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Biogerontology 4: 193–202, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
193
Research article
Effect of Epitalon on biomarkers of aging, life span and spontaneous
tumor incidence in female Swiss-derived SHR mice
Vladimir N. Anisimov
1,∗
, Vladimir Kh. Khavinson
2
, Irina G. Popovich
1
, Mark A. Zabezhinski
1
,
Irina N. Alimova
1
, Svetlana V. Rosenfeld
3
, Natalia Yu. Zavarzina
1
, Anna V. Semenchenko
4
&
Anatoli I. Yashin
4
1
Department of Carcinogenesis and Oncogerontology, N.N. Petrov Research Institute o f Oncology, Pesoch ny-2, St.
Petersburg, 197758, Russia;
2
St. Petersburg Institute of Bioregulation and Gerontology, 3, pr. Dynamo, 197110,
Russia;
3
I.P. Pavlov State Medical University, Lev Tolstoy S tr., 6/8, St. Petersburg, 197022, Russia;
4
Max-Planck
Institute for Demographic Research, Doberaner Str., 114, Rostock 18057, Germany;
∗
Author for correspondence
(e-mail: aging@mail.ru; fax: +7-812-596-8947)
Received 7 November 2002; accepted in revised form 2 January 2003
Key words: chromosome aberrations, Epitalon, estrous cycle, life span, SHR mice, spontaneous tumors
Abstract
From the age of 3 months until their natural deaths, female outbred Swiss-derived SHR mice were subcutaneously
injected on 5 consecutive days every month with 0.1 ml of normal saline (control) or with 1.0 µg/mouse
(∼30–40 µg/kg) of tetrapeptide Epitalon
(Ala-Glu-Asp-Gly) dissolved in 0.1 ml saline. There were 54 mice in
each group. The results of this study show that treatment with Epitalon did not influence food consumption, body
weight or mean life span of mice. However, it slowed down the age-related switching-off of estrous function and
decreased the frequency of chromosome aberrations in bone marrow cells (by 17.1%, P < 0.05). It also increased
by 13.3% the life span of the last 10% of the survivors (P < 0.01) and by 12.3% the maximum life span in
comparison with the control group. We also found that treatment with Epitalon did not influence total spontaneous
tumor incidence, but inhibited the development of leukemia (6.0-fold), as compared with the control group. The
data obtained suggest a geroprotector activity of Epitalon and the safety of its long-term administration in mice.
Introduction
The search for new effective and safe means to prevent
premature aging is one of the priorities in geronto-
logy (Anisimov 2001; Butler et al. 2002; De Grey
et al. 2002). During the last decade a number of
reports have appeared on the role of the pineal gland
in aging (Armstrong and Redman 1991; Reiter 1995;
Reppert and Weaver 1995; Pierpaoli 1998; Reiter et
al. 2002). A modulating effect of the pineal gland
on the neuroendocrine and the immune system was
shown to change during aging (Arendt 1995). Pinea-
lectomized rats showed a reduced life span (Malm et
al. 1956; Reiter et al. 1999), whereas the adminis-
tration of the pineal hormone melatonin to rodents or
syngeneic transplantation of pineal glands from young
donors into the thymus or in situ of old mice prolonged
the life span of the recipients (Pierpaoli and Regelson
1994; Lesnikov and Pierpaoli 1994; Anisimov et al.
2001b; Oxenkrug et al. 2001). Most investigators
invoked melatonin as a primary mediator of the endo-
crine capabilities of the pineal gland. However, some
of the effects of the pineal gland might have obvi-
ously resulted from pineal peptide secretion (Benson
1977; Bartsch et al. 1992; Yuwiler and Brammer
1993; Arendt 1995). Some crude peptide extracts or
purified peptides isolated from pineal glands were
shown to have antigonadotropic, metabolic and anti-
194
tumor activity (Anisimov et al. 1994; Bartsch et al.
1992; Lapin and Ebels 1979). One of the complex
peptide bioregulators isolated from the pineal gland,
Epithalamin
, was shown to slow down aging rate,
prolong life span in fruit flies, mice and rats, and
inhibit spontaneous and induced carcinogenesis in
rodents (Anisimov et al. 1994; Khavinson et al. 2001c;
Khavinson 2002).
