Mutation Research 527 (2003) 57–66

Effect of caloric restriction on Hprt lymphocyte

mutation in aging rats
Anane Aidoo a,∗ , Roberta A. Mittelstaedt a , Michelle E. Bishop a ,
Lascelles E. Lyn-Cook a , Yi-Ju Chen b , Peter Duffy a , Robert H. Heflich a
a U.S. FDA Jefferson Laboratories, Division of Genetic & Reproductive Toxicology, National Center for Toxicological Research,
3900 NCTR Road, Jefferson, AR 72079, USA
b U.S. FDA Jefferson Laboratories, Division of Biometry & Risk Assessment, National Center for Toxicological Research,

Jefferson, AR 72079, USA

Received 1 November 2002; received in revised form 7 March 2003; accepted 18 March 2003

Abstract
Caloric restriction (CR) reduces tumor incidence and retards aging in laboratory animals, including non-human primates.
Because of the relationships among mutation, disease susceptibility, and aging, we investigated whether or not CR affects the
accumulation of somatic cell mutations in aging animals. Starting at approximately 2 months of age, male CD rats (Harlan
Sprague–Dawley-derived) were placed on different levels of dietary intake: ad libitum (AL) feeding, and 90% (10% CR), 75%
(25% CR) and 60% (40% CR) of the total calories consumed by AL animals. At 3, 6, 12, and 24 months after the beginning
of CR, Hprt mutant frequencies (MFs) were determined. The MFs measured in spleen lymphocytes from AL and CR rats
sacrificed at 3 months of dietary restriction were similar for all dietary groups. However, the MFs at 6, 12, and 24 months of
CR were significantly higher in AL-fed rats compared with animals on 40% CR: (4.5 ± 0.4) × 10−6 versus (3.3 ± 0.3) × 10−6
(P = 0.032) in 6 months CR rats; (10.3 ± 2.3) × 10−6 versus (7.3 ± 1.2) × 10−6 in 12 months CR rats (P = 0.04), and
(18.3 ± 3.2) × 10−6 versus (7.8 ± 1.0) × 10−6 (P = 0.001) in 24 months CR rats. In addition, rats receiving 25% CR for 24
months had a MF, (10.7 ± 2.0) × 10−6 , between the 40% CR and AL rats. Multiplex PCR of the Hprt gene in mutant clones
from 12 and 24 months 40% CR rats and the corresponding AL rats detected deletions in 42% of CR mutants and 19% of AL
mutants. Because of the difference in Hprt MF in the two groups, the estimated MF associated with deletions in CR rats was
similar to the deletion MF in AL rats. This observation implies that the lower MF in CR rats is due to a reduction in smaller
Hprt mutations (i.e. base substitutions and frameshifts). The pattern of smaller Hprt mutations from AL rats suggests that
many were produced by reactive oxygen species (ROS). The results indicate that CR reduces the accumulation of spontaneous
somatic cell mutation in aging rats, especially those caused by base substitutions and frameshifts.
Published by Elsevier Science B.V.
Keywords: ad libitum; Caloric restriction; Hprt mutation; Aging; Reactive oxygen species

1. Introduction

The only established model demonstrating a direct

∗ Corresponding author. Tel.: +1-870-543-7331; effect of nutrition on aging is that of caloric restric-
fax: +1-870-543-7379. tion (CR). CR is the balanced reduction of the pro-
E-mail address: aaidoo@nctr.fda.gov (A. Aidoo). tein, carbohydrate and fat content of the diet without

0027-5107/03/$ – see front matter. Published by Elsevier Science B.V.

