Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 11, pp. 1930-1937 © The Author(s) 2024. This article is an open access publication.

1930

Behavioral Features and Blood Enzyme Activity

in Offspring of Rats Conceived

from an Alcohol-Intoxicated Father

Sergey K. Sudakov

1,a

*, Natalya G. Bogdanova

, Galina A. Nazarova

and Nikolai N. Zolotov

Departement of Normal Physiology, I. M. Sechenov First Moscow State Medical University, 119991 Moscow, Russia

Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies,

P.K. Anokhin Research Institute of Normal Physiology, 125315 Moscow, Russia

e-mail: s-sudakov@mail.ru

Received May 22, 2024

Revised July 16, 2024

Accepted August 6, 2024

Abstract— Quite often, conception of a child occurs after consuming small doses of alcohol. However, effect of

this factor on offspring has not been studied at all. The aim of this study was to examine the level of motor

activity, anxiety-like and depressive-like behavior, sensitivity to analgesic effect of ethanol, as well as activity

of the enzymes DPP-IV, PEP, and ADG in the blood of rats whose fathers received ethanol immediately before

mating. As a result of the conducted experiments, it was found that the males conceived by the intoxicated

fathers have significant differences in behavior compared to control animals. Thus, motor activity in the rats

conceived by males under the influence of alcohol was 2-2.5 times less intense; they exhibited decreased sever-

ity of the anxiety-like and depressive-like behavior. In such animals, activity of DPP-IV and ADG was increased

and activity of PEP in the blood was reduced. In the rats conceived by the fathers under the influence of

alcohol, analgesic effect of ethanol was decreased, and there was also reduction in response of the activities

of ADG, DPP-IV, and PEP enzymes to ethanol administration. It is assumed that a single use of ethanol by

male rats immediately before mating leads to the decrease in methylation of the paternal inherited genes in

offspring. As a result, activity of a number of enzymes could change, which leads to the change in the balance

of neuropeptides involved in mediation of animal behavior.

DOI: 10.1134/S0006297924110075

Keywords: ethanol, offspring, alcohol dehydrogenase, peptidases, blood plasma, fluorimetric analysis, open field,

elevated plus maze, hot plate

Abbreviations: ADG, alcohol dehydrogenase; DPP-IV, dipep-

tidyl peptidase-IV; PEP, prolyl endopeptidase.

* To whom correspondence should be addressed.

INTRODUCTION

It has been shown that alcohol consumption

during pregnancy negatively affects behavior of the

offsprings  [1,  2]. There are also data that significant

and persistent overconsumption of ethanol by the fa-

ther prior to conception could affect behavior of the

offsprings. In particular, offsprings of the alcoholised

mice exhibited attention deficit and hyperactivity  [3],

hypoactivity  [4], learning disabilities  [5], decrease of

the anxiety level  [6], decrease of sensitivity to ethanol

[7], and decrease of alcohol craving  [8]. According to

unofficial data no less than 20% of all conceptions

in the world, and in Russia especially, occurs after

consumption of moderated doses of ethanol. How-

ever, effects of this factor on behavior of the future

offsprings have been not investigated sufficiently.

We have found only two studies devoted to the effects

of consumption of a single moderate dose of ethanol

by the male before mating. It has been reported that

the offsprings demonstrated developmental prob-

lems  [9], as well as that the offsprings demonstrated

much less of the risk-assessment behavior and were

more aggressive that the ‘normal’ animals [10].

BEHAVIOR OF RATS CONCEIVED IN ALCOHOL INFLUENCE 1931

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

Behavior of humans and animals is determined

mainly by the processes mediated by neuromediators

and neuromodulators in the central nervous system.

Neuropeptides play an important role in these pro-

cesses with their levels depending significantly on

activity of the enzymes of their degradation. In par-

ticular, dipeptidyl peptidase-IV (DPP-IV) participates

in degradation of such peptides as growth factors,

chemokines, incretins, neuropeptides, and vasoactive

peptides. It has been shown that DPP-IV is involved in

formation of ethanol tolerance and dependence [11].