Tetrapeptide Epitalon
(Ala-Glu-Asp-Gly, mole-
cular weight 390.35 dalton) was designed on the
basis of Epithalamin amino acid analysis and synthe-
sized (Khavinson et al. 2000). The geroprotective
activity of Epitalon was studied in three strains of
Drosophila melanogaster (Khavinson et al. 2000;
Mylnikov and Lyubimova 2000). Epitalon increased
the life span of imagoes significantly by 11–16%
when applied at unprecedentedly low concentrations
– from 0.001 × 10
−6
to 5 × 10
−6
wt% of the cul-
ture medium. A recent study by us demonstrated a
geroprotective effect of long-term Epitalon adminis-
tration in female inbred CBA mice (Anisimov et al.
2001a). The bioregulator slowed down aging of the
reproductive function, inhibited free radical processes,
and decreased total spontaneous tumor incidence in
female CBA mice (Anisimov et al. 2001a). Epitalon
inhibited mammary carcinogenesis and metastasis in
transgenic HER-2/neu mice (Anisimov et al. 2002b)
and colon and small intestine carcinogenesis induced
by 1,2-dimethylhydrazine in rats (Anisimov et al.
2002a). Administration of Epitalon to young (6–8
years old) and senescent (20–26 years old) female
monkeys Macaca mula tta restored the evening level
of melatonin and the circadian rhythm of cortisol in
the blood serum of senescent monkeys (Khavinson et
al. 2001a).
This paper presents data on the effect of Epitalon
on life span, estrous function, incidence of chromo-
some aberration in the bone marrow cells and spon-
taneous tumorigenesis in outbred Swiss-derived SHR
mice.
Materials and methods
Animals
Female outbred Swiss-derived SHR 2-month-old mice
(108 specimens) were purchased from the Rappolovo
Animal Farm of the Russian Academy of Medical
Sciences (St. Petersburg). The mice were kept in
polypropylene cages (30 × 21 × 9 cm), 5 mice to a
cage, at a temperature of 22 ± 2
◦
C. A regimen was
followed of 12 hours of light and 12 hours of dark.
The animals received sterilized standard laboratory
feed (Anisimov et al. 2003) and tap water ad libitum.
Mice were checked daily by animal care personnel and
weekly by a veterinarian. The study was conducted
in accordance with the regulations for ensuring the
humane treatment of animals under the approval of
the Committee on Animal Research of the N.N. Petrov
Research Institute of Oncology.
Experiment
At the age of 3 months the mice were randomly
divided into two groups, 54 animals in each, and they
were individually marked. Mice of the control group
were subcutaneously injected with 0.1 ml of 0.9%
normal saline for 5 consecutive days every month,
whereas the mice of the second group received subcu-
taneously 1.0 µg of Epitalon dissolved in 0.1 ml
of saline. This treatment dosage and regimen were
effective for the inhibition of spontaneous tumorigen-
esis in female CBA mice (Anisimov et al. 2001a).
Epitalon was synthesised in St. Petersburg, Institute
of Bioregulation and Gerontology, by E.I. Grigoriev
and was 99.8% pure. Four intact female SHR mice
were euthanized at the age of three months to evaluate
the initial level of chromosome aberrations. Addition-
ally four mice from each group were euthanized at the
age of 12 months for a cytogenetic study of chromo-
some aberrations in bone marrow cells (see below).
Once every 3 months, simultaneously with weighing,
the amount of food consumed was measured. Thirty
grams of food were given in each cage after cleaning
and twenty-four hours thereafter the food that had
not been consumed was collected from each cage and
weighed. The mean amount of food (grams) consumed
per mouse during this day was calculated for each
group.
Once every three months, vaginal smears taken
daily for two weeks from the animals were examined
cytologically to estimate the phases of their estrous
functions. In the same period, the rectal body tempera-
tures of the mice were measured with an electronic
thermometer, TPEM (KMIZ, Russia). Animals were
observed until their natural death. The date of each
death was recorded, and the mean life span, the age by
which 90% of the animals died, and the maximum life
span were estimated.
195
Cytogenetic study
Chromosomal aberrations in bone marrow cells were
studied by a modified Ford’s method, described by
Rosenfeld et al. (2001). Mice were sacrificed by
ether anaesthesia. Both femurs of each mouse were
dissected and bone marrow cells were flushed gently
with 0.56% KCl solution into a centrifuge tube. Cells
were treated for 20 min with hypotonic solution and
fixed with an ethanol : acetic acid mixture (3:1). Slides
were stained with 4% acetoorseine: 20–30 well spread
anaphases were analyzed for each animal and cells
with chromosome breaks, acentric fragments, and
other aberrations were evaluated at 1000× magnifi-
cation under a light microscope (Leitz, Germany).
