JEMS · MMS

DNAging Genetic Instability and Aging

ELSEVIER

Mutation

Research

33X (IYYS)

51-57

Effect of aging on spontaneous micronucleus frequencies in peripheral blood of nine mouse strains: the results of the 7th collaborative study organized by CSGMT/JEMS * MMS* Sei-ichi Sato ‘.I, Masako Taketomi “, Madoka Nakajima “*I, Michiyo Kitazawa b, Hiroyasu Shimada ‘, Satoru Itoh ‘, Miyuki Igarashi ‘, Naohiko Higashikuni d, Shizuyo Sutou d, Yu F. Sasaki ‘, Makoto Hayashi ‘.‘,*, Toshio Sofuni f, Takafumi Higashiguchi g, Shinji Nito g, Yasushi Kondo g, Sachiko Honda h,l, Mikiko Hayashi h, Yasuhiro Shinagawa h, Eiichi Nakajima ‘, Yoshie Oka ‘, Kayoko Shimoi j, Yumiko Hokabe j, Akira Morita j, Naohide Kinae j, Masaki Takeuchi k, Haruyoshi Hirono k, Eiji Yamamura k, Koichi Tamai ’ ” Toxicology Research Laboratories, C‘entrtd Phurmaceuttcul Reseurch Institute, Japan To&co Inc., 23 Nakogi, Hatano 257, Japan h Biosafety Reseurch Center, Foods, Drugs und Pesttcides. 5-82-2, Arahamu Shioshinden, Fukude-rho, Iwata-gun, Shizuoku 437-12 Jupnn ‘ Drug Safety Reseurch Center. Der elopmcnt Research Laboratones Datrchi Phurmrtceutical Co., Ltd., I- 16-13, Kitakasai, Edogawu-ku. Tokyo 134, Jupun ” Itohum Centrul Research Institute, I-2 Kuhoguoka. Moriya. Ibaraki 302.01, Japan Faculty of C’hemicul and Btologucd Engineering, Huchmohe N~t~onulCollege of Technology, Tumonoki Uwunotai 16-1, Hachinohe, Aomori 039. I I, Jupan ’ Dit ision of Genrttc~.r und Mutugenests, .Nationul lnstttute of Heulth Scrences, I-IX-1 Kamiyoga, Setagayu-ku, Tokyo 158, Japan ’ Safety Reseurch Luhorutory. Tunuhe Seiyaku Co., Ltd., 16-89. Kashtmu 3.Chome. Yodogawa-ku. Osaku 532, Japan h To~mcr Institute of Health, 17-l. Nakatatkoyamu, Kosugr-machi, Toyama 939-03, Japan ’ Pharmaceutrruls Research Center, Toyoho Co.. Ltd., 2-l-I Katata, Ohtsu, Shiga 520-02. Japan phoratory of Food Hygiene, School of Food and Nutritionul Sciemes, Unir~ersity of Shizuoka, S2-1. Yada, Shizuoka-shi, 422, Japan Toxicology Luhorutories, Yoshitomi Phurmuceuticul Industrres. Ltd., 955 Koiwai, Yoshitomi-cho, Chikujo-gun, Fukuoka-ken 871, Jupun ’ Depurtmrnt of .~luttrgmetrr. Heulth S~~rences Research Instrtute. 106. (;odo-cho, Hodoguyu-ku, Yokohama 240, Japan Accepted

The Collaborative Study Group for the Micronucleus (CSGMT). a subgroup of the Mammalian Mutagenesis Group (MMS), a suborganization in the Environmental gen Society of Japan (JEMS). 0921~8734/95/$09.50 SSDI 0921.8734(95)0001

P lYY5 Elsevier I-Y

Science

15 May

Test Study Muta-

B.V. All right\

1995

* Corresponding author. Tel: + 81.3-3700-1141[435]; Fax: + 81-3-3700-2348; E-mail hayashi(anihs.go.jp. ’ Member of organizing committee; S.S.: organizer-in-chief. resetved

S. Sam

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et ul. ,/Mtmttron

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Abstract The spontaneous frequencies of micronucleatedreticulocytes (MNRETs) were examined monthly over the life spans of animals belonging to nine mousestrainsfor the 7th collaborative study organized by the CSGMT/JEMS . MMS. Both sexes of the BDFl strain and femalesof the A/J strain showeda statistically significant increasein mean spontaneous MNRET frequency in their last month of life, suggesting the possibility of strain-specific, age-dependent chromosomal instability. SAMP6/Tan, an accelerated senescence-prone strain, showed the same tendency, although it was not statistically significant. The other strains studied, ddY, CD-l, B6C3F1, SAMRl, and MS/Ae, did not showsignificantage-relateddifferencesin meanof MNRET frequencies.More extensivestatistical

analysesare underway, and the outcomeswill be reported separately. Keywords:

