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Toxicologic Pathology, 31:589–603, 2003
Copyright C
by the Society of Toxicologic Pathology
ISSN: 0192-6233 print / 1533-1601 online
DOI: 10.1080/01926230390257885
Effects of Exogenous Melatonin—A Review
VLADIMIR N. ANISIMOV
Department of Carcinogenesis and Oncogerontology, N.N. Petrov Research Institute of Oncology, St. Petersburg 197758, Russia
ABSTRACT
The results of studies on the effect of pineal indole hormone melatonin on the life span of mice, rats, fruit flies, and worms are critically reviewed.
In mice, long-term administration of melatonin was followed by an increase in their life span in 12 experiments and had no effect in 8 of 20 different
experiments. In D. melanogaster, the supplementation of melatonin to the nutrient medium during developmental stages gave contradictory results,
but when melatonin was added to food throughout the life span, an increase in the longevity of fruit flies has been observed. Melatonin decreased the
survival of C. elegans but increased the clonal life span of planaria Paramecium tertaurelia. Available data suggest antioxidant and atherogenic effects
of melatonin. Melatonin alone turned out to be neither toxic nor mutagenic in the Ames test and revealed clastogenic activity in high concentration
in the COMET assay. Melatonin inhibits mutagenesis induced by irradiation and by indirect chemical mutagens and inhibits the development of
spontaneous and chemical-induced tumors in mice and rats. Further studies and clinical trials are needed to verify that melatonin is both safe and has
geroprotector efficacy for humans.
Keywords. Melatonin; life span; longevity; tumorigenesis; mouse; rat; fruit fly; worm.
INTRODUCTION
Growing expansion of so-called antiaging remedies pro-
moted by a variety of companies promising increased human
longevity induced hot debate on antiaging medicine in the
scientific community (29, 45, 46). Discussing what is known
about the possibility of slowing, stopping, or reversing aging
in animals and how this might be applied to humans, Butler
et al (29) concluded that there is no convincing evidence that
the administration of any specific compound, natural or ar-
tificial, can globally slow aging in people, or even in mice
and rats (29). Nevertheless, there are available data on the
means to increase the life span of laboratory animals, includ-
ing caloric restriction, some genetic manipulations, and ad-
ministration of hormones, chemical substances, stem cells,
and so on. Melatonin is among the most promoted candi-
dates of “geroprotectors” which means “preventing aging,”
Using the key word “melatonin” in a MEDLINE search, we
found close to 10,000 papers, using “melatonin and aging,”
we found more than 500. What do we really know about
the effects of this hormone on life span and age-associated
processes, including pathology?
Melatonin (N-acetyl-5-methoxy-tryptamine) is the main
pineal hormone synthesized from tryptophan, predominantly
at night (17). Melatonin is critical for the regulation of circa-
dian and seasonal changes in various aspects of physiology
and neuroendocrine function (17, 88, 110, 170). As age ad-
vances, the nocturnal production of melatonin decreases in
animals of various species, including in humans (160, 161,
176). A pinealectomy resulted in the reduction in life span in
rats (87, 130) whereas grafting a pineal gland from young
donors into the thymus of old syngenetic mice or in situ
into pinealectomized old mice prolongs the life span of the
Address correspondence to: Vladimir N. Anisimov, Department of Car-
cinogenesis and Oncogerontology, N.N. Petrov Research Institute of On-
cology, Pesochny-2, St. Petersburg 197758, Russia; e-mail: aging@mail.ru;
geros@land.ru
recipients (77, 117). The capacity of melatonin to extend life
span is a hot topic (12, 18, 67, 68, 114, 128, 129, 133, 165).
In this work the results of studies on the effect of adminis-
tration of exogenous melatonin to mice, rats, fruit flies, and
worms are reviewed.
EFFECT OF MELATONIN ON THE LONGEVITY OF MICE
Romanenko (134) administered melatonin subcutaneously
into C57BL/6 mice (both males and females) at a single dose
2.5 mg/mouse (∼80 mg/kg) twice a week for 5 months, start-
ing at the age of 1.5 months. The mean life span of 25 control
mice was 22 months, whereas it was 19 months in the mela-
tonin group (n=45). Almost 98% of melatonin-treated mice
and 32% of control mice developed leukemia. In another set
of experiments, C57BL/6 and C57Br mice (both males and
females) were subjected to the same treatment, but its dura-
tion was restricted to 2.5 months. The treatment reduced the
mean life span of C57BL/6 mice from 17 to 13.5 months,
and of CC57Br mice from 17 to 15 months. Leukemia was
detected in 70% of melatonin-treated C57BL/6 mice versus
14% of the control mice, and in 78% C57Br mice versus 38%
of the control mice (135).
Pierpaoli and Maestroni (115) were the first to demon-
strate life span extension induced by treatment with exoge-
nous melatonin. In November 1985 they started a daily ad-
ministration of melatonin with drinking water (10 mg/L) into
10 male C57BL/6J mice aged about 19 months. Ten control
mice received a 0.01% solution of ethanol in the drinking
water. Melatonin was given from 18.00 hrs to 8.30 hrs. After
5 months, control mice became bold, and less active and had
decreased body weight. The treatment with melatonin pre-
vented body weight loss. The mean life span of mice under
the influence of melatonin increased by 20%.
In 1991 Pierpaoli et al (117) reported the results of 3 new
experiments with melatonin. In all of them melatonin was
given with drinking water (10 mg/L) during the night. Fif-
teen female C3H/He mice were given melatonin starting at
the age of 12 months. Fourteen mice of the same strain served
589
590 ANISIMOV TOXICOLOGIC PATHOLOGY
as a control. The treatment failed to increase longevity of the
C3H/He mice and increased the incidence of spontaneous
tumors (lympho- and reticulosarcomas and ovarian tumors).
It is worth noting that female C3H/He mice are character-
ized by a high incidence of spontaneous mammary carci-
noma (148); however, the authors did not provide any data
on mammary tumors in this report. Analysis of presented sur-
vival curves has shown that the mice exposed to melatonin
lived 2 months less than controls. In the second series of the
experiment, melatonin was given day or night to female NZB
(New Zealand Black) mice, who are characterized by a high
incidence of autoimmune hemolytic anemia, nephrosclero-
sis, and systemic reticulo-cell tumors types A and B. Each
group included 10 mice, and melatonin was given from the
age of 4 months. The administration of the hormone during
the day failed to influence survival, and all mice of this group
died before the age of 20 months (in the control group, be-
fore the age of 19 months). The night exposure to melatonin
reduced the mortality of NZB mice: at the age of 20 months,
4 out of 10 mice were alive, and 2 of them survived to the age
of 22 months. The last mouse died 4 months after the death
of the last mouse in the control group. In the third series,
the mean life span of 20 male C57BL/6J mice was 743 ±
84 days, whereas the mean life span of 15 males treated with
melatonin from the age of 19 months was 871 ±118 days.
Treatment with melatonin failed to modify body weight as
compared with the controls.
In 1994 Pierpaoli and Regelson (116) summarized their
own previous data and presented some new results. Mela-
tonin (10 mg/L) was given with drinking water during the
night (between 18.00 and 08.30 hours) to female BALB/c
mice aged 15 months. The mean life span of 26 control mice
was 715 days, whereas the mean life span of the 12 mice ex-
posed to melatonin was 843 days (+18%). The maximum life
span was 27.2 months in the control group and 29.4 months
in the treated group. There was no difference in the body
weight between the groups. In another experiment melatonin
(10 mg/L) was given with drinking water at night to male
BLAB/c mice starting at age 18 months (118). Animals were
sacrificed in small groups 4, 7 and 8 months after the be-
ginning of the experiment. Eight months after the beginning
of the experiment the weight of thymus, adrenal glands,and
testicles of mice treated with melatonin was significantly dif-
ferent from the age-matched controls but was similar to the
younger control mice. Similar changes were observed in the
number of peripheral blood leucocytes, and the levels of zinc,
testosterone, and thyroid hormones. The authors believe that
the cyclic administration of melatonin has a positive influence
on animals in keeping the endocrine and thymico-lymphoid
organs young. It is worthy noting that only a few animals
were in each group.