Prolyl endopeptidase (PEP) participates in degra-

dation of numerous peptide hormones and neuropep-

tides. Role of PEP in effects of ethanol on organisms

of humans and animals is still poorly investigated.

Activities of DPP-IV and PEP could serve as mark-

ers of aggressive behavior  [12], levels of anxiety  [13]

and depression[14]. It was shown that the individuals

diagnosed with alcoholism have extremely low levels

of DPP-IV and PEP [15].

Alcohol dehydrogenase (ADG) is the enzyme cleav-

ing endogenous and exogenous ethanol. Role of this

enzyme in formation of ethanol sensitivity has been

investigated in great detail [16].

Based on the information presented above, goal

of this study was investigation of the level of motor

activity, anxiety-like and depression-like behavior,

sensitivity to analgesic effect of ethanol, as well as

activity of the DPP-IV, PEP, and ADG enzymes in the

blood of rats conceived by the fathers that consumed

ethanol immediately prior to mating.

MATERIALS AND METHODS

Animals. Wistar rats obtained from Stolbovaya

Laboratory Animal Facility (Moscow, Russia) were used

in the study. Animals were kept in ventilated Tecniplast

green line 1500U cages (Tecniplast, Italy) with natural

corn bedding (Zolotoy Kot, Russia), 4-5 animals per

cage with free access to water and combined food

(3 kcal/g; Profgryzun, Moscow, Russia) at average tem-

perature 21°C, average humidity 20%, with illumina-

tion (90lux) from 20:00 to 08:00.

Mating procedure. Rats with body mass 210-240  g

(males) and 170-200  g (females) at the age of 2-3months

were used in the experiments. Three groups of males

and females (4 animals of each sex per group) were

formed with equal average level of motor activity in

the groups. A female in the estrus state was placed

with a male for 24  h. At the time of mating each cage

contained 1 male and 1 female.

Males of the 1st (control) group were adminis-

tered water intragastrically. Males of the 2nd and 3rd

groups received immediately prior to mating a 20%

ethanol solution at a dose 0.5 or 1.5  g/kg intragastri-

cally through a gastric tube. Males received solution

immediately after they started exhibiting mating be-

havior, and were placed back to the cage after ethanol

administration. Female rats did not receive anything.

In each group, three females out of four produced

offsprings.

Examination of offsprings. As a result of mat-

ing of control animals 15 males and 16 females were

born. Mating of the fathers that received 0.5  g/kg

of ethanol produced 16 males and 16 females. Only

males were used in further experiments. Behavior of

the 46 males conceived under conditions described

above was examined from the age of 2 months to the

age of 3  months. Levels of motor activity was tested

sequentially every 3-5 days, as well as their behaviour

in the open field test, elevated plus maze test, and tail

suspension test was examined. And finally, sensitivity

of the rat offsprings to analgesic effects of ethanol was

tested in the hot plate test. Seventy-two hours after

administration of ethanol and determination of its

analgesic effect, animals were decapitated and blood

samples were taken for biochemical tests.

Determination of the level of motor activity.

Rats were individually placed into experimental

Phenomaster chambers (TSE Systems, Germany) for

60  min, where their total horizontal motor activity was

determined automatically, as well as motor activity in

the center and number of rearing during 60min with

10 min intervals. The experimental chambers were

identical to the housing cages in which rats were

kept to minimize effects of stress. Experiments were

carried out from 11:00 to 15:00 in the absence of il-

lumination. Simultaneously 8 Phenomaster chambers

were used.

Open field test behavioral assessment. “Open

field” comprised a round 98-cm diameter arena sur-

rounded with 31-cm high walls (OpenScience, Russia,

model TS0501-R). Arena was separated with lines into

7 central and 12 peripheral sectors. Arena was illu-

minated uniformly with a medical lamp (arena sur-

face illuminance 450 lux). At the beginning a rat was

placed into the center of arena and horizontal motor

activities were monitored (total number of crossed

sections), number of rearings, and number of center

location within five minutes.

Determination of anxiety-like and depression-

like behavior in the elevated plus maze test (EPM).