doi:10.1016/S0027-5107(03)00072-1
58 A. Aidoo et al. / Mutation Research 527 (2003) 57–66
reduction of its vitamin content [1]. Numerous stud-
ies on CR in rodents, non-human primates, and other
organisms indicate that this nutritional intervention
retards the aging process, as evidenced by increased
longevity, reduced pathology, as well as mainte-
nance of physiological function in a more youthful
state [1–3]. A reduction in calorie intake dramati-
cally slows or delays the development of spontaneous
and chemically-induced tumors in rodents [4–8]. In
addition, CR delays spontaneous tumorigenesis in
p53-deficient mice that are genetically engineered to
develop tumors early in life [9], suggesting a possible
role for CR in cancer prevention. Although CR has
great promise for delaying age-related diseases and the
extension of mean, as well as maximum life span [10],
the mechanisms underlying the health benefits asso-
ciated with this dietary strategy have not been fully
elucidated.
Major contributing factors that have been pro-
posed for the effects of CR include modulation of
gene expression, apoptosis, cell turnover, and free
radical-induced DNA damage [5,11–14]. Of these
processes, the attenuation of oxidative damage by CR
has been the main focus of research. The accumula-
tion of random oxidative damage to DNA, lipids, and
proteins [15–17] gives rise to abnormal cellular and
physiological changes, and CR may reduce this free
radical damage. In support of this hypothesis, expo-
sure to superoxide dismutase and catalase mimetics
that modulate free radical damage is associated with
an increase in life span [18].
The presence of reactive oxygen species (ROS)
arising in cells primarily as a by-product of normal
metabolic activities is thought to influence the etiol-
ogy of age-related diseases such as cancer [19–21].
ROS-induced formation of DNA adducts could lead to
mutations [22,23]. DNA sequence alterations that oc-
cur in the absence of any obvious external mutagenic
exposure are believed to play a major role in carcino-
genesis, genetic disease, and aging [24–27]. However,
despite the role of mutations in aging and disease,
there have been few studies that have evaluated the
effects of CR on spontaneous mutation as animals
age. A previous study by Dempsey et al. [28] mea-
sured mutant frequency (MF) in mice for a period of
12 months and indicated that CR was effective at re-
ducing the age-associated accumulation of mutation.
In the present study, we have determined spontaneous
MFs and mutation spectra in CD rats maintained on
different levels of CR over a 24-month period.
2. Materials and methods
2.1. Animals and feeding regimen
The animal care and dietary procedures employed
in this study were reported previously [29]. Our In-
stitutional Animal Care and Use Committee approved
the study protocol. The original founder stocks of
CD Sprague–Dawley rats [CRl:CD(SD)BR] were ob-
tained from Charles River Laboratory in 1972 and
the breeding colony established from these animals
has been maintained in a specific pathogen-free envi-
ronment at the National Center for Toxicological Re-
search. The average body weight of rats in this colony
has remained constant over several decades [29]. The
male rats that were used in this study were maintained
at 23 ◦ C, and conditioned to a 12 h light/12 h dark cy-
cle with lights on from 6:00 to 18:00 h daily. An ad
libitum (AL) feeding regimen was used for all animals
from the time they were weaned (at 3 weeks of age)
until the time they entered the experimental protocol.
The animals were housed singly in standard rat cages,
and received NIH-31 diet and water AL. The ingre-
dients, specifications, and the nutrient composition of
the NIH-31 diet, as well as the rodent nutrient require-
ments, were the same as previously published [1].
At approximately 2 months of age, the test animals
were separated into four groups: a control group that
continued to receive food AL, and three CR groups
which were assigned to rations 90% (10% CR), 75%
