Mutagenesis, Vol. 18, No. 3, 273-275,
May 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
The effect of aging on the results of the rat micronucleus assay
Central Research Laboratory, SSP Co. Ltd, 1143 Nanpeidai, Narita, Chiba 286-8511, Japan, 1 Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan and 2 National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
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Abstract |
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We conducted the micronucleus assay in 3-, 5-, 7-, 9-, 11- and 13-week-old male rats to determine whether the results varied with age. We administered cyclophosphamide orally at 0 (vehicle), 5, 10 or 20 mg/kg, twice, 24 h apart, to five rats per dosage group and collected bone marrow and peripheral blood 24 h after the second treatment. We observed an age-related decrease in micronucleus-inducing activity in both polychromatic erythrocytes (bone marrow) and reticulocytes (peripheral blood), which we attributed to an age-related decrease in hematopoiesis. In spite of the age-related decrease in sensitivity to the model chemical studied here, all age groups tested showed positive responses. We concluded that the rat is suitable for the micronucleus assay regardless of age.
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Introduction |
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The mouse is conventionally used for the micronucleus assay while the rat is usually used for general toxicologic and toxicokinetic studies. If micronucleus induction can be evaluated in the same rats that are used for general toxicology studies, a lot of information can be gathered concomitantly. We explored the possibility of incorporating micronucleus assays into 28 day repeat dose general toxicology studies, where the effect of aging could be important, using the same animals (Hamada et al., 2001
). The Collaborative Study Group for the Micronucleus Test (CSGMT), a working group of the Mammalian Mutagenicity Study Group (MMS), which is a subgroup of the Japanese Environmental Mutagen Society (JEMS), previously evaluated the effects of aging on the micronucleus assay in mice (CSGMT, 1995a; Sato et al., 1995; Hamada et al., 1996; Higashikuni and Sutou, 1996). In the present study, we investigated the aging effects in rats using cyclophosphamide (CP) as the model chemical. |
Materials and methods |
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Chemicals
Cyclophosphamide (CAS no. 50-18-0, lot no. 9014), obtained from Shionogi & Co. Ltd (Osaka, Japan), was dissolved in distilled water immediately before use.
Animals
Male Crj:CD(SD)IGS rats 3, 5, 7, 9, 11 and 13 weeks old were purchased from Charles River Japan Inc. They were given commercial pellets and tap water ad libitum throughout the acclimatization and experimental periods and were subjected to a 12 h light/dark cycle.
Micronucleus assay protocols
To perform the micronucleus assay using bone marrow and peripheral blood simultaneously in the same animal, a double dosing regimen (CSGMT, 1990, 1995b) was used. CP dissolved in distilled water at 0 (vehicle), 5, 10 or 20 mg/kg was administered orally twice, 24 h apart, to five rats per dosage group. Both peripheral blood and bone marrow were collected 24 h after the second treatment. Acridine orange staining (Hayashi et al., 1983) was used for analysis. Micronucleated polychromatic erythrocyte (MNPCE), micronucleated normochromatic erythrocyte (MNNCE) and micronucleated reticulocyte (MNRET) frequencies were recorded based on the observation of 2000 polychromatic erythrocytes (PCEs), 2000 normochromatic erythrocytes (NCEs) or 2000 reticulocytes (RETs). Cytotoxicity was evaluated based on the observation of 300 bone marrow erythrocytes or 2000 peripheral blood reticulocytes.
Statistical analysis
To compare the responses of each dosage group with those of the concurrent control group, we used Kastenbaum and Bowman’s tables (Kastenbaum and Bowman, 1970) for the frequency of MNPCEs, MNRETs and MNNCEs and the t-test for the frequency of PCEs and RETs.
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Results |
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Table I
shows the results of both assays. In the bone marrow assay, the MNPCE frequency increased significantly in the treated animals in all dosage and age groups. The magnitude of this increase decreased with increasing age in the 10 and 20 mg/kg/day groups. There were no significant increases in MNNCE frequency in any group treated at 5 mg/kg/day. The PCE frequency was highest in the 3-week-old age group and decreased significantly at all doses in the 9-, 11- and 13-week-old groups. The decreases were age-related.
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In the peripheral blood micronucleus assay, the MNRET frequency was increased significantly in all dosage groups by CP at 5, 7, 11 and 13 weeks of age. The magnitude of the increases decreased in an age-related manner within the 5, 10 and 20 mg/kg/day groups up to 7–9 weeks of age, but not in the vehicle control groups. There was no age- or dose-dependent increase in the frequencies of MNNCE. The RET frequency decreased significantly in treated animals at all dosages in the 3-, 7- and 9-week-old groups and at 20 mg/kg/day in all age groups, relative to the 3-week-old group. An age-related decrease was seen in all dosage groups.
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Discussion |
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There was a tendency towards an age-related decrease in micronucleus assay sensitivity to CP in the rat bone marrow assay. The sensitivity of the assay may depend on the metabolic activity of cytochrome P450 2C enzyme (CYP2C), which metabolizes CP to a genotoxic intermediate (Clarke and Waxman, 1989
), but this needs further evaluation. Yajima et al. (1993a–c) and Handa and Yajima (1995) speculated that the increased MNPCE and MNRET frequencies are the result of errors that occur in the processes of denucleation, differentiation and repair of genetic damage. They also suggested a close relationship between erythropoiesis and micronucleus induction. In the present study, there was a close relationship between PCE and MNPCE frequencies at the same CP dose (Figure 1). These results suggested that the young animals, which had a high level of erythropoietic activity, tended to show high micronucleus induction. We confirmed an age-related reduction in sensitivity to CP, although all dosages induced MNPCE significantly. At 5 mg/kg/day, CP did not significantly increase MNNCE frequency in any age group. These results might suggest that the micronucleus-inducing activity of compounds could be evaluated in bone marrow cells regardless of age of rat if MNPCEs, but not MNNCEs, were scored.
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The results of the peripheral blood assay were similar to those of the bone marrow assay. The micronucleus-inducing activity of compounds could be evaluated regardless of age if MNRETs, but not MNNCEs, were scored. A good correlation between RET and MNRET frequencies was seen within the same CP dosages, but the peripheral blood data varied more widely than the bone marrow data (Figure 2
), probably because some circulating micronucleated erythrocytes were destroyed in the spleen (Schlegel and Macgregor, 1982, 1984; Schlegel et al., 1986; Hayashi et al., 1992), but that needs further evaluation.
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The effect of aging on the micronucleus assay was not so clear in the mouse (Hamada et al., 1996
) as in this study of the rat. The lowering of erythropoiesis with age in the mouse was less (Hamada et al., 1999) than in the rat, which was assumed to be the primary cause of the small effect of aging in the mouse micronucleus assay. In summary, these results suggest that the effect of aging in the micronucleus assay depends mainly on age-related changes in erythropoietic function and the chemical induction of micronuclei in bone marrow cells can be evaluated by scoring MNPCEs in the bone marrow or MNRETs in the peripheral blood in the rat regardless of age, with marginal age-dependent differences in sensitivity.
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Acknowledgments |
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We are grateful to the Tsutikawa Memorial Fund for Study in Mammalian Mutagenicity for partially sponsoring this study.
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Notes |
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3 To whom correspondence should be addressed. Tel: +81 476 27 1511; Fax: +81 476 26 7948; Email: shuichi.hamada{at}ssp.co.jp
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References |
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Received on August 22, 2002; revised on October 7, 2002; accepted on October 8, 2002.
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