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DOI 10.11603/ijmmr.2413-6077.2022.1.12924

ANTIOXIDANT PROTECTION IN WHITE RATS ON THE BACKGROUND  
OF DRINKING WATER USE WITH EXCESSIVE NITRATE CONCENTRATION

M. V. Danchyshyn, O. V. Lototska 
I. HORBACHEVSKY TERNOPIL NATIONAL MEDICAL UNIVERSITY, TERNOPIL, UKRAINE

Background. The problem of nitrate pollution in terms of its scale and consequences has reached a global 
level. Approximately one-third of groundwater bodies for which information is currently available exceed the 
maximum allowable concentrations of nitrates in drinking water.

Objectives. The aim of the research is to study the features of antioxidant protection in the body of white 
rats on the background of drinking water with excessive concentrations of nitrates for 30 days regarding the age.

Methods. The experiment was performed on 2 groups of outbred white female rats of different age (mature 
and immature), 30 animals each, divided into 5 subgroups, which differed in the quality of drinking water they 
consumed independently. In a 30-day study, nitrates were given to rats with drinking water at a dose of 50, 150, 
250, 500 mg of sodium nitrate per liter. The animals of the control group drank water from the city water supply. 
The effect of nitrates on superoxide dismutase (SOD), catalase (CAT) and ceruloplasmin (CP) in the serum of rats 
was evaluated.

Results. The research has shown increased SOD, CAT and CP in blood serum on the background of drinking 
water with a nitrate concentration of 150 to 500 mg/l in the experimental groups of animals of both ages. 
According to the research, the most significant changes in parameters are caused by drinking water with nitrates 
at a concentration of 500 mg/l. More significant changes were in immature animals then the mature ones.

Conclusions. Excessive concentrations of nitrates in drinking water adversely affect the body of experimental 
animals causing activation of the studied parameters of the antioxidant system.

KEYWORDS: drinking water; nitrates; superoxide dismutase; catalase; ceruloplasmin.

*Corresponding author: Maryana Danchyshyn, PhD student, 
I. Horbachevsky Ternopil National Medical University, Ternopil, 
46002, Ukraine. 
E-mail: danchyshyn@tdmu.edu.ua

International Journal of Medicine and Medical Research 
2022, Volume 8, Issue 1, p. 67-73
copyright © 2022, TNMU, All Rights Reserved

M. V. Danchyshyn et al.

Introduction
The problem of nitrate pollution in terms of 

its scale and consequences has reached the 
global level. It consists in total pollution of the 
biosphere, deterioration of plant agricultural 
products and drinking water, negative impact 
on the human body, especially young children 
and farm animals. Approximately one third of 
the groundwater bodies, for which information 
is currently available, exceeds the maximum 
permissible concentrations (MPC) of nitrates in 
drinking water that is equal to 50 mg/dm3. 
Monitoring of nitrate levels in groundwater in 
different districts of Ternopil region showed 
that the maximum concentration ranges from 
1.1 to 7.3 times or from 55 to 365 mg/l and even 
more [1, 2]. This is due to the widespread use 
of nitrogen-containing organic and inorganic 
fertilizers in agriculture. However, unfortunately, 
residents of houses have been using such water 
for drinking for many years, because nitrates, 

even their excessive concentrations are not felt 
or tasted by humans.

Nitrates are non-toxic, but entering the 
gastrointestinal tract they are reduced to 
nitrites, which due to their oxidizing properties 
are ten times more toxic than nitrates. Nitrites 
are absorbed into the blood, and consequently 
reach tissues. The main mechanism of nitrite 
toxicity is the oxidation of Fe2+ to Fe3+, the 
conversion of hemoglobin into methemoglobin 
(MetHb) [3].

The use of nitrates with drinking water 
affects the nervous and cardiovascular systems, 
development of embryos; it can cause colorectal 
cancer, thyroid disease and neural tube defects 
[4, 1]. When systematically ingested, they can 
lead to disorders of the endocrine and central 
nervous system, blood circulation, etc. [5].

