Full text of DOI:10.1093/gerona/56.7.B311

Journal of Gerontology: BIOLOGICAL SCIENCES
Copyright 2001 by The Gerontological Society of America
2001, Vol. 56A, No. 7, B311–B323
Melatonin Increases Both Life Span and Tumor
Incidence in Female CBA Mice
Vladimir N. Anisimov,1 Natalia Y. Zavarzina,1 Mark A. Zabezhinski,1 Irina G. Popovich,1
Olga A. Zimina,3 Anastasia V. Shtylick,2 Alexander V. Arutjunyan,3 Tatiana I. Oparina,3
Valentina M. Prokopenko,3 Anatoli I. Mikhalski,4 and Anatoli I. Yashin5
1N. N. Petrov Research Institute of Oncology, 2St. Petersburg State University, and 3D. O. Ott Research Institute of Obstetrics
and Gynecology, St. Petersburg, Russia.
4Institute of Control Sciences of the Russian Academy of Sciences, Moscow, Russia.
5Max-Planck Institute for Demographic Research, Rostock, Germany.
From the age of 6 months until their natural deaths, female CBA mice were given melatonin
with their drinking water (20 mg/l) for 5 consecutive days every month. Intact mice served as
controls. The results of this study show that the consumption of melatonin did not significantly
influence food consumption, but it did increase the body weight of older mice; it did not influ-
ence physical strength or the presence of fatigue; it decreased locomotor activity and body tem-
perature; it inhibited free radical processes in serum, brain, and liver; it slowed down the age-
related switching-off of estrous function; and it increased life span. However, we also found that
treatment with the used dose of melatonin increased spontaneous tumor incidence in mice. For
this reason, we concluded that it would be premature to recommend melatonin as a geroprotec-
tor for long-term use.
HE search for an effective drug that prevents premature
aging and increases active life span is an important di-
At the same time, in several studies, melatonin failed to
show any effect on life span (18,24,30,31). Moreover, long-
term treatment with melatonin was followed by an increase
of tumor incidence in some mouse strains (18,31,32).
T
rection of research in modern gerontology (1,2). The diffi-
culties of this search are related to the lack of theoretical
background as well as to insufficient information regarding
the safety and adverse effects of existing geroprotectors.
Some of these geroprotectors, for example, antioxidants,
chelating agents, and growth hormone, which are capable of
increasing the average life span in animals, can promote
cancer development or suppress the reproductive function
under certain conditions (3,4).
During the past decade, a number of reports, sometimes
contradictory, appeared concerning the role of the pineal
gland in aging (5–10). Melatonin (N-acetyl-5-methoxy-
triptamine) is the main pineal hormone synthesized from
tryptophan, predominantly at night time (11). It has a wide
spectrum of physiological effects on endocrine and repro-
ductive functions (8,11,12). As age advances, the nocturnal
production of melatonin decreases in animals of various
species, including humans (8,13,14). The performance of a
pinealectomy on rats produced a reduced life span (15,16),
whereas the administration of melatonin to mice, rats, fruit
flies, or planaria led in some cases to life extension (17–24).
The grafting of a pineal gland from young donors into the
thymus of old mice or in situ into pinealectomized old mice
prolonged the life span of the recipients (18,25). Many stud-
ies show that melatonin inhibits tumor growth both in vivo
and in vitro (26,27,28). The interest in all these observations
was significantly increased after the discovery of the antiox-
idant activity of melatonin, both in vitro and in vivo (8,29).
There are strong critical comments on the antiaging effects
of melatonin in mice. These comments mainly relate to the
observation that murine strains used in some studies do not
synthesize melatonin as a result of a genetic defect (BALB/c,
NZB, and C57BL/6). In fact, a shortening of the life span
was observed in melatonin-producing mouse strain C3H/He
(9,33). Later it was shown that the pineal gland did produce
melatonin in the above-mentioned mouse strains with genetic
defects, but the production night peak was very short, so its
presence was difficult to detect (34). At the same time, a crit-
ical review of available data on the effect of melatonin on the
life span and tumor incidence in rodents has shown that prac-
tically all studies are invalid from the point of view of the cur-
rent guidelines for long-term testing of chemicals for carcino-
genic safety (35,36) and, to some extent, from the point of
view of the correctness of the gerontological experiment (37).
Risk assessment is a set of decision rules widely used in
the United States and other countries for identifying and
quantifying the risk of chemicals, causing adverse effects in
humans, primarily cancer (38). According to the Interna-
tional Agency for Research on Cancer and the U.S. National
Toxicology Program, exposure must start a few weeks after
animals are weaned, and surviving animals must be sacri-
ficed at 18 months (mice and hamsters) or at 24 months
(rats). Some problems inherent to this protocol are currently
under discussion (39–42). The 2-year assay is significant for
B311

