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1 (6) 2024
Alfraganus
xalqaro ilmiy jurnali
THE MECHANISMS OF THE
TOXIC EFFECT OF INTRAUTERINE
AND EARLY POSTNATAL EXPOSURE
TO PESTICIDES ON THE DEVELOPMENT
OF THE IMMUNE SYSTEM OF OFFSPRING
Abstract:
The aim of the study was to iden fy the mechanisms of the toxic effect of intrauterine and early postnatal
exposure to pes cides on the development of the immune system of offspring. Experiments were performed on white
adult female rats, which are obtained respec vely pes cides cyhalothrin or fipronil during the pregnancy and lacta on
periods. The offspring from experimental and control animals have been studied on days 3, 7, 14, 21 and 30 days a�er birth
using morphology, electron microscopy, immunohistochemistry and biochemistry methods. In was shown, that the toxic
effect of pes cide exposure through the maternal organism during pregnancy and lacta on is manifested in the form of a
viola on of the growth and forma on of the thymus, as well as thymus-dependent zones of the peripheral organs of the
immune system. In the mechanism of immunotoxic effect of pes cides a leading role plays the imbalance between
prolifera on and cells apoptosis of the thymus, caused both by direct toxic effects of drugs, and by the development of
hypothyroidism and oxida ve stress.
Аннотация
:
Целью исследования было выявление механизмов токсического воздействия внутриутробного и
раннего постнатального воздействия пестицидов на развитие иммунной системы потомства. Эксперименты
проводились на белых взрослых крысах-самках, которые получали соответственно пестициды цигалотрин или
фипронил в периоды беременности и лактации. Потомство от экспериментальных и контрольных животных
изучали на 3, 7, 14, 21 и 30-й дни после рождения с использованием методов морфологии, электронной
микроскопии, иммуногистохимии и биохимии. В работе было показано, что токсический эффект воздействия
пестицидов через материнский организм во время беременности и лактации проявляется в виде нарушения роста
и формирования тимуса, а также тимусзависимых зон периферических органов иммунной системы. В механизме
иммунотоксического действия пестицидов ведущую роль играет дисбаланс между пролиферацией и апоптозом
клеток тимуса, вызванный как прямым токсическим воздействием лекарственных препаратов, так и развитием
гипотиреоза и окислительного стресса.
Annotatsiya:
Tadqiqotning maqsadi pes tsidlarning intrauterin va tug'ruqdan keyingi erta ta'sirining nasl immun
zimining rivojlanishiga toksik ta'sir mexanizmlarini aniqlash edi. Tajribalar homiladorlik va laktatsiya davrida mos ravishda
cygalotrin yoki fipronil pes tsidlarini olgan oq ka alar urg'ochi kalamushlarida o'tkazildi. Eksperimental va nazorat
hayvonlarining avlodlari tug'ilgandan keyingi 3, 7, 14, 21 va 30-kunlarda morfologiya, elektron mikroskopiya,
immunohistokimyo va biokimyo usullaridan foydalangan holda o'rganilgan.
Ishda homiladorlik va laktatsiya davrida
onaning tanasi orqali pes tsid ta
'
sirining toksik ta
'
siri musning o
'
sishi va shakllanishining buzilishi, shuningdek immunitet
zimining periferik organlarining musga bog
'
liq zonalari sifa da namoyon bo
'
lishi ko
'
rsa lgan. Pes tsidlarning
immunotoksik ta
'
sir mexanizmida mus hujayralarining ko
'
payishi va apoptozi o
'
rtasidagi nomutanosiblik etakchi ro
'
l
o
'
ynaydi, bu dorilarning to
'
g
'
ridan-to
'
g
'
ri toksik ta
'
siri, shuningdek hipo roidizm va oksidlovchi stressning rivojlanishi
na jasida yuzaga keladi.
Keywords:
pes cides; developmental immunotoxicity; postnatal ontogenesis; thymus; immune system; apoptosis;
cells prolifera on.
Zokirova Nargiza Bahodirovna
Doctor of Medical Sciences of the Department of Histology,
Pathology and Embriology «ALFRAGANUS» UNIVERSIT
Y
ORCID: 0000-0001-9554-7073 e-mail: nzokirova72@gmail.com
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INTRODUCTION
T
he immune system, along with
the endocrine and nervous
systems, is extremely sensitive to
the effects of various environmental
pollutants. Pesticides are one of the most
common pollutants of the environment.
Widespread use of pesticides in
agriculture, in everyday life and public
health causes their entry into the human
div and animals. Adverse effects
of pesticides on the immune system
are defined as immunotoxicity, which
implies the violation of certain functions
of the immune system under the influence
of a toxic substance [1]. However, the
immunotoxic effect of pesticides is not
limited to the direct exposure of toxic
substances to immune cells, causing
their death and immunosuppression.