In addition, it is established that nitrites in 
the presence of amines can form N-nitrosamines, 
which have carcinogenic activity. Nitrates can 
also inhibit the enzyme systems involved in 
tissue respiration. As a result, a large number 
of free radicals is formed. At the same time, at 



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the cellular level the processes of free radical 
oxidation of phospholipids of biological cell 
membranes take place that lead to development 
of oxidative stress [6].

This can be overcome by reducing the 
intensity of lipid peroxidation (LPO) in the body 
with antioxidant system (AOS) which controls 
and inhibits all stages of reactions of free 
radicals capable of damaging the cell from their 
initiation to formation of hydroperoxides and 
malonic dial [7].

This protective system combines a number 
of substances of different nature; each of them 
acts in close contact with its other structural 
elements, harmoniously complements, and in 
many cases enhances the action of each other. 
The main antioxidants of blood plasma are 
superoxide dismutase (SOD), catalase (CAT) 
and ceruloplasmin (CP) [8, 9]. When an 
organism is in unfavorable living conditions, in 
particular ecological, or when it is exposed to 
adverse factors the activity of these antioxidants 
increases sharply [10].

Therefore, the aim of the research was to 
study the features of antioxidant protection in 
the body of white rats on the background of 
drinking water with excessive concentrations 
of nitrates for 30 days regarding the age.

Methods
The study was conducted on 60 outbred 

white female rats which were kept on the 
standard diet of the vivarium of I. Horbachevsky 
Ternopil National Medical University. Animals 
were divided into 2 age categories: the 1st ca-
tegory – 30 mature animals weighing 180­200 g 
and the 2nd – 30 immature animals weighing 
60-80 g. The experimental animals of each age 
category were divided into 5 groups with 6 rats 
in each. The groups were selected by rando-
mization. The animals of control group drank 
drinking water from the city water supply. The 
rats of the 1st group drank drinking water with 
nitrate content of 50 mg/l, the 2nd – 150 mg/l, 
the 3rd – 250 mg/l, the 4th – 500 mg/l. To create 
model aqueous solutions, drinking water from 
city water supply was used; taking into account 
background concentrations, sodium nitrate 
(NaNO3) was added into it to create aqueous 
solution with nitrates at a dose of 50, 150, 250, 
500 mg per liter. The experiments were per-
formed in accordance with the European Con-
vention for the Protection of Vertebrate Animals 
Used for Research Purposes (Strasbourg, 1986) 
[12] and the norms of biomedical ethics and 
the “General Ethical Principles of Animal Expe-

riments” adopted by the First National Congress 
on Bioethics (Kyiv, 2001).

The animals were taken out of the experi-
ment by bloodletting under thiopental-sodium 
anesthesia in 30 days after the beginning of the 
experiment.

The activity of catalase (CAT), ceruloplasmin 
(CP) and superoxide dismutase (SOD) was de-
termined in the serum by means of a semi-
automatic biochemical analyzer Humalyzer 
2000 using standard kits by HUMAN diagnostics 
(Wiesbaden, Germany). The kits used for eva-
luation of CAT, CP and SOD were adapted for 
their determination in animal serum. The re-
sults were statistically processed using compu-
ter programs STATISTICA 10.0 and Excel 2010. 
The choice of statistical research method was 
based on the correct distribution of the studied 
features. Further pairwise comparison of 
groups was performed using the Mann-Whitney 
U­test, the statistical significance p<0.05.

Results 
The results of our studies showed that when 

drinking water with different concentrations of 
nitrates, the AOS of the experimental animals 
of both ages underwent significant changes.

The key enzyme of AOS is SOD. It was found 
that the level of SOD, CAT and CP in the 1st 
experimental group of both age categories did 
not differ significantly from the data in the 
control group.