Lenz et al (76) injected melatonin into female NZB/W
mice in a single dose 100 µg/mouse (2–3.5 mg/kg) daily for
9 months in the morning (between 08.00 and 10.00 hours) or
in the evening (between 17.00 and 19.00 hours), starting at
the age of 8 months. There were 15 mice in each group. It
was shown that morning injections of melatonin significantly
(p<0.001) increased the survival time of mice, whereas the
evening injections failed to influence their survival. Twenty
percent of the control mice survived 34 weeks, whereas 65%
of the mice exposed to the morning injections of melatonin
survived this age. Unfortunately, the observation was finished
before the natural death of all the animals, and no complete
necropsy had been performed.
Mocchegiani et al (95) administered melatonin (10 mg/L)
with drinking water to 50 male Balb/c mice starting at the age
of 18 months. Fifty other mice were given water with supple-
mentation of zinc sulfate (22 mg/L), and 50 mice served as the
control group. The treatment with melatonin and zinc signif-
icantly shifted the survival curves to the right and increased
maximal life span of mice by 2 and 3 months, respectively.
Unfortunately, no numerical data on mean and maximum life
span was presented. Both compounds failed to influence food
consumption or body weight dynamics.
Conti and Maestroni (36, 37) have studied the effect of
melatonin on the longevity of female non-obese diabetic
(NOD) mice with a high incidence of insulin-dependent di-
abetes. One of the mouse groups (n=25) was neonatally
pinealectomized; the second group (n=30) was given sub-
cutaneous injections of melatonin in a single dose of 4 mg/kg
at 16.30 hours 5 times a week from the age of 4 weeks until
the age of 38 weeks. Mice of the third group were injected
with bovine serum (PBS) as a control to the second group. In
the fourth group mice were given melatonin (10 mg/L) with
drinking water during the night 5 times a week from the age
of 4 weeks until the age of 38 weeks. The fifth group included
29 intact mice. The survival of pinealectomized mice was sig-
nificantly reduced due to autoimmune diabetes. At the age of
32 weeks only 8% of the mice in this group survived. In the
control group 65.6% of mice died before the age of 50 weeks.
Long-term injections with melatonin slow down diabetes de-
velopment and mortality. Only 10% of mice in this group
died before the age of 50 weeks. Administration of mela-
tonin with drinking water was less effective than injections:
At the end of the observation period 58.8% of melatonin-
treated mice survived, whereas 34.5% of the control group
survived ( p<0.0019). Thus, pinealectomy accelerated the
development of diabetes and reduced the life span of mice,
whereas the treatment with melatonin slowed down the dis-
ease development and increased the longevity (37).
Oxenkrug et al (106) studied the effect of long-term admin-
istration of melatonin and N-acetylserotonin (NAS) in male
and female C3H mice. Melatonin was given with drinking wa-
ter during the night at the daily dose of 20 mg/L (2.5 mg/kg)
starting at the age of 1 month. The increased life span in the
male mice was about 20% greater than that of control ani-
mals ( p<0.01), but this did not affect the life span of female
mice.
The ability of melatonin to alter disease incidence and
longevity was studied in adult male C57BL/6 mice (80). Mice
were fed lab chow supplemented with melatonin (11 ppm)
starting at the age of 18 months. Melatonin failed to influence
the body weight and food consumption as well as the mor-
tality of mice. Fifty percent of the control group mice died
by the age of 26.5 months, and 50% of the melatonin-treated
mice by the age of 26.7 months. Survival curves were not
presented. Some of the animals were sacrificed at the age of
24 months (cohort 1) or at the age of 50% mortality (cohort
2). The third cohort included mice that died before the age of
2 years. There were 20 control and 20 melatonin-treated mice
in cohort 1; 7 and 13 mice, correspondingly, in cohort 2; and
28 and 30 mice, correspondingly, in cohort 3. The authors
Vol. 31, No. 6, 2003 EFFECTS OF EXOGENOUS MELATONIN 591
claimed that diet supplementation with melatonin initiated
during middle age did not appear to affect age-associated le-
sion patterns, lesion burden, or longevity in male C57BL/6
mice. Data presented in the paper showed that the incidence of
lymphomas was similar in the control and melatonin-treated
mice in cohort 3 (21.1% and 23.3%, respectively), however,
in cohort 2 the incidence of lymphomas was 28.6% in controls
and 77.9% in melatonin-treated animals.
In our experiments (12), 50 female CBA mice from the
age of 6 months until their natural deaths were given mela-
tonin with their drinking water (20 mg/L) for 5 consecutive
days every month. Fifty intact mice served as controls. The
results of this study show that the consumption of melatonin
did not significantly influence food intake, but it did increase
the body weight of older mice; it did not influence physical
strength or the presence of fatigue; it decreased locomotion
activity and body temperature; it inhibited free radical pro-
cesses in the serum, brain, and liver; and it slowed down the
age-related switching-off of estrous function. The survival
rate dynamics were similar in both groups up to the age of
22 months. Afterward, a pronounced decrease in mortality
rate was observed under the influence of melatonin. Under
the influence of melatonin the number of mice that reached
the age of 24 months increased 5.4-fold in comparison with
the controls ( p<0.001)(data not shown). Thus, the survival
curve of mice treated with melatonin was shifted to the right
as compared to the control animals. The mean life span of
mice treated with melatonin was increased slightly compared
with controls (5.4%; p<0.05). The life span in the last 10%
of the mice increased by the duration of melatonin treatment
(2 months). The maximum life span was extended by almost
4 months under the effect of melatonin (Table 1). Treatment
with melatonin was followed by a 4.3-fold increase in malig-
nant tumor incidence in comparison with that of the control
TABLE 1.—The effect of melatonin on life span and spontaneous tumor inci-
dence in female CBA mice.
Parameters Controls Melatonin, 20 mg/L
Number of mice 50 50
Mean life span, days (±S.E.) 685 ±9.2 722 ±12.6∗∗
Mean life span of last 10% of survivors, 738 ±1.1 793 ±18.6∗∗
days (±S.E.)
Maximum life span, days 740 867
Number of tumor-bearing mice 14 (30%) 17 (34%)
Number of malignant tumor-bearing mice 3 (6%) 13 (26%)∗
Total number of tumors 20 22
Total number of malignant tumors 3 15
Localisation and type of tumors
Mammary gland
Adenoma 1 0
Adenocarcinoma 3a4
Lymphoma 0 5∗
Lung
Adenoma 10b4∗∗∗
Adenocarcinoma 0 5∗
Other sites
Benign 3c3d
Malignant 0 1e
aTwo mice developed two tumors at this site.
bOne mouse had two lung adenomas.
cOvarian hemangiomas.
dOvarian hemangiomas.
eUterine leiomyoscarcoma.
∗p<0.001 (Fischer’s exact and X2tests).
∗∗ p<0.05 (student’sttest).
∗∗∗ p<0.01 (Fischer’s exact and X2tests).
group ( p<0.001). 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 develop-
ment of lung adenomas in animals treated with melatonin was
decreased in comparison with that of the controls ( p<0.01).
No essential impact of melatonin upon the neoplasia of other
localizations was registered (Table 1).
In a recent experiment conducted in our laboratory, mela-
tonin (2 mg/L or 20 mg/L) was given with drinking water
for 5 consecutive days every month to female Swiss-derived
SHR mice starting at the age of 3 months (15). There were
50 mice in each group. Fifty intact mice served as controls.