B312
ANISIMOV ET AL.
observation of the carcinogenic potential of the majority of
agents. However, in some cases, if animals have been ex-
posed to a tested agent from a young age and are sacrificed
at the age of 2 years, an underestimation of the carcinogenic
potential of nongenotoxic agents and tumor promoters is
possible (43). It may be important to reevaluate standard
2-year protocols for the long-term assay for carcinogenicity,
and it may be recommended to keep animals until their nat-
ural deaths. Although this is not profitable from an econom-
ical point of view, it is more reliable scientifically (1,43),
particularly for studies that evaluate the life extension po-
tential of pharmacological interventions (1,37,43).
Often in experiments with rodents, melatonin is given to
a small number of animals (10–20); the treatments start
when the animals are old; the observations stop when 50%
of the animals have died or at some other voluntary time,
but not at the natural death of the last survivor; an autopsy
and a correct pathomorphological examination sometimes
are not performed; the body weight gain and food consump-
tion of the animals are not monitored; and so on.
In this paper we present the results of a study of the ef-
fects of melatonin on life span and spontaneous tumor inci-
dence. The study was designed to avoid the drawbacks and
limitations mentioned above. Some additional biomarkers
of aging (estrous function, body temperature, locomotor ac-
tivity, physical strength, and fatigue) were monitored as
well. The effect of melatonin on free radical processes was
also evaluated in the same study. Female CBA mice were
selected for the study because of unequivocal evidence of
pineal melatonin production in this mouse strain (33).
vidually marked. Mice of one group were given melatonin
(Sigma, St. Louis, MS) with tap water (20 mg/l) during the
night (from 6 PM to 9 AM), 5 days a week each month, until
their natural deaths. This dose of melatonin has been used
for life extension and carcinogenesis inhibition in several
studies (19,27). Melatonin was dissolved in several drops of
96% ethanol and diluted with sterile tap water to a relevant
concentration. A fresh melatonin solution was prepared
each third day. All bottles containing melatonin were made
from dark glass. The other 60 mice were kept intact and
served as a control group.
Ten mice from each group were euthanized in 24 hours
after the first course of melatonin administration for bio-
chemical study. Their blood sera, livers, and brains were
immediately frozen with liquid nitrogen and kept at ꢃ20ꢂC.
The remainder of the animals were weighed monthly with
an electronic balance. For the animals of each group, a
mean body weight and its standard errors, as well as the
slope of the linear regression of age-related body weight
gain and its standard errors, were defined. Additionally, the
mice were divided into 3 classes: lean (ꢄ28 g), medium,
(29–33 g) and fat (ꢅ34 g). The mice in each group that were
operationally defined as fat, medium, and lean were defined
on the 6th, 12th, and 15th months of the experiment. Once
every 3 months, simultaneously with weighing, the amount
of drinking water and consumed food was measured and the
rate of consumed tap water (milliliters) and food (grams)
per mouse and per body weight unit was calculated.
Once every 3 months, vaginal smears taken daily for 2
weeks from the animals were cytologically examined to esti-
mate the phases of their estrous functions. In the same period,
rectal body temperatures of the mice were measured with an
electronic thermometer, TPEM (KMIZ, Russia), and the
physical activity of each mouse was estimated. After 12
months of treatment, an assessment of muscular strength and
fatigability was performed as well. The animals were ob-
served until their natural deaths. The date of each death was
registered, and the mean life span, the age at which 90% of
the animals died, and the maximal life span were determined.
METHODS
Animals
One hundred twenty female CBA 4-month-old mice 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 ꢁ 2ꢂC. A regimen of 12
hours of light and 12 hours of dark was followed. The ani-
mals received sterilized standard laboratory chow (44) and
tap water ad libitum. Mice were checked daily by animal
care personnel and weekly by a veterinarian. The study was
carried out 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 Re-
search Institute of Oncology.
Method of estimating physical activity of mice in the
“open field” test.—Animals of each group were placed one
by one in a plastic chamber measuring 30 ꢀ 21 ꢀ 9 cm, at
the bottom of which squares (5 ꢀ 5 cm) were drawn: 5
squares in length and 4 squares in breadth. Each mouse was
observed moving in the cage, and its locomotor parameters
were estimated: (1) the number of crossed squares in the
field (a square was considered crossed if the animal stepped
over its border at least with 2 paws); (2) the number of verti-
cal sets (when the animal rose to its hind paws); and (3) the
duration of grooming reaction of muzzle, body, and genita-
Experiment
At the age of 6 months, all the mice were randomly di-
vided into 2 groups, 60 animals in each, and they were indi-
Table 1. Body Weight Gain Dynamics in Female CBA Mice Treated With Melatonin
Body Weight (g)
Group
6 mo
9 mo
12 mo
15 mo
18 mo
21 mo
Control
Melatonin
21.4 ꢁ 0.25
20.9 ꢁ 0.66
24.5 ꢁ 0.32
24.5 ꢁ 0.29
29.0 ꢁ 0.56
29.6 ꢁ 0.43
28.9 ꢁ 0.60
30.4 ꢁ 0.47*
29.7 ꢁ 0.78
32.2 ꢁ 0.66*
30.8 ꢁ 1.18
32.7 ꢁ 0.77
*The difference with controls is significant: p ꢆ .05.