The increase in SOD activity in other groups 
of experimental rats of both ages compare to 
the controls was directly proportional to the 
concentration of nitrates in water. The level of 
SOD in the 2nd group of mature animals in-
creased significantly by 43.7%, in the 3rd – by 
56.2% and in the 4th – by 62.5% (Fig. 1). 

In the immature animals, a more significant 
increase was evidenced. Thus, in the 2nd group 
the activity of SOD significantly increased by 
61.1%, in the 3rd – by 94.4%. However, the activity 
of SOD increased the most in the 4th group – by 
116.6% (Fig. 2).

SOD always works in conjunction with CAT, 
which converts hydrogen peroxide to water and 
molecular oxygen. When determining the acti-
vity of CAT in the serum of mature animals it 
increased by 16.0% in the 2nd group, in the 3rd – 
by 30.5% (p<0.05), and in the 4th group – by 
97.2% (p<0.05) compare to the control (Fig. 3). 

In the immature animals, insignificant 
increase in the activity of this parameter was 
found in the 2nd and 3rd groups (by 6.7% and 
18.3%, respectively). However, in the animals 

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Fig. 1. The level of superoxide dismutase in the serum of mature experimental animals when drinking 
water with different concentrations of nitrates (unit/g).
Note: * – the differences are significant comparing to the control group (p<0.05). Error bars indicate ± standard deviation of 
the mean after three replicates.

Fig. 2. The level of superoxide dismutase in the serum of immature experimental animals when drinking 
water with different concentrations of nitrates (unit/g).
Note: * – the differences are significant comparing to the control group (p<0.05). Error bars indicate ± standard deviation of 
the mean after three replicates.

* 

* * 

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Control Group 1 Group 2 Group 3 Group 4

Th
e 

le
ve

l o
f s

up
er

ox
id

e 
di

sm
ut

as
e,

 
un

it/
g 

* 

* 
* 

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

Control Group 1 Group 2 Group 3 Group 4

Th
e 

le
ve

l o
f s

up
er

ox
id

e 
di

sm
ut

as
e,

 
un

it/
g 

of the 4th group the parameter increased by 
28.8% (p<0.05) compare to the control group 
(Fig. 4).

The nonenzymatic part of AOS was assessed 
for the presence of СP in the blood serum, 
which is also one of the main antioxidants in 
the human’s body. A main feature of this 
protein is its high stability to the toxic action of 
reactive oxygen species which allows it to 
maintain biological activity under conditions of 
their intensive generation [7, 8].

Increased level of CP in both age categories 
was established; it differed not much. In the 2nd 

group of mature animals the parameter in-
creased by 22.1%, in the 3rd group – by 23.4%, 
in the 4th group – by 53.3% (p<0.05) (Fig.5).

The number of SR in the immature animals 
of the 2nd group increased by 28.4%, in the 3rd – 
by 39.0% (p<0.05), in the 4th – by 61.0% (p<0.05) 
(Fig. 6).

Discussion
According to the literature the antioxidant 

substance in the cell is present at low con cen-
trations and significantly reduces or prevents 
oxidation of the oxidizable substrate. The most 

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efficient enzymatic antioxidants contain glu­
tathione peroxidase, catalase and superoxide 
dismutase. SOD is one of the most potent 
intracellular enzymatic antioxidants, and it 
catalyzes the conversion of superoxide anions 
to dioxygen and hydrogen peroxide. The 
hydrogen peroxide is removed by catalase. This 
enzyme is present in the peroxisome of aerobic 
cells and is very efficient in promoting the 
conversion of hydrogen peroxide to water and 
molecular oxygen [13]. An increase in the acti-
vity of CAT and SOD is usually observed in cases 
of environmental pollutants since SOD-CAT 
system represents the first line of defense 

against oxidative stress [14, 15]. According to 
Krishnamoorthy and Sangeetha [16], sodium 
nitrite (300 mg/kg body weight) significantly 
increased the lipoperoxidation and the activities 
of liver marker enzymes such as alanine amino-
transferase, aspartate aminotransferase, acid 
phosphatase and lactate dehydrogenase. 