Pathomorphological examination
All animals that died, or were sacrificed when
moribund, were autopsied and their skin and internal
organs were examined. Neoplasias were classified
according to the recommendations of the International
Agency of Research on Cancer (IARC) as ‘fatal’ (i.e.,
those that directly caused the death of the animal)
or ‘incidental’ (in cases where the animal died of a
different cause) (Gart et al. 1986). All tumors, as
well as tissues and organs with suspected tumors,
were excised and fixed in 10% neutral formalin.
After routine histological processing, tissues were
embedded in paraffin. Thin, 5–7 µm histological
sections were stained with hematoxylin-eosine and
examined microscopically. The experimental group
to which the mouse belonged was blinded. Tumors
were classified according to IARC recommendations
(Turusov and Mohr 1994).
Statistics
Experimental results were statistically processed
by the methods of variation statistics (Goubler
1978). The significance of discrepancies was defined
according to Student’s t-criterion, Fischer’s exact
method, χ
2
-analysis, and the non-parametric criterion
of Wilcoxon–Mann–Whitney (Goubler 1978). To
estimate discrepancies in neoplasm incidence, an
IARC method of combined contingency tables calcu-
lated individually for the fatal and incidental tumors
(Gart et al. 1986). For survival analysis, Cox’s method
(Cox and Oakes 1996) was used. All reported test
values for survival analyses are two-sided.
Survival models and estimations
The mathematical model used to describe survival is
the Gompertz model with the survival function:
S(x) = exp
−
β
α
exp(αx) − 1
where parameters α and β are associated with demo-
graphic aging and initial mortality rate, respectively.
Parameters for the model were estimated from data
using the maximum likelihood method implemented
in the GAUSS statistical system (Gauss System 1994).
Confidence intervals for the aging rate parameter
estimates were calculated using log-likelihood func-
tions (Cox and Oakes 1994).
Results
Age-related body weight dynamics
Mean values of body weight for mice at different ages
in the control and treated with Epitalon groups are
displayed in Table 1. The body weight of the mice
in both groups increased with age, exceeding by 13
months the body weight of 3-month-old animals by
44.5% in the control group (P < 0.001), and by 48.5%
in the group given Epitalon (P < 0.01). There were no
differences in the body weight between groups at any
period of observation.
Age-related dynamics of food consumption
Measurements showed that the amount of food
consumed by the mice in the control (saline) group
was practically stable from the age of 5 months to the
age of 16 months, increasing slightly at the age of 18
months. Mice treated with Epitalon consumed more
food from the 5th to the 16th months of their life than
the control group (Table 2).
Age-related dynamics of estrous function in mice
The estrous function in the animals of both age groups
was examined every three months, starting when the
mice were three months old. The following para-
meters of estrous function were estimated: the length
of the estrus, the relative rate of estrous cycle phases
(in percent); and the relative number of short (< 5
days) and long (> 5 days) estrous cycles. The relative
number of animals with regular cycles and irregular
196
Table 1. Body weight gain dynamics in female SHR mice treated with saline or Epitalon
Group Body weight (g)
3 mo 5 mo 7 mo 9 mo 11 mo 13 mo 16 mo 18 mo
Saline 24.7 ± 0.29 27.8 ± 0.63 30.9 ± 0.85 32.4 ± 1.30 34.3 ± 1.24 35.7 ± 1.67 34.0 ± 1.73 32.4 ± 1.50
Epitalon 24.1 ± 0.41 28.1 ± 0.40 30.1 ± 0.74 33.2 ± 1.04 34.4 ± 1.08 35.8 ± 1.68 33.9 ± 1.67 30.7 ± 1.49
Table 2. Food consumption dynamics in female SHR mice treated with saline or Epitalin
Group Daily food consumption (g/mouse)
3 mo 5 mo 7 mo 9 mo 11 mo 13 mo 16 mo 18 mo
Saline 4.6 ± 0.03 2.8 ± 0.05 3.3 ± 0.02 2.8 ± 0.03 3.1 ± 0.01 3.4 ± 0.03 3.8 ± 0.06 5.3 ± 0.15
Epitalon 4.3 ± 0.17 4.9 ± 0.15
∗
5.4 ± 0.09
∗
5.6 ± 0.13
∗
5.6 ± 0.22
∗
5.7 ± 0.24
∗
5.9 ± 0.32
∗
5.8 ± 0.47
The difference from the saline group is significant.