MELATONIN INCREASES LIFE AND TUMORIGENESIS
B313
Table 2. The Distribution of Mice According to the Body
Weight in Groups Treated and Not Treated With Melatonin
blind process. Tumors were classified in accordance with
IARC recommendations (45).
Number of Mice in Body Weight Classes (%)
Biochemical Tests
Lean
Medium
Fat
The generation of reactive oxygen species (ROS) was
studied in brain and liver homogenates and in blood serum
according to the method of peroxide luminol-dependent
chemiluminescence (46) on an Emilite EL-1105 chemilumi-
nometer (BioChimMac, Moscow, Russia). The intensity of
peroxide lipid oxidation was estimated by the content of di-
ene conjugates and Schiff’s bases (47). Total antioxidant ac-
tivity (AOA) of the tissues was evaluated by the method of
riboflavin chemoluminescence registration (48). Simulta-
neously, the activity of Cu-, Zn-superoxide dismutase
(SOD) was estimated by means of suppressing Nitro Blue
Tetrasodium with biological material (49).
Group
(ꢄ28 g)
(29–33 g)
(ꢅ34 g)
At the Age of 12 Mo
Control
Melatonin
At the Age of 18 Mo
Control
Melatonin
At the Age of 21 Mo
Control
38
24.5
54
59.2
8
16.3
38
21.7
58
60.9
4
17.4
25
11.9
67
54.8
8
Melatonin
33.4*
*The difference with controls is significant: p ꢆ .05.
lia. As a way to exclude the possibility of smell-associated
orientation reaction, the chamber floor was wiped with a wet
cloth after each animal. The mice were tested at the age of 6,
9, 12, and 18 months in the daytime from 10 AM to 5 PM.
Statistics
Experimental results were statistically processed by the
method of variation statistics (50). Parameters of the regres-
sion equation for the curves of age-related body weight dy-
namics were calculated. The significance of the discrepan-
cies was defined according to Student’s t criterion, Fischer’s
exact method, a chi-square analysis, and a nonparametric
criterion of Wilcoxon-Mann-Whitney (50). For discrepan-
cies in neoplasm incidence to be estimated, an IARC method
of combined contingency tables calculated individually for
the fatal and incidental tumors (35) as well as a prevalence
analysis (51,52) were applied. For survival analysis, Cox’s
method (53) was used for testing two groups’ equality, and
Tarone’s (54) life table test was used. All reported p values
for the survival analyses are two sided.
Method of studying muscular strength and physical
fatigability of the mice.—The mice were suspended on a
string stretched to an altitude of 80 cm, so that they would
hang by the string, clutching at it with their front paws. The
time until the moment of their fatigue and fall was regis-
tered in seconds. In 20 minutes the mice were suspended
again and the time for which they managed to hold on was
measured. A discrepancy between these 2 indices was re-
garded as a parameter of physical restoration.
Pathomorphological Examination
All the animals that died or that were sacrificed when
moribund were autopsied. At autopsy their skin and all the
internal organs were examined. Revealed neoplasia were
classified according to the recommendations of the Interna-
tional Agency of Research on Cancer (IARC) as “fatal”
(i.e., those that directly caused the death of the animal) or as
“incidental” (for the cases in which the animal died of a dif-
ferent cause) (35). All the tumors, as well as the tissues and
organs with suspected tumor development, were excised
and fixed in 10% neutral formalin. After routine histological
processing, the tissues were embedded in paraffin. Thin, 5–7
ꢇm histological sections were stained with hematoxylin-
eosine and were microscopically examined; regarding the
experimental group to which the mice belonged, this was a
Mathematical Models and Estimations
Two mathematical models were used to describe survival
under the treatment. The first model is the traditional Gom-
pertz model with survival function




β
α
S(x) = exp –--- [exp(αx) – 1]


where parameters ꢈ and ꢉ are associated with the aging rate
and the initial mortality rate, respectively. Parameter ꢈ is
often characterized by the value of mortality rate doubling
time (MRDT), calculated as ln(2)/ꢈ.
The second model was used to estimate changes in the ag-
ing rate in the two-stage model. The two-stage model uses
Table 3. Food Consumption Dynamics in CBA Mice Treated and Not Treated With Melatonin
Daily Food Consumption (g)
Group
Unit
6 mo
9 mo
12 mo
15 mo
18 mo
21 mo
Control
g
2.5 ꢁ 0.21
11.7 ꢁ 0.98
2.7 ꢁ 0.11
12.8 ꢁ 0.39
3.4 ꢁ 0.21
13.8 ꢁ 0.86
4.0 ꢁ 0.10
16.5 ꢁ 0.33
3.1 ꢁ 0.12
10.8 ꢁ 0.41
3.3 ꢁ 0.19
11.1 ꢁ 0.86
3.1 ꢁ 0.23
10.6 ꢁ 0.80
3.2 ꢁ 0.11
10.5 ꢁ 0.44
2.8 ꢁ 0.14
9.6 ꢁ 0.47
2.7 ꢁ 0.06
8.5 ꢁ 0.22
3.2 ꢁ 0.08
10.3 ꢁ 0.26
2.9 ꢁ 0.04
9.0 ꢁ 0.24
g/100 g of body weight
g
g/100 g of body weight
Melatonin

B314
ANISIMOV ET AL.
Figure 1. Effect of melatonin on age-related dynamics of square
crossing (“open field” test) in female CBA mice.
Figure 3. Effect of melatonin in age-related dynamics of the
grooming reaction in female CBA mice.
two different values of aging rate during the life span. The
model is constructed from two Gompertz models as follows:
melatonin. The mean body weight of mice exposed to mela-
tonin was increased in comparison with those in the control
group at the age of 15 and 18 months (p ꢆ .05). Parameters
of the regression equation for body weight gain in the con-
trols were 1.108 ꢁ 0.062; those in the group that received
melatonin were ꢃ0.906 ꢁ 0.033 (p ꢆ .01).
It must be emphasized that the individual body weights of
the animals of all groups varied considerably, and the num-
ber of mice weighing much less or more than the mean indi-
ces for the group differed with age. After the subdivision of
mice of each group into 3 body weight classes—lean (ꢄ28
g), medium (29–33 g), and fat (ꢅ34 g)—it appeared that at
every age the number of medium-weighing mice did not
differ among the groups. At the same time, the number of
lean mice treated with melatonin was relatively smaller than
that in the control group, whereas the number of fat experi-
mental animals exceeded that in the control group (Table 2).
β
α




1
-----
S(x) = exp – [exp(α x) – 1]
x ≤ x*

1
1

β
α




1
-----
= exp – [exp(α x*) – 1]