Analyzing the dynamics of changes in the 
activity of AOS in the serum of rats, significantly 
higher SOD against CAT and CP were established. 
The SOD interrupts the chains of oxygen-de-
pendent free radical reactions, causing dismu-
tation of the superoxide anion radical with the 
release of hydrogen peroxide, which may 

Fig. 3. The level of catalase in the serum of mature animals when drinking water with different concentra-
tions of nitrates (mcat/l).
Note: * – the differences are significant comparing to the control group (p<0.05). Error bars indicate ± standard deviation of 
the mean after three replicates.

Fig. 4. The level of catalase in the serum of immature animals when drinking water with different concen-
trations of nitrates (mcat/l).
Note: * – the differences are significant comparing to the control group (p<0.05). Error bars indicate ± standard deviation of 
the mean after three replicates.

* 

* 

0
1
2
3
4
5
6
7
8
9

10

Control Group 1 Group 2 Group 3 Group 4

Th
e 

le
ve

l o
f c

at
al

as
e,

 m
ca

t/
l  

* 

0

1

2

3

4

5

6

7

8

9

Control Group 1 Group 2 Group 3 Group 4

Th
e 

le
ve

l o
f c

at
al

as
e,

 m
ca

t/
l 

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precede formation of the most toxic hydroxyl 
radical (OH-) and triplet oxygen [10]. Increased 
activity of SOD is an additional damaging factor 
and may cause pathological processes as a 
result of increased cytotoxic action of hydrogen 
peroxide, formed as a result of dismutation of 
superoxide [8]. The increase of CAT in serum 
does not exclude the presence of cytolysis on 
the background of drinking water with different 
concentrations of nitrates, because CAT is an 
intracellular enzyme and its increased activity 

in the blood occurs in cases of increased per-
meability of cell membranes.

CP neutralizes free radicals and protects cell 
membranes due to its ability to inhibit lipid 
peroxidase. It prevents activation of the floor, 
formation of toxic iron ions and damage to cell 
membranes. This enzyme acts as an “extra-
cellular superoxide dismutase” neutralizing 
superoxide anions in the extracellular envi-
ronment, blood, amniotic and cerebrospinal 
fluid, muscle, kidney and myocardium. CP 
increases with iron deficiency and excessive 

Fig. 5. The level of ceruloplasmin in the serum of mature animals when drinking water with different con-
centrations of nitrates (mg/l).
Note: * – the differences are significant comparing to the control group (p<0.05). Error bars indicate ± standard deviation of 
the mean after three replicates.

Fig. 6. The level of ceruloplasmin in the serum of immature animals when drinking water with different 
concentrations of nitrates (mg/l).
Note: * – the differences are significant comparing to the control group (p<0.05). Error bars indicate ± standard deviation of 
the mean after three replicates.

* 

0

2

4

6

8

10

12

14

Control Group 1 Group 2 Group 3 Group 4

Th
e 

le
ve

l o
f c

er
ul

op
la

sm
in
, m

g/
l 

* 

* 

0

2

4

6

8

10

12

Control Group 1 Group 2 Group 3 Group 4

Th
e 

le
ve

l o
f c

er
ul

op
la

sm
in
, m

g/
l 

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copper, in response to hypoxia, insulin, throm-
bin, estradiol, proinflammatory cytokines [11].

Increased oxidation processes are extremely 
crucial in the genesis of many pathological 
conditions, including inflammatory lesions of 
various tissues. In case of increased intake of 
xenobiotics, depletion of antioxidant depots 
and influence of negative factors, oxidative 
stress develops; it is characterized by pro-
oxidant and antioxidant disbalance and de-
velopment of oxidative damage [17].

