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47
BIOMEDICAL SCIENCES
ISSN 2413-6077. IJMMR 2016 Vol. 2 Issue 2
doI 10.11603/IJMMR.2413-6077.2016.2.7036
AgE RELATEd OXIdATIvE PROcEssEs ANd ENdOgENOUs
INTOXIcATION dYNAmIcs Of RATs ARTER TOBAccO smOKE
AffEcTION
P. G. Lyhatskyy, O. B. Rytsyk, L. S. Fira, O. Z. Yaremchuk
I. HORBACHEVSKY TERNOPIL STATE MEDICAL UNIVERSITY, TERNOPIL, UKRAINE
Background. In an experiment on rats in the content of reactive oxygen species and lipid peroxidation
products in blood and liver of rats of different age groups after 45-day affection with tobacco smoke was studied.
Objective. The study was aimed to investigate the rate of reactive oxygen species formation, especially the
processes of lipid peroxidation and degree of endogenous intoxication in rats of different age groups in terms
of 45 day affection with tobacco smoke.
Methods. The content of ROS was determined in blood neutrophils method gradient centrifugation, the
activity of free radical processes in rats evaluated the content of TBA - active products (TBA - AP), the degree of
endogenous intoxication - the content average molecular weight (AMW) of the two factions – MW1 (dominated
by chain amino acids) and MW2 (dominated by aromatic amino acids). Blood, blood serum and liver of the
experimental rats were used for the investigation. It was prepared 10% homogenate in saline from liver tissue.
Results. We have noticed that the destruction of rats by tobacco smoke for 45 days caused the increase of
ROS in blood content, which is the result of toxic effect on the body. The immature rats were the most sensitive
to the affection, which ROS contents in neutrophils increased in 2.75 times till the end of the experiment, in the
mature animals it was in 1.65 times higher than in the intacts, in the senile it was higher in 2.43 times than normal
rate. It was found that the level of oxygen metabolites and TBA-active products increased during the experiment
(on the 15th, 30th and 45th day of toxicity).
Conclusions. The most pronounced changes were inherent for the immature rats. Accumulation of active
toxic metabolites was conducted with endogenous intoxication intensifying that was proved by the high content
of catabolism products in the body – middle mass molecules that are likely to grow in serum of rats of all age
groups.
KEY WORDS: oxidative processes, lipid peroxidation, reactive oxygen species, endogenous in-
toxication, tobacco smoke, rats.
Introduction
At present tobacco smoke is one of the most
common anthropogenic agents that has a wide
range of effects on morphofunctional status of
various systems of the body [8, 10, 15, 16]. The
share of tobacco smoke in the overall air
pollution is quite considerable and each year
continues to rise, so it is one of powerful
polluters of the environment. It is established
that the basis of the pathogenic action of
contaminated pollutants or cigarette smoke air
is the oxidant aggression on the mucosa of
respiratory tract reactive oxygen species, nitro-
gen dioxide and sulfur and other free radicals,
which cause the activation of lipid peroxidation
and damage of biological membranes [3, 11].
Smoking generates the reactive oxygen species
(ROS: O2
•, O2
1, OH, H2O2, etc.), which are impor-
tant for many physiological and biochemical
processes: the regulation of vascular tone, cell
proliferation, prostaglandins synthesis, signals
transmission from intercellular signalling mo-
lecules of regulatory system that control the
expression of phagocytes genes antimicrobial
action [6, 9, 14]. Under the inuence of extreme
factors of various origin (chemical contami-
nation, ionizing radiation, hyper- and hypoxia,
toxic substances, inflammatory processes)
formation of ROS in organisms is enhanced [12,
Corresponding author: Petro Lyhatskyy, Department of Medi-
cal Biochemistry, I. Horbachevsky Ternopil State Medical
University, 1 Maidan Voli, Ternopil, Ukraine, 46001
Phone number: +380352254784
E-mail: luhatsky@tdmu.edu.ua
International Journal of Medicine and Medical Research
2016, Volume 2, Issue 2, p. 47-51
copyright © 2016, TSMU, All Rights Reserved
P. g. Lyhatskyy et al.
48
BIOMEDICAL SCIENCES
ISSN 2413-6077. IJMMR 2016 Vol. 2 Issue 2
13, 17, 18]. The last causes free radical oxidation
activation that leads to increased lipid peroxi-
dation (LPO), oxidative modication of proteins
(OMP), degradation of nucleic acids, carbo-
hydrates, increasing of endogenous intoxication
in the organism.
However, in literature there is no denitive
data on the rate of formation of ROS and pro-
cesses of lipid peroxidation activation in rats of
different ages after prolonged exposure with
tobacco smoke.