“The Elevated Plus Maze” apparatus (Columbus In-

struments, USA) was used in the study. It comprises a

cross-shaped platform with four arms (length– 50cm,

width – 15  cm). Two opposite arms have non-trans-

parent high walls, while other two are open. Height

of the walls in closed arms is 43  cm. Labyrinth is

raised to the height of 75  cm. Surface area of the

central part of the apparatus is 15  cm

. Illumination

of the labyrinth surface is 90  lux. A rat was placed

SUDAKOV et al.1932

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

for 5 min into the platform and number of crossing

from section to section was measured, as well as

time spent in the center, in the closed and open arms

of the apparatus.

Determination of the level depression-like and

in the tail suspension test. The test involves hanging

a rat with adhesive tape and special attachment up-

side down by the tail for 6  min (TSE Systems, Tail Sus-

pension Monitor, 303590 series). Duration(s) and num-

ber of immobilization periods and passive movements

were detected. Energetic whole-body movements and

rotation around the axis were considered as active

movement. Slow movements with one or two paws

and head movement, such as swaying and whisker

twitching were considered as passive movements. All

other motion-fewer periods were considered as immo-

bilization periods.

Determination of sensitivity to analgesic effect

of ethanol. Animals from all three groups for this

test were randomly classified according the level of

motor activity and separated into two subgroups:

20% ethanol solution at the dose 1.5  g/kg was intra-

gastrically administered to the rats from the first sub-

group 30  min prior to the test. Rats from the second

subgroup received isotonic solution of sodium chlo-

ride. Each animal was placed onto the surface of

a“Hot plate” instrument (TSE Systems) heated to 56°C.

Latency period until animal started licking paws was

measured.

Determination of enzyme activity. Sample prepa-

ration. Activities of ADG, PEP, and DPP-IV were deter-

mined in blood plasma samples collected into tubes

with EDTA. Samples were centrifuged for 10  min

at 3000 rpm. Plasma samples were transferred into

clean tubes and stored at –70°C before use in the ex

periments.

Fluorometric method for determination activity

of PEP and DPP-IV. Method is based on fluorometric

determination of released in the course of enzymatic

reaction 7-amino-4-methylcoumarin (AMC) from the

peptide Z-Ala-Pro-AMC (for PEP) or from the peptide

Gly-Pro-AMC (for DPP-IV), which has different fluores-

cence spectrum from the fluorescence spectra of the

peptides. Hydrolysis of the substrates was recorded af-

ter 30-min incubation at 37°C using a LS-5B spectroflu-

orimeter (Perkin-Elmer, USA). Amount of the released

7-amino-4-methylcoumarin was determined from the

level of fluorescence. Specific activity of enzymes was

calculated using equation (1):

A = [(E−C)/(S−B)]*t–1*v–1, (1)

where E – fluorescence of the sample (380/460  nm);

C – fluorescence of the mixture containing 0.05ml of

each substrate and enzyme, 1.9  ml of Tris-HCl-buffer

(prepared from Tris base Serva, Germany) (pH 8.0)

supplemented with 1  mM of each EDTA-Na

(Reanal,

Hungary), dithiothreitol (Serva) and NP-40 (Sigma,

USA) and 1  ml of acetate buffer (pH  4.0); B – fluores-

cence of the mixture containing 0.05  ml of substrate

solution, 1.95  ml Tris-HCl-buffer (pH  8.0) supplement-

ed with 1  mM of each EDTA-Na

, dithiothreitol, and

NP-40 and 1  ml of acetate buffer (pH  4.0); S– fluores-

cence of the mixture containing 0.05  ml of substrate

solution, 1.93  ml of Tris-HCl-buffer (pH  8.0) supple-

mented with 1  mM of each EDTA-Na

, dithiothreitol,

and NP-40, 1  ml of acetate buffer (pH  4.0), and 0.02  ml

of 7-amino-4-methylcoumarin solution (2  nmol, Serva).

Reaction was stopped by addition of 1 ml of acetate

buffer (pH 4.0) to the incubation mixture.