In a number of cases, pesticides cause
a number of metabolic changes and
are inducers of the development of
autoimmune reactions and abnormal
hypersensitivity of the organism [2; 3].
The mechanism of immunotoxic effects
of pesticides is complex and diverse. It is
shown that many pyrethroid pesticides
lead to a decrease in the concentration
of immunoglobulins, interleukins and
interferon [7; 8]. Of particular interest
is the developmental immunotoxicity,
when pesticides or other environmental
toxicants have an adverse effect on the
developing immune system of the fetus
or newborn. It is known that the fetus
and newborns are particularly sensitive
to the action of various environmental
toxicants. In experiments with zebrafish
(Danio rerio) embryos, pyrethroid
pesticide cypermethrin has been shown
to cause immune disorders as a result
of the induction of apoptosis of cells
[5]. Another pesticide, carbendazim,
also induced cell apoptosis and caused
immune and endocrine disorders in
zebrafish (Danio rerio) in embryo
development stage [4]. Mechanisms
of
developmental
immunotoxicity
of pesticides, especially in mammals
and humans, are diverse and remain
unclear. In our previous studies it
has been shown that many modern
pesticides in rats through the organism
of a pregnant mother can have a negative
effect on the development of the fetus
and the newborn [9; 10]. A pesticide
from the pyrethroid class of lambda-
cyhalothrin and an insecticide from the
class of benzopyrazoles fipronil had an
endocrine-disrupting action in the form
of hypothyroidism in the mother and
offspring [11; 12]. The same animals
showed oxidative stress in the form of
an increase in the degree of free radical
oxidation, as well as a pronounced
induction of apoptosis of cells in the
organs of offspring [9; 10; 13; 14]. All this
served as the basis for further studies of
the mechanisms of immunotoxicity of
development in the immune system of
offspring.
The aim
of the study was to identify
the mechanisms of the toxic effect of
intrauterine and early postnatal exposure
to pesticides on the development of the
immune system of offspring.
MATERIAL AND METHODS
Experiments were performed on
nulliparous, white adult female rats,
which were divided into 3 groups of 30
animals each. Two groups of animals for
30 days daily per os obtained respectively
pesticides lambda-cyhalothrin (LCT,
8 mg/kg), or fipronil (FPN, 3.6 mg/
kg). The third group receiving only the
same volume of sterile saline served as
a control. The following day, the females
were coupled to healthy males for
fertilization. Pregnancy was monitored
for the presence of sperm in vaginal
smears. Exposure of pesticides was
continued incessantly during pregnancy
and after delivery until the end of
lactation. Offspring obtained from the
experimental and control females were
studied in dynamics on days 7, 14, 21
and 30 after birth. Thymus (Th), spleen
(Sp), mesenteric lymph nodes (Mln)
and thyroid gland (Tg) were studied by
morphometric and electron microscopic
techniques. For immunohistochemical
studies of proliferation and apoptotic
cells paraffin sections of thymus and
thyroid gland have been used [13].
Apoptotic cells were detected using
a rabbit monoclonal antidiv to
fragments of caspase-3 proteins and
the family of p-53 (manufactured by
Thermo Scientific, USA). Proliferation
cells have been detected using a rabbit
monoclonal antidiv to protein Ki-67
(manufactured by Thermo Scientific,
USA). Further the numbers of labeled
proliferating, and apoptotic cells
counted on 1000 – 5000 total cells and
calculated an index of proliferation and
apoptosis, that is expressed in parts per
thousand. Furthermore, in the blood
serum of offspring was determined
level of thyroxine (T4), triiodothyronine
(T3), thyroid stimulating hormone
(TSH) of pituitary. Besides, biochemical
determination of the status of lipid
peroxidation and antioxidant enzyme
levels in the liver tissue were carried out
[9; 10]. All digital data were processed
by the method of variation statistics.
Statistical significance between control
and experimental groups was compared
using the Student’s test and P values
<0.05 were considered significant.
RESULTS AND DISCUSSION
Morphometric studies have shown
that exposure to pesticides through the
maternal organism significantly slows
the rate of postnatal growth and the
formation of the immune system of
offspring. Thus, the growth rate of the
thymus lobule area under the influence
of LCT by 10-15%, and when FPN is
applied, it is 15-30% lagging behind
the control parameters (P1, P2 <0.05).
The area occupied by the cortical zone
of the thymus decreased by 15-25%
and 20-40%, accordingly. In the lymph
nodes and spleen, a marked lag in the
formation of thymus-dependent zones
(T-zones) of organs was noted. Electron
microscopy in the thymus showed a
marked decrease in the secretory activity
of epithelio-reticular cells. And, on the
contrary, especially in the cortical zone
of the thymus, high functional activity
of macrophages was noted. In their
cytoplasm, numerous heterophagosomes
with remnants of destroyed thymocytes
were found. All this indicated that
exposure to pesticides in the embryonic
and early postembryonic periods had an
immunotoxic effect on the development
of the immune system of the offspring.