Accordingly, the study was aimed to inves-
tigate the rate of reactive oxygen species for-
ma tion, especially the processes of lipid
peroxidation and degree of endogenous
intoxication in rats of different age groups in
terms of 45-day affection with tobacco smoke.
Material and Methods
The experiments were conducted on white
outbred male rats, which were kept on a vi-
varium standard diet of Ternopil State Medical
University. The rats were divided into three age
groups: the rst — immature, weight 60–80 g,
the second — mature, weight 180–200 g and
the third — senile, weight 300–320 g. Each age
group consisted of two subgroups: an intact
control (C) and an experimental group (E). The
rats of the experimental groups were affected
with tobacco smoke during 45 days. The mod-
el of the chronic smoke was created by means
of airtight chamber volume of 30 litres that
allowed animals to fumigate free behaviour.
Tobacco smoke was formed by smoking of 6
cigarettes Prima sribna (synia) (0.6 mg of nico-
tine and 8mg of tar), was served into it through
openings in the chamber. Six animals were si-
multaneously in the chamber during 6 minutes.
The animals of the control group were also 6
minutes in a sealed chamber, but were not
subjected to smoke.
In 15, 30 and 45 days after the beginning of
the affecting the animals with tobacco smoke,
they were taken out of the experiment by eu-
thanasia, which was performed with thiopental
anaesthesia.
Blood, blood serum and liver of the experi-
mental rats were used for investigation. 10%
homogenate in saline of liver tissue was pre-
pared.
The content of ROS was determined by blood
neutrophils method [6], the activity of free radi-
cal processes in rats evaluated the content of
TBA-active products (TBA-AP) [5], the degree of
endogenous intoxication — the average mo-
lecular weight (AMW) content of two factions [2,
7]: MW1 (dominated by chain amino acids) and
MW2 (dominated by aromatic amino acids).
We followed general principles of animal
experiments in the research that were appro-
ved at the National Congress on Bioethics (Kyiv,
Ukraine, 2001) and consistent with the provi-
sions of the European Convention for the Pro-
tection of vertebrate animals used for experi-
mental and other scientic purposes (Stras-
bourg, France, 1985) [4]. Parametric (according
to Student) and non-parametric (according to
Wilcoxon) methods for data statistical analysis
were used. Changes were considered as sig-
nicant at p≤0.05.
Results and Discussion
Any stress reaction normally is accompanied
by a brief increase in the number of ROS [6].
This is due to adaptation to extreme conditions
in which ROS play a role of a secondary mes-
senger participating in signal transduction and
activation of transcription factors and related
genes, including those encoding enzymes
antioxidants.
We noticed that the affection of the rats by
tobacco smoke during 45 days caused the
increase of ROS in blood content (Table 1),
which is the result of toxic factor on the body.
The immature rats were the most sensitive to
the affection, the ROS contents in neutrophils
increased in 2.75 times at the end of the
experiment, in the mature animals it was in 1.65
times higher than in the intacts, in the senile it
was in 2.43 times higher than normal rate.
It is established that under the inuence of
various extreme factors (chemical contami-
Table 1. The contents of ROS (%) in blood neutrophils of the rats affected
with tobacco smoke of all ages (M±m; n=72)
Research time,
days
Groups of the experimental animals
immature rats mature rats senile rats
intact rats 15.06±0.71 18.47±0.22 19.87±0.86
15th day of the affection 17.19±0.83 28.58±2.53* 25.38±1.95
30th day of the affection 39.25±1.29* 29.54±0.50* 41.89±0.78*
45th day of the affection 41.52±2.37* 30.63±0.66* 48.32±0.57*
Note: * — differences between the intact rats and the rats affected with tobacco smoke (p≤0.05).
P. g. Lyhatskyy et al.
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ISSN 2413-6077. IJMMR 2016 Vol. 2 Issue 2
nation, ionizing radiation, hyper- and hypoxia,
toxic substances, inammatory processes) the
formation of ROS in organisms is enhanced.
Among the reasons that causes of the increased
production of ROS are: violation of electron
transport in the respiratory chain of mitochon-
dria and electron transport chain microsomes,
the intensication of synthesis and oxidation
of catecholamines, the increased degradation
adenylate nucleotides and activation of
xanthine oxidase, the emergence of a pool of
catalytically active metal ions of variable valence
(especially Fe2+), the synthesis of prostaglandins
from arachidonic acid (reactions catalysed
cyclooxygenase and lipoxygenase), the acti-
vation of inducible form of nitric oxide synthase,
the increased activity of phagocytes [14].
The intensication of free radical oxidation
reactions is one of the manifestations of oxygen
metabolites toxic effects. Free radical oxidation
is a universal mechanism, which controls the
most important homeostatic physical and che-
mi cal parameters of cells: strength, integrity and
selective permeability of cell membranes [3].