1
1

β
α
2


-----
exp – {exp[α (x – x*)] – 1} ,
x > x*
2


2
where x* is the day separating the first and the second
phases of the life span, and S(x) is a survival function. It is
worth noting that our two-stages model provides a descrip-
tion of the survival of a single organism, not a population.
Parameters for the models were estimated from empirical
data by use of the maximum likelihood method imple-
mented in the Gauss statistical system (55). Confidence in-
tervals for the aging rate parameter estimates were calcu-
lated by profiling the log-likelihood function (53).
Age-Related Dynamics of Water and
Food Consumption
The amount of drinking (tap) water was stable during the
entire period of observations, that is 5.3 ꢁ 1.2 ml/mouse per
24 hours and 3.8 ꢁ 1.1 ml/mouse per night. There were no
differences in this parameter between the control and mela-
tonin-treated animals. Regular measurements did not reveal
any significant differences in the amount of consumed food
between groups of controls and melatonin-treated animals,
both at a rate per mouse and per unit of its body weight (Ta-
ble 3). The amount of food consumed by the mice varied in
different age periods. Thereby, both the periods of increased
and decreased food consumption were registered. These
variations were similar in both groups. The obtained data in-
dicated that the greater weight of mice treated with melato-
nin was not caused by the hormone’s impact on food con-
sumption by the animals.
RESULTS
Age-Related Body Weight Dynamics
Mean values of body weight for mice at different ages in
the control and melatonin-treated groups are displayed in
Table 1. As demonstrated in the table, the body weight of
the mice in both groups increased with age, exceeding by 21
months the body weight of 6-month-old animals by 43.9%
in the control group and by 56.5% in the group that received
Effect of Melatonin
Physical activity of mice.—In the initial test, physical
activity of the animals (or locomotor activity, which would
be more appropriate in this case) was evaluated. The test
consisted of defining the number of crossed squares in the
field and appeared to be most energy consuming. The ob-
tained data (Figure 1) demonstrate that the control mice re-
vealed the highest activity according to this parameter at the
age of 6 and 9 months. In the primary test, mice treated with
Figure 2. Effect of melatonin on age-related dynamics of vertical
sets (“open field” test) in female CBA mice.

MELATONIN INCREASES LIFE AND TUMORIGENESIS
B315
Table 4. Parameters of Muscular Strength and Fatigue Measured in 18-Month-Old CBA Mice Treated and Not Treated With Melatonin
Relative Body Weight Held by the Mice in a Unit of Time (g/s)
Group
Measurement 1
Measurement 2
Sum 1 ꢋ 2
Difference 2 ꢃ 1
Body Weight ꢄ 28 g
Control
0.52 ꢁ 0.16
0.56 ꢁ 0.21
0.26 ꢁ 0.06
0.18 ꢁ 0.04
0.78 ꢁ 0.19
0.75 ꢁ 0.23
ꢃ0.21 ꢁ 0.15
ꢃ0.36 ꢁ 0.20
Melatonin
Body Weight 29–33 g
Control
0.54 ꢁ 0.15
0.54 ꢁ 0.15
0.38 ꢁ 0.06
0.33 ꢁ 0.05
0.92 ꢁ 0.17
0.89 ꢁ 0.15
ꢃ0.17 ꢁ 0.15
ꢃ0.22 ꢁ 0.1
Melatonin
Body Weight ꢅ 34 g
Control
4.97 ꢁ 2.04
1.64 ꢁ 1.01
3.18 ꢁ 1.22
1.54 ꢁ 0.83
8.15 ꢁ 0.82
3.18 ꢁ 1.83
ꢃ1.79 ꢁ 3.26
ꢃ0.15 ꢁ 0.26
Melatonin
melatonin crossed a significantly fewer number of squares
in comparison with the controls (p ꢆ .001). Subsequently,
when the mice reached the age of 12 and 18 months, the dis-
crepancies in the number of crossed squares in the field be-
came statistically insignificant. A study of the dynamics of
this parameter revealed a rather predictable age-related de-
crease in locomotor activity. This decrease was especially
pronounced at the age of 9 months—in comparison with the
age of 6 months, a significant lessening of activity was ob-
served, by 41% in the control group and by 30% in the
group exposed to melatonin. At the age of 12 months, only
the control mice demonstrated a significantly smaller num-
ber of crossed squares in the field in comparison with 9-month-
old animals (p ꢆ .05).
Results on the second parameter of locomotor activity—
the number of vertical sets—were similar to those on the
previous parameter: the control animals showed the maxi-
mum activity (Figure 2). However, in the melatonin-treated
group these discrepancies were significant only at the age of
6 months. Age-related dynamics of the number of vertical
sets were analogous to those of the number of crossed
squares in the field: in both tests, a significant decrease by
the age of 9 months was registered.
Grooming reaction should refer not to locomotor activity
(progressive movement) but rather to physical activity, be-
cause the animal moved, though without changing its place.
The test on this reaction was not too energy consuming. The
control mice showed the highest activity according to this
parameter and a number of other parameters studied sepa-
rately (Figure 3). Time of grooming in the case of melatonin
administration significantly differed from that in the control
only at the age of 6 months; subsequently (at the age of 9
and 12 months) the duration of the studied reaction did not
significantly differ from that in the control. Age-related dy-
namics according to this parameter appeared to differ from
those according to the above-regarded parameter to a cer-
tain extent. In particular, by the age of 9 months, a certain
increase in grooming duration was observed; in the last test,
18-month-old animals were washing themselves a little
longer in comparison with 12-month old mice.
The conducted observations prompted a conclusion that
locomotor activity in the control mice decreased with age,
whereas melatonin caused a regular lessening in locomotor
activity over the course of life. On the whole, these data
suggest a slight decrease in the physical activity of mice ex-
posed to melatonin.
Table 5. Age-Related Dynamics of Estrous Functional Parameters in Mice Treated and Not Treated With Melatonin
No. of Estrous Cycles
Rate of Separate Phases
of Estrous Cycles (%)
Short
(ꢆ4 d)
Long
(ꢊ4 d)
No. of Mice
No. of
Mice
Length of
Estrous Cycle (d)
Total
No.
Age (mo)
P ꢋ M
E
D
No.
%
No.
%
PE
AE
Control
6
9
12
15
30
30
30
28
25
4.86 ꢁ 0.31
4.59 ꢁ 0.24
4.86 ꢁ 0.25
5.48 ꢁ 0.43
5.71 ꢁ 0.39
18
12
10
8
39
42
43
41
36
43
46
47
51
51
57
60
59
31
31
28
31
27
13
9
49
52
46
42
29
29
32
18
22
51
48
54
58
1
0
0
0
0
0
0
0
1
4
18
13
29*
71*
Melatonin
6
9
12
15
18
30
30
30
30
27
4.35 ꢁ 0.25
4.57 ꢁ 0.22
4.65 ꢁ 0.31
5.23 ꢁ 0.29
5.13 ꢁ 0.38
18
7
6
7
15
36
36
45
44
31
46
57
49
49
54
53
54
51
40
38
29
30
28
14
17
55
56
55
35
45
24
24
23
26
21
45
44
45
65
55
0
0
0
0
1
0
0
0
0
4
Note: P ꢌ proestrus; E ꢌ estrus; M ꢌ metaestrus; D ꢌ diestrus; PE ꢌ persistent estrus; AE ꢌ anestrus.
*The difference with the age of 6 mo is significant; p ꢆ .05.