∗
– P < 0.001 (Student’s t-test).
cycles (persistent estrus and anestrus) were also calcu-
lated. Judging by the data presented in Table 3, the
length of estrous cycle in the control female SHR mice
increased with advancing age (P < 0.05; Student’s t-
test). Thus, no essential age-related alterations in the
rate of estrous cycle phases were observed. However,
the relative number of short estrous cycles decreased
significantly with age (37.1% at the age of 3 months,
9.4% at the age of 12 months (P < 0.05; Fischer’s
exact test) and zero at the age of 15 months, whereas
the number of long cycles rose (5.1% at the age of 6
months and 36% at the age of 15 months, P < 0.05;
Fischer’s exact test).
In the group of mice exposed to Epitalon the length
of estrous cycles did not change with the age of the
animals and decreased in comparison with the age-
matched controls at the age of 15 months (P < 0.05).
There was no significant age-related decrease in the
number of short cycles, or an increase in the number of
long cycles. The number of mice with regular cycles
did not change significantly with age in both groups
(Table 3).
Age-related dynamics of body temperature in mice
Data on body temperature alterations in the mice
exposed to saline or Epitalon are presented in Table 4.
The control mice and mice treated with Epitalon
revealed a significant decrease in body temperature
with age, both on the whole (irrespective of the estrous
cycle phases) and in any of the phases. No cyclic
alterations in rectal body temperature during the estrus
cycle were observed in mice of the control group, but
the temperature at diestrus was significantly higher
than that in estrus in mice treated with Epitalon at
the age of 15 months (P < 0.05). It should be noted
that the average body temperature in the mice treated
with Epitalon was not significantly different from the
control mice during the entire period of observation
(Table 4).
Chromosome aberrations in mouse bone marrow cells
The incidence of chromosome aberrations in bone
marrow cells of 3-month-old female SHR was 2.1
± 0.29%. At the age of 12 months this parameter
increased to 8.2 ± 0.41% (P < 0.001; Wilcoxon–
Mann–Whitney test) in the group injected with saline.
In mice treated from the age of 3 months with Epitalon
the incidence of chromosome aberrations at the age of
12 months was 6.8 ± 0.21 (– 17.1%; P < 0.05).
Survival and longevity of female SHR mice
Survival dynamics in the mice treated with either
saline or DSIP are demonstrated in Table 5 and
Figure 1. The survival dynamics were in general
similar in all groups up to the age of 22 months.
However, thereafter, the number of survivors was
much higher in Epitalon-treated groups.
The last mouse in the control group died at the
age of 739 days (24.3 months), whereas in the groups
treated with Epitalon 12% of mice survived to this
age, and the maximum life span was 830 days (27.3
months, + 12.3%). The mean life span of mice
treated with Epitalon did not change as compared with
controls. However, the life span in the last 10% of the
197
Table 3. Age-related dynamics of estrous functional parameters in SHR mice treated with saline or Epitalon
Age No. Length of Rate of separate phases Rate of estrous cylces (%) Rate of Rate of
(mo) of estrous cycle of estrous cylce (%) mice with mice with
mice (days) E D P + M < 5 d 5–7 d > 7 d regular irregular
cycles (%) cycles (%)
Saline
3285.68± 0.19 42.4 54.7 2.9 31.7 55.0 13.3 88.0 12.0
6225.38± 0.19 45.4 50.5 4.1 30.8 64.1 5.1 95.5 4.5
9225.77± 0.30 34.2 62.4 3.4 25.6 59.0 15.4 95.5 4.5
12 22 6.25 ± 0.26 47.1 51.3 1.6 9.4
∗∗
81.2 9.4 95.5 4.5
15 17 6.84 ± 0.26
∗
34.2 65.1 0.7 0
∗∗
64.0 36.0
∗∗
88.2 11.8
Epitalon
3505.87± 0.24 42.8 53.1 4.1 29.1 47.3 23.6 100 0
6485.81± 0.23 46.5 48.9 4.6 16.2 75.7 8.1 91.4 8.6
9365.33± 0.21 51.5 46.1 2.4 35.7 54.8 9.5 95.5 4.5
12 20 5.96 ± 0.34 48.9 49.2 1.9 22.2 59.3 18.5 94.7 5.3
15 15 5.87 ± 0.33
a
43.3 54.4 2.3 10.7 78.6 10.7 85.7 14.3
Note: E = estrus; D = diestrus; P = proestrus; M = metaestrus.
