression at the early stage of carcinogenesis is important and we are going forward withfurther investigations by changing conditions and using real-time PCR.

160

•

~

~"--.:"L-JSTP/IFS TP IATP 200_4

Posters

P-53
Region-specific global gene expression analysis in the microdissected
hypothalamic medial preoptic area of rat neonates exposed perinatally todi(2-ethylhexyl)phthalate
Kyoung-Youl LEE', Makoto SHIBUTANI 1, Hironori TAKAGI', Natsurni KATO' , Shu TAKIGAMI 1,
Masao HIROSE 1
National Institute of Health Sciences, Tokyo 158-0098, Japan'
Phthalate esters affect male sexual differentiation in rats exposed prenatally or perinatally by affecting
perinatal testosterone surge due to testicular toxicity. To identify genes linked to disruption of brain sexual
differentiation by phthalate esters, a region-specific global gene expression analysis was performed in the
medial preoptic area (MPOA) rnicrodissected from hypothalamus of rat neonates at postnatal day (PND) 2
following maternal exposure to di(2-ethylhexyl)phthalate (DEHP; 6000 ppm) in diet from gestational day 15.
For comparison, a group of neonates were exposed to flutarnide (FA; 250 µg/pup, s.c.) at birth. At PND 2,
brains of animals (3 males and 3 females per group) were fixed with methacarn and paraffin-embedded.
Total RNA (50 ng) extracted from rnicrodissected MPOA was subjected to two-round in vitro transcription,
hybridized with Affymetrix GeneChip Rat Genome U34A Array containing 8000 probes, and analyzed theexpression data by GeneSpring software ver. 5 (Silicon Genetics). In the MPOA, about 3600 genes showedpresence call in both sexes. Among these genes, a total of 5% showed sex difference in the expression (>2fold) in untreated controls, with 62 and 124 genes showing higher expression in males and females,
respectively. By DEHP-treatment, up- or down-regulation (>2-fold) was observed with 24 and 21 genes inmales, and 31 and 418 genes in females, respectively. Among them, only one and 2 genes showed up- or
down-regulation commonly to both sexes, respectively. On the other hand, 2 of up-regulated genes by DEHP
in males showed down-regulation in females and 2 other genes showed opposite expression pattern. Amonggenes showing a sex difference with higher expression in males, 7 genes showed down-regulation in males
and 5 genes showed up-regulation in females by DEHP. Similarly, among genes showing sex difference withhigher expression in females, 22 genes showed down-regulation in females and 10 genes showed upregulation in males by DEHP. By single injection of FA, up- or down-regulated genes were found to be 6each in males, and 9 and 29 in females, respectively. Only cyclin G 1 showed up-regulation commonly to FA
and DEHP in males, and apolipoprotein E and death-associated like kinase showed down-regulationcommonly to both chemicals in females. In summary, we identified genes showing altered expression byperinatal DEHP exposure with apparent sex difference or common to the neonatal FA-treatment to be
candidates playing role(s) for central endocrine disruption by DEHP.

JSTP/IFSTP IATP- 200~

Posters

P-54
Differential global gene expression in rat brown and white adipose tissues
Akira UNAMP, Yasuo SHINOHARA2 , Kazuaki KAJIM0T0 2 , Kenjiro TSUBOTA', Yoshimasa OKAZAKI1,
Shiro FUJIHIRA', Masahiro MATSUMOTO 1, Yuji OISHI', Yoshinobu BABA 3
Toxicology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Osaka 532-8514, Japan'
Institute for Genome Research, The University of Tokushima2
Faculty of Pharmaceutical Sciences, The University of Tokushima3
It is well known that white and brown adipocytes are present in mammals and that those function as counter
actors in energy metabolism. The physiological role of white adipocytes is to accumulate excess energy asfat, and that of brown adipocytes is its expenditure as heat. Increasing in adipose cell number and size resultsin obesity, which is one of risk factors for hypertension, hyperlipidemia and diabetes. Recently, some
proteins that participate in the physiological function of these adipocytes have been noticed as new drug
targets. Therefore, to clarify the molecular mechanisms of energy metabolism in adipocytes is considered tobe very useful to develop the therapy for obesity. In the present study, we analyzed gene expression profiles
in brown (BAT) and white (WAT) adipose tissues of rat using a high-density cDNA microarray tocharacterize unknown gene associating with energy metabolism in those tissues.
Microarray analysis showed that 499 cDNA/ESTs were expressed at least 5-fold higher or lower in BAT thanin WAT. Genes encoding uncoupling protein 1 (UCPl), muscle-type camitine palmitoyltransferase (M-CPTI),
carnitine carrier (CC) and some other proteins involved in energy metabolism were highly expressed in BAT.
Most of the genes encoding mitochondrial proteins, such as subunits of ATP synthase, cytochrome c oxidase
and NADH dehydrogenase, were higher expressed in BAT than WAT, which might be dependent on thecellular content of mitochondria in BAT and WAT. However, several genes encoding mitochondrial protein,· such as liver mitochondrial aldehyde dehydrogenase and dicarboxylate cruTier, were remarkably lower
expressed in BAT than in WAT. Furthermore, we examined the expression levels of proteins which showed
higher transcript levels in BAT by Western blotting. UCPl , M-CPTI and CC were higher expressed in BAT
than in WAT even in protein levels, whereas expression levels of two protein subunits of redox complex and
ATP complex were not markedly different between two adipose tissues. Taken together, it suggested that
expression levels of these subunits would be regulated in dependence on expression levels of the othersubunits of the complexes. These results may provide further insight for clarifying the unique energy
metabolism in BAT.

162

JSTP/IFSTP IATP 2004

Posters

P-55
Microarray analysis of hepatocellular adenoma induced by di(2-ethylhexyl)
phthalate in rasH2 mice
Kaoru TOYOSAWA1, Miwako HARADA2, Katsuji NAKAN02, Kohji TANAKA1, Nobuo MATSUOKA 1
Safety Research Laboratories, Drug Research Division, Dainippon Pharmaceutical Co., Ltd.,
Osaka 564-0053, Japan 1
Pharmacology & Microbiology Research Laboratories, Drug Research Division, Dainippon PharmaceuticalCo., Ltd. 2
We have reported that Di(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, induces hepatocellularadenomas in transgenic mice carrying a human prototype c-Ha-ras gene (rasH2 mice), there are no
mutations of the transgene found in the hepatocellular adenoma induced by DEHP, and the mRNA levels of
the transgene and peroxisome proliferator activated receptor (PPAR) a are essentially equal between thehepatocellular adenoma and adjacent liver tissue (1,2).
In the present study, comprehensive analyses were performed using microarrays to find genes related totumorigenesis. Gene expressions related to fatty acid metabolism and peroxisome increased in the DEHPtreated liver in comparison with the control liver, whereas they decreased in the hepatocellular adenoma incomparison with the adjacent liver tissue, and those related to cell cycle increased unequivocally in thehepatocellular adenoma in comparison with the adjacent liver tissue. Phenotypic expressions reflecting thesegene expressions will be reported together on a poster presentation.
1) K.Toyosawa et al. Toxicologic Pathology 29:458-466, 2001.
2) K.Toyosawa et al. Cancer Letter 192:199-203, 2003.

JSTP/IFSTP IATP 200~

Posters

P-56
Mechanistic analyses using chip array analysis on inhibited uterine
tumorigenesis in rasH2 mice initiated with N-ethyl-N-nitrosourea followed bydietary treatment of ethinylestradiol
Takao WATANABE 1, Kayo SUMIDA2, Tomoko MUTOH 1, Miwa OKAMURA', Mitsuyoshi MOT0 1,
Yoko KASHIDA1, Kunitoshi MITSUMORI1Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, Tokyo 183-8509,
Japan'
Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd. 2
Estrogen has a potent tumor promoting effect in the uterus, liver and kidney of experimental animals. In theuteri , estrogen stimulates DNA synthesis and cell proliferation in the luminal and glandular epithelia ofrodent uteri in vitro, and induces the expression of genes such as c-fos, c-jun, c-myc, c-Ha-ras, epidermal
growth factor (EGF), EGF receptor, and estrogen receptor in mammalian uteri. Although, to date, several
experiments on the uterine carcinogenesis have been introduced in rats and mice, links between hormonal
carcinogenesis promoted by estrogens and overexpression of such oncogenes have yet to be proven. In ourprevious studies in which N -ethyl-N -nitrosourea (END) was intraperitoneally given to transgenic micecarrying a human prototype c-Ha-ras gene (rasH2 mice), the incidence of uterine adenocarcinomas, atypical
hyperplasias and endometrial hyperplasias were significantly increased in rasH2 mice, as compared to thosein non-transgenic mice with initiation of ENU. On the other hand, the incidences of uterine tumors such as
adenocarcinomas, atypical hyperplasias and endometrial hyperpl asias in END-initiated rasH2 mice followedby dietary treatment of 2.5 ppm ethinylestradiol (EE) for 26 weeks were completely inhibited, whereas
significantly high incidences of those in END-initiated ICR mice fo llowed by dietary treatment of 2.5 ppm
EE fo r 26 weeks were observed. In the present study, in order to clarify this paradoxical inhibitory
mechanism of uterine tumors in rasH2 mice by 2.5 ppm EE treatment, the gene expression patterns fromuterine tissues in rasH2 mice given EE for 6 weeks after ENU initiation were examined using high density
DNA microanay (Affymetrix, Santa Clara, CA) in comparison with those in ICR mice subjected to the same
treatment. As a result, the decreases of m-RNA expression for ER alpha, CYPlBland EGF receptor, and theincrease of 17-beta hydroxysteroid dehydrogenase, CYP3A16 were detected. These results, thus, suggest thatthe alterations of these m-RNAs may be related to the inhibitory mechanism for uterine tumorigenesis in
END-initiated rasH2 mice followed by 2.5 ppm EE treatment. Additional molecular pathologicalinvestigations are now in progress in our laboratory.

t64

JSTP/IFSTP IATP 2004

Posters

P-57
Tumor induction by colon carcinogen in rat gastric mucosa featuring intestinal
metaplasia caused by X-irradiation
Shoji KASHIWABARA1 , Naoki KASHIMOT01, Toshihiro UESAKA 1, Osamu KATOH 1,
Keiji WAKABAYASHI2, Hiromitsu WATANABE 1
Department of Cellular Biology, Research Institute for Radiation Biology and Medicine,
Hiroshima University, Hiroshima 734-8553, Japan 1
National Cancer Center Research Institute2
Based on investigations in humans, intestinal metaplastic changes in the stomach have been considered to beprecancerous lesions or a predisposing condition for differentiated gastric carcinoma development. However,
we have experimentally reported an inverse relationship between intestinal metaplasia, with or without
Paneth cells, and gastric tumor development, and we have established that the presence of intestinal
metaplasia does not promote induction of gastric neoplasia by N-methyl-N' -nitro-N-nitrosoguanidine(MNNG) or N-methylnitrosourea (MNU) in rats. Nakagawa et al. have reported that colorectal mucosa
implanted into the glandular stomach is like the intrinsic large intestine sensitive to tumorigenesis caused by
1,2-dimethylhydrazine (DMH), in contrast to the normal gastric mucosa. The present study was designed to
examine whether intestinal metaplasia might be a target for colon carcinogen-induction of malignant tumors
in the glandular stomach. Male 5-week-old Crj:CD, Ctj:Wistar and F344/DuCrj rats were X-irradiated with a
total of 20 Gy in two equal fractions with a 3-day interval. Beginning 16 weeks after the first irradiation,
DMH was injected i.m. at a dose of 20 mg/kg body weight weekly for 10 times to CD and Wistar rats.
Azoxymethane (AOM) was injected i.m. at a dose of 15 mg/kg body weight weekly for 3 weeks and 2arnino-1-methyl-6-phenylimidazo[4,5-b ]pyridine (PhIP) was given every 2 days, 3 times per week, for a total
10 doses of 75 mg/kg body weight by intragastric intubation to F344 rats. Twelve months after the initialcarcinogen treatment, tumors in the pylorus of the glandular stomach were observed in 2 (3 lesions) of 30
CD rats, 5 of 23 Wistar rats and 4 of 29 animals in the X-rays+AOM groups and 4 of 25 F344 rats receiving
X-rays+PhIP. No such lesions were found in the DMH, AOM, PhIP or X-ray alone or nontreated groups.
Large intestinal tumors developed in the colon carcinogen treated groups. Skin, pancreatic and kidney tumors
developed in the X-inadiated groups. While we must consider the alternative possibility that the effects ofirradiation and DMH and other colon carcinogens on glandular stomach epithelial cells are additive orsynergistic, it appears likely that intestinal mucosa! stem cell(s) are susceptible to colon carcinogenesis,
independently of the administration route or their location. In summary, the presence of intestinal metaplasia,
with or without Paneth cells, may increase the sensitivity of the stomach to the induction of tumors by
carcinogens like DMH, AOM or PhIP, but not MNNG or MNU.

JSTP/IFSTP IATP

:zoo~

Posters

P-58
Expression of peroxisome pro/iterator-activated receptor gamma inhelicobacter pylori- associated mouse gastric cancer tissue and humangastric cancer cells.
Dae-Yong KIM', Sang-Yeon OH' , Ki Taek NAM 2 , Beom Seok HAM 2 , Dong Deuk JANG2 , Ki-Hwa YANG2 ,Ki-Baik HAHM 3
Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University 1
National Institute of Toxicological Research 2
Genome Research Center for Gastroenterology, Ajou University School of Medicine3
Peroxisome proliferator-activated receptor (PPAR) is nuclear hormone receptors that can be activated by avariety of compounds. Two PPAR gamma isoforms are expressed at the protein level in mouse, gamma 1
and gamma 2. And PPAR gamma is intimately associated with cell differentiation and proliferation. Basedon the recent study established mice model of H. pylori-associated gastric cancer, the aim of this study is toinvestigate the expression pattern of PPAR gamma in mouse gastric cancer tissues and human gastric cancer
cell Jines. To generate stomach cancer, female C57BL/6 mice were treated with N-methyl-N-nitrosourea(MNU) and H. pylori. All mice were sacrificed at the 50th week after carcinogen treatment, andhistopathology, immunohistochemistry, and western blotting for PPAR gamma was performed. In vitro
experiment was performed using gastric cancer cells and administration of PPAR gamma ligands and H.
pylori. Cell viability and death after PPAR gamma ligand treatment was assessed. PPAR gamma protein was
expressed normal gastric epithelial cells, and more highly expressed in gastric adenoma and adenocarcinoma.
PPAR gamma protein expression in gastric adenocarcinoma was higher than adenoma. In gastric cancer cells,
PPAR gamma protein expression decreased in treatment of PPAR gamma agonists. However, when wetreated PPAR gamma antagonist, PPAR gamma protein was more highly expressed. The expression of RXR
alpha heterodimerized with PPAR gamma was decreased in treatment of PPAR gamma antagonist. PPAR
gamma agonists induced apoptosis in gastric cancer cells, but not PPAR gamma antagonist.

166

JSTP/IFSTP IATP 2004

Posters

P-59
Time-course and quantitative study of the relationship between transforming
growth factor-alpha and glutathione S-transferase placental form expression
during rat chemical hepatocarcinogenesis
Mitsuaki KITAN0 1, Jutaro WADA 1, Yutaka ARIKI 1, Masanori KAT0 1, Hideki WANIBUCHI2,
Keiichirou MORIMURA 2 , Kazunori HOSOE 1, Shoji FUKUSHIMA2
Life Science Research Laboratories, Life Science RD Center, Kaneka Corporation, Hyogo 676-8688, Japan 1
Department of Pathology, Osaka City University Medical SchooF
The expression of transforming growth factor-alpha (TGF- alpha) during progression from glutathione Stransferase placental form (GST-P) positive foci to neoplasms in rat chemical hepatocarcinogenesis wasinvestigated. One hundred F344 rats were given a single intraperitoneal injection of diethylnitrosamine(DEN) (200 mg/kg body wt.) and subjected to two-thirds partial hepatectomy at week 3. Commencing 2
weeks from the sta1t, sodium phenobarbital (PB) at doses of O or 500 ppm was fed to the rats for 48 weeks.
Groups of ten rats given DEN only and ten rats given DEN and PB were killed at weeks 4, 8, 16, 32, 48
and their livers were immunohistochemically examined for expression of GST-P, TGF- alpha, and epidermal
growth factor receptor (EGF-R). TGF- alpha positive foci were observed from week 4. They were seen
within GST-P positive foci and were much fewer than these latter. The numbers of TGF- alpha positive focidid not increase from weeks 4 to 48, but their areas did show increment at weeks 32 and 48. Almost all of
the tumors observed at week 32 and 48 were positive for TGF- alpha (98 %). PB administration enhanced theareas of TGF- alpha positive foci. EGF-R overexpression was observed in TGF- alpha positive lesions. Ourdata suggest that TGF- alpha positive foci observed in the early stage of rat hepatocarcinogenesis mightdevelop into tumors in the progression stage.

Posters
P-60
Enhancing effects of combined treatment with IQ and sodium nitrite on colonand liver carcinogenesis in rats
Yasuki KITAMURA1, Kazushi OKAZAK.11, Akiyoshi NISHIKAWA1, Keita KANK.1 1, Takashi UMEMURA 1,
Takayoshi IMAZAWA 1, Masao HIROSE'
Division of Pathology, National Institute of Health Sciences, Tokyo 158-8501, Japan'[Objectives] Combined effects of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a heterocyclic amine, andsodium nitrite (NaN02) were examined in a rat two-stage carcinogenesis model targeting the liver and colon.
[Methods] Experiment 1: For the initiation treatment, male 6-week-old F344 rats were given a single
intraperitoneal injection of 200 mg/kg body weight of diethylnitrosamine (DEN) and subcutaneous injections(four times) of 40 mg/kg body weight of 1,2-dimethylhydrazine (DMH) during initial 2 weeks. Then,
animals were administrated O or 300 ppm IQ in diet and/or 0, 0.1 or 0.2% NaN02 in drinking water for 27
weeks. All surviving animals were killed at week 29 for the histopathological examination. Experiment 2:
Male 6-week-old F344 rats were given IQ and/or NaN02 under the same conditions as Experiment 1, for
one week. All animals were sacrificed and their liver, lung and colon (mucosa only) were sampled foranalyzing 8-oxodeoxyguanosine (8-0HdG) levels by HPLC with an electrochemical detection system andbromodeoxyuridine (BrdU)-labeling index (LI) by immunohistochemical procedure.
[Results] Experiment 1: In both liver and colon, incidences and multiplicities of benign and malignant
tumors in the groups given IQ were significantly higher than those in the groups receiving the initiation
treatment alone. The combined treatment with IQ and NaN02 synergistically enhanced colon carcinogenesis
and also tended to enhance hepatocarcinogenesis. Interestingly, incidences of lung tumors in the group given
IQ (73%) were significantly increased as compared with the initiation alone group (5%) irrespective of
combined treatment with NaN02. Experiment 2: In both liver and colon, the 8-0HdG levels were increasedby the co-treatment with IQ and NaN02. In colon, particularly, the 8-0HdG levels and BrdU-LI in the group
given 0.2% NaN02 concomitantly with IQ were significantly higher as compared with the group given IQ
alone.
[Conclusion] These results indicated that NaN02 enhanced carcinogenic potential of IQ in the liver andcolon and oxidative DNA damage might be involved in the observed enhancement of carcinogenesis. In
addition, it is suggested that IQ is a potent lung carcinogen in rats.

Posters
P-61
Involvement of the altered regulation of pre-mRNA splicing in the liver
carcinogenic processes
Masakazu TAKAHASHI 1, Fumiyuki UEMATSU 1, Midori YOSHIDA1 , Maki IGARASHI 1,
Naoto WATANABE 1, Akihiko MAEKAWA2, Dai NAKAE 1
Department of Pathology, Sasaki Institute, Sasaki Foundation, Tokyo 101-0062, Japan 1
Director, Sasaki Institute, Sasaki Foundation, Japan 2
Introduction: Altered expression of genes and their products is crucially involved in carcinogenesis. Such
events are partly ascribed to the aberrant pre-mRNA splicing, and aberrant spliced mRNAs of specific genes
and their product proteins are frequently detected in a variety of malignancy cases. Its rationale remains
largely obscure. The present study was thus conducted to examine the involvement of the altered regulationof pre-mRNA splicing in hepatocarcinogenesis. Nagase analbuminemic rat (NAR) have a 7-base-pair deletionat the 5' splice site of the HI intron of the albumin gene, and the normal albumin generation is abolished. In
this rat, however, the aberrantly spliced mRNA and protein of albumin are generated in the liver by aging or
the administration of hepatocarcinogen, 3' -meyhyl-4-dimethylaminoazobenzene (3 ' -Me-DAB). There wereincreased amount of exon H-skipped (delta H), exons G and H-skipped (delta GH), and exons H and Iskipped (delta HI) mRNAs and aberrant 60-kDa of rat albumin. NAR can thus serve as an useful tool in thepresent study.
Methods: Seven weeks old male NAR were divided into four groups. Groups 1 and 2 received the control
CE2 diet alone and containing 0.06% of 3' -Me-DAB , respectively, while groups 3 and 4 received cholinesupplemented (CSAA) and choline-deficient, L-amino acid-defined (CDAA) diets, respectively, for 18 weeks.
Serum albumin concentration was measured by single radial immunodiffusion. In the liver, numbers andsizes of glutathione S-transferase placental form (GST-P)-positive, preneoplastic lesions and numbers ofalbumin-positive hepatocytes were determined by immunohistochemicaly, and mRNA expression of albumin,alternative splicing factor (ASF), 30kDa arginine-serine-rich protein (SRp30) and 55kDa arginine-serine-richprotein (SRp55) was examined by RT-PCR.
Results and Discussion: Numbers and sizes of GST-P-positive lesions were significantly higher in groups 2
and 4 than in 1 and 3, respectively. Serum albumin content, number of albumin-positive hepatocytes, andmRNA expression of aberrantly spliced albumin, ASF and SRp55, but not SRp30, significantly increased
only in group 2. These results indicate that, 3'-Me-DAB causes aberrant pre-mRNA splicing by virtue of its
altered regulation, and thus suggest that the altered regulation of pre-mRNA splicing may be involved in theliver carcinogenic processes .

Posters
P-62
Genetic alterations in the Catnb gene but not the H-ras gene in hepatocellularneoplasms and hepatoblastomas of 86C3F1 mice following exposure todiethanolamine for 2 years
Shim-mo HAYASHI 1, Thai Vu TON 2 , Hue-Hua L. HONG2 , Richard D. IRWIN 2 , Joseph K. HASEMAN 2 ,Theodora R. DEVEREUX2 , Robert C. SILLS 2
Pfizer Inc., Worldwide Safety Sciences, Taketoyo, Aichi 470-2393, Japan'
National Institute of Environmental Health Sciences 2
The present study characterized the immunohistochemical localization of ~-catenin protein in hepatocellularneoplasms and hepatoblastomas in B6C3Fl mice exposed to diethanolamine (DEA) for 2 years andevaluated genetic alterations in the Catnb and H-ras genes which are known to play important roles in thepathogenesis of liver malignancies. Genomic DNA was isolated from paraffin sections of each liver tumor.
Catnb exon 2 (corresponds to exon 3 in human) genetic alterations were identified in 18/18 (100%)
hepatoblastomas from DEA exposed mice. Deletion mutations (15/18, 83 %) were identified more frequently
than point mutations (6/18, 33%) in hepatoblastomas. Eleven of 34 (32%) hepatocellular adenomas andcarcinomas from DEA treated mice had mutations in exon 2 of the ~-catenin gene, while only 1 of 10
spontaneous neoplasms had a deletion mutation of codon 5-6. Common to all liver neoplasms (hepatocellularadenomas, carcinomas and hepatoblastomas) was membrane staining for the ~-catenin protein, while
cytoplasmic and nuclear staining was observed only in hepatoblastomas. The lack of H-ras mutations inhepatocellular neoplasms and hepatoblastomas suggests that the ras signal transduction pathway is not
involved in the development of liver tumors following DEA exposure which is different from that of
spontaneous liver tumors that often contain H-ras mutations.

170

-

-

~

,_,.

JSTP/IFSTP IATP 2004

Posters

P-63
Carcinogenicity of phenobarbital in Mmh!OGG1 knockout mice
Anna KINOSHITA 1, Hideki WANIBUCHI 1, Keiichirou MORIMURA 1, Takayuki YUNOKI1,
Shoji FUKUSHIMA 1
Department of Pathology, Osaka City University Medical School, Osaka 545-8585, Japan 1
The carcinogenic potential of phenobarbital (PB), was assessed in a mouse line carrying a mutant Mmhallele of the Mmh/OGGJ gene. Mmh homozygous and wild type male and female 10-week-old mice weretreated with PB in diet at a dose of 500 ppm for 70 weeks. Non-treated knockout mice developed only lungtumors at a low rate. An increase of total tumor incidence and multiplicity in mutant, but not in the wild
type, male (25%) and predominantly female (60%) mice was induced by PB treatment as compared tocontrol (15%). Hepatocellular carcinomas were found only in PB-treated mutant mice, while wild typeanimals developed only liver adenomas. In PB-treated but not in control Mmh/OGGJ -1- females, lymphomas
were observed (35%). The 8-hydroxydeoxyguanosine (8-0HdG) level was significantly elevated in the liverof all mutants as compared to wild type mice. Moreover, 8-0HdG levels and cell proliferation was
significantly increased by PB in the liver of Mmh/OGGJ ·1-. Microarray analysis of the livers of control Mmh/
OGG 1-1- mice revealed a significant underexpression of several oncogenes, genes involved in xenobiotic
metabolism, intracellular kinase network, DNA damage and repair, and encoded extracellular matrix andcytoskeleton proteins. PB treatment of Mmh/OGG 1-1- mice resulted in significant overexpression of genes
involved in xenobiotic metabolism, DNA damage and repair (females), encoded extracellular matrix proteins
and omithine decarboxylase as compared to both Mmh/OGG 1-1- and PB-treated Mmh/OGG 1 +I+ controls.
These results indicate that PB is carcinogenic in Mmh/OGGJ mutant mice, which might be related to theincreased level of non-repaired DNA oxidative base modifications.

JSTP/IFSTP IATP .2(l'O~

PostersP-64
Oxidative stress and aberrant DNA methylation in cancer
Yashige KOTAKE 1, Kiyoshi ASADA 1, Hong SANG 1, Robert H. BROYLES 1, Robert A. FLOYD 1
Oklahoma Medical Research Foundation, Oklahoma 73104, USA 1
The relationship between cytosine methylation and 8-hydroxyguanine (8-0HdG) formation in DNA is notwell understood. In vitro, the presence of 8-0HdG in the CG sequence in synthetic oligonucleotides has
been shown to interfere with maintenance DNA methyltransferase activity which results in aberrant
methylation in replicated strands. We hypothesize that 8-0HdG-mediated hyper- or hypo-methylation occurs
in carcinogenesis in vivo in CG islands within the promoter region of crucial genes. Therefore, the objectiveof this study was to show that the 8-0HdG level in DNA correlates with aberrant methylation in vivo in ahepatocarcinogenesis model, and that decrease in 8-0HdG by antioxidant treatment leads to normalization of
methylation status. In a choline-deficient (CD) diet induced hepatocarcinogenesis model in rats, we haveshown that: 1) there is an increase in 8-0HdG level in liver DNA; 2) the 8-0HdG increase was prevented byco-feeding with the synthetic antioxidant, free radical trap phenyl N-t-butylnitrone (PBN); and 3) PBN
inhibits formation of frank hepatocellular carcinoma (75-week feeding) as well as preneoplastic foci (16week feeding). In addition, previous studies in this model demonstrated that aberrant DNA methylation
occurred in global DNA and in specific genes. We will determine the methylation status of global DNA andof the promotor of four specific genes (c-myc, p16, GST-P, and SOD2) by using 5-methylcytidine HPLC,ELISA and bisulfite sequencing. Liver genomic DNA was obtained from non-tumor liver tissues in fourgroups of rats (5 animals each group): group 1) fed choline deficient L-amino acid defined (CDAA) diet
(Dyets Inc); group 2) fed CDAA diet plus 0.03 % PBN in food; group 3) fed choline sufficient L-amino aciddiet (CSAA); and group 4) fed CSAA diet plus 0.03% PBN. The feeding period was 30 weeks. Preliminary
results in p16 and SOD2 show the tendency that methylation status of PBN treated group is closer to control
groups than to CDAA group. We anticipate the results which indicate that PBN treatment prevent aberrant
DNA methylation and experiments are in progress. Supported in part by NIH CA-082506 .

JSTP/IFSTP IATP 2004.

Posters

P-65
Methylation-associated silencing of p53 responsive gene 2 and Fibril/in 2 inhuman pancreatic cancers
Atsushi HAGIHARA1, Kazuaki MIYAMOT02, Junichi FURUTA2 , Shuichi SEKI3, Shoji FUKUSHIMA1,Toshikazu USHIJIMA 2
Department of Pathology, Osaka City University Medical School, Osaka 545-8585 , Japan'
Carcinogenesis Division, National Cancer Center Research Institute2
Internal Medicine, Osaka City University Medical School3
Pancreatic cancer is one of the most refractory neoplasms, with the average 5-year survival being less than
20 %. In addition to the anatomical location of the pancreas that makes early detection of pancreatic cancers
very difficult, several genetic and biological characteristics, such as the presence of severe chromosomal
instability and the strong involvement of stromal tissues, are considered to underlie their poor prognosis. In
this study, aberrant methylation in human pancreatic cancers was searched for, using the genome scanningtechnique, methylation-sensitive-representational difference analysis (MS-RDA). CpG islands (CGis) in the 5'
regions of 30 genes were found to be methylated in at least one of seven pancreatic cancer cell lines, while
unmethylated in two human pancreatic ductal epithelial cell lines. Among these, four genes, p53 responsive
gene 2 (PRG2/MG50) , Fibrillin 2 (FBN2), ras protein-specific guanine nucleotide-releasing factor 2(RASGRF2), and Thrombomodulin (THBD), were found to be abundantly expressed in the ductal cell lines,
while the others were expressed in small amounts. Expression of the first four genes was lost in cancer celllines with methylation, and their expression was restored by 5-aza-2' -deoxycytidine, showing their silencing.
In seven and nine of 12 primary pancreatic adenocarcinomas, respectively, the CGis of the PRG2 and FBN2
were methylated. These data suggested that PRG2 and FBN2 could be involved in pancreatic cancer
development and progression.

JSTP/IFSTP IATP 200~

Posters

P-66
Lack of influence of testicular castration or sialoadenectomy on sodium
L-ascorbate promotion of urinary bladder carcinogenesis in male F344 rats
Satoru MORI1, Takashi MURAI1, Keiichirou MORIMURA 1, Hideki WANIBUCHI 1, Shoji FUKUSHIMA'
Deparment of Pathology, Osaka City Univercity Medical School, Osaka 545-8585, Japan 'The study was designed to investigate whether testicular castration (TC) or sialoadenectomy (SE) can
infl uence sodium L-ascorbate (Na-AsA)-promoting effects on urinary bladder carcinogenesis in male F344/
DuCtj rats. The animals, 6-weeks-old at the commencement, were given 0.05 % N-butyl-N- (4-hydroxybutyl)
nitrosamine (BBN) in their drinking water for 4 weeks and then underwent TC or SE. Thereafter theyreceived basal diet with or without Na-AsA supplement for 32 weeks. Na-AsA increased significantly
urinary pH and concentrations of sodium ion and total ascorbic acid. TC significantly decreased the relative
organ weights of accessory sexual glands, kidneys and livers whereas SE was without effect on these organs.
Both decreased very slightly BEN-induced carcinogenesis, but did not influence the Na-AsA -promoting
effects. The results indicate that Na-AsA -promotion of two-stage urinary bladder carcinogenesis does notdepend on the presence of testes or salivary glands.

174

JSTP/IFSTP IATP .. 2(l04Posters

P-67
Effect of amiloride and ouabain on promotion by sodiium L-ascorbate in twostage rat urinary bladder carcinogenesis
Takashi MURAI', Hideyuki MIYAUCHI 1, Satoshi IN0UE 1, Takeki UEHARA1, Toshiyuki MARUYAMA1,
Satoru MORI2, Hideki WANIBUCHF, Shoji FUKUSHIMA2
Developmental Research Laboratories, Shionogi & Co., Ltd., Osaka 561-0825, Japan 1
First Department of Pathology Osaka City University Medical Schoo!2
The effects of arniloride (AM), which is a potent inhibitor of the Na+ffi+exchanger, and ouabain (OU), which
is a potent inhibitor of the Na+/K+-ATPase on the promotion by sodium L-ascorbate (Na-AsA) in two-stage
rat urinary bladder carcinogenesis were examined to evaluate the potency of promotion mechanism of NaAsA. In Experiment 1, F344/DuC1j rats were initiated with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine(BBN) in drinking water for 4 weeks or left intact and subsequently given basal MF diet with or without a
5% Na-AsA for 24 weeks. During the administration period of Na-AsA, the initiated or intact rats were
given 6.6 mg/kg AM subcutaneously three times a week. In the rats treated with both BBN and Na-AsA,
AM treatment delayed the occurrence of the occult blood in urine and hematuria and significantly decreased
the incidence of them. Although the AM treatment did not influence urinary pH, the concentration of sodium
ion in the urine decreased in the rats given AM. The increase in urinary bladder weight due to the neoplasia
and the number of carcinoma per rat were significantly inhibited by AM treatment. In Experiment 2, thesimilar two-stage bladder carcinogenesis protocol as in Experiment 1 was carried out to evaluate the effects
of OU and to confirm the effect of AM on the second stage of urinary bladder carcinogenesis. The OU
treatment showed significant decrease of body weight, but did not affect any urinalysis data.
Histopathological evaluation of urinary bladder revealed that the number of urinary bladder carcinoma per rat
was significantly inhibited by AM. OU showed slightly increase in size of carcinoma. The effect of AM on
the initiation by BBN alone in Experiment 3 or the effects of AM and OU on the promotion by Na-AsA
alone in Experiments 4 and 5 was examined in the shorter observation period than Experiments 1 and 2. As
the results, only AM inhibited cell proliferation by Na-AsA, although AM did not affect mitogenesis by
BBN. These results indicate that the oscillation of intracellular Na+ which induce cell proliferation is
involved in the promotion stage by Na-AsA in two-stage rat urinary bladder carcinogenesis.

JSTP/IFSTP IATP · -200·~

Posters

P-68
Susceptibility of p27kipt knockout mice on N-butyl-N-(4-hydroxybutyl)
nitrosamine-induced urinary bladder carcinogenesis
Toshiya MURASAK.11, Kumiko OGAWA 1, Atsuya HIKOSAKA1, Satoshi SUGIURA1,
Seishiro TAKAHASHI', Tomoyuki SHIRAI 1
Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of
Medical Sciences, Aichi 467-8601, Japan 1
p27 kipI is a cyclin dependent kinase (CDK) inhibitor regulating the cell cycle. It is present abundantly andubiquitously in quiescent cells but declines in proliferating cells in response to mitogenic stimulation by
growth factors and cytokines. Although mutations of the p27kipJ gene are rare in human tumors, decrease in
p27kipI protein correlates with a poor prognosis in breast, colon, stomach, prostate and bladder cancers.
Our previous study on chemical-induced rat urinary bladder carcinogenesis revealed that cell cycle regulationof p27kipI is maintained in hyperplastic lesions but lost in carcinomas, presumably contributing to theirdisordered proliferation.
In the present study for further elucidation the role of p27kipI in urinary bladder carcinogenesis, alteration ofsusceptibility to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), a rodent urinary bladder carcinogen, wasassessed in p27kipI knockout mice.
Male and female mice of three p27 genotypes, i.e. nullizygous (p27 -1-), heterozygous (p27+1-) and wild (p27+1+), originating from the C57BL/6 strain were at the age of 6 weeks divided into one group of 18-20 mice
given drinking water containing 0.05 % BBN for 10 weeks and the other group of 11-15 mice receiving plain
drinking water. At the 201h experimental week, all mice were killed under ether anesthesia and their urinary
bladders, kidneys, livers and spleens were excised. They were weighed and bladders and kidneys were
processed for histological examination.
Simple hyperplasia or papillary or nodular (PN) hyperplasia, putative premalignant lesions of murine
urothelia, occurred in all mice receiving the BBN treatment. Carcinomas were observed in 100, 85 and 80%
of male p27 -1-, p27+1- and p27+t+ mice, respectively. The incidences of carcinomas in female groups weremuch lower at 39, 15 and 0% respectively, demonstrating a significant difference among the three p27
genotypes. In both male and female groups, quantitative assessment with an image analyzer, revealed larger
tumors in p27 -1- than p27+t+ mice, while p27+1- mice had intermediate-sized lesions although no significant
difference were seen.
In conclusion, loss of p27 clearly increases the susceptibility to BBN-induced mice minary bladdercarcinogenesis.

176

··-~

~~

JSTP/IFSTP IATP 2004

PostersP-69
Promoting effect of sodium L-ascorbate on N-butyl-N-(4-hydroxybutyl)
nitrosamine-induced renal pelvic carcinogenesis in SD/cShi rats of both sexes
Takashi MURAI', Hideyuki MIYAUCHP, Satoshi INOUE 1, Takeki UEHARA1, Toshiyuki MARUYAMA 1,
Akihiro K0IDE 2 , Yukio MORI2, Satoru MORP, Hideki WANIBUCHP, Shoji FUKUSHIMA 3
Developmental Research Laboratories, Shionogi & Co., Ltd., Osaka 561-0825, Japan 1
Laboratory of Radiochemistry, Gifu Pharmaceutical University 2
Department of Pathology, Osaka City University Medical SchooI3
Susceptibility to the promoting effects of sodium L-ascorbate (Na-AsA) on the development of pelvis andurinary bladder tumors in male and female SD/cShi rats, featuring spontaneous hydronephrosis, was· investigated. Rats received 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in their drinking water for
4 weeks and subsequently given basal diet with or without a 5% Na-AsA supplement for 32 weeks.
Histopathological examination revealed the promoting effect of Na-AsA on not only the development ofurinary bladder tumors but also renal pelvic tumors in the animals of both sexes in this two-stage
carcinogenesis experiment, the effect being more prominent in males. Administration of either BBN or NaAsA alone also induced papillomas and papillary or nodular hyperplasia of renal pelvis or urinary bladder,
respectively, in male but not female rats. However, the 5-bromo-2' -deoxyuridine-labeling index of urotheliumin the pelvis and bladder increased slightly in male rats and significantly in female rats given Na-AsA alone
for 8 weeks. N-butyl-N-(3-carboxypropyl)nitrosamine, which is a metabolite of BBN and proximate
carcinogen, was found more in the urine of urinary bladder than that of renal pelvis. These results indicatethat the urothelium of the renal pelvis and urinary bladder in SD/cShi rats is susceptible to promoting effectsof Na-AsA in the present two stage model urinary tract carcinogenesis, with the urinary bladder of male rats
as the most sensitive organ.

JSTP/IFSTP IATP 20·0~

Posters

P-70
Renal cell carcinoma cell lines established from Nihon rat
Izumi MATSUMOT0 1, Tadayoshi UEDA 1, Kazuyasu KIJIMA1, Youko HIRAYAMA2, Hiroaki MITANI2,
Kazuo OKIMOT0 1, Kohji TANAKA 1, Okio HIN0 2 , Nobuo MATSUOKA 1
Drug Research Division, Dainippon Pharmaceutical Co., Ltd., Osaka 564-0053, Japan 1
Depaitment of Experimental Pathology, Cancer Institute2
A novel rat model of hereditary renal cell carcinoma (RC) was discovered in a rat colony of the SpragueDawley strain in Japan, and thus was named "Nihon rat". The phenotype of RCs in Nihon rats is clear cell
type predominant, the phenotype being common in human. The mutation gene is located on the chromosome
10, and finally predisposing gene was identified. LOH was detected, fitting Knudson's "two hit" model andproving us with insights into a novel tumor suppressor gene. To further investigate the RCs in Nihon rats,
cell lines were isolated and established.
A RC was aseptically removed from a 10-month-old male rat, finely minced and incubated in Dulbeco's
modified Eagle media supplemented with 10% fetal calf serum. The outgrowth of cells was selected forepithelial cells that were harvested and passaged to establish cell colonies showing a strong ability of
proliferation. Seven cell lines were finally isolated from these colonies using the limited dilution method.
Characterization of the cell lines included growth curves, morphological examination with a light and anelectron microscope, immunohistochemical staining for renal epithelial cells, chromosome analysis, gene
analysis and tumorigenicity in nude mice. These cell lines will provide insights into a novel tumor
suppressor gene involved renal carcinogenesis.