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ANISIMOV ET AL.
Table 6. Body Temperature Dynamics in CBA Mice Treated and Not Treated With Melatonin
Mean Body Temperature (ꢂC)
Total Cycle
Age (mo)
(w/out Phase Subdiv.)
Estrus
Diestrus
Metaestrus ꢋ Proestrus
Control
9
12
15
18
37.42 ꢁ 0.062
37.67 ꢁ 0.092*
37.37 ꢁ 0.120
37.13 ꢁ 0.077
35.8 ꢁ 1.600
37.7 ꢁ 0.200
34.96 ꢁ 2.469
37.11 ꢁ 0.167
37.48 ꢁ 0.112
37.73 ꢁ 0.080
37.37 ꢁ 0.183
37.18 ꢁ 0.111
37.4 ꢁ 0.207
37.31 ꢁ 0.234
37.26 ꢁ 0.219
37.12 ꢁ 0.136
Melatonin
9
12
15
18
38.23 ꢁ 0.033†
38.21 ꢁ 0.049
38.24 ꢁ 0.054***†
37.70 ꢁ 0.048***
36.08 ꢁ 0.130***†
36.97 ꢁ 0.181***
38.25 ꢁ 0.061†
37.70 ꢁ 0.046***
36.04 ꢁ 0.088***†
37.01 ꢁ 0.104***
37.83 ꢁ 0.103**
36.04 ꢁ 0.146***
37.04 ꢁ 0.107***
37.47 ꢁ 0.115***
35.96 ꢁ 0.214***†
37.05 ꢁ 0.206**
*The difference with the age of 12 mo is significant: p ꢆ .05; **p ꢆ .01; ***p ꢆ .001.
†The difference with the controls of the corresponding age: p ꢆ .01.
Muscular strength and physical fatigability of mice.—
A study of the muscular strength, fatigability, and ability to
recover strength in both the experimental and control mice
was carried out on 18-month-old animals, 12 months after
the start of the experiment. Measurements indicated a great
individual variability of the mice. Treatment with melatonin
failed to modify these parameters (Table 4).
long-term administration of melatonin slows down age-
related changes in estrous function.
Age-related dynamics of body temperature in mice.—
Data on body temperature alterations in the mice exposed to
melatonin are presented in Table 6. The control mice re-
vealed a pronounced increase in rectal body temperature
during diestrus in comparison with estrus from the 9th to the
15th month of life, which was caused by the functioning of
the corpora lutea in the ovaries during diestrus. Rectal body
temperature increase during diestrus was not observed in
18-month-old control mice: the body temperature indices
remained constant irrespective of the cycle phase. The con-
trol mice did not reveal any significant alterations in body
temperature with age, both on the whole (irrespective of the
estrous cycle phases) and in any of the phases. No cyclic al-
terations in rectal body temperature during the estrous phase
or its age-related changes were observed in mice treated
with melatonin. It should be noted that the average body
temperature in the mice of this group at the age of 9 months
was higher, and at the age of 15 and 18 months was lower,
than that in the controls during the phase of diestrus of the
estrous cycle (Table 6).
Age-related dynamics of estrous function in mice.—
Investigations of the estrous function in the animals of both
age groups were performed every 3 months, starting when
the mice were 6 months of age. The following parameters of
estrous function were estimated: the length of estrus, the rel-
ative rate of estrous cycle phases (in percent); and the rela-
tive number of short (ꢆ4 days) and long (ꢅ4 days) estrous cy-
cles. The relative number of animals with regular cycles,
persistent estrus and anestrus were calculated as well.
Judging by the data presented in Table 5, the length of es-
trous cycle in the control female mice was stable with the
advance in age. Thus, no essential age-related alterations in
the rate of estrous cycle phases were observed. However,
the relative number of short estrous cycles significantly de-
creased with age (49% at the age of 6 months and 29% at
the age of 18 months; p ꢆ .05), whereas the number of long
cycles rose (51% and 71%, respectively). In the controls of
the oldest age groups, anestrus was registered, which was
not observed in younger animals. In the group of mice
treated with melatonin, no effects were observed on age-
related dynamics of both the length of the estrous cycle and
the rate of separate phases of the estrous cycle as compared
with the control group. However, the number of both short
and long estrous cycles was practically same at the age of 6
and 18 months (Table 5). Thus, these data suggest that the
Survival and longevity of female CBA mice.—Survival
rate dynamics in the mice treated with melatonin are pro-
vided in Table 7. As shown in the table, the survival rate dy-
namics were similar in both groups up to the age of 22
months. Afterward, a pronounced decrease in mortality rate
was observed under the effect of melatonin. Under the influ-
ence of melatonin the number of mice that reached the age
of 24 months increased 5.4-fold in comparison with the con-
trols (p ꢆ .001). Thus, the survival curve of mice treated
Table 7. Survival Distribution of Female CBA Mice Exposed and Not Exposed to Melatonin
No. of Survivors
Group
12 mo
14 mo
16 mo
17 mo
18 mo
19 mo
20 mo
21 mo
22 mo
23 mo
24 mo
25 mo
26 mo
28 mo
29 mo
Control
Melatonin
50
50
50
49
49
49
48
49
47
49
45
46
43
46
42
46
40
41
33
41*
7
38*
0
12*
0
1
0
1
0
0
*The difference with the control group is significant: p ꢆ .001.

