The Toxicologist - Society of Toxicology

1403 GENOTOXICITY AND PRE-NEOPLASTIC LESIONS
INDUCED BY 2, 4-DIAMINOTOLUENE IN THE LIVER
OF F344 GPT DELTA TRANSGENIC RATS.
T. Nohmi 1 , N. Toyoda-Hokaiwado 1 , T. Inoue 2 , K. Masumura 1 , H. Hayashi 3 , Y.
Kawamura 3 , Y. Kurata 3 , M. Takamune 1 , M. Yamada 1 , H. Sanada 4 , T.
Umemura 2 and A. Nishikawa 2 . 1 Division of Genetics and Mutagenesis, National
Institute of Health Sciences, Setagata, Tokyo, Japan, 2 Division of Pathology, National
Institute of Health Sciences, Setagata, Tokyo, Japan, 3 Meiji Seika Kaisha, Ltd.,
Yokohama, Kanagawa, Japan and 4 Safety Research Department, Central Research
Laboratories, Kaken Pharmaceutical Co., Ltd., Fujieda, Shizuoka, Japan.
Transgenic rodent genotoxicity assays are valuable tools to examine genotoxicity of
chemicals in vivo. Recently, we have established Fischer 344 gpt delta rats by backcrossing
Sprague Dawley gpt delta rats with F344 rats for 15 generations. To begin
validation of F344 gpt delta rats, we examined the genotoxicity and hepatotoxicity
of 2,4-diaminotoluene (2,4-DAT) and 2,6-diaminotoluene (2,6-DAT). Although
both compounds are genotoxic in the Ames/Salmonella assay, only 2,4-DAT induces
tumors in rat livers. Male F344 gpt delta rats were fed diet containing 2,4-
DAT at doses of 125, 250, or 500 ppm for 13 weeks, or 2,6-DAT at a dose of 500
ppm for the same period. Mutation frequencies (MFs) of base substitutions, mainly
at G:C base pairs, were significantly increased in the livers of 2,4-DAT-treated rats
at all three doses. MFs of deletions, mainly one-base deletions, were also increased
at 250 and 500 ppm. In contrast, virtually no induction of genotoxicity was identified
in the kidneys of 2,4-DAT-treated rats or in the livers of 2,6-DAT-treated rats.
Micronucleus assays in the bone marrow were negative with both 2,4-DAT and
2,6-DAT. GST-P-positive foci were detected in the livers of rats treated with 2,4-
DAT at a dose of 500 ppm, but not in those treated with 2,6-DAT. These results
suggest that genotoxicity should be examined in target organs of rats where carcinogenicity
is identified (liver in this case) and also that gpt delta transgenic rats
may be useful to integrate in vivo genotoxicity and pathological toxicity assays,
thereby reducing numbers of animals for safety evaluation.
1404 CHEMOPREVENTIVE EFFECTS OF SILYMARIN ON 1,
2-DIMETHYLHYDRAZINE-INDUCED MUTAGENESIS
AND CARCINOGENESIS IN THE COLON OF GPT
DELTA TRANSGENIC RATS.
K. Masumura 1 , N. Toyoda-Hokaiwado 1 , Y. Yasui 2 , M. Muramatsu 1, 3 , M.
Takamune 1 , M. Yamada 1 , T. Tanaka 4 and T. Nohmi 1 . 1 Division of Genetics and
Mutagenesis, National Institute of Health Sciences, Tokyo, Japan, 2 School of
Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan, 3 Tokyo
University of Pharmacy and Life Sciences, Tokyo, Japan and 4 Tohkai Cytopathology
Institute, Gifu, Japan. Sponsor: A. Nishikawa.
Silymarin, a natural flavonoid from the milk thistle is a prospective chemopreventive
agent. We investigated potential chemopreventive effects of silymarin against
carcinogenicity and genotoxicity induced by 1,2-dimethylhydrazine (DMH) plus
dextran sodium sulfate (DSS) in the colon of F344 gpt delta transgenic rats. In the
rat model, transgene lambda EG10 DNA carrying the gpt gene of E. coli is integrated
in the chromosome as a reporter for detection of point mutations. Therefore,
it is possible to examine anti-genotoxic effects of chemicals in any organs of rats.
Seven week-old male F344 gpt delta rats were given a single subcutaneous injection
of 40 mg/kg DMH, and followed by 1.5% DSS in drinking water for a week. They
were fed diets containing 100 or 500 ppm silymarin for 4 weeks, starting one week
before DMH injection. At week 4 after the DMH injection, the gpt mutant frequencies
(MFs) in the colon were increased about 120-fold by DMH/DSS treatments.
Silymarin suppressed the induced MFs by 30%, although the reduction was
not statistically significant. The number of preneoplastic lesions, i.e., aberrant
crypto foci, induced in DMH/DSS groups was significantly decreased by feeding
with silymarin. At week 32, DMH/DSS treatments induced colon tumors substantially
and silymarin significantly suppressed the tumor formation. In vitro, silymarin
reduced genotoxicity of DMH by 80% in S. typhimurium YG7108, a sensitive
strain to alkylating agents. These results suggest that silymarin is able to
suppress DMH/DSS-induced colon carcinogenesis when fed at the initiation phase
via inhibition of genotoxicity of DMH at least in part, and also that F344 gpt delta
rats are useful to examine the mechanisms of chemopreventive agents in the target
organs of carcinogenesis.