178

JSTP/IFSTP IATP 2004

Posters

P-71
Strain-specific mammary proliferative lesion development following lifetime
oral administration of ochratoxin A in DA and Lewis rats
Yong-Soon LEE', Woo-Chan SON2 , Kyung-Sun KANG 3
Department of Public Health, Seoul National University, Seoul 151-742, Korea (South)'Huntingdon Life Sciences Pathology2
Department of Public Health, Seoul National University 3
OTA, a potent nephrotoxin in several species, is a renal carcinogen in animals and is implicated in the
etiology of BEN. The NTP classified OTA as having clear evidence of carcinogenic activity, based on
uncommon tubular adenomas and tubular cell carcinomas of the kidney and multiple fibroadenomas of the
mammary gland, seen in the rat. As shown previously (Castegnaro et al., Int J Cancer 1998;77:70.5),
induction of renal tumors by OTA is sex- and strain-specific in DA and Lewis rats, with DA males being
most responsive and DA females being resistant; however, that report was confined to the kidney and urinary
tract. To obtain OTA induced tumorigenic information in rats, we administered OTA (0.4 mg/kg) by oral
gavage to both DA and Lewis rats for their lifetimes and extended the investigation to complete
histopathology of all tissues and organs. We also observed the characteristic renal tumor that is highly strainand sex-specific, and there were increased incidences of proliferative mammary lesions in Lewis rats but not
in DA rats, indicating that these were also strain-specific. In view of the NTP report of OTA treatmentrelated mammary fibroadenoma in F344 rats, we observed increased mammary proliferative lesions in Lewis
rats but not in DA rats. Our results suggest that OTA may play some role in mammary tumor development insome rat strains. Key words : ochratoxin A; renal tumor; mammary gland; fibroadenoma; DA rat; Lewis rat

JSTP/IFSTP IATP.~200~

Posters

P-72
Strain difference in spontaneous development of uterine adenocarcinoma
between donryu and Fischer344(F344)rats
Takaharu NAGAOKA1, Hiroaki MIYAJIMA1, Akihiko MAEKAWA2
Shin Nippon Biomedical Laboratories, LTD. Kagoshima 891-1394, Japan 1
Sasaki Institute2
Strain difference in spontaneous development of uterine adenocarcinoma was investigated between Donryu
and F344 rats. 1. Incidence of uterine endometrial proliferative lesions. In Donryu rats, the incidence ofendometrial adenocarcinoma, the most commonly occurring condition, was 35.1 %. Endometrial hyperplasia
also developed at a high incidence. Conversely, in F344 rats, endometrial stromal polyp was the mostfrequently observed lesion, at an incidence density of 21.2 %. The incidence of endometrial adnocarcinoma
was less than 1 %. 2. Characteristics in histology of endometrial adenocarcinoma. In Donryu rats,
endometrial adenocarcinomas were well differentiated at 51.3%, moderately differentiated at 38.5%, andpoorly differentiated at 10.3 %. In F344 rats, 60% were moderately differentiated and 40% were poorly
differentiated. 3. Expression of p53 positive phenotype in endometrial adenocarcinoma. "P53 Positive
phenotype" was only observed in the well-differentiated adenocarcinomas in Donryu rats. Conversely, in F
344 rats, the majority of adenocarcinomas and hyperplasias were positive, regardless of the level ofdifferentiation. 4. Changes in sequential hormone production. Sequential analyses of plasma gonad steroid
levels determined that there was a significant link between the increase in the ratio of estrogen/progesterone
and the higher incidence in the development of endometrial adenocarcinomas in Donryu rats. However,changes in hormone production were not evident in F344 rats. 5. Incidence of endometrial proliferative
lesions in androgenized animals. Incidences of 15. 7 % for endometrial adenocarcinomas and of 51.1 % forendometrial hyperplasias were detected in Donryu rats. Conversely, in F344 rats, only endometrial
hyperplasia was observed, at an incidence of 45.7%. The findings in the two rat strains stated above, indicate
a clear strain difference in the spontaneous development of uterine proliferative lesions. The incidences of
adenocarcinomas in Donryu and F344 rats were high and low, respectively. In addition, the imbalance ofhormone production and the aberrant expression of p53 phenotype may be related to the development ofendornetrial carcinogenesis.

180

JSTP/IFSTP IATP 2.QO..~

Posters

P-73
Expression profile of estrogen receptor- a -related signaling pathways in thedevelopment of endometrial adenocarcinomas in Donryu rats
Midori YOSHIDA1, Takasumi SHIMOMOT0 1, Yutaka HATANAKA3, Takuji MIHARA 3,
Akihiko MAEKAWA 2, Dai NAKAE 1
Department of Pathology, Sasaki Institute, Sasaki Foundation, Tokyo 101 -0062, Japan 1
Director, Sasaki Institute, Sasaki Foundation 2
BioMedical Science Department, Dako Cytomation Co., Ltd. 3
Endometrial adenocarcinoma is a common malignant tumor in women. For its improvement of diagnosis,
management and prevention, molecular profiling is essential but not as yet sufficiently elucidated. In Donryu
rats, endometrial adenocarcinomas are spontaneously developed and have various similarities regarding
morphological and biological properties to the human counterparts, and are also inducible by intrauterine
treatment of N -ethyl-N '-nitro-N -nistroguanidine (ENNG). The underlying molecular pathway in rats,
however, has been largely obscure. In this context, we investigated gene expressions of estrogen receptor(ER)-a-related signaling pathways and their product proteins in endometria1 proliferating lesions in rats.
Assessed materials were histopathologically confirmed normal epithelia, hyperplasias and well- and poorlydifferentiated adenocarcinomas in Donryu rats induced by the ENNG. Immunohistochemical expressions forproteins were examined and image-analyzed using the automated cellular scanning system (Ario! SL-50,
USA). The assessed proteins were ER-a, proliferating cell nuclear antigen (PCNA), Bcl-2, epidermal growth
factor receptor (EGFR), insulin-like growth factor binding protein 5 (IGF-BP5), transforming growth factor(TGF)- ~, PTEN/MMACl (PTEN), p53 (both wild and mutated), ~-catenin and phosphorylated p44/42 MAP
kinase (MAPK/Erkl/2). RT-PCR was performed to assess mRNA expressions of the IGF-BP5 , EGFR, Bcl-2
and PTEN genes in normal epithelium and hyperplasia tissues obtained by a laser capture microdissection
after the confirmation of sufficient mRNA expression of GAPDH gene.
ER-a was expressed in the normal epithelia, hyperplasias and well-differentiated adenocarcinomas with asimilar intensity but not in poorly-differentiated ones . PCNA-positive cells increased with the progression of
proliferating lesions. p53 was only detected in poorly-differentiated adenocarcinomas. Nuclear MAPK/Erkl /2
was expressed in poorly-differentiated adenocarcinomas and slightly scattered at periphery of hyperplasias.
PTEN was similarly expressed among all categories. Bcl-2, EGFR, IGF-BP5 , TGF-~ or nuclear ~-catenin
was not detected in any cases. No mRNA expressions of assessed genes were detected with the exception of
PTEN expressed in both normal epithelia and hyperplasias.
These results indicate the close correlation between expressions of mRNA and proteins and suggest that: 1)
Expression of ERa might be related to endometrial carcinogenesis in rats and the malignant progression ofendometrial adenocarcinomas is related to the loss of hormone dependency; 2) the alteration on p53 is a late
event during endometrial carcinogenesis, and 3) the alteration on the mitogen-activated protein kinasecascade plays some roles in both of its early and late stages. These findings are relevant to the humanendometriod adenocarcinomas.

JSTP/IFSTP IATP 20·0~

Posters

P-74
Constitutive expression of cox-2 in thyroid follicular epithelial cells and its
significant reduction during DHPN-induced carcinogenesis in rats
Toshio IMAI 1, Mai HASUMURAI, Jun-ichi ONOSE 1, Makoto UEDA', Tamotsu TAKIZAWA',
Young-Man CHO', Masao HIROSE'
National Institute of Health Sciences, Tokyo 158-8501, Japan'
In human thyroid follicular epithelial cells, constitutive expression of cyclooxygenase-2 (COX-2) has been
demonstrated (Smith et al., 1999). To assess the role of COX-2 during rat thyroid carcinogenesis, weevaluated its expression in normal rat thyroid tissue and the proliferative lesions induced by goitrogens withor without N-bis(2-hydroxypropyl)nitrosamine (DHPN)-initiation. DHPN (2800 mg/kg body weight) was
subcutaneously injected once to 40 male 6-week-old F344 rats. One week after the initiation, goitrogens suchas propylthiouracil (PTU, 0.003%) and sulfadimethoxine (SDM, 0.1%) were administered in drinking waterfor 4 or 10 weeks, and then thyroid samples were collected for histopathology, Western blot analysis andimmunohistochemistry for COX-2. Goitrogen alone and basal diet alone groups were also placed. At week 4,
multiple focal follicular cell hyperplasias and adenomas were frequently observed in DHPN + PTU and
DHPN + SDM groups. In DHPN + SDM and SDM alone groups, severe inflammation with fibrosis in the
thyroid capsule with migrated follicular epithelial cells into the capsule were also observed. At week 10,
increased incidences and multiplicities of focal hyperplasias and adenomas in DHPN + PTU and DHPN +SDM groups were observed. In addition, invasive adenocarcinomas to capsule were also detected in 3 of 10
rats of the DHPN + SDM group. These carcinomas were suggested to originate in the focal hyperplasias or
adenomas adjacent to the capsule. Western blot analysis and immunohistochemistry for COX-2 revealed theCOX-2 expression in the normal follicular epithelial cells. In the proliferative lesions observed in DHPN +PTU and DHPN + SDM groups as well as the migrated follicular epithelial cells into the capsule in DHPN
+ SDM and SDM alone groups, COX-2 reactivity was frequently reduced or negative. These results
suggested that capsular inflammation and reduction of COX-2 expression in the preneoplastic and neoplastic
lesions play roles in the development of invasive adenocarcinomas and promotion/progression of rat thyroid
carcinogenesis, respectively.

JSTP/IFSTP IATP.2 004

Posters

P-75
PCNA labeled index of spontaneous lung proliferating lesion in rats
Masayuki MITSUI 1, Midori YOSHIDA 2 , Kasuke NAGAN0 3, Toshifumi TSU JIU CHI 1,
Hiroshi MARUYAMA4, Masahiro TSUTSUMI'
Department of Oncological Pathology, Cancer Center, Nara Medical University, Osaka 556-0005 , Japan'
Depaitment of Pathology, Sasaki Laboratory 2
Division of Pathology, Japan Bioassay Research Center3
Department of Pathology, Hoshigaoka Koseinenkin Hospital 4
Some endpoint markers such as gamma glutamyl-transferase (GGT)-positive phenotype has been reported to
examine lung cai·cinogenicity in the lung proliferating lesions of rats, however the GGT histochemical
staining can not react to formaldehyde-fixed lung tissues. Therefore, it is searching for more conventional
markers available to routine formaldehyde fixative. Proliferating cell nuclear antigen (PCNA;36 kDa protein
acting as a subunit of DNA polymerase delta) is one of the most common indicator to evaluate cellproliferating activity or tumor malignancy in many tissue and/or organs. In the present study, we investigatedusefulness of PCNA labeling index to evaluate relationship between malignancy and morphologicalvariations in spontaneous lung proliferating lesions of rats.
Material and Method : Samples were formaldehyde-fixed and paraffin-embedded sections, and supplied
from vai·ious types of lung proliferating lesions in F344 rats of control groups of carcinogenicity studies . The
lesions classified into hyperplasia, adenoma and adenocarcinoma were sub-divided by proliferating pattern(tubular, papillai·y and solid), nuclear atypism and existence of active stroma using HE staining. The nuclear
atypism was characterized by enlai·ged atypical nucleus with mai·ked nucleolus and rough and coarse
chromatin granules, and active stroma was consisted with fibroblast, inflammatory cells and collagen fiber.
Serial sections of each sample were made and incubated with PCNA antibody (DAKO Epos-anti-PCNA
antigen) after heating at 100° C for 5 minutes in distilled water to retrieve antigen. PCNA labeled index(LI)in the lung proliferating lesions were countered after visualization by DAB and shown as percents.
Result and discussion: The LI in lung proliferating lesions were sequentially increased with malignancy
from hyperplasia to adenocarcinoma via adenoma. Based on the sub-division of lesions, nucleai· atypism was
highly correlated with PCNA labeled cells. In addition, there was a tendency that LI was increased withactivity stroma. On the other hand, there was no correlation between the LI and all proliferating pattern(tubular, papillary and solid).
Conclusion: These results demonstrate that evaluation of PCNA labeled cells is a useful and convenient
marker to estimate malignancy of spontaneous lung proliferating lesions in rats.

JSTP/IFSTP IATP ·200~

Posters

P-76
The role of polymerase beta mediated DNA repair in skin tumorigenesis: using
DNA polymerase beta over-expressed transgenic mouse
Kazuo HAKOI 1, Raymond W TENNANT2, Ronald E CANNON2
Drug Safety Research Lab., Taiho Pharmaceutical Co., Ltd., Tokushima 771 -0194, Japan 1
Cancer Biology Group, National Center for Toxicogenomics, National Institute of Environmental Health
Sciences 2
Transgenic mice engineered to over express DNA polymerase beta were initiated with a single topical doseof 7,12-dimethylbenz [a] anthracene (DMBA) followed by 25 weeks of promotion with 12-0tetradecanoylphorbol-13-acetate (TPA) two times per week to investigate the effect of DNA polymerase beta
on skin tumorigenesis. Non-transgenic isogenic mice were used as a genotypic control. Induced skin tumors
were observed for 52 weeks post initiation to investigate benign and malignant tumor fo1mation.
The incidence of keratoacanthoma and papilloma with codon 61 H- ras mutation was determined to besignificantly higher in male mice that over expressed polymerase beta as compared to all other genotypes
and genders. The incidence of squamous cell carcinoma, BrdU labeling index, expression of COX-2 and P53
were no different across genotypes or genders. These data indicate that the over expression of DNApolymerase beta does not effect the malignant conversion under this experimental paradigm. These data also
indicate that type of tumors induced is not dependant on genotype or gender.
The conclusion from these results that a male associated effect enhances the incidence of skin tumors
induced by a two-stage protocol in transgenic mice that over express DNA polymerase beta.

JSTP/IFSTP IATP 2004

Posters

P-77
Possible relationships between immunosuppressive effects andcarcinogenicity of the synthetic gestagens with glucocorticoid-like effects inrodents
Shigeru HISADA 1, Toshi HORIUCHI 1
Toxicology & Pharmacokinetics Research Department, Kawasaki 213-8522, Japan 1
The ultimate end point of immunotoxicy is increased susceptibilities to infection and tumorigenseis. In thepresent study we discus the relationship between weak immunosuppression and tumorigenesis observed in
the compounds with glucocorticoid-like effect.
Medroxyprogesterone acetate (MPA), a synthetic gestagen , has a glucocorticoid-like effect and the female
Crj:CD(SD) rats receiving MPA at the dose levels of 1 and 10 mg/kg for 4 weeks showed atrophy of theadrenal (decreased weight and cortical atrophy) , atrophy of the thymus (decreased weight and cortical
atrophy), atrophy of the spleen (decreased weight and atrophy of periarteriolar lymphoid sheath), slightly
decreased numbers of CDS and CD8-possitive cells in the peripheral blood but no changes in antiovoalubumin(OVA) antibody production. On the other hand, another synthetic gestagen osateron acetate(TZP-4238) has a rather week glucocorticoid-like effect and the female Crj:CD(SD) rats receiving TZP-4238
at the dose levels of 0.1 , 1 and 10 mg/kg showed a slightly decreased weight of the adrenal but noimmunosuppressive changes were observed including organ weights and histopathology of the thymus,
spleen and lymph nodes , flowcytometry of peripheral blood and anti-OVA antibody production.
The results of the carcinogenicity testing of MPA and TZP-4238 were as follows.
1) Female F344 rats fed MPA in the diet at 300 ppm for 104 weeks showed a statistically significant
increase in lethality of mononuclear cell leukemia (MCL) accompanying decreased weight of the adrenal
with cortical atrophy and decreased weight of the spleen with atrophy of periarteriolar lymphoid sheath.
2) Males and females of F344 rats fed TZP-4238 in the diet at 6, 30 and 150 ppm for 104 weeks showed astatistically significant increase in lethality of MCL accompanying decreased weight of the adrenal withcortical atrophy and decreased weight of the spleen with atrophy of periarteriolar lymphoid sheath at 150
ppm.
3) An additional study in which female F344 rats were fed TZP-4238 in the diet at 150 ppm for 104 weeks
revealed no changes in lethality of MCL and weight of the adrenal.
4) Female Crj:CD(SD) rats and female ACI/N rats fed TZP-4238 in the diet at 150 ppm for 104 weeks
showed no increases in incidence of tumors although the weight of the adrenal was decreased.
Based on these results, it seemed likely that synthetic gestagens with glucocorticoid-like effects induced
MCL in the MCL-prone F344 rats when adrenocortical atrophy was apparent. These steroids showed little
effects on antibody production but might affect NK cell activity.

JSTP/IFSTP IATP-~200ij

Posters

P-78
Chemopreventive effects of plant derived compounds in a rat medium term
liver bioassay and on hepatocellular carcinoma cell lines and gene expression
analysis by a microarray
Hiroyuki OHNISHI 1, Kazunari TSUJIMURA 1,2 , Makoto ASAMOT0 1, Masanori KURIBAYASHI 1•3,
Shuga SUZUKI1, Tadashi OGIS0 1, Tomoyuki SHIRAI1
Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of
Medical Sciences, Aichi 467-8601, Japan 1
Chemicals Evaluation and Research Institute, Chemicals Assessment Center2
Ono Pharmaceutical Co . Ltd., Safety Research 3
Recently, it has repeatedly been shown that compounds derived from several plants of various origins havechemopreventive effects on carcinogenesis. In the present study we investigated the effects of chemicals inthis category, such as nobiletin, garcinol, auraptene, ~-cryptoxanthin hesperidin rich pulp (CHRP), and 1' acetoxychavicol acetate (ACA), expected to have inhibitory effects on liver carcinogenesis, using a rat
medium term liver bioassay system and hepatocellular carcinoma (HCC) cell lines. Six week-old male F344
rats were given a single dose of diethylnitrosamine (DEN; 200 mg/kg b.w. , i.p.), then during experimental
weeks 2 to 8, test chemicals were given in the diet (0.05% ). At week3, all rats were subjected to a two-thirds
partial hepatictomy. At week 8, the experiment was terminated and the livers were taken for analysis ofdevelopment of glutathione S-transferase (GST-P) positive liver foci, putative preneoplastic lesions, with theaid of an image analyzer after immunohistochemical staining. There were no significant differences in thenumbers and areas of GST-P positive foci between the controls and any of the chemical-treated groups.
Then, we evaluated the effect of chemicals on the growth of HCC cell lines, HepG2 and MHlCl, to see
whether they might suppress the progression stage of hepatocarcinogenesis. Nobiletin had an inhibitory effecton the growth of HepG2 and MHlCl cells and promoted apoptosis. Furthermore, we also performed
microarray analysis to detect alteration of gene expression in HCC cells and found a group of genes whose
expression pattern was specifically altered with the nobiletin treatment. These genes might point tomechanisms of chemoprevention by nobiletin. Our results suggest that nobiletin might have chemopreventive
potential regarding progression of hepatocellular carcinoma. In conclusion, while the chemicals did not
suppress the development of GST-P foci, nobiletin might have a suppressive action on the progression stageof hepatocarcinogenesis.

186

JSTP/IFSTP IATP 200~

Posters

P-79
The inhibitory effects of arctiin on F344 rat multiorgan carcinogenesis
Masaharu MOKU 1, Hideki WANIBUCID 1, Jin Seok KANG 1, Saki NAIT0 1, Anna KINOSHITA 1,
Shoji FUKUSHIMA 1
Department of Pathology, Osaka City University Medical School, Osaka 545-8585, Japan 1
Arctiin is a naturally occurring lignan which is extracted from the seeds of burdock (Arctium Lappa). Somekinds of lignans have been shown to have phytoestrogenic characteristics and chemopreventive effects on
chemically induced tumors of different organs such as liver, skin and lung in the rat. In the present
experiment, we evaluated inhibitory effects of arctiin on rat multiorgan carcinogenesis model. Eighty four, 6
week-old, male, F344 rats were divided into 4 groups (groups 1-4) and given initiation treatment with
diethylnit:rosarnine (DEN)(lOOmg/kg body weight, i.p., once at the start), n-methylnitrosourea (MNU)(20mg/
kg body weight, i.p., 4times for first 2weeks), 1,2-dimethylhydrazine (DMH)(40mg/kg body weight, s.c.,4times for following2weeks), N-butyl-N-(4-hydroxybutyl)nitrosarnine (BBN)(0.05%in drinking water, during
weeks land 2) and 2,2-dihydroxy-di-n-propylnitrosarnine (DHPN)(O. l %in drinking water, during weeks 3 and
4) (DMBDD treatment). Another18rats, divided into 2 non-initiated groups (groups 5 and 6), were given thevehicle alone. After DMBDD treatment, rats in groups 1 to 4 received 0, 0.004, 0.02 and 0.1 % arctiin in
NIH-07 (soy bean free) diet respectively. Groups 5 and 6 received O and 0.1 % arctiin in their diet. All
surviving rats were killed under ether anesthesia at week33and all organs were excised and subjected topathological examination. Arctiin has no toxity in all experimental dose. In high dose (0.1 %) group, bothincidence and multiplicity of bladder carcinoma were significantly decreased as compared with control
group. No any significant effect was observed in other organs. Our results indicate that arctiin exerts
chemopreventive effects in the bladder of the F344 rats in multiorgan carcinogenesis model.

JSTP/IFSTP IATI,-,~200fl

Posters

P-80
Chemopreventive effect of PJJ-34 in rat multi-organ carcinogenesis model
Chiharu YAMAGUCHI1, Sakai KUNIYOSI 1, Takasi YAMAGUCHI1, Tomoko NAKATA1, Reiko TANAKA 1,
Hideki WANIBUCHF, Anna KINOSITA2, Keiichirou MORIMURA 2 , Shoji FUKUSIMA2
Department of Medicinal Chemistry, Osaka University of Parmaceutical Science, Osaka 569-1094, Japan 1
Department of Pathology, Osaka City University Medical School2
Our efforts to search for tumor-chemopreventive agents have been focused in constituents from coniferous
trees plants. An extract of picea jezonesis, 13a,14a-epocy-3b-methoxyserratan-21b(PJJ-34), is a serratane-type
triterpenoid. PJJ-34 is reported to show inhibitory effects on skin carcinogenesis with the Epstein-Barr virus
early antigen activation test. In this experiment, we examined the modifying effect of PJJ-34 on rat multiorgan bioassay. A total of 70 male 6 weeks old F344/DuCrj rats were divided randomly into 5 groups. (20rats each for groups 1-3: 5rats each for groups 4-5). Animals in groups 1-3 were treated sequentially with NNitrosodiethylamine (lOOmg/kg.b.w. i.p., single dose at the commencement), N-Methyl-N-nitrosourea (20mg/
kg.b.w. i.p., 4 times, until 2weeks), 1,2-DimethylhydrazineDihydrochloride (40mg/kg.b.w. s.c, 6 times, during
weeks 2 and 6), N-n-Butyl-N-butan-4-ol-nitrosarnine (0.05% in the drinking water, until 2weeks) andDiisopropunolnitrosamine (0.1 % in the drinking water, during weeks 2 and 6). Groups 4 and 5 were given
vehicle as control. After the completion of 5 carcinogens treatment (DMBDD treatment) groupsl,4 andgroup2 were administered PJJ-34 at the concentration of 10 and 5mg/kg.b.w. i.g., up to the end of theexperiment, respectively. All rats were killed under the ether anesthesia at weeks 30 from the start of theexperiment, all major organs were excised, and subjected to histological and imrnunohistcemical analysis.
Relative weights of liver, kidney and spleen did not demonstrate any significant differences among thegroups. The incidence of lung tumor was 72, 55 and 84% for groupl, 2 and 3, respectively. Statisticalsignificance was observed between group2 and 3. The incidence of colon tumor was 22, 45 and 58% forgroupl, 2 and 3, respectively and statistical significance was detected between groupl and 3. Moreover, theincidence of thyroid tumor of group2 (10%) was significantly decreased, compared with that of group3(53 %). PJJ-34 showed inhibitory effect of tumor formation in several organs on this rat carcinogenesis
model. Immunohistcernical analysis for PCNA and cyclinD 1 were performed with lung tissue. The
percentage value of positive stain on normal appearance mucosa was reduced in groups 1 and 2, comparedwith group3 . These results might suggest that PJJ-34 has an inhibitory effect, based on the cell cycle
regulation. To further investigate the mechanism of inhibitory effect of PJJ-34, cDNA rnicroarray analysis on
short-term carcinogenesis model is now in progress.

188

JSTP/IFSTP IATP ·2oofl

Posters

P-81
Dietary protocatechuic acid inhibits progression of chemically induced rat
tongue carcinogenesis
Rikako SUZUKI 1•2 , Hiroyuki KOHN0 1, Shigeyuki SUGIE 1, Takuji TANAKA 1
Department of Pathology, Kanazawa Medical University, Ishikawa 920-0293, Japan 1
Research Fellow of the Japan Society for the Promotion of Science2
The frequent consumption of edible plants is associated with a low incidence of a variety of cancer.
Protocatechuic acid (PCA), a simple phenolic acid, is a constituent of many edible plants. Our previous
experiment demonstrated that PCA inhibits the development of 4-nitroquinoline I-oxide (4-NQO)-induced
tongue carcinogenesis when fed during the initiation and promotion phases. The compound can also inhibit
chemical carcinogenesis in other organs. In the present study, we investigated the modifying effects of
dietary administration of PCA during the progression phase of tongue carcinogenesis initiated with 4-NQO
in male F344 rats. For tumor progression, we developed a new animal model, where rats initiated by 4-weektreatment of 20 ppm 4-NQO in drinking water and thereafter received four cycles of 20 ppm 4-NQO (onecycle: 2 weeks of 4-NQO followed by 2 weeks of tap water, starting at 14 weeks after the initiation) to
induce advanced tongue cancer. The treatment enabled to induce high incidence of tongue cancer with orwithout metastases of tongue cancer in lungs, when compared with rats given the initiation treatment alone,suggesting that this model can be used for tongue tumor progression study. Starting two weeks before thecycle treatment with 4-NQO, animals were fed the 2000 ppm PCA containing diet and continued on this diet
until the end of the study. At the termination of the experiment (week 32), the incidences of tongue
neoplasms and preneoplastic lesions, and total polyamine levels in the tongue tissue, and cell proliferation
activity estimated by morphometric analysis of silver-stained nucleolar organizer regions' protein werecompared among the groups. Feeding with PCA containing diet during the progression phase significantly
reduced the incidence of tongue squamous cell carcinoma (31 % inhibitory by PCA, P<0.05) andpreneoplasia (hyperplasia and dysplasia) (29% inhibitory by PCA, P<0.001) . Rats given PCA-containing diethave no metastasis. In addition, PCA feeding significantly lowered polyamine levels of the tongue at the endof the experiment (week 32): 3.80 ± 0.44 in the group initiated with 4-NQO + cycle treatment with 4-NQO;
and 3.23 ± 0.34 in the group initiated with 4-NQO + cycle treatment with 4-NQO + PCA (P<0.001). Our
results suggest that a simple phenolic acid PCA has inhibitory effect on 4-NQO-induced rat tongue
carcinogenesis when given during the progression phase of tumorigenesis, and such inhibition might berelated to suppression of cell proliferation by PCA.

JSTP/IFSTP IATP ·200~

Posters

P-82
Chemopreventive effect of FBRA on N-nitrosomethylbenzylamine-induced
esophageal tumorigenesis in rats
Toshiya KUN0 1, Yoshinobu HIROSE', Yasuhiro YAMADA', Keiko SAKATA', Nami KITAORJl,
Akira HARN, Hideki MORI'
Department of Tumor Pathology, Gifu 500-8705, Japan 1
FBRA (Fermented brown rice by Aspergillus Orizae) is a natural product occurring in rice germ withproperties associated with reduced liver and colon cancer risk in rat. Modifying effects of FBRA during theinitiation or post-initiation phase of N -nitrosomethylbenzylamine (NMBA)- induced esophageal
tumorigenesis were investigated in male F344 rats. A total of 150 rats were used. Five-week-old rats weredivided into 7 groups, and at 7 weeks of age, rats in groups 1 through 5 received 15 s.c. injections of
NMBA over the course of 5 weeks (0.5mg/kg /dose, three dose /week). Rats in groups 2 and 4 were given
the diet containing 5 and 10% FBRA, respectively, for 7 weeks (starting at 6 weeks of age until 1week after
the last dosing of the carcinogen). They were switched to the basal diet and maintained on that until thetermination. Staiting 1 week after the final injection of NMBA, rats in groups 3 and 5 were fed the dietmixed with 5 and 10% FBRA, respectively, until the termination. Group 6 was given the diet containing
10% FBRA throughout the experiment, and group 7 was kept on basal diet alone and served as an untreatedcontrol. All rats sacrificed at 32 weeks were examined for tumor formation, and esophageal tumor were
processed for histopathological evaluation. In group 1, the incidence of total neoplasmas was 88%. The
incidences of esophageal papilloma in groups 2-5 were 69, 76, 69 and 62%, respectively. The incidence of
total esophageal neoplasms in group 5 was significantly less than that in group 1 (P<0.05). Multiplicity of
total neoplasms in group 3 and 5 (l .16+/-0.99, 0.81 +/-0.85, respectively) were significantly smaller than thatin group 1 (l.77+/-1.07) (P<0.05). These findings indicate that FBRA inhibits NMBA-induced esophageal
tumorigenesis when given the post initiation phase rather than initiation phase. Incidence and multiplicity of
preneoplastic lesions and proliferative activity of each lesion are just exploring. These data will bedemonstrated at the meeting.

190

.

-~ --~-~

JSTP/IFSTP IATP .2Jl04

Posters

P-83
Acyclic retinoid inhibits the aberrant crypt foci formation induced bydimethylhydrazine in the rat colon
Masumi SUZUI', Takahiro SHIMIZU 1, Takamitsu MORIOKA1, Viengvansay NABANDITH 1,
Morihiko INAMINE 1, Tatsuya KANESHIRO 1, Tatsuya KINJ0 1, Naoki Y0SHIMI1
Department of Pathology, University of the Ryukyus Faculty of Medicine, Okinawa 903-0215, Japan 1
Acyclic retinoid (ACR), a novel synthetic retinoid, has recently been demonstrated by us to inhibit thegrowth of human hepatoma cells by inhibiting in the levels of expression of the cell cycle related molecules
thus this agent may be useful in the chemoprenevtion and therapy of hepatoma and possibly other humanmalignancies (Suzui, M. et al. Cancer Res. 62: 3997-4006, 2002). Therefore, in the present studies weinvestigated the possible chemopreventive effect of dietary administration of ACR on rat colon
carcinogenesis. Aberrant crypt foci (ACF) are putative preneoplestic lesions of colon carcinogenesis thus
these lesions are widely utilized as useful proliferating biomarkers. The number of ACF in rat colonicepithelium was examined. A total of 35 male F344 rats were randomly divided into the following 5
experimental groups. At 5 weeks of age, rats in groups 1, 2, and 3 were weekly given s.c. injections of a
carcinogen dimethylhydrazine (DMH, 40 mg/kg body weight) for 2 weeks to induce ACF, rats in groups 2
and 3 were fed a diet containing ACR at concentrations of 50 and 150 ppm, respectively, throughout theexperiment. Rats in group 4 were served as untreated controls. Rats in group 5 were fed a diet containing
150 ppm ACR. All animals were sacrificed at 5 weeks after the start of the experiment. Dietaryadministration of ACR caused a significant decrease in number of ACF at both doses (P<0.003), whencompared with the group treated with DMH alone (group 1). No toxic effect caused by ACR was observedin all organs examined. These findings suggest that ACR may be a promising chemopreventive agent forhuman colon carcinogenesis. These novel effects of ACR provide further evidence that ACR may be avaluable compound in the chemoprevention and therapy of various malignancies.

JSTP/IFSTP IATP ,.200~

Posters

P-84
Time course observation on inhibitory effects of hepato-proliferative lesions inrats induced by a liver-selective thyromimetic, KAT-681
Morimichi HAYASHI1, Toru TAMURA' , Yuji OKUHARA1 , Tatsuya NAGASAWA1, Tsuyoshi KITAMURA',
Junji KURODA1, Nobuo SHIBATA', Kunitoshi MITSUMORF
Kissei Pharmaceutical Co., Ltd., Nagano 399-8305, Japan 1
Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology 2
Our previous study showed that a liver-selective thyrornimetic, KAT-681 (KAT), inhibited the development of
hepato-proliferative lesions positive for glutathione S-transferase placental form (GST-P) in rats that were
induced by diethylnitrosamine (DEN)-initiation treatment followed by promotion treatment with 2acetylarninofluorene (2-AAF) and partial hepatectomy. To clarify the inhibitory effect, a time course
observation on the hepato-proliferative lesions in rats treated with KAT was performed in the present study.
For the induction of hepato-proliferative lesions, male F344 rats were treated with 2-AAF (7 .5 mg/kg/day)by gavage twice a day from Weeks 2 to 4 and a partial hepatectomy at Week 3 after single intraperitoneal
inj ection of DEN (150 mg/kg). From 5 weeks after completion of 2-AAF administration, rats were treatedorall y with KAT at the dose of 0.25 mg/kg/day for up to 3 weeks. These animals were sacrificed on Days 0,
1, 2, 4, 7, 14 or 21 of KAT treatment, and a quantitative morphometric analysis using a computer-assisted
image processor was performed on the GST-P-positive lesions such as altered hepatic foci (AHF) andhepatocellular adenomas (HCAs). In addition, the labeling indices of proliferating cell nuclear antigen(PCNA) and single-strand DNA (ssDNA) were measured as a proliferative index (PI) and an apoptotic index(AI) in the GST-P-positive lesions, respectively. The total area and number, except for mean size, of AHF
were significantly decreased from Days 4 or 7 to 21 of KAT treatment. Those of HCAs did not show any
significant changes, except for the reduction of mean size on Day 14. The PI was significantly increased in
AHF, but not in HCAs, with a peak on Day 2. The AI showed no significant changes in both AHF and
HCAs. These results suggest that the inhibitory effect of KAT on hepatic proliferative lesions is attributable
to the reduction in the total area and number of AHF.

192

JSTP/IFSTP IATP -2004

Posters

P-85
Knockout of iNOS gene does not prevent hepatocarcinogenesis caused by acholine-deficient, I-amino acid-defined (CDAA) diet in mice
Ayumi DENDA1 , Nao MURATA1, Toshifumi TSUJIUCHI 1, Masahiro TSUTSUMI1, Dai NAKAE2 ,Yoichi KONISHI 1, Hiraki KUNIYASU 1
Department of Oncological Pathology, Nara Medical University, Nara 634-8521 , Japan 1
Department of Pathology, Sasaki Institute 2
Inflammation has been postulated to involve in human carcinogenesis, one of key molecules in which beinga frequently and highly expressed inducible NO synthase (iNOS) during inflammation. NO produced by
iNOS, can cause DNA damages and thus gene alterations, and can promote growth of tumors throughinhibition of apoptosis and enhancement of angiogenesis and invasion. Moreover, tumors of several organs
including colon in humans and animals have been reported to express iNOS, in fact, with iNOS geneknockout preventing colon carcinogenesis in min mice. In human livers, especially of patients infected with
HCV, iNOS is reportedly highly expressed with the development of cirrhosis and hepatocellular carcinomas(HCC). Nevertheless, roles of iNOS in hepatocarcinogenesis remain to be elucidated. A CDAA diet causes
hepatocarcinogenesis associated with development of fatty liver, fibrosis and cirrhosis in rats and mice,
which is the only animal model possessing similar histological sequences to those of humanhepatocarcinogenesis, especially of that caused by HCV. Recently, in this rat model, we have foundexpression of iNOS protein in the livers during hepatocarcinogenesis. In the present study, in order to seek
into roles of iNOS in the hepatocarcinogenesis, hepatocarcinogenicity of a CDAA diet in iNOS -/- mice was
examined.
[Methods] Male 7-8 weeks old wild C57BL/6J (Japan Clea) and iNOS -/- (NOS2 tm/Lau, Jackson) mice, 60
each, were fed a CDAA or the control choline-supplemented (CSAA) diet for 21, 44 and 80 weeks.
Development of prenoplstic and neoplastic liver lesions was histologically examined, and expression of iNOS
by RT-PCR and immunohistiochemistry. Serum NOx levels assessed by the method of Miranda.
[Results] RT-PCR analysis in wild mice revealed that iNOS was hardly expressed in control livers whereas
iNOS was moderately expressed in the livers fed a CDAA diet for 21 and 44 weeks, and in the resected
tumors and surroundings at 80 weeks . Immunohistochemically iNOS protein was weakly - moderately
positive in steatotic hepatocytes scatteringly and some of altered foci at 21 and 44 weeks, and in adenomas
and HCCs at 80 weeks. Serum NOx levels in iNOS -/- mice were significantly lower than those of wild at
80 weeks. Nevertheless, no significant differences were observed between wild and iNOS -/- mice, in theincidence and multiplicity of altered foci, adenomas and HCCs at 80 weeks. These results suggested that
iNOS might not work as a driving force for the hepatocarcinogenesis at least in the present CDAA dietmodel.

JSTP/IFSTP IATP jl)O~

Posters

P-86
Modifying effects of Terminalia catappa on azoxymethane-induced colon
carcinogenesis in male F344 rats
Takamitsu MORI0KA 1, Masumi SUZUI1, Viengvansay NABANDITH1, Morihiko INAMINE 1,
Tatsuya KANESHIROt, Tatsuya KINJ0 1, Takahiro SHIMIZU1, Miyuki AONAHATN, Naoki YOSHIMI1
Tumor Pathology, University of the Ryukyus Faculty of Medicine, Okinawa 903-0215, Japan 1
The modifying effects of dietary administration of Terminalia catappa (TC) was investigated on the rat
colon carcinogenesis induced by azoxymethane (AOM). The number of aben-ant crypt foci (ACF) and ~catenin accumulated crypts (BCAC) in the colon, and proliferating cell nuclear antigen (PCNA) labelingindex in the colonic epithelium were examined in a total of 36 male F344 rats. At 6 weeks of age, rats ingroups 1, 2 and 3 were given s.c. injections of AOM once a week for 2 weeks at a concentration of 20 mg/
kg body weight. One week before the first injection of AOM, rats in groups 2 and 3 were fed a dietcontaining TC at concentrations of 0.02 and 0.1 %, respectively, throughout the experiment. Rats in group 4
were fed a diet containing 0.1 % TC. Rats in group 5 were served as untreated controls. All animals weresacrificed at the week 5 after the start of the experiment. Oral administration of TC at both doses
significantly decreased the numbers of both ACF (P <0.05 for 0.02% TC, P <0.005 for 0.1 % TC) and BCAC/
cm 2/rat (P<0.05 for both 0.02 and 0.1 % TC), when compared with the control group (group 1). PCNA
labeling indices of rats in groups 2 and 3 were also significantly lower than that of rats treated with AOM
alone (P<0.001 for 0.02% TC, P<0.005 for 0.1% TC). These results suggest that TC has a potent
chemopreventive effect for colon carcinogenesis and this effect may be associated with the inhibition of thedevelopment of ACF and BCAC.

194

'

·---~

JSTP/IFSTP IATP 2004

Posters

P-87
Lovastatin inhibits tumor growth and metastasis in a mouse mammary
carcinoma model by p53-independent mitochondrial-mediated induction of
apoptosis
Masa-aki SHIBATA', Yuko ITO', Junji MORIMOT0 2, Yoshinori OTSUKI'
Department of Anatomy & Biology, Osaka Medical College, Osaka 569-8686, Japan 1
Laboratory Animal Center, Osaka Medical College2
The effects of lovastatin, a potent inhibitor of HMG CoA reductase, were studied in a mouse metastatic
mammary cancer model incorporating mutation in p53 . Mammary tumors, induced by inoculation ofsyngeneic BALB/c mice with BJMC3879 cells, were subsequently treated with lovastatin at 0, 25 and 50 mg/kg three times a week. Significantly reduced tumor volumes were observed for the 25 and 50 mg/kg groups
by experimental week 2 and thereafter throughout the 6-week study. There was an associated increase in
TUNEL-detected apoptosis and a decrease in DNA synthesis, as assessed by BrdU incorporation. Lovastatin
at 50 mg/kg significantly inhibited lung metastasis. In a separate in vitro study, flow cytometric analyses ofmammary cancer cells treated with lovastatin showed arrest at G 1 as well as decreases in S and G2/M
fractions and a significant elevation of p53-independent apoptosis. Apoptosis was further confirmed byultrastructural analysis demonstrating fragmented nuclei with condensed chromatin that occasionally assumeda characteristic half-moon shape. Elevated caspase-3, -8 and -9 activities were observed in lovastatin-treatedcells. Furthermore, lovastatin decreased the mitochondrial membrane potential with subsequent release ofcytochrome c. These results suggest that lovastatin may be useful both as an adjuvant therapy in and achemopreventative of breast cancers having p53 mutations due to its suppression of DNA synthesis andinduction of p53-independent mitochondrial-mediated apoptosis.