The treatment with melatonin did not significantly influence
food consumption, but its administration at the lower dose
did decrease the body weight of mice; it slowed down the
age-related switching-off of estrous function; and it did not in-
fluence the frequency of chromosome aberrations in the bone
marrow cells. The last mouse in the control group died at the
age of 772 days (25.4 months). In groups treated with mela-
tonin at doses of 2 and 20 mg/L, 6% and 12% of mice survived
this age. The maximum life span was 881 days (29 months)
and 890 days (29.3 months) in melatonin-treated groups, re-
spectively. The mean life span of mice treated with melatonin
was not increased compared with controls. The life span in the
last 10% of survivors increased with melatonin treatment (by
56 days at a dose of 2 mg/L and 86 days at the dose of 20 mg/L;
see Table 2). These data suggest that the mice are heteroge-
neous in their susceptibility to melatonin and some of them
are more susceptible to its geroprotective effect. Treatment
with melatonin at a dose of 2 mg/L was followed by a 1.9-fold
decrease in total number of tumors, and a 2.2-fold decrease
in total number of malignant tumors as compared with the
controls ( p<0.01). Incidence of mammary carcinomas with
TABLE 2.—The effect of melatonin on life span and spontaneous tumor inci-
dence in female SHR mice.
Melatonin, Melatonin,
Parameters Controls 2 mg/L 20 mg/L
Number of mice 50 50 50
Mean life span, days (±S.E.) 479 ±30 479 ±25 495 ±29
Mean life span of last 10% 759 ±8 815 ±29 845 ±13∗∗
of survivors, days (±S.E.)
Maximum life span, days 772 881 890
Number of tumor-bearing mice 21 (42%) 11 (22%)∗19 (38%)
Number of malignant 20 (40%) 9 (18%)∗15 (30%)
tumor-bearing mice
Total number of tumors 26 17 23
Total number of malignant tumors 22 11 18
Localization and type of tumors
Mammary gland
Adenocarcinoma 13 (26%) 3 (6%)∗12a(24%)
Leukemia 7 6 3
Lung
Adenoma —13
Adenocarcinoma 1 3 —
Other sites
Benign 4b4c2d
Malignant 1e
aThree mice developed 2 tumors of this site.
bUterine polyps, ovarian cyst, skin papilloma.
cUterine polyp, 3 ovarian cysts.
dOvarian cyst; granuleso-cell ovarian tumor.
eAdenocarcinoma of uterus.
∗p<0.01 (Fischer’s exact and X2tests).
∗∗ p<0.01 (student’sttest).
592 ANISIMOV TOXICOLOGIC PATHOLOGY
the lower dose of melatonin decreased 4.3-fold (p<0.01).
The incidence of lung metastases of mammary carcinomas
decreased 4-fold. The mean latent period of mammary carci-
nomas was significantly ( p<0.01) increased (2 months) in
mice treated with 2 mg/L melatonin compared with controls.
There was no significant difference in the incidence of any
other tumors between mice treated with the lower melatonin
dose and controls. There was no effect of treatment with
the higher dose of melatonin (20 mg/L) on the total inci-
dence of tumors or on the incidence of tumors at any site
(Table 2).
The effect of melatonin was studied in our laboratory us-
ing a senescence-accelerated mouse model (SAM) (136). Fe-
male SAMP-1 and SAMR-1 mice were given melatonin with
their drinking water (20 mg/L) for 5 consecutive days every
month until their natural death. There was no significant ef-
fect of melatonin on the mean life span or spontaneous tu-
mor incidence in senescence-accelerated SAMP-1 mice and
senescence-resistant strain SAMR-1 mice.
Female FVB/N transgenic HER-2/neu mice were treated
at night with melatonin (20 mg/L) in their drinking water
in 2 regimens: 5 consecutive days every month (interrupted
regimen) or 5 days a week without 3-week-long intervals
(constant regimen), both starting at the age of 2 months and
prolonged until their natural death (14). The interrupted ad-
ministration of melatonin failed to influence the life span
in the mice; however the constant treatment was followed
by a slight reduction (by 14.2%, p<0.05) of the mean life
span and of the maximum life span (by 16.4%) as compared
with the controls. At the same time, the constant treatment
with melatonin inhibited mammary carcinoma development
and metastasis in comparison to the control. The constant
treatment with melatonin reduced age-related disturbances
in estrous function in HER-2/neu transgenic mice.
In Table 3 we summarized available data on survival and
tumor incidence in mice exposed to long-term treatment
with melatonin. Melatonin did not induce malignancies in
male C57BL/6 mice when administered at 10 mg/L (1.5–
2.0 mg/kg) in the night drinking water from 19 months
(115, 117). Lipman et al (80) observed lymphomas in 77.9%
of male C57BL/6 mice that received melatonin with food
(11 ppm or 68 µg/kg) from the age of 18 months and survived
to a 50% mortality (26.5 months). In controls only 28.6%
of mice developed lymphomas. Leukemia was detected in
70%–98% of C57BL/6 mice and 78% of CC57Br mice (both
males and females) treated subcutaneously with melatonin
at a dose of 2.5 mg/mouse (∼80 mg/kg) twice a week for
of 2.5–5 months in contrast to 14% in control C57BL/6 and
38% in control CC57Br mice (134, 135). Thus, when mela-
tonin was administered in a significantly high dose (about
80 mg/kg), it induced lymphomas and leukemias in C57BL/6
mice. At low dose (from 68 mg/kg to 1.5–2.0 mg/kg) it in-
duced these cancers only in one small group of 7 mice. In
our previous study with CBA mice, melatonin given in night
drinking water in an interrupted regimen at a relatively low
dose (3–3.5 mg/kg) was carcinogenic. Lymphomas and lung
adenocarcinomas developed in CBA mice treated with mela-
tonin, whereas no such malignancies developed in controls
(12). In female SHR mice, melatonin given approximately
in the same dose (20 mg/L; 2.7–3.3 mg/kg) failed to signifi-
cantly increase the total incidence of tumors or tumors at any
localization. Strain differences in susceptibility to chemical
carcinogens are well known (166, 169).
There is one strong critical comment on the results of long-
term experiments with melatonin in mice. It was claimed
that murine strains used in some studies do not synthesize
melatonin as a result of a genetic defect (BALB/c, NZB,
and C57BL/6) (56). 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 (37). It is worth
noting that the major signal transduction cascades in the
pineal gland did not differ between melatonin-proficient C3H
mice and melatonin-deficient C57BL mice (149).
Thus, with exception of the results of Romanenko’s stud-
ies, which used a very large dose of the compound being ad-
ministered into mice of various strains regardless of sex and
the age of mice at the beginning of the treatment, melatonin
increased the life span in 12 of 20 experiments (60%) and had
no effect in 8 experiments (40%); (p>0.05) (Table 4). In
males, 4 out of 5 experiments resulted in life extension with
melatonin, while in females 8 experiments were positive and
7 negative. If the treatment was started early, longevity was
increased in 8 of 14 studies, and no effect was observed in
6 studies. In general, there is no significant evidence of a
geroprotective effect of melatonin in mice. However, among
4 experiments that were conducted according to the standard
protocol (50 animals per group), 3 resulted in an increase in
life span under the influence of melatonin.
EFFECT OF MELATONIN ON THE LONGEVITY OF RATS
In an experiment with male CD rats, melatonin was given
with drinking water (4 mg/L) during the entire day start-
ing at 11–13 months of age for 16 months (103). Additional
groups of rats received drinking water with a melatonin antag-
onist M-(1,4-dinitrophenyl)-5-methoxytryptamin (ML-23) at
a dose of 0.4 mg/L or a combination of melatonin and ML-
23 in the same doses. Observation was stopped when the rats
reached 26–29 months. Seven of 16 rats (44%) of the con-
trol group survived to this age, whereas 13 or 15 rats (87%)
reached this age in the group exposed to melatonin alone.