Spectrophotometric method for determination of

ADG activity. Activity of ADG in blood plasma was

determined with the modified method suggested by

Mezey et al.  [17]. For this a 50-µl plasma samples

were mixed with 400  µl of glycine buffer (pH8.8), 3%

ethanol solution, and NAD solution (3  mg/ml, Fluka,

Switzerland); next each sample was incubated in a

water bath for 60  min at 37°C followed by measuring

optical density at 340 nm with a DU-50 spectropho-

tometer (Beckman, USA). Activity of ADG (µmol NADH/

ml*min) was calculated using molar extinction coef-

ficient of the reduced form of pyridine nucleotides

(6.22  mmol/ml/cm).

Statistical data processing. Statistical data pro-

cessing was carried out according to algorithms of

Statistica 13.0 program with testing normality of data

distribution using Shapiro–Wilk test. In the case of

normal distribution one-way ANOVA test was used to

compare mean values of several independent data sets

with following comparison of the mean values using

Duncan’s test; in the case of non-normal data distribu-

tion non-parametric one-way Kruskal–Wallis test was

used followed by post  hoc analysis using non-para-

metric Mann–Whitney U-test. Data are presented as a

M  ±  SEM, where M– is a mean value, SEM– standard

error of the mean.

RESULTS

Behavioral changes. It was shown that the males

conceived by the fathers in the state of intoxication

exhibited significant differences in behavior in com-

parison with the normal animals. In particular, mo-

tor activity of the rats conceived by the fathers in

the state of intoxication was 2-2.5-fold less intense:

U  =  36.00, p  =  0.000997 in comparison of the ‘Control’

group with the ‘Ethanol 0.5  g/kg’ group and U  =  62.00,

p  =  0.001642– in comparison with the ‘Ethanol 1.5g/kg’

group (Fig. 1a).

It was also found out in the ‘Open field’ test that

the rats conceived by the ethanol-affected fathers

BEHAVIOR OF RATS CONCEIVED IN ALCOHOL INFLUENCE 1933

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

Fig. 1. Changes in behavior of rat males conceived by the fathers in intoxicated state. a) Motor activity (arb. units) in ex-

perimental chambers Phenomaster; b) horizontal motor activity (arb. unit) in the ‘open field’ test; c) vertical motor activity

(rearing) in the ‘open field’ test; d)duration of staying in open arms of EPM(s); e) duration of rat immobilization in the tail

suspension test (s). Significant differences from the control are presented (non-parametric Mann–Whitney U-test).

exhibited lower horizontal motor activity (significant

difference from the control, U  =  19.50, p  =  0.000027 for

the group ‘Ethanol 0.5  g/kg’; U  =  33.50, p  =  0.000018 for

the group ‘Ethanol 1.5  g/kg’, Fig.  1b) and vertical mo-

tor activity (significant difference from the control,

U  =  32.50, p  =  0.000478 for the group ‘Ethanol 0.5  g/kg’;

U  =  78.00, p  =  0.00995 for the group ‘Ethanol 1.5  g/kg’,

Fig. 1c).

Time spent in the open arms of the elevated plus

maze (EPM) by the males conceived by the fathers

exposed to 1.5  g/kg of ethanol increased more than

5-fold (U  =  14.50, p  =  0.00001) (Fig. 1d).

In the ‘Tail suspension test’, the rats conceived

by the fathers intoxicated with alcohol demonstrated

reduced immobilization time (significant difference

from the control, U  =  49.50, p  =  0.007544 for the group

‘Ethanol 0.5  g/kg’; U  =  77.50, p  =  0.008991 for the group

‘Ethanol 1.5  g/kg’, Fig. 1e).

It was revealed that pain sensitivity of the rats

conceived by the fathers under effect of alcohol

SUDAKOV et al.1934

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

Fig. 2. Latency of paw licking in the ‘Hot plate’ test.