Posters
P-88
Prevention of H. pylori associated gastric carcinogenesis in N-methyl-Nnitrosourea-treated Mongolian gerbils using concentrate of Japanese apricot(Ume)
Harunari TANAKA1, Ken-ichi INADA2, Toru NIWA 1, Takafumi OTSUKA1, Mitsuo GOT0 1,
Hirotoshi UTSUNOMIYA 3 , Tetsuya TSUKAMOT0 1, Masae TATEMATSU 1
Division of Oncological Pathology, Aichi Cancer Center Research Institute, Aichi 464-8681, Japan 1
1st Department of Pathology, Fujita Health University 2
Department of Pathology, Wakayama Medical University School of Medicine3[Background] Helicobacter pylori (H. pylori) is considered to be a cause of gastric and duodenal ulcer, andits persistent infection is an important risk factor of gastric cancer. Since H. pylori infection has been treatedwith antibiotics in combination with a proton pump inhibitor, several problems have been issued such as
adverse effects or emergence of resistant bacteria. Some natural foodstuffs such as garlic, cinnamon, andgreen tea are known to have bactericidal effect and have been shown suppression effect against growth of H.
pylori and H. pylori induced gastritis. A Japanese traditional folk medicine, concentrate of Japanese apricot(CJA, Ume, Prunus mume), is said to have bactericidal effect and to be effective to sickness of a digestiveorgan. Thus, we investigated whether CJA was effective to relieve gastritis and N -methyl-N -nitrosourea(MNU)-induced gastric carcinogenesis using H. pylori infected Mongolian gerbils.
[Methods] One hundred and forty one 5-week-old male Mongolian gerbils were divided into 8 groups. They
were treated with 30 ppm MNU every other week for ten weeks (groups A, B, C, and D), inoculated with H.
pylori intragastrically (groups A, B, C, E, F, and G) and administered CJA at 3 (groups A and E) or 1%(groups B and F) . Animals in groups C, D, G, and H received no CJA. Gerbils were sacrificed at 52
experimental week. Stomach mucosae were routinely processed and histologically evaluated for severity of
inflammation using modified Sydney system and emergence of tumors.
[Results] Inflammation in stomach mucosae were suppressed significantly (P<0.0001) by administration of
CJA at both 3 and 1%. Thirteen adenocarcinomas were found in 9 animals (9/25 =36%) in MNU+H. pylori
group (group C) and 1 cancer in 1 animal (7%=1/15) from MNU alone group (group D). CJA wassignificantly effective (P<0 .005) to reduce incidence of adenocarcinomas in both 3 and 1% CJA groups; nocancers were found in group A (0/30) and in group B (0/24). No tumors were found in control groups(groups E, F, G, and H).
[Conclusion] CJA could be effective to alleviate gastric inflammation and consequently to prevent gastriccarcinogenesis associated with H. pylori infection.

196

JSTP/IFSTP IATP 200.~

Posters

P-89
The effect of N-acetylecysteine on low dose 2-amino-3,Bdimethylimidazo[4,5-f]quinoxaline(Me/Qx)-induced rat hepatocarcinogenesis
Natsuko MIYAZI 1, Hideki WANIBUCHI 1, Masaharu MOKU 1, Keiichirou MORIMURA 1,
Yoshiaki TAGAWA 1, Shoji FUKUSHIMA 1
Department of Pathology, Osaka City University Medical School, Osaka 545-8585 , Japan'
Human is exposed to low dose MeIQx in cooking meat and fish. In this study, we examined whether Nacetylecysteine (NAC), one of water soluble organosulfur components contained in garlic, can prevent low
dose MeIQx-induced hepatocarcinogenesis. 320 F344/Ducrj male rats at the age of 21 days were devided
to 14 groups. The rats in groups 1 to 7 were given 1.5 ml/mg NAC drinking water. The rats in groups 8 to
14 were given tap water. They were fed a diet containing 0, 0.01, 0.1 , 1, 10, 30, 100 ppm of MeIQx for 32
weeks. The numbers of GST-P possive foci in the liver increased significantly between O ppm rats and 30
ppm, 100 ppm treated rats, however no significant difference was observed compared with NAC treatedgroup and MeIQx alone group, respectively. The 8-0H formation, PCNA expression, Glutathione Stransferase activity and Glutathione levels were no significantly different. In conclusion, NAC has no
chemopreventive effect in this model.

JSTP/IFSTP IATP 200fl

Posters

P-90
Glycogen granule, glycogen phosphorylase, and CYP1 after exposure to DMBA
in rat liver
Shin WAKUI1, Masakuni FURUSAT02
Department of Toxicologic Pathology, Azabu University School of Veterinary Medicine, Kanagawa 229-8501,
Japan 1
Department of Pathology, Kyorin University School of Medicine2
The changes in rat liver after a single oral dose of 7,12-dimethylbenz[a]anthracene (DMBA) were examined.
Ultrastructually, glycogen granules of centrilobular hepatocytes increased with time to peak on Day 1. After
this, they sharply decreased on Day 2, and on Days 5-10 returned to the normal levels. Proliferation ofsmooth endoplasmic reticulum (sER) of the centrilobular hepatocytes also gradually increased with time topeak on Day 2, after which it decreased, and on Days 5-10 returned to the normal levels. While the activityof hepatic glycogen phosphorylase a was decreased at 6-12 hrs and on Day 1, on Days 2-10, it was elevatedto a level similar to the normal levels. Thus, the high proliferation of sER and the glycogenolysis
concurrently occurred on Day 2 following DMBA administration. DMBA is an indirect carcinogen thatenlists the host metabolism to produce its ultimate carcinogenic form, and it is known that this metabolism isconducted by cytochrome P450 lAl and/or lBl(CYPl). It has been recognized that the increase ofglycogenolysis, glucolysis, is required to sustain the induction levels of CYPl. Moreover a proliferation ofsER may be associated with increased quantities of CYPl activity in the liver. Expression of liver CYPlmRNA was observed at 6 hr and gradually increased with time to peak on Days 1-2, but then disappearedon Day 5. Expression of CYPlAl protein was first observed at 12 hr, peaked on Day 2 and decreased on
Day 5, while expression of CYPlBl protein was first observed on Day 2 and decreased on Day 5. Resultsfrom the present study indicate that exposure of rats to DMBA could induce a reversible initial change in thehepatic glycogen metabolism. Moreover, the glycogenolysis of hepatocytes with high proliferation of sER
and the high CYPl enzyme production that occmTed concmTently on Day 2. Therefore DMBA metabolism
might occur in rat liver substantially on Day 2 following DMBA administration.

198

JSTP/IFSTP IATP-··2tl021

Posters

P-91
Comprehensive gene expression profiles of hepatocellular carcinomas
induced in rats fed a choline-deficient, L-amino acid-defined diet
Furniyuk:i UEMATSU 1, Naoto WATANABE2 , Maki IGARASHF, Masakazu TAKAHASHI',
Midori YOSHIDA 1, Ak:ihiko MAEKAWA3, Dai NAKAE 'Department of Pathology, Sasaki Institute, Sasaki Foundation, Tokyo 101-0062, Japan 1
Department of Food and Nutritional Sciences, Graduate School of Agriculture, Tokyo University ofAgriculture 2
Director, Sasaki Institute, Sasaki Foundation3
A choline-deficient, L-arnino acid-defined (CDAA) diet induces hepatocellular carcinomas (HCCs) in rats at
a high rate through the endogenous molecular mechanisms that are different from the exogenous mechanisms
due to carcinogenic xenobiotics but remain still largely obscure. The morphological, biochemical andmolecular biological characters of the carcinogenic processes of rat hepatocarcinogenesis by the CDAA dietresemble, at least partly, those of human hepatocarcinogenesis cases including that derived from viral chronic
hepatitis. On the other hand, human carcinogenesis in general occurs by virtue of not only extrinsic but also
intrinsic factors . It is thus meaningful to elucidate the molecular mechanisms underlying the CDAA dietassociated rat hepatocarcinogenesis. In this context, the present study assessed comprehensive gene
expression profiles of HCCs induced in rats fed the CDAA diet.
Total RNA was extracted from 5 HCCs and their surrounding non-cancerous liver tissues of 5 individual
male Fischer 344 rats fed the CDAA diet from the age of 6 weeks for 70 weeks, and from 5 liver tissues of
5 individual strain-, sex- and age-matched untreated rats. The RNA was then subjected to an oligonucleotide
rnicroarray analysis involving 3757 genes. After normalization, 148 genes were detected to be differentiallyexpressed among the 3 classes of tissues by an ANOVA procedure. Based on the differences in theexpression patterns, these genes were classified into 4 clusters either by hierarchical or k-means clustering
procedures. The members of each cluster became identical by these 2 clustering procedures, and the numbersof the genes in Clusters 1, 2, 3 and 4 were 54, 18, 47 and 29, respectively.
While each cluster included genes with various functions, notable genes were related to apoptosis,
transcriptional regulation and alternative splicing. Among these, several changes were suggested to be humanrelevant. For instance, two CYP isozymes were down-regulated in HCCs compared to their surrounding livertissues, while these genes are down-regulated also in human HCCs. The tcfl gene encoding hepatocyte
nuclear factor 1 was down-regulated in HCCs compared to the untreated liver tissues, and the transcription
factor plays crucial roles in the development and malignant conversion of human hepatocellular adenomas. It
is thus indicated that the present results provide information contributable to elucidate humanhepatocarcinogeic mechanisms and to draw an evidence-based strategy to control human HCCs.

JSTP/IFSTP IATl1~200~

Posters

P-92
Enhanced preneoplastic lesion induction in the livers of gap junction
disrupted transgenic rats
Naomi HOKAIWAD0 1, Makoto ASAMOT0 1, Kazunari TSUJIMURA' ·2 , Kumiko OGAWA1,
Tadashi OGIS0 1, Tomoyuki SHIRAI1

Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of
Medical Sciences, Aichi 467-8601, Japan 1
Chemicals Evaluation and Research Institute, Chemicals Assessment center2
We have established transgenic (Tg) rats with a dominant negative mutant of connexin 32 gene under controlof the albumin promoter, in order to investigate the role of gap junctions in hepatocarcinogenesis in vivo.
Connexins are subunits of gap junction channels, which allow direct transfer of ions, secondary messenger
molecules and other metabolites between contacting cells. Gap junctions are reported to be involved in tissue
homeostasis, embryonic development and control of cell proliferation. In the livers of the male Tg rats, thenorm al membrane localization of endogenous connexin 32 protein appeared disturbed and gap junction
capac ity measured by scrape dye-transfer assay in vivo was found to be markedly decreased as comparedwith th e wild-type case. In normal rats, d-galactosamine and carbon tetrachloride induced severe necrosis ofhepatocytes and marked elevation of serum AST and ALT, while, only scattered single cell necrosis ofhepatocytes and slight elevation of enzymes (AST and ALT) were detected in the Tg animals. These findings
suggest that the Tg rats have marked resistance to hepatotoxicity of these compounds . Important roles of gap
junctional intercellular communication (GJIC) in multistage carcinogenesis are supported by several pieces ofevidence. Gap junctional proteins are often decreased in tumor tissue, and overexpression of connexinssuppresses tumorigenicity. Furthermore, connexin 32 knock out mice demonstrate increased susceptibility
with regard to hepatocarcinogenesis. Therefore we examined the effects of functional inhibition of gap
junction on hepatocarcinogenesis in our Tg rats. Animals were given a single intraperitoneal administrationof 200 mg/kg diethylnitrosamine (DEN) at 8 weeks of age and killed sequentially at time intervals up to 20
weeks later. Liver tissues were taken for histological and immunohistochemical examination and RT-PCR
analysis. Glutathione S-transferase placental form (GST-P) positive foci were semiquantatively measured withthe aid of an image analyzer. No differences between Tg and wild rats in DEN-induced inflammatory
responses and necrotic changes were revealed by histological assessment. In male Tg rats, a significant
increase in the numbers and areas of GST-P positive foci was evident at 20 weeks compared with the wild
type case. Interestingly, there was also a clear tendency for Tg rats to develop larger foci. These results
indicate that GJIC plays important roles in the development of liver preneoplastic lesions. Now a one year
long-term experiment is underway in order to analyse differences in tumorigenic sensitivity between Tg andwild rats at the level of hepatocellular tumor development.

200

JSTP/IFSTP IATP 20Q4

PostersP-93
In situ hybridization of interferon-gamma mRNA in concanavalin A-induced
hepatitis in mice using the polymerase chain reaction-derived cRNA probes
Hideki TANAKA1, Atsushi FUKUNARI 1, Tomomichi IWAKI 1, Shiro TAKAGI 1
Mitsubishi Pharma Corporation, Kanagawa 227-0033 , Japan'
The information obtained by immunohistochemist:ry (IHC) regarding intracellular protein synthesis might belimited; especially cytokines are rapidly secreted and hardly deposited in cells. As in situ hybridization (ISH)using cRNA probes (riboprobes) detects gene transcription products, many laboratories have reported successin hybridizing cytokines mRNA. However, constriction of plasmid templates for the synthesis of riboprobeswith phage RNA polymerases is often a complicating and time-consuming step. Additionally, since mostinvestigations use frozen sections for ISH, some problems exist, i.e. poor structural resolution and/or
instability of mRNA. Recently, a rapid and efficient generation of the polymerase chain reaction (PCR)derived riboprobe template has been introduced, and the riboprobes can be applied in ISH to routinely fixedand paraffin-embedded sections. Concanavalin A (Con A) is known to induce T-cell activation and an acutehepatitis, and this experimental system is reported to provide a model for investigating the regulation of a setof cytokines. We thus analyzed the spatial and temporal expression of interferon-gamma (IFN-y) using amouse model of hepatitis induced by Con A injection. Con A (20mg/kg) was administered to male BALB/cmice via the tail vein. Total RNA was extracted from the injured liver 8 h after Con A-injection. Reverse
transcription PCR was performed for mouse IFN-ygene. T7 promoter adaptor was directly added to IFN-ycDNA-specific PCR product by using a PCR promoter addition kit. This cDNA was then amplified by PCR
using IFN-ygene-specific primer in combination with T7 promoter adaptor primer sequence and used as atemplate of the riboprobes . Digoxigenin-labeled riboprobes were synthesized by in vitro transcription method
using RNA polymerase. ISH was pe1formed using formalin-fixed and paraffin-embedded sections. Striking
increase in ISH signals of IFN-y were recognized in lymphoid cells within the hepatic sinusoids during 2 12 h after Con A-injection. Time course of change in the signals was well correlated to that of the plasma
IFN-ylevels. The present results indicate that ISH using the PCR-derived riboprobes is a reproducible andefficient technique even in the case of detecting cytokines, very small amounts of biomarkers.

JSTP/IFSTP IATP-·2ou~

Posters

P-94
Rapid induction of type-1 diabetes mellitus by X-irradiation in Long-Evans
Agouti (LEA) rats
Tokiko NAKAI1, Akiko KUBO', Biko TAKISHITA 1, Tamotsu TAKIZAWA1, Takeshi TSUCHIGAUCHI 1,
Keiji KODAMA', Keisuke IZUMI1
Department of Molecular and Environmental Pathology, The University of Tokushima School of Medicine,
Tokushima 770-8503, Japan 1
The Long-Evans Agouti (LEA) rat established from a Long-Evans rat closed colony has been used for thecontrol strain of the Long-Evans Cinnamon (LEC) rat, an animal model of Wilson's disease. It is reported
that both LEA and LEC rats are sensitive to ionizing radiation. Initially, the aim of the present study was to
examine the effect of X-irradiation on carcinogenesis. In Experiment 1, male F344, LEC and LEA rats (n=15) were X-irradiated at 6 and 8 weeks of age (Group 1) and at 6 we_eks of age (Group 2) at a dose of 2 Gy/day. Control rats were sham-irradiated (Group 3). In Experiment 2, male LEC and LEA rats (n=9) wereinadiated at 6 and 8 weeks of age at a dose of 2 Gy/day and the control rats were sham-irradiated. At week
6, rats were submitted to an oral 2 g/kg glucose tolerance test. In Experiment 1, all LEA rats irradiated 4 Gy
became diabetic by week 10, and the survival rates were 7/15 (47%) at week 10 and 3/15 (20%) at week 30.
Thi1teen of 15 (87%) LEA rats irradiated 2 Gy showed glycosuria by week 30 and the survival rate was 13/
15 (87%). Fourteen % of the control LEA rats showed glycosuria at week 30, but hyperglycemia andglycosuria did not appear in F344 and LEC rats in any group. Histologically, the pancreas in X-irradiated
LEA rats showed islet cell loss with hemosiderosis. In Experiment 2, 30-min postglucose load blood glucose
in all non-irradiated LEA rats were diabetic levels (159-249 mg/dl). By 4 Gy irradiation, fasting blood
glucose in 3 of 8 LEA rats and 30-min blood glucose in 5 of 8 LEA rats were 250 mg/dl, but not in LEC
rats. Serum insulin levels in LEA rats were decreased more severely by irradiation. In conclusion, LEA rats
are prone to develop type-I diabetes mellitus and X-irradiation accelerates the development of the disease.
Radiation-induced islet injury may be a cause of diabetes in LEA rats.

202

JSTP/IFSTP IATP 2004

Posters

P-95
Porcine-serum-induced hepatic fibrosis in Brown Norway and Wistar rats
Yasuko BABA1, Koji UETSUKA 1, Hiroyuki NAKAYAMA1, Kunio DOI1
Department of Veterinary Pathology, Graduate School of Agricultural and Life Science, The University ofTokyo, Tokyo 113-8657, Japan 1[Background] In human, hepatic fibrosis and cirrhosis are severe diseases. Many hepatic fibrosis models
have been developed, but detailed fibrogenic process and mechanisms of hepatic fibrosis are still obscure.
Porcine serum (PS) induces hepatic fibrosis with little hepatocyte damage, although many models accompany
prominent hepatic damage.
[Aim] Strain differences in PS-induced hepatic fibrosis were examined in Brown Norway (BN) and Wistar
rats.
[Materials and Methods] Twenty-four 6-week-old male rats each of BN and Wistar strains were used. The
rats of each strain were divided into two groups. One group was injected i.p. with 0.5ml sterile PS twice aweek for 1, 2, 3 and 4 weeks and the other group was treated with physiological saline in the same way andserved as controls. At 1, 2, 3 and 4 weeks, three rats of each group were killed by blood sampling fromabdominal aorta under ether anesthesia at 24 hr after the last PS or saline injection. Liver was divided into
two pieces. One piece was fixed by 10% neutral buffered formalin and the other was frozen by liquid
nitrogen. Four-µm paraffin sections were stained with hematoxylin and eosin. Total RNA was extracted fromfrozen liver and used for Affymetrix GeneChip microarray analysis, and reverse transcription-polymerase
chain reaction (RT-PCR).
[Results] Histologically, the development of PS-induced hepatic fibrosis differed between BN rats and Wistar
rats, and formation of hepatic fibrosis occurred earlier in the former. In GeneChip microarray analysis, theexpression of genes linked to antigen presentation (CD74, major histocompatibility complex (MHC) class IIalpha chain and beta chain) was elevated earlier in BN rats than Wistar rats. The changes in the expressionof these genes were confirmed by RT-PCR in BN rats, but not in Wistar rats.
[Discussion] CD 74 and MHC class II are well known to be closely correlated with exotic-antigen
presentation and antibody production. Therefore, the present results suggest that antigen presentation is an
important process in PS-induced hepatic fibrosis and that the difference in the development of hepatic
fibrosis between BN and Wistar rats may be at least due to the difference in the MHC class II-related
antigen presentation.

20

JSTP/IFSTP IATP-~200~

Posters

P-96
Induction of mRNA related with hepatic injury response to oxidative stimuli inhepatocytes and non-parenchymal cells
Toshinobu YAMAMOTO I, Hiroyuki UTSUMI1, Naoko SHIMADA', Tetsu OGATA', Naohisa TSUTSUI'
Toxicology Laboratory, Mitsubishi Pharma Corporation, Chiba 292-0818, Japan'
Oxidative stress and inflammatory reactions may lead and promote hepatic injury. However, the gene
expression of hepatocytes and non-parenchymal cells response to oxidative stimuli was not well known. In
the present study, two liver injury models induced oxidative stress, acetaminophen-administration or ischemia/repe1fusion, were designed, and the mRNA expression (oxidative and inflammatory related genes: H0-1,
HSP70, iNOS, TNF a, IL-1 ~, and IL-6) of liver were analyzed by RT-PCR. Moreover, hepatocytes and non-parenchymal cells were divided by collagenase perfusion method, and both cells were suspected to RT-PCR
analysis.
Treatment of male SD rats with a hepatotoxic dose of acetaminophen (1000 mg/kg, p.o.) resulted in an
induction of H0-1, HSP-70, iNOS, TNF a, IL-1 ~. and IL-6 in the liver. Under the experimental condition,
HSP70 was mainly increased in both hepatocytes and non-parenchymal cells in contrast to mRNA of H0-1,
which was rather induced in hepatocytes. Other mRNAs, iNOS and inflammatory cytokines, were mainly
increased in the non-parenchymal cells. Elevation of ALT in serum, and centrilobular necrosis of hepatocytes
were also observed.
In ischemia(45 min)/reperfusion experiments, HSP70 and IL-6 were induced in liver at 1hr reperfusion. At
this point, ALT in serum was elevated but no histopathological change was observed in liver. At 3hr
reperfusion, H0-1, HSP70, iNOS and IL-6 were induced in liver. Elevation of ALT in serum, and periportal
necrosis of hepatocytes were also observed. H0-1 was increased in hepatocytes, and iNOS and IL-6 wereincreased in non-parenchymal cells. While, HSP-70 was mainly induced in both hepatocytes and nonparenchymall cells.
These observations suggest that oxidative stress causes a common spectrum of changes in gene of liver. In
those two injured liver models, hepatocytes would be more sensitive than non-parenchymal cells to theeffects of oxidative stress on H0-1. In contrast, non-parenchymal cells would be more sensitive thanhepatocytes on iNOS and inflammatory cytokines.

JSTP/IFSTP IATP · 2oofl

PostersP-97
Persistence of liver cirrhosis is associated with altered location of a -smoothmuscle actin ( a -SMA) positive cells
Jin Seok KANG 1, Hideki WANIBUCHI 1, Keiichirou MORIMURA1 , Elsayed I. SALIM 1,
Shoji FUKUSHIMA 1
Department of Pathology, Osaka City University, Osaka 545-8585, Japan 1
Liver cirrhosis is one of the strongest risk factors for hepatocellular carcinoma in human. To investigate
whether liver cirrhosis is irreversible after cessation of stimuli, we used thioacetamide (TAA)-induced livercirrhosis model. Male F344 rats were randomly allocated to three groups. Animals of group 1 (n= l5) werereceived normal water as control, and those of group 2 (n=15) and group 3 (n=15) were received 0.015 % or
0.03 % TAA in drinking water for 12 weeks, respectively. During the treatment of TAA, there were fouranimals death in group 3, but there were no death in group 1 and group 2. At 12 weeks, 10 animals ofgroups 1 and group 2, and 6 animals of group 3 were sacrificed, and remaining animals were maintained fora further 4 weeks without TAA treatment, and were sacrificed. Liver cirrhosis was induced in 30% and100% of animals in group 2 and group 3, respectively, at 12 weeks of TAA treatment, and this lesion was
also found at 4 weeks after cessation of TAA treatment. Collagen contents of liver in group 2 and group 3
were significantly increased at 12 weeks (p<0.01), and there was significant increase of collagen content ingroup 3 after cessation of TAA treatment (p<0.05). Number and area of GST-P positive foci and 8-0HdG
formation in group 3 after cessation of TAA treatment showed significant higher level compared with that ofcontrol (p<0.01, p<0.01 , and p<0.05 , respectively). Although PCNA index of hepatocytes in group 2 andgroup 3 were reduced, those of non-parenchymal cells were increased significantly after cessation of TAA
treatment (p<0.01). RT-PCR analysis of a-smooth muscle actin (a.-SMA) also showed significant higher
level in group 3 after cessation of TAA treatment (p<0.05). Immunohistochemical staining of a.-SMA
represented that positive cells were mainly existed around regenerating hepatic nodules at 12 weeks of TAA
treatment, however, they were focused into enlarged portal areas consisting of plentiful fibrous tissues andpseudo-bile ductular cells after cessation of TAA treatment. Taken together, we conclude liver cirrhosis isirreversible at 4 weeks after cessation of TAA treatment, and we suggest that persistence of liver cirrhosis
could be associated with the proliferation of non-parenchymal cells and altered location of a.-SMA positive
cells.

JSTP/IFSTP IATP lOO~

Posters

P-98
Spontaneous development of maxillary incisor lesions in aged poly(ADPribose) polymerase-1 (Parp-1) knockout mice
Osamu KUSUOKA1 , Masahiro TSUTSUMI2, Toshifumi TSUJIUCHI2, Kazutoshi TAMURA I,
Kohsuke HORIGUCHI 1, Kazumi SHIRAIWA3, Nobuo KAMADA4, Hitoshi NAKAGAMA5,
Takashi SUGIMURA5, Mitsuko MASUTANI5
Gotemba Laboratories, Bozo Research Center Inc. , Shizuoka 412-0039, Japan'
Department of Oncological Pathology, Cancer Center, Nara Medical University 2
Institute for Life Science Research, Asahi Kasei Corporation 3
Chugai Research Institute for Medical Sciences, Inc. 4
Biochemistry Division, National Cancer Center Research Institute5
Parp-1 is activated by DNA strand breaks and is involved in DNA repair and maintenance of genomic
stability. Furthermore, alterations of gene-transcription, apoptosis, and cell differentiation have been reported
in Parp-J-1- deficient cells and animals. In the present study, a spontaneous occurrence of maxillary incisor
lesions in aged Parp-1 -1- mice is documented.
[Materials and methods] Parp-1-1- and Parp -J+I+ mice of a mixed genetic background of ICR and 129Sv
were used in this study. Mice were killed and autopsied at the ages of 16 (5 each of Parp-J-1- and Parp-J+I+mice), 41 (7 Parp-1-1- mice and 5 Parp-J+I+ mice) and 110 weeks (9 Parp-J-1- mice and 13 Parp -J+I+ mice).
Organs were fixed in neutralized 10% formalin solution. Histopathological analysis of incisors was
performed after decalcification by a formic acid/formalin solution, and the routine process of embedding thetissue in paraffin and staining sections with hematoxylin and eosin.
[Results and discussion] Maxillary incisor lesion was observed in 9 out of 9 Parp-1-1- mice and 4 of 13
Parp-J+I+ mice at the age of 110 weeks. No pathological changes were observed either in 16 or 41 week-old
Parp-J-1- and Parp-1 +I+ mice. Lesions were observed in the pulp cavity and characterized as a disarrangementof odontoblast, hypertrophy of dentin, and distortion of tooth architecture caused by denticles impacting. The
incidence of incisor alteration was higher in Parp- J -1- than Parp-1 +I+ mice, and much severe changes wereobserved in Parp-J -1- mice. These results suggest that Parp-1 deficiency may be attributable to thedevelopment of dental dysplasia in mice by perturbing normal tooth development during the aging process.

JSTP/IFSTP IATP 2004

Posters

P-99
Pancreas anomaly and intestinal tumorsin the mouse small eye mutants, Pax6
Sey3H and Pax6Sey4H
Yumiko NITTA 1
Res. Inst. Radiat. Biol. Med., Hiroshima University, Hiroshima 734-8553, Japan 1
Deletions of the central regions of chromosome 2 are associated with radiation-induced acute myeloid
leukemias in mice. While, there are some murine small eye mutants, which genetically delete the central
segments of chromosome 2. Expecting a predisposition to acute myeloid leukemia, we examined thetumorigenicity of two small eye mutants, Pax6Sey3H and Pax6Sey4H. Both mutants were missing a segmentof 3.2Mb between 106.0Mb and 109.2Mb from the centromere, where the Wilm's tumor 1 (Wtl),
Reticulocarbin (Ren), Paired box gene 6 (Pax6), Elongation protein homolog 4 (Elp4) and other fourteen
novel genes are located. E!-rays and N-methyl-N-nitrosourea shortened the latency but did not increase thefrequency of the hematopoetic tumors in the mutants. Hemizygous deletions of the 3.2Mb-segment ofchromosome 2 did not contribute to the development of hematopoietic tumors. Instead, both mutants
developed intestinal tumors spontaneously (42.9% and 52.0% for Pax6Sey3H and Pax6Sey4H, respectively),
which were not observed in normal sibs. They impaired to elevate the blood glucose level when loaded withinsulin, no matter how the glucagons level was normal. They showed some morphological anomaly of the
Wirsung duct. Chronic pancreatitis or fatty degeneration of the pancreas was observed with age.
Hemizygosiy of the Pax6 gene might contribute to the intestinal tumorigenicity.

JSTP/IFSTP IATP--~20061

Posters

P-100
Establishment and characterization of cancer cell lines from glandular
stomach carcinoma induced by MNU in p53-deficient mice
Masami YAMAMOT0 1, Hayao NAKANISH1 1, Tetsuya TSUKAMOT0 1, Akihiro HIRATA1, Hiroki SAKAF,
Norimitsu SHIRAI 1, Takeshi IIDAKA 1, Tokuma YANAF, Toshiaki MASEGI2, Masae TATEMATSU 1
Laboratory of Pathology, Aichi Cancer Center Research Institute, Aichi 464-8681, Japan 1
Department of Veterinary Pathology, Gifu University 2[Purpose] We previously established stomach cancer cell lines from N-methyl-N-nitrosourea (MNU)-induced
glandular stomach carcinoma in BALB/c mouse, which were non-tumorigenic. In the present study, we
newly established additional malignant cancer cell lines with both tumorigenecity and metastatic potentialfrom MNU-induced glandular stomach carcinoma in p53 knock out (KO) mouse.
[Materials and Methods] Mouse glandular stomach carcinomas generated in a male p53 (+/-) KO mouse of C
57BL strain at 30 weeks after oral administration of 30 ppm MNU for alternate 10 weeks (for total exposureof 5 weeks) were used as a tumor source in this study. From this tumor, we established two cloned cell lines,
designated MGT53-1E and MGT53-1G. Biological characteristics of these cell lines, including growth,morphology, tumorigenicity, metastatic potentials and genotype were examined.
[Results] The primary glandular stomach tumor was a well-differentiated adenocarcinoma. MGT53-1E cells
were keratin positive and grew as an epithelial monolayer in culture, whereas MGT53-1G cells were keratin
positive but contained abundant giant cells . Flow cytometry analysis revealed that DNA content of MGT53-1
G cells was significantly larger than MGT53-1E cells. The genotype of the primary stomach tumor was p53(+/-), but cell lines showed loss of wild type allele. When subcutaneously injected into a p53 (+/-) KO
mouse, both cell lines produced tumors with 100% tumor take rate and without difference in growth rate.
Spontaneous lung and lymph node metastasis were observed in both cell lines, but MGT53 -1E cells showed
hi gher metastatic potential than MGT53- l G cells. Histology of both subcutaneous tumors in p53 (+/-) KOmice were moderately differentiated adenocarcinoma with undifferentiated or sarcomatous change.
[Conclusion] These cell lines derived from glandular stomach adenocarcinoma in a p53 (+/-) KO mouseproved highly malignant and phenotypically unstable in terms of appearance of giant cells and sarcomatouscomponent. These results suggest that these malignant phenotypes are related to the genetic instability via p
53 inactivation. These cell lines would be useful model for the malignant progression of gastric cancer.

208

JSTP/IFSTP IATP 2004

PostersP-101
Influence of p53 gene deficiency on spontaneous tumor development in TCRs·1-;
ps3·1· mice
Kana HASHIM0T01, Shoichi KAD01, Kazumi UCHIDA1, Shin IWATA', Yuriko NAGATA',
Minoru AND0 1, Hideyuki FUNABASHI 1, Masaharu ONOUE 1
Yakult Central Institute for Microbiological Research, Tokyo 186-8650, Japan 1
TCR~;-'-/p53·1· mice (T cell receptor ~ chain and p53 gene-deficient mice = Double Knock Out mice; DKOmice) develop adenocarcinoma in the large intestine by the age of 17 weeks at a rate of 100%. We havedemonstrated that adenocarcinoma of DKO mice is initiated by TCR ~ chain gene deficiency, and p53 genedeficiency accelerates the tumorigenesis. DKO mice develop not only adenocarcinoma of the large intestine,
but also spontaneous tumors in lymphatic tissues, soft tissues and many other organs, although thoroughhistopathological analysis of these tumors has not yet been performed. We investigated non-treated male
DKO mice (mean age; 17 .1 weeks), to examine the occurrence rate and histological tumor type of tumors
other than adenocarcinoma in the large intestine, and compared them with those reported in the literature on
p53·1· mice. We also investigated non-treated male TCR~-1-Jp53+i+ mice (mean age; 17.0 weeks), to examine
the influence of p53 gene deficiency on the tumorigenesis of spontaneous tumors in DKO mice.
Lymphoma was observed in 45 % (21/47) of DKO mice, and the histological type was malignant lymphoma,
lymphoblastic type. It was confirmed by imrnunohistochemical study that the tumor cells were of T cellorigin, as they were CD3 £ antibody positive and CD45Ra antibody negative. On the other hand, sarcomas
were observed in 15% (7/47) of DKO mice, and the tumor type was leiomyosarcoma (3/7),
hemangiosarcoma (2/7) or liposarcoma (1/7). Other than sarcomas, teratoma of the testis was observed in
one case. These results on the occurrence rate and tumor type correspond to the literature on lymphomas andsarcomas of p53·1- mice. These findings indicate that deficiency of the p53 gene plays a critical role in thetumorigenesis of tumors other than adenocarcinoma of the large intestine in DKO mice, and deficiency of
the TCR ~ chain gene has a small influence on it.

209

JSTP/IFSTP IATP,2DO~

Posters

P-102
Progression of glomerulonephritis in cynomolgus monkeys caused by
autoimmunity to NC1 domain of type JV collagen [K35}
Masatoshi YAMAMOT0 1, Iori ITAGAKJI, Masarni HIRUMA 1, Yoshihiro TAKEI1, Norio MUT01,
Flordeliza P. DE VILLA2, Tsukao YOKOYAMA3, Syunji HATTORI4, Hidekazu SHIGEMATSU5
Ina Research Inc., Ina-shi, Nagano, 399-4501 Japan, Nagano 399-4501, Japan'
INA RESEARCH PHILIPPINES, INC. 2
Collagen Research Center3
Nippi Reseach Institute of Biomatrix4
Department of Pathology, Shinsyu University School of Medicine5
K35, NCl domain of type IV collagen, is an antigen purified from bovine glomerular basement membrane(GBM). Ina Research and INARP have developed a primate model of human autoimmune anti-GBM
nephritis. The procedure involves immunization of K35. The purpose of this study is to evaluate thehistopathological features in the renal tissues.
[Materials and methods] To induce the glomerulonephritis (GN), two female cynomolgus monkeys (Macaca
fas cicularis) were immunized once with K35 (4 mg/monkey) in Freund's complete adjuvant by intradermal
injections to dorsal skin. Two additional females were likewise immunized at 1 mg and boosted at 3 mg
three weeks later. All animals were observed for clinical signs daily. Determinations were conducted onserum anti-NCl antibody titers, urinalysis, hematology and blood chemistry. Renal tissues were collected 4
weeks (biopsy sample) and 6 weeks after the single or first immunization, respectively. Paraffin sections
were stained with HE, PAS and PAM for morphological evaluations. Additional sections were also stained byindirect immunoperoxidase method to detect the expression of CD68, as a macrophage marker and a -SMA
(a-smooth muscle actin), as a phenotypic marker of myofibroblasts. Immunofluorescent staining was
conducted to detect IgG, IgA and C3 from cryostat sections.
[Results] No unusual clinical signs were evident. Circulating anti-NCl antibodies were first detected 2
weeks after the single or first K35 immunization. High titers were evident in all animals at Weeks 3 to 6.
Proteinuria and hematuria were detected from Weeks 4 to 6. Anemia and low serum albumin were evident at
Week 6. Autologous IgG deposition was noted along the GBM in all animals at Weeks 4 and 6.
Intraglomerular hypercellularity with focal and segmental GBM destruction and extracapillary efflux of fibrin
were evident in three animals at Week 4. These animals developed slight to marked crescentic andnecrotizing glomerulonephritis at Week 6. Epithelioid cell proliferation was also noted in the Bowman's
space and tubular lumens. In addition, CD68-positive cells and a -SMA-positive cells were detected in theglomeruli and peri-glomerular regions as well as in the cortical interstitium.
[Conclusion] These findings suggest that the macrophage accumulation and transdifferentiation of glomerular
and tubular cells may play a critical role in the progression of more severe renal damage.

210

Posters
P-103
Histological changes of pancreatic islets in Zucker Diabetic Fatty rats, with
progress in the type 2 diabetes-like disease
Naoshi SHIMOTI 1, Wakako TOGA1, Satoko TOMI0KA 1, Haruyo SUGIYAMA 1, Kaori SASA 1,
Nobutaka DEMURA1, Haruko KAMEDA1, Yusuke NAGAE 1
Preclinical Development Department, Novartis Pharma K. K., lbaraki 300-2611, Japan 1
The Zucker Diabetic Fatty (ZDF) rat is known as an animal model for type 2 diabetes mellitus. The ZDF rat
shows the progressive hyperinsulinemia, followed by hypoinsulinemia with severe hyperglycemia. Thisphenomenon suggests that the ZDF rat is available as a spontaneous model of beta cell dysfunction. This
study was carried out to characterize the morphologic features of spontaneous changes in pancreatic islets
observed in the ZDF rat at the early and progressed stages of diabetes.
Five weeks old male ZDF rats were purchased from Charles River Genetic Models Inc. , as well as ZDF leanrats as control animals. They were bred in an animal room under well-controlled conditions, and fed with
Purina diet. Plasma glucose and insulin levels were monitored. Animals were sacrificed under anesthesia 30
minutes after injections of 5-bromo 2' -deoxyuridene (BrdU), at 11 and 16 weeks of age, respectively. The
pancreases were removed, weighed, fixed with 10% buffered formalin , embedded in paraffin and sectioned at
5 µm . Staining was performed with hematoxylin and eosin and Masson 's trichrome stain for lightmicroscopic examination. Immunohistochemical approaches using anti-insulin, anti-ssDNA and anti-BrdU
antibodies were also applied.
Prominent histological findings observed in the pancreas of the ZDF rats were hyperplasia and/or fibrosis of
the islets. Size of islets evidently increased when compared with those in lean rats. Irregular and obscureislet margins and fibrous tissues within and/or surrounding islets were noted. Entrapped exocrine pancreatic
tissues were often seen within the islets. Ratio of the insulin-positive area to the whole islet's area was
reduced, which suggested a dysfunction of the beta cells. The ssDNA positive islet cells in the ZDF rats
were observed at higher rate than in the lean rats, though BrdU labeling indices remained unchanged.
Noteworthiness of this study was also the observation of insulin positive cells in the outside of the islets
such as ductal epithelial cells and endocrine acinar cells. The present results, therefore, imply the potential
ability of the redifferentiation of the non-endocrine cells to the beta cell-like endocrine cells in the ZDF rat
pancreas.

