No change in spontaneous mutation frequency or specificity in dietary restricted mice
Gregory R.Stuart2,
Yoshimitsu Oda1,
Johan G.de Boer and
Barry W.Glickman
Centre for Environmental Health, University of Victoria, PO Box 3020, Victoria, BC, Canada V8W 3N5 and
1 Osaka Prefectural Institute of Public Health, 3-69 Nakamichi 1-Chome, Higashinari-ku, Osaka 537-0025, Japan
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Abstract
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It is well known that dietary restricted rodents live longer and are generally healthier than their ad libitum fed counterparts, with fewer tumors. Additionally, while dietary restriction appears to reduce the frequency of chemically induced mutation in laboratory animals, relatively little is known regarding the effect of dietary restriction on spontaneous mutational events. Although spontaneous mutation rates are generally low compared with chemically induced events, spontaneous mutations accumulate in most tissues over the lifetime of the animal and are therefore expected to contribute significantly to spontaneous neoplasia. It is generally presumed that dietary restriction results in less oxidative damage and a lowering of the mutation frequency. Here we report the results of dietary restriction on mutation frequency and specificity in lacI transgenic mice aged 6 and 12 months. Unexpectedly, no changes were observed in either the frequency or specificity of mutation in dietary restricted mice, compared with ad libitum controls. We therefore conclude that dietary restriction appears to have no appreciable effect on spontaneous mutation, at least in chromosomal DNA.
Abbreviations: MF, mutant frequencies; MS, mutational spectra.
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Introduction
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Caloric restriction (a reduction in calories with supplementation to provide essential micronutrients) and dietary restriction (a simple reduction in food intake) are known to dramatically reduce the incidence of spontaneous and chemically induced neoplasias in and to extend the lifespan of rodents (13). Calorie (dietary) restricted rodents are also believed to experience a reduction in premutagenic DNA lesions and mitotic indices and an enhancement in the efficiency of DNA repair (4,5). Consequently, it has been generally presumed that the frequency of spontaneous mutations in calorie (dietary) restricted rodents would be depressed, relative to age-matched ad libitum fed controls. While there is some indication that dietary restriction has little effect on spontaneous mutant frequencies (MF) (3), little if anything is known regarding the effect of dietary restriction on spontaneous mutational spectra (MS). The study of the factors which contribute to the frequency and specificity of mutation in vivo is facilitated through the use of transgenic rodent mutational assays. We therefore investigated the effect of dietary restriction on spontaneous MF and specificity in liver from Big Blue® lacI transgenic mice (6) aged 6 and 12 months.
Control male C57BL/6 Big Blue® lacI transgenic mice were provided with food (Purina Mouse Chow 5015; Ralston Purina Co., St Louis, MO) and water ad libitum, while male dietary restricted mice were provided with ~70% by weight of the diet consumed by the ad libitum group, starting at age 11 weeks. The degree of dietary restriction in the present study was slightly less severe than the 60% restriction (of ad libitum levels) often used in dietary restriction studies. Nevertheless, the body weights of the dietary restricted mice were 51 and 77% of those of their ad libitum controls at ages 6 and 12 months, respectively (Table I
), confirming that the mice were truly dietary restricted. Similarly, James and Muskhelishvili (7) reported that their 60% calorie restricted B6C3F1 mice weighed 69% as much as the ad libitum controls at age 12 months. In a review, Tannenbaum and Silverstone (2) indicated that carcinogenesis is affected by even small degrees of calorie restriction, with the degree of inhibition dependent on the extent of restriction. Similarly, Turturro et al. (8) reported a linear relationship between the body weight of dietary restricted male B6C3F1 mice at 13.5 months of age and the incidence of liver tumors at age 25.5 months. Thus, it was unlikely that we would fail to observe any potential effects of 70% dietary restriction on MF and MS, rather than if we had utilized a 60% dietary restriction protocol.
MF and specificity were determined in the lacI transgene recovered from chromosomal DNA from liver, as previously described (9,10). Liver was selected for study for several reasons: liver is a slowly proliferating tissue, in which spontaneous MF continue to increase with age in adult mice (9); lacI spontaneous (and induced) MF and MS are exceptionally well characterized in mouse liver; finally, dietary restriction decreases the incidence of spontaneous and induced liver tumors, as noted above. In ad libitum (control) mice, the MF in liver at 12 months of age was significantly increased compared with mice aged 1.5 and 6 months (Table I
). However, we found no significant change in the MF in the lacI transgene recovered from mice of the same age when ad libitum fed mice were compared with dietary restricted animals. The question might be raised as to the time at which effects of dietary restriction on MF can first be observed. In this regard, Casciano et al. (3) began dietary restriction of their F344 rats at age 10 weeks, followed by aflatoxin B1 treatment at age 16 weeks. This brief period of dietary restriction was sufficient to significantly decrease aflatoxin-induced MF in splenic lymphocytes. Based in part on these observations, we believe that we would have detected a significant change in spontaneous MF in our dietary restricted mice relative to control mice, noting that dietary restriction began at age 11 weeks, and had therefore continued for ~15 and ~41 weeks by ages 6 and 12 months, respectively.
As we have previously demonstrated that mutational spectra can be sensitive indicators of changes in mutational outcome despite inconsequential changes in MF (11), we sequenced mutant lacI genes recovered from ad libitum fed and dietary restricted mice (Table II
). Only slight differences were noted in the mutational spectra from ad libitum fed and dietary restricted mice; for example, the frequency of A:T
G:C transition mutations appeared to be lower in dietary restricted mice compared with ad libitum fed mice, though not significantly. Indeed, when complete mutational spectra (Table II
) from ad libitum fed and dietary restricted mice were compared using the AdamsSkopek test (12) (data not shown), overall there were no significant changes (P > 0.05) in mutational spectra, suggesting that dietary restriction does not alter the distribution of spontaneous mutagenesis. Furthermore, we noted no differences in the contribution of G:C
T:A or A:T
C:G transversions (13) to the spectra of either ad libitum fed or dietary restricted animals or reference mutational spectra (9,14), indicating that oxidative DNA damage is unlikely to be a major contributor to the observed MF. This contradicts specific predictions arising from aging and diet models (15). Collectively, our data indicate that dietary restriction has a minimal influence on spontaneous mutational processes in nuclear DNA of somatic cells. We tentatively conclude that the well-established effects of dietary restriction on the physiology, longevity and neoplastic status of rodents are likely to be attributable to factors other than modulation of spontaneous mutation in chromosomal DNA, with the following caveats. Firstly, although the Big Blue® assay is exceptionally sensitive to base substitution mutations and small deletions, larger deletions and chromosomal aberrations are likely to remain undetected. Secondly, although the effects of dietary restriction on spontaneous mutation were negligible in this study, the effects of dietary restriction on end points such as cancer incidence might be manifested due to uncharacterized effects on non-genetic factors and cellular organelles, including mitochondria, and cellular macromolecules, including proteins and RNA (16).
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Acknowledgments
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We wish to acknowledge the technical assistance provided at various times by Heather Erfle, Adlane Ferreira, James Holcroft, Ken Sojonky, Erika Thorleifson and David Walsh. We also thank the referee's for their thoughtful reviews. The support of The Cancer Research Society Inc. (Montreal, Quebec, Canada) for G.R.S. is gratefully acknowledged.
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Notes
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2 To whom correspondence should be addressed Email: gstuart{at}uvic.ca

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Received June 25, 1999;
revised September 17, 1999;
accepted October 8, 1999.