Mechanisms of Ageing and Development
120 (2000) 141 – 149
www.elsevier.com/locate/mechagedev

Effect of epitalon on the lifespan increase in
Drosophila melanogaster
V.Kh. Khavinson a, D.M. Izmaylov b,*, L.K. Obukhova b,
V.V. Malinin a
a
St. Petersburg Institute of Bioregulation and Gerontology,
North-West Branch of the Russian Academy of Medical Sciences, 3 Prospect Dynamo,
St. Petersburg 197110, Russia
b
N.M. Emanuel Institute of Biochemical Physics of the Russian Academy of Sciences,
4 Kosygin Street, Moscow 117977, Russia

Received 14 May 2000; received in revised form 11 September 2000; accepted 14 September 2000

Abstract
The geroprotector activity of epitalon, a synthetic tetrapeptide Ala – Glu – Asp – Gly, was
studied on the Drosophila melanogaster wild strain Canton-S. The substance was added to
the culture medium only at the developmental stage (from egg to larva). Epitalon signiﬁcantly increased the lifespan (LS) of imagoes by 11 – 16% when applied at unprecedented low
concentrations — from 0.001 ×10 – 6 to 5 ×10 – 6 wt.% of culture medium for males and from
0.01× 10 – 6 to 0.1×10 – 6 wt.% of culture medium for females. The increase in LS did not
depend on the substance dose. Effective concentrations of epitalon were 16 000 – 80 000 000
times lower than those of melatonin. The possible mechanisms of the antioxidant and
regulatory effects of epitalon are discussed. © 2000 Published by Elsevier Science Ireland
Ltd.
Keywords: Epitalon; Ultralow concentrations; Lifespan; Drosophila melanogaster

1. Introduction
Studying the biological activity of peptide regulators is one of the most promising
ﬁelds of modern experimental gerontology. These studies started with a long-term
* Corresponding author. Tel.: +7-959-397254; fax: +7-951-374101.
E-mail address: izm-dm@mail.ru (D.M. Izmaylov).
0047-6374/00/$ - see front matter © 2000 Published by Elsevier Science Ireland Ltd.
PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 2 1 7 - 7

142

V.K. Kha6inson et al. / Mechanisms of Ageing and De6elopment 120 (2000) 141–149

investigation of the roles of epithalamin (a pineal gland preparation) and melatonin
(a pineal gland hormone) in the structural and functional homeostasis of living
systems (Anisimov et al., 1994).
The purpose of this study was to estimate the geroprotector effect of epitalon, a
synthetic tetrapeptide Ala – Glu – Asp–Gly obtained by means of targeted construction based on amino acid analysis of epithalamin (Khavinson and Morozov, 1999).
The data obtained indicated that the preparation increased the mean lifespan
(MLS) of adult Drosophila melanogaster when added to the nutrient medium only
at the developmental stage at concentrations as unprecedentedly low as 0.001×
10 – 6 wt.%. In addition, we found that the geroprotector effect, namely, an 11–16%
increase in MLS, was almost independent of the substance concentration within the
range from 0.001×10 – 6 to 5× 10 – 6 wt.%.