JSTP/IFSTP IATii 200~

Posters

P-104
The mouse rasH2/BHT model as an in vivo rapid assay for lung carcinogens
Takashi UMEMURA1, Yukio KODAMA2, Keita KANKJl, Yasuki KITAMURA ', Takayoshi IMAZAWA 1,
Akiyoshi NISHIKAWA 1, Masao HIR0SE 1
Division of Pathology, National Institute of Health Sciences, Tokyo 158-8501 , Japan 1
Division of Toxicology, National Institute of Health Sciences 2
We have demonstrated the applicability of a 9-week in vivo two-stage model for screening lung cancer
initiating agents, using transgenic mice carrying the human prototype c-Ha-ras gene (rasH2 mice) andbutylhydroxytoluene (BHT) as a potent lung tumor-promoter (rasH2/BHT model). The first aim of thepresent study was to establish a standard protocol for this model, especially with regard to appropriate BHT
administration. Subsequently, according to the established protocol, validation studies were performed withwell-known genotoxic murine lung carcinogens. [Methods] Experiment 1 Male rasH2 mice received bygavage one to six consecutive administrations of 400 mg/kg of BHT. The interval of administration was set
to be 1 week. Controls were treated with com oil alone. They were killed 3 days after each final treatment
with BHT. All animals were intraperitoneally injected with bromodeoxyuridine (BrdU: 100 mg/kg) once 2 h
before killing. Experiment 2 Male and female rasH2 mice were intraperitoneally injected with urethane (UR)at a dose of 250 mg/kg and then given weekly administration of BHT at a dose of 400 mg/kg starting 1
week after the UR treatment for 3, 5 or 8 weeks. Control animals were given com oil instead of BHT. All
animals were killed 9 weeks after the UR treatment. Experiment 3 Validation was tested with six chemicals
as follows: 3-methylcholanthrene (100 mg/kg), N-ethyl-N-nitrosourea (120 mg/kg), dimethylnitrosamine (15mg/kg), diethylnitrosamine (100 mg/kg), benzo(a )pyrene (80 mg/kg), 7, 12-dimethylbenz(a )anthracene (5 mg/kg). [Results and Discussion] Whereas the BrdU-labeling index (LI) of alveolar type II cells was increased
dramatically with the initial BHT administration, the efficacy was gradually diminished with further
exposures, and finally six BHT treatments failed to induce cell proliferation. In fact, in Experiment 2 using
UR as an initiator, although up to five consecutive doses of BHT were able to enhance the promoting effects
in terms of lung adenoma yield, no more increment was evident with further treatments. Therefore, it can beproposed that as a standard protocol for rasH2/BHT model rasH2 mice are treated with test chemicals,
starting 1 week later given BHT weekly five times at a dose of 400 mg/kg by gavage, and then killed at
week 9. Under this protocol, initiating activities of all the chemicals examined in Experiment 3 were
substantially detectable. Thus, the rasH2/BHT model could be a powerful tool to detect environmental
carcinogens acting as initiating agents to the lung.

Posters
P-105
The utility of EHBR for toxicity study of organic anion drugs
Chitose KUWAYAMA 1, Hiroyasu NABA1, Chihaya KAKINUMA 1, Takuo OGIHARA 1, Shuhei OHNISHI1,
Akihito SHIMOI 1

Phamaceutical Research Center, Mochida Phamacetical Co., Ltd., Tokyo 160-8515 , Japan 1
Some organic anion drugs are actively transported to bile by the action of multidrug resistance-associatedprotein 2 (MRP2), which is a major ATP-dependent canalicular organic anion transporter. EHBR (Eizai
hyperbilirubinemic rat) is characterized by the deficiency of MRP2, and show higher plasma concentrationsof organic anion drugs than the normal rats when treated with those drugs . Hence, we assessed the utility of
EHBR for toxicity study from the results of pravastatin or methotrexate induced toxicities . [Method] 12weeks old EHBRs were used. SDs (Sprague-Dawley rats) were also prepared for comparison. Study 1:
Animals were administrated orally with pravastatin at 200mg/kg/day or 400mg/kg/day for 7 days or 14 days.
Skeltal muscles were examined histopathologically and plasma concentration of pravastatin was determined.
Study 2:Animals were administrated orally with methotrexate at 0.6mg/kg/day for 7 days. Main organs were
examined histopathologically, and plasma concentration of methotrexate was determined. [Result] In bothstudies, plasma concentrations of drugs in EHBRs showed higher than those in SDs. Study 1:EHBRs with
200mg/kg/day for 14 days showed rhabdomyolysis, but SDs didn't. Study 2:EHBRs with 0.6mg/kg/day for 7
days showed severe affections in liver, hematopoietic organs and digestive tracts, but SDs showed slightaffections in bone 1rnmow and thymus. [Discussion] From the result of the toxicity study for organic anion
drugs, EHBRs showed higher sensitivities than SDs. Taking these facts together, we concluded that EHBR isuseful for the evaluation of toxicities induced by organic anion drugs.

JSTP/IFSTP IATP ·z~
OOfl

Posters

P-106
Morphological evaluation of bone dynamics using in vivo micro-CT analysis
and histomorphometry in mice osteoporosis model
Anbo XIANG 1, Masahiro KANEMATSU1, Mana MITAMURA1 , Hideo KIKKAWA 1, Kiyoshi KOBAYASHI1,
Hiroyuki HIGASHIYAMA1, Mine KINOSHITA 1, Satoshi ASAN0 1
Pharmacology Department, GlaxoSmithKline KK., Ibaraki 300-4247, Japan 1
For the purpose of comprehensive evaluate the bone dynamics in mice osteoporosis model, we quantified theparameters of bone resorption and formation, the bone architecture and the trabecular connectivity by using
in vivo micro-CT and histomorphometry. Furthermore, the influence of repetitive X-ray irradiation by microCT in vivo scanning on the immunological function, haemopoiesis function and bone density was examinedin time course study. The 6-week-old female mice of BALB/c strain were either sham or ovariectomized(OVX) on dayO. The OVX mice were daily treated with vehicle or Estradiol-l 7beta (E2) for 2 weeks. On
day 14, the left femur bones were isolated and then analyzed using micro-CT and resin embedded bone
tissue specimens. For the detection and analysis of osteoclasts and osteoblasts, we applied TRAP (tartrateresistant acid phosphatase)/ALP (alkaline phosphatase) double staining. Using morphometric analysis, TRAP
and ALP positive cells were quantified. For the in vivo micro-CT study, the body of each mouse wasprotected by lead sheet, and only the left femur of mice was scanned by micro-CT (X-ray: 1.68 Gy/min for2.78 min) for once irradiation on day O or twice irradiation on day O and 7. On day 14, the animals were
killed and the left femur bones were scanned. As the results, the number of osteoclasts per bone surface, oneof the parameter revealed bone resorption, were significantly increased in OVX group, and prevented by E2
administration. In both of micro-CT and morphometrical analysis, the significant decrease of bone volume,
collapse of trabecular bone was observed in the same way. In addition, the deterioration of connectivity oftrabecular bone was observed in OVX mice compared with sham mice using micro-CT analysis. These
alterations induced by OVX were recovered to the sham level after E2 treatment. In the study of in vivo
micro-CT scanning, X-ray irradiation did not affect the body weight change, spleen weight, bone marrowcell number and hematological parameters. The bone volume did not change in once irradiation group, but
significantly decreased in twice irradiation group. Micro-CT and histomorphometry could be the high content
and high throughput (micro-CT) tools for evaluating the effect of compounds in the models of osteoporosis
and related bone diseases. Using in vivo micro-CT, high sensitive examination would be applied in thesemodels for the development of chemicals for bone disease and the detection of bone toxicity.

JSTP/IFSTP IATI'-~2004

Posters

P-107
Expression of a system L amino acid transporter at testis of rats after prenatal
exposure to di(n-butyl)phtalate
Tomoko MUT0 1, Yoshikatsu KANAI 1, Hitoshi ENDOU 1
Department of Pharmacology and Toxicology, Kyorin University, Tokyo 181-8611, Japan 1
L-type amino acid transporter 1 (LATl) has been proposed to be one of the major nutrient transport system.
The expression of LATl in rat testis following prenatal exposure to di(n-butyl) phtalate (DBP) was
examined. SD rats were injected (i.g.) with lg of DBP/kg or vehicle, on days 12-21 postconception. At 25 to
65 days old, testicular weights of DBP group were significantly lower, and showed degenerated surniniferous
tubules with Leydig cells hyperplasia and/or Leydig cell tumor lesions . At 100 to 150 days old, testicularweight and many suminiferous tubules and Leydig cells were similar to those of the vehicle group. But somenumber of Leydig cells hyperplasia and/or Leydig cell tumors were also observed. A polyclonal affinity
purified antibody against the C-terminus of LATl was generated in chickens and used in
imrnunohistochemical analysis. At 25 to 65 days old of DBP group, LATl expressed on the hyperplastic and/or neoplastic Leydig cells and the capillary endothelial cells. At 100 to 150 days old of DBP group, noexpression of LATl revealed in testis except Leydig cells hyperplasia and/or Leydig cell tumor lesions .
Previous studies have been showed the DBP induced reversible Leydig cell hyperplasia that is involved in anunderproductivity of testosterone in Leydig cells. These results suggest that the expression of LATl might beinvolved in Leydig cells proliferation and malignant transformation as well as in amino acid transport in rat
testis induced by prenatal exposure to DBP.

JSTP/IFSTP IATP-~200~

Posters

P-108
Infiltrating COB+ T and NK cells, and IL-1 O and TGF- p 1 cytokines expression inchemically induced neoplasias in a medium-term alternative bioassay using
male Wistar rats
Ana Lucia Tozzi SPINARDI-BARBISAN 1, Luis Fernando BARBISAN 1, Joao Lauro Viana de CAMARGO',Maria Aparecida Marchesan RODRIGUES'
Dept. Pathology, UNESP Medical School, SP 18618-000, Brazil 1
An initiation-promotion assay with Wistar rats was adopted by the Brazilian Institute for the Environment(IBAMA) as a source of evidence of the carcinogenic potential of pesticides. This bioassay protocol has beenoriginally proposed as an alternative/complementary assay for the standard 2-year rodent assay for chemical
carcinogenesis. In order to better understand this model, the present study aimed to evaluate the CD8+ T andnatural killer (NK) infiltrating cells and the expression of interleukin-IO (IL-10) and transforming growth
factor-beta 1 (TGF-betal) in the chemically induced neoplasias. Male Wistar rats were sequentially treatedwith N-nitrosodiethylamine, N-methy 1-N-nitrosourea, N-butyl-N-( 4-hydroxybutyl)nitrosamine, dihydroxy-diN-propy lnitrosamine and 1,2-dimethylhydrazine during 4 weeks. Two groups were subsequently exposed
through diet to phenobarbital (0.05 %) or 2-acetylarninofluorene (0.01 %) for 25 weeks. An untreated group
was used as control. Immune cells and cytokines were immunohistochernically evaluated in neoplasias and insurrounding normal tissues of the liver, kidney, lung, and small and large intestines. When compared to therespective normal tissues an increased number of NK cells was verified infiltrating the colon, lung andkidney neoplasias, being the number of CD8+ T cells decreased in the intestine and lung neoplasias.
Expression of IL-10 was found mainly in tumors of kidney and small and large intestines. TGF-beta 1 was
expressed mainly in liver and kidney tumors. The results indicate a differential immune activity withinneoplastic and in normal tissues which may be dependent of tumor development and characterizes its
microenvironment.

216

Posters
P-109
Green fluorescent protein (GFP) as a marker of aryl hydrocarbon receptor(AHR) function in transgenic zebrafish (Danio rerio)
Seung H. SEOK 1, Jong-H. PARK' , Sun A. CHO', Min W. BAEK', Hui Y. LEE 1, Dong J. KIM 1,
Yong S. LEE 2, Jae H. PARK 1
Department of Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University,
Seoul 151-742, Korea (South) 1
Department of Public Health, College of Veterinary Medicine, Seoul National University 2
Zebrafish (Dania rerio) is a very attractive experimental model system because it has short generation time
(12 weeks), long life span (2-3 years), and relatively small diploid genome. We are developing transgenic
zebrafish lines in which DNA motifs that respond to selected environmental pollutants are capable ofactivating a reporter gene that can be easily assayed . Aromatic hydrocarbon response elements (AHREs)
respond to numerous polycyclic hydrocarbons and halogenated coplanar molecules. Each of these substances
is known to be bioconcentrated in fish to varying degrees and would act upon the ARRE motif and turn on
the green fluorescent protein (GFP) reporter gene. ARRE is a ligand-activated transcription factor thatmediates the toxic actions of environmental contaminants such as beta-naphthoflavone (BNF). Induction ofcytochrome P4501Al (CYPlAl) is an early biomarker of ARRE activation. Injected fish exposed to BNF
exhibited induction of GFP in the liver and vertebrae of zebrafish compared to vehicle controls, which did
not express GFP.

Posters
P-110
Pathology of Minamata disease (methylmercury poisoning) - special reference
to the peripheral nerves
Komyo ET01, Akira YASUTAKE 1, Motohiro TAKEYA2, Michio AKIMA 3
National Institute for Minamata Disease, Kumamoto 867-0008, Japan 1
Graduate School for Medicial and Pharmaceutical Science, Kumamoto University 2
Toho University, School of Medicine3
Minamata disease was first discovered about 50 years ago in Minamata Bay, Kumamoto Prefecture, Japan. A
study group of Kumamoto University School of Medicine found that the disease was caused by humaningestion of a large amount of methylmercury (MeHg) contaminated fish or shellfish from Minamata Bay.
The mercury came from a nearby factory that manufactured acetaldehyde and vinyl compounds. The factory
used manganese dioxide as a cocatalyst from 1932 to 1951 in its acetaldehyde production plant. In 1951, thefactory changed the cocatalyst used in the process of producing acetaldehyde from manganese dioxide toferric sulfide. This resulted in the production of MeHg chloride, which the factory dumped directory into
Minamata Bay from 1951 to 1968. Over those 17 years, Minamata Bay was contaminated with a high level
MeHg in their meat and internal organs.
Sensory disturbance is a very important symptom of Minamata disease patients with constrictive visual
field s, hearing disturbance, ataxia, and dysarthria. It is well known that peripheral neuropathy is a common
but non-specific clinical and histopathological entity. The peripheral nervous system alone was found tosusceptible to MeHg in rodents, while swine and common marmosets showed lesions both in the central andperipheral nervous system similar to those we have seen in Minamata disease.
To evaluate peripheral nerve involvement in Minamata disease patients, we examined the peripheral nerves ofhuman autopsy and control cases, and also experimental MeHg poisoning in common marmosets. For
histochemical examination, we used antibodies to PG-Ml , KP-1 (CD 68, macrophage), protein product 9.5(PGP-9.5), neurofilament 210 (NF-210) and S-100. The infiltration of macrophages in the spinal posteriorroot was stronger than the anterior root in the Minamata disease patients, and also the infiltration ofmacrophages was prominent in the sciatic nerves of severe cases of MeHg poisoning in the common
marmosets. Spinal posterior root showed stronger degeneration than the anterior root by the staining of PGP9 .5 and NE-210. Schwann cells' proliferation is prominent in the spinal posterior root.
Our studies of MeHg poisoning in common marmosets and the autopsy studies on patients with Minamata
disease showed peripheral nerve damage, which at least in part contribute to the clinical sensory symptoms.
We conclude that pathogenesis sensory nerve lesions were derived from axonal degeneration and thepathological changes of the spinal posterior ganglions. For the study of the complete or incomplete
degeneration of peripheral nerves, it is important to use an electron microscope.

JSTP/IFSTP IATP ioo4

Posters

P-111
Mechanisms and disease control of N-methyl-N-nitrosourea-induced retinaldegeneration in animals
Katsuhiko YOSHIZAWA', Katsuji KIUCHP, Kaei MORIGUCHP, Yuji OISHI', Nobuaki SHIKATA2,
Airo TSUBURA2
Department of Toxicologic Pathology, Toxicology Research Laboratories, Fujisawa Pharmaceutical Co. , Ltd.,
Osaka 532-8514, Japan 1
Department of Pathology II, Kansai Medical University 2
Department of Ophthalmology, Kansai Medical University 3
Retinitis pigmentosa (RP) is a human disease characterized by loss of photoreceptor cells leading to visual
disturbance and eventually to blindness. Establishment of reliable animal models is essential for better
understanding of this disease and approaches to therapeutic intervention. N-Methyl-N-nitrosourea (MNU)induced retinal degeneration is highly reproducible, and involves photoreceptor cell loss that ends
approximately 7 days after a single systemic administration of MNU to mice (60mg/kg), rats (60 to 75mg/kg), hamsters (90mg/kg), shrews (65mg/kg), and monkeys (40mg/kg). Although the triggering mechanismsof pathogenesis are different and the apoptosis cascade may differ from human RP, MNU-induced
photoreceptor cell loss is due to apoptosis with down-modulation of Bcl-2 protein, up-regulation of Bax
protein, and activation of caspase families. Therapeutic approaches to control MNU-induced photoreceptor
cell loss have been evaluated. The first strategy we attempt to suppress photoreceptor cell loss was shutting
down the apoptosis cascade; caspase-3 inhibitor (4000ng Ac-DEVD-CHO injected intravitreally twice)partially suppressed disease progression. Next, we tested the ability of nicotinarnide (a water-soluble B-groupvitamin; 25 tolOOOmg/kg nicotinamide injected subcutaneously) to expect damaged DNA repair or by othermechanisms, and found to be effective. Also, dietary supplementation of 9.5 % docosahexaenoic acidcounteracted photoreceptor cell loss. Suppression of MNU-induced photoreceptor apoptosis in animals may
give therapeutic information for human RP control.
Key words: Retinitis pigmentosa, Retinal degeneration, Caspase-3 inhibitor, Apoptosis, Nicotinamide,
Docosahexaenoic acid, N-Methyl-N-nitrosourea
References:
1.Tsubura A, Yoshizawa K, Miki H, et al. Phylogenetic and ontogenetic study of retinal lesions induced by N
-methyl-N-nitrosourea in animals. Anim. Eye Res. 17: 97-103, 1998 (in Japanese; abstract in English).
2.Yoshizawa K, Nambu H, Yang J, et al. Machanisms of photoreceptor cell apoptosis induced by N-methyl-N-nitrosourea in Sprague-Dawley rats. Lab. Invest. 79: 1359-1367, 1999.
3. Yoshizawa K, Yang J, Senzaki H, et al. Caspase-3 inhibitor rescues N-methyl-N-nitrosourea -induced
retinal degeneration in Sparague-Dawley rats. Exp. Eye Res. 71: 629-635 , 2000.
4. Kiuchi K, Yoshizawa K, Shikata N, et al. Nicotinarnide prevents N-methyl-N-nitrosourea- induced
photoreceptor cell apoptosis in Sprague-Dawley rats and C57BL mice. Exp. Eye Res. 74: 383-392, 2002.
5. Mo1iguchi K, Yuri T, Yoshizawa K, et al. Dietary docosahexaenoic acid protects against N-methyl-Nnitrosourea-induced retinal degeneration in rats. Exp. Eye Res. 77: 167-173, 2003 .

JSTP/IFSTP IATP 20021

Posters

P-112
Neuronal cell death in the fetal rat spinal cord following NMDA-treatment todams
Hirotake TAK.AP, Tsuneo IT0 1, Kiyoka KATSUYAMA 1, Masarni SUZUKI1, Kei-ich KATAYAMA2,
Kunio DOF
Safety Assessment Department, Chugai Pharmaceutical Co., Ltd., Shizuoka 412-8513, Japan 1
Department of Veterynary Pathology, Graduate School of Agricultural and Life Sciences, The University ofTokyo 2The N-methyl-D-aspartate receptor (NMDAR), which is one of the glutamate receptors, is considered to havea close relation with synaptic plasticity in the rat brain. Recently we clarified that apoptosis was induced in
the fetal rat brain following NMDA administration to the dams (Exp Toxic Pathol 2003; 55: 33-37). In
addition, it is also reported that NMDARs are expressed in the spinal cord as well as in the brain. In thisstudy, we carried out a histopathological study on the fetal rat spinal cord after NMDA administration todams.
[Materials and Methods] Pregnant rats at embryonal day of 20 (E20) were injected with 150 mg/kg of
NMDA, NMDAR agonist, intraperitoneally. Dams were sacrified at 24 hours after the treatment, and thefetuses were collected. The spinal cords from the fetuses were fixed in 10% neutral-buffered formalin.
Paraffin sections were stained with hematoxylin and eosin. Some cases were stained for the TUNEL method,a widely used method for the in situ detection of apoptotic nuclei. Immunohistochemical staining for MAP-2
was performed for the control group.
[Results and Discussions]In the control group, morphological differences were observed between the dorsal
and ventral neurons in the spinal cord. The neurons in the dorsal horn, which were positive for MAP-2, were
small and round-shaped with large oval nuclei and scant cytoplasm. On the other hand, many of the cells inthe ventral horn, which were also positive for MAP-2, were large and multiangular-shaped with relatively
abundant cytoplasm. In the NMDA-treated group, a large number of cells characterized by pyknotic orkaryorrhectic nuclei, almost all of which were stained by the TUNEL method, were mainly detected in thedorsal horn while a smaller number was observed in the ventral horn. The cytoplasm of these cells waseosinophilic and somewhat glanular. Thus, differences in the morphology and the sensitivity for NMDA wereseen between the dorsal and ventral neurons in the fetal spinal cord. Differences in the stage of celldifferentiation and/or distribution of NMDARs among the regions may contribute to the different changes
observed in the present study.

220

JSTP/IFSTP IATP 2QQ4

Posters

P-113
Analysis of cell cycle, migration and apoptosis of neural stem cells in the 5azacytidine (5AzC)-treated rat fetal brain
Masaki UENO', Kei-ichi KATAYAMA', Hiroyuki NAKAYAMA', Kunio DOI'
Department of Veterinary Pathology, The University of Tokyo, Tokyo 113-8657, Japan'(Background) 5-azacytidine (5AzC), a cytidine analogue, is suggested to induce p53-dependent apoptosis
and cell cycle arrest in the rat fetal brain. In the fetal brain, neural stem cells are proliferating in theventricular zone (VZ), the area around the ventricles. In VZ, the nuclei of neural stem cells undergo a typicalmigration, "elevator movements", whose positions are co1Telated with the cell cycle phases. Namely, S phasenuclei located in the outer area of VZ translocate inward during G2 phase and mitosis occurs at theventricular surface. Then, the nuclei migrate outward during G 1 phase and enter into S phase again. In thepresent study, to clarify the detailed mechanism of 5AzC-toxicity to the neural stem cells, we
histopathologically examined the change of their migration and further used flow cytometric methods forinvestigating the alteration of cell cycle distribution and the cell cycle position of apoptotic cells.
(Materials and methods) 5AzC (10 mg/kg) was injected intraperitoneally (i.p.) into pregnant Jcl:Wistar rats
on day 13 of gestation and sacrificed at 1, 3, 6, 9, 12, and 24 h after treatment. Forebrains of fetuses wereobtained under stereoscopic microscope, and then subjected to flow cytometric methods. Cells were stainedwith propidium iodide (PI) for cell cycle analysis and detection of fragmented DNA was performed using
TUNEL method. For detecting migration, we used BrdU-incorporating method. In brief, 5AzC (10 mg/kg)
and BrdU (20 mg/kg) were injected into pregnant rats i.p simultaneously and BrdU-positive cells in the fetal
brain were sequentially examined immunohistochemically.
(Results) In the BrdU-incorporating method, BrdU-positive neural stem cells in S phase firstly migrated
inward and remarkably accumulated along the ventricle at 6 h. Then, they migrated outward and underwent
apoptosis from 9 to 24 h. The migration mostly delayed from 6 to 12 h compared with controls. Theseobservations suggested that 5AzC induced cell cycle arrest in some phases and apoptosis after M phase.
Using flow cytometric methods, G2/M phase cells were remarkably increased at 6 to 9 h and then decreased,suggesting an occurrence of cell cycle an·est in G2/M phase. Apoptotic cells were increased at 9 to 24 h. The
cells undergoing apoptosis were mainly in G 1 phase, however apoptosis was also observed in G2/M and S
phase cells.
(Conclusion) In the rat fetal brain, 5AzC induced cell cycle arrest in G2/M phase and apoptosis mainly in G
1 phase. This was coincided with the delay of nuclei migration.

221

•

·~r

-~

JSTP/IFSTP IATP 200"

Posters

P-114
T-2 toxin-induced changes in the rat fetal brain
Shinya SEHATA1, Naoki KIYOSAWA2, Toshihiko MAKIN0 2 , Fusako ATSUMI2, Kazumi IT0 2,
Takashi YAMOT0 2 , Munihiro TERANISHI2, Sunao MANABE2, Koji UETSUKA1, Hiroyuki NAKAYAMA1,
Kunio DOI'
Department of Veterinary Pathology, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Tokyo 113-8657, Japan 1
Medicinal Safety Research Laboratories, Sankyo Co. , Ltd. 2
T-2 toxin is a trichothecene mycotoxin produced by various species of Fusarium spp . We reported that T-2
toxin induces apoptotic cell death in the lymphoid tissues, gastrointestines, liver, placenta and fetal tissues in
the pregnant rats at 24 or 48 hours after treatment (HAT). The mechanism of T-2 toxin-induced toxicity is
still unclear. In the present study, we focused on the changes observed in the fetal brain as one of the target
organs of T-2 toxin-induced fetotoxicity. The purpose of the present study was to investigate sequential
morphological changes and gene expression changes in the fetal brain induced by T-2 toxin.
Wistar rats (3 rats/group) on day 13 of gestation were orally administered with T-2 toxin at a single dosageof 2 mg/kg. At 1, 3, 6, 9, 12 and 24 HAT, rats were sacrificed. The fetal brain was fixed andhistopathological examination was conducted. TUNEL staining was performed, and morphometry was donein the telencephalon (labeling index: number of positive cells/500 cells). Microarray analysis was performed
for the samples at 6, 12, and 24 HAT using the Affymettix Rat Genome U34A oligonucleotide chip
(Affymetrix, USA). Real time RT-PCR was also performed for selected genes.
Sequential histopathological examination showed that the number of apoptotic neuroepithelium in thetelencephalon was increased by T-2 toxin. Morphometry using TUNEL staining showed that the labelingindex was increased from 1 HAT. The highest labeling index was observed at 12 HAT. From thehistopathological results, we selected the time point (6, 12 and 24 HAT) for the microarray analysis. We are
now conducting the analysis, and will also perform a real time RT-PCR analysis for some selected genes
obtained from the results of microarray analysis. We will present the result at the meeting.

222

'

....

f't.-~<V"l'ir

JSTP/IFSTP IATP 2004

Posters

P-115
Examination of discriminative diagnosis of brain tumors difficult to be stainedwith hematoxylin-eosin
Yoshihumi KANEKO 1, Akiko IKEDA1, Makiko TAKAHASHI 1, Sumihisa SUEYOSHI 1, Yasukazu SAT0 1,
Yoshihiro MASUMOT0 1, Kiyoyuki TSURU 1

Kyorin Pharmaceutical Co., Ltd. Research Center, Tochigi 329-0114, Japan 1
Generally, lesions suspected of tumors as well as non-tumor lesions are diagnosed on morphologicalstandpoints with stained tissue preparations. Discriminative diagnosis between difficult-to-discriminate lesions
requires additional information, for example, based on some special staining or other technique. We
examined several staining methods to select an applicable one for such diagnosis of well-known brain tumors(atrocytoma and oligodendroglioma) in rats, in combination with ultra-structural observation on an electron
microscope. Brain tumors were induced to pregnant rats by giving a transplacental dose of ethyl nitroso-urea(ENU, 50 mg/kg) during the days 14 to 20 of gestation and then discriminatively diagnosed on theirmorphological observation. Animals found dead were included in these examinations to consider theirpostmortem changes .
Examination showed that Luxol fast blue (LFB) and Aldehyde fuchsin (AF) each stained characteristicallythe cytoplasm of astrocytoma in survivals, and distinguished between astrocytoma and oligodendroglioma. In
addition, transmission electron microscopy revealed that Rosental fibers were present in astrocytoma, andthat in oligodendroglioma, intermediate filaments were present and rough endoplasmic reticula (RERs)
increased. Lipid markers were traced but found not useful for discriminative diagnosis, because
phosphatidylinositol 4-monophosphate (PIP) was located in the cytoplasm of both tumors andphosphatidylinositol 4,5-biphosphate (PIP2) also in the nuclear membrane of both tumors. Meanwhile, LFB
stained the cytoplasm of astrocytoma in dead animals whereas AF did not. Transmission electron microscopy
revealed that characteristic ultrastructures were present in both tumors in dead animals but less than insurvivals. Grial fibrillary acidic protein (GFAP:astrocytoma's marker) and Ki-67 (marker for cell malignancy)
were found not useful for tumor discrimination in this study.
In conclusion, the results suggest that conventional LFB and AF staining methods may be used for
distinction between astrocytoma and oligodendroglioma in survival animals, and that LFB may be used fortheir distinction in dead animals within one day after death.

JSTP/IFSTP IATP ,,20.0 4

Posters

P-116
A spontaneous cholesterol granuloma of choroid plexus in a beagle dog
Shuji HAYASHI1, Satoshi SUZUKI' , Fumiko NINOMIYA 1, Kazuo HAKOI 1, Shuji YAMAGUCHI1,
Kenji IRIMURA 1, Shoji FUKUSHIMA2
Drug Safety Research Laboratory, Taiho Pharmaceutical Co ., LTD. , Tokushima 771 -0194, Japan 1
Depaitment of Pathology, Osaka City University Medical School2
A cholesterol granuloma of the choroid plexus was identified within the fourth ventricle of a male 72-monthold beagle dog. No remarkable clinical signs were observed. Macroscopically, a nodule was 5 x 4 x 3 mm insize and tan in color. Some pearl-like spots were observed in the nodule. The aqueduct of the mid brain wasslightly dilated. Other remarkable findings were not observed. Histopathologically, the nodule was mainlycomposed of numerous cholesterol clefts, lipid-laden macrophages, granulomatous tissues and cystic
structures in the choroid plexus. Focal hemorrhage, mononuclear inflammatory cell infiltration, hemosiderinladen macrophages and giant cells were scattered. In addition, proliferation, hyalinization and calcification ofconnective tissue, and edema in interstitium were occasionally observed. The cystic structures were linedwith cu boidal, columnar or stratified squamous epithelium. The lumens of the cysts were filled withcholesterol clefts, lipid-laden macrophages, erythrocytes, neutrophils, eosinophilic serous fluids and celldebris. Immunohistochemically, the epithelial cells of cystic structures were positive for cytokeratin, and thespindle cells around cholesterol clefts were positive for smooth muscle actin. No adhesions and invasions tothe surrounding nervous tissues were observed. This is a rai·e case report of the cholesterol granuloma of thechoroid plexus in a beagle dog.

224

JSTP/IFSTP IATP 2{)04

Posters

P-117
A study on the developmental process of distal axonopathy caused byacrylamide
Naofurni TAKAHASHI1, Maki KUWAHARA 1, Yukiko TAKEUCHI 1, Toshinori YOSHIDA1,
Akiko ENOMOT0 1, Nobuaki NAKASHIMA1, Yasufumi SHUTOH 1, Sayaka ISHIMINE 1,
Toshiaki KITAZAWA3, Keizo MAITA2, Takanori HARADA 1

Toxicology Division II, The Institute of Environmental Toxicology, Ibaraki 303-0043, Japan 1
Division of Study Planning and Consultation, The Institute of Environmental Toxicology 2Contract and Research Management Division, The Institute of Environmental Toxicology 3
Acrylarnide (ACR) is a prototypical neurotoxicant that produces distal axonopathy and is recommended byregulatory agencies as a positive control substance for neurotoxicological studies of chemicals. We conducteda 28-day validation study of ACR in rats. In the validation study, ACR was repeatedly administered bygavage to male and female Crj:Wistar rats at dose levels of 0, 10, 20, and 40 mg/kg/day, but the duration of
exposure to the high-dose level was shortened to 14 days because of severe intoxication. As the results ofdetailed clinical observations and functional tests, dysfunctions of the central-peripheral nervous andneuromuscular systems were observed in the 40 and 20 mg/kg/day groups. Neuropathological examinations
revealed moderate necrosis of Purkinje cells and slight axonal degeneration of peripheral nerves in the 40 mg/kg/day group. Minimal Purkinje cell necrosis and severe axonal degeneration were also observed in the 20
mg/kg/day. These results were consistent with previous reports. It is generally believed that ACR primarily
produces nerve cell damage in the central nervous system (CNS) when exposed to a high-dose level, whereas
a low-dose exposure induces more prevalent lesions in the peripheral nervous system (PNS). However, recent
investigations by Lehning et al. have suggested that ACR intoxication may produce early and progressive
nerve terminal degeneration regardless of dose levels, and distal axonal degeneration is a secondary effectrelated to duration of exposure. Thus, the toxic profile of ACR at high- and low-dose levels is still open to
discussion. In order to confirm this toxic profile, we have conducted a further 14-day study of ACR in rats at
a dose of 50 mg/kg/day. In the additional study, animals showing abnormal gait were selected based on its
severity and killed at a certain interval during the study to examine the developmental process of CNS
lesions caused by ACR. A contemporary amino-cupric-silver stain method was used to detect degeneration ofneuronal dendrites, cell bodies, terminals and axon tracts. In addition, electron microscopic examination was
conducted. Histopathologically, degeneration of Purkinje cells was observed in the ACR-treated rats and theoccurrence of the lesion was consistent with the time when the abnormal gait appeared. Detailed examinationof the cerebrum is now underway.

JSTP/IFSTP IATP "2001

Posters

P-118
Screening test for central neurotoxicity by a newly developed human hybrid
neuronMin-Cheol LEE1, JK RYU2 , SU KIM 2, KH YANG 3
Department of Pathology, Chonnam National University Medical School, Chonnam 501-190, Korea (South)'Brain Disease Research Center, Ajou University 2
Department of General Toxicology National Toxicology Institute3
A human hybrid neuronal cell line Al has been generated by somatic fusion between a human fetal cerebral
neuron and a human neuroblastoma cell, and tested neurotoxicity for beta amyloid. RT-PCR,
imrnunochernical, and electrophysiological studies of the hybrid cells indicated that the cells express
faithfully of morphological, imrnunochernical, physiological, and genetic features of human cerebral neurons.
Al hybrid neurons express neuron-specific markers such as neurofilament-L (NF-L), NF-M, NF-H, MAP-2,
and beta tubulin III. Al human hybrid neurons express messages for various cytokines and cytokine
receptors which are similar to parental human CNS neurons and different from the other parental cell line,
SK-SH-SYSY neuroblastoma. Al hybrid neurons also express messages for choline acetyltransferase (ChAT),tyrosine hydroxylase (TH), and glutarnic acid decarboxylase (GAD), indicating that they could differentiate
into various subsets of neuronal types. Whole-cell patch clamp experiments showed that Al hybrid neurons
expressed Na+ currents, which were completely blocked by tetrodotoxin. In addition, depolarizing andhyperpolarizing voltage clamp steps evoked respective outward and inward K+ currents in these cells. WhenAl hybrid neurons were exposed to beta amyloid for 72 hr, there was three-fold increase in TUNEL positive
cells over controls, indicating that beta amyloid is neurotoxic to Al hybrid neurons. The present study
indicates that the Al human hybrid neuronal cell line should serve as a valuable cell for the screening ofcentral neurotoxicity in vitro as well as for the studies of biology, physiology, and pathology of humanneurons in health and disease.

226

JSTP/IFSTP ·1ATP 2004

Posters

P-119
Developmental neurotoxicity of domoic acid in rat
Min-Cheol LEE 1, YS KIM 1, JY W00 1, J KIM1, KH YANG 2
Department of Pathology, Chonnam National University Medical School, Chonnam 501-190, Korea (South) 1
Department of General Toxicology National Toxicology Institute2
Domoic acid (DA), an amnestic shellfish poisoning, has been known a kainate-receptor agonist resulting
excitotoxic injury of the brain. It has been known that the hippocampus had well-developed glutamate
receptors. To investigate neuronal injury of the hippocampus, DA (0.8, 1.0, 2.0, 2.5 mg/Kg) was
administrated intravenously via tail vein in the different developmental periods of rats consisting of fetal
stage; EIS, neonatal stage; Pl4, actively growing stage; P35 , and young adult stage; P70. Histopathologicaland immunohistochemical studies, ultrastructural studies, and Western blot analysis were performed from the
hippocampus at 3, 7, 14, and 28 days after DA treatment. Presence of neurobahavioural abnormalities andelectroencephalography were evaluated at 6 weeks after DA administration. Acute mortality rate was 40-50%
in rats treated with 2.5 mg/Kg of DA. Histopathologic features of neuronal injury of the hipocampus
demonstrated in all developmental periods of rats, and significantly severe injury observed in growing andyoung adult stage rats and over 1.0 mg/Kg of DA treated rats. There were two types of neuronal injury,
swollen or shrunken degenerating neurons, especially in the CAI and CA2 regions. The shrunken neurons
positively stained with c-FOS , and some of the neurons demonstrated ISEL positivity indicating apoptotic
cell death. Electron microscopically, clumping of heterochromatin of nucleus and shrinkage of cytoplasmic
organelles with increased number of mitochondria, and subsequent neuronal condensation were noted.
Western blot analysis revealed amplification of Bax, Bcl-2, and Caspase 3 proteins on 3 to 7 days after DA
administration. Various stages of convulsive seizures with epileptic spikes were developed. In conclusion,
neurotoxicity of DA was developed all of the experimental period from fetal to young adult stage, especiallyadministrated dosages over 1.0 mg/Kg. The hippocampal neuronal injury was predominant on CAI and CA2
regions which resulted abnormal convulsive seizures. DA-induced seizures might be a valuable animal model
to study epileptogenesis related to excitotoxic injury.

JSTP/IFSTP IATP 2ooi

Posters

P-120
Newly-established tumor lines from a spontaneous malignant schwannoma in
F344 rats
Jyoji YAMATE 1, Hisae YASUI 1, Mitsuru KUWAMURA1, Takao KOTANI 1, Sadashige SAKUMA1,
Jonathan LAMARRE 2
Laboratory of Veterinary Pathology, Osaka Prefecture University, Osaka 599-8531, Japan 1
Department of Biomedical Sciences, University of Guelph2
F344 rats are an inbred strain with genetically homogeneous nature, and have been widely used in chronic
toxicity and carcinogenecity studies. In order to develop animal tumor models, we have attempted toestablish various transplantable tumor lines from spontaneous tumors found in F344 rats in terms of studies
on biological behavior, morphological characteristics and paraneoplastic manifestation in relation toneoplasia. Schwann cells are derived from the neuroectodermal neural crest that can differentiate towards
meso-ectoderm in embryogenesis. The histogenesis and cellular characteristics of Schwann cell tumors
remain to be investigated. The frequency of naturally occurring malignant schwannomas in the soft tissue ofF344 rats, that had been maintained to 2 years of age, was less than 4%. In this study, transplantable tumor(KE) and cloned cell (KE-Fl 1) lines were established from a spontaneous malignant schwannoma found in a
24-month-old F344 rat. The primary tumor and KE tumors consisted of oval or spindle cells arranged in illdefined bundles. Cultured KE-Fl 1 cells exhibited polygonal or spindle configurations.
Immunohistochemically, neoplastic cells in KE and KE-Fll reacted to vimentin, S-100 protein, NSE, MBP,
and GFAP in varying degrees, indicating neurogenic features; occasional cells reacted to alpha-smoothmuscle actin. Cells positive for lysosomal enzymes (acid phosphatase and non-specific esterase), and EDI(rat macrophage- specific) were observed in KE-Fll , and electron microscopically, cells with many
Iysosomes were frequently present, indicating expression of macrophage-like phenotypes. Bioassay analysis
revealed that KE-Fl 1 cells produced high levels of NGF. DNA synthesis was inhibited by TGF-betal
addition, and Northern blot analysis revealed that expression of c-myc, a cell cycle-related immediate early
gene, was depressed by TGF- betal. Likely, TGF-betal is a factor capable of inhibiting cellular growth of
Schwann cells. mRNA expression of the low density lipoprotein receptor-related protein (LRP) was seen in
KE-Fl l cells by Northern blot analysis, and the level was decreased by lipopolysacccharide (LPS) treatment.
LRP may be attributable to regulation of Schwann cell functions. KE-Fl 1 cells seeded on laminin-coateddishes exhibited more extended cytoplasmic projections than on collagen type I-coated dishes. The present
study provides evidence that biological properties of malignant schwannoma-derived cells might be affectedby exogenous factors such as TGF-betal , LPS and laminin. These tumor lines would become useful forstudies on pathobiological characteristics of Schwann cells.