The toxic effect is manifested in the
slowing of the growth and formation of
the thymus, a decrease in the secretory
activity of the epithelio-reticular cells,
and the intensification of destruction and
death of thymocytes within the thymus.
As a result of violation of the regulatory
function of the thymus, in the peripheral
organs of the immune system (Sp, Mln),
the formation of T-zones of these organs
is slowed down.
Our morphometric studies have
shown various disturbances in the
dynamics of growth and formation of
the thyroid gland of offspring under the
influence of pesticides. The growth rate
of the total area of the epithelium of the
follicles under the influence of LCT by
10-17%, and with the action of FPN, 15-
30% lag behind the control parameters
(P1, P2 <0.05). Accordingly, the area
occupied by the colloid decreased by
15-25% and 20-40%. The slowing down
of the growth rate and the formation
of the structural and functional unit
of the thyroid gland - follicle, is most
pronounced when exposed to FPN,
compared with the action of LCT.
However, the negative effect of pesticides
was not limited only to inhibition of
follicle formation. It was found that the
effect of pesticides leads to a decrease in
the area of the follicle as a whole due to
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Alfraganus
xalqaro ilmiy jurnali
a decrease in the areas of the epithelium
of the follicle and thyrocyte. The growth
rate of the height and the average
area of the thyrocyte when exposed
to LCT by 10-20%, and when exposed
to FPN - by 15-30% lagged behind the
control parameters (P1, P2 <0.05). The
slowing of growth and formation of
the thyroid gland was accompanied by
a significant decrease in the functional
activity of the organ. Despite the high
levels of thyroid-stimulating hormone
(TSH), the concentration of thyroxine
(T4), triiodothyronine (T3) remained
significantly lower compared to the
control. Moreover, the most pronounced
hypothyroidism was observed in
offspring under the influence of FPN in
comparison with LCT.
The effect of pesticides led to
inhibition of the proliferative activity of
cells of both thymus and thyroid gland.
On the 7th day after birth, the index of
thymus cells proliferation under the
action of LCT in 1.5 times, and when
exposed to FPN 1.8 times decreased in
comparison with the control (P <0.05).
A similar decrease in the proliferation
index was found in the thyroid gland.
A significant decrease in the cell
proliferation index in both organs
persisted up to 21 days after birth. Thus,
exposure to pesticides led to inhibition
of proliferative activity of cells in both
the immune and endocrine organs of
the offspring. In this case, the negative
effect of FPN was more pronounced in
comparison with LCT. Data of a different
nature were obtained when calculating
the apoptosis index of cells in the thymus
and thyroid gland. Intrauterine and
early postnatal exposure to pesticides
led to a significant increase in the degree
of apoptosis of cells of the thyroid gland
and thymus. In the thyroid gland of the
offspring, under the influence of LCT,
the apoptosis index was 3.5-4 times, and
when FPN was 4.5-5 times higher than the
control group in all periods of the study
(P <0.05). A similar significant increase
in the apoptosis index at all times of the
study was observed in the thymus of the
experimental animals. Thus, exposure to
pesticides led to a significant increase in
the degree of apoptosis of cells of both
the thyroid gland and thymus.
The obtained data show that the toxic
effect of pesticides on the development
of the immune system of the offspring
(the developmental immunotoxicity) is
due to a number of metabolic changes
in organs and tissues. First of all, it is
endocrine-disrupting, more precisely,
thyroid- disrupting effect of pesticides,
which leads to hypothyroidism in the
mother and offspring [11; 12; 14]. Further,
it is necessary to emphasize the role of
oxidative stress, as the main inducer
of apoptosis of cells [9; 10; 13]. Recent
data show that thyroid hormones also
have a high anti-apoptotic effect, which
opens great prospects for the regulation
of apoptosis in various diseases [6]. All
this makes it possible to consider that
the intensity of induction of apoptosis
in our experiments is to a certain extent
determined by the degree of thyroid
dysfunction and the weakening of the
proliferation-stimulating
and
anti-
apoptotic effects of its hormones.
Consequently, the induction of apoptosis
in the experimental progeny is caused
not only by the direct toxic effect of
pesticides, but also largely mediated
by the weakening of the anti-apoptotic
function of thyroid hormones due
to hypothyroidism and the resulting
oxidative stress in the form of an increase
in the number of free radicals.
Conclusions:
The toxic effect of
pesticide exposure through the maternal
organism during pregnancy and lactation
is manifested in the form of a violation of
the growth and formation of the thymus,
as well as thymus-dependent zones of the
peripheral organs of the immune system.
In the mechanism of immunotoxic effect
of pesticides plays a leading role the
imbalance between proliferation and
cells apoptosis of the thymus, caused
both by direct toxic effects of drugs, and
by the development of hypothyroidism
and oxidative stress.
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