2. Materials and methods
In this study, Drosophila melanogaster of a wild strain, Canton-S, was used. All
the experiments were conducted under constant conditions of 25°C, 75% relative
humidity, and light/dark cycle of 0:24.
Two types of culture medium were prepared: one for the developmental stage
and one for determination of adult LS. The culture medium of the ﬁrst type
contained 1000 ml water, 120 g yeast, 100 g sucrose, and 15 g agar. The culture
medium of the second type contained 1000 ml water, 100 g cornmeal, 100 g sucrose,
and 4.5 g agar. After 30 min cooking, the hot medium was poured into 100× 25
mm glass vials whose surfaces were seeded with a suspension of living yeast and
dried at room temperature for 24 h.
An outbreeding cultivation protocol was followed continuously and uniformly
for all experiments. Randomly chosen 5-day virginal imagoes were pair mated for
1 day, and the next generation was observed. A total of 50–80 pairs were used to
obtain each successive generation. After each eclosion, randomly chosen 5-day
insects were crossed to obtain the next generation.
As a geroprotector, we applied the peptide Ala–Glu–Asp–Gly in solution form,
produced by S. Petersburg Institute of Bioregulation and Gerontology (Khavinson
and Morozov, 1999). The peptide as sterile (pure) solution (1 mg in 1 ml of isotonic
solution) was introduced into the culture medium cooled down to 50–60°C, and the
mixture was then thoroughly stirred. The concentration of peptide was calculated
per unit mass of culture medium. For experimental investigation ﬁve concentrations
of epitalon were used: from 0.001 ×10 − 6 to 5 × 10 − 6% of culture medium weight.
The life span (LS) was registered as follows. A total of 90 virgin ﬂies of both
sexes were randomly chosen from each eclosion, and ten ﬂies of each sex were
placed separately in each vial. In the survival experiments, the culture medium was
replaced three times a week.
As LS distribution parameters, we used mean values and standard errors.
Standard t-test was used to compare the differences between mean values (Zar,
1984).

V.K. Kha6inson et al. / Mechanisms of Ageing and De6elopment 120 (2000) 141–149

143

Table 1
Mean life-span (MLS) and standard error (S.E.) in control and epitalon-treated groups
No.

Treatment

Concentration
× 10−6%

MLS 9S.D. days
(relative effect, %)
Males

1

Control
Epitalon
Control
Epitalon
Control
Epitalon
Control
Epitalon
Control
Epitalon

2
3
4
5

a
b

0.001
0.01
0.1
1.0
5.0

Females

35.419 1.31
41.30 9 1.40 (+16.6)a
32.55 91.14
36.47 9 1.09 (+12.0)a
31.449 1.35
34.859 1.18 (+10.8)a
29.999 1.38b
33.809 1.28 (+12.8)a,b
33.90 91.14
37.74 91.16 (+11.3)a

32.0791.07
34.3690.98 (+7.1)
31.2290.91
35.4191.07 (+13.4)a
31.4291.10
35.1391.23 (+11.8)a
32.3991.17b
33.43+1.23 (+3.2)b
36.8791.40
38.0491.56 (+3.2)

MLS difference is estimated as signiﬁcant (t-test, PB0.05).
Average data, original data are in Table 2.

3. Results
Table 1 shows the characteristics of LS distribution, including mean values,
standard errors of the mean (Zar, 1984), and changes in MLS (expressed as a
percentage) after epitalon treatment.

Fig. 1. Relative effect of epitalon treatment on mean life-span in D. melanogaster males.

144

V.K. Kha6inson et al. / Mechanisms of Ageing and De6elopment 120 (2000) 141–149

Fig. 2. Survival curves in D. melanogaster males (epitalon concentration): (a) 0.001 × 10 − 6%;
(b) 0.01× 10 − 6%; (c) 0.1× 10 − 6%; and (d) 5×10 − 6%.

V.K. Kha6inson et al. / Mechanisms of Ageing and De6elopment 120 (2000) 141–149

145

Fig. 2. (Continued)