Surprisingly, 6 of the 10 exposed to the melatonin antagonist
ML-23 survived; and 8 of the 10 rats treated with a combi-
nation of melatonin and ML-23 survived. Body weight was
similar in all groups. Five of the 7 control rats had pneumonia
at necropsy, whereas there were no cases of pneumonia in the
group treated with melatonin or ML-23. The serum level of
testosterone was 2.8 times higher in rats treated with mela-
tonin as compared with the controls. The authors believed that
melatonin antagonist ML-23 induces chronic deprivation of
melatonin receptors followed by their hypersensitivity to the
melatonin. It is worth noting that there was a small number
of animals per group in this experiment and the experiment
was terminated before the natural death of all the animals.
Meredith et al (93) studied the effect of lifelong supplemen-
tation with melatonin on reproductive senescence. Holtzman
rats were divided into three treatments on day 10 after birth.
Treatment 1 pups had access to water, whereas treatment 2
and 3 pups had access to water containing 10 mg/L melatonin
only at night (treatment 2) or continuously (treatment 3).
Estrous cycles and weights of pups were monitored dur-
ing the experiment; ovaries were removed for histology at
TABLE 3.—Summary of experiments on the effect of melatonin on life span and spontaneous tumor incidence in mice.
Effects of melatonin on
Strain Sex
Nos. of
mice C/M
Age at the start
of treatment, mo Treatment with melatonin
Age at the end
of observation Mean life span Tumor incidence References
Balb/c Female 26/12 15 10 mg/L in night
drinking water
ND +18% No data Pierpaoli and Regelson,
1994
Balb/c Male 50/50 18 10 mg/L in night
drinking water
ND Shift to right of
the survival
curve
No data Mocchegiani et al,
1998
C57BL/6 Male & female 25/45 1.5 2.5 mg/mouse s.c. twice
awk×5mo
22 mo −13% Increased Romanenko, 1983
C57BL/6 Male & female 29/57 1.5 2.5 mg/mouse s.c. twice
awk×2.5 mo
22 mo −20.6% Increased Romanenko, 1985
C57Br Male & female 26/57 1.5 2.5 mg/mouse s.c. twice
awk×2.5 mo
22 mo −12% Increased Romanenko, 1985
C57BL/6J Male 10/10 19 10 mg/L in night
drinking water
ND +20% No data Pierpaoli and
Maestroni, 1987
C57BL/6 Male 20/15 19 10 mg/L in night
drinking water
ND +17% No data Pierpaoli et al, 1991
C57BL/6 Male a: 20/20
b: 7/13
c: 38/30
18 11 ppm (68 µg/kg) with
lab chow ad libitum
a: 24 mo
b: 50%
survival
c: died <2y
No effect a: No effect
b: Increased
c: No effect
Lipman et al, 1998
CBA Female 50/50 6 20 mg/L in night
drinking water
ND +5% Increased Anisimov et al, 2001
C3H Male
Female
20/20
20/20
1
8
2.5 mg/kg/day in night
drinking water
23 mo
27 mo
+20%
No effect
No data Oxenkrug et al, 2001
C3H/He Female 14/15 12 10 mg/L in night
drinking water
ND No effect Increased Pierpaoli et al, 1991
C3H/Jax Female 16/39 3 wk 25–50 mkg/ mouse/d
with drinking water
12 mo No data Decreased Subramanian and
Kothari, 1991
HER-2 /neu Female 30/27 /22 2 2.5 mg/kg/ day in night
drinking water 5 d/w
monthly or constantly
ND M1: No effect
M2: −13%
M1: No effect
M2: Decreased
Anisimov et al, 2002
NOD Female 25/30 1 4 mg/kg s.c. at 4:30 PM,
5 times a wk, 4–38 wk
50 wk C:32% survivors
M:90% survivors
No data Conti and Maestroni,
1998
NOD Female 29/17 1 10 mg/L in night
drinking water, 5
times a wk, 4–38 wk
50 wk +17% No data Conti and Maestroni,
1998
NZB Female 10/10 4 10 mg/L in night
drinking water
20 mo Survivors:
C:0
M: 40%
No data Pierpaoli et al, 1991
NZB/W Female 15/15/ 15 8 2–3.5 mg/kg s.c.daily at
8–10 hrs (M1) or at
17–19 hrs (M2) ×
9mo
34 wk Survivors:
C: 20%
M1: 60%
M2: 60%
No data Lenz et al, 1995
SAMP-1 Female 20/23 3 20 mg/L in night
drinking water
ND No effect No effect Rosenfeld, 2002
SAMR-1 Female 14/19 3 20 mg/L in night
drinking water
ND No effect No effect Rosenfeld, 2002
SHR Female 50/50/50 3 2 or 20 mg/L in night
drinking water
ND M1: No effect
M2: +3 mo. MLS
M1: −1.9-fold
M2: No effect
Anisimov et al, 2003
C=control group; M =melatonin-treated group; MLS =maximum life span; ND =animals survived until natural death; NOD =non-obese diabetic; SAMP-1 =senescence accelerated mouse-prone; SAMR-1 =senescence accelerated
mouse-resistant.
593
594 ANISIMOV TOXICOLOGIC PATHOLOGY
TABLE 4.—Melatonin and life span in mice: Summary of results.
Effect
Start of Number of No effect
Sex treatment∗experimentsaIncrease or decrease
Males Early 1 1 0
Late 4 3 1
Total 5 4 1
Females Early 13 7 6
Late 2 1 1
Total 15 8 7
Total Early 14 8 6
Late 6 4 2
Total 20 12 8
∗Early—Start of treatment before the age of 12 months. Late—start of treatment at the
age of 14 months and older.
aEach group with new dose, schedule of treatment, or mouse strain was evaluated as a
new experiment.
75 and 380 days of age. Vaginal opening in treatment 2 was
delayed ( p<0.01) compared with treatments 1 and 3, but
there was no difference (p>0.05) among treatments in per-
centage of normal length estrous cycles from vaginal opening
to 75 days of age. There were fewer (p<0.001) abnormal-
length estrous cycles from 180 to 380 days of age in treatment
2 pups as compared with treatments 1 or 3 pups. There was
no effect of treatment (p>0.05) on number of primordial
follicles. The authors concluded that treatment at night, but
not continuous supplementation with melatonin, delayed pu-
berty and reproductive senescence without any effect on the
number of primordial follicles. In general, these data are in
agreement with our observations on the slow-down of age-
related switching-off of reproductive function.
EFFECT OF MELATONIN ON THE LONGEVITY OF FRUIT FLIES
It was shown that melatonin is synthesized in Drosophila
melanogaster (51), and arylalkylamine N-acetyltransferase,
a key enzyme in melatonin biosynthesis, was identified in the
insect nervous system and the gut (1, 63).
We have studied the effect of melatonin on longevity in
D. melanogaster strain HEM (8). Melatonin was added to the
nutrition medium (100 µg/ml) during the second and third age
of larvae for survival analysis; the flies were passed every 3–
7 days. Exposure to melatonin was followed by a decrease in
the level of conjugated hydroperoxides and ketodienes in fe-
males and failed to influence the activity of catalase in males,
but increased it in females by 24% (p<0.02) and failed to
influence the Cu,Zn-superoxide dismutase (SOD) activity in
both males and females. It was shown that melatonin did not
influence the life span of this strain of fruit flies.
The life span of D. melanogaster wild strain Canton-S
was studied under the effect of melatonin at a concentration
of 800 mg/L (67). The hormone was introduced into a nutri-
ent medium only at the larva stages. Five experiments with
melatonin have shown a variation in the effect of melatonin
on life span: The mean life span in males varied from 10.0%
to 18.5%, whereas in females it varied from 2.3% to 12.1%.
An inverse correlation was observed between the change in
life span after melatonin supplementation and the life span
in the corresponding control group. For a relative low life
span in a population from which the control and experimen-
tal group were formed, the geroprotector effect of melatonin
was the most distinct; for a relatively high life span, the
effect of melatonin was either not detected or appeared as
a reduction in life span due to its toxic effect.