A signicant increase of ROS, which was
evidenced after poisoning the animals with
tobacco smoke, caused the intensication of
free radical oxidation including lipid peroxi da-
tion. As one of the indicators of lipid peroxidation,
the contents of TBAAP signicantly increased
in serum and liver of the rats after tobacco
smoke intoxication (Table 2). In serum of the
im mature animals on the 15th day of the intoxi-
cation, this indicator increased by 30% (changes
were not signicant). The other age groups in
this period were more sensitive: the TBAAP
content in serum increased by 87% in the ma-
ture animals and by 58% in the senile (p≤0.05).
The intoxication with tobacco smoke during
45 days caused the signicant activation of lipid
peroxidation, which was evidenced by consi-
derable increase of TBA-AP content in serum of
the animals of all experimental groups (in the
immature rats the indicator increased in 2
times, in the immature — in 2.1 times, in the
senile — in 1.8 times).
In liver of the experimental animals a similar
increase of investigational product lipid per-
oxidation was observed during the experiment.
By the end of tobacco smoke toxicity (on the
45th day of the research), the increase of TBA-AP
content in liver of the mature and senile animals
in 1.8 times respectively was evidenced. The
immature animals were more sensitive to this
indicator and the content of intermediate
peroxidation increased in 3.2 times in the test
organ. Perhaps this is due to insufficient
decontaminating of liver, including oxidation
processes and microsomal enzymes that took
part in it. In pathological processes the average
molecular weight (AMW) is quite important,
some its fraction exhibit high biological activity
[1]. The spectrum of pathological action of
these compounds has a different character:
they break the physical and chemical properties
of cell membranes, inhibit tissue respiration
and oxidative phosphorylation, inhibit ATP
activity and DNA synthesis, activate lipid per-
oxidation and reduce the activity of antioxidant
defence cells, stimulate cellular proliferation
and inhibiting the process of apoptosis [2].
AMW accumulation is a consequence of the
activation of catabolic processes and reduction
of liver detoxification function. Metabolic
syndrome intoxication is developing, which
leads to severe functional impairment and
morphological damage to various organs and
systems. Activation of ROS-formation processes
and lipid peroxidation causes increased pro-
teolysis reactions in the body affected with
tobacco smoke, accompanied by accumulation
of both factions AMW — AMW1 (dominated by
chain amino acids) and AMW2 (dominated by
aromatic amino acids). The results of the
Table 2. The content of TBA-AP in blood serum (mmol/L) and liver (mmol/kg)
of the rats in dynamics of tobacco smoke affection (M±m; n=72)
Research time,
days
Groups of the experimental animals
immature rats mature rats senile rats
blood serum
Intact rats 3.28±0.23 1.85±0.14 2.35±0.14
15th day of the affection 4.28±0.31 3.47±0.29* 3.71±0.28*
30th day of the affection 4.85±0.36* 4.28±0.31* 4.42±0.28*
45th day of the affection 6.62±0.08* 3.94±0.27* 4.23±0.17*
Liver
Intact rats 15.49±1.28 14.42±0.71 16.55±0.98
15th day of the affection 18.69±1.28 23.50±1.35* 25.12±2.09*
30th day of the affection 30.86±1.55* 28.73±0.78* 29.06±1.46*
45th day of the affection 49.66±3.38* 26.49±1.51* 29.69±2.39*
P. g. Lyhatskyy et al.
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ISSN 2413-6077. IJMMR 2016 Vol. 2 Issue 2
research of this indicator in blood serum are
presented in Table 3.
After 15-day toxicity with tobacco smoke in
blood serum of the immature rats the AMW1
content increased by 35%, AMW2 – by 38%. By
the end of the experiment, these indices in-
creased in 3 and 2.2 times respectively in this
age group. At the end of the research in the
mature rats the AMW1-content in blood serum
was in 3 times higher than in the intact animals,
AMW2-content was in 1.7 times higher than
normal. Similarly, the increased content of MSM
in the senile rats in 45 days after the tobacco
intoxication in blood serum of this age group
the AMW1-content was in 2.8 times higher than
the level of the intact control, the AMW2-content
exceeded the level of the healthy animals in 1,9
times. Analysing the processes of catabolism
in the affected organism we can notice that they
were activated in all age groups of rats simi-
larly. Obviously, tissue hypoxia is the primary
in the development of destructive processes in
the organism after tobacco smoke toxicity due
to the breach of oxygen transport.
Conclusions
Due to the chronic affection with tobacco
smoke on the rats of different ages the massive
formation of reactive oxygen species in neutro-
phils causes activation of lipid peroxidation and
permanent changes of endogenous intoxica-
tion (the increase of TBA-active products in
blood serum and liver of the effected rats, the
accumulation of average molecular weight).