Note that epitalon did not affect the duration of the D. melanogaster developmental stages from egg to imago, which generally suggests an absence of a genotoxic
(mutagenic) effect of the substance.
Epitalon had a signiﬁcant geroprotector effect on males at all concentrations
studied: their MLSs signiﬁcantly increased by 10.8–16.6% in different experimental
groups. Fig. 1 shows the results obtained in the form of a diagram. The corresponding survival curves for the control and experimental groups are shown in Fig.
2(a– d). As is seen from the ﬁgure, the difference between the experimental and
control survival curves in each experiment are mostly restricted to the segment of
the mass mortality, with the initial plateau and the maximal LS being unaffected.
An experiment with an epitalon concentration of 1.0× 10 – 6% was carried out in
triplicate (Table 2). We found that, at the same concentration of the substance, the
MLS may be considerably (by 21.8%), moderately (by 13.3%), or only slightly and
nonsigniﬁcantly increased (by 3.3%) compared to the control value. The average
MLS changes in the three experiments were +12.8 and + 3.2% for males and
females, respectively (Table 1, row 4). Fig. 3(a–c) show survival curves for
experimental and control males after treatment with 1.0× 10 – 6% epitalon. It is
worth noting an obvious trend: the lower the control MLS, the higher the relative
epitalon effect on MLS in the experimental group. Similar reverse relationships
between the geroprotector effect and the varying viability in the control population
were found earlier and described in detail for 4-hydroxy-TEMPO (Izmaylov and
Obukhova, 1996) and melatonin (Izmaylov and Obukhova, 1999).
In females, epitalon was only effective at concentrations of 0.01 × 10 – 6, 0.1 × 10 – 6
and once at 0.1×10 – 6%, at which the MLS was signiﬁcantly increased by 13.4 and
11.8% (Table 1) and 9.8% (Table 2), respectively. At other concentrations, differences between the experimental and control groups were not statistically signiﬁcant.

146

V.K. Kha6inson et al. / Mechanisms of Ageing and De6elopment 120 (2000) 141–149

Fig. 3. Survival curves in D. melanogaster males. (a – c) epitalon concentration, 1 ×10 − 6% (three
replays).

V.K. Kha6inson et al. / Mechanisms of Ageing and De6elopment 120 (2000) 141–149

147

Table 2
Mean life-span (MLS) and standard error (S.E.) in control and epitalon-treated groups (concentration
1.0×10−6%)
No.

Treatment

Concentration

MLS 9 S.D. days (relative effect, %)

−6

×10

%
Males

4.1

4.2

4.3

a

Females
32.32 9 0.97
32.5990.84
(+1.0)
36.17 9 1.23
35.76 91.35
(−1.1)
28.67 90.93
31.49 91.28
(+9.8)a

Control
Epitalon

1.0

30.31 91.33
31.30 9 1.03 (+3.3)

Control
Epitalon

1.0

32.45 9 1.41
36.77 9 1.44 (+13.3)a

Control
Epitalon

1.0

27.20 9 1.35
33.14 91.32 (+21.8)a

MLS difference is estimated as signiﬁcant (t-test, PB0.05).

4. Discussion
The data obtained are fundamentally new and unique due to the ultralow
concentrations of the peptide preparation.
The literature data indicate that concentrations of 10 – 3 –10 – 1% are the lower limit
for the geroprotector effects of most substances on D. melanogaster. This concerns
both synthetic and natural substances. Examples of the former are Gerovital,
3-hydroxypyridine, Dibunol, Centrophenoxin (Obukhova et al., 1979; Obukhova
and Emanuel, 1984; Frolkis and Muradyan, 1988), and 4-hydroxy-TEMPO (Izmaylov and Obukhova, 1996). The latter are vitamins A, E, and C (Baker, 1993; Massie
et al., 1993); hormones (Hochschild, 1971), lactic and gluconic acids, glutathione
(Obukhova and Emanuel, 1984; Frolkis and Muradyan, 1988), melatonin, and
epithalamin (Anisimov et al., 1994, 1997, 1998; Izmaylov and Obukhova, 1999;
Khavinson and Morozov, 1999). For example, melatonin increased MLS to about
the same extent as epitalon (by 13–16%) in similar experiments, with its optimum
concentration being 80 000 ×10 – 6% (Izmaylov and Obukhova, 1999). In other
words, epitalon produced the same effect as melatonin at 16 000–80 000 000-fold
lower concentrations.
Our data indicate that the effective concentration range of epitalon for D.
melanogaster males is unprecedentedly wide: from 0.001× 10 – 6 to 5 × 10 – 6% (the
upper and lower limits differ by 5000 times). Note that the highest and the lowest
effective concentrations yielded almost the same signiﬁcant effect (on average, a
13% increase in MLS), i.e. the effect did not depend on the dose of the substance.
Analysis of the possible mechanism of epitalon action is of interest. For this
purpose, it should ﬁrst be noted that MLS was changed in adult populations,
whereas only larvae were treated with epitalon. Between these stages, the insects
undergo a complete metamorphosis, with intense lysis of larval and pupal tissues