(25% CR), and 60% (40% CR) of the AL regimen. A
40% CR diet was included in this experiment so that
the results from the present study could be compared
with those previously published [29–31]. The 25%
CR diet was chosen because this regimen has been
promoted and used by the pharmaceutical industry for
chronic bioassay studies [2,32]. Furthermore, the 10%
level was selected because little is known about the
effects of a small reduction in calories on life span.
All rats were fed at 10:00 h daily, which corre-
sponded to 4 h after lights on. The AL rats were
offered more food than they could consume in 24 h,
whereas the CR rats were fed defined portions of
food that were reduced in amount and calories. CR
 A. Aidoo et al. / Mutation Research 527 (2003) 57–66
Fig. 1. One group of male CD rats was maintained on NIH31 ad libitum (AL) (䊏). In the other group, the rats were switched to caloric
restriction (CR) diets ( ) at 2 months of age. Rats were sacrificed at the times indicated by double arrows (↔) and analyzed for lymphocyte
Hprt mutant frequency.
was accomplished by the reduction of all nutrients,
except vitamins which were fortified at 1.67 times the
concentration of the NIH-31 standard diet formula-
tion to ensure adequate vitamin supplementation for
the most restricted rats [1]. The animals remained in
their respective nutritional groups for 3, 6, 12, or 24
months prior to sacrifice. Other rats not in the above
groups were killed 2 weeks after birth (Fig. 1).
2.2. Mutant and mutation analysis
At sacrifice, spleens were rapidly removed and
lymphocytes were isolated using an Accu-Paque
separation gradient (Accurate Chemical and Scien-
tific, Westbury, NY). The Hprt assay was then per-
formed using previously described methods [33–36].
In brief, two sets of 96-well round-bottom mi-
crotiter plates were inoculated with 200 ␮l per well
of culture medium (supplemented RPMI 1640) con-
taining 0.25 ␮g/ml ionomycin, 10.0 ng/ml phorbol
12-myristate 13-acetate, 20% conditioned medium
and 35 ␮M 2-mercaptoethanol. In plates used to estab-
lish the cloning efficiency (CE) under non-selective
conditions, four lymphocytes and 5 × 104 irradiated
(40 Gy from a 60 CO source) lymphocytes were added
to each well. In the selection plates, 5 × 104 lympho-
cytes and 2.5 ␮g/ml 6-thioguanine (6TG) were added
to all the wells. After incubating the plates for 10–14
days, the wells were scored for colony formation,
and the CEs were calculated using Poisson statistics
to determine the frequency of 6-thioguanine-resistant
59
(6TGr ) T-lymphocytes. Hprt mutants from 6TGr
colonies were expanded to ≥5 × 105 cells for molec-
ular analysis of mutations [35]. Multiplex PCR was
performed on DNA released from half of the cells
from the expanded colonies as described previously
[36] in order to screen for large alterations in the Hprt
gene. Briefly, the nine Hprt exons and k-ras exon 2
(used as a PCR control) were amplified in nine prod-
ucts (Hprt exons 7 and 8 were amplified in a single
product) using primers specific for intronic sequence.
Smaller Hprt sequence alterations were analyzed by
RT-PCR of Hprt mRNA extracted from the remain-
ing cells from expanded colonies, followed by DNA
sequence analysis. The PCR conditions and primers
used for this analysis were described previously [36].
In addition, Hprt exons 2, 3, and 7 and 8 were am-
plified separately from the genomic DNA of some of
the mutants with the same primers used in the mul-
tiplex PCR. These products were sequenced in order
to identify point mutations and frameshifts.
2.3. Statistical analysis
A one-way analysis of variance (ANOVA) was
performed on the duration of restriction and degree
of restriction using the Statistical Analysis System
(SAS). The two-sided Dunnett’s test for multiple
comparisons was conducted in the comparisons with
the AL (control group). A Dunnett’s T-statistic for
multiple comparisons was equal to the two indepen-
dent t-test statistic when the number of total groups
60 A. Aidoo et al. / Mutation Research 527 (2003) 57–66