Table 1. Activity of the ADG, DPP-IV, and PEP enzymes in rat blood plasma

Saline/Ethanol

ADG (µmol/mg/min)

Control Ethanol 0.5 g/kg Ethanol 1.5 g/kg

Saline 19.073 ± 0.91 19.475 ± 0.70

21.798 ± 0.96*

MS = 4.2465, df = 42.0

p = 0.0428

Ethanol 1.5 g/kg

17.896 ± 0.77

MS = 4.2465, df = 42.00

p = 0.0027

16.969 ± 0.61

MS = 4.2465, df = 42.00

p = 0.0104

19.819 ± 0.71

DPP-IV (nmol/mg/min)

control ethanol 0.5 g/kg ethanol 1.5 g/kg

Saline 2.694 ± 0.15 2.943 ± 0.14

3.269 ± 0.09*

U = 12.00,

p = 0.0379

Ethanol 1.5 g/kg

3.077 ± 0.2

U = 13.00,

p = 0.0488

3.134 ± 0.11 3.355 ± 0.12

PEP (nmol/mg/min)

control ethanol 0.5 g/kg ethanol 1.5 g/kg

Saline 0.946 ± 0.04 0.955 ± 0.03

0.789 ± 0.01*

U = 7.00,

p = 0.0069

Ethanol 1.5 g/kg

0.774 ± 0.02

U = 6.00,

p = 0.0046

0.804 ± 0.03

U = 5.00,

p = 0.0029

0.799 ± 0.01

Note. *  Significant differences between the groups of offsprings conceived by the fathers exposed to ethanol and

conceived by the fathers that received water; # significant differences between the animals that received saline

and ethanol in each group of offsprings.

BEHAVIOR OF RATS CONCEIVED IN ALCOHOL INFLUENCE 1935

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

does not differ significantly from the control rats.

Administration of 1.5  g/kg of ethanol resulted in signif-

icant increase of the latency of paw licking (analgesia)

both in the control animals and in the rats conceived

by the fathers exposed to 0.5  g/kg of ethanol. However,

no analgesic effect of ethanol was observed in the rats

conceived by the fathers that received ethanol at the

dose 1.5 g/kg (Fig. 2).

Changes in enzyme activities. Activity of ADG

in the animals conceived by the fathers exposed to

1.5  g/kg of ethanol was significantly higher than in the

animals of all other groups. Activity of ADG signifi-

cantly decreased after ethanol administration in all

groups (Table 1).

Activity of DPP-IV in the animals conceived by the

fathers exposed to 1.5  g/kg of ethanol was significantly

higher than in the animals of all other groups. Follow-

ing ethanol administration activity of DPP-IV increased

significantly only in the control animals (Table 1).

Activity of PEP in the animals conceived by the

fathers exposed to 1.5  g/kg of ethanol was significantly

lower than in the animals of all other groups. Follow-

ing ethanol administration activity of PEP decreased

significantly in the animals of the control group and

in the animals conceived by the fathers exposed to

ethanol at the dose 0.5  g/kg. No decrease of the activ-

ity of PEP after ethanol administration was observed

in the rats conceived by the fathers exposed to 1.5  g/kg

of ethanol (Table 1).

Comparison of the activities of ADG was carried

out using one-way ANOVA, which followed by the

comparison of mean values of dispersion complex us-

ing Duncan’s test. Comparison of activities of other en-

zymes was carried out using non-parametric one-way

dispersion analysis using Kruskal–Wallis test followed

by posterior paired analysis based on Mann–Whitney

U-test.

DISCUSSION

We demonstrated pronounced differences in the

behavior of male rats with fathers exposed to minor

or moderate doses of ethanol prior to mating. In par-

ticular, decrease of the level of motor activity was ob-

served both in the Phenomaster apparatus and in the

‘Open field test’. Previously hypoactivity of mice was

shown for the animals with fathers exposed to long-

term action of ethanol prior to mating  [4]. We also

showed increase of the time spent in the open arms of

the elevated plus maze by the animals with intoxicat-

ed fathers, which indicates reduced levels of anxiety.