228

JSTP/IFSTP IATP 200·~

Posters

P-121
Spontaneous malignant schwannoma in F344 rat
Bang Hyun KIM', Dong Deuk JANG', Mina CHOI', Beom Seok HAN', Ki Taek NAM', Chul Kyu KIM',
Kook Kyoung LEE 2, Ki Dae PARK 1, Wan Seob CHO', Ki Hwa YANG'
Department of General Toxicology, National Institute of Toxicological Research, Seoul 122-704, Korea(South)'Department of Veterinary Medicine, Cheju National University 2
During the carcinogenicity study with diisodecyl phthalate (DIOP), we incidentally found one male F344 rat
which showed dyspnea and severe flexured lumbar vertebrae in the 0.2 % DIOP treated group at the age of
23 week. Two white to yellow masses were observed in the subcutis of the lumbosacral region and the left
inguinal region of abdominal cavity, 25*17*8 mm and 16*14*8 mm in size, respectively. Microscopic
findings were varied . Highly cellular and spindle cells were growing in sweeping fascicles, herringbone
pattern, and locally rossette-like pattern. They were pleomorphic and had more than 2 nucleoli.
Characteristically, extensive necrosis was observed over the whole masses. Imrnunohistochernically, theneoplastic cells were positive for S-100 but negative for smooth muscle actin, desrnin , etc. According tothese results, these masses in male F344 rat were diagnosed as malignant schwannoma. This case might be agood reference of malignant schwannoma.

JSTP/IFSTP IAfl>~2·00~

Posters

P-122
Histopathological characterization of nasal airway lesions induced byintranasal challenge with ovalbumin in sensitized guinea pigs
Emiko KUWASAKI 1, Koshirou KATO KU 1, Takafumi OSHIKATA1 , Yutaka NAKAHARA1,
Hideyuki WATANABE1, Masao HAMAMURA1, Kosuke MORIZUMF
Department of Pathology, Panapharm Laboratories Co. , Ltd., Kumamoto 869-0425, Japan 1
Department of Parmacology, Panapharm Laboratories Co. , Ltd. 2
Allergic rhinitis induced by challenge with ovalbumin in sensitized guinea pigs is commonly known. The
histopathological examination of the nasal airway lesions for the above pathological model was, however,shown in very few reports. We made an allergic rhinitis model induced by intranasal challenge with
ovalbumin in sensitized guinea pigs. In this study, we investigated the histopathological features due to thesuggested time-dependent nasal airway lesions. Male guinea pigs, 6 weeks old, were sensitized by a single
subcutaneous administration of saline containing ovalbumin and aluminum hydroxide as the adjuvant. The
sensitized animals were challenged by intranasal instillation with ovalbumin 14 days after the sensitization,and they received a second challenge with ovalbumin 7 days after the first challenge. 5 animals wereeuthanized at 0.5, 4 and 8 hours after the second challenge. The nasal cavity, the trachea and the lung were
fixed in 10% buffered formalin. The nasal cavity was decalcified in 10% EDTA. The paraffin sections ofthese organs were stained with hematoxylin and eosin, Luna, Alcian blue pH2.5 and periodic acid-Schiff,and these sections were processed for light microscopy. All guinea pigs challenged with the above method
were observed to have eosinophil infiltration in the mucosa of the nasal cavity. At 0.5 hours after the second
challenge, eosinophils infiltrated into the lamina propria underling the respiratory epithelia of the nasal
septum, the ventral wall and the nasoturbinates. At 4 and 8 hours after the second challenge, eosinophils
infiltrated from the lamina propria into the respiratory epithelia. Eosinophil infiltration into the lamina
propria and the respiratory epithelia of the nasal septum, the ventral wall and nasoturbinates tended to be
most intense 8 hours after the second challenge. At 4 and 8 hours after the second challenge, eosinophils
ex udated in the nasal cavity. At all time points, several mast cells, stained positively with Alcian blue pH2.5,
were fo und in the lamina propria of the nasal septum. The results of this study suggested that theeosinophils left the lumen of the nasal cavity from the lamina propria through the respiratory epithelia in thismodel. Eosinophil infiltration in this model was similar to that in human allergic rhinitis. In addition, thetime of eosinophil infiltration in this model was in agreement with the time of late nasal airway responses inallergic rhinitis.

JSTP/IFSTP IATP 200~

Posters

P-123
Hydroxyurea(HU)-induced apoptosis and changes in apoptosis-related genes
expression in the mouse fetal lung
Gye-Hyeong W00 1, Eun-jung BAK2, Koji UETSUKA 1, Hiroyuki NAKAYAMA1, Kunio DOI'
Department of Veterinary Pathology, The University of Tokyo, Tokyo 113-8657, Japan'
Department of Biomedical Science, The University of Tokyo 2
Hydroxyurea (HU), a potent mammalian teratogen, affects proliferating embryonic cells and inhibits DNA
synthesis. The teratogenic potential of HU has been well known in experimental animals for several decades.
In human beings, cases of respiratory distress have been reported. In this study, we assessed thecytotoxicologic effects of HU on the fetal lung by exposing pregnant mice to HU on day 13 of gestation.
The number of TUNEL-positive cells began to increase at 3 hours after treatment (HAT), peaked at 6 HAT,
and rapidly decreased at 24 HAT. This positive reactivity for TUNEL was seen mainly in mesenchymal cells.
On the other hand, the number of immunohistochemically p53-positive cells peaked at 3 HAT and decreased
at 12 HAT, and the expression of p53 mRNA was significantly elevated at 6 HAT. In addition, theexpression of apoptosis-related genes (p21 and bax) was also significantly elevated. Significant increase in
the expression of p21 mRNA was detected at 1 HAT to 12 HAT and that of bax mRNA was noted at 6
HAT. These results suggest that p53 may play an important role in HU-induced apoptosis in the fetal lung.

JSTP/IFSTP IATP · 200~

Posters

P-124
In vivo detection of lung tumors in mice by high-resolution X-ray
microtomography
Horst WEILER' , Kris MEURRENS 2 , Nora M. DE CLERCK3, Andrei A. P0STNOV3, Piter M. TERPSTRA 2
Philip Morris Research Laboratories, GmbH, Cologne 51149, Germany'
Philip Morris Research Laboratories Bvba2
University of Antwerp 3
The major application of micro-CT in biomedical research has been the imaging of calcified tissues. In vivo
high-resolution X-ray microtomography (micro-CT) is a promising technique for the non-invasive imaging ofsoft tissues of small laboratory animals. For example, detection and evaluation of structural lung disorders
has up to now required many animals to be killed at many timepoints, and for emerging lesions, such as
small tumors, time-consuming step-serial histological sectioning is unavoidable. As a possible alternative, theapp lication of X-ray micro-CT to detect lung tumors was investigated in live mice at an early and moreadvanced stage of tumor development. A/J mice were treated with urethane 1000 mg/kg bodyweight ip andexamined at 3.5 and 9 months post treatment. Healthy age-matched groups of mice were used as control.
The chest area of anesthetized mice was scanned for lung tumors by X-ray micro-CT (Skyscan 1076,
Aartselaar, Belgium) without gating for cardiac or respiratory motion. Positioning the mice on their backs in
the X-ray micro-CT and banding the chest area reduced motion artifacts. Scans lasted 17 minutes per mouse:spatial resolution was 35 µm and radiation load was 0.4 Gy. All mice survived the procedure with noapparent ill effects. Reconstructed virtual CT cross sections of the lungs (Feldkampf cone beam algorithm)
confirmed the macroscopic evaluation. Tumors in the hilar region or at the periphery of the lungs were moredifficult to localize on the CT cross sections. In step-serial sections (every 100 µm ) from one pair of lungs,
the tumors detected by CT were seen plus a few additional, very small tumors (~ 150 µm diameter) . The invivo X-ray micro-CT method opens broad perspectives for non-invasive imaging. Continued improvements inresolution and detection in soft tissue imaging may make this a powerful research tool for monitoring tumor
development and progression.

.

..f ....>•

JSTP/IFSTP IATP 20011

Posters

P-125
Eosinophilic change of the lining cells of the nasal cavity in rasH2 miceadministered di(2-ethylhexyl)phthalate
Takatoshi KOUJITANI 1, Kaoru TOYOSAWA1, Izumi MATSUMOT0 1, Kazuo OKIMOT0 1, Marni KOUCHP,
Koji KUROKP, Kohji TANAKN, Nobuo MATSUOKA 1
Safety Research Laboratories, Dainippon Pharmaceutical Co., Ltd., Osaka 564-0053, Japan 1[Introduction] Administration of Di(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, induces
hepatocellular adenomas and enhances eosinophilic change of the respiratory epithelium in the nasal cavity
in transgenic mice carrying a human prototype ·c-Ha-ras gene (rasH2 mice)(l). In the present study,
eosinophilic change in the olfactory region of the nasal cavity was examined in addition to the lesion in the
respiratory region.
[Materials and methods] Thirty-three male rasH2 mice were purchased at 6-week old . After 2-week
acclimation period, mice were divided into a control group (n=12) and a DEHP group (n=21). Mice in the
DEHP group were treated DEHP 6000 ppm in diet for 26 weeks. At necropsy, the head was fixed in 10%
neutral buffered formalin , and decalcified by 5% formic acid solution. Thereafter, the nasal cavity was
trimmed according to the method described by Nagano et al (2). Sections from the nasal cavity were stainedwith hematoxylin and eosin, PAS , Alcian blue, Toluidine blue and Masson-trichrome, and examined
microscopically.
[Results] Respiratory region: Eosinophilic change characterized by appearance of eosinophilic globules wasobserved in 10 of 21 mice in the control group and in all of 21 mice in the DEHP group, and respiratory
metaplasia of glands in 11 of 21 in the DEHP group. Histologic grade of the eosinophilic change wasgreater in the DEHP group than in the control group. Eosinophilic globules in the respiratory epithelium
were stained in red-orange by Masson-trichrome, however, were negative by PAS , Alcian blue and Toluidine
blue. Olfactory region: No histopathological change was found in the control group. Eosinophilic change wasobserved in all of 21 mice, and respiratory metaplasia in 4 of 21 mice in the DEHP group. The eosinophilicchange in the olfactory region was exclusively located in the olfactory epithelium of the dorsal meatus in thenasal cavity. Eosinophilic globules in the olfactory epithelium were stained in pale-purple by Massontrichrome, however, were negative by PAS , Alcian blue and Toluidine blue.
1) K. Toyosawa et al. Toxicol Pathol 29:458-466, 2001.
2) K. Nagano et al. J Toxicol Pathol 1:115-127, 1988.

JSTP/IFSTP IATP ·200~

Posters

P-126
Biological persistence and pathological changes of potassium octatitanate of
two different shapes.
Akira OGAMI 1, Takako OYABU1, Yasuo MORIMOT01, Hiroshi YAMAT0 1, Izumi AKIYAMA1,
Isamu TANAKA 1
Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health,Fukuoka 807-8555, Japan'
The biological effects (bio-persistence and pathological changes) in the rat lung were evaluated with two
different types of potassium octatitanate. The materials used in this study are; Potassium Octatitanate whisker(PTl), and Potassium Octatitanate Particle (PTP). The rats had the intratracheal instillation of PTl and PTP
with dose of 2mg were sacrificed at the time of 3days, 1week, 1 month, 3 months and 6 months after the
instillation. The bio-persistence of PTP was much shorter than PTlin the lung. Inflammation score showedthat the inflammation of PTl after instillation was much more persistent than in that of PTP. These results
suggests that the scale-shaped potassium octatitanate is much less hazardous to the rat lung than the fibershaped.

234

JSTP/IFSTP IATJf'2ooi

Posters

P-127
Sequential change in acute lung damage due to intratracheal instillation of
Quartz in F344 male rats establishment of a biological bioassay for detectionof lung toxicity by fine particles
Masanao YOKOHIRA 1, Hijiri TAKEUCHI1, Keiko YAMAKAWA 1, Kousuke SA00 1, Mico IKEDA 1,
Yoko HOSOTANI1, Zeng YU 1, Kyoko HOSOKAWA 1, Katsumi IMAIDA1, Makoto SHIRAISHF
Department of Onco-Pathology, Kagawa University, Kagawa 761-0793, Japan 1
Diagnostic Pathology and Cytology Institute, Shikoku Cytopathologic Institutes 2
There are many toxicants in our environment, including air pollutants, and it is an urgent priority toestablish biological bioassay for detection of hazards of fine particles, which can be inhaled into deep lungtissue by human being. Quartz instillation is known to produce an inflammatory reaction followed byhistological changes characteristic of lung fibrosis. In order to establish an appropriate bioassay for detectionof lung damage after particle inhalation, sequential histopathological changes were here examined using
Quartz as a typical lung toxic chemical.
A total of 50 F344 male, 10-week-old rats, were separated into two groups. 25 rats were exposed byintratracheal instillation to Quartz (DQ-12, 4mg/rat) suspended in saline (0.2ml) and subgroups of 5 rats
were killed on Days 1, 3, 7, 14, and 28. The remaining rats were exposed by intratracheal instillation tosaline (0.2ml) as a control group and 5 rats were killed on the same days as for the Quartz exposure group.
Histopathological changes were observed as follows ; neutrophil infiltration in the walls and spaces of thealveoli, histiocyte infiltration, pulmonary edema and pulmonary fibrosis, and restructure of the walls of thealveoli in all lungs of Quartz treated rats. These parameters could be scored. For detection of acuteinflammatory changes, neutrophil infiltration in the alveoli appeared to be an important parameter.
Furthermore, as subacute inflammatory changes, histiocyte infiltration in the alveoli, areas of pulmonary
edema and pulmonary fibrosis and restructure of the walls of the alveoli were readily identifiable. Lungs ofrats in the Quartz treated group killed on Day 1 demonstrated the strongest signs of acute inflammation,
whereas, lungs of rats killed on Day 28 had the most pronounced evidence of subacute inflammation.
Additionally, granulation with giant cells and macrophages in the alveoli were significantly observed.
According to the results, Days 1 and 28 after intratracheal instillation of test fine particles were suggested tobe the most appropriate days for detection of acute and subacute inflammatory changes, respectively. This
bioassay may be useful for detection of acute or subacute lung toxicity due to inhaled fine particles.

Posters
P-128
lmmunohistochemical demonstration of IL-13 protein in rat late athmatic
response model
Kiyoshi KOBAYASHI' , Osamu KURUSU1, Hiroyuki HIGASHIYAMA 1, Anbo XIANG', Hideo KIKKAWA 1,
Satoshi ASANO', Mine KINOSHITA'
Pharmacology Department, High Throughput Biology, TRL, GlaxoSmithKline K.K., Tsukuba 300-4247,
Japan'[INTRODUCTION] IL-13 is an immunoregulatory and effector cytokine in the allergic diseases such asbronchial athma. A variety of immune and non-immune cells are known as IL-13 producers. In the present
study, we immunohistochemically analyzed the IL-13 expressing cells in the lung tissue of ovalbumin(OVA)sensitized and challenged Brown-Nowway (BN) rat known as athma model.
[MATERIALS AND METHODS] Nine weeks old BN rats were intraperitoneally injected with OVA at adose of 100 mg/kg in Al(OH)3 (100 mg/kg) in 0.9% saline on 3 consecutive days. Three weeks after the
final OVA injection, rats were exposed to aerosol containing OVA at 1% (w/v) for 20 minutes. Animals were
killed under anesthesia 24 h after aerosol exposure. Inflammatory cell counting and ELIZA analysis for IL13 protein was performed using bronchioalveolar lavage fluid (BALF). In a separate experiment, lung tissue
was removed, fixed in zinc fixative, embedded in paraffin followed by histological and immunohistochemical
analysis using anti-rat IL-13 monoclonal antibody.
[RESULTS] In OVA-challenged rats, number of eosinophils and neutrophils in BALF was markedly elevatedbeing accompanied by the elevation of IL-13 protein concentration. Histological examination revealed
perivascular, peribronchiolar and alveolar infiltration of inflammatory cells consisting of neut:rophils,
eosinophils and macrophages. Immunohistochernically, eosinohils infiltrated in alveolar spaces were strongly
positive for IL-13 whereas those in perivascular/peribronchiolar area were negative or scantly positive. In
addition, alveolar macrophages were also stained positive for IL-13. Phagocytosis of degenerated eosinophils
and other materials by alveolar macrophages were observed suggesting the possible IL-13-positive reaction
in macrophages due to the phagocytotic reactoin .
[DISCUSSION] IL-13 is known to play an important role in various allergic diseases, and eosinophil
infiltration is one of the most characteristic findings in athma. Thus, IL-13 production by eosinophils withadvances in alveolar infiltration may play an important role in the pathogenesis of present animal modelthrough an autocrine mechanism.

236

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,1•111•

JSTP/IFSTP IATP 20021

Posters

P-129
One case of pleural mesothelioma in male F3441DuCrj rat
Hideki SENOH 1, Tetsuya TAKEUCHI 1, Yumi UMEDA 1, Taku KATAGIRI 1, Shigetoshi AIS0 1,
Kasuke NAGAN0 1
Japan Bioassay Research Center, Kanagawa 257-0015 , Japan 1
A case of mesothelioma spontaneously developing in the thoracic cavity of a male F344/DuCrj rat (Charles
River Japan, Kanagawa), found from 5,000 males used in 25 studies of 2-yr carcinogenic bioassay carriedout following the Japanese Guideline in the past 20 years is presented.
The present case was survived up to the end of a 2-yr study and received a complete necropsy. Grossobservation revealed numerous white nodules, from 2 to 5 mm in size, on the pleural smface of lungs, thediaphragm and the mediastinum accompanied by hemorrhagic fluid in the thoracic cavity. No abnormal
finding was seen in the abdominal or scrotal cavity. Specimens of nodules were fixed in 10% bufferedformalin solution and embedded in paraffin. Each 5 µm thick sections were stained with hematoxylin andeosin, and examined microscopically.
Multiple nodules developing on the pulmonary surface, parietal thoracic wall and diaphragmatic surface
showed same histological characteristics as same as mesotheliomas, usually occurring in the abdominal or
scrotal cavity of male rats. Nodules were composed of both cuboidal epithelial-like cells and mesenchymal
components. Proliferation of cuboidal epithelial-like cells were seen covering the surface areas and in the
inner part of the mesenchymal area. The epithelial-like growth was mostly arranged in a single layer,occasionally showing a papillary projection. However, multiple layer-arrangement and solid growth were also
observed. Mesenchymal components, mainly much of fibrous tissue, were found in the almost all areas.
The thymus was atrophic, but remained intact. The microscopic examination confirmed that no mesothelioma
was found in the abdominal or scrotal cavity of the present case.
From the above-described findings, this tumor was diagnosed as a rare mesothelioma probably originatedfrom the pleura in the rat.

237

JSTP/IFSTP IATP 200fl

Posters

P-130
Incidence of cardiomyopathy in rats treated with Ph/P varies with the rat strain
Shoji KASHIWABARA 1, Naoki KASHIM0T0 1, Toshihiro UESAKA1, Osamu KATOH',
Keiji WAKABAYASHF, Hiromitsu WATANABE'
Department of Cellular Biology, Research Institute for Radiation Biology and Medicine,
Hiroshima University, Hiroshima 734-8553, Japan 1
National Cancer Center Research Institute2
It is well known that 2-amino- l-methyl-6-phenylimidazo[4,5-b ]pyridine (PhIP) is a heterocyclic amine
derived from cooked meat and a potent environmental carcinogen for rat colon, prostate and mammary
glands. We have reported that the cardiomyopathy was induced in Donryu rats treated with PhIP. The aim of
this study is to clarify the rate of cardiomyopathy induced by PhIP in several strains of rats.
[Methods] The following seven strains of rats were used in this study: Crj:Donryu, Crj:CD, Crj:Wistar, WKY/NCrj, SHR/NCrj, LEW/Crj and BN/Crj. Each strain was divided into two groups. One was used as a PhIP
treatment group and the other as a non-treatment group. PhIP (75 mg/kg/day) was administered by gavage 3
times per week at two-day interval to a total of 10 doses. Saline was administered as a control instead of
PhIP. Animals in a mo1ibund state were killed and autopsied, and all others were killed 53 weeks after the
initial treatment. The incidence of cardiomyopathy in each strain was investigated.
[Results and summary] No significant difference in the mean survival was observed among the groups,
except for in the Donryu rats. Donryu rats began to die due to cardiomyopathy after the first PhIP treatment,reaching 45% at 98 days and 80% at 310 day s. The body weights of Donryu, CD, LEW and BN rats in thetreated groups were significantly decreased in comparison with non-treatment. The relative heart weights(heart weight/body weight) of LEW/Crj in the treated groups were significantly greater than those in the non-treated groups . The blood pressure showed no significant difference between the treated and non-treatedgroups of each strain. Cardiomyopathy was observed to develop only in the Donryu strain. These results
indicate that induction of cardiomyopathy by administration of PhIP occurs only in Donryu, but not in anyof the other treated strains.

238

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•

::-"7V..Jl'""-....-a11

JSTP/IFSTP IATP 2004

Posters

P-131
Histopathological effect of Enalapril maleate on fetal heart development in rat.
A.rash KHAKI1, Iraj SOHRABI HAGHDOST 1, Amir Afshin KHAKI2, Mahnaz HAYDARP
Department of Veterinary Pathology, Islamic Azad University, Tabriz/Iran, Tabriz 51388, Iran 1
Department of Histology in Medical College of Tabriz University/Iran2
Department of Pharmacology, Shahid Beheshty Medical College Tehran/Iran 3
Enalapril maleate is an antihypertensive drug, which could reduce blood pressure by dilating blood vessels
and diminishing systolic of diastoles pressure. Since there is little information about enalapril maleate side
effect on of heart rat embryo. This preliminary study was planed to see what kind of changes occurs indevelopment of heart in Rat embryo. The aim of present study was to determine the histopathological effects
of enalapril maleate on heart of rat fetal. Twenty male Wistar Rat & twenty female Wistar Rats were selected& matched together & after pregnancy, randomly divided into two groups; control (n= lO) and test (n= lO) .
The test group has been received 0.4mg/kg (PO) enalapril maleate daily during pregnancy. However, thecontrol group just received plate. After delivery, heart tissue of fetal in both groups were taken and prepared
for light microscopy. Staining method was H&E. Microscopic study of heart tissue slices showed that in thetest group myocardial cell were smaller and nucleus were denser when controlled to control group. Weightof fetal in test group was reduced in compared to control group (P<0.05). Since enalapril maleate had side
effect on heart of rat fetal. It is not suggested that enalapril maleate has been used during pregnancy inhuman.

JSTP/IFSTP IATP 200~

Posters

P-132
Human heart glutamate receptors (GluRs) - possible effector sites: An
opportunity for drug discovery.
Olga PULID0 1, John VEIN0T2 , Ruedi MUELLER 1, Meghan KAVANAGH 1, Colin ROUSSEAUX3,
Santokh GILL1.
Pathology Section, Toxicology Research Division, Food Directorate Bureau of Chemical Safety, HPFB,
Health Canada, Ontario KIA OU, Canada 1
Department of Pathology and Laboratory Medicine, University of Ottawa, Civic Hospital Carnpus2
Department of Cellular and Molecular Medicine, Faculty of medicine, University of Ottawa3
Excitatory amino acids (EAAs) mediate their effects through the glutamate receptors (GluRs) in the brain.
GluRs play an important role in the pathobiology of a variety of neuropsychiatric conditions and in theirtreatment; they are also central to the neurotoxicity of EAAs such as domoic and kainic acid. Previously wedemonstrated the presence of GluRs in the heart of rats and nonhuman primates, where they are
preferentially localized within the different components of the conducting system, nerve terminals andintramural ganglia cells. The findings of severe cardiac lesions in Californian sea lions that died fromdomoic acid (DA) intoxication suggest that DA and other glutamate analogs may be potentially cardiotoxic
and these effects may be mediated through the GluRs. Since the GluRs are present in cardiac and otherperipheral tissues, we have hypothesized that EAAs can exert systemic effects through a common mechanism
mediated by GluRs. We investigated the presence of GluRs in human hearts using irnmunohistochemistry.
Unstained histological preparations of human heart tissues were obtained from archived specimens collectedduring postmortem examinations following the standard autopsy procedures established at the Ottawa Civic
Hospital. All tissues were fixed in formaldehyde, embedded in paraffin, sectioned at 5-6µ mounted on
salinated slides and stored until stained. Preliminary results showed that several subtypes (ionotropic andmetabotropic) of GluRs tested are differentially expressed in all the human hearts assessed, and the
distribution and stain intensity varieg with of each receptor subtype. In contrast to non-human primates,
GluRs are more widely expressed in humans, where the specialized structures of the conducting system, theworking myocardium, the wall of blood vessels, intramural nerves and ganglia cells stained for GLuRs.
Slides stained with H&E were used for the histopathological assessment and for the anatomical identificationof specific cardiac structures such as the different components of the conducting system. This is the firstreport of GluRs in human heart. Hence, they suggest that in human these receptors may be involved on
important cardiac functions such as contraction, rhythm, coronary circulation and thus the pathobiology ofsome cardiac disease. Further functional and pharmacological characterization of these GluRs in the heart
could lead to the development of therapeutic agents designed specifically for heart GluRs.
*This study was supported by a grant from the Office of the Chief Scientist and approved by the Health
Canada Research Ethics Board

240

JSTP/IFSTP IATP 20·0~

Posters

P-133
Enzyme- and immuno-histochemical changes in rat salivary glands induced bytheophylline
Satoru KATIKAWA1, Kenji NAKAN01, Aisuke NII1, Hiroyuki NAKAYAMA 2, Kunio D0I2
Safety Research Labs., Yamanouchi Pharmaceutical Co. , Ltd., Tokyo 174-8511, Japan'
Laboratory of Veterinary Pathology, Graduate School of Agricultural and Life Sciences, The University ofTokyo 2Introduction Theophylline (1,3-dimethylxanthine), a phosphodiesterase inhibitor, is widely used as a smoothmuscle relaxant and its efficacy may be associated with increased cellular cAMP as a result of inhibition ofphosphodiesterase. A high dosage of theophylline is also known to induce salivary gland hypertrophy. It waspreviously shown that a single administration of theophylline induces salivation and time-dependent
decreases in organ weight and the number of secretory granules in the salivary glands up to 4 hours after thetreatment. However, the precise mechanisms of salivary changes by theophylline still remain unclear. In thepresent study, the changes in salivary glands induced by theophylline were examined using immuno- andenzyme-histochemical methods.
Materials and Methods Male F344 rats were given theophylline at 50 mg/kg intraperitoneally, sacrificed at 1
and 4 hours after the treatment, and the submaxillary and parotid glands were excised (n=3). As a vehicle
control, physiological saline was given in the same fashion. Enzymehistochemistry for phosphodiesterase and5' -nucleotidase was performed using frozen sections. Immunohistochemistry for aquaporin 5, a water
channel protein, and a-amylase was done using paraffin sections.
Results In the submaxillary glands of the control group, the activity of phosphodiesterase was diffusely
detected and that of 5' -nucleotidase was observed in the peri-acinar and peri-intercalated duct areas. The
expression of aquaporin 5 was evident in the luminal area of the acinus and intercalated duct. Alpha-amylase
was not detected. At 4 hours after the treatment, the activity of phosphodiesterase was reduced, while that of
5' -nucleotidase was increased. Aquaporin 5 signals were increased and also detected in the cytoplasm ofacinar cells. In the parotid glands of the control group, the activities of phosphodiesterase and 5 ' nucleotidase were detected exclusively in the intercalated ducts. Alpha-amylase was expressed in thesecretory vesicles. Aquaporin 5 was not detected in any portion of the parotid glands. In the theophylline
group, the signals of a-amylase were reduced time-dependently, while the activities of the two enzymes were
unchanged.
Discussion Reduced activity of phosphodiesterase by theophylline was demonstrated in the submaxillary
glands. Subsequently increased cAMP might induce the expression of water channels, leading to saliva
secretion. By contrast, the activity of phosphodiesterase was unchanged in the parotid glands despite the time-dependent reduction of a-amylase. Therefore, there might be different mechanisms of saliva secretion anddifferent responses to theophylline between the submaxillary and parotid glands.

JSTP/IFSTP IATP ·200~

Posters

P-134
Histopathological study of the time course changes in the PTHrP-induced
incisor lesions of rats.
Atsuhiko KAT0 1, Masami SUZUKI1, Yayoi KARASAWA1, Tetsuro SUGIM0T01, Kunio D0I2
Safety Assessment Dept.Chugai Pharmaceutical Co., Ltd. , Shizuoka 412-8513, Japan 1
Department of Veterinary Pathology, Faculty of Agriculture, University of Tokyo 2
Parathyroid hormone related peptide (PTHrP) was known as a causative factor of humoral hypercalcemia ofmalignancy (HHM). Previously we have reported our HHM model showed incisor fracture withsymmetrically localized odontoblast lesions. In this study, the early response of odontoblats to elevated
PTHrP, its progression, and relation to the fracture are described, and the possible role of PTHrP in theodontogenesis is discussed. Twenty HHM nude rats were prepared by implantation of PTHrP-expressing
human tumor cell line (LC-6), and each 5 of them were sacrificed at 2, 5, 8 and 10 weeks after implantation,
along with 3 untreated control animals. The plasma and incisors were collected for measurements of blood
parameters (Ca2+, PTHrP), and for preparation of paraffin embedded H&E staining sections, which were
evaluated histopathologicaly and histomorphmetricaly. In histomorphometry analysis, both columnar- andhigh-columnar-odontoblastic cell heights were measured in each sampling point. Blood Ca2+ and PTHrPlevels were increased in week-5 and later HHM rats. All week-8 and -10 HHM rats fractured its incisor in 7
weeks after tumor implantation. In histopathology, sh01tening of odontoblast was observed in week-5 andlater HHM rats. In week-8 and -10 HHM rats, shortening of odontoblast was accompanied by thinning ofdentin, and was the only change, which reached to the fracture area. In histomorphometry, cell heights weredecreased in culumnar-odontoblats of week-5 , and in both columnar- and high-columnar-odontoblasts ofweek-8 and -10 HHM rats. From these results, as an earliest response to the elevated PTHrP, columnarodontoblasts are decreased its cell height. This change is progressed to more mature high-columnar
phenotype, and accompanied by thinning of dentin in later stage. Finally, thinning of dentin deteriorate thestrength of the incisor, and is caused fractures. Since it is known PTHrP delays the maturation ofchondrocytes, it is suggested that the response of odontoblasts to elevated PTHrP is not mere decrease of thecell height, but a maturation delay.

242

JSTP/IFSTP IATP 2004

Posters

P-135
Spontaneous ectopic sebaceous glands (Fordyce's granules) in the oral
mucosa of Sprague-Dawley rats
Masako IMAOKA1, Hiroshi SATOH 1, Kiyonori KAP, Tetsuyo KAJIMURA2 , Kazuhisa FURUHAMA'
Drug Safety Research Laboratory, Daiichi Pharmaceutical Co., Ltd., Tokyo 134-8630, Japan'
Research Planning & Administration Department, Daiichi Pharmaceutical Co., Ltd. 2
Although Sprague-Dawley rats are most frequently used in numerous toxicological studies, there have been
few reports dealing with the spontaneous occurrence of ectopic sebaceous glands (Fordyce's granules) in theoral tissue so far. Hence, to clarify the morphological characteristics of Fordyce's granules and the incidence
in this strain, 110 male and 110 female CD(SD)IGS rats aged 19 to 112 weeks were examined
microscopically. As results, Fordyce's granules were identified in the upper molar gingiva, and most of themwere located around the first and third molars. The granules consisted of one or more sebaceous glands
without hairs and hair follicles, and had some lobules comprising acini with adipose vacuoles. The sho1t
excretory duct was lined with stratified squamous cells and drained to the gingival surface. The sebum
constituting disintegrated acinar cells in the duct was also noted. In some cases, the granules wereaccompanied by cystic dilatation in the ducts and/or inflammatory reactions, without neoplastic or preneoplastic lesions. The overall incidence (9.1 %) of Fordyce's granules in males was higher than that (0.9%)
in females, and it somewhat increased with ascending ages, demonstrating sex- or age-specific difference.
Based on the above findings, the nature of Fordyce's granules seen in CD(SD)IGS rats was similar to thoseof other strains and humans.

JSTP/IFSTP IATl>. "200~Posters

P-136
Acquisition of a gastric or duodenal phenotype on heterotropic transplantationof esophagus diaphragm, trachea and bladder tissues in F344 rats
Hiromitsu WATANABE 1, Takashi OCHIYA 2 , Seiichi KAWAMATA3, Tomoyuki KUROSE 3 ,Yoko KOMINAMI1, Masayo NISHIKI 1, Atsushi SASAKI 1, Miho SHIRAISHI1, Rina GON 1,
Masaomi HAYASHI 1, Shoji KASHIWABARA1, Naoki KASHIMOT0 1, Toshihiro UESAKA 1,
Osamu KATOH 1
Department of Cellular Biology, Research Institute for Radiation Biology & Medicine, Hiroshima University,
Hiroshima 734-8553, Japan 1
Department of Cancer Metastesis, National Institute of Cancer2
Institute of Health Science, Faculty of Medicine, Hiroshima University 3
Cell differentiation is very important but not well understood. We have reported that intestinal metaplasia canbe induced by X-irradiation in rats, and proposed that an elevation of the pH of gastric juice due to thedisappearance of parietal cells in the fundic gland mucosa is one of the principal factors responsible for its
development. We also found that grafted colon mucosa differentiates into pseudogastric mucosa in the gastricregion and, more recently, that female stomach grafts differentiate into 'intestine' with goblet cells in themale duodenum. In the present study the possibility that such trans-differentiation might similarly occur withother tissues transplanted into the fundus or the duodenum in rats was tested. Pieces of esophagus,
diaphragm and trachea from 8-week-old male F344 rats were transplanted into the gastric fundus or
duodenum of females and examined especially after 3 or 6 months. While the diaphragm was notrecognizable as a muscle layer in either the stomach or the duodenum , the esophagus and trachea persisted,
the latter with the presence of cartilage. Esophagu s grafts transplanted into the glandular stomach andduodenum, trans-differentiated into gastric and duodenal mucosa, respectively. Pieces of bladder weretransplanted into the duodenum and observed after 1, 3, 6 and 12 months. Goblet cells with alcian-blue
positive mucin appeared in bladder tissue implanted into the duodenum three months after operation. Six
months after the operation, their numbers had increased and cytoplasm alkaline phosphatase (ALP) positivity
was noted. Twelve months after operation number of goblet cells were decreased and ALP was not observed.
Some donors were used in transgenic F344 GFP rats. Positive cells against anti-GFP antibody were observedin recipient tissues. Gastrointestinal and also bladder stem cells may have multipotential ability for
differentiation and to be able to trans-differentiate when transplanted into different environments in thegastrointestinal tract.

JSTP/IFSTP IATP 200~

Posters

P-137
Proliferative changes in upper digestive duct of rats treated with a single doseof Alloxan
Kiyokazu OZAKI 1, Yasushi K0DAMA 1, Tetsuro MATSUURA 1, Isao NARAMA 1
Research Institute of Drug Safety, Setsunan University, Osaka 573-0101, Japan 1
Male rats of WBN/Kob strain are one of the diabetic model animals that develop long-lasting diabetic
symptoms and some complications from about 40 weeks after birth without any treatment. In order toaccelerate the diabetic condition, animals were received a single dose of Alloxan and developed proliferative
lesions of squamous epithelium in forestomach. The lesions of forestomach progressed to squamous cell
carcinoma 50 weeks after Alloxan treatment. Histopathologic and immunohistochemical examinations were
performed to clarify carcinogenic mechanisms and distribution of digestive lesions.
Methods & Materials
Male and female WBN/Kob rats, 7-36 weeks old, were dosed singly with Alloxan (50 and 40mg/kg BW
respectively) via tail vein. The animals were sacrificed 50 weeks after dosing. Immunohistochemistry wasapplied in addition to routine histopathological examination.
Results
Hyperplasia of squamous epithelium was observed in the central part of tongue, hard palate, posterior partsof esophagus and whole part of forestomach , and was often associated with hyperkeratosis in esophagus andforestomach. About 20% of animals had squamous cell carcinoma (SCC) in the forestomach, and someanimals also had SCC in palate and/or posterior parts of esophagus. SCC was well-differentiated but invaded
into submucosal and/or muscular layer, and metastases to regional lymphonodus was also observed in somecases . Proliferative changes of squamous epithelium were constantly accompanied with erosion or ulceration,and moderate to severe infiltration of lymphocytes and plasma cells in underlying mucosal propria. Candida
albicans and E. coli infection were also frequent in the superficial mucosal layer. In cancer tissue, E Cadherin and s-Catenin showed partial loss, but p53 and Ras/p21 were not amplified. COX-2 was positive
for fibroblast and macrophage in the lamina propria near to the lumen without any topographic relation to
the neoplastic transformation, and was negative for neoplastic cells. iNOS was positive in suppurative
inflammatory lesion, but was negative for neoplastic cells.
Conclusion
It is strongly suggested that inflammatory process caused by fungous and bacterial infection play importantrole in the pathogenesis of Alloxan-induced squamous cell carcinoma.

JSTP/IFSTP IATP l_OOfl

Posters

P-138
Gastric carcinoma in mustard gas factory workers in the Ohkuno island
Takamitsu SASAK.1 1, Tomonori SASAHIRA 1, Wataru YASUF, Hiraki KUNIYASU 1
Department of Oncological Pathology, Nara Medical University Cancer Center, Nara 634-8521, Japan'
Department of Molecular Pathology, Hiroshima University Graduate School of Biomedical Sciences2
Gastric carcinomas occurred in persons who had worked in the mustard gas factory during World War II are
subjected. In follow up study, 56 mustard gas workers and 91 non-mustard gas workers in Ohkuno island
showed higher frequency of well-differentiated type adenocarcinoma than patients treated in National Cancer
Center Hospital (as a control). However, no significant difference was found between mustard gas workers
and non-mustard gas workers. Five cases of mustard gas workers who were received pathological diagnosis
on the resected materials were examined immunohistochernically. All cases showed no abnormal
acc umulation of p53 protein. No point mutation was confirmed by genome sequencing on exon 5-8 of the p
53 gene. E-cadherin production was reduced in all cases. Four cases showed reduced production of betacatenin, and one cases showed abnormal accumulation of beta-catenin in cytosol, whereas nuclear staining ofbeta-catenin was not detected in all cases. By methylation sensitive restriction enzyme assay showed DNA
methylation of E-cadherin promoter. These data suggested that gastric carcinoma in mustard gas workers
might possess distinctive properties on genetic and epigenetic alterations from usual cases.

4

JSTP/IFSTP IATl,- 2004

Posters

P-139
Gastric carcinoma in a Japanese macaque (Macaca fuscata)
Tokuma YANAI', Masato KOBAYASHI', Shunji GOTOU2, Akino KATOU2 , Hiroki SAKAI',
Akihiro HIRATA 1, Toshiaki MASEGI'
Department of Veterinary Pathology, Gifu University, Gifu 501-1193, Japan'
Primate Research Institute, Kyoto University 2
Gastric carcinoma is one of the most common malignant tumors in humans, especially in Japanese. The
tumors frequently occur in the lesser curvature of the antropyloric region, and less frequent in the cardiac
region in humans. There have been only a few repo1ts of gastric carcinomas in nonhuman primates whichhave a lot of similarity in anatomy and physiology with humans. We examined a case of gastric carcinoma
occun-ed in the cardiac region in a Japanese macaque, and tried to compare it's morphological features tothose in humans. An 18-year-old male Japanese macaque had gradual weight loss with occasional vomits
about 1 year before death. The animal became to have frequent vomits and severe anorexia 2 weeks before
death. Grossly, there was a tumor mass, 2 cm in the diameter, in the esophagus-cardia (EC) junction with asevere degree of cardial stenosis. The tumor bulged iffegularly with a severe ulcer on the surface, and was ill-defined in the mucosa. Histologically, there was an infiltrative growth of poorly differentiated carcinoma inthe mucosa of the cardiac region, invading deeply through the muscularis mucosae to the submucosa andmuscular layers. The tumor cells showed marked cell atypia with various-sized nuclei and prominent
nucleoli, and formed occasional glandular structures and squamous differentiation. There were frequent
mitotic figures in the tumor. The tumor cells contained mucinous material in the cytoplasm, when the Alcian
blue stain and periodic acid- Schiff (PAS) reaction were applied. There was no neoplasm except the gastrictumor. This tumor was diagnosed as gastric carcinoma originating from the cardiac region based on the siteof occurrence and morphological features. We previously reported an advanced gastric carcinoma originatingfrom the cardiac region, characterized by infiltrative growth with prominent glandular formation in a
Brazza's guenon (Cercopithecus neglectus).1) Other three cases of gastric tumors (one adenocarcinoma andtwo squamous cell carcinomas) were reported in the literature, all of which were 01iginated from the cardia.
It is still uncertain why gastric carcinomas favored to occur in the cardiac region in nonhuman primates ? 1)
Yanai T, Noda A, Sakai H, Murata K, Hama N, Isowa K, Masegi T; Advanced gasllic carcinoma in a deBrazza's guenon(Cercopithecus neglectus) J. Med. Primatol 1997; 26: 257-259

JSTP/IFSTP IATP ~-200~

PostersP-140
One case of Ito cell tumor in female F3441DuCrj rat
Yumi UMEDA1, Tetsuya TAKEUCHI1, Hideki SENOH1, Taku KATAGIRI1, Shigetoshi AIS0 1,
Kasuke NAGAN0 1
Japan Bioassay Research Center, Kanagawa 257-0015, Japan 1
One case of Ito cell tumor, spontaneously developing in the liver of a female F344/DuCrj rat (Charles River
Japan, Kanagawa), found from 5,000 females used in 25 studies of 2-yr carcinogenic bioassay can-ied out
following the Japanese Guideline is presented. The present case was survived up to the end of the 2-yr study
and received a complete necropsy. A mass, approximately 5 cm in size and with varying colors from white
to red, was observed in the central lobe of liver. Multiple white areas were also found in the lungs.
Organs were fixed in 10% buffered formalin solution and embedded in paraffin. Each 5 µm thick sections
were stained with hematoxylin and eosin, and examined microscopically. Unicentric large mass was foundhistologically in the liver and well demarcated from the sun-ounding liver tissue at the low magnification.
This nodule was predominantly composed of clear stained cells having a round, ovoid or spindle-shaped
nucleus. The cytoplasm, varying from abundant to scanty and often having vacuoles, exhibited chromophobic
tinctorial appearance in hematoxylin and eosin staining. The cell border was not clear. Mitotic figures wereoccasionally seen.
The fluorescence microscopic examination on frozen sections of the liver mass revealed fluorescent contents
in the cytoplasm of clear cells, suggesting a vitamin A being characteristic of the Ito cell. These clear cells
were an-anged in a cord-like structure covered with the sinusoidal endothelium. A part of cell-cords wasfound to be continuous with the sun-ounding hepatic cell cords at high-power magnification. A few nest ofclear cells with the cord-like structure was found to grow in parenchymal hepatocytes associated with thesinusoidal endothelia and Ito cells keeping the normal liver architecture. Prominent amount of collagenous
matrix was observed among the cord-like components of the tumor.
Several small foci consisting of clear cells were observed in the lungs and regarded as metastasis of thistumor.
From the above-described findings, this tumor was diagnosed as a rare Ito cell tumor in the rat liver.