Recently, the effect of melatonin on life span was stud-
ied in the D. melanogaster Oregon wild strain (27). Mela-
tonin, added daily to the nutrition medium at a concentra-
tion of 100 µg/ml during the entire experiment, significantly
increased the life span of flies. The maximum life span was
61.2 days in the control group and 81.5 days in the melatonin-
fed group (+33.2%). Relative to the controls, the percentage
of the melatonin-fed flies was 19.3% in the onset of 90% mor-
tality and 13.5% in median life span. Authors have shown also
that melatonin treatment increased the resistance of fruit flies
to superoxide-mediated toxicity of paraquat and to thermal
stress.
These data suggest if melatonin was added to the fruit flies’
food throughout their life span, there would be a statistically
significant increase in their the longevity.
EFFECT OF MELATONIN ON THE LONGEVITY OF WORMS
Bakaev et al (19) have studied the effect of various doses
of melatonin on the life span of nematode Caenorhabditis
elegans. Three- to five-day-old adult nematodes (Bristol, N2)
were kept 4 hours in a melatonin-free nutrient medium with
E. coli and then transferred into standard vessels with mela-
tonin. The solution was changed each day. The temperature
was +21◦C and the animals were kept in constant darkness.
The mean life span of C. elegans in the control group was
23.7 ±1.8 days and the maximum life span was 32 days. Con-
centrations of 10 mg/L and 100 mg/L of melatonin failed to
influence the life span of the worms. However, at concentra-
tions of from 1 to 100 g/L the hormone significantly decreased
the mean life span (by 31% to 55.7%, p<0.05).
It was shown that melatonin-synthesizing enzyme activi-
ties and melatonin level has a circadian rhythm in planarians
(66). Melatonin supplementation in dim red light at a dose of
10 mg/L of nutrient media per day, followed by incubation of
23 hours of darkness, increased the mean clonal life span of
an aerobic single-cell organism, planaria Paramecium tertau-
relia, in days by percentages ranging from 20.8% to 24.2%
(158). The maximum clonal life span also was increased in
melatonin-supplemented cells, from 14.8% to 24.0% over
controls. It is worth noting that the increase in the concen-
tration of melatonin in the nutrient medium was followed by
a decrease in the life span of planaria. The mean clonal life
span in fissions was not significantly increased in melatonin-
supplemented cells, with values ranging from 6.0% to 15.5%
over controls. The authors suggested that the geroprotector
effect of melatonin on worms depends on its capacity to scav-
enge free radicals in cells. Thus, at a concentration of 10 mg/L
melatonin failed to influence the life span in the round worm
C. elegans but increased it in planaria. In both the species
increased doses of melatonin had a toxic effect and reduced
the life span of worms.
EFFECT OF PINEAL TRANSPLANTATION ON LONGEVITY
Syngeneic transplantation of the pineal gland from young
(3–4-month-old) donors into the region of the thymus of
old (16–22-month-old) BALB/c, C57BL/6 and (BALB/c ×
C57BL/6)F1 mice was followed by an increase in their life
span. In control groups the maximum life span was 26
months, whereas some mice with transplanted pineal glands
Vol. 31, No. 6, 2003 EFFECTS OF EXOGENOUS MELATONIN 595
survived 31 and 33 months. The mean life span in these mice
was increased by 17%–27% (116). The authors noted the
normalization of the morphological structure of the thymus
and thyroid gland after implantation of the pineal gland into
the thymus. It is worth noting that there were only 3–7 ani-
mals in each group.
Transplantation of the pineal gland from young male Wis-
tar rats to old (18-month-old) recipients into the region of thy-
mus was followed by an increase in survival of the animals
(118). Fifty percent of the control rats survived 24 months
whereas 70% of the rats with implanted pineal glands sur-
vived 24 months. The survival at the age of 26 months was
20% and 50%, respectively. The authors reported that the
weight of thymus, spleen, testicles, adrenal glands; the num-
ber of lymphocytes in peripheral blood; and testosterone in
sera in rats bearing pineal gland implants were similar to
those in young animals.
In another experiment the pineal glands of young (4-
month-old) and old (18-month-old) female BALB/c mice
were surgically removed and stereotaxically implanted re-
ciprocally in situ (77). The mean life span of the 30 sham-
operated mice was 719 ±32 days and it was 1021 ±56 days
in the old group with implanted pineal glands from young
donors (n=10, p<0.01). If the pineal glands from the old
donors were implanted into young recipients, the mean life
span was reduced to 510 ±36 days (n=10, p<0.002).
Thus, implantation of young pineal glands into old mice in-
creases their survival by 6 months, whereas implantation of
old pineal glands to young recipients shortened their life span.
The authors believe that these results suggest the concept of
the pineal gland as a “clock of aging.”It is worth noting that
transplantation of the pineal gland from 2–4-month-old male
mice into 18-month-old animals was followed by an increase
in the weight of their testicles (118).
The results of the experiments with pineal transplantation
are impressive; however, few animals were used in these stud-
ies and a synthesis or secretion of melatonin by grafted pineal
gland was never evaluated.
MELATONIN AS AN ANTIOXIDANT
According to the free radical theory of aging, various ox-
idative reactions occuring in the organism (mainly in mito-
chondria) generate free radicals as a byproduct, which cause
multiple lesions in macromolecules (nucleic acids, proteins,
and lipids), resulting in aging. This theory explains not only
the mechanism of aging per se, but also a wide variety of
age associated pathology, including cancer. Recent evidence
suggests that key mechanisms of both aging and cancer are
linked via endogenous stress-induced DNA damage caused
by reactive oxygen species (59, 60, 70, 147).
Since 1993, when melatonin was first discovered to be
a free radical scavenger (154), there have been many pa-
pers published confirming the ability of melatonin to pro-
tect DNA from free radical damage (Table 5). There is evi-
dence that melatonin in vitro directly scavenges OH, H2O2,
singlet oxygen (O·
2-), and inhibits lipid peroxidation. Mela-
tonin stimulates a number of antioxidative enzymes, includ-
ing SOD, glutathione peroxidase, glutathione reductase, and
catalase. It was shown that melatonin enhances intracellular
glutathione levels by stimulating this rate-limiting enzyme
in its synthesis, γ-glutamylcysteine synthase, and inhibits
TABLE 5.—Effect of melatonin on free radical processes.
Radical or reactive oxygen Effect of
specimens, enzyme melatonin References
Reactive oxygen specimens
Hydroxyl radical Decreased Tan et al (1993)
Singlet oxygen Decreased Cagnoli et al (1995)
Hydrogen peroxide Decreased Barlow-Walden et al (1995)
Nitric oxide Decreased Pozo et al (1994)
Peroxyl radical Decreased Pieri et al (1994)
Peroxynitrite Decreased Gilad et al (1997)
Products of oxidation
Malone aldehyde Decreased Coto-Montes and Hardeland
(1999)
Ketodiene Decreased Cuzzocrea et al (1999)
Diene conjugate Decreased Anisimov et al (1996)
Schiff’s bases Decreased Anisimov et al (1996)
CO-derivatives of amino acids Increased Anisimov et al (1997)
8-oxyguanine Decreased Coto-Montes and Hardeland
(1999)
8-hydroxy-2-deoxyguanosine Decreased Tang et al (1998),
Qi et al (2000)
Enzymes of antioxidative defense system
Cu,Zn-superoxide dismutase Increased Antolin et al (1996)
Catalase Increased Barlow-Walden et al (1995)
Gluthatione peroxidase Increased Pablos et al (1998)
Glutathione reductase Increased Urata et al (1999)
γ-Glutamylcystein synthase Increased Pierrefiche, Laborit (1995)
Glucoso-6-phosphate Increased Cuzzocrea et al (1999)
dehydrogenase
Myeloperoxidase Increased Cuzzocrea et al (1999)
Prooxidant enzymes
Nitric oxide synthase Decreased Pozo et al (1994)
the proxidative enzymes nitric oxide synthase and lipoxyge-
nase. There is evidence that melatonin stabilizes microsomal
membranes, thereby probably helping them resist oxidative
damage (69). Melatonin has been shown to increase the effi-
ciency of the electron transport chain and, as a consequence,
to reduce electron leakage and the generation of free radicals
(131). It was shown that melatonin reduced the formation of
8-hydroxy-2-deoxyguanosine, a damaged DNA product, 60
to 70 times more effectively than some classic antioxidants
(ascorbate and α-tocopherol) (125). Thus, melatonin acts as
a direct scavenger of free radicals and can detoxify both re-
active oxygen and reactive nitrogen species and indirectly
increase the activity of antioxidative defense systems (129,
131, 132, 156). However, although most papers confirm the
antioxidant potential of melatonin, under some conditions
melatonin can be a prooxidant (34, 104, 113, 127, 153, 180).