was two in the comparison. A two-sided P-value of CR (10 and 25%) after 24 months of CR revealed
was used to determine if the restriction level had a non-significant reductions in MF as compared with
significant effect at a 5.0% confidence level. the AL rats, although the MF in the 10% CR rats was
significantly (P = 0.05) different from the 40% CR
group.
3. Results Hprt mutant lymphocytes from 6TGr clones were
collected and expanded to determine any differences
Spleen lymphocytes were isolated from AL and in the types and frequency of mutations associated
CR rats and cultured to calculate the percent CE with a reduction in calorie intake. DNA from cultured
(%CE) under non-selective conditions and the 6TGr clones from 14- and 26-month-old rats was analyzed
T-lymphocyte MF as previously described [33]. for relatively large deletions in the Hprt gene by a mul-
The MF calculated for 2-week-old rats (prior to tiplex PCR assay [36]. The fraction of mutant clones
weaning and prior to the imposition of CR) was from the 40% CR rats with deletions in the Hprt gene
(0.7 ± 0.1) × 10−6 . The non-selected %CE and the was approximately twice that of mutants from the AL
6TGr T-lymphocyte MFs determined for the dietary rats (Table 2). Also the fraction of mutants with dele-
groups and the neonates are shown in Table 1. Al- tions for the two diets did not change with age; 42%
though the non-selected %CE decreased for all the of clones from CR animals of both ages had deletions,
groups as the animals aged, there was no consistent while 16 and 24% of clones from AL animals had dele-
difference between AL and CR rats during the pe- tions at 14 and 26 months of age, respectively. Using
riod of restriction. In contrast, the Hprt MF increased these data to calculate the Hprt lymphocyte MF asso-
with the age of both AL and CR animals, with the ciated with deletions and ’point’ mutations (e.g. base
greater increase occurring for the AL rats. Pairwise
comparisons for AL and 40% CR rats at 6, 12, and 24 Table 2
months of CR were significant (P = 0.032–0.001). Analysis of mutant clones from CD rats for Hprt deletion mutation
Evaluation of the effects of the intermediate levels by multiplex PCR
Deleted AL rats CR rats
Table 1 products(s)
Effect of caloric restriction (CR) on Hprt mutant frequency (MF) Number Total Number Total
and cloning efficiency (CE) of lymphocytes from aging male CD of clones clones (%) of clones clones (%)
rats Exon 1 3 2
Months Months of Number %CE MF (×10−6 ) Exons 1–2 0 1
of age CR (% CR) of rats Par. exons 1–2a 0 1
Exon 2 0 2
0.5 0 5 10.0 ± 1.1 0.7 ± 0.1
Par. exon 2a 0 1
5 0 28 8.9 ± 0.3 2.9 ± 0.3 Exons 2–9 0 1
3 (10) 5 9.7 ± 0.4 2.3 ± 0.4 Exon 3 1 0
3 (25) 4 9.5 ± 0.6 2.7 ± 1.3 Exons 3–9 1 0
3 (40) 30 9.8 ± 0.3 2.9 ± 0.3 Par. exon 4a 2 0
Exon 5 0 1
8 0 15 9.7 ± 0.4 4.5 ± 0.4
Par. exons 7–8a 0 1
6 (40) 16 10.7 ± 0.5 3.3 ± 0.3a
Exons 7–9 1 0
14 0 26 9.2 ± 0.3 10.3 ± 2.3 Exon 9 0 1
12 (40) 26 7.7 ± 0.6 7.3 ± 1.2b Exons 1–9 2 9
26 0 19 4.6 ± 0.5 18.3 ± 3.2 Total deletions 10 19 20 42
24 (10) 12 3.6 ± 0.3 18.2 ± 3.5
None missing 43 81 28 58
24 (25) 12 4.4 ± 0.6 10.7 ± 2.0
24 (40) 26 5.6 ± 0.5 7.8 ± 1.0c,d Total analyzed 53 48
a Different from the AL response, P = 0.032. Analysis performed on clones from 12 and 24 months 40% CR
b Different from the AL response, P = 0.043. rats and corresponding AL rats. Clonality of mutants was not
c Different from the AL response, P = 0.001. considered.
d Different from the 10% CR response, P = 0.05. a Partial deletion as evidenced by shortened PCR product.
 A. Aidoo et al. / Mutation Research 527 (2003) 57–66 61