JSTP/IFSTP IATP 2004

Posters

P-141
Effects of bandage of the torso to hepatomegaly induced by phenobarbital andclofibrate in rats
Kazuhiro HAYAKAWA1 , Satoru HOSOKAWA1, Toyohiko AOK.1 1, Atsushi INAGAMI 1, Akira IN0MATA1 ,Jiro SONODA1, Satoru MOTOOKA 1, George LOSOS 2, Kazuo TSUKIDATE 1
Drug Safety Research Laboratories, Eisai Co. , Ltd. , Japan, Gifu 501 -6195 , Japan 1
Asia Pacific Consulting Company2
There are many drugs which induce hepatomegaly. In some cases of hepatomegaly we have observed focal
coagulative necrosis usually confined to focal subcapsular areas of the liver. The lesions are compatible withfocal ischemic necrosis but it is difficult to confirm if these focal lesions are primary or secondary drug
effects. It has been demonstrated that in experiments which require wrapping of the torso by a bandage focal
ischemic liver lesions characterized by centrilobular coagulative necrosis with inflammatory cell infiltrationor fibrosis, and these were generally located near capsular surface (Parker GA: Toxicol Pathol, 23 , 507-512,
1995). It was postulated that some forms of physical manipulation predispose to the development of focal
ischemic liver lesions in the cases of drug-induced hepatomegaly in toxicity studies. A study was conducted
applying a bandage around the abdomen of rats administered either phenobarbital (PB) or clofibrate (CF) to
induce hepatomegaly. Thirty male SD rats were divided into 6 groups and 100 mg/kg of PB and 300 mg/kg
of CF were administered orally for 11 days to each treatment group; the groups were: non-bandage control,
bandage control, non-bandage PB , bandage PB , non-bandage CF and bandage CF groups. The control group
received an equivalent volume of the vehicle (corn oil). The bandage was applied during the final 4-days (4hours in first day, and 1 hour per day for 3-days). Liver enzymes, ALT and AST, were measured and liver
was weighed and all liver lobes were processed for microscopic examination. Local subcapsular ischemic
liver necrosis associated with ALT/AST elevation were observed in all bandage groups including control, but
the severity of the lesions was clearly greater in the PB/CF groups in which the liver weights were 1.3 to 1.5
times higher than control group. Histopathologically the liver lesions were characterized by centrilobular
coagulative necrosis with inflammatory cell infiltration predominantly located in the subcapsular areas, andin severe case the necrosis extended between central veins without affecting portal areas. In less severe case,hyaline droplet in hepatocytes, possibly derived from serum protein, and centrilobular mononuclear cell
infiltration were observed in subcapsular regions, and centrilobular hepatic vacuolation was also observed insome bandage PB group. These findings indicated that the liver lesions were ischemic in nature. There wereno necrotic lesions in non-bandaged groups. Theses results indicate that physical manipulation of abdomen
can induce focal subcapsular ischemic liver lesions in the toxicity studies of drugs that cause hepatomegaly.

JSTP/IFSTP IATP 20'iJ21

Posters

P-142
Different susceptibilities of rat liver lobes in carbon tetrachloride-induced
hepatotoxicity
Takeki UEHARA' , Takashi MURAI', Satoshi INOUE', Akira TOUCH!', Satoru MORI',
Toshiyuki MARUYAMA'
Developmental Research Laboratories, Shionogi & Co., Ltd., Osaka 561-0825, Japan'
It is well known that carbon tetrachloride (CC14) is metabolically activated with cytochrome P-450 (CYP),
and the reactive intermediates cause hepatocellular damage. It is also well established CCl4-induced
hepatotoxicity is enhanced by the pretreatment with several agents that induce hepatic CYP activity. We havepreviously demonstrated that the differences in hepatic CYP activity among the lobes are closely correlated
to the differences of hepatotoxic effect of CC14 among various lobes in the rat liver. In the present study, weinvestigated the correlation between hepatic CYP activity and CCl4-induced hepatotoxicity enhanced by the
pretreatment with two typical hepatic enzyme-inducing agents, phenobarbital (PB) or beta-naphthoflavone(NF) among the lobes.
Male Wister rats (10-11 weeks old) were allocated to 6 groups: (1) vehicle-treated alone, (2) PB-pretreatedalone, (3) NF-pretreated alone, (4) CCl4-treated alone, (5) PB-pretreated + CCl4-treated and (6) NF-pretreated+ CCl4-treated. Animals were given a single oral administration of CC14 (0.1 mL/kg, dissolved in liquidparaffin) or vehicle alone following the pretreatment, intraperitoneal injection for 3 days of PB (40 mg/kg,
dissolved in sterile saline) or NF (40 mg/kg, dissolved in sesame oil) . One day after the CCl4-administration,
all animals were necropsied, and the liver samples were obtained from the left and the median lobes. Eachsample was used for histopathological examination and 7-alkoxycoumarin 0 -dealkylase activities assay toevaluate the CYP activity.
PB-pretreatment significantly enhanced 7-alkoxycoumarin 0 -dealkylase activities as compared to the vehicle
group, and induced hepatocellular hypertrophy in centrilobular region. NF-pretreatment significantly
enhanced some of the enzyme activities without any histopathological change. The enzyme activities in the
median lobe were higher than those in the left lobe in both the vehicle and PB groups. All CCl4-treatedanimals revealed typical liver damage, such as the presence of hydropic degeneration and necrosis incentrilobular hepatocytes, and the depression of the enzyme activities. PB -pretreatment remarkably enhanced
the extent of the liver damage, which was also greater in the median than in the left lobe. NF-pretreatment
only slightly enhanced the extent of the liver damage.
These results indicate that different susceptibilities among the lobes in CCl4-induced liver damage may beclosely related to the heterogeneity of hepatic CYP activity.

250

.

,-··· ---JS TP/IFS TP IATP 2().04

Posters

P-143
Characterization of drug metabolic reaction involved in the acquired resistanceto bromobenzene-induced hepatotoxicity
Kohji TANAKA1, Satoko SATO', Isao IGARASHI 1, Naoki KIYOSAWA 1, Toshiyuki WATANABE',
Munehiro TERANISHI', Sunao MANABE'
Medicinal Safety Research Laboratories, Sankyo Co., Ltd., Shizuoka 437-0065, Japan'
Previously, it was demonstrated that the initial hepatic insult caused by bromobenzene (BB) was no longer
detected in rats despite subsequent dosing, indicating that the liver acquired resistance to BB-induced
hepatotoxicity. Changes in drug metabolic reaction including phase I (CYP), phase II (GST) and phase III(drug transporters) contributed to the resistance with the greatest contribution considered to be from anincreased phase II response. In this study, changes in drug metabolic reactions were characterized by hepatic
localization of the proteins using immunohistochernistry. The experimental design used in a previous study,
in which rats had acquired resistance to BB-induced hepatotoxicity (The Japanese Society of Toxicological
Pathology, 17th annual meeting, 2001: The Japanese Society of Toxicology, 30th annual meeting, 2003) was
employed. Briefly, F344 rats were treated intraperitoneally with BB (225 mg/kg). At 24 hr post-dose, hepatic
injury was confirmed by measuring AST values in the blood obtained from the tail vein, and then dosing
was continued at the same dose for an additional 8 days. Immunohistochemical staining using antibodies was
conducted in the liver on Days 4 and 8. As a result, positive staining for CYP3A and 2E antibodies wasobserved in the central zone of the hepatic lobule on both days. However, the staining intensity of CYP3A
and 2E was weaker in the whole hepatic lobule of the treatment groups than in that of the vehicle-treatedcontrols. Regarding GST antibodies, strong intensity of GST staining was observed in the central-to-mid
zonal hepatocytes. There were no remarkable differences in the intensity between Days 4 and 8. Decreased
CYP 3A and 2E, increased GST and multidrug-resistance protein (MRP) 3 were observed as changes inprotein content and mRNA levels. Thus, the results in this study corresponded with the changes in protein
content and mRNA levels of CYP and GST. In conclusion, GST induction was observed centrilobularly, but
CYP inhibition was not observed in a specific zone of the hepatic lobule. Presently, changes in protein
content and immunohistochemical localization of drug transporters involved in the phase III response arebeing examined.

251

JSTP/IFSTP IATP ·2'l)Oi

Posters

P-144
Depletion of tumor-infiltrating macrophages is associated with amphoterin
expression in colon cancer
Tomonori SASAHIRA1, Takamitsu SASAKI' , Hiroki KUNIYASU 1
Department of Oncological Pathology, Nara Medical University Cancer Center, Nara 634-8521, Japan 1
Macrophage infiltration into colon cancer and amphoterin expression in cancer cells was examined in 42
subserosa-invading human colon cancers. The mean number of infiltrating macrophages was significantly
higher in Dukes' B cases than that in Dukes' C cases (p=0.0065). Tumors with few infiltrating macrophages(macrophage depletion) were significantly more frequent in Dukes' C cases than that in Dukes' B cases (p=0.0014). No Dukes' C cases with relevant macrophage infiltration showed macrophage-cancer cell contact,
whereas 5 Dukes' B cases showed such contact (p<0.0001). In human colon cancer cells implanted in thececum of nude mice, KM12SM (high metastatic) tumors yielded less macrophage infiltration and more liver
metastases than were yielded by KM12C (low metastatic) tumors (14 ± 3 vs. 78 ± 32 and 24 ± 6 vs. 5 ± 3 perliver, respectively). Amphoterin expression was detected at high frequency in both Dukes' B and C cases (p=0.0684). In macrophage-depleted cases, frequency of amphoterin expression was significantly higher thanthat in non-depleted cases (p=0.0015). To confirm biological effects of arnphoterin on macrophages, cellseeded Boyden-chamber assay was done. Infiltration of PMA-treated U937 monocytes through the KM12SM
cell-layer was increased by pretreatment of KM12SM cells with amphoterin antisense Soligodeoxynucleotides exposure. Moreover, extracted amphoterin inhibited PMA-U937 monocytes infiltration
in a dose-dependent manner. Thus, amphoterin may play an important role in the inhibition of macrophage
infiltration into colon cancer.

252

JSTP/IFSTP IATP 2(l°O~

Posters

P-145
A new medium-term bioassay system for the detection of colon
carcinogenesis modifiers
Young-Man CH0 1, Jun-ichi ONOSE 1, Toshio IMAI 1, Mai HASUMURA1 , Makoto UEDA 1, Masao HIR0SE 1
Division of Pathology, National Institute of Health Sciences, Tokyo 158-8501, Japan 1
Bioassay systems utilizing aberrant crypt foci (ACF) as a presumable preneoplastic marker have been widelyused for the detection of carcinogens/modifiers for colon carcinogenesis within a short period in rats.
However, it was documented that in number of cases, ACF do not correlate with tumorigenic potential of test
chemicals. Furthermore, some compound which reduced the ACF induction, have been found to enhance thedevelopment of colon cancers. In these circumstances, we recently established a rapid colorectal 2-stage
carcinogenesis model in rats initiated with 1,2-dimethylhydrazine (DMH) followed by dextran sodium sulfate(DSS), applying neoplastic lesions as endpoint markers. In the present study, we validated this new modelusing known colon carcinogenesis modifiers. F344 male rats were given three s.c. injections of DMH (40 mg/kg b.w.) in a week and administered drinking water containing 1% DSS ad libitum for a week after the
initiation period. Chemicals used were 0.3% deoxycholic acid (DCA) as a promoter, 1.5% 1hydroxyanthraquinone (1-HA) as a carcinogen, and 0.04% nimensulide and 2% lactoferrin as inhibitors ofcarcinogenesis. They were dietary administered from 3rd week to 20th week of the experiment. Animals
were killed at weeks 10 and 20. At the 10th week of the experiment, the number of ACF in the control
group was 20.7 ± 4.5/rat, and it was significantly (p<0.01) decreased and increased in the group treated withnimensulide (13.7 ± 3.6) and DCA (31.1 ± 11.2), respectively, but lactofen-in (19.1 ± 6.18) and I-HA (24.1 ±26.4) showed no effect. Histopathologically, dysplastic foci , adenomas and adenocarcinomas were observedin all groups. At week 10, in I-HA group, the incidence of adenomas and the multiplicity of both dysplastic
foci and adenomas were significantly (p<0.05 or 0.01) increased, while in nimensulide and lactofen-ingroups, the incidence and combined multiplicity of adenomas adenocarcinomas were significantly (p<0.05 or0.01) decreased as compared to controls. At week 20, both the incidences and multiplicities of theproliferative lesions obviously increased in DCA and 1-HA groups and decreased in nimensulide andlactofen-in groups. Particularly the multiplicities of adenomas and adenocarcinomas were significantly (p<0.01) increased in 1-HA group, while the incidence and multiplicity of adenocarcinomas were decreased innimensulide group (p<0.01) as compared with control. It is concluded that this medium-term bioassay system
is useful for the detection of promoters and inhibitors of colorectal carcinogenesis.

253

JSTP/IFSTP IAT:P . 200~

Posters

P-146
Renal damage in newborn rats treated with p-Cumylphenol
Yuko YAMAGUCHI 1, Nobuo NISHIMURA 2 , Megumi YAHATA2, Hiroshi EDAMOT0 1,
Shinichiro IKEZAKI1, Kazutoshi TAMURA1, Eiichi KAMATA4, Makoto EMA4, Ryuichi HASEGAWA 3
Division of Pathology, Gotemba Laboratory, Bozo Research Center Inc., Shizuoka 412-0039, Japan 1
Division of The Second Laboratory for Safety Evaluation, Gotemba Laboratory, Bozo Reseach Center Inc. 2
Division of Medicinal Safety Sciences, National Institute of Health Sciences 3
Division of Risk Assessment, Biological Safety Research Center, National Institute of Health Sciences4
To explore renal toxicity of p-Cumylphenol (PCP) in newborn rats [Ctj:CD(SD)IGS], single and repeateddosing studies were conducted. In the single dosing study a total of 50 newborn rats, 4-days old, were given
doses of 0, 300 and 600mg/kg/day, and were terminated at 1, 3 and 7 days after treatment. Seven males and3 females at 600mg/kg died by 24 hours after administration. These animals showed tubular damage
including slight dilatation of collecting ducts and renal tubules, and slight necrosis of papillary ducts andneutrophilic infiltration in the tip of papilla. Similar changes were observed on days 1 and 3 in each treatedgroup, while no animals showed histological change on day 7 in any group.
In the repeated dosing study a total of 50 rats were given doses of 300mg/kg/day from 4 days to 21 days
after bi1th, and were terminated on days 4, 8, 15 and 18, and 7 days after withdrawal. On days 4 and 8,
slight dilated and hyperplastic collecting ducts were frequently observed in medulla, prominently in inner
medulla, and also neutrophils infiltrated in the tip of papilla. On days 15 and 18, marked cystic dilated ducts
were mainly observed in outer medulla, tending to be progressive with time. These cystic dilated ducts were
lined by hyperplastic epithelial cells and occasionally contained exfoliated epithelial cells. The collecting
ducts in papilla were dilated, but not with cystic formation or hyperplastic epithelial cells. Focal neutrophilic
infiltration in the tip of papilla was also noted in some animals on day 15. Seven days after withdrawal,
although the dilated ducts in outer medulla were still persistent, they were slightly reduced in size withdecrease of epithelial cell density. However, in cortex, basophilic tubules with lymphoid infiltration appeared
multifocally. Similar change was noted in our previous study, in which the lesions existed at 9 weeks after
withdrawal when cystic dilated ducts were mostly diminished.
In conclusion, a single high dose of PCP induced papillary damage, and it may not be related to form
polycystic lesions. Predilection site of polycystic lesions was a collecting duct in outer medulla, and thelesions may be primarily associated with proliferation of epithelial cells, although pathogenesis of cystic
lesions including a hyperplastic change and after-withdrawal changes in cortex are still undetermined.

254

Posters
P-147
A photographic spectrum of non-neoplastic renal tubule and transitional
epithelial structures within selected renal neoplasm of the rat
John Curtis SEELY', Gordon C HARD 2Experimental Pathology Laboratories, Inc., Research Triangle Park, North Carolina 27709, USA'
Consultant in Toxicology, Pathology, Carcinogenesis. 2
The identification and characterization of non-neoplastic renal tubule and transitional epithelial structures areimportant in the diagnosis of several tumors of the rat kindly;i.e.,renal mesenchymal tumor (RMT) andnephroblastoma(NB). Controversy still exists over the interpretation of these structures. Some pathologist
consider these structures to represent pre-existing and entrapped renal tubules or transitional epithelium
which have undergone hyperplastic and/or metaplastic (embryonal) transformation. Other pathologists
consider these st:Iuctures to represent a neoplastic component of the tumor. Indeed, these are some tumors inwhich these structures represent a challenging and often puzzling dilemma for the pathologist. We present a
number of microphotographs which illustrate the spectrum of changes that we encountered during a review
of large database of RMT and NB. Our diagnostic criteria contend that these structures represent a
hyperplastic to metaplastic transformation. Although the mechanism for these changes are not known, theunique relationship during renal development between the primitive metanephric mesenchyme and theepithelial structures derived from the ureteric bud might support a hypothesis of a 'genetic component' or thepresence of 'tumor factors' which could account for the spectrum of changes observed within theseneoplasms.

255

JSTP/IFSTP IATP -2.DO~

Posters

P-148
Effect of water deprivation or furosemide on cefalotin and glycerol-induced
proximal tubular toxicity in rats
Miyoko OKADA', Naoki OHYAMA' , Shun-ichiro ISHII', Naoya MASUTOMI 1, Fumiko SANO',Jiro SUGIMOTO', Shiro TAKAGI'
Toxicology Laboratory, Mitsubishi Pharma Corporation, Chiba 292-0818, Japan'
In combination with cephalosporin antibiotics and glycerol are known to cause acute renal failure such as
proximal tubular necrosis. Dehydration has been reported to be an important risk factor to induce acute renal
failure. In this study, we investigated the effect of water deprivation or a diuretic drug, furosemide, on
cefalotin and glycerol-induced proximal tubular toxicity in rats. Experiments were performed in six groups
using male CD(SD)IGS rats aged between 6- and 11-week-old; Group 1: physiological saline was injected
intravenously (i.v.) as controls, Group 2: cefalotin sodium (CET) was injected i.v. at a dose of 3000 mg/kg,
Group 3: glycerol (GLY) was injected subcutaneously (s.c.) at a dose of 1000 mg/kg, Group 4: furosemide(FUR) was injected s.c. at doses of 20, 50, 100 or 200 mg/kg, Group 5: CET (2000 or 3000 mg/kg) wereconcomitantly treated with GLY (1000 mg/kg), Group 6: FUR (20, 50, 100 or 200 mg/kg) were combinedwith CET (2000 mg/kg) and GLY (1000 mg/kg). All administrations were performed at once on the sameday. All groups, except Group 6, had a water-supplied or -deprived subgroups. Animals in the water-deprived
condition were deprived of water 24 hours before administration to the sacrifice. After 24 hours of the final
admin istration, all animals were killed under anesthesia and the kidneys were examined histopathologically.
In Gro up 1, no treatment-related change was observed in the water-supplied or -deprived rats. In Groups 2
and 3, slight degeneration/necrosis of proximal tubular (PT) epithelium was noted in the water-deprived rats
while no treatment-related change was seen in the water-supplied rats. In Group 4, slight PT damages wereobserved in the water-supplied rats at 200 mg/kg of FUR and slight to moderate changes were seen in thewater-deprived rats at all dose groups of FUR. In Group 5, slight PT degeneration/necrosis was noted in thewater-supplied rats and slight to severe changes were observed in the water-deprived rats. In Group 6, slight
to severe renal damages were noted in rats at all dose groups of FUR. It is concluded that the water-deprived
condition or FUR-treatment enhances the PT damage induced by CET or GLY alone, or the combination of
CET and GLY.

256

JSTP/IFSTP IATP 2004

Posters

P-149
Pathological observation of karyomegalic cells in the kidney on male rats
treated with Paecilomyces japonica
Yong-Bum KIM1, Chang-Su HA1, Hwa-Young SON2 , Sung-Whan CH0 2 , Boo-Hyon KANG 1
Korea Institute of Toxicology, KRICT, Yuseong, Daejeon 305-600, Korea (South) 1
College of Veterinary Medicine, Chungnam National University 2
Background. Paecilomyces japonica(P. japonica) is a fungus used as a traditional medicine in China, Japanand Korea. We recently observed that P. japonica induced renal failure with karyomegaly. The purpose ofthis study was to investigate the pathogenesis of karyomegaly induced by P. japonica and the possibility as aprogenitor of kidney tumors.
Methods. 40 male SD rats, 5 weeks old, were orally treated with P. japonica at dose levels of 0, 250, 500 or
1000 mg/kg/day for 5 weeks. Kidneys were observed with light and electron microscopy.
Immunohistochemical detection of proliferating cell nuclear antigen(PCNA) and bromodeoxyuridine(BrdU)
were performed to assess renal epithelium proliferation. To detect apoptosis, TUNEL method was adopted.
Results. Karyomegaly was observed in the outer stripe of outer medulla in all treatment groups.
Karyomegalic cells contained enlarged, irregularly shaped nuclei, showed invaginations of nuclear membrane
and formed apparent nuclear inclusions. The number of mitotic cells significantly increased in all treatmentgroups (15,6, 24,1 and 5,3) compared with control group (0.2). The number of PCNA(5.4, 8.8 and 5.2 timesof control) and BrdU (2.9, 3.1 and 2.5 times of control)-positive cells significantly increased in all treatmentgroups, but the cells with karyomegalic nuclei had a lower rate of positivity on both BrdU and PCNA
reaction. TUNEL-positive cells significantly increased in all treatment groups (13, 17 and 22.7) comparedwith control group (1.7). Karyomegalic cells with inclusions showed predominantly positive immunoreaction
with the TUNEL method.
Conclusions. The karyomegalic renal tubular cells underwent neither abnormal cell division nor highproliferation rates, in spite of sharing enlarged nuclei of tumor -like morphology. The possibility of a renal
tumor progenitor from karyomegaly was considered to be low.

JSTP/IFSTP IATr··_z:oo~

Posters

P-150
Long-term observation of anti-Thy-1 glomerulonephritis in uninephrectomized
rats: Relationship between morphological changes and renal function
Shunji NAKATSUIT 1, Kenjiro TSUBOTA1, Yoshimasa OKAZAKI1, Masahiro MATSUMOT0 1,
Shiro FUJIHIRA1, Yuji OISHI 1, Masayuki TOMITA 2 , Hajime SOGABE2 , Shoko NAKAZAT0 2 ,Hiroshi KAWACHI3, Fujio SHIMIZU3
Toxicology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd. , Osaka 532-8514, Japan 1
Medicinal Biology Research Laboratories, Fujisawa Pharmaceutical Co., Ltd. 2
Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences 3
A progressive renal injury accompanied by continuous proteinurea is induced by administration of anti-Thy-I
antibody to unilaterally nephrectomized rats; however, the mechanism of progression to end-stage kidney has
not fu lly understood. In the present study, we investigated this progressive renal injury in uninephrectomized
rats for 47 weeks, and discussed the relationship between morphological changes and renal functionalparameters.
Methods. A progressive glomerulonephritis was induced in unilaterally nephrectomized Wistar rats (8 weeks
of age) with a single injection of anti-Thy- I antibody (1 mg). Animals were sacrificed on Day 2 and Weeks
1, 2, 4, 6 and 8, then every 4 weeks by 47 weeks after the injection of the antibody. Urine and blood
samples were collected to determine proteinurea, blood urea nitrogen (BUN) and serum creatinine
concentrations. Kidneys were processed for light and electron microscopy according to standard procedures.
Paraffin sections were stained with hematoxylin-eosin and periodic acid-Schiff (PAS), andimmunohistochemistry was performed using anti-desmin, anti-alpha-smooth muscle actin and anti-vimentin
antibodies.
Results. Initial stage (Day 2 to Week 8 after the injection): Urinary protein excretion peaked on Week 1 andthen decreased gradually, but it began to increase again after 4 weeks. Histologically, glomerular mesangial
cell proliferation and matrix expansion were observed on Week 1, followed by interstitial inflammation andtubular regeneration by Week 8. Glomerular alpha-smooth muscle actin expression was strongly appeared on
week 1 and then decreased thereafter. In contrast, glomernlar desmin expression was noted from Weeks 2 to
8. Later stage (Weeks 12 to 47 after the injection): Progressive proteinurea was noted during the observation
period until 47 weeks, and high levels of BUN and serum creatinine were maintained from Weeks 12 to 47.
After 24 weeks, tubulointerstitial lesions including interstitial fibrosis and tubular atrophy was remarkably
appeared in addition to glomerular screrotic changes.
Conclusion. These data suggest that the renal functional changes in the early stage in this model areassociated with the morphological changes of glomerular epitherial and mesangial cells, and that the
tubulointerstitial lesions are closely involved in the deterioration of renal function in the later stage.

258

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'7

.:-•J•_....rr

JSTP/IFSTP IATP 2004

Posters

P-151
Spontaneous glomerulonephritis in cynomolgus monkeys
Xiuying YANG 1, Shigeru SATAKE 1, Yasuhiro KAMIMURA' , Kimiaki HIRAKAWA 1, Hiroshi MAEDA 1,
Hiroaki MIYAJIMA 1
Shin Nippon Biomedical Laboratories, Ltd., Kagoshima 891 -1394, Japan'[Background] Spontaneously occurring glomerulonephritis, to our knowledge, is very rare in non-humanprimates and has not been documented in cynomolgus monkeys. We present 7 cases of glomerulonephritis incynomolgus monkeys that we encountered during safety studies .
[Materials and Methods] The kidneys of 7 cynomolgus monkeys aged 4-6 years old were collected from thecontrol groups of repeated studies. They were sectioned routinely and stained with H.E., PAM, PAS and
Masson trichrome. Imrnunohistochemical staining was perfo1med with IgG. Sections for electron microscopic
examination were prepared from paraffin-embedded specimens.
[Results] Histologically, the glomeruli were characterized by expansion of the mesangial areas withhypercellularity and increased mesangial matrix. Except for 1 case, there were no remarkable changes in the
tubules or intersitium. Immunohistochemically, granular IgG deposition was confirmed in 2 cases. Increased
mesangial cells and matrix, infiltration of the macrophages in the mesangial areas, electron dense deposits inthe mesangial and paramesangial areas were confirmed by electron microscopy.
[Conclusion] As in humans, immune-mediated glomerulonephritis can spontaneously occur in cynomolgus
monkeys, which have histological, imrnunohistologial and electron microscopic features that are similar tohumans. Since monkeys used in safety studies are usually very young, the occurrence of the disease isconsidered rare. This accounts for the infrequent documentation of the disease.

JSTP/IFSTP IATP 2001

Posters

P-152
Spontaneous collagen glomerulopathy in a cynomolgus monkey
Kae FUJISAWA!, Nobuo TAKASU 1, Noriko TSUCHIYA' , Shuuichi MATSUSHIMA' , Haruhisa INAGAKI2,
Mikinori TORII 1
Developmental Research Laboratories, Shionogi & Co., Ltd., Osaka 561-0825, Japan 'Discovery Research Laboratories, Shionogi & Co., Ltd. 2
Collagen glomerulopathy is one of rare glomerular lesions and has been known in human, however, it has
not been reported in monkey to our knowledge. We report here light microscopic and electron microscopic
findin gs in a young male cynomolgus monkey that was diagnosed as collagen glomerulopathy byhistopathologic examination.
An intact male cynomolgus monkey showed mild proteinuria and hypoproteinemia at 4 years and 5 monthsof age and was necropsied under anesthesia with sodium pentobarbital. Kidneys were fixed in 10% neutral
buffered formalin, routinely processed and embedded in paraffin. Paraffin section was stained withhematoxylin and eosin, Mallory azan, Congo-red, Periodic Acid-Schiff reagent (PAS) and anti-type III
collagen antibody for immunohistochemistry. Electron microscopic examination was routinely carried out
using a piece of formalin-fixed kidney tissue. Kidneys were macroscopically pale and granular on thesurface. Histopathology revealed diffuse glomerular swelling accompanying with deposition on eosinophilic
amorphous material in the glomerular basement membrane and mesangial area. Amorphous material was
negative for amyloid and PAS staining, but was positive for collagen fiber by azan staining.
Immunohistochemistry revealed strongly positive reaction with anti-type III collagen . Mesangial area waswidened, but there was no increase in number of mesangial cells. There was no remarkable lesion in theefferent/afferent artery, small or medium-sized artery. Mild tubular degeneration was only observed in thecortex, occasionally with cholesterin crystal and interstitial lymphocytic infiltration. Electron microscopy
revealed various-sized collagen fibrils with periodicity in the glomerular basement membrane and mesangial
area.
These findings draw a conclusion of spontaneous collagen glomerulopathy in a young cynomolgus monkey.

260

JSTP/IFSTP IATP .20021

Posters

P-153
Expression of somatostatin mRNA and peptides in C-ce/1 tumours of the
thyroid gland in Han Wistar rats
Andrew PILLING 1, Stewart JONES 2 , John TURTON 3
Huntingdon Life Sciences, Huntingdon PE28 4HS, UK 1
GlaxoSrnithK.line2
School of Pharmacy, University of London 3
C-cell tumours are among the most common spontaneous neoplasms of the laboratory rat. With the exceptionof calcitonin, little attention has been paid to the secretory peptides of C-cells and their possible role in thedevelopment of neoplasia. Of these peptides, somatostatin (SS) is of particular interest because it has beenshown to regulate the growth and secretion of thyroid follicular cells in vitro. In the present study, in situ
hybridization and immunohistochernistry were used to investigate the expression of SS rnRNA and SS
peptides, in both normal C-cells and a range of spontaneous proliferative C-cell lesions in the Han Wistar
rat. It was confirmed that a small minority of C-cells in the normal rat thyroid gland produce and store SS
peptides, however approximately half of all C-cell adenomas and C-cell carcinomas carcinomas stained
positively for SS rnRNA and peptides. SS expression was also observed in all metastatic deposits ofcarcinomas in drainage lymph nodes. The discrepancy in the extent of SS expression between the normal
and neoplastic C-cell population can possibly be attributed to the lack of differentiation of the neoplastic
cells, with the assumption of a more primitive phenotype that is capable of producing alternative peptides. In
addition, the mean percentage of cells that stained positively for SS rnRNA and peptides was significantly
higher in smaller (4mm) suggesting that the presence of SS was exerting a growth controlling influence onthese lesions.

JSTP/IFSTP IATP ·200~

Posters

P-154
A spontaneous dwarf mutation in Br/Han:WIST@Jcl (GALAS) rat Histopathological characteristics of the endocrine system
Takuya DOI1, Masato NAMIK.1 1, Michiko ASHINA1, Naoto TOYOTA1, Hiroko KOKOSHIMA1,
Yuki TOMONARI 1, Junko SAT0 1, Takeshi KANN01, Yumi WAK0 1, Minoru TSUCHITANI1
Mitsubishi Chemical Safety Institute Ltd., Ibaraki 314-0255, Japan 1
We found a spontaneous dwarf mutation in BrlHan:WIST@Jcl(GALAS) rats. This report describes thehistopathological characteristics of the endocrine system in the affected animals.
[Background] Two males produced from externally normal parent BrlHan:WIST@Jcl(GALAS) rats,
purchased from Clea Japan Inc. (Shizuoka, Japan), showed external abnormality (small body, delicate fur,micrognathia, brachyury, and exophthaloms). One of them was successfully mated with a normal littermate,and dwarf and normal rats were produced. Presently, we know that the dwarf animal was produced bybrother-sister mating between normal littermates.
[Material & methods] Eight- and forty-five-week-old dwarf (D) rats, 45-week-old normal littermates (NL),and eight- and forty-five-week-old normal (N) rats purchased from Clea Japan Inc. (Shizuoka, Japan) wereused in the present study. The thyroid, parathyroid, pituitary, adrenal and pancreas were removed, fixed in
l 0% neutral phosphate-buffered formalin , embedded in paraffin, sectioned and stained with hematoxylin andeosin for microscopic examination.
[Result] Body weights of the D rats were markedly lighter than those of the same age NL and N rats.
Macroscopically, an increased size of the thyroid in the D and NL rats, and discoloration (grayish) of thepituitary anterior lobe were observed in the D rats. Microscopically, the following changes were observed.
Thyroid: Little colloid formation and decreased follicular size in the D rats . Vacuolar change in thyroid
fo llicular cells in the D and NL rats. Pituitary: Few acidophilic cells in the D rats. Adrenal: Marked
lipofuscin deposition in 45-week-old Drats only. Parathyroid and pancreas: No remarkable change.
[Discussion] Little colloid formation and decreased follicular size in the thyroid of the D rats indicated thatthey had been affected by hypothyroidism. The rdw rat was reported by Koto et al., which is a hereditary
dwarf model caused by hypothyroidism derived from Wistar-imamichi strain (Experimental Animals 37: 21 30, 1988). The vacuolar change in thyroid follicular cells in the D rats was similar to the change caused byretention of thyroglobulin in the dilated rER in the rdw rat (Anatomical Record 259: 60-66, 2000). Vacuolarchange, however, is occasionally observed i.µ BrlHan:WIST@Jcl(GALAS) rat, and is not accompanied withhormonal abnormality (Journal of Toxicologic Pathology 14: 253 -257, 2001). In the present study, vacuolarchange was also observed in the NL rats. Hence, detailed analysis is needed to determine whether or not thevacuolar change in the D rats is related to hypothyroidism.

262

JSTP/IFSTP IATP 2004

Posters

P-155
Association of adrenal pheochromocytoma and lung pathology in inhalation
studies with particulate compounds in the male F344 rat--the National
Toxicology Program experience.
Keisuke OZAK.1 1, Joseph K. HASEMAN2 , James R. HAILEY3, Robert R. MARONPOT3 , Abraham NYSKA 3
Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., Osaka 554-8558, Japan 1
Biostatistics Branch, National Institute of Environmental Health Sciences (NIEHS) 2
Laboratory of Experiment Patholgy, National Institute of Environmental Health Sciences (NIEHS)3
Systemic hypoxemia, occun-ing in space-occupying lung pathologies such as inflammation and neoplasms.
reduces the gas exchange area and stimulates catecholamine secretion from the adrenal medulla wherechronic endocrine hyperactivity may lead to hyperplasia and neoplasia. We investigated the possible
correlation between nonneoplastic chronic pulmonary lesions and adrenal pheochromocytoma in 9 recent,
NTP, 2-year particulate inhalation studies in male F344 rats. Re-evaluation for chronic active inflammation,interstitial fibrosis, alveolar epithelial hyperplasia, squamous metaplasia, proteinosis, and histiocytosis
revealed significant associations of pheochromocytoma only with the severity of inflammation and fibrosis.
Nickel oxide, cobalt sulfate, indium phosphide, talc, and nickel subsulfide studies showed chemical-related
incidences of adrenal pheochromocytoma and significant (p < 0.01) associations with inflammation andfibrosis. Gallium arsenide, vanadium pentoxide, molybdenum trioxide, and nickel sulfate hexahydrate studies
revealed an increased incidence and/or severity of nonneoplastic lung lesions, but no increased incidence ofpheochromocytoma. Although gallium arsenide and molybdenum trioxide showed no dose-related increase inpheochromocvtoma, a significant (p < 0.01) correlation of the latter with the severity of fibrosis andinflammation occurred. In the vanadium pentoxide and nickel sulfate hexahydrate studies, no relationshipbetween nonneoplastic lung lesions and pheochromocytoma was manifested. Our investigation assessed thestrength of these various associations and supports the possible roles of 2 chronic pulmonary lesions-fibrosis
and inflammation-and hypoxemia in the induction of pheochromocytoma in the F344 male rat.

JSTP/IFSTP IATP 20021

Posters

P-156
Renal toxicity induced by folic acid is directly responsible for the enhancementof male reproductive toxicity of di (n-butyl) phthalate in male rats
Toshio ICHIHARA 1, Tomoko TSUTSUMI', Mayumi KAWABE 1, Hiroko YOSHINO', Makoto ASAMOT0 2 ,Syugo SUZUKI2, Tomoyuki SHIRAI2
Daiyu-kai Institute of Medical Science, Aichi 491-0113, Japan'
Department of Experimental Pathology and Tumor Biology, Nagoya City University Graduate School of
Medical Sciences2
Di (n-butyl) phthalate (DBP) is a phthalate ester with extensive industrial use in plastic piping, various
varnishes and lacquers, safety glass, nail polishes, paper coatings, dental materials pharmaceuticals, andplastic food wrapping, there is a high potential for human exposure in work place and home environment.
DBP has been shown to possess antiandrogenic action, which results in testicular atrophy (seminiferous
degeneration, Leydig cell hyperplasia and adenomas) and blocks male reproductive development. The
pharmacologic actions of chemicals may be modified, strengthened or diminished, under certain pathologic
conditions. For example, decreased hepatic or renal function can influence chemical activation /detoxification or excretion resulting in toxicity of a given chemical. Usually, bioassay for toxicity ofchemicals are carried out using health experimental animals and there is always a question as to how
observed toxicities would be modified under pathologic conditions such as lowered liver or renal functions .
This type of data might be very important for risk evaluation and risk management for human health. In thisstudy, we investigated whether testicular toxicity of DBP is influenced by diminished renal function. To
generate an experimental condition reflecting chronic renal disease in man, six week old male F344 rats
were given 5 consecutive weekly subcutaneous injections of folic acid at a dose of 300 mg/kg and then thediet containing 1200, 5000 and 20000 ppm of DBP for 4 weeks. These concentrations roughly correspond to60, 250 and 1000 mg/kg/day/rat, respectively. Urine volume, specific gravity and osmotic pressure were alsomeasured as markers of renal function. All animals were killed under anesthesia at experimental week 9 andblood samples were collected from the aorta for measurement of serum creatinine and BUN. And
spermatogenesis (account of sperm, sperm mobility and morphological abnormality) were investigated. The
liver, kidneys, testes, epididymis and prostate of each rat were weighed after removal. Folic acid clearly
induced interstitial nephritis accompanied by impairment of renal function. Enhanced testicular toxicity withseminiferous degeneration, diminished spermatogenesis and increase in the number of morphologicallyabnormal sperm was evident in rats given folic acid and then 20000 ppm DBP as compared to those given
DBP alone. The data suggest that DBP male reproductive toxicity can be increased by renal dysfunction. We
thank Dr. Shibutani, Division of Pathology, National Institute of Health Sciences, Tokyo, for his kindcomments and discussion for this study.