This phenomenon was also described previously for

the offsprings with fathers chronically exposed to eth-

anol prior to mating  [6]. In addition, the results of our

experiments demonstrated that administration of eth-

anol at the dose 1.5  g/kg to the fathers prior to mating

results in producing offspring with reduced depres-

sion-like response to stress and decrease of sensitivity

to analgesic effect of ethanol. Decrease of sensitivity

to ethanol was also shown previously for the offspring

of chronically intoxicated mice  [7]. Effects on depres-

sion have not been demonstrated previously. Hence,

administration of a single minor or moderate dose of

ethanol to the rat males immediately prior to mating

cause the same behavioral changes as in the offspring

of the fathers with chronic exposure to alcohol.

Activity of ADG in the blood of animals with fa-

thers under the influence of alcohol was higher than

in the control rats. It has been shown that ADG could

participate in the mechanisms of analgesia induced

by administration of ethanol  [18], hence, decrease of

sensitivity to analgesic effect of ethanol in the ani-

mals conceived by the intoxicated fathers could be

explained by this fact.

The results of our experiments do not allow re-

vealing causative association between the activity of

enzymes and behavioral changes. However, we could

suggest that the increase of activity of DPP-IV and de-

crease of activity of PEP in the blood of animals con-

ceived by the fathers under the alcohol influence facil-

itate changes of the ratio of regulatory neuropeptides

and peptide hormones, which, in turn, could cause be-

havioral changes. In order to find out what particular

changes occur with the changes in activities of DPP-IV

and PEP, further experiments are required. Neverthe-

less, certain suggestions could be made. In particular,

decrease of the activity of PEP in blood could result

in increase of the level of thyrotropin-releasing hor-

mone in blood, which exerts an antidepressant effect

[19,  20]. It was shown previously in the experiments

with mice that the synthetic inhibitors of PEP have an

antidepressant activity [21].

Increase of the DPP-IV activity in offspring males

could indicate decrease of concentration of the sub-

stance  P (SP) affecting perception of pain via modula-

tion of SP concentration at the periphery and activation

of the peripheral NK1 receptors by the peptide  [22].

Endomorphin-2 is another important substrate of

DPP-IV  [23]. It is likely that DPP-IV modulates analgesia

induced by endomorphin-2. The third important sub-

strate of DPP-IV is neuropeptide  Y (NPY)  [24], which

plays a very important role in regulation of affective

state, reaction to stress, and pathogenesis of a number

of diseases, including depression.

In addition, we have found out that the offspring

males conceived by the fathers under alcohol influ-

ence exhibit suppression of the reaction of enzyme

activities on ethanol administration. While in the con-

trol animals’ administration of ethanol results in in-

crease of the DPP-IV activity and decrease of the PEP

activity, the offspring of the intoxicated fathers do not

SUDAKOV et al.1936

BIOCHEMISTRY (Moscow) Vol. 89 No. 11 2024

exhibit such response. This could be manifestation of

the general trend: offspring of the males exposed to

ethanol prior to mating are less sensitive to most of

the ethanol effects [7].

It was shown that chronic consumption of eth-

anol by the mouse males results in the decrease of

methylation of imprinted genes in spermatozoa, in-

cluding genes of neurotrophic factors  [25]. This could

result in the change of functioning of limbic system in

the brain and, as a result, to the change in animal be-

havior. Considering that the single-dose administration

of ethanol exerts the same effect as the chronic one,

it cannot be ruled out that the observed in this study

changes in the offspring of the intoxicated fathers

could be associated with this epigenetic mechanism.

As a result, activity of a number of enzymes could

change, which, in turn, could result in the change of

the balance of neuropeptides participating in media-

tion of the animal behavior.

Contributions. S.K.S. concept and supervision of

the study; N.G.B. and G.A.N. conducting experiments;

S.K.S. and N.N.Z. discussion of the results and writing

text of the paper; N.G.B. editing text of the paper.

Funding. This work was supported by ongoing in-

stitutional funding. No additional grants to carry out

or direct this particular research were obtained.

Ethics declarations. All applicable international,

national, and/or institutional guidelines for the care

and use of animals were followed. The authors of this

work declare that they have no conflicts of interest.

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