MELATONIN,DNADAMAGE AND MUTAGENESIS
There is evidence of an age-related accumulation of spon-
taneous mutations in somatic and germ cells (163, 174,
137). Accumulation with age of some spontaneous muta-
tions or mutations evoked by endogenous mutagens can in-
duce genome instability and, hence, increase the sensitivity
to carcinogens and/or tumor promoters. It has been shown
that clonally expanded mtDNA mutations accumulate with
age in normal human tissues (35). The finding that deleted
mtDNA accumulated in human muscle tissue as well as evi-
dence for partially duplicated mtDNA in aged human tissues
(26) allow us to suggest the important role of clonal expan-
sion of mutant mtDNA in the age-related increase of systemic
oxidative stress in the whole organism (44). A significant
trend toward increasing the frequency of p53 mutations with
596 ANISIMOV TOXICOLOGIC PATHOLOGY
advancing age was found in some normal and malignant tis-
sues (105, 78). Simpson (1997) suggests that the aging human
body accumulates enough mutations to account for multistep
carcinogenesis by selection of preexisting mutations.
Melatonin has been found to inhibit X-ray-induced muta-
genesis in human and mouse lymphocytes in vitro (171, 172,
173), to reduce Cis-platinum-induced genetic damage in the
bone marrow of mice (72), to decrease hepatic DNA adduct
formation caused by safrole in rats (155), and to protect
rat hepatocytes from chromium (VI)-induced DNA single-
strand breaks in vitro (152). We studied the effect of mela-
tonin on the induction of chromosome aberrations and sperm
head anomalies in mice treated with cyclophosphamide, 1,2-
dimethylhydrazine, and N-nitrosomethylurea and found that
melatonin inhibited greatly the mutagenicity of these carcino-
gens (97).
Since melatonin can protect cells directly as an antioxi-
dant and indirectly through receptor-mediated activation of
antioxidative enzymes, we applied the Ames test system
in vitro (98). Some mutagens used are known to damage
DNA through several mechanisms. In order to test these mu-
tagens, we used different strains of Salmonella typhimurium
to detect different mechanisms, in a nontoxic range of con-
centrations, using the preincubation assay. The strains TA 97
and TA 98 are sensitive to different frameshift mutagens. TA
100 detects G-A base-pair substitutions. TA 102 is sensitive
to mutagens damaging A-T pairs, mainly through free rad-
ical formation and cross-linking agents (89). We supposed
that because melatonin is an antioxidant it could affect mu-
tational events that resulted from oxidative damage and so
could inhibit revertant colony formation in the stain TA 102
specially designed to detect this type of lesion. Thus, mela-
tonin alone turned out to be neither toxic nor mutagenic in
the Ames test.
The single cell gel glectrophoresis assay (SCGE assay or
COMET assay) was used to evaluate the clastogenic effect
of melatonin. It was shown that melatonin is clastogenic at
the highest concentration tested (100 µM) in the SCGE assay
(98).
As oxidative mutagens we used DMH, bleomycin (with
S9-mix), and mitomycin C (without S9-mix), which are
believed to generate oxygen radical species (25, 86). Fur-
thermore, mitomycin C acts as a bifunctional alkylating
agent (41). Additionally, we tested 8 other intercalat-
ing and alkylating agents, both direct-acting and requir-
ing metabolic activation. The 12 mutagens used were
7,12-dimethylbenz(a)anthracene (DMBA); benzo(a)pyrene
(BP); 2-aminofluorene (AF); 1,2-dimethylhydrazine (DMH);
bleomycin; cyclophosphamide (CP); 4-nitroquinoline-N-
oxide (NQO); 2,4,7-trinitro-9-fluorenone (TNF); 9-amino-
acridine (AA); N-nitrosomethylurea (NMU); mitomycin C;
and sodium azide tested in the absence or in the presence of
S9 mix. In the four Salmonella typhimurium tester strains
TA 97, TA 98, TA 100, and TA 102, melatonin significantly
reduced the mutagenicity of chemicals that require S9 acti-
vation. In the SCGE assay performed on CHO cells, prein-
cubation with melatonin led to a strong inhibition of clas-
togenic activities of DMBA and CP, and to a lesser extent
with BP and NMU. With mitomycin C, melatonin exacer-
bated responses in both tests. It must be noted that mela-
tonin was effective as an antimutagen only at extremely high
doses (0.25–2µmol/plate) (98). The modifying activity of
melatonin was not related to the mechanism of action of
the mutagens, i.e., frameshift mutations or base-pair substi-
tutions. As melatonin displays its protective effect on pro-
mutagens, we can only speculate that it can also exert its
influence on the metabolic activation process, perhaps by in-
hibiting the cytochrome P 450-dependent mono-oxygenase
enzyme system of the S9-mix. Another explanation for the
S9- mediated effect of melatonin could be that the antimuta-
genic effect results from a metabolite of melatonin, such as
6-hydroxymelatonin. 6-hydroxymelatonin was found to be
able to inhibit the thiobarbituric-induced lipoperoxidation in
mouse brain homogenates, which is imputed to lead to the
production of oxygen free radicals (120).
Generally, the results of the SCGE assay are in accordance
with those of the Ames test. Melatonin modulates the clas-
togenicity of DMBA, BP, and cyclophosphamide. Compared
with the data obtained in the Ames test, our data showed that
melatonin inhibited the clastogenicity of the chemicals at
lower concentrations (0.1–1 nM). When melatonin is com-
bined with mitomycin C, a significant dose-related exacer-
bating effect has been observed in both tests (98). The avail-
able data shows that melatonin may play an important role
in defending cells from DNA damage induced not only by
oxidative mutagens but also by different alkylating agents.
MELATONIN AND APOPTOSIS
In the following series of studies it was shown that mela-
tonin inhibits apoptosis in brain cells induced by reactive
oxygen species (ROS) (31), kainate (55), amyloid β-peptide
related to Alzheimer’s disease (108, 144), neuromediators
(146) and neurotoxins (64), but not by N-methyl-D-aspartate
(55), staurosporine, or the neurotoxin ethylcholinazyridine
(61). It was suggested that the protective effect of melatonin
on neurocyte apoptosis depends on the model used and is
not mediated by caspase-dependent programmed cell death,
but includes prevention of glycolysation derivative-induced
necrosis (61). In some cases melatonin can enhance the dam-
age of neurons in primary culture (61). Melatonin supple-
mentation suppresses NO-induced apoptosis via induction
of Bcl-2 expression in immortalized pineal PGT-beta cells
(183). A similar pathway of the inhibitory effect of melatonin
on apoptosis is induced by ischemic neuronal injury (151).
It was shown that low doses of melatonin (10−7–10−9M)
inhibit apoptosis in both dexamethasone-treated cultured thy-
mocytes and the intact thymus (140). This effect of melatonin
was mediated by its inhibitory influence on the proliferation
of thymocytes (141). Long-term (8 months) administration
of melatonin at a daily dose of 40–50 µg per mouse prevents
thymic involution. This effect was mediated by the inhibitory
effect of melatonin on dexamethazone-induced apoptosis in
thymocytes and splenocytes (123). Administration of mela-
tonin in drinking water (15 mg/L) to mice for 40 days also
attenuated apoptotic thymocyte death caused by free radicals
and stimulated thymocyte proliferation (159).