MELATONIN INCREASES LIFE AND TUMORIGENESIS
B317
Table 8. Parameters of Life Span in Female CBA Mice
Treated and Not Treated With Melatonin
Controls
Melatonin
Parameters
n ꢌ 50
n ꢌ 50
Mean life span, d (mean ꢁ SE)
Median
685 ꢁ 9.2
705
722 ꢁ 12.6*
747
Mean life span of last 10% of survivors
Maximum life span
738 ꢁ 1.1
740
793 ꢁ 18.6*
867
*The difference with control is significant: p ꢆ .05.
with melatonin was shifted to the right as compared with
that in the control animals. The mean life span of mice
treated with melatonin was slightly increased compared
with controls (ꢋ5.4%, p ꢆ .05). The life span in the last
10% of the mice increased as a function of the duration of
melatonin treatment (by 2 months). The maximum life span
expanded by almost 4 months under the effect of melatonin
(Table 8).
Figure 4. Proportion of survived mice for fatal tumor (melatonin
treated, —; control group, ---).
2.3 months as compared with that of the control group (Fig-
ure 4; Table 10).
Spontaneous tumor development in female CBA
mice.—The total tumor incidence in the control female
mice was 30%. Lung adenomas and mammary carcinomas
developed most frequently, which corresponded to the on-
cological characteristics of CBA mice (56). The treatment
with melatonin was followed by a 20% increase in malig-
nant tumor incidence in comparison with that of the control
group (p ꢆ .001) (Table 9). Five cases of lymphomas and 5
cases of lung adenocarcinomas were observed in the group
treated with melatonin, whereas no cases of similar tumors
were found in the control group. At the same time, the de-
velopment of lung adenomas in animals treated with mela-
tonin was decreased in comparison with that of the controls
(p ꢆ .01). No essential impact of melatonin upon the neo-
plasia of other localization was registered (Table 9). It is
worth noting that the mean life span of fatal tumor-bearing
mice in the group treated with melatonin was increased by
Mathematical Models and Estimations on 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 five different contexts: (1) for all animals
in each group (total cases); (2) for total tumor-free animals;
(3) for total tumor-bearing mice; (4) for fatal tumor-free
mice, and (5) for fatal tumor-bearing mice. We composed
the groups of animals without consideration of possible ef-
fects caused by dependence between these groups. The one-
stage model (the traditional Gompertz model) shows a
slight slow down in the aging rate (calculated as ꢈ in the
Gompertz equation) under the influence of melatonin and a
significant reduction in parameter ꢈ for “fatal tumor-bear-
ing mice” context (by three-fold; p ꢆ .05). This could be in-
terpreted as a slow down in the growth rate of fatal tumors.
The limitations of the Gompertz model are demonstrated in
Figure 5, presenting the observed and modeled survival for
total tumor-free mice. Applying the same aging rate value to
the whole life span, we obviously underestimated the mor-
tality rate for ages between 725 and 775 days. The two-
stage model allows us to use two different values of aging
rate during the life span. Figure 6 shows that this model
gives good approximation for the mortality process. We
used the two-stages model to give a numerical interpretation
for observed survival curves. The simple Gompertz model
fails to fit the curves, with a rather long initial plateau. We
interpret this as the presence of two stages: a “negligible
mortality” stage and a “Gompertz-like mortality” stage. The
“join point” was estimated by the use of the maximum like-
lihood method, together with other parameters.
Table 9. Incidence, Localization, and Type of Tumors in Female
CBA Mice Treated and Not Treated With Melatonin
Control
Melatonin
Parameters
n ꢌ 50
n ꢌ 50
No. of Tumor-Bearing Mice
No. of Malignant Tumor-Bearing Mice
Total no. of Tumors
Total no. of Malignant Tumors
Localization and Type of Tumors:
Mammary gland
15 (30%)
3 (6%)
20
3
17 (34%)
13 (26%)*
22
15
Adenoma
Adenocarcinoma
1
0
4
5 (3†)
Lungs
Adenoma
Adenocarcinoma
11 (10‡)
0
4
5*
Calculations of ꢈ and MRDT in the two-stage model
show that at the first phase of the life span (before the age of
700 days) melatonin treatment has no significant effect on
the aging rate parameter, ꢈ, which also renders insignificant
the seeming increase of the MRDT, calculated as ln(2)/ꢈ.
However, at the second phase of life, the aging rates for the
total cases and the total tumor-free mice have a clear ten-
dency to increase in the case of melatonin treatment. The
parameters for the fatal tumor-free mice were significantly
Uterus
Leiomyosarcoma
Lymphoma
Vessel wall: hemangioma
0
0
3
1
5*
3
Note: Parenthetical information denotes the number of mice with this tumor.
*The difference with the control group is significant: p ꢆ .001.
†Two mice developed 2 tumors of this site.
‡One mouse had 2 lung adenomas.