Immature rats were the most sensitive to to-
bacco smoke; metabolic disorders were the
most pronounced as well.
Table 3. AMW content in blood serum (cu/L) of the rats in dynamics
of tobacco smoke affection (M±m; n=72)
Research time,
days
Groups of the experimental animals
immature rats mature rats senile rats
AMW1
Intact rats 14.00±1.15 11.00±0.85 13.66±0.61
15th day of the affection 19.00±1.52 17.66±1.20* 18.00±1.55
30th day of the affection 37.00±1.12* 31.00±1.12* 35.66±0.61*
45th day of the affection 41.66±0.95* 33.00±1.52* 38.00±1.03*
AMW2
Intact rats 16.66±0.84 12.67±0.84 15.33±0.67
15th day of the affection 23.00±2.29 15.33±0.66 20.33±1.82
30th day of the affection 28.67±0.67* 18.66±0.67* 23.33±0.99*
45th day of the affection 36.33±1.20* 21.66±1.20* 29.66±1.20
References
1. Andreychyn SM, Holomasha TM. The current
idea of endogenous metabolic intoxication. Infectious
diseases 2012; 1 (67): 84–88.
2. Nikolskaya VA, DanylchenkoYuD, Memeto-
va ZN. Biochemical aspects of consideration on the
role of middle mass molecules in the body. Scien-
tists’notes of V.I. Vernadsky Taurian National Uni-
versity. Series “Biology, Chemistry” 2013; 26 (65):
139–145.
3. Hryshchuk LA, Marushchak MI. Dynamics of
lipid peroxidation and antioxidant protection in rats
under acute lung injury. Tuberculosis, pulmonary
diseases, HIV infection 2011; 2 (5): 16–20.
4. Doctor’s Ethics and Human Rights: regulations
on the use of animals in biomedical experiments.
Experimental and clinical physiology and biochemistry
2003; 2 (22): 108–109.
5. Lushchak VI, Bahniukova TV, Lushchak OV.
Indicators of oxidative stress. Tiobarbiturativ pro-
ducts and protein carbonyl groups. Ukrainian
Biochemistry journal 2004; 26: 136–141.
6. Maruschak MI. Reactive oxygen intermediatesin
the development and progression of acute lung
injury in an experiment. Med. himiya 2012; 14 (1):
104–108.
7. Nikolaichyk VV, Kirkovskiy VV, Main VM et al.
Middle molecules – education and methods for
determining. Laboratory case 1989; 8: 31–33.
8. Krasovskyi KS, Andreyeva TI, Hryhorenko AA,
Butylska NA. Evaluation of smoking frequency in
P. g. Lyhatskyy et al.
51
BIOMEDICAL SCIENCES
ISSN 2413-6077. IJMMR 2016 Vol. 2 Issue 2
Ukraine. Bulletin of Social Hygiene and Organisation
of Health Care of Ukraine 2009; 2: 91–99.
9. Pikas OB.Current level of cigarette smoking,
its effect on humans diseases incidence. Bukovyna
Medical Journal 2015; 4 (76): 227–230.
10. Balakirieva OM, Bondar TV, Halich YuP et al.
The frequency and trends in tobacco, alcohol, drug
abuse among the youth in Ukraine. O.Yaremenko
Ukrainian Instituteof Social Research. K.: OBNOVA
2011; 176.
11. Sakharova HM, Sakharov HM, Antonov AN.
Prevention the tobacco epidemic – the preservation
of human health. Prevention of diseases and health
care. Preventive medicine 2010; 13 (6): 3–7.
12. Tiazhka OV, Vanhanova TO. Passive smoking
for toddlers. Transport Health of Ukraine 2012; 1:
93–99.
13. Chuchalin AH. Respiratory diseases and
smoking. Therapeutic archive 2009; 3: 5–9.
14. Haussmann HJ et al. Comparison of fresh and
room-aged cigarette sidestream smoke in a sub-
chronic inhalation study on rats. Toxicol Sci 1998; 1:
100–116.
15. Domagala-Kulawik J. Effects of cigarette
smoke on the lung and systemic immunity. J Physiol
Pharmacol 2008; 6: 19–34.
16. Wirth N [et al]. Respiratory diseases related
to passive smoking. Rev Mal Respir 2009; 6: 667–678.
17. Wipi H [et al]. Secondhand smoke exposure
among women and children: evidence from 31
countries. Am J Public Health 2008; 4: 672–679.
18. Wipi H, Samet JM. Global economic and
health benets of tobacco control: part 1. Clin Phar
macol Ther 2009; 3: 263–271.
Received: 2016-12-16
P. g. Lyhatskyy et al.