Administration of melatonin in night drinking water
(20 mg/L) to rats exposed to 1,2-dimethylhydrazine failed
to influence an apoptotic index in normal colon mucosa but
significantly (by 1.8 times) increased it in colon tumors (10).
Treatment with the potent hepatocarcinogen aflatoxin B1 lead
to direct or indirect caspase-3 activation and consequently
Vol. 31, No. 6, 2003 EFFECTS OF EXOGENOUS MELATONIN 597
to apoptosis in the liver of rats. Treating rats with mela-
tonin enhances their hepatic antioxidant/detoxification sys-
tem, which consequently reduces the apoptotic rate and
necrobiotic changes in their liver (91).
MELATONIN EFFECTS ON GLUCOSE AND LIPID METABOLISM
The insulin/insulin-like growth factor-1 (IGF-1) signaling
pathway plays a fundamentally important role in animal phys-
iology influencing longevity, reproduction, and diapause in
many species (48, 71, 101, 162). Despite the large number of
studies, the role of melatonin on glucose metabolism remains
rather controversial. There are reports on the effects of the
lack of melatonin on insulin action in rats (23), on stimulation
of the blood glucose and insulin level in rats (50), and on a
marked decrease of insulin secretion by rat pancreatic islets in
response to glucose in vitro (111). In postmenopausal women,
oral administration of 1 mg of melatonin reduced glucose
tolerance and insulin sensitivity (30). Melatonin administra-
tion at middle age (0.2 or 0.4 mg/L in drinking water) sta-
tistically significantly decreased visceral fat, plasma insulin,
IGF-1 and leptin levels in rats (126, 181) whereas a pinealec-
tomy significantly increased them (32). Melatonin enhanced
the utilization of liver carbohydrates but suppressed hepatic
lipolysis in rats (99). Long-term treatment with melatonin
reduced hyperinsulinemia, hypertriglyceridemia, and hyper-
leptinemia and restored hepatic delta-5 desaturase activity in
type 2 diabetic rats (102). Melatonin augmented significantly
the level of total, free, and esterified cholesterol, as well as
high-density lipoprotein cholesterol in the blood of rats (50).
Supplementation of a diet with melatonin led to the increase
of the surface of atherosclerotic lesions in the proximal aorta
of hypercholesterinemic mice and increased the susceptibility
to ex vivo oxidation of atherogenic lipoproteins isolated from
the plasma during the fasting period (153). However, there
are reports on the inhibitory effect of high doses of melatonin
on oxidation of low-density lipids in rodents and humans (49,
57, 175) (see also section 7.) Thus, these observations should
be taken into consideration when long-term treatment with
melatonin is recommended. Further investigations should be
performed on animals and humans to clarify the effect of
melatonin on carbohydrate and lipid metabolism.
MELATONIN AND THE IMMUNE SYSTEM
Immunopharmacological activity of melatonin has been
demonstrated in various experimental models. Treatment
with melatonin increases the production of antibodies to
sheep erythrocytes and immune response to primary immu-
nization with T-dependent antigens (84). There is evidence
of melatonin involvement in complex relationships between
the nervous and endocrine systems (58, 79, 85). There are
melatonin membrane receptors on helper (Th). Activation of
melatonin receptors leads to an increase in the release of some
types of Th1 cytokines, including γ-interferon, interleukin-
1, and opioid cytokines related to interleukin-4 and dinor-
phine (58, 85). Melatonin at physiological concentrations
stimulates production of interleukins-1, -6 and -12 in hu-
man monocytes. These mediators can prevent stress-induced
immunodepression defending mice from virus- and bacteria-
induced lethal diseases (85). An important chain in the mech-
anism of the influence of melatonin on hemopoiesis includes
the effect of melatonin-induced opioids on opioid receptors at
stromal macrophages of bone marrow (85). It is worth noting
that γ-interferon and colony-stimulating factors (CSFs) can
modulate a production of melatonin in the pineal gland (85).
Thus, melatonin may be considered an important endogenous
neuroimmunomodulator and a potential immunotherapeutic
agent.
EFFECT OF MELATONIN ON GENE EXPRESSION
The available data on the genomic effect of melatonin is
rather scarce. In a cytogenetic study, it was shown that a
pinealectomy was followed by a decrease in ribosomal gene
activity in rats (109). Menendez-Pelaez et al (92) reported
that treatment with melatonin decreases the level of mRNA
for the rate-limiting enzyme in porphyrin synthesis, and 5-
aminolevulinate synthase, in the Harderian glands of Syrian
hamsters. Melatonin decreased the levels of mRNA for his-
tone H4 and prevented age-related decrease in mRNA for Bcl-
2, but not for p53, in thymocytes of mice (141, 142). Mela-
tonin also increased the mRNA for some antioxidant enzymes
(Mn-SOD, Cu,Zn-SOD) in the Harderian gland of Syrian
hamsters (16). Administration of melatonin caused a marked
increase in relative mRNA levels for Mn-SOD, Cu,Zn-SOD
and glutathione peroxidase in rat cerebral cortexes (74). Us-
ing the GT1-7 cell line, an in vitro model of gonadotropin-
releasing hormone (GnRH)-secreting neurons of hypothala-
mus, Roy and Belsham (138) showed that melatonin induced
protein kinase C activity 1.65-fold over the basal level and ac-
tivated c-fos and junB mRNA expression. Melatonin (1 nM)
significantly downregulates the gonadotropin-releasing hor-
mone mRNA (139). Male C57 mice were injected in the
morning with melatonin (5 mg/kg) for 10 days. The level of
gene expression in the splenocytes and peritoneal exudate
cells (PEC) was analyzed with the reverse transcription-
polymerase chain reaction (82). It was observed that mela-
tonin upregulated the level of gene expression of transform-
ing growth factor-α, macrophage-colony stimulating factor
(M-CSF), tumor necrosis factor-α(TNFα) the stem cell fac-
tor in PEC, and interleukin-1β, M-CSF, TNFα, interferon-γ,
and the stem cell factor in splenocytes. Melatonin reduced
the transcription of genes correlated to T lymphocyte acti-
vation (HLA-DBR, thymosin-β10) and to lymphokine ac-
tivated killer (LAK) activity (thymosin-β, tumor rejection
antigen—TRA 1), nRap 2) in human lymphocyte culture (33).
Administration of dietary melatonin (200 ppm) to 26-month-
old mice for 6 weeks resulted in a reduction of the basal
level of cytokine mRNA levels (interleukin-6 and TNFα)
to values found in 5-month-old mice (143). Implantation of
melatonin tubes did not affect the expression of three clock
genes, qPer2, qPer3, and qClock, in the suprachismatic nu-
cleus (SCN) of Japanese quail, however, it may act on the
mechanisms of synchronization among SCN oscillatory cells
(182). These findings are in agreement with data that the SCN
is the target site for the effect of exogenous melatonin on the
amplitude of the endogenous melatonin rhythm (28). Supple-
mentation of 1 nM melatonin into cultural media inhibited
the cell proliferation of breast cancer cells MCF-7, coinci-
dent with a significant increase in the expression of p53 as
well as p21WAF1 (90). In transgenic HER-2/neu mice, treat-
ment with melatonin inhibited mammary tumor development
and downregulated the expression of HER-2/neu oncogene in
mammary tumors (21).