Difference from the parameters at the age of 6 months in the same group:
∗
P < 0.05 (Student’s t-test);
∗∗
P < 0.05 (Fischer’s exact test).
Difference from the corresponding age in the control group:
a
P < 0.05 (Student’s t-test).
Table 4. Body temperature dynamics in SHR mice treated with saline or Epitalon
Age (mo) Number Total cycle Mean body temperature (
◦
C)
of mice (without phase Estrus Diestrus Metaestrus +
sub-division) proestrus
Saline
7 22 39.95 ± 0.14 39.80 ± 0.30 40.00 ± 0.20 39.40 ± 0.10
12 22 38.83 ± 0.18
a
38.73 ± 0.30
b
38.90 ± 0.24
a
–
15 17 38.73 ± 0.17
a
38.68 ± 0.30
b
38.75 ± 0.20
a
–
17 12 38.05 ± 0.24
a
37.40 38.10 ± 0.30
a
–
19 10 37.70 ± 0.11
a
37.60 ± 0.10
a
37.70 ± 0.14
a
–
Epitalon
7 23 40.68 ± 0.16 40.60 ± 0.20 40.40 ± 0.40 40.96 ± 0.20
12 20 38.90 ± 0.22
a
39.07 ± 0.30
a
38.90 ± 0.30
a
–
15 15 39.07 ± 0.17
a
38.20 ± 0.10
a
39.10 ± 0.20
b,c
17 12 38.28 ± 0.21
a
38.75 ± 0.30
a
38.10 ± 0.30
a
19 10 37.32 ± 0.14
a
37.10 37.40 ± 0.15
a
–
Difference from the age of 7 months in the same group is significant:
a
P < 0.01;
b
P < 0.05 (Student’s t-test).
Difference with the parameter at phase estrus of the same group and age:
c
P < 0.05 (Student’s t-test).
Table 5. Survival distribution of female SHR mice treated with saline or Epitalon
Group No. of survivors at the age of:
3 4 6 8 10 12 14 16 18 20 22 24 26 27 28
mo mo mo mo mo mo mo mo mo mo mo mo mo mo mo
Saline 50503836363433282016102 0 0 0
Epitalon50493936343332251714117 3 3 0
198
Figure 1. Effect of Epitalon on the survival curves of female SHR mice. Y-axis: number of mice as percentage of total.
Table 6. Parameters of life span in female SHR mice treated with
saline or Epitalon
Parameters Saline Epitalon
Number of mice 50 50
Mean life span, days (M ± S.E.) 456 ± 29 455 ± 31
Median 512 487
Mean life span of last 709 ± 10.8 803 ± 15.0
∗
10% of survivors, days (+ 13.3%)
Maximum life span, days 739 830
(+ 12.3%)
Differences from saline is significant:
∗
P < 0.01 (Student’s t-test).
mice increased for the duration of Epitalon treatment
by 3.1 months (+ 13.3%, P < 0.01; Student’s t-test)
(Table 6).
Spontaneous tumor development in female S HR mice
The total tumor incidence in the control female
mice was 36%. Mammary carcinomas and leukemias
developed most frequently, corresponding to the onco-
logical characteristics of female SHR mice (Anisimov
et al. 1989). The treatment with Epitalon failed to
influence the total or malignant tumor incidence in
comparison with that of the control group. However,
the incidence of leukemias during the treatment with
Epitalon decreased 6-fold (P < 0.01; Fischer’s exact
test). There was no significant difference in the incid-
ence of any other tumors between the group of mice
treated with the peptide and saline (Table 7). The treat-
ment with Epitalon significantly shifted to right the
total tumor yield curve as compared with the control
group (Figure 2).