148

V.K. Kha6inson et al. / Mechanisms of Ageing and De6elopment 120 (2000) 141–149

and formation of adult tissues out of the small amounts of cells contained in
imaginal disks (Ashburner, 1989). Only a structure capable of template duplication,
i.e. nuclear DNA, may ensure a larva–imago informational succession. Therefore,
epitalon affects the larval genetic material so that the adult MLS is changed. We
observed earlier similar phenomena in experiments with 3-hydroxypyridine
(Obukhova et al., 1979), melatonin (Izmaylov and Obukhova, 1999), and 4-hydroxy-TEMPO (Izmaylov and Obukhova, 1996).
Differences between males and females with respect to the dose–effect relationship apparently reﬂect the effect of epitalon on the female reproductive function,
which masks the ‘pure’ effect of epitalon on LS. Results of other studies using
melatonin (Anisimov et al., 1998; Izmaylov and Obukhova, 1999) also suggest
differential sensitivity of males and females to the substance. As in the present
work, female MLS in these experiments was changed to a lesser degree than male
MLS. These effects of the pineal gland peptides call for further study.
Apparently, epitalon does not scavenge free radicals in the body of D.
melanogaster as antioxidant geroprotectors do. In other words, the geroprotector
effect of ultralow concentrations of this substance is not accounted for by an
antioxidant mechanism. The effect obtained is most likely to be determined by
optimization of vital functions due to the regulatory properties of short-lived
peptides (Ashmarin and Kamenskaya, 1988), which are characterized by long-term
action. Short-lived peptides are involved in an evolutionarily ancient regulatory
mechanism and are widespread in lower organisms (Ashmarin and Kamenskaya,
1988).
The unusual pattern of the concentration–effect relationship with a large plateau
(from 0.001× 10 – 6 to 5× 10 – 6%) reﬂects the characteristic dynamics of interaction
between a small number of molecules and their targets. There are other examples
where the effect does not depend on concentration within a wide range (Burlakova,
1994). This is determined by fundamentally different kinetic patterns of relationships between biological structures and ultralow amounts of active substances
(Burlakova, 1994), rather than speciﬁcity of the substances (antioxidants, herbicides, immune response modiﬁers, toxins, etc.) or the objects.
Consequences of epitalon metabolism after it enters the digestive tract of a D.
melanogaster larva deserves special consideration. Is the geroprotector effect of the
substance determined by the properties of the tetrapeptide itself or the products of
its decomposition, i.e. dipeptides or amino acids? The tetrapeptide undoubtedly
undergoes enzymatic hydrolysis (Ashburner, 1989). However, the products of the
possible degradation of epitalon could not noticeably affect the general metabolic
amino acid ﬂow in the insect body, taking into account the low concentrations of
epitalon in the medium. In addition, the Ala–Glu–Asp–Gly sequence is obviously
not unique and may be found in native proteins; therefore, hydrolysis of food
proteins might yield the same tetrapeptide. The geroprotector properties of epitalon
found in this study apparently result from its interaction with speciﬁc receptors, the
location of which in the body of D. melanogaster larva requires further
investigation.

V.K. Kha6inson et al. / Mechanisms of Ageing and De6elopment 120 (2000) 141–149

149

In order to estimate the geroprotector effectiveness of epitalon by means of
extrapolation, let us make the following assumptions. A larva weighing
1.5 mg
(Church and Robertson, 1966) eats approximately ﬁve times its own weight (Chiang
and Hodson, 1950), i.e. 7.5 mg, while kept on the culture medium. This amount of
the medium contains 7.5 ×10 – 11 mg of epitalon, given its concentration C=
0.001× 10 – 6%. An average human weighs 75 kg. If we set up a proportion, we will
ﬁnd that the effective dose of epitalon for humans is
0.075 mg or 0.001 mg/kg
body weight.