Table 3
Analysis of the contribution of deletion mutation to Hprt lymphocyte MFs in AL and CR rats
Feeding regimen Months of age Hprt lymphocyte Fraction of mutants Calculated deletion Calculated ‘point’
MFa due to deletionb MF MF
AL 14 10.3 × 10−6 0.16 1.6 × 10−6 8.7 × 10−6
26 18.3 × 10−6 0.24 4.4 × 10−6 13.9 × 10−6
40% CR 14 7.3 × 10−6 0.42 3.1 × 10−6 4.2 × 10−6
26 7.8 × 10−6 0.42 3.3 × 10−6 4.5 × 10−6
a Data from Table 1.
b From multiplex PCR analysis of 6TGr clones (Table 2).

Table 4
Distribution of mutations identified in 6TGr lymphocytes from AL and 40% CR ratsa
Position (exon)b Mutationc Sequence contextd Amino acid change

Mutations from AL rats

74 (ex 2) C→T ATA CCT → CTT AAT Pro → Leu
100 (ex 2) A→T GAA AAG → TAG GTG Lys → amber
116 (ex 2) A→C CCT CAT → CCT GGA His → Pro
122/126 (ex 2) +GATT GGA CT/G → CTG ATT GAT Frameshift
I2, −2 A→G ctgcag → cggG AC
139/140 (ex 3) +G ACT G/AA → GGA AAG Frameshift
188 (ex 3) T→G ATT GTG → GGG GCC Val → Gly
191/193 (ex 3) −C GCC CTC → /TCT GTG Frameshift
194 (ex 3) T→C GCC CTC → CCC TGT Leu → Pro
214 (ex 3) T→A GGC TAT → AAT AAG Tyr → Asn
494 (ex 7) T→A CTG GTG → GAG AAA Val → glu
569 (ex 8) G→T GTT GGA → GTA TAT Gly → Val
580 (ex 8) G→T CTT GAC → TAC TAT Asp → Tyr
593 (ex 8) A→C GAG CAC → CCC TTC His → Pro
605 (ex 8) T→A GAT TTG → TAG AAT Leu → amber
Mutations from CR rats
43–83 (ex 2) −41 bp GAA CCA · · · · · · · · · TAT GCT
58 (ex 2) G→C CTA GAT → CAT TTA Asp → His
72–74 (ex 2) −3 bp ATA CCT → AT/T AAT -Pro
I1, −5 T→A tctttt → agcagA
134/135 (ex 3) +G cag/G AC → agG GAC Frameshift
211 (ex 3) G→C GGG GGC → CGC TAT Gly → Arg
211–220 (ex 3) −10 bp GGG GGC TAT AAG TTC TTT
215 (ex 3) A→G GGC TAT → TGT AAG Tyr → Cys
I3, +1 G→A TGT gta → ataagt
G→C TGT gta → ctaagt
527/528 (ex 7) +A CCA/GAC → AGA CTg Frameshift
565 (ex 8) G→T GTT GTT→TTT GGA Val → Phe
a
Presumed splicing mutations (i.e. exon deletions detected by RT-PCR analysis) not included.
b
cDNA sequence and numbering is from Jensen et al. [64]; intron (I) sequence is from Chen et al. [65] and is numbered back (indicated
by a minus sign) from the 5 end of the neighboring exon or forward (indicated by a plus sign) from the 3 end.
c Sequence of non-transcribed stand is shown.
d Bases in the coding region shown in capital letters and grouped by codon. ‘/’ marks the position of a frameshift. Altered/added/deleted

bases are underlined. Altered position assigned arbitrarily when ambiguous.

62 A. Aidoo et al. / Mutation Research 527 (2003) 57–66

substitutions and frameshifts) indicates that the fre- of the few mutations identified, G:C → A:T transi-
quency of Hprt lymphocyte mutants caused by dele- tions occurred with higher frequency (28%) in AL rats
tion was low and relatively similar for CR and AL rats compared to 40% CR rats (8%), while A:T → G:C
(Table 3). In contrast, the MF associated with point transitions were similar in both dietary groups. The
mutation in AL rats was over twice that in CR rats. frequency of transversion mutation was nearly equal in
Relatively small sequence changes were determined AL and CR rats, however, the transversions in CR rats
by RT-PCR of Hprt mRNA from expanded mutants were dominated by G:C → C:G (25%), a relatively
that did not contain genomic deletions by multiplex rare mutation in AL rats (6%). These apparent differ-
PCR. Eighty-six percent of these mutants produced an ences in mutation patterns, however, did not produce