Mathematical model and estimations of survival of
tumor-free and tumor- bearing mice
A mathematical analysis of the survival data of the
mice from the control and melatonin- treated groups
has been done separately for three different contexts:
(1) for all animals in each group (total cases); (2) for
fatal tumor-bearing mice, and (3) for fatal tumor-free
mice. We composed the groups of animals without
consideration of possible effects caused by depend-
ence between these groups. The Gompertz model
shows a slowdown (by 29.0.4%) of the population
aging rate (calculated as α in the Gompertz equa-
tion) and a corresponding increase in MRTD under
the influence of Epitalon. The mortality rate in the
group of fatal tumor-free mice treated with Epithalon
was decreased by 32.5% as compared with the controls
(P < 0.05, Table 8).
199
Table 7. Incidence, localization and type of tumors in female SHR mice treated and not
treated with Epitalon
Parameters Saline Epitalon
Number of mice 50 50
Number of tumor-bearing mice 18 (36%) 16 (32%)
Number of malignant tumor-bearing mice 15 (30%) 16 (32%)
Total number of tumors 25 22
Total number of malignant tumors 18 20
Number of tumors per tumor-bearing mice 1.38 1.38
Mean life span of fatal-tumor bearing mice, days 549 ± 29 551 ± 21
Mean life span of fatal tumor-free animals, days 416 ± 38 410 ± 43
Localization and type of tumors:
Mammary gland: adenocarcinoma 12 (10)
a
17 (13)
b
Nos. of metastases 7 6
Leukemia 6 1
∗
Lung: adenoma 1 0
Adenocarcinoma 0 2
Utery: polyp 1 1
Ovary: cyst 5 1
a
Two mice had two mammary tumors each.
b
Four mice had two mammary tumors each.
Differences from saline is significant:
∗
P < 0.05 (Fischer’s exact test).
Figure 2. Effect of Epitalon on total tumor yield curve in female SHR mice. Y-axis: number of tumor-bearing mice as percentage of total.
200
Table 8. Parameters of life span in female SHR mice treated with saline or Epitalon
Group Total no. of cases Fatal tumor-bearing mice Fatal tumor-free mice
Number of mice
Saline 50 15 35
Epitalon 50 16 34
Mean life span (days)
Saline 456 ± 29 549 ± 29 416 ± 38
Epitalon 455 ± 31 551 ± 21 410 ± 43
Mean life span of the last 10% of survivors (days)
Saline 709 ± 10 731 ± 8 692 ± 7
Epitalon 803 ± 15
∗
695 ± 16 827 ± 2
∗∗
Aging rate α × 10
3
(days
−1
)
Saline 4.55 (4.43; 4.86) 9.15 (9.02; 10.4) 2.80 (2.74; 3.20)
Epitalon 3.23 (3.15; 3.46)# 11.8 (11.3; 11.35)# 1.89 (1.82; 2.16)#
MRDT (days)
Saline 152.41 75.71 247.71
Epitalon 214.69# 58.91# 367.1#
Note: Mean life spans are given as mean ± standard error; 95% confidence limits are given in paren-
theses; MRDT = mortality rate doubling time.
Difference from controls is significant:
a
P < 0.05 (Fischer’s exact test);
∗
P < 0.05;
∗∗
P < 0.001
(Student’s t-test);
#
P 0.05 (Cox’s method).
Discussion
The results of our study show that the long-term
administration of Epitalon slows down demographic
aging rate, increases survival and maximum life span
and decreases the development of spontaneous leuke-
mias in female SHR mice.
Treatment with Epitalon increased the food con-
sumption in comparison to the controls between the
5th and 16th months of their life, but the body
weight was similar in both the control and Epitalon-
treated group. Administration of Epitalon to female
CBA mice failed to influence the body weight, and
increased food consumption at the age of 12 months
(Anisimov et al. 2001a). No significant differences
in the age-related dynamics of body temperature
between both groups were observed in SHR mice. In
CBA mice a slight decrease in the body temperature
under the influence of Epitalon has been observed
(Anisimov et al. 2001a). Thus, it could be suggested
that increased food consumption induced by Epitalon
was not followed by an increase in the basal meta-
bolic rate, because both the body weight and the body
temperature were not different from the controls.
The administration of Epitalon was followed by
a slowing down of the age-related disturbances in
estrous function in female SHR mice. These obser-
vations are in agreement with data obtained with
Epitalon in other strains of mice – CBA and FVB/N
(Anisimov et al. 2001a, 2002b). It is worth nothing
that long-term administration of melatonin or pineal
peptide preparation Epithalamin was also followed
by a slowdown of age-related switching-off of repro-
ductive function in SHR, C3H/Sn and CBA mice and
rats (Anisimov et al. 1989, 1998, 2001a, b; Meredith
et al. 2000).