264

JSTP/IFSTP IATP 2004'-°i(t·Posters

P-157
Motional and pathological analysis for rat testicular toxicity induced by boricacid
Masakazu KAKUNI 1, Naoya KIMOTO 1, Tsuyoshi TAKEDA1 , Masato MIZUTANI 1, Kazuo SUZUKI 1,
Hitoshi SAT0 1, Katsumi TAKABA1, Takuji HARA 1
Toxicological Research Laboratories, Kyowa Hakko Kogyo Co., Ltd. , Yamaguchi 755-8501 , Japan 1
Boric acid is known to induce the male reproductive toxicity. We had some supportive data of the male
reproductive toxicity induced by boric acid with motional and pathological analysis. In the study boric acidwas administered daily by oral gavage to male Crj:CD(SD)IGS rats of 6 wks old at dose level of 500 mg/kg
for 4 weeks. In necropsy, testes and epididymidis were macroscopically atrophic, and the weights of theseorgans were significantly decreased as compared with control group . Motional analysis, which evaluated
Percentage of motile sperm, Curvilinear velocity, Linearity, Amplitude of lateral head displacement (A.L.H.)
max, Amplitude of lateral head displacement (A.L.H.) mean and Beat cross frequency, revealed thatsignificant decrease was observed in Percentage of motile sperm, Linearity and Beat cross frequency. A
further histopathological examination is in progress.

JSTP/IFSTP IATP 2004

Posters

P-158
Early ultrastructural changes of the Sertoli cells with vacuole formations
Yoshiaki SAIT0 1, Kenji USUMI 1, Noriko OSAWA1, Tomoko NAGATA 1
Food and Drug Safety Center, Hatano Research Institute, Kanagawa 257-8523, Japan 1
Among histological alterations of the testis caused by various chemicals in the toxicity studies, we
frequently observed vacuoles in the germ cell linings as the first noticeable changes and recognized later as
scattered groups of degeneration and/or depletion of germ cells that assumed to originate from Sertoli celldegeneration. To investigate early ultrastructural changes on the Sertoli cells that accompany with vacuoles
as well as grouped depletion and/or degeneration of germ cells under light microscopy, we chose threechemicals as follows; di-(2- ethylhexyl)phthalate (A), (-)-hydroxycitric acid-calcium salt (B) andaminophenylnorharman (C). Rats of Sprague-Dawley strain (Crj:CD SPF) were purchased and used forexperiments at the age of 10-11 weeks. After a single oral administration of 2800 mg/kg of A, rats wereanesthetized with sodium pentobarbital at 3, 6, 24 and 48 hours after dosing. Rats treated with single oral
dosing of 100 or 200 mg/kg of C were anesthetized at 1, 2, 3, 4 or 5 days after dosing and for B, 0.13, 0.66
or 3.31 % containing powder diet (CE-2, CLEA Japan) were given for 28 days and were anesthetized. The
testes of all animals were fixed by perfusion via aorta with fixatives, postfixed with osmium tetroxide andembedded in epoxy resin and subjected to the light and electron microscopic observations . Under lightmicroscopy, vacuoles were observed 6 hours after administration of A, 2 days after treatment of 200 mg/kg
group and 4 days after treatment of 100 mg/kg groups of C and 28 days treatment of B. By electron
microscopy, dilation of the rough endoplasmic reticulum (ER) associated with ectoplasmic specialization
which consisted of the blood-testicular barrier was observed in animals treated with A, dilation of
extracellular space as well as fragmentation of the cell membrane were observed in B and dilation of smooth
ER of the Sertoli cells were recognized in C. From these results, vacuolar formation in the Sertoli cells
may possibly be caused by different mechanisms among these chemicals, though the light microscopy
showed similar morphological changes. Thus, it is important to examine early ultrastructural changes of the
Sertoli cells to clarify pathogenesis of the testicular toxicity.

266

Posters
P-159
International advancement in education in veterinary & comparative pathology
D. Reid PATTERSON'
Reid Patterson Consulting, Inc., Grayslake 60030, USA 1
With over 3,000 member-pathologists in more than 80 countries around the world, the C. L. Davis, D.V.M.
Foundation has become the most effective educational institution in veterinary and comparative pathology inthe world. Founded in 1970 by an endowment from the family of famed American pathologist and educator,
Charles Louis Davis, this charitable, not-for-profit corporation has recruited over 100 renowned educatorsand researchers in pathology from Canada, Europe, the United Kingdom and the United States. This Facultyof Discussants has produced hundreds of video-recorded lectures for its members and institutions andpersonally present at 12 highly acclaimed annual symposia, seminars and workshops in Africa, Europe andthe U.S. Topics range from toxicological pathology, to avian, wildlife, domestic and companion animal, andcomparative pathology, plus unique lectures in mutant animal models, laboratory animal medicine, molecularpathology , medical photography and pharmaceutical safety assessment. Presentations accompany thoseorganized such as the American College of Veterinary Pathologists, the Society of Toxicologic Pathology, theEuropean Society of Veterinary Pathology, the Royal College of Pathologists, the American Association of
Zoo Veterinarians, and the Armed Forces Institute of Pathology, or are hosted independently. To advanceknowledge in zoo and wildlife species, the Foundation established a decade ago a pathology research
program, in collaboration with the American Association for Zoo Veterinarians. The Foundation has thelargest collection of pathology specimens of any single organization, enabling an extensive circulation of
study sets and the establishment of 6 independent study centers within the U.S. With a mission to advancethe study of the diseases of animals and the comparison of diseases manifested by diverse species ofanimals, including humans, the Foundation strives to improve the health and well being of animals andhumans throughout the world. To realize this mission, the Foundation seeks to share its wealth of
information to all 404 veterinary schools in the world, to to sponsor externship programs that have alreadybenefited 46 international veterinary students and to recognize the exceptional teaching and/or scholarship ofover 550 international veterinarians. This poster is offered to understand interest within Asia for participationin this unique educational program. The Foundation is presently offering seminars within Eastern Europe andSouth America. A recent Foundation conference in Vietnam was well received. The Foundation is excitedabout sharing with scholars in Asia.

JSTP/IFSTP IATP 200~

Posters

P-160
Histopathology, immunocytochemistry and electron microscopy of
spontaneous uveal melanoma in two HAN Wistar rats
Alexandra MORAN 1, Christopher BARTON 1
Pathology Department, Covance Laboratories, Harrogate HG3 lPY, UK 1
Uveal melanoma in rats are unilateral neuroectodermal tumours derived from uveal melanocytes. They occur
in numerous species of animals, including laboratory animals, and are most frequently recorded in cats.
Here, we describe the histopathologic, immunocytochemical and electron microscopic characteristics of uveal
melanomas in two HAN Wistar rats.
The tumours occurred in two, one year old HAN Wistar rats that were involved in a long term drug study.
Fresh tissues were routinely fixed and processed. Paraffin embedded sections were stained with haematoxylin
and eosin. Additional sections were irnrnunohistochemically examined for reactivity to Vimentin, S 100,
Neuron Specific Enolase (NSE), Melan A, Ki-67 and Proliferating Cell Nuclear Antibody (PCNA). In
addition, sections were also processed for Transmission Electron Microscopy.
Histologic examination in each case revealed an unencapsulated, infiltrative, densely cellular mass composedof sheets and nests of cells supported by a fine fibrovascular stroma. The tumour cells were round tospindloid, with variably distinct cell borders. Occasional cells contained a dark brown granular pigment
within their cytoplasm. The tumour cells were positive for Vimentin , S100, NSE, and Melan A. Cell
proliferation was apparent with both Ki-67 and PCNA.
Using histopathology, immunohistochemistry and electron microscopy, these tumours were conclusively
diagnosed as malignant uveal melonomas. These are rel atively rare tumours in laboratory rodents and to ourknowledge this is the first report combining these techniques in the diagnosis of this tumour in HAN Wistar
rats.

JSTP/IFSTP IATP 200~

Posters

P-161
Case report: naturally occuring proliferative vitreoretinopathy with multiple
ocular alterations in a rabbit
Osamu KATSUTA1, Takeshi YAMAGUCHI', Kenji TAKASE'
Drug Safety/Drug Metabolism Group, Research and Development Center, Santen Pharmaceutical Co., Ltd.,Nara 630-0101 , Japan'
One of the most serious complications of retinal detachment is proliferative vitreoretinopathy (PVR). The
pathogenesis of the disease is explained in humans as follows: After retinal detachment, the cells derived
from the retinal pigmental epithelium infiltrate in the vitreous cavity through a retinal hole and retinal glial
cells grow through the internal limiting membrane. These cells make preretinal proliferative membranes. The
membrane exerts a contraction on the retina, resulting in the retinal folding and its entire detachment. Thisdisease often occurs in failures in retinal surgery. Recently, we came across a young rabbit with naturally
occuring PVR. Now, we describe the pathological features of this case.
Animal: A six-week-old male Japanese white rabbit (Kbl: JW) (Conv.) purchased from Kitayama Labes Co .,Ltd., Nagano, Japan.
Clinical findings: The animal showed protrusion of both eyes with unilateral pupillary dilatation of the left
eye at the time of arrival. This case also did not react to light stimulation. So this was regarded as one ofcongenital glaucomas like buphthelmos, and was sacrificed at eight weeks of age. Ophthalmologic
examinations were not performed.
Necropsy findings: The intraocular examination revealed retinal detachment of both eyes and a whitish
coagulated substance on the posterior surface of the lens. In the left eye, the subretinal space was filled witha whitish gelatinous substance. Other organs and tissues were normal.
Microscopic findings: In both eyes, major findings were heavy foldings of the detached retina, posterior
subcapsular cataracts with partial rupture, preretinal proliferative membranes, and choroidal hypoplasias. The
retina of both eyes showed a funnel-shaped detachment. The retinal foldings were connected to each otherby fibrous membrane. The membrane contained a few pigmented cells, glial cell-clusters, and well-developed
capillaries. Preretinal neovasculization was conspicuous in the right eye. Marked serous exudation wasobserved under the retinal space in the left eye. No particular findings were seen in the anterior part of theeyes.
Based on these observations, this case was diagnosed as a late stage PVR. However, we could not explain
the pathogenesis of this case. Of course this case has not received any surgery. It is possible that the PVR ofthis case occurred bilaterally almost at the same time. Congenital disorders such as congenital cataracts
might be associated with the retinal detachment. Also, in some degree the choroidal hypoplasia might beconcerned with the event. Further examination is necessary to define the pathogenesis.

-

• -

f

•

-

JSTP/IFSTP IATP 200~

Posters

P-162
Histopathological findings of cleft palate in rat embryos induced bytriamcinolone acetonide
Satoshi FURUKAWN , Koji USUDA 1, Masayoshi ABE 1, Izumi OGAWA 1
Biological Research Laboratories, Nissan Chemical Industries, LTD., Saitama 349-0294, Japan'
Triamcinolone acetonide (TAC), a synthetic glucocorticoid, induces cleft palate resulting from poor
development of palatal shelves in mice. However, TAC has no effect on medial edge epithelial cells (MEE
cells) in secondary palatal shelves. In the present study, we examined the relationship between thepathogenesis of cleft palate and the effects on MEE cells and palatal mesenchymal cells in rat embryos
exposed to TAC. Pregnant Wistar Hannover rats were given TAC intramuscularly at 0.5 mg/kg at gestation
days (Day) 12, 13, and 14, then embryos were harvested on Days 14.5, 15, and 16. The effects of TAC wereas fo llows; an inhibition of palatal mesenchymal cell proliferation on Day 14.5, a decrease in the density ofpalatal mesenchymal cells and expression of epidermal growth factor (EGF) receptors in MEE cells on Day
15, and a reduction of stratified squamous differentiation of MEE cells with expression of cytokeratin and
EGF receptors on Day 16. These findings indicated that TAC inhibited the proliferation of mesenchymal
cells and affected the differentiation of MEE cells into stratified squamous epithelia in the palatal shelves ofrat embryos. However, these stratified squamous MEE cells partially fused with each other. Thus, we
suspected that a major contributing factor to the formation of TAC-induced cleft palate might not be thealtered differentiation of MEE cells, but the inhibition of mesenchymal cell proliferation.

270

JSTP/IFSTP IATP ·~t()OZI

Posters

P-163
Spontaneous neural crest tumors of pinna in mice
Hiroshi EDAMOT0 1, Mariko NAGATANI 1, Rie ANDO', Sachiko TAMAI', Kenichiro KASAHARA',
Shuzo OKAZAKI', Kazutoshi TAMURA 1
Division of Pathology, Gotemba Laboratory, Bozo Research Center Inc. Shizuoka, 412-0039, Japan'
Division of the Second Laboratory for Safety Evaluation, Gotemba Laboratory, Bozo Research Center Inc. 2
Neural crest tumor (NCT) in pinna, also referred to as a melanotic / amelanotic melanoma, is a rare tumor inmice. The purpose of the present study is to provide information about the frequency and some aspects ofthe pathological features of NCTs arising spontaneously at pinna in mice. A total of 7688 B6C3F, mice usedin long-term studies including studies for background data collection in our facility were surveyed. The
pinna NCTs occurred in 0.2% of males and 0.5 % of females. The earliest occurrence was recorded at 87
weeks of age in a male, which was observed externally as a solitary small nodular lesion. Most NCTs aroseat the basal portion of the pinna as a solid tumor pale in color. It is noteworthy that most NCTs wereobserved on the side of ear-tag application. Some NCTs grew rapidly with partial necrosis and ulceration,and the biggest reached to 35x30x25mm. Seventeen out of 30 (54.8%) mice died from NCTs including
eating disturbance from the markedly grown tumor. Histologically, most tumors were mainly composed ofspindle cells which proliferated with interlacing fascicular and striform patterns. Moreover, round andpleomorphic cells which had a large nucleus or multiple atypical nuclei were also seen in various ratios.
Mitosis also varied in the cases. In 8 out of 30 (26.7%) cases a few melanin pigments were observed but
they could not be detected in other cases. However, various numbers of premelanosomes and/or
melanosomes were confirmed in all cases examined electron microscopically. Almost all tumor cells
destructively invaded the surrounding tissues, sometime involving the eyes. Distant metastasis was observedin 8 out of 30 (26.7%) cases, and the main sites of metastasis were lungs, liver, kidneys, submandibularlymph nodes and heart, and only a few cases were seen in spleen, adrenals, thoracic cavity and tongue.
Although pinna NCT is a rare tumor, it often kills the animals directly or indirectly, and persistent and/or
repeated inflammation and regeneration caused by trauma may play a role in development of tumors.

JSTP/IFSTP IATP _2Q04

Posters

P-164
Histological changes in the dorsal skin of YPC mice irradiated with UVB for 4
weeks
Taro OKADA 1, Akira YASOSHIMA 1, Kaoru TAKAN0 2 , Junichiro MATSUDA2, Koji UETSUKA 1,
Hiroyuki NAKAYAMA 1, Kunio DOI1
Department of Veterinary Pathology, Graduate School of Agricultural and Life Sciences,
the University of Tokyo, Tokyo 113-8657, Japan 1
Department of Veterinary Science, National Institute of Infectious Diseases2[Background] YPC is an autosomal recessive mutant mouse strain which expresses poor and wavy hair coatand curly whiskers, and has been detected first in a female of Swiss albino mice colony at National Instituteof Infectious Diseases, Japan. In this study, YPC mice were irradiated with ultraviolet (UV) B daily for up to
4 weeks, and histological changes in the dorsal skin were examined.
[Materials and Methods] A total of 16 seven-week-old mice (male:8; female:8) were irradiated with UVB ata daily dose of 3 or 6 kJ/m2 x 6 days/week for 2 or 4 weeks(w). They were sacrificed 2 hours after
subcutaneous injection of bromodeoxyuridine (BrdU; 50mg/kg), and dorsal skin samples were served tohistological, immunohistochemical (cytokeratin 10 (CKlO), involucrin, and BrdU), and ultrastructual studies.
[Results] In mice irradiated with 6kJ/m2 UVB, thickening of the epidermis and infundibulum of hair follicles
due to proliferation of keratinocytes and dermal thickening due to proliferation of connective tissues wereobserved in both sexes at 2w. At 4w, infundibular keratinocytes became more proliferated and some cystic
structures were observed in those areas . Cystic structures were more frequently found in females than inmales. Many keratinocytes making up these structures were labeled with BrdU, and keratinocytes lining theinside surface of the cyst were positive for CKlO and involucrin. Ultrastructually, infudibular keratinocytes
showed cornification as seen in the epidermis, and some of these keratinocytes anchoring the basement
membrane had small cytoplasmic projections into dermis. Degeneration of collagen fibers and vessels was
also observed in the upper dermis. On the other hand, in mice irradiated with 3kJ/m2 UVB, epidermal anddermal thickening was observed at 2w and 4w. However, no changes were observed in the infundibulum andhair structures.
[Concl usion] In this study, the thickening of the epidermis and infundibulum with formation of cystic
structures and dermal thickening were found in the dorsal skin of YPC mice exposed to DVB-irradiation for
4 weeks. In addition, degeneration of collagen fibers and vessels in the dermis were also observed. In
addition, proliferation of keratinocytes was induced not only in the epidermis but also in the infundibulum,and such phenomena have not been reported yet. Thus, YPC is considered to be a useful experimental
animal in the field of photodermatology.

272

JSTP/IFSTP IATP 200~

Posters

P-165
Effects of scratching on the onset and exacerbation of dermatitis in NC/Ngamice
Yutaka NAKANISHI 1, Atsushi NAKAMURA1, Yuki HASHIMOT0 2 , Iwao ARAI2, Hiroto MIYATA 1,
Masaaki KIMURA 1
Toxicology Laboratory, Medical Research Laboratories, Taisho Pharmaceutical Co., Ltd., Saitama 331-9530,
Japan 1
Phamacology Laboratory, Medical Research Laboratories, Taisho Pharmaceutical Co., Ltd. 2
Introduction:
NC/Nga mouse is known as an atopic dermatitis model. The onset of dermatitis in NC/Nga mouse admits atconventional breeding, whereas it doesn't admit at SPF (specific pathogen free) breeding. The scratching
behavior increases immediately after conventional breeding, since then the onset of dermatitis is observed in
NC/Nga mouse. Although it is thought exacerbation of atopic dermatitis in human relate to scratching
behavior, the proof of evidence using experimental animals is not understood. So we established anexperimental model to avoid the effect of scratching by claw cutting in NC/Nga mouse at conventional
breeding. In this study, to estimate the effect of scratching on the dermatitis pathogenesis in NC/Nga mouse,
we carried out histopathological verification.
Method:Animal experiment: The dermatitis was induced after conventional breeding in NC/Nga mice (male, 4 weeks,Charles River). The dermatitis was observed macroscopically, and judged with skin severity score (minimum
0 to maximum 12). The claw of their hinds was cut with toenail clippers and nail files twice in a week. The
experimental design was as the following.
Prophylactic test: Divided the NC/Nga mice at 4 weeks age into 3 groups.
i) SPF breeding, ii) conventional breeding and iii) claw cutting + conventional breeding. These mice weresacrificed after 8 weeks breeding.
Therapeutic test: Divided the NC/Nga mice with severe dermatitis at 15 weeks age into 2 groups.
i) conventional breeding and ii) claw cutting + conventional breeding. These mice were sacrificed after 7
weeks breeding.
Histopathological study: The back skin in NC/Nga mice were fixed and embedded by conventional methods,
and stained with hematoxylin and eosin(H.E.) and toluidine blue.
Result and discussion:
In prophylactic test, the scratching behavior in NC/Nga mice increased immediately after conventional
breeding, and then the dermatitis was observed macroscopically within 4 weeks. In therapeutic test, thedermatitis improved macroscopically for one week. Microscopically, several findings including acanthosis,
ulcer/erosion, crust, inflammatory cell infiltration, infestation of ectparasite, fibrosis were noted. These
histopathological findings gave a good correlation with skin severity score. Skin severity score of dermatitis
showed improvement by claw cutting in these test. Furthermore, both of acanthosis and inflammatory cell
infiltration were also improved histopathologically on treatment with claw cutting. These results showed thatscratching behavior plays an important role for dermatitis pathogenesis in NC/Nga mice as well as atopic
dermatitis in human.

.

-----~-

JSTP/IFSTP IATP 2·00~

Posters

P-166
Morphological changes of auricular tissue in the mouse TNCB-repeatedapplication model.
Chie TAK.ADA' , Daisuke HARADA', Yukihito TSUKUM0 1, Toyoko KASHIWAGI 1, Haruhiko MANABE 1,
Satoshi NISHIKAWA', Kazuo SUZUKI', Katsumi TAKABA'
Kyowa Hakka Kogyo Co.,Ltd., Shizuoka 411 -8731, Japan 1
2,4,6-trinitro-l-chlorobenzene (TNCB)-repeated application model is well-known as chronic dermatitis model.
However, it has not been reported the pathological analysis of this model in detail. In this study, we
examined morphological changes of auricular tissue in the mouse TNCB-repeated application model.
Right ear auricles of BALB/c mice were sensitized with 0.3 w/v%TNCB and challenged three times per
week for 21 days starting from 7 days after sensitization (first challenge: day 0). The animals were sacrificed
at 0, 3, 6, 9, 24 and 48 hours after each challenge on days 0, 7, 9, 14 and 21, and at 48 hours after eachchallenge on days 2 and 16. The right auricles were removed and were fixed in 10 % buffered formalin.
Then, the tissues were embedded in paraffin. The sections were stained with Hematoxilin and Eosin, and fastgreen FCF for light microscopy examination.
Crust formation, erosion, microabscess in epidermis, inflammatory cell infiltration into epidermis and dermis,and edema in dermis were observed and/or getting more serious from day 7. On the other hand, thethickening of epidermis and eosinophil infiltration increased gradually from the first challenge. Some ofthem were time-dependent changes. Microabscess and increased eosinophils were most prominent at 6 to 9h
after each challenge. Erosion and crust formation attained the maximum magnitude at 24 hours, and edema
at 6 hours, respectively.
This model indicated that the lesions aggravated abruptly from day 7 except for the thickening of epidermis
and eosinophil infiltration.

274

JSTP/IFSTP IATP ·20011

Posters

P-167
Application of special staining to decalcified skeletal tissues in routine toxicity
studies
Aisuke NII 1, Kenji NAKANO', Takanori HANADA 1
Safety Research Laboratories, Yamanouchi Pharmaceutical Co. , Ltd. , Tokyo 174-8511, Japan'
Although relatively rare compared with other major organs, the skeletal system can be a target in toxicity
studies of drug candidates. The skeletal system is different from other organs in that it needs to be
decalcified before tissue preparation. Decalcified and paraffin embedded sections stained with hematoxylin
and eosin (H&E) are routinely used in toxicity studies. Other special techniques including non-decalcified
sections, X-ray microanalysis, bone labeling with fluorochrome markers such as tetracycline etc., are notusually incorporated in study design. We here present the usefulness of some special staining for decalcified
skeletal sections.
The important points when examining bone slides are cellular components and the intercellular matrix. For
the former, there are three major types of cells: osteoblasts, osteocytes, and osteoclasts. Toxic effects on
osteoblasts and osteoclasts are often encountered and sometimes result in lesions which resemble metabolicbone diseases such as osteomalacia, osteoporosis, osteopetrosis, etc. Alkaline phosphatase (ALP) and tartrateresistant acid phosphatase (TRAP) are the best-known histochemical markers of the osteoblast and osteoclast,respectively. For the osteoclast, some macrophage markers for immunohistochemistry and lectin
histochemistry are also useful. It is infrequent that the osteocyte is affected; however, the changes of the cell,once occurred, are easily recognized since the cell is located in the lacunae and difficult to be eliminated.
Osteocyte processes can be appreciated by the network structure of bone canaliculi using silver staining like
Bodian's method. Apoptosis of the cellular components can be seen by TUNEL or single-strand DNA
staining.
For the intercellular bone matrix, information on inorganic components is limited due to decalcification. For
organic matrices, immunohistochemistry can be applied for collagens, while that for non-collagenous
proteins is uncommon. Osteoid layers on the bone surface are seen with Masson's trichrome, and cement
lines in the trabeculae with Giemsa or alcian blue-PAS stain. Cartilagenous components are stained withalcian blue or toluidine blue. In addition, even in H&E stained sections, birefringent collagen fibers can beobserved with a polarized microscopy. For drug-induced skeletal changes, several special staining can beapplied to decalcified sections in routine toxicity studies, and the obtained results will be help in evaluating
the toxicity. Photomicrographs of some staining will be presented.

•~,&",,., .._ _ ,JSTP/IFSTP IATP ·200~

Posters

P-168
Histopathological characterization of the skeletal myopathy in rasH2 mice
Takayuki TSUCHIYA 1, Fumiko SAN0 1, Kazuhiro GOT0 1, Mamoru MUTAI 1, Toshimi USUI2,
Jiro SUGIMIT0 1, Shiro TAKAGI 1
Toxicology Laboratory, Mitsubishi Pharma Corporation, Chiba 292-0818, Japan 1
Central Institute of Experimental Animals 2
Skeletal myopathy was found approximately 100% in the rasH2 mice. Microscopically, this is essentiallysimilar to the features of muscular dystrophy. To confirm the detail features of rasH2 skeletal myopathy, HE,
Gomori trichrome and NADH-TR stain and electron microscopic examinations were applied to the muscles
obtained from male rasH2 and non-transgenic littermates aged 10-13 weeks. To evaluate the possible
involvement of dystrophin and its associated protein in the pathogenesis of this skeletal myopathy, theexpression of dystrophin and cx-sarcoglycan was assessed immunohistochemically. As the result, variation of
the muscle fiber size was widespread in the skeletal muscle. Scattered degeneration/necrosis and regenerationof muscle fibers were observed. Myopathic grouping of regenerating muscle fibers was not a feature of therasH2 mice myopathy in this age. The results of NADH-TR stain demonstrated no fiber-type grouping thatsuggests denervation of the muscle fiber. Muscle fibers showing disorganized intermyofibrillar network andnecrotic change were clearly demonstrated in NADH-TR stain. No specific morphological changes, like rod
structure or tubular aggregation suggesting specific type of myopathy, were noted in Gomori trichrome and
NADH-TR stains. Immunohistochemically, the expression pattern of dystrophin and cx-sarcoglycan (positivearound the rim of muscle fibers) were comparable between rasH2 and non-transgenic mice.
Electronmicroscopically, occasional muscle fiber degeneration/regeneration and invaded phagocytic cells wereobserved. These results indicated that myopathic changes in rasH2 mice is based on the gradual progress ofdegeneration and regeneration process of muscle fibers without specific structural changes seen in thespecific type of human myopathy. Abnormality of dystrophin and cx-sarcoglycan, major components ofdystrophin-glycoprotein complex, was not considered to be involved in the pathogenesis of myopathic
changes in rasH2 mice. Therefore, the mechanism of muscular degeneration in the rasH2 mice is thought tobe different from that of dystrophin-deficient model such as the mdx mice.

276

JSTP/IFSTP IATP 2004

Posters

P-169
Spontaneous degenerative lesions of the femoral growth plate in rats
Shinichiro IKEZAK.1 1, Osamu KUSUOKA1, Mizuho TAKAGI', Katsuhiko WARITA 1,
Maiko TSURUKAME 1, Kayoko KUDO', Sachiko TAMAI', Atsushi NAKAMURA'
Division of Pathology, Bozo Research Center Inc., Shizuoka 419-0101, Japan'
Degenerative lesions of cartilage, such as chondromucinous degeneration (CMD) or chondromucinous cystic
degeneration, are often seen in the sternum but also in articular and growth plate of the femur in rats.
Histologically, the lesions consist of vaiious stages of progression from beginning of dissolution of the
cartilage to lysis with cystic formation. It is reported that the incidence of CMD increases with advancing
age, with lesions first becoming noticeable at about 130-180 days of age. However, we have recently foundthat similar changes spontaneously occur in distal growth plate of the femur of young adult Crj:CD(SD)IGS
rats. The present study was performed to clarify the histo1ical information about the spontaneous
degenerative lesions in rats. Over two thousand male or female rats between 8 and 45 weeks of age, which
had been purchased from commercial breeders as Crj:CD(SD)IGS, were surveyed laying emphasis on
incidence of the degenerative lesions in distal growth plate of the femur. These animals were collected from
non-treatment and vehicle control groups in various toxicological studies and were divided into four different
age groups of 8 (group 1), 10-14 (group 2), 19-23 (group 3) and 32-45 (group 4) weeks old. All samples
were cut into sagittal sections and stained with hematoxilin and eosin for microscopical examination.
Histology chai·acte1istics of the degenerative lesions in this study were: (a) lysis of cartilage matrix in growthplate, (b) disorder of differentiation in the chondrocytic columns, (c) resulting in disai-rangement of thegrowth plate line. These lesions generally occun-ed in opposite side of the patella and in rats of over 10
weeks of age. The incidence of the lesions in groups 1 to 4 by the above criteria were respectively 0, 1.7,
10.5 and 7 % in male, and 0, 0.7, 2, and 3.4% in female. Thus lesions in males showed a tendency to occur
more frequently compared with females. The incidence in rats over 19 weeks of age was higher than that inrats under 14 weeks of age. Furthermore, the present survey showed the degenerative lesions of cartilage
such as CMD have also occurred in younger rats than that in the past reports, although they are of low
incidence.

Posters
P-170
Early pathophysiological feature of arthropathy in juvenile dogs induced byofloxacin, a quinolone antimicrobial agent
Koichi YABE1, Hiroshi SATOH 1, Toshimasa JIND0 1, Tadaki SUGAWARA 1, Kazuhisa FURUHAMA1,
Masanobu GORY0 2 , Kosuke OKADA2
Drug Safety Research Lab., Daiichi Pharmaceutical Co. , Ltd., Tokyo 134-8630, Japan 1
Veterinary Pathology, Iwate University 2
Arthropathy in dogs induced by ofloxacin, a quinolone antimicrobial agent, was pathophysiologicallyinvestigated to elucidate the early morphological characteristics in both in vivo and in vitro systems. In thein vivo studies, ofloxacin was orally administered once or twice at 20 mg/kg/day to male juvenile (3-monthold) or adult (36-month-old) dogs. Unlike adult dogs, unifocal or multifocal fluid-filled vesicles were
macroscopically observed on the articular surfaces of juvenile dogs from 24 hours after a single treatment
with ofloxacin. Microscopically, fissures or cavity formations in the middle zone of the articular cartilage
were noted only in juvenile dogs. Further, the cartilage matrix from the abnormal area to the articular surface
showed a decreased safranin-0 staining intensity, suggesting proteoglycan depletion . Ultrastructurally,chondrocytes in the middle zone of juvenile dogs displayed dilatation of the cisternae in the roughendoplasmic reticulum as an initial hallmark, in conjunction with depletion in the proteoglycan granules andedema in the territorial matrix. In the in vitro studies, chondrocytes isolated from the articular cartilage ofnaive juvenile dogs were exposed to ofloxacin at 6.3 to 100 µg/rnl for 24 hours. Although no changes werenoted in the DNA synthesis, protein synthesis or proteoglycan release at concentrations of up to 100 µg/rnl,
the proteoglycan synthesis was evidently decreased in a dose-dependent manner from 12.5 µg/rnl. The results
obtained suggest that the inhibitory action of ofloxacin on proteoglycan syntheses in the chondrocytes would
largely contribute to the early morphological changes in the articular cartilage of the juvenile dog, andconsequently the proteoglycan depletion in the matrix may play a critical role in the cavity formation,
preceded by dilatation of the cisternae in the rough endoplasmic reticulum.

278

JSTP/IFSTP IATP 2004

Posters

P-171
Organ calcification in monkeys treated intradermally with rhPTH1-34 for
6 months followed by a 1-month recovery period
Jihong YANG', Peng WANG', Xiangting XU 1, Guoping XU 1, Ling ZHANG' , Liuyi ZHANG'
Department of Pathology, Yunnan Pharmacological Laboratories of Natural Products, Kunming Medical
College, Kunming City P. R. 650031, China (PRC)'
ABSTRACT:Parathyroid hormone (PTH) is the key endocrine factor regulating extracellular Ca2+
homeostasis. In recent years, PTH has been used for prevention and treatment of osteoporosis. Humanparathyroid hormone 1-34 (rhPTHl-34) is the segment which includes the extracellular Ca2+ homeostasis
function.
Objective:The purpose of this 6-month study was to investigate the potential toxicity of rhPTHi-34
administered once daily in Cynomolgus monkeys (macaqa fascicularis) for safety evaluation. Based upon theclinical dose, rhPTH1-34 was administered via intradermal (i.d.) injection for six months at dose levels 5, 15,
and 45µg/kg/day. Solvent administered i.d. to an additional group of monkeys served as control. The solvent
control group and the rhPTH1.34 groups at 5 and 15µg/kg/day consisted of six monkeys (3F, 3M), while therhPTH1 .34 group at 45µg/kg/day included seven monkeys (3F, 4M).
Histopathology Results:One male monkey in the high dose group, showing diffuse multifocal calcificationof heart and kidneys, was sacrificed after 1 l 7days.
CD After 6-month treatment followed by I-month recovery, calcium deposits, verified in situ by the staining
methods of von Kossa and Dahl, were observed in various regions of cortex and medulla of the kidneys in
one monkey of the median dose and two monkeys of the high dose.
® Bone with decreased bony spicules of decreased size was not demonstrated in vertebrae, sternum, ribs
and thighbone.
® After 6-month treatment the number of chief cells of the parathyroid glands was noted to decrease withan accompanying interstitial fibrosis in altogether three monkeys of the 15µg/kg/day and 45µg/kg/day
groups. One monkey in the high dose group showed atrophy of the parathyroid glands, but following 1month recovery this was restored to normal. No changes were observed in the solvent control group or the
rhPTHi -34 5µg/kg/day group.
Conclusion:6-month administration at doses of 15µg/kg/day and 45µg/kg/day of rhPTHl-34 would induce
lesions in organs of Cynomolgus monkeys. Despite the fact that the clinical dose, 0.8µg/kg/day, is in thesafety range, great care has to be exerted during administration of rhPTHl-34 to avoid organ lesions.

JSTP/IFSTP IATP 2·00~

Posters

P-172
Physiological parameters and background histopathology findings fromchronic (2 year) carcinogenicity studies in the HAN wistar rat
Christopher BARTON 1
Department of Pathology, Covance Laboratories, North Yorkshire HG31PY, UK 1
The HAN Wistar rat is becoming increasingly popular for use on regulatory toxicology studies.
The HAN rat strain tends to have a lower incidence of spontaneous background histopathology, whencompared with some other rat strains; most notably those tumours involving endocrine and hormoneassociated tissues.
The HAN rat generally has a low mortality rate, with good survival over the course of a 2 year study andthe potential for reducing group sizes. In addition, the small adult body weight can result in considerable
reductions in bulk drug requirement.
There are few publications detailing basic physiological data: Bodyweight gain, food consumption, survival
data and spontaneous tumour incidences for the HAN Wistar rat in chronic (2 year) carcinogenicity studies.
The control data in this poster presentation are from carcinogenicity studies conducted recently at CovanceLaboratories, Harrogate, England

280

Author Index

281

AUTHOR INDEXS : Symposium

A
ABDUL-HAMID, Amani
ABE, Masayoshi
AISAKI, Ken-Ichi
AISO, Shigetoshi
AKIMA, Michio
AKIYAMA, Izumi
ANDO, Minoru
ANDO, Rie
AOKI, Toyohiko
AONAHATA, Miyuki
ARAI, Iwao
ARIKI, Yutaka
ASADA, Kiyoshi
ASAMOTO, Makoto
ASANO, Satoshi
ASHIDA, Hitoshi
ASHINA, Michiko
ATKIN, Thelda
ATSUMI, Fusako

P-32
P-162
P-45
P-1 29, P-140
P-110
P-126
P-101
P-163
P-141
P-86
P-165
P-59
P-64
S2-l-l , P-16, P-24, P-47
P-48, P-78, P-92, P-156
P-106, P-128
P-37
P-154
S3-4
P-114

B
BABA, Yasuko
BABA, Yoshinobu
BAEK, Min won
BAK, Eun-Jung
BARBISAN, Luis Fernando
BARTON, Christopher
BLANCHARD, Terrell
BOONYAPHIPHAT, Pleumjit
BOUCHIHA-OLSON, Sophie
BROWN, Corrie
BROYLES, Robert H.