Hprt cDNA PCR product, and 19% of these products significant differences in the overall spectra of muta-
contained alterations in their cDNA sequence. Addi- tions for AL and CR rats, with or without the addition
tional mutations were identified in mutants that pro- of the previously published AL mutations (P = 0.12).
duced no or inconclusive cDNA sequence information
by using genomic DNA from the mutants to amplify
and sequence Hprt exons 2, 3, 7 and 8. Analysis of a 4. Discussion
database of human HPRT mutations [37] indicates that
these are the exons most likely to contain mutations. Current attempts to slow the aging process and the
The mutations identified in 6TGr lymphocytes from accompanying onset of disease have focused on the
AL and CR rats are listed in Table 4. In order to aug- modulation of DNA damage that could be responsible
ment the relatively low number of mutations and in- for the accumulation of specific mutations. Although
crease the power of the analysis, additional mutations dietary antioxidants have been extensively used in
from previous Hprt lymphocyte mutation analyses in model intervention studies, by far the best results are
AL rats [38,39] are included with the mutations from achieved using CR. CR diets, if begun early enough
Table 4 in the data summary in Table 5. On the basis during the life of the organism, result in a substan-
tial reduction of disease morbidity, and produce an in-
Table 5 creased lifespan [10,40]. It is unlikely that CR as used
Summary of independent mutations in the lymphocyte Hprt gene in animal studies will be adopted by humans; however,
of mutants from AL and 40% CR rats understanding the mechanisms by which CR exerts
Mutation AL ratsa CR rats its benefits may shed light on intervention strategies
Number of Percent Number of Percent for reducing obesity and age-related diseases such as
mutations mutations cancer [41,42]. The development of cancer and other
Transitions diseases is believed to be influenced, if not caused, by
G:C → A:T 10 28 1 8 mutations. Thus, the purpose of the present study was
A:T → G:C 3 2 1 8 to determine if spontaneous mutations measured in ag-
Transversions ing rats are modulated by maintaining the animals on
G:C → T:A 3 8 1 8 different levels of CR.
G:C → C:G 2 6 3 25 Our results indicate that Hprt lymphocyte MF in-
A:T → C:G 4 11 0 0
creased with age in rats fed AL, thus confirming
A:T → T:A 6 17 1 8
published data on the age-related accumulation of
−1 or −2 frameshift 1 3 0 0 Hprt mutation in rodents and humans [28,43–45].
+1 or +2 frameshift 3 8 2 17
Deletions/insertions 4 11 3 25
CR significantly lowered the cumulative effects of
AL feeding on mutation. Although the 25% level of
Total mutations 36 12
restriction reduced MFs somewhat, only the 40% CR
Suspected splicing mutants by RT-PCR analyses and deletion mu- diet produced significant effects on MF. These results
tations detected by multiplex PCR are not included. No difference are generally consistent with data on physiological and
between the two profiles (P = 0.12) using the test of Adams and
pathological parameters measured in these same rats
Skopek [66].
a Includes 21 mutations previously reported for AL Big Blue® [1], which indicate that CR results in decreased body
rats [38] and Fischer 344 rats [39]. weight and higher survival. In contrast to what was
 A. Aidoo et al. / Mutation Research 527 (2003) 57–66 63