Micronucleus; Aging; CSGMT/JEMS

MMS

1. Introduction

2. Materials and methods

Decreasing chromosomal stability in human aging and carcinogenesis is a subject of current research interest (Fenech and Morley, 1985; Nisitani et al., 1990). Takeda et al. (1981) developed an accelerated-senescence model from the AKR/J mouse strain (SAMPl, SAMRl), which show an age-dependent chromosomal aberration frequency (Nisitani et al., 1990). It is of interest. therefore, to know whether experimental animals show age-dependent chromosomal instability. The peripheral blood micronucleus assay is a reliable index of in vivo chromosomal damage (MacGregor et al., 1980). Recently, Hayashi et al. (1990) reported a simplified method for the preparation and observation of micronuclei in mouse peripheral blood reticulocytes using acridine orange (AO) coated glassslides. The usefulness and reliability of this method was demonstrated by the 5th Collaborative Study (CSGMT, 1992). The method enabled us to monitor micronucleus frequencies over the life span of individual animals. CSGMT/JEMS . MMS has investigated the influence of a number of factors involved in the micronucleus test (CSGMT, 1986, 1988, 1990, 1992; Hayashi et al.. 1989). In the present collaborative study. we monitored the frequency of micronucleated reticulocytes (MNRETs) in mouse peripheral blood to evaluate chromosomal instability over the animal’s life span. Nine strains, including the acceleratedsenescencemodels, were selected, and 11 laboratories participated.

2.1. Animals

Male and female CD-1 (ICR) (Charles River Japan Inc., Atsugi, Japan), ddY (Japan SLC Inc., Shizuoka, Japan), BDFl (Charles River Japan Inc., Atsugi or Japan SLC Inc., Shizuoka, Japan), B6C3Fl (Charles River Japan, Inc., Atsugi, Japan), and A/J (Japan SLC Inc., Shizuoka, Japan) mice were purchased at 3 weeks of age. Male and female MS/Ae (mutagen-sensitive mouse) were bred at Itoham Central Research Inst., Moriya, Japan. Male and female SAMP6/Tan, male SAMPl (accelerated senescence-prone strains), and male SAMRl (senescence-resistant strains) mice, which were developed from the AKR/J strain by Takeda et al. (1981). were bred at the laboratories where the micronucleus assayswere performed. The mice were acclimated for at least one week. They were 4 weeks old at the start of the study, and we used lo-16 mice of each sex of each strain except SAMPl and SAMRl, for which only males were used. Mice were given commercial pellet and tap water ad libitum throughout the acclimation and experimental periods and were subjected to a 12 hour light/dark cycle. 2.2. Slide preparation

and analysis

Approximately 5 ~1 blood were collected from the ventral tail blood vessel. All animals were sampled prior to 4 weeks of age and monthly

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Fig. 1. The monthly Institute of Health:

0

number Laboratory

6

12

18

24

Months of outwork and the mean spontaneous MNRET 8: Yoshitomi Pharmaceutical Industries. Ltd.

thereafter. The peripheral blood micronucleus test was performed according to Hayashi et al. (1990) using AO-coated glass slides. At least 1000 reticulocytes (RETs) per animal per sample time were analyzed by fluorescence microscopy. and

30

frequencies

36

in ddY

mice.

Laboratory

A: Toyama

the MNRET frequencies were recorded. The mean difference in MNRET frequencies between the first and last sample were analyzed for each group by conditional binomial analysis (Kastenbaum and Bowman, 1970).

20 E 2 'S L 2

15 10

04 0

"

6

12

1R

24

Months Fig. 2. The monthly number ot survivors and the mean spontaneous MNRET Yoshitomi Pharmaceutical Industries. Ltd.: Laboratory C: Toyobo Co.. Ltd.

30

36

frequencies

in CD-1

(ICR)

mice.

Laboratory

B:

3. Results

RET frequencies varied between sexes and among laboratories from the onset of the study (Fig. 3) but was more pronounced after 15 months. At two of three laboratories, the mean spontaneous MNRET frequencies were elevated at the end the study (laboratory E: male from 1.4 to 5.0 MNRETs/lOOO RETs. female from 1.6 to 3.3; laboratory-A: male from 2.1 to 3.1, female from 1.2 to 4.1) (Table 1). B6C3Fl mice of both sexes were studied at two laboratories. The spontaneous MNRET frequencies of this strain were stable with regard to sample time, laboratory, and sex except for male mice at one laboratory (Fig. 4). An extremely high mean spontaneous MNRET frequency (6.7 MNRETs/lOOO RETs) was recorded at the 27th month for the 7 mice still alive at that time. Groups of this strain showed higher mean MNRET frequencies at the end of the study than at the beginning, except for females at one of the laboratories (Table 1); the differences, however, were not statistically significant. Both sexes of SAMP6/Tan and male SAMPl (an accelerated senescence-prone strains), and male SAMRl (senescence-resistant strain) were