References
Anisimov, V.N., Khavinson, V.Kh., Morozov, V.G., 1994. Twenty years of study on effect of pineal
peptide preparation: epithalimin in experimental geerontology and oncology. Ann. NY Acad. Sci.
719, 483–493.
Anisimov, V.N., Mylnikov, S.V., Oparina, T.I., Khavinson, V.Kh., 1997. Effect of melatonin and pineal
peptide preparation epithalamin on life span and free radical oxidation in D. melanogaster. Mech.
Ageing Dev. 97, 81–91.
Anisimov, V.N., Mylnikov, S.V., Khavinson, V.Kh., 1998. Pineal peptide preparation increases the life
span of fruit ﬂies, mice and rats. Mech. Ageing Dev. 103, 123 – 132.
Ashburner, M., 1989. Drosophila: A laboratory handbook, Cold Spring Harbor Laboratory.
Ashmarin, I.P., Kamenskaya, M.A., 1988. Neuropeptides in synaptic transmission. VINITI, Itogi Nauki
i Tehniki, Ser. Phisiol. Cheloveka i Zhivotnyh, Moscow (in Russian).
Baker, G.T., 1993. Effect of various antioxidants on aging in Drosophila. Toxicol. Ind. Health 9,
163–186.
Burlakova, E.B., 1994. The effect of extra-low doses. Heralds Russian Acad. Sci. 64, 425 – 431.
Chiang, H.C., Hodson, A.C., 1950. An analytical study of population growth in Drosophila
melanogaster. Ecol. Monog. 20, 173– 206.
Church, R.B., Robertson, F.W., 1966. A biochemical study of the growth of Drosophila melanogaster.
J. Exp. Zool. 162, 337–352.
Frolkis, V.V., Muradyan, Kh.K., 1988. Experimental methods of life prolongation, Nauka, Leningrad,
pp. 181–188.
Hochschild, R., 1971. Effect of membrane stabilizing drugs of mortality in Drosophila melanogaster.
Exp. Gerontol. 6, 133–151.
Izmaylov, D.M., Obukhova, L.K., 1996. Geroprotector efﬁciency depends on viability of control
population: life span investigation in D. melanogaster. Mech. Ageing Dev. 91, 155 – 164.
Izmaylov, D.M., Obukhova, L.K., 1999. Geroprotector effectiveness of melatonin: investigation of
lifespan of Drosophila melanogaster. Mech. Ageing Dev. 106, 233 – 240.
Khavinson, V.Kh., Morozov V.G., 1999. Novel approaches to the prevention and treatment of
age-associated pathology using peptide bioregulators-epithalamin and thymalin. Abstract of the
Pan-American Congress on Gerontology and Geriatrics, San Antonio, p. 46.
Massie, H.R., Aiello, V.R., Williams, T.R., Baird, M.B., Hough, J.L., 1993. Effect of vitamin A on
longevity. Exp. Gerontol. 28, 601–610.
Obukhova, L.K., Nakaidze, N.Sh., Serebryany, A.M., Smirnov, L.D., Akif’ev, A.P., 1979. Experimental
analysis of the mechanism of aging in Drosophila melanogaster. Exp. Gerontol. 14, 335 – 342.
Obukhova, L.K., Emaneul, N.M., 1984. Molecular mechanisms of delaying senescence with antioxidants. In: Emanuel, N.M., Nadzharyan, T.L. (Eds.), General Problems of Biology, Biological
Problems of Aging, VINITI, Moscow, vol. 6, pp. 44 – 80.
Zar, J.H., 1984. Biostatistical Analysis, second ed. Prentice-Hall, Englewood cliffs, NJ.

.

