Американский Научный Журнал STATE OF GLUTATHIONE ANTIOXIDANT SYSTEM IN BLOOD LYMPHOCYTES AT OVARIAN CANCER (12-15)

Activation of lipid peroxidation (LP) processes in blood lymphocytes of women with ovarian cancer was shown. Thus, in the control group the concentration of malonic dialdehyde (MDA) in blood lymphocytes was (62.1±4.5) μmol/mg protein. This value increases significantly up to (96.9±7.4) μmol/mg protein at ovarian cancer (OC). A similar situation is observed when determining the concentration of MDA in serum. Thus, in the control group it reaches (7.5±0.6) μmol/L. When OC, the processes of LP are intensified in 1.6 times relative to the control group. Simultaneously, with the intensification of LP processes the corresponding changes in the activity of enzymes of the glutathione antioxidant system were revealed. It was shown that in practically healthy women the concentration of glutathione is (17.8±1.5) nmol/mg protein. At OC, this value increases in 1.4 times and reaches (25.3±2.2) nmol/mg protein. Also a significant difference in glutathione peroxidase activities was revealed. In the control group it is (161.8±12.7) nmol GSH/min∙mg protein. At OC this value decreases in 1.7 times up to (96.1±8.8) nmol GSH/min∙mg of protein. Regarding the activity of glutathione reductase, in the lymphocytes of the control group it is equal to (51.9±5.1) nmol NADPH/min∙mg protein. At OC, this activity is reduced to (35.1±3.1) nmol NADPH/min∙mg protein, that is in 1.4 times lower than in control group. The glutathione transferases (GSTs) activity of the control group is (114.9±9.2) nmol GSH/min∙mg protein. With the development of OC (stage III-IV) the activity of GSTs increases to (138.8±11.6) nmol GSH/min∙mg of protein, that is in 1.2 times more than in control, however these changes are not statistically significant. Thus, OC significantly intensifies LP processes in both lymphocytes and blood plasma. Simultaneously, the development of OC significantly reduces the activity of two major antioxidant enzymes - glutathione peroxidase and glutathione reductase. Скачать в формате PDF
12 American Scientific Journal № ( 42 ) / 2020
МЕДИЦИНСКИЕ НАУКИ

STATE OF GLUTATHIONE ANTIOXIDANT SYSTEM IN BLOOD LYMPHOCYTES
AT OVARIAN CANCER

Barylyak R.,
Graduate Student, Department of Medical Biology,
Assistant Professor, Department of Anesthesiology and Intensive Care,
Danylo Halytsky Lviv N ational Medical University, Lviv, Ukraine
Onufrovych O.,
Assistant Professor, PhD, Department of Medical Biology,
Danylo Halytsky Lviv National Medical University, Lviv, Ukraine
Fafula R.,
Professor, D Sci, Department of Biophisics,
Danylo Halytsky Lviv National Medical University, Lviv, Ukraine
Vorobets D.,
Professor, D Sci, Department of Urology,
Danylo Halytsk y Lviv National Medical University, Lviv, Ukraine
Vorobets Z.
Professor, D Sci, Department of Medical Biology,
Danylo Halytsky Lviv National Medical University, Lviv, Ukraine

Abstract . Activation of lipid peroxidation (LP) processes in blood lymphocyt es of women with ovarian
cancer wa s shown. Thus, in the control group the concentration of malonic dialdehyde (MDA) in blood
lymphocytes was ( 62.1±4.5) μmol/mg protein. This value increases significantly up to (96 .9±7 .4) μmol/mg protein
at ovarian cancer (OC). A similar situation is observed when determining the concentration of MDA in serum.
Thus, in the control group it reaches (7.5±0.6) μmol/ L. When OC, the processes of LP are intensified in 1.6 times
relative to the control group. Simul taneously , with the intensification of LP processes the corresponding changes
in the activity of enzymes of the glutathione antioxidant system were revealed. It was shown that in practically
healthy women the concentration of glutathione is (17.8±1.5) nmol /mg protein . At OC, this value increases in 1.4
times and reaches (25 .3±2 .2) nmol/mg protein . Also a significant difference in glutathione peroxidase activitie s
was revealed. In the control group it is (161.8±12.7) nmol GSH/min∙mg protein. At OC this value decreases in 1.7
times up to (96.1±8.8) nmol GSH/min∙mg of protein. Regarding the activity of glutathione reductase, in the
lymphocytes of the control group i t is equal to (51.9±5.1) nmol NADPH/min∙mg protein. At OC, this activity is
reduced to (35.1±3.1) nmol NADPH/min∙mg protein, that is in 1.4 times lower than in control group. The
glutathione transferases (GSTs) activity of the control group is (114.9±9.2) nmol GSH/min∙mg protein. With the
development of OC (stage III -IV) the activity of GSTs increases to (138.8±11.6) nmol GSH/min∙mg of protein,
that is in 1.2 times more than in control, however these changes are not statistically significant. Thus, OC
signi ficantly intensifies LP processes in both lymphocytes and blood plasma. Simultaneously , the devel opment of
OC significantly reduces the activity of two major antioxidant enzymes - glutathione peroxidase and glutathione
reductase.
Key words : ovarian cancer , lymphocytes, malonic dialdehyde , glutathione, glutathione peroxidase,
glutathione reductase, glu tathione -S transpherase.

Introduction. According to modern ideas, the
development of pathological processes in the organism
is accompanied by a violation of the antioxidant
protection mechanisms of cells [1 -3]. During tumor
growth, oxidative free radical processes are initiated in
particular in phospholipids of cell membranes
containing polyunsaturated fatty acids. Intensification
of lipid peroxidation leads t o the accumulation of toxic
products which leads to DNA damage , gen e mutations
and reducing the body's resistance which are
considered the initial stages of carcinogenesis [2, 4, 7,
12]. However, the buffer capacity of the antioxidant
system under normal c onditions is quite large and is
provided by various components. An important role
among the antioxidant system of the cell is occupied by
the glutathione system which consists of both
enzymatic (glutathione peroxidase, glutathione
reductase, glutathione S-transferase) and non -
enzymatic (reduced glutathione) components [5, 9-11].
Although the glutathione system has been the the
subject of many studies there is no consensus in the
literature regarding to its role in malignant growth.
Because intracellular lym phocyte metabolism is based
on the physiological and biochemical ab ility of these
cells to respond rapidly to any changes in homeostasis
of the organism the modulation of enzyme activity in
lymphocytes occurs much earlier than their
morphological and funct ional parameters change.
This allows to use the status of lymphocyt es as a
"metabolic mirror" of the organism . We suggest that
peripheral blood lymphocytes may be a convenient and
adequate model for studying the peculiarities of the
functioning of regulato ry systems in both healthy
individuals and at neoplastic ovarian tr ansformation.
Therefore, analysis of the functional state of the
glutathione system in blood lymphocytes at ovarian

American Scientific Journal № ( 42 ) / 2020 13

cancer will reveal its role in the pathogenesis and
prognosis of the dise ase.
Material and methods. The study was performed
on blood lympho cytes of practically healthy women
and patients with neoplastic ovarian changes. The total
number of practically (clinically) healthy women
representative by age (mean age 53.8 5.4 years) w as
40 people. This group was formed by volunteers from
the staff of Danylo Halytsky Lviv National Medical
University and employees of the Lviv State Regional
oncology treatment and diagnostic Center .
The group of women with ovarian cancer (stage
III -IV) c onsisted of 78 women aged 24 -75 years (