The aging process predisposes cells to accumulate
mutations, some of which are necessary for initia-
tion of tumor growth in target tissues (Vijg 2000;
Bodyak et al. 2002). The incidence of chromosome
aberrations increases with age in different strains of
mice (Crowley and Curtis 1963; Sato et al. 1995).
Previously we found age-related increases in chro-
mosome aberrations in bone marrow cells and in
primary spermatocytes in male SHR mice (Rosenfeld
et al. 2001). In this study we observed a significant
increase in the frequency of chromosome aberrations
in the bone marrow cells in 12-month-old female SHR
mice compared to 3-month-old specimens. Long-term
treatment with Epitalon significantly decreased the
age-associated increase in chromosome aberrations in
female SHR mice. It was shown earlier that Epitalon
201
also inhibited the incidence of chromosome aberration
in one-year-old senescence-accelerated mice (Rosen-
feld et al. 2002).
The long-term administration of Epitalon failed to
influence total spontaneous tumor incidence in female
SHR mice, but it significantly inhibited the develop-
ment of leukemias (P < 0.001) (Table 7). Treatment
with Epitalon inhibited the growth of transplanted
sarcoma M-1 (Khavinson et al. 2001b), decreased
spontaneous tumor incidence in female CBA mice
(mainly lung adenomas) (Anisimov et al. 2001a)
and in HER-2/neu transgenic mice (Anisimov et al.
2002b). It also inhibited colon carcinogenesis induced
by 1,2-dimethylhydtrazine in rats (Anisimov et al.
2002a). It possible to suggest that the capacity of
Epitalon to prevent the development of spontaneous
leukemias in female SHR mice can be related to its
antioxidative activity. It has been shown that Epitalon
inhibits free radical processes in D. melanogaster and
CBA mice (Anisimov et al. 2001a; Khavinson and
Mylnikov 2000).
Our observation of the positive effect of Epitalon
on the life span of SHR mice is in agreement with
observations of similar activities of Epithalamin in
SHR and C3H/Sn mice and rats (Anisimov et al. 1989,
1994) and of Epitalon in female CBA mice (Anisimov
et al. 2001a). The effective concentration of Epitalon
was 1000–5000 times less than that of Epithalamin.
In experimens with two strains of D. melanogaster,
Epithalon treatment was followed by an increase in
their mean life span (Khavinson and Mylnikov 2000).
It is noteworthy that effective concentrations of the
tetrapeptide were 1000 times less than of Epitha-
lamin and 16,000–80 × 10
6
times less than that of
melatonin (Khavinson and Mylnikov 2000). Epithalon
increased catalase activity and decreased the level of
conjugated hydroperoxides in fruit flies (Mylnikov
and Lyubimova 2000). The results of this investiga-
tion agree with data obtained in previous observations
on the safety of long-term administration of peptide
preparations isolated from the pineal gland on their
geroprotective and anti-tumour effects (Anisimov et
al. 1994; Khavinson et al. 2001c; Khavinson 2002).
Thus, the results obtained confirm a geroprotective
potential of the peptide preparation Epithalon (Ala-
Glu-Asp-Gly).
In the heart of CBA mice, the expression of 15,247
transcripts from the cDNA library was studied by the
microarray technique (Anisimov S et al. 2002). The
analysis of the results of hybridizing the cDNA clone
containing microarrays with the heart samples of the
control and Epitalon-exposed mice revealed intensi-
fied expression of 194 clones and reduced expression
of 48 clones. The analysis identified the multiple genes
involved in cell division (14 genes), cell signaling/
communication (14), cell structure/motility (6), cell/
organism defense (13), gene/protein expression (17),
metabolism (11), and genes encoded by mitochon-
drial DNA (5). These subgroups may include gene
products that can explain some of the physiological
effects described above, and form a molecular basis
for the geroprotective effects of these peptides.
Acknowledgements
This study was supported by grants 99-04-48023 and
02-04-07573 from the Russian Foundation for Basic
Research. The authors are very grateful to James W.
Vaupel for the opportunity to use the facilities of the
Max Planck Institute for Demographic Research to
complete this paper, and to I. I. Mikhailova and O.V.
Novikova for their excellent technical assistance.
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