found for lymphocyte MF, however, the animals in the tron transport complexes in bleomycin-exposed rats
10 and 25% CR groups had a survival rate equivalent [54].
to that of 40% CR rats. Also, the percentage of animals ROS-induced DNA damage gives rise to several
with tumors at 26 months of age was higher in the AL types of base substitution mutations [55–57]. Further-
group than all of the CR groups, approximately 95%
for the AL group compared with about 65% for the
different levels of CR (Duffy, P., unpublished data).
In addition to a higher tumor burden and reduced life
span, gross morphological examination of the internal
tissues/organs of AL rats at sacrifice revealed a vari-
ety of pathological manifestations including enlarged
spleens, hearts, kidneys, and lungs. Most of the organs
were covered with fatty tissues. In some animals, the
spleen was fused to either the left kidney or the liver,
and fluid was retained in the thoracic and abdominal
areas. The external appearance of AL rats was char-
acterized by patchy, browning hair. These aberrations
were essentially absent among CR rats (unpublished
observations). The lack of an exact correspondence
between Hprt lymphocyte mutation, tumorigenesis
and other pathologies in the intermediate CR groups
could be due to the relatively small number of rats in
these groups that were assayed for mutation.
In a similar study that measured lymphocyte Hprt
MF in mice, it also was observed that 40% CR de-
creased the accumulation of mutation [28]. These ob-
servations in mice and the results of our study with
rats indicate that CR is antimutagenic, which may par-
tially account for its ability to reduce tumor burden
and other effects of aging. CR has pleiotropic biolog-
ical effects that could reduce MF, including the en-
hancement of DNA repair, decreasing the rate of cell
division, altering growth factor levels and the expres-
sion of cancer-related genes, and reducing oxidative
DNA damage [14,46–49]. In the case of the specific
gene mutations measured in this study, it is also con-
ceivable that CR might act to increase the negative se-
lection for Hprt lymphocyte mutations that are known
to occur in mice [50]. The ability of CR to control
oxidative DNA damage is of great interest not only
because ROS are ubiquitous in cells, but also because
the metabolism of exogenous agents by cytochrome
P-450 enzymes releases free radicals [51]. In previ-
ous studies in our laboratory, we found that CR re-
duced the MFs induced by treating rats with aflatoxin
B1 and bleomycin, compounds known to release free
radicals [52,53]. In a parallel study, CR also mod-
ulated the activity of antioxidant enzymes and elec-
more, the concentration of 8-oxo-2 -deoxyguanosine
(8-oxo-dG), a marker for oxidative DNA damage and
a lesion that results in base substitution mutation, is re-
duced in both nuclear and mitochondrial DNA by CR
[58]. Our multiplex PCR analysis indicated that the
MF caused by relatively large deletion mutations was
similar in AL and CR rats. Thus, we conclude that the
reduced MF in CR rats is mainly due to a lower fre-
quency of small-scale sequence alterations, mutations
of a type consistent with ROS-induced mutation. In or-
der to gain further insight into the effect of CR on mu-
tation induction, we examined mutant clones from CR
and AL rats for small-scale mutations. This analysis
was disappointing because of the few mutations that
were identified. The low yield of mutations was due to
a combination of problems: a low percentage of clones
expanded sufficiently for analysis (ranging from 10 to
50% for clones from individual rats), and a relatively
low frequency of RT-PCR products and genomic DNA
PCR products contained mutations. These problems
may be partially due to working with mutant cells
from relatively old rats, i.e. 14–26 months of age, that
appear to grow more poorly and express Hprt mRNA
to a lesser extent than mutants from younger animals
(unpublished observations). The absence of mutations
in many of the RT-PCR products and genomic PCR
products that were amplified from mutant clones is
harder to explain, but may be due to mutations oc-
curring outside the coding region of the gene or gene
inactivation by epigenetic effects (i.e. gene silencing).
Pooling data from previous analyses of small-scale
Hprt lymphocyte mutations (mainly from 3 to 6
month-old rats [38,39]) with the mutations found in
the present study enabled us to assemble a reason-
ably sized set of 36 mutations for AL rats (Table 5).
There was no indication of clonal amplification (the
same mutation isolated multiple times from a single
animal) from these analyses, suggesting that ampli-
fication of mutant clones was not involved in the
higher lymphocyte mutant frequency seen in AL
rats. Three types of point mutations, G:C → A:T
transition, and G:C → T:A and A:T → T:A transver-
sion, accounted for 53% of the mutations from AL
rats. These three mutations are also among the most
64 A. Aidoo et al. / Mutation Research 527 (2003) 57–66
commonly found mutations in the Human HPRT
Mutant Database [59]. In contrast, G:C → A:T tran-
sition and G:C → T:A and A:T → T:A transitions
accounted for only 25% of the 12 mutations isolated
from CR rats. This may be of interest because these
three mutations have been associated with specific
types of ROS-mediated DNA damage. For example,
the widely studied oxidative DNA adduct, 8-oxo-dG,
induces G:C → T:A transversion by misreplication
[60]. Similarly, 5-hydroxydeoxycytosine is responsi-
ble for ROS-induced G:C → A:T transition mutation
[57]. In addition, hydrogen peroxide, a precursor of
the potent hydroxyl radical, induces both G:C → A:T
and G:C → T:A substitution [61–63]. Taken together,
these three mutations could be considered a molecular
signature of oxidative DNA damage. Therefore, with-
out offering definitive evidence, the data are at least
consistent with the hypothesis that CR reduces the for-
mation of oxidative DNA damage leading to mutation.
In summary, the results of this study indicate that
the increase in MF associated with the AL feed-
ing of rats was significantly reduced by CR. Since
the contribution of relatively large deletions to the
MF was similar for AL and CR rats, we conclude
that CR reduces lymphocyte Hprt MF by reduc-
ing the frequency of mutations caused by relatively
small sequence alterations. Although our analysis of
small-scale mutations was of limited success, a large
fraction of the mutations detected in AL rats were
of the type associated with oxidative DNA damage,
and the frequency of these mutations was lower in
CR rats. The influence of CR on the accumulation of
spontaneous somatic mutation in our study displays a
general correlation with documented CR effects, such
as reduced pathology and increased lifespan, and sug-
gests a relationship between the antimutagenic and
the anticarcinogenic and antiaging actions of CR. CR
may be impractical for humans; however it deserves
attention because it could lead to the formulation of
pharmacological mimetics or physical strategies that
may limit obesity and postpone age-related diseases.
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