The results are summarized in Figs. 1-6 and all individual data are available by the corresponding author on request. Generally, MNRET frequencies were stable for approximately 15 months, at which time the animals started to die due to aging. The MNRET frequencies started to vary at this time, although a tendency to increase was not apparent. Male and female ddY mice were studied at two laboratories. As Fig. 1 shows, the MNRET frequencies were stable for IS months, and then the animals began to die. After 15 months, mean spontaneous MNRET frequencies varied with rcgard to sample time, laboratory, and sex. but no age-dependent increase of MNRET frequency was seen. Both sexes of CD-I (ICR) mice were studied at two laboratories (Fig. 2). The mean spontaneous MNRET frequencies of this strain remained constant at approximately 1.5 MNRETs per 1000 RETs for the animal’s lift span. Male and female BDFl mice were studied at three laboratories. The mean spontaneous MN-

04 0

6

I?

IX

24

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6

I2

1x

24

30

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Months

Fig. 3. The monthly number ot w~c~vor~ and the mean spontaneous MNRET frequencies in BDFI mice. Laboratory B: Yoshitomi Pharmaceutical Industries: Lahoratol> D: Tanabe Seiyaku Co.. Ltd.: Lahorator-y E: Biosafety Research Center. Foods, Drugs and Pesticides.

01 0

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24

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MNRET

frequencies

in BK3Fl

Months

Fig. 4. The monthly number of wrwors and the mean spontaneous Tobacco Inc.: Laboratoni G: Daiichi Pharmaceutical Co., Ltd.

studied at two laboratories (Fig. 5). Generally. the mean MNRET frequencies were variable for sample times, sub-strains, and sex. The last data point, which shows a very high MNRET frequency (10 MNRETs/lOOO RETs), describes a

OJ 0

Fig. 5. The monthly number mice. Laboratory D: Tanabe

6

12

mice.

Laboratory

F: Japan

single survivor. The 50% survival time for SAMP6/Tan and SAMPl males were 14 and 15 months, respectively, shorter, as expected, than those of other strains. The 50% survival times of SAMP6/Tan and SAMRl females were 2.5 and

Y I8

24

30

Months of survwors and the mean spontaneous MNRET frequencies Sciyaku Co., Ltd.; Laboratory H: University of Shizuoka.

36

in SAMP6/Tan,

SAMRl,

and SAMPl

56

01 0

Fig. 6. The monthly number Tanabe Seiyaku. Co., Ltd.:

6

12

18

14

Months ot sutwvors and the mean spontaneous MNRET Laboratory 1: ltoham Foods Inc.

22 months, respectively. and not different from those of other strains. The spontaneous MNRET frequencies for MS/Ae and A/J mice of both sexes varied among sample times, especially after the animals started

Table Initial

I and final

mean i S.D. of MNRET

Strain

Laboratory

ddY ddY CD-l CD-I BDFI BDFI BDFI B6C3F I B6C3FI SAMPh/Tan SAMPl SAMRI MS/Ae

A .I B B (‘ I:, B E F G D H II I D

frequencies

for each sex of each mouse

in MS/Ae

and A/J

mice. Laboratory

D:

strain Female

Start

A/J

frequencies

36

to die by senescence (Fig. 6). Several older male A/J mice showed more than 10 MNRETs/lOOO RETs. These strains also showed a tendency to have higher mean MNRET frequencies at the end of their lives than at the beginning.

Male

I.7 + 1.8 * I.8 + 2.5 * 1.4* I.8 2 2.1 & I.1 i 1.7 * 3.1 + I.1 + 2.7 f 2.‘) t 3.1 f

30

1.4 0.9 1.9 I.0 I.1 1.6 I.1 I.0 I.7 I.4 0.6 I.5 I.7 I.3

Final

Start

I.1 1.6 1.3 I.6 5.0 2.1 3.1 I .x 2.6 4.0 I.? 7.x 2.3 5.7

I.0 I.3 I.6 2.9 1.6 2.4 1.2 I .2 2.3 2.6

ti i i i + -ff ~+ -f+ f i +

I.0 I.6 I.0 I.5 2.2 + I.1 I.5 0.x 77 -.3. I 1.2 2.4 I .h 3.5 .