P-95 *
P-54
P-40, P-109
P-123
P-108
P- 160, P-172*
S3-4
P-44*
S3-4
S4-4*
P-64

C
P-108
CAMARGO, Joao Lauro Viana De
CANNON, Ronald E
P-76
P-31
CAO, Xueyuan
CHIBA, Yuko
P-9
P-29, P-30*, P-40, P-109
CHO, Sun A
CHO, Sung-Whan
P-149
P-121
CHO , Wan Seob
P-74, P-145*
CHO, Young-Man
CHOI, Mina
P-121
CHOI, Young Jin
P-34
COHEN, Samuel M .
Plenary Lecture*
COUGHLIN, James R.
L-1 *
L-6*
CROUCH, Cristopher N.
CUNNINGHAM, Michael L.
S2-2 Chair, S2- l-4*

D
DE CLERCK, Nora M.
DE VILLA, Flordeliza P.
DEMURA, Nobutaka
DENDA, Ayumi

P-124
P-102
P-103
S2-l-3 , P-85*

DESCOTES, Jacques
DEVEREUX, Theodora R.
DOI, Kenichiro
DOI, Kunio

DOI, Takuya
DOI, Yuko
DUAN, J. D .
DUNN, Dale

P : Poster

S2-2-3*
P-62
P-18*, P-35
S4 Chair,P-95 , P- 112,P-113
P-114, P-123, P-133, P -134
P-164
P-154*
P-11*, P-12, P-24
IATP
S3-4

E
EDAMOTO, Hiroshi
EMA , Makoto
ENDOU, Hitoshi
ENOMOTO, Akiko
ETO, Komyo

P-146, P-163 *
P-146
P-107
S2-2-2, P-5, P-117
P-110*F
P-13*
FENNER-CRISP, Penelope A
S4 Chair
FINCH, John
FLOYD, Eugenia
S 1-2-3*
FLOYD, Robert A.
P-64
P-54, P-150
FUJIHIRA, Shiro
P-152*FUJISAWA, Kae
FUJIWARA, Osamu
P-24
S 1-2-4
FUKAMACHI, Katsumi
FUKUNARI, Atsushi
P-93
Sl-1-1, P-11, P-14, P-15
FUKUSHIMA, Shoji
P-22
P-18, P-20, P-21 ,P-46
P-28, P-32, P-35,
P-66
P-59, P-63, P-65,
P-67 , P-69, P-79 ,P-80
P-89 , P-97, P-116
P-101
FUNABASHI, Hideyuki
P-21
FUNAE, Yoshihiko
P-135, P-170
FURUHAMA, Kazuhisa
FURUKAWA, Satoshi
P-162*P-90
FURUSATO, Masakuni
P-65
FURUTA, Junichi
P-47
FUTAKUCHI, Mitsuru

G
GERMANN, Paul-GeorgGERMOLEC, Dori
GILL, Santokh
GON, Rina
GORYO, Masanobu
GOTO, EmikoGOTO, Kazuhiro
GOTO, Mitsuo
GOTOU, Shunji

S2-2 Chair
S2-2-4*
P-132
P-136
P-170
P-43
P-168
P-88
P-139

H
HA, Chang-su
HAGIHARA, Atsushi

P-149
P-65*

HAGIWARA, Akihiro
P-19*
HAHM, Ki-Baik
P-58
HAHN, Sean
S3 -4
P-155
HAILEY, James R.
P-20, P-76*, P-116
HAKOI, Kazuo
HAMAGUCHI, Tetsuya
IATP
P- 122
HAMAMURA, Masao
Sl-2-4, P-58, P-121
HAN, Beom Seok
P-34
HAN , Kyung Ja
HANADA, Takanori
P-167
HARA, Akira
P-82
P-157
HARA, Takuji
HARADA, Daisuke
P-166
HARADA, Miwako
P-55
S2-2-2* , P-5, P-7 , P-9, P-117
HARADA, Takanori
P-147
HARD , Gordon C
HARRISON, David
S4-2*
HASEGAWA, Ryuichi
P-146
P-62, P-155
HASEMAN, Joseph K.
HASHIMOTO, Kana
P-101 *HASHIMOTO, Yuki
P- 165
P-74, P-145
HASUMURA, Mai
HATA, Junko
P-28*
P-37, P-73
HATANAKA, Yutaka
P-102
HATTORI, Syunji
HAYAKAWA, Kazuhiro
P-141 *
P-7, P-9
HAYASHI, Koichi
P-136
HAYASHI, Masaomi
HAYASHI, Morimichi
P-84*
HAYASHI, Shim-mo
P-62*
HAYASHI, Shuji
P-116*P-131
HAYDARI, Mahnaz
HIBINO, Tsutomu
P-36*
HIGASHIYAMA, Hiroyuki
P-106, P-I28
HIKOSAKA, Atsuya
P-68
Memorial Keynote Lecture*, P-70
HINO, OkioHIRAKAWA, Kimiaki
P-151
HIRATA, Akihiro
P-17 \ P-100, P-139
HIRAYA MA, Youko
P-70
S 1-1 Cair, S 1-1-2, P-25, P-50
HIROSE, Masao
P-53, P-60, P-74, P-104
P-145, L-1 *HIROSE, Yoshinobu
P-82
P-10, P-12
HIROTA, Takeshi
HIRUMA , Masarni
P-102
HISADA, Shigeru
P-77*
HOKAIWADO, Naomi
P-16, P-92'''
HONG, Hue-Hua L.
P-62
P-23
HONG, Jin Tae
HORIGUCHI, Kohsuke
P-98
HORII, Ikuo
S3 Chair
HORIUCHI, Toshi
P-77
P-14, P- 15
HOSHI, Manabu
HOSOE, Kazuno1i
P-59
HOSOKAWA, Kyoko
P-39, P-127
P- 141HOSOKAWA, Satoru
P-39 , P-127
HOSOTANI, Yoko
S2-l-2HOSYUYAMA, Satsuki

I
IATROPOULOS, Michael
ICHIHARA, Toshia

IATP*
P-19, P-156*

IGARASHI, Isao
IGARASHI, Katsuhide
IGARASHI, Maki
IIDAKA, Takeshi
IKEDA, Akiko
IKEDA, Mico
IKEZAKI, Shinichiro
IMAI, Kiyoshi
IMAI, Norio
IMAI, Toshia!MAIDA, Katsurni
IMAOKA, Masako
IMAZAWA, Takayoshi
INADA, Ken-Ichi
INAGAKI, Haruhisa
INAGAMI, Atsushi
INAGUMA, Shingo
INAMINE, Morihiko
INOMATA, Akira
INOUE, Satoshi
INSKEEP, William
IRIMURA, Kenji
IRWIN, Richard D.
ISHII, Shun-Ichiro
ISHIKAWA , Kayoko
ISHIMINE, Sayaka
ITAGAKI, I01i
ITO, Kazumi
ITO, Nobuyuki
ITO, Tsuneo
ITO, Ts uyoshi
ITO, Yuko
IWAKI, Tomomichi
IWATA, ShinIZUMI, Kei suke

P-51, P-143
P-45
P-37* , P-38 , P-61 , P-91
P- 17, P-100
P-41*, P-115
P-39*, P-127
P-146, P-169*
P-1
P-10, P-11 , P-19
P-74*, P-145
P-39, P-1 27
P- 135*
P-25, P-60, P-104
P-88
P-1 52P- 141
P-16, P-48
P-83 , P-86
P-141
P-67, P-69, P-142
S3-4
P-116
P-62
P-148
P-51
P-117
P-102
P-49, P-51, P-114
P-33
P-112
P-1*
P-87
P-93
P-101
P-94

J
JANG, Dong DeukJEFFREY, A . M.
JINDO, Toshimasa
JOHNSON, Keith
JONES , Stewart

P-58, P-121
IATP
P-170
S2-2-4P- 153K
KADO, Shoichi
KAI, Kiyonori
KAJIKAWA, Satoru
KAJIMOTO, Kazuaki
KAJIMURA, Tetsuyo
KAKINUMA, Chihaya
KAKUNI, Masakazu
KAMADA, Nobuo
KAMATA, Eiichi
KAMEDA, Haruko
KAMIMURA, Yasuhiro
KANAI, Yoshikatsu
KANBORI, Miyuki
KANDORI, Hitoshi
KANEKO, Yoshihumi
KANEMATSU, Masahiro
KANESHIRO, Tatsuya

P-101
P-135
P- 133*
P-54
P-135
P-105
P-20, P-157*
P-98
P-146
P-103
P-151
P-107
P-49, P-51
P-16*
P-41 , P-115'~
P-106
P-83, P-86

KANG, Boo-Hyon
KANG, Chang Suk
KANG, Jin Seok
KANG, Jong-Koo
KANG , Kyung-Sun
KANKI, Keita
KANNO, Jun
KANNO, Takeshi
KARASAWA, Yayoi
KASAHARA, Kenichiro
KASAHARA, Yoshinori
KASHIDA, Yoko
KASHIMOTO, Naoki
KASHIWABARA, Shoji
KASHIWAGI, Toyoko
KASHON, Michael
KATAGIRI, Taku
KATAYAMA, Kei-Ichi
KATI, Ayumi
KATO, Atsuhiko
KATO, Masanori
KATO, Natsumi
KATOH, Osamu
KATOKU, Koshirou
KATOU, Akino
KATSUDA, Shin-Ichi
KATSUTA, Osamu
KATSUYAMA, Kiyoka
KAVANAGH, Meghan
KAWABE, Mayumi
KAWACHI, Hiroshi
KAWAGUCHI, Hiroaki
KAWAMATA, Seiichi
KAWANO, Yukiko
KHAKI, Amir Afshin
KHAKI, Arash
KIJIMA, Kazuyasu
KIKKAWA, Hideo
KIKUCHI, Kaoru
KIM, Bang Hyun
KIM, Chuel Kyu
KIM, Chui Kyu
KIM, Dae Joong
KIM, Dae-Yong
KIM, Dong-Jae
KIM, J
KIM, So-Hyun
KIM, Tae Myoung
KIM, SU
KIM, Yong Gu
KIM, Yong-Bum
KIM, YSKIMOTO, Naoya
KIMURA, Masaaki
KINJO, Tatsuya
KINOSHITA, AnnaKINOSHITA, MineKINOUCHI, Shigemi
KITAnMA, Natsuki
KITAMURA, Tsuyoshi
KITAMURA, Yasuki
KITANO, Mitsuaki
KITAORI, Nami

P-149
P-34*
P-79, P-97*
P-23
P-71
P-25, P-60, P-104
P-45*
P-154
P-134
P-163
P-28
P-26, P-27, P-56
P-57, P-130, P-136
P-57 *, P-130*, P-1 36
P-166
S2-2-4P-129, P-140
P-1 12, P-113
P-36
P-134*
P-59
P-50, P-53
P-57, P-130, P-136
P-122
P-139
P-43
P-161 *
P-112
P-132
P-10, P-156
P-150
P-8*, P-52
P-1 36S2-l -2
P-131
P-131 *
P-70
P-106, P-128
P-46
P-121 *
S 1-2-4
P-121
P-23 *
P-58*
P-29, P-30, P-40, P-109
P-119
P-29
P-23
P-11 8P-34
P-1 49*P-119
P-1 57
P-42, P-165
P-83, P-86
P-14, P-18, P-63*, P-79, P-80
P-106, P-128
P-39
P-28
P-84
P-25, P-60*, P-104
P-59*
P-82

KITAZAWA, Toshiaki
KIUCHI, Katsuji
KIYOSAWA, Naoki
KOBAYASHI, Kiyoshi
KOBAYASHI, Masato
KODAMA, Keiji
KODAMA, Yasushi
KODAMA, Yukio
KOHNO, Hiroyuki
KOIDE, Akihiro
KOJIMA, Sayuri
KOKOSHIMA, Hiroko
KOMINAMI, Yoko
KOMMINENI, C
KONISHI, Yoichi
KOSAKA, Tadashi
KOSYK, Oksana
KOTAKE, Yashige
KOTANI, Takao
KOUCHI, Marni
KOUTITANI, Takatoshi
KUBO, Akiko
KUDO, Kayoko
KUNIYASU, Hiroki
KUNIYOSI, Sakai
KUNO, Toshiya
KUPER, C Frieke
KURIBAYASHI, Masanori
KURODA, Junji
KUROKI, Koji
KUROSE, Tomoyuki
KURUSU, Osamu
KUSHIDA, Masahiko
KUSUOKA, Osamu
KUWAHARA, Maki
KUWAMURA, Mitsuru
KUWASAKI, EmikoKUWAYAMA, Chitose

P-5, P-117
P-111
P-49, P-51*, P-114, P-143
P-106, P-128*
P-139
P-94
P-137
P-104
P-Y, P-6, P-81
P-69
S2-2-2, P-9
P-1 54P-136
S2-2-4S2-l-3 , P-85
S2-2-2, P-5 , P-7, P-9
S2-l-3
P-64*
P-120
P-125
P-125*
P-94
P-169
P-85, P-138, P-144
P-80
P-82*
S2-2-4P-16, P-24*, P-78
P-84
P-125
P-136
P-128
P-22*, P-46
P-98*, P-169
S2-2-2, P-5 *, P-7, P-9, P-117
P-120
P-122*
P-105 *L
LAMARRE, Jonathan
LEE, Beom Jun
LEE, Hui Young
LEE, In Soo
LEE, Kook Kyoung
LEE, Kyoung-Youl
LEE, Min-Cheol
LEE, Yong-Soon
LEE, Young-Soon
LOSOS , George
LUSTER, Michael

P-120
P-23
P-29, P-30, P-40*, P-109
P-30
P-121
P-50, P-53 *P-118*, P-119*
P-40, P-71 *, P-109
P-29
P-141
S2-2-4M
MACHIDA, Noboru
MAEDA, Hiroshi
MAEKAWA, Akihiko
MAITA, Keizo
MAKINO, Toshihiko
MANABE, Haruhiko

P-26, P-27
P-151
P-1 , P-37, P-38,
P-61
P-72, P-73, P-91
S2-2 Chair, S2-2-2, P-5
P-7,
P-9, P-117
P-114
P-166

MANABE, Sunao
MAR, Mei-Heng
MARONPOT, Robert R.
MARUYAMA, Hiroshi
MARUYAMA, Toshiyuki
MASEGI, Toshiaki
MASUMOTO, Yoshihiro
MASUMURA, Ken-ichi
MASUTANI, Mitsuko
MASUTOMI, Naoya
MATSUDA, Junichiro
MATSUMOTO, Izumi
MATSUMOTO, Masahiro
MATSUNUMA, Naochika
MATSUO, Masatoshi
MATSUOKA, Nobuo
MATSUOKA, Yoichiro
MATSUSHIMA, Shuuichi
MATSUURA, Tetsuro
MATUYAMA, Junya
MCBURNEY, Michael W.
MENSE, Mark
MEURRENS , Kris
MIHARA, Takuji
MIKAMI, Nobutoshi
MIKAMI, Nobuyoshi
MITAMURA, Mana
MITANI, Hiroaki
MITOMA, Hideo
MITSUI, Masayuki
MITSUMORI, Kunitoshi
MIYAJIMA, Hiroaki
MIYAMOTO, Kazuaki
MIYAMOTO, Koichi ro
MIYATA, Hiroto
MIYATA, KaoriMIYAUCHI, Hideyuki
MIYAZI, Natsuko
MIZUTANI, Masato
MOKU, Masaharu
MOON, Yeon Sook
MOORE, Malcolm A.
MORAN , Alexandra
MORAWITZ, Gerd
MORI, Hideki
MORI, Satoru
MORI, Yu kio
MORIGUCHI, Kaei
MORIMOTO, Junji
MORIMOTO, Yasuo
MORIMURA, Keiichirou

MORIOKA, Takamitsu
MORIZUMI, Kosuke
MOTO, Mitsuyoshi
MOTOOKA, Satoru
MUELLER, Ruedi
MURAI, Takashi
MURASAKI, Toshiya
MURATA, Nao
MURO!, Takako

286

P-49, P-51, P- 114, P-143
P-37
P-155
P-75
P-67, P-69, P-142
P-17 , P-100, P-139
P-41, P-115
P-25
P-98
P-148
P-164
P-70*, P-125
P-54, P-150
P-49, P-51
P-4
P-55 , P-70, P-125
IATP, Sl-2-4
P-152
P-137
P-36
Sl -1-4*
S3-4*
P-124
P-73
P-46
S2- l- l
P-106
P-70
P-2
P-75 *
P-26, P-27, P-56, P-84, L-8*
P-72, P- 15 1
P-65
P-8, P-52*
P-42*, P-1 65
P-4*
P-67, P-69
P-15 , P-89*
P- 157
P-79*, P-89
P-34
P-33
P-160*
S3 Chair
P-82
P-66*, P-67 , P-69, P-142
P-69
P-111
P-87
P-126
Sl -1-1 , P-11 , P-14*, P-15
P-19 , P-21 , P-22, P-28
P-32, P-59, P-63 , P-66
P-80, P-89, P-97, L-5 *P-83, P-86*
P-122
P-26*, P-27 , P-56
P-141
P-132
P-66, P-67*, P-69'\ P-142
P-68 *P-85
P-2

MUTAI, Mamoru
MUTO, Norio
MUTO, Tomoko
MUTOH, Tomoko

P-168
P-102
P-107*
P-26, P-56

N
NABA, Hiroyasu
NABAE, Kyoko
NABANDITH, Viengvansay
NAGAE, Yusuke
NAGANO, Kasuke
NAGAO, Tetsuji
NAGAOKA, Takaharu
NAGASAWA, Tatsuya
NAGATA, Tomoko
NAGATA, Yuriko
NAGATANI, Mariko
NAITO, Akihiro
NAITO, Saki
NAKAE, Dai

NAKAGAMA, Hitoshi
NAKAHARA, Yutaka
NAKAI, Tokiko
NAKAMURA, Atsushi
NAKANISHI, Hayao
NAKANISHI, Yutaka
NAKANO, Katsuji
NAKANO, Kenji
NAKASHIMA, Nobuaki
NAKATA, Tomoko
NAKATSUTI, Nori o
NAKATSUJI, Shunji
NAKAYAMA, Hiroyuki
NAKAYAMA, Koji
NAKAZATO, Shoko
NAM, Ki Taek
NAMIKI, Masato
NARAMA, Isao
NII, Aisuke
NIINO, Noriyo
NINOMIYA, Fumiko
NISHIKAWA, Akiyoshi
NISHIKAWA, Satoshi
NISHIKI, Masayo
NISHIMORI, Miki
NISHIMURA, Nobuo
NITTA, Yurniko
NIWA, TomNODA, Shuji
NOHMI, Takehiko
NOLAN, Judith A.
NYSKA, Abraham

P-105
P-10*, P-11 , P-12, P-24
P-83, P-86
P-103
P-75, P-129, P-140
S2-2-l *
P-72*
P-84
P-158
P-101
P-163
IATP, Sl-2-4
P-15*, P-79
S2- l -3, P-1, P-37 , P-38
P-61 , P-73, P-85, P-91
L-9*
P-98
P-122
P-94*
P-42, P-165 , P-169
P-100
P-42, P-165*
P-55
P-133 , P-167
S2-2-2, P-5, P-117
P-80
S4-l *
P-150*
P-95, P-113, P-114, P-123
P-133, P-164
S2- l-2
P-150
P-58, P-121
P-154
P-137
P-133 , P-167*
P-49, P-51
P-116
Sl-1 -2*, P-25 , P-60, P-104
P-166
P-136
P-43 *
P-146
P-99*
P-88
P-2
P-25
S3-3 *
S2-2-4, P-155

0
OCHIYA, Takashi
OCHOA, Ricardo
OEDA, Kenji
OGAMI, Akira

P-136
S3 -2
P-46
P-126*

OGATA, Tetsu
OGAWA, Izumi
OGAWA, Kumiko
OGIHARA, Takuo
OGISO, Tadashi
OH, Sang-Yeon
OHASHI, Nobuyuki
OHNISHI, Hiroyuki
OHNISHI, Shuhei
OHNO, Rie
OHTA, RyoOHTSUKA, Ryoichi
OHYAMA, Naoki
OISHI, Yuji
OKADA, Kosuke
OKADA, Miyoko
OKADA, Taro
OKAMURA, Miwa
OKAZAKI, Kazushi
OKAZAKI, Shuzo
OKAZAKI, Yoshimasa
OKAZAKI, Yoshimitsu
OKIMOTO, Kazuo
OKUHARA, Yuji
OKUMURA, Hiroki
OKUNO, Yasuyoshi
ONISHI, Takamasa
ONO, Atsushi
ONOSE, Jun-Ichi
ONOUE, Masaharu
OSADA, Mayuko
OSAWA, Noriko
OSHIKATA, Takafumi
OTSUKA, Masanori
OTSUKA, Takafurni
OTSUKI, YoshinoriOYABU, Takako
OZAKI, Keisuke
OZAKI, Kiyokazu

P-96
P-162
P-12, P-47, P-48 , P-68 , P-92
P- 105
P-24, P-78, P-92
P-58
S3- l *
P-78*
P- 105
P-42
P-1
S2-2-2, P-7*, P-9
P-148
P-54, P-111 , P- 150
P-170
P-148*
P-164*
P-26, P-27* , P-56
P-60
P-163
P-54, P-150
P-43
P-70, P-125
P-84
P-1
P-4, P-22, P-46
Sl-2-4
P-45
P-74, P- 145
P-101
P-21
P- 158P-122
S2-l-l , S2-1-2, P-2
P-88
P-87
P-126
P-22, P-46, P-155 *P-137*

p
PARK, Cheol BeomSl-2-4, P-23
PARK, Jae-Hak
P-29, P-30, P-40, P-109
PARK, Jong-Hwan
P-29*, P-30, P-40, P-109
P-121
PARK, Ki Dae
P-29
PARK, Yong-Ho
PATTERSON, D. Reid
IATP*, P-159*
PATTON, Dorothy E
P-13
PAUMEN, Michael
L-4 *P-153 *PILLING, Andrew
S2-2-4PORTIER, Christopher
POSTNOV, Andrei A.
P-124
POWELL, Christine
S2-l -3
PRUIMBOOM-BREES, Ingrid
S3-2*
PUATANACHOKCHAI, Rawiwan
P-18, P-20, P-21*
PULIDO, Olga
P-132*
PUTTAWIBUL, Puttisak
P-44

R
RODRIGUES , Maria Aparecida Marchesan
P-108
Sl -1 Chair, P-132
ROUSSEAUX, Colin

RUSYN, Ivan
RYAN, Anne M.
RYU, JK

S2-l-3*Sl-1-3*
P-118

s
SAITO, Koichi
SAITO, Yoshiaki
SAKA, Machiko
SAKAI, Hiroki
SAKAMOTO, Satoko
SAKATA, Keiko
SAKUMA, Kyoko
SAKUMA, Sadashige
SALIM, Elsayed I.
SANDERS, James E.
SANG, Hong
SANO, Fumiko
SANO, Masashi
SAOO, Kousuke
SASA, Kaori
SASAHIRA, Tomonori
SASAKI, Atsushi
SASAKI, Junya
SASAKI, Takamitsu
SATAKE, Shigeru
SATO, Hidetaka
SATO, Hitoshi
SATO, Junko
SATO, Satoko
SATO, Takayuki
SATO, Yasukazu
SATO, Yuichi
SATOH, Hiroshi
SAWAKI, Masakuni
SEELY, John Curtis
SEHATA, Shinya
SEKI, Shuichi
SEKIJIMA, Masaru
SENOH, Hideki
SEOK, Seung H.
SHEN, Jun
SHIBATA, Masa-Aki
SHIBATA, Nobuo
SHIBUTANI, Makoto
SHIGEMATSU, Hidekazu
SHIKATA, Nobuaki
SHIM, Sang In
SHIMADA, Naoko
SHIMAZAKI, Kei-Ichi
SHIMIZU, Fujio
SHIMIZU, Takahiro
SHIMOI, Akihito
SHIMOJI, Naoshi
SHIMOMOTO, Takasumi
SHINOHARA, Yasuo
SHINOHARA, Yasuro
SHIOTA, Kunio
SHIRAI, Norimitsu
SHIRAI, Tomoyuki

S2-l-2, P-46
P-158*
S2-2-2
P-17 , P-100, P-139
P-2
P-82
P-49, P-51
P-120
P-18, P-32*, P-35 , P-97
L-7 *
P-64
P-148 , P-168
P-4
P-39 , P-127
P-103
P-138, P-144*
P-136
S2-2-2
P-138*, P-144
P-151
P-43
P-157
P-154
P-143
P-49
P-115
L-6*
P-135, P-170
P-2*
IATP*, P-147*
P-114*
P-65
S2- l -l, S2-l-2
P-129* , P-140
P-29, P-30, P-40, P-109*
P-35 *
P-87*
P-84
P-50* , P-53
P-102
P-111
P-34
P-96
L-2 *
P-150
P-83 , P-86
P-43, P-105
P-103 *
P-73
P-54
S2- l-2
S4-3 *
P-17, P-100
S2- l Chair, S2- l - l *, S2-l-2
P-10, P-12, P-16,
P-24
P-33, P-47, P-48 , P-68
P-78, P-92, P-156

P-127SHIRAISHI, Makoto
P-136
SHIRAISHI, Miho
P-98
SHIRAIWA, Kazumi
P-42
SHIRANE, Rika
P-117
SHUTOH, Yasufumi
P-62
SILLS, Robert C.
P-150
SOGABE, Hajime
P-8
SOHDA, Masakazu
P-131
SOHRABI, HAGHDOST, Iraj
SON , Hwa-Young
P-149
P-71
SON , Woo-Chan
P-141
SONODA, Jiro
SPINARDI-BARBISAN, Ana Lucia Tozzi
P-108*P-44
SUDHIKARAN, Wanna
P-41, P-115
SUEYOSHI, Sumihisa
P-170
SUGAWARA, Tadaki
P-3, P-6, P-81
SUGIE, Shigeyuki
P-148, P-168
SUGIMOTO, Jiro
P-134
SUGIMOTO, Tetsuro
P-98
SUGIMURA, Takashi
P-68
SUGIURA, Satoshi
P-103
SUGIYAMA, Haruyo
P-22, P-46*
SUKATA, Tokuo
S2-l-2, P-46, P-56
SUMIDA, Kayo
P-83*, P-86
SUZUI , Masumi
SUZUKI, Kazuo
P- 157, P-166
P-112, P-134
SUZUKI, Masami
P-3 , P-6, P-81 *SUZUKI, Rikako
SUZUKI, Satoshi
P-116
P-10, P-47*, P-48, P-78, P-156
SUZUKI, Shugo

T
TACHIKAWA, Tetsuhiko
TAGAWA, Yoshiaki
TAGUCHI, Shuhei
TAKABA, Katsumi
TAKADA, Chie
TAKAGI, Hironori
TAKAGI , Mizuho
TAKAGI , Shiro
TAKAHASHI, Makiko
TAKAHASHI, Masakazu
TAKAHASHI, Naofurni
TAKAHASHI, Seishiro
TAKAI, Hirotake
TAKAKURA, Saori
TAKANO, Kaoru
TAKASE, Kenji
TAKASU, Nobuo
TAKASUKA, Nobuo
TAKATSUKI, Mineo
TAKEDA, Makio
TAKEDA, Tsuyoshi
TAKE!, Yoshihiro
TAKETO, Makoto MarkTAKEUCHI, Hijiri
TAKEUCHI, Tetsuya
TAKEUCHI, Yukiko
TAKEYA, Motohiro
TAKIGAMI, Shu
TAKISHITA, Eiko
TAKIZAWA, Tamotsu

288

L-3*
P-89
P-8 , P-52
P-157, P-166
P-166*
P-50, P-53
P-169
P-93, P-148, P-168
P-41, P-115
P-37, P-38, P-61*, P-91
S2-2-2, P-5, P-7, P-9, P-117*
P-16, P-68
P-112*
P-2
P-164
P-161
P-152
IATP, S 1-2-4
P-2
S2-2-2, P-7
P-157
P-102
S 1-2-2*
P-39, P-127
P-129, P-140
S2-2-2, P-5, P-7, P-9*, P-117
P-110
P-50, P-53
P-94
P-74, P-94

TAMAI, Sachiko
P-163, P-169
P-11, P-19, P-24
TAMANO, Seiko
TAMURA, Azusa
P-28
TAMURA, Kazutoshi
P-98, P-146, P-163, L-6*
TAMURA, Torn
P-84
TANAKA, HarunariP-31, P-88*
TANAKA, Hideki
P-93*
TANAKA, Isamu
P-126
TANAKA, Katsuaki
L-2*
TANAKA, Kohji
P-55, P-70, P-125, P-1 43*
TANAKA, Reiko
P-80
TANAKA, Takuji
P-3, P-6*, P-81
TATEMATSU, Masae
P-17, P-31, P-88, P-100
S3 -2
TENGOWSKI, MarkP-76
TENNANT, Raymond W
P-114, P-143
TERANISHI, Munehiro
P-1 24TERPSTRA, Piter M.
THAMAVIT, Witaya
P-33*
The ILSI RSI WORKGROUP
P-13
THONGSUKSAI, Paramee
P-44
TODA, Yousuke
P-11
TOGA, Wakako
P-103
TOMIOKA, Satoko
P-103
TOMITA, Masayuki
P-150
TOMIYAMA, Naruto
S2-2-2TOMONARI, Yuki
P-154
TON, Thai Vu
P-62
TORII, Mikinori
P-152
P- 142
TOUCH!, Akira
P-55*, P-125
TOYOSAWA, Kaoru
P-154
TOYOTA, Naoto
P-54, P-150
TSUBOTA, Kenjiro
P-111
TSUBURA, Airo
TSUCHIGAUCHI, Takeshi
P-94
TSUCHITANI, Minoru
P-154
TSUCHIYA, Noriko
P-152
TSUCHIYA, Takayuki
P-168*TSUDA, Hiroyuki
IATP*, Sl -2-4*
S2-1 - l, S2-1-2, P-47, P-48*
TSUJIMURA, Kazunari
P-78, P-92
TSUJIUCHI, Toshifumi
P-75, P-85, P-9 8
TSUKAMOTO, Tetsuya
P-17, P-31 *, P-88, P-100
TSUKIDATE, Kazuo
P-141
P-166
TSUKUMO, Yukihito
P-41, P-115
TSURU, Kiyoyuki
TSURUKAME, Maiko
P-169
TSUTSUI, Naohisa
P-96
P-75, P-85, P-98
TSUTSUMI, Masahiro
TSUTSUMI, Tomoko
P-156
P-153
TURTON, Johnu
UCHIDA, Kazumi
UEDA, Makoto
UEDA, Shinobu
UEDA, Tadayoshi
UEHARA, Takeki
UEMATSU, Fumiyuki
UENO, Masaki
UESAKA, Toshihiro
UETSUKA, Koji
UMEDA, Yumi

P-101
P-74, P-145
IATP, Sl-2-4
P-70
P-67, P-69, P-142*
S2-l-2, P-37, P-38, P-61, P-91*
P-113*
P-57, P-130, P-136
P-95, P-114, P-123, P-164
P-129 , P-140':'

UMEKITA, Yoshihisa
UMEMURA, Takashi
UNAMI, Akira
UNO, Hiroshi
USHIJIMA, Toshikazu
USUDA, Koji
USUI, Toshimi
USUMI, Kenji
UTSUMI, Hiroyuki
UTSUNOMIYA, Hirotoshi
UWAGAWA, Satoshi

P-52
P-25 , P-60, P-104'-'
P-54*
P-28
P-65
P-162
P- 168
P-158
P-96
P-88
P-22, P-46

V
VEINOT, John
VOS, Joseph G.

P-132
IATP*

w
WADA, Jutaro
WAKABAYASHI, Keiji
WAKO, Yumi
WAKUI, Shin
WANG, Jianqing
WANG, Peng
WANIBUCHI, Hideki

WARD, Jerrold M.
WARITA, Katsuhiko
WATANABE, Hideyuki
WATANABE, Hiromitsu
WATANABE, Naoto
WATANABE, Takao
WATANABE, Toshiyuki
WEI, MinWEILER, Horst
WESTER, Pieter W.
WILLIAMS, Gary M.
WOO, Gye-Hyeong
WOO, JY

P-59
P-57, P-130
P-154
P-90'''
P-24
P- 171Sl-1-1 *, P-11 , P-14, P-18
P-19, P-20, P-21, P-22
P-32, P-35 , P-59, P-63
P-66, P-67 , P-69, P-79
P-80, P-89, P-97
S 1-2 Chair, S 1-2-1 *P-169
P-1 22
P-57 , P-130, P-136*
P-37 , P-38 *, P-61 , P-91
P-26, P-56*
P-143
P-35
P-124*IATP*
IATP*
P-123 *P-119

X
XIANG, Anbo
XU, Guoping
XU, Xiangting

P-106*, P-128
P-171
P-171

y
YABE, Koichi
YABUSHITA, Setsuko
YAHATA, Megumi
YAK.ABE, Yoshikuni
YAMADA, Yasuhiro
YAMAGUCHI, Chiharu
YAMAGUCHI, Satoru
YAMAGUCHI, Shuji
YAMAGUCHI, Takasi
YAMAGUCHI, Takeshi
YAMAGUCHI, Yuko

P-170*
P-4
P-146
S2-l-2
P-82
P-80*
P-7
P-116
P-80
P-161
P-146*YAMAKAWA, Keiko
YAMAMOTO, Masami
YAMAMOTO, Masatoshi
YAMAMOTO, Naoki
YAMAMOTO, Toshinobu
YAMANAKA, Hidenori
YAMASAKI, Kanji
YAMATE, Jyoji
YAMATO, Hiroshi
YAMOTO, Takashi
YANAGIDA, Takamasa
YANAI, Tokuma
YANG, KH
YANG, Jihong
YANG, Ki-Hwa
YANG, Xiuying
YASOSHIMA, Akira
YASUI, Hisae
YASUI, Wataru
YASUTAKE, Akira
YOKOHIRA, Masanao
YOKOYAMA, Tsukao
YOO, Hwan Soo
YOSHIDA, Hiroki
YOSHIDA, Midori
YOSHIDA, Toshinori
YOSHIMI, Naoki
YOSHINO, Hiroko
YOSHIZAWA, KatsuhikoYU, Zeng
YUN, Young Won
YUNOKI, Takayuki

P-39, P-127
P-17, P-31 , P-100*
P-102*
P-36
P-96*
S2- l -2*P-2
P-120*
P-126
P-49*, P-51, P-114
P-36
P-17, P-100, P- 139*
P- 118, P-119
P-171 *
P-58, P-121
P-151 *
P-164
P-120
P-138
P-110
P-39, P-127*
P-102
P-23
P-8 , P-52
P-37, P-38, P-61
P-1,
P-73 *, P-75 , P-91
S2-2-2, P-5, P-9, P-117
P-83 , P-86
P-10, P-12*, P-19, P-156
P- 111 *P-127
P-23
P-20*, P-63z
ZEISEL, Steven H
ZENG, Yu
ZHANG , Ling
ZHANG, Liuyi

P-37
P-39
P-171
P-171

MEMO

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Integrated Solution for Protein Screening & Identification

A complete system for protein difference analysis

Ettan DIGE
Ettan DIGE system uses multiplexing. the simultaneous co-separation of multiple,

fluorescently labelled samples including an internal standard on a single gel.
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yy:.;t,b. /X:1'7tif1'I/A~i'C~t±
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De&lgnated drug:pn,wlptlon n,qulred
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Indications, dosage, administration and precautions for use,
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Manufacturer & Distributor (product tnfonnatlon)e

TAIHO Pharmaceutical Co.,Ltd.
1·27 Kandanlshlkl-eho, Chlyoda·ku, Tokyo 101-8444.Japan

Daiyu-kai Institute of Medical Science,
DIMS , has developed a medium-term

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The unique approach and the reliability ofour data have won us an outstanding reputationworldwide. We also provide other valuable
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<Subcontract menu>
We conduct experimental safety tests of chemical compounds

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1. Medium-term carcinogenesis bioassays(Liver, urinary bladder, Stomach, Kidney, Lung, Thyroids, Nasal cavity,
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4. General toxicity study (Single dose and repeated dose study)
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6. Natural metastasis (lung metastasis of liver cancer) model
Using this model useful data for the evaluation of thepharmacological effects of metastasis inhibitors can be obtained.

DAIYU-KAI INSTITUTEOF
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DIMS
64 Goura, Nishiazai, Azai-cho, Ichinomiya, 491-0113, Japan
Phone: +8 1-586-51-1201 Fax: +8 1-586-51-5634
E-mail: query@daiyu-kai.com
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Tel 075 211 2021 I Kyoto)Tel 03 5256 M36 !Tokyo)~1'~B6r,/lil,~/lil,l!~Mi. c:p~1!1Jl'¥~'dl.\lim~Mi·ll¥® ·t§ffl~*OOim~·illl;lUtill.\lim~~·i-1:!.lll*ll.\lim~~· 5J!Jil:'..:!§*00im~ Mi·5£.1,i<~*OOim~~.~llE*ll.\lim~Mi· 7'l..;JL,::\='-*00im~~· llE!f311l.\JitO)~t1.J~l!~Mi·OTC!l.\lim~Mi- /!li/lil,l!~Mi~:i:tt~l!~Mi-f-O)itll

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Tel.058-392-6222 Fax. 058-392-2432

Web site
http ://www.dakocytomation.com/

Distributores in more than 50 cou ntries

AntibodyExpression System(E coi, Insect cell, Mammalian cell)

Gene Profiling- DNA chip
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3-6-10 AZUSAWA, ITABASHI, TOKYO 174-8505 TEL:81-3·3968-1192 FAX:81-3-3968-4863

fbi@oyc.co.jp

we f o r ~~A!3 '6t_lol-~~"e 1" A1'~~c.k
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No one knows where the key to the cure might be found. At Kyowo Hakko, Japan's leading pharmaceutical and biotechn o logy company,
our employees regularly study soil samples from the most unexpected places-hoping to one day find the enzyme or microorgonism that wi ll lead to a cure.
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CTBR - Building Relationships Based on Trust and Commitment
CTBR is pleased to have been involved with the Japanese health care industry for more than
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CTBR Pathology Experience and Expertise
•
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13 Veterinary Pathologists (9 ACVP Certified)
160 pathology scientific and technical staff
Pathology evaluation on more than 550 GLP studies per year
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used with PCR licenses ava ilable from Applied Biosystems. Its use with Authorized Reagents also provides a limi ted PCR license in accorda nce with the labelrights accompanying such reagents. Purchase of this instrument does not convey any right to practice the 5' nuclease assay or any of the othe r rea l-time
methods covered by patents owned by Roche or Applied Biosystems."~ -91]:;J) ~1"7.tfjfc it~ it*B:$:~%~~B:$:~%~

TEL. 03-3271 -8553 FAX. 03-3271-7282 TEL. 077-565-6979 FAX. 077-565-6978

TaKaRa 7?=1.JJl,~itt-1--5-1':::.t~ &.i:-:::i~ ,z(7)ttffia'ilt.i: Z::11frAii:wr~(7)ffit.fsr,t:t L, ;1:9 o
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TEL (0978) 72-0454
FAX (0978) 72-2320

E-Mail: biopathology@muj .biglobe.ne .jp

Shin Nippon Biomedical Laboratories, Ltd.

:£ ~tt ffl '.9l j:ijj

SNBL USA, Ltd.

Drug Safety Research Laboratories(Kagoshima)(WA. USA)The SNBL Group undertakes all processes of pharmaceuticaldevelopment from pre-clinical studies to clinical trials (CRO, SMO, andPhase I trials), and through Translational Research, supports bridging
from the earliest stages of drug production and medical techniques.

Contact:
Headquarters, Drug Safety Research Laboratories
2438 Miyanoura, Yoshida-cho, Kagoshima-gun, Kagoshima 891-1394, Japan
TEL +81 99 294 2600 FAX +81 99 294 3619
Pharmacokinetics and Bioanalysis Center
Kainan Intelligence Park, 16-1 Minami-Akasaka, Kainan, Wakayama 642-0017, Japan
TEL +81 73 483 8881 FAX +81 73 483 7377
Tokyo Office
Toho Twintower Building 6F, 1-5-2 Yuraku-cho, Chiyoda-ku, Tokyo 100-0006, Japan
TEL +81 3 3500 5045 FAX +81 3 3500 5046
Osaka Office
Sumitomo Mitsui Banking Corporation Korat-bashi Building, 2-1-1 Fushimi-cho,
Chuo-ku, Osaka 541-0044, Japan
TEL +8166233 8411 FAX +8166233 8412
SNBL USA, Ltd.
6605 Merill Creek Parkway Everett, WA 98203
TEL +1 425 407 0121 FAX +1 425 407 8601

http://www.snbl.com

e-mail: info@snbl.co.jp

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The most trusted name in histology.
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Proven Reliability
SAKURA

Sakura Finetek U.S .A., Inc.
1750 West 214th Street
Torrance, CA 90501 U.S.A.
Phone:(800)725-8723
Fax: (310)972-7888
www.sakuraus.com

Sakura Finetek Japan Co., Ltd.
I-7 Honcho 2-chome, Nihombashi
Chuo-ku, Tokyo 103-0023 JAPAN
Phone:(03)3270-1666
Fax: (03)3270-2779
www.sakurajp.co.jp

Sakura Finetek Europe B.V.
Hoge Rijndijk 48a
2382 AT Zoete1woude
The Netherlands
Phone:+3 1-7 1-58 98 300
Fax: +31 -71-58 98 488
www.sakuraeu.com

Schedule for Conference
Sunday February 15. 2004
Regi stra tion

M .a in Ha ll

Reception
11.allRoon, 30 1

Room
40 1 +4 02

Roon, 5 0 1

Roon"l 502

Other

10:00a.m
I I :OOa rn

1 2:0 0pn,

11 :30am
I :OOp n,
JSTP
Mi c r oexn1n in ntion

2:00 p,n

Ex p l nnn ti o n

Opening Rcni i1.rks

3:00p m
5:00pm
4:00pmJSTP

JATP
Educn ti onul

5:00p mBoard orDirecto r s

Sessio.n

Meet in g( Ro o m 505)

6:00 p rn

7:00 prn

Wc l co1nc
8:00 pm

RcccpHon(Portopia l-:lo tc l)

9:00p mMonday. February 16. 2004
Regist:rnt-ion

Mnin "F-la ll

R ece ption
Ho ll

Roon'l 3 0l

R oo n:i
40 1-+402

PosterPosterSe t - up

Set-up

PosterOiscus:1-ion

Poster
Di....:c u ssio 1:tRoon, 30 1

R oo n,401 + 402

R oo n, 5 0.1

Roon'l 502

Luncheon
Seminar 2

Luncheon
Seminar 3

Roon, 50 L

Roon, 5 0 2

Otb e 1-

Lunc h eon
Seminm- S

L un c heon
Seminar 6

J STP Coun c il
( R oo m 5 0 5)

Other

8:00u ,n
9:00am
1 0:00an,

Syrnposiun'J
1 1 :OOa rn

1-1

8:3 0am

1 2:00 pn,

L unc heon
Scrnina r 1

I :OOpn,

Exhibi t i on
2:00 pn,J o hn Fucclni

6: 00pm
3: 00pn,Mcn,orin ll<.cvaOlc 1.,.ecturc

4:00pn,Syr1,posi um
1-2

5:00pn,
6:00pn,Tuesday. Feb1·uary 17. 2004
Re g i s troti o n

Mnin J-1.n ll

Rece pti o n
Ha ll

8: QQaJTl

9:00on,Symposi um

10 :00an,

2- 1

I I :OOa n,
8:3 0am

1 2:00 prn

Luncheon
Seminnr4

1 :00p m

Exhibition

Poster
Discussion

2:00 p ,n
6:00pn'l
3:00 p,nPosterDisc ussion

Plenn.ry
Lecture4:00p n,Symp si un:i
2-2

5:00 pn,
6:00 pn,
7:00p n,Ga.la

8:Q Qp 1TI(Portopin H o te l)

R e cep6on

Wednesday. February 18. 2004
Reg i stration

Mn.in Ho.II

Recept"i On
Ha ll

R oo n, 30 1

R oo m40 1 + 402

PosterDi~cussion

Poster
Discuss-ion

PostorRcmovu lPosterR.omovalRoon, 50 1

R oo n, 502

Luncheon
Sem in n r 8

L un c h eo n
Seminar 9

8: 0QaJTI

9:00a ,nyn:iposh,m

1 0:00an,

3

I I : OOan,

8:30a..m
JSTP Oc nc r-.a l McctinH

1 2: 00pn,

Luo c heon
Sc1n.innr 7

I :OOp n,

Ex hlbiti o 11

2:00 pm

5:00pn::i
3: 00pn,Syn1posi1.u::n
4

4:00p n ,5: 00pn,C l o,i ing R.c1nnrks

Ot h er