B318
ANISIMOV ET AL.
Table 10. Parameters of Life Span in Female CBA Mice Treated and Not Treated With Melatonin
Total No.
of Cases
Total No.
Tumor-Free Mice
Total No.
Tumor-Bearing Mice
Fatal No.
Tumor-Free Mice
Fatal No.
Tumor-Bearing Mice
Group
Number of Mice
Controls
Melatonin
50
50
35
33
15
17
47
37*
3
13*
Mean life span (days)
Controls
Melatonin
685 ꢁ 9.2
722 ꢁ 12.6*
688 ꢁ 10.6
722 ꢁ 17.0
678 ꢁ 18.8
722 ꢁ 17.7
688 ꢁ 9.6
724 ꢁ 15.1*
645 ꢁ 20.6
716 ꢁ 23.0*
Mean Life Span of the Last 10% of Survivors (days)
Controls
738 ꢁ 1.1
736 ꢁ 2.4
725 ꢁ 4.6
738 ꢁ 1.1
645 ꢁ 20.1
Melatonin
793 ꢁ 18.6*
770 ꢁ 2.2**
789 ꢁ 19.8**
770 ꢁ 2.2**
788 ꢁ 20.0**
One-Stage Model
Aging Rate ꢈ ꢀ 103 (daysꢃ1
Controls
Melatonin
)
30 (23; 38)
19 (15; 26)
32 (23; 36)
43 (32; 47)
27 (17; 37)
15 (11; 21)
31 (24; 39)
41 (30; 45)
42 (22; 48)
14 (9; 20)*
MRDT (days)
Controls
Melatonin
23.1
36.5
21.7
16.1
25.7
46.2
22.4
16.9
16.5
49.5
Two-Stage Model
Aging Rate ꢈ ꢀ 103 (daysꢃ1), Stage I
Controls
Melatonin
6 (0; 13)
4 (0; 12)
3 (0; 13)
1 (0; 9)
10 (0; 25)
14 (3; 50)
2 (0; 10)
1 (0; 9)
42 (12; 120)
15 (4; 30)
MRDT (days), Stage I
Controls
Melatonin
Aging Rate ꢈ ꢀ 103 (dayꢃ1), Stage II
Controls
Melatonin
115.5
173.3
231.0
593.1
69.3
49.5
346.6
593.1
16.5
46.2
34 (6; 61)
76 (56; 97)
34 (3; 62)
83 (60; 108)
35 (0; 93)
8 (0; 20)
34 (6; 61)
90 (65; 118)*
—
10 (0; 22)
MRDT (days), Stage II
Controls
Melatonin
20.4
9.1
20.4
8.3
19.8
86.6
20.4
7.7
—
69.3
Note: Mean life spans are given as mean ꢁ standard error; 95% confidence limits are given in parentheses; MRDT ꢌ mortality rate doubling time.
*The difference with controls is significant: p ꢆ .05; **p ꢆ .001.
(p ꢆ .05) modified: ꢈ was increased by 2.6-fold whereas
MRDT was decreased by 2.6-fold. A comparison of Figures
5A and 5B shows that mortality of the elder melatonin-
treated mice directly related to the development of fatal tu-
mors in the second half of their life.
water significantly increases both the survival and malig-
nant tumor incidence in female CBA mice. The effect of
melatonin on survival does not relate to its influence on
food consumption. No reduction in food consumption was
observed. Moreover, body weight was increased in mice
treated with melatonin as compared with that in the control
group. These results are in concert with other observations
(23,31). A positive correlation between excessive body
weight and tumor incidence is observed in rodents and in
human females (57,58). Melatonin failed to modify physical
strength and fatigue and slightly decreased the locomotor
activity of mice. The sedative (sleep) effect of melatonin is
well known (11,14). Long-term treatment with melatonin
was followed by a slowing down of the aging of the repro-
ductive system in female CBA mice. A similar observation
in female rats was recently reported (59).
It is worth noting that, in our study, melatonin induced a
decrease of body temperature in mice (Table 6). It has been
shown in the literature that a decline of body temperature
caused by the slowing down of metabolic processes in an
organism is followed by an extension of life (60–62). Other
effects of melatonin on life span could be related to its anti-
oxidant potential (8,29). Our results confirm these observa-
tions.
Effect of Melatonin on Free Radical Processes in Mice
The comparison of the data on free radical processes gen-
erated in various organs revealed the most intensive genera-
tion of ROS in blood serum, where it twice exceeded the cor-
responding indices in liver and brain. Thus, the level of
antioxidant activity in blood sera was significantly lower
than that found in livers and brains (Table 11). The treatment
with melatonin was followed by a significant decrease in lu-
minol-induced chemiluminescence and a significant increase
of total antioxidant activity in the serum, but not in the brain
or liver of mice. Melatonin treatments inhibited lipid peroxi-
dation (decreased the level of both diene conjugates and
Schiff’s bases) in brain and liver tissue. In the serum, mela-
tonin treatment was followed by a decrease in the level of di-
ene conjugates, but not Schiff’s bases. Melatonin treatment
failed to modify the activity of SOD in any tested tissue.
DISCUSSION
An important result of our study is the increase of malig-
nant tumor incidence under the influence of melatonin in fe-
male CBA mice. In Table 12 we summarized the available
The results of our study show that long-term night admin-
istration of melatonin at a daily dose of 20 mg/l in drinking