Final * * * f + * * + i i

0.8 1.2 I.4 1.0 1.4 1.2 I.3 0.9 1.3 1.5

3.3 i I.5 2.1 t I.2

0.7 k 1.9 * I.1 * 3.3 * 3.3 f 2.1 * 4.1i2.2 1.7 i I .9 * 3.x *

0.6 I.5 1.2 3.5 1.8 * * 2.0 ** 1.2 1.3 2.9

2.2 f I.4 2.9 k 1.9

“ Laboratories A: Toyama Institute of Ilealth; B: Yoshitomi Pharmaceutical Industries, Ltd.; C: Toyobo Co., Ltd.; D: Tanabe Seiyaku Co., Ltd.; E: Bioaafety Research Center, Food, Drugs and Pesticides; F: Japan Tobacco Inc.: G: Daiichi Pharmaceutical Co., Ltd.: H: University of Shiruoka: I: Itoham Foods Inc. . ’ p i 0.01 (conditional binomial test).

S. Sato et al. /Mutation

Discussion

Generally, spontaneous MNRET frequencies did not vary with sample time, laboratory, or sex for approximately 1.5 months. They started to vary with the commencement of death by senescence. This tendency may merely reflect the decrease of sample size (number of animals per group) due to mortality by aging. Although, this could be a reflection of true chromosomal instability. More extensive statistical analysis of the data is needed to clarify which is the more likely explanation. It is conceivable that serial sampling of peripheral blood itself may introduce variation in spontaneous MNRET frequencies. This seems unlikely, however, because many mice showed stable spontaneous MNRET frequencies throughout their life-span. Therefore, there appears to be no serious technical problem in this long term study using the peripheral blood sampling method, although there were occasional difficulties in obtaining an adequate volume of blood. Among the nine mouse strains studied, A/J, BDFl, and SAMP6/Tan showed higher mean spontaneous MNRET frequencies at the final observation than at the initial one. The differences were statistically significant in the first two strains (p < O.Ol), but not in the third (p = 0.08). This result suggests that chromosomal instability and/or aneuploid induction relating to senescence is probable in some mouse strains. Other strains did not show this tendency, and the spontaneous MNRET frequencies were almost identical at the beginning and the end of the study. Nisitani et al. (1990) reported age-related changes in the frequency of chromosomal aberrations in the bone marrow cells of some mouse strains: an age-dependent increment in chromosomal aberrations was more evident in the bone marrow cells of SAMPl and SAMRl mice than in those of A/J and C57BL/6 mice. Our present data for A/J mice agree with Nisitani et al. (1990); SAMP6/Tan mice showed the same tendency, but the difference was not statistically significant (C57BL/6 mice were not studied here). Based on our present observations, we conclude that the spontaneous MNRET frequencies

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338 (1995)

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57

of the nine mouse strains studied here were stable over time, at least until the mice began to die of old age (approximately 15 months), among laboratories, and for both sexes. Three of the nine strains showed an increase in spontaneous MNRET frequencies toward the end of their lifespan. More extensive statistical analysis are underway, and the outcomes will be reported separately.

References CSGMT (Collaborative Study Group for the Micronucleus Test) (1986) Sex difference in the micronucleus test, Mutation Res., 172, 151-163. CSGMT (1988) Strain difference in the micronucleus test, Mutation Res., 204, 307-316. CSGMT (1990) Single versus multiple dosing in the micronucleus test: the summary of the 4th collaborative study by CSGMT/JEMS.MMS, Mutation Res., 234, 205-222. CSGMT (1992) Micronucleus test with mouse peripheral blood erythrocytes by acridine orange supravital staining: the summary report of the 5th collaborative study by CSGMT/JEMS.MMS, Mutation Res., 278, 83-98. Fenech, M. and A.A. Morley (1985) The effect of donor on spontaneous and induced micronuclei, Mutation Res., 148, 99-10s. Hayashi, M., T. Morita, Y. Kodama, T. Sofuni and M. Ishidate Jr. (1990) The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides, Mutation Res., 245, 245-249. Hayashi, M., S. Sutou, H. Shimada, S. Sato, Y.F. Sasaki and A. Wakata (1989) Difference between intraperitoneal and oral gavage application in the micronucleus test, The 3rd collaborative study by CSGMT/JEMS.MMS, Mutation Res., 223, 329-344. Kastenbaum, M.A. and K.O. Bowman (1970) Tables for determining the statistical significance of mutation frequencies, Mutation Res., 9, 527-549. MacGregor, J.T., CM. Wehr and D.H. Gould (1980) Clastogen-induced micronuclei in peripheral blood erythrocytes: the basis of an improved micronucleus test, Environ. Mutagen., 2, 509-514. Nisitani, S.M. Hosokawa, M.S. Sasaki, K. Yasuoka, H. Naiki, T. Matsushita and T. Takeda (1990) Acceleration of chromosome aberration in senescence-accelerated strains of mice, Mutation Res., 237, 221-228. Takeda, T., M. Hosokawa, S. Takeshita, M. Irino, K. Higuchi, T. Matsushita, Y. Tomita, K. Yasuhira, H. Hamamoto, K. Shim& M. Ishii and T. Yamamura (1981) A new murine model of accelerated senescence, Mech. Ageing Dev., 17, 183-194.