1405 EXAMINING THE MUTAGENICITY OF ETHYL
METHANESULFONATE IN MICE USING THE PIG-A,
HPRT, AND GPT DELTA ASSAYS.
X. Cao, J. G. Shaddock, R. A. Mittelstaedt, V. N. Dobrovolsky, S. D. Shelton,
M. Manjanatha and R. H. Heflich. DGMT, NCTR/U.S. FDA, Jefferson, AR.
Sponsor: T. Chen.
Ethyl methanesulfonate (EMS) has recently undergone a comprehensive cancer risk
assessment that included dose-response measurements of LacZ mutation in
MutaMouse. This assessment indicated that EMS had a no-effect threshold for
genotoxicity of at least 25 mg/kg/day. However, the MutaMouse data were characterized
by unusually high backgrounds which may have compromised the quantitative
risk estimate. Recognizing that assay sensitivity is affected by fold-increases
above background, we conducted a series of preliminary in vivo experiments to examine
the mutagenicity of EMS in mice using reporter systems with low backgrounds,
including Pig-a mutation in RBCs, Hprt mutation in lymphocytes, and
transgene mutation induction in gpt delta mice. Male C57BL/6 mice (or gpt delta
C57BL/6 transgenic mice) were treated with an acute dose of 360 mg/kg of EMS
via oral gavage. Blood samples were obtained on Days -1, 15, 30, and 45 for Pig-a
mutation analysis and the spleens were collected on Day 45 for Hprt mutation
assay. For the gpt delta assay, the transgenic animals were sacrificed on Day 28 and
the bone marrow, spleen, liver, lung, kidney, and small intestine were harvested for
mutant analysis. The Hprt assay and spleen gpt assay had the greatest responses
with ca. 8-fold increases in mutant frequency (MF) over the background in treated
animals. In addition to the spleen, bone marrow also exhibited a statistically significant
response to the EMS treatment with a fold induction of 2.3. Results with the
Pig-a assay revealed a mutant phenotype erythrocyte response on Day 15 with a
fold induction of 6.6. Taken together, these findings suggest that different endpoints
provide different levels of sensitivity for detecting the genotoxicity of EMS.
Therefore, utilizing a combination of assays with low backgrounds may benefit the
dose-response genotoxicity evaluations of weak mutagens such as EMS. Ongoing
studies are testing this hypothesis.
1406 MANIFESTATION AND PERSISTENCE OF PIG-A
MUTANT FREQUENCY IN C57BL/6 MICE TREATED
WITH DIFFERENT DOSES OF ENU.
J. A. Bhalli, M. G. Pearce, V. N. Dobrovolsky and R. H. Heflich. NCTR, U.S.
FDA, Jefferson, AR. Sponsor: B. Parsons.
Treating rats with single doses of N-ethyl-N-nitrosourea (ENU) results in a timedependent
accumulation of Pig-a-mutant peripheral red blood cells (RBCs), reaching
a plateau at about 6-weeks posttreatment and resulting in a nearly linear doseresponse,
with the magnitude of the induced response persisting for at least 26
weeks. In the present study, we have conducted a detailed evaluation of ENU-induced
Pig-a mutant manifestation and persistence in the mouse. Groups of 5 male
C57BL/6 mice were given single i.p. injections of seven different doses of ENU: 0
(vehicle control), 10, 25, 45, 70, 100 and 140 mg/kg ENU. Blood samples were
collected one day prior to treatment and at 2, 4, 6, 8, 12, 20 and 26 weeks posttreatment
and analyzed for CD24Neg RBCs and CD24Neg reticulocytes (RETs)
(Pig-a mutants) using a FACSCanto-II flow cytometer. Mean CD24Neg frequencies
in vehicle-treated mice ranged from 0×10−6 to 1.8×10−6 for RBCs and
0×10−6 to 4×10−6 for RETs, and displayed no time-related trends. For RETs,
maximum Pig-a mutant frequencies were observed at the 2nd week after ENU
treatment, whereas maximum Pig-a mutant frequencies for RBCs were observed at
the 4th week after treatment; mutant (CD24Neg) RET frequencies were consistently
approx. twice those of RBC frequencies throughout the study period. After
the maximum responses were reached, the CD24Neg RBC and RET frequencies in
ENU-treated mice began to slowly decrease with time. Although significant, dosedependent
increases in Pig-a mutant frequency were observed for all doses of ENU,
the dose-response appeared to be sublinear at lower doses. In addition, the absolute
magnitude of the ENU-induced Pig-a mutant RBC response in mice was about 2-
3-fold less than previously detected in rats. The data from this study indicate that
the ENU-induced Pig-a mutant RBC frequency in mice and rats differ in magnitude,
manifestation kinetics, and persistence.
1407 NITROXIDES TEMPO AND TEMPOL INDUCE TK
MUTATIONS IN MOUSE LYMPHOMA CELLS.
X. Guo 1 , L. Guo 2 , M. M. Moore 1 and N. Mei 1 . 1 Division of Genetic and
Molecular Toxicology, NCTR/FDA, Jefferson, AR and 2 Division of Biochemical
Toxicology, NCTR/FDA, Jefferson, AR.
Low molecular weight nitroxides, Tempo and Tempol (4-OH-Tempo), are stable
free radical compounds widely used throughout chemistry and biochemistry as
process intermediates. Due to their antioxidant abilities and protective properties in
SOT 2011 ANNUAL MEETING 301