598 ANISIMOV TOXICOLOGIC PATHOLOGY
To identify early molecular events regulated by mela-
tonin, gene expression profiles were studied in hearts of
melatonin-treated CBA mice in comparison with the control
using cDNA gene expression arrays (15,247 cDNA clone
set, NIA, USA) (2). The dose and schedule of the treatment
were similar to those used in the long-term study (12). Thus,
10 mice received melatonin (20 mg/L) overnight for 5 days,
and 10 mice served as a control. All mice were sacrificed on
the sixth day and their hearts were withdrawn to be frozen
immediately in a liquid nitrogen. Comparative analysis of
cDNA gene expression arrays hybridised with heart RNA
samples from control and melatonin-treated mice has shown
that the primary effectors are the genes controlling the cell cy-
cle, cell/organism defense, protein expression, and transport.
Melatonin also increased the expression of some mitochon-
drial genes (16S, cytochrome coxidases 1 and 3 [COX1 and
COX3], and NADH dehydrohenase 1[ND1]), which agrees
with its ability to inhibit free radical processes (see section 7).
Using differential display RT-PCR, Prunet-Marcassus et al-
showed that the cytochrome b gene is also a putative target for
melatonin in brown adipocytes of Syberian hamsters (124).
A significant effect of melatonin on the expression of
some oncogenesis-related genes was detected (2). While ex-
pression of myeloblastosis oncogene-like 1 (Mybl1) was
downregulated by melatonin (exceeded the 2-fold confidence
level), melatonin upregulated an expression of RAS p21 pro-
tein activator 1, Enigma homolog 2, and myeloid/lymphoid or
mixed-lineage leukemia (trithorax [Drosophila]) homolog),
translocated to 3 (MLLT3). Of a great interest is the effect
of melatonin on a large number of genes related to calcium
exchange, such as cullins, Kcnn4 and Dcamkl1, calmod-
ulin, calbindin, Kcnn2 and Kcnn4. Whereas the expression of
cullin-1 in the mouse heart is downregulated, that of cullin-
5 is highly upregulated, and expression of cullins-2 and -3
is not altered significantly. The cullin family, comprising at
least six members, is involved in ubiquinone-mediated pro-
tein degradation required for cell-cycle progress through the
G1 and S phases. Nevertheless, cullin-1, but not other mem-
bers of the cullin family, is generally thought to be implicated
in SCFs (Skp1-cullin-F-box protein ligase complexes), which
control the ubiquinone-dependent degradation of G1 cyclins
and inhibitors of cyclin-dependent kinases, thus playing an
important role in cell proliferation and differentiation (47,
75, 94). Like the effects of other proteins of this family, the
effect of cullin-5 is mediated by a SKP1/F-box-independent
mechanism. It is believed that melatonin may influence tumor
growth by interfering with calcium binding and blocking the
formation of the MAPs/calmodulin and tubulin/calmodulin
complexes to prevent cytoskeletal degradation (24). Four
serine/threonine kinases (Pctk3, FUSED, TOPK, and Stk11)
with expression increased by both peptides can be found in the
same functional category (cell signaling/communication) (2).
At least one of these, Stk11 kinase with an unclear function,
hasanticarcinogenic effects and mutations that lead to the
Peutz-Jeghers syndrome, which is associated with high risk
of tumor development in multiple localizations (62). Thus,
these data present direct evidence for the various effect of
melatonin on the expression of different genes in vivo.
Specific gene expression profiles are associated with the
aging process in animals and humans (83, 121, 178, 179).
Using whole-genome DNA microarrays, Lund et al (83)
have shown a decrease in heat shock gene expression and
an increase in the expression of insulin-like genes, leading to
decreases in insulin signaling gene expression during aging
in C. elegans. Pletscher et al (121) found no evidence relating
age-dependent changes in transcript level to genes located in
specific regions of the chromosome, and they found no sup-
port for systemic gene disregulation with advancing age in
D. melanogaster. However the authors did find that caloric
restriction results in rapid downregulation of a large number
of genes involved in cell growth and maintenance. Wein-
druch et al (178) have shown that the aging process in mice is
characterized by the activation of an adaptive stress response
consistent with increased level of reactive oxygen species in
both the skeletal muscle and brain. The aging process in mice
is characterized by reductions in the expression of biosyn-
thetic enzyme genes and genes involved in protein turnover.
Caloric restriction induced genes are involved in fatty acid
metabolism, glycolysis, and gluconeogenesis. Available data
on the effects of melatonin on gene expression, primarily mi-
tochondrial genes, suggest that some of them may be respon-
sible for the capacity of the hormone to prevent age-related
disturbances in the organism as well as for its adverse effect.
Further studies are need in this direction.
CONCLUSION
Analysis of available data on the effect of melatonin on
longevity supports the hypothesis of the geroprotective ef-
fect of melatonin. At the same time, a critical review of real
results has shown that the majority of studies are invalid from
the point of view of the current guidelines for long-term test-
ing of chemicals for carcinogenic safety (52, 53) and, to some
extent, from the point of view of the correctness of the geron-
tological experiment (177). Often in reviewed experiments
with rodents, melatonin was given to a small number of ani-
mals (10–20); the treatments started when the animals were
old; the observations stopped when the animals reach the age
of 50% mortality or at some other voluntary time, but not
at the natural death of the last survivor; an autopsy and a
correct pathomorphological examination sometimes was not
performed; the body weight gain and food consumption of
the animals were not monitored; and so on. We believe that
the study of the long-term effects of melatonin at a variety
of doses in different strains and species (e.g., in rats) will be
useful for making a conclusion on its safety. In adequately de-
signed experiments (50 animals per group), melatonin given
during the night in relatively small doses (2.5–3 mg/kg) de-
layed the onset of age-related disturbances in estrous function
and increased the survival of the animals.
There are data on the suppressive effect of melatonin on
the development of spontaneous mammary carcinogenesis
and that induced by chemical agents and ionizing radiation
in mice and rats (38, 39, 96), spontaneous endometrial ade-
nocarcinomas in BDII/Han rats (43), colon carcinogenesis
induced by 1,2-dimethylhydrazine in rats (9, 4), DMBA-
induced carcinogenesis of the uterine cervix and vagina
in mice (11), and preneoplastic liver lesions induced by
N-nitrosodiethylamine in rats (65). The positive effect of
melatonin in the treatment of advanced cancer patients has
been observed (81).
Thus, melatonin has two faces:it is both a potent gero-
protector, anticarcinogen, and inhibitor of tumor growth
Vol. 31, No. 6, 2003 EFFECTS OF EXOGENOUS MELATONIN 599
in vivo and in vitro, and in some models it may induce tu-
mors and promote tumor growth. There are no contradictions
between data on the carcinogenic and anticarcinogenic poten-
tial of melatonin. Some antioxidants, including natural ones
(e.g., α-tocopherol), have both geroprotector and tumorigenic
potential and could be potent anticarcinogens as well (see
3, 5). The results of administration of melatonin to peri-
menopausal women are promising (22). At the same time
there are real data on the adverse effects of melatonin. They
were summarized recently (168) as follows: melatonin may
cause infertility, hypothermia, and retinal damage; it reduces
sex drive in males; leads to high blood pressure, diabetes, and
cancer; and it can induce or deepen depression in susceptible
individuals. It was noted that melatonin may be dangerous for
people with cardiovascular risk factors. It should not be taken
by people with immune-system disorders (including severe
allergies), autoimmune diseases (such as rheumatoid arthri-
tis), immunosystem malignancies (e.g., lymphoma), severe
mental illness, or by those taking steroids. Thus, we believe
that further studies and clinical trials are needed to evaluate
both the efficacy and the safety for humans of melatonin—
this is still a very intriguing and two-faced hormone.
ACKNOWLEDGEMENTS
This article was written in the framework of the project
Cancer Rates Over Age, Time, and Place at the Max-Planck
Institute for Demographic Research, Rostock, Germany, and
was supported in part by grant #01-04-07007 and #02-04-
07573 from the Russian Foundation for Basic Research. The
author is very grateful to James W. Vaupel for the oppor-
tunity to use the facilities of the Max Planck Institute for
Demographic Research to complete this paper.
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