Conclusions
The study allowed drawing the conclusion 

that excessive concentrations of nitrates in 
drinking water adversely affected the body of 
experimental animals causing activation of the 
studied parameters of the antioxidant system. 
As a result, the increased activity of SOD, CAT 

and CP in blood serum against consumption of 
drinking water with concentration of nitrates 
from 150 to 500 mg/l within 30 days in the 
experimental groups of both age categories 
has been revealed. The most pronounced 
changes in the parameters were regarding 
drinking water with nitrates at a concentration 
of 500 mg/l. The changes were more pronounced 
in immature animals then in the mature ones.

Conflict of Interests
The authors declare no conflict of interest.
Acknowledgements
This research received no external funding.
Autho’s Contributions
Maryana Danchyshyn – investigation, data 

curation, formal analysis, writing – original 
draft, writing – reviewing and editing; Olena 
Lototska – conceptualization, methodology, 
writing – reviewing and editing.

ОСОБЛИВОСТІ АНТИОКСИДАНТНОГО ЗАХИСТУ В ОРГАНІЗМІ БІЛИХ 
ЩУРІВ НА ТЛІ ВЖИВАННЯ ПИТНОЇ ВОДИ З ПОНАДНОРМАТИВНОЮ 
КОНЦЕНТРАЦІЄЮ НІТРАТІВ 

М. В. Данчишин, о. В Лотоцька
ТЕРНОПІЛЬСЬКИЙ НАЦІОНАЛЬНИЙ МЕДИЧНИЙ УНІВЕРСИТЕТ ІМЕНІ І. Я. ГОРБАЧЕВСЬКОГО МОЗ УКРАЇНИ, 

ТЕРНОПІЛЬ, УКРАЇНА

Вступ. Проблема забруднення довкілля нітратами за своїми масштабами та наслідками вийшла 
на глобальний рівень. Приблизно в одній третині підземних водних об’єктів, щодо яких наразі доступна 
інформація, перевищено гранично допустимі концентрації вмісту нітратів у питній воді. 

Мeтою цієї роботи стало вивчення особливості антиоксидантного захисту в організмі білих щурів 
на тлі вживання питної води з понаднормативною концентрацією нітратів впродовж 30 днів в 
залежності від віку. 

Матеріали. Експеримент проводили на 2 групах безпородних білих самок-щурів різного віку 
(статевозрілих та статевонезрілих) по 30 особин у кожній, розділених на 5 підгруп, які відрізнялися за 
якістю питної води, яку вони споживали самостійно. У 30-денному дослідженні нітрати давали щурам 
у питній воді в дозі 50, 150, 250, 500 мг нітрату натрію на літр. Тварини контрольної групи пили воду 
з міського водопроводу. Оцінювали вплив нітратів на активність супероксиддисмутази (СОД), каталази 
та церулоплазміну (СР) в сироватці крові щурів.

Результати. У результаті досліджень виявлено підвищену активність СОД, каталази та СР у 
сироватці крові на тлі вживання питної води з концентрацією нітратів від 150 до 500 мг/л у 
піддослідних групах обох вікових категорій. Встановлено, що найбільш виражені зміни показників 
викликає вживання питної води з нітратами у концентрації 500 мг/л. Більш виражені зміни були у 
статево незрілих тварин. 

Висновки. Присутність нітратів у питній воді у понаднормативних концентраціях негативно 
впливає на організм піддослідних тварин, викликаючи активацію досліджуваних показників 
антиоксидантної системи. 

КЛЮчоВі сЛоВа: питна вода; нітрати; супероксиддисмутаза; каталаза; церулоплазмін.

Information about the authors 
Maryana Danchyshyn, PhD student, I. Horbachevsky Ternopil National Medical University, Ternopil, 

Ukraine
https://orcid.org/0000­0002­7422­2883, e­mail: danchyshyn@tdmu.edu.ua
Olena Lototska, Professor, I. Horbachevsky Ternopil National Medical University, Ternopil, Ukraine
https://orcid.org/0000­0002­1393­7914, e­mail: lototska@tdmu.edu.ua

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Received 18 May 2022; revised 29 May 2022;  
accepted 10 June 2022.

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