MELATONIN INCREASES LIFE AND TUMORIGENESIS
B319
Figure 6. Gompertz and two-stage models for survival among total
tumor-free mice (proportion survived mice, —; modeled survival, . . .).
dose of melatonin in these experiments could be estimated
as 0.8–2.0 mg/kg of body weight. It is worth noting that
Pierpaoli and colleagues (18) did not report any cases of
spontaneous mammary adenocarcinomas in C3H/He mice
observed at 12 months of age. Subramanian and Kothari
(63) reported that 62.5% of intact female C3H/Jax mice de-
veloped mammary adenocarcinomas at the age of 12 months.
A similar incidence of spontaneous mammary adenocarci-
nomas was observed in female C3H mice of different sub-
lines, for example, C3H/He and C3H/Sn (3,64,65).
Figure 5. Number of deaths among control, A, and melatonin-
treated, B, mice (tumor free: ꢀ; non-fatal tumor: ; fatal tumor: ꢁ).
Melatonin did not induce any malignancies in male
C57BL/6 mice when it was administered at a dose of 10 mg/l
(ꢀ1.5–2.0 mg/kg) in the night drinking water from the age
of 19 months (17,18). However, Lipman and colleagues
(31) observed lymphomas in 77.9% of male C57BL/6 mice
that received melatonin with food (11 ppm or 68 mg/kg)
from the age of 18 months to 50% survival at 26.5 months,
whereas in control groups only 28.6% mice developed lym-
phomas. Leukemias were detected in 70–98% of C57BL/6
mice and in 78% of CC57Br mice (both males and females)
that were treated subcutaneously with melatonin at a dose of
2.5 mg/mouse (ꢀ80 mg/kg) twice a week for a duration of
2.5–5 months (32,66). The number of C57BL/6 mice in the
study of Pierpaoli and Maestroni (17) and in the study of
data on survival and tumor incidence in mice and rats ex-
posed to long-term treatment with melatonin. There are
findings of lymphosarcomas and reticulosarcomas and ova-
rian carcinomas in female C3H/He mice that have received
melatonin at night hours starting at the age of 12 months
(18). However, Subramanian and Kothari (63) reported a
suppressive effect of melatonin on the development of
spontaneous mammary carcinomas in female C3H/Jax mice
treated from the age of 3 weeks until the age of 12 months.
Pierpaoli and colleagues (18) started the treatment with mel-
atonin in mice that were older than 12 months. The daily
Table 11. Effect of Melatonin on Free Radical Processes in Female CBA Mice
Parameters
Brain
Control
Melatonin
Luminol-induced chemiluminescence (ꢀ 104 units/mg of protein)
94.1 ꢁ 7.9
3.1 ꢁ 0.19
22.29 ꢁ 0.87
388 ꢁ 25
76.9 ꢁ 7.7
3.56 ꢁ 0.25
19.02 ꢁ 0.65*
276 ꢁ 13*
Total antioxidant activity (units/mg of protein)
Diene conjugates (nM/g of tissue)
Schiff’s bases (units/g of tissue)
SOD activity (units/mg of protein)
23.4 ꢁ 1.8
23.5 ꢁ 1.7
Liver
Luminol-induced chemiluminescence (ꢀ 104 units/mg of protein)
Total antioxidant activity (units/mg of protein)
Diene conjugates (nM/g of tissue)
78.4 ꢁ 10.4
4.72 ꢁ 0.45
66.37 ꢁ 2.17
543 ꢁ 14
79.4 ꢁ 6.8
4.81 ꢁ 0.20
59.58 ꢁ 1.52*
459 ꢁ 19**
34.5 ꢁ 1.7
Schiff’s bases (units/g of tissue)
SOD activity (units/mg of protein)
35.5 ꢁ 1.6
Serum
Luminol-induced chemiluminescence (ꢀ 104 units/mg of protein)
Total antioxidant activity (units/mg of protein)
Diene conjugates (nM/g of tissue)
224.0 ꢁ 29.8
1.40 ꢁ 0.15
4.36 ꢁ 0.43
29.2 ꢁ 3.2
126.3 ꢁ 23.8*
1.86 ꢁ 0.07***
2.47 ꢁ 0.52*
26.2 ꢁ 2.5
Schiff’s bases (units/g of tissue)
Note: SOD ꢌ superoxide dismutase.
*The difference with control is significant: p ꢆ .05; **p ꢆ .01; ***p ꢆ .001.

B320
ANISIMOV ET AL.

MELATONIN INCREASES LIFE AND TUMORIGENESIS
B321
Pierpaoli and colleagues (18) was small, and no data on tu-
mor development were reported. Romanenko (32,66) and
Lipman and colleagues (31) used many more animals in
their studies. Assuming that the body weight of mice was
16–30 g in the studies of Romanenko (32,66), we could esti-
mate the dose of melatonin as 80–120 mg/kg of body
weight. In our study, melatonin that was given in night
drinking water in an interrupted (course) regimen in a rela-
tively low dose (3–3.5 mg/kg) was carcinogenic as well. We
believe that the testing of an effect of melatonin in a variety
of doses (including much smaller doses that have been used
in our study and other studies) will be useful in coming to a
more exact conclusion on the carcinogenicity of melatonin.
A clastogenic effect of melatonin may be involved in the
mechanism of its carcinogenic effect. It was shown that in
pharmacological doses melatonin induces DNA damage
and cleavage in hamster ovarian cells in a Single Cell Gel
Electrophoresis (COMET) assay (69). In contrast, melato-
nin has not been shown to be mutagenic using the Ames test
(69,70) and inhibits mutagenesis induced by some cytostat-
ics and ionizing irradiation (29,71,72). A treatment with
melatonin inhibits development of spontaneous endometrial
adenocarcinomas in BDII/Han rats (68), mammary car-
cinogenesis induced by 7,12-dimethylbenzo(a)anthracene
(DMBA) or N-nitrosomethylurea in rats (26,28), colon car-
cinogenesis induced by 1,2-dimethylhydrazine in rats (27)
and DMBA-induced carcinogenesis of the uterine cervix
and vagina in mice (73).
There are no principal controversies between the data
on the carcinogenic and anticarcinogenic potential of mel-
atonin. Some antioxidants, including natural ones (e.g.,
ꢈ-tocopherol) have both geroprotector and tumorigenic po-
tential and could be potent anticarcinogens as well (for a re-
view, see 3,4). It is worth noting that melatonin treatment
accelerated aging rate parameters (ꢈ and MRDT) in the sec-
ond phase of life as estimated in the two-stage model. In our
previous observations it was shown that there is a positive
correlation between the aging rate (estimated as ꢈ) of the
population and the malignant tumor incidence in the same
population (3,4). In other words, those geroprotectors that
increase the rate of aging induce an increase in malignant
tumor incidence; however, those that decrease the aging rate
inhibit spontaneous carcinogenesis. These results make it
premature to recommend melatonin for wide use as an anti-
aging drug.
Acknowledgments
This study was supported by Grant 99-04-48023 from the Russian Foun-
dation for Basic Research. We thank James W. Vaupel for the opportunity
to use the facilities of the Max-Planck Institute for Demographic Research,
Germany, to complete this study, and I. I. Mikhailova, E. V. Solomatina,
and O. V. Novikova for excellent technical assistance.
Address correspondence to Professor V. N. Anisimov, Laboratory of
Carcinogenesis and Aging, N. N. Petrov Research Institute of Oncology,
Pesochny-2, St. Petersburg 197758, Russia. E-mail: aging@mail.ru
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Received April 7, 2000
Accepted February 22, 2001
Decision Editor: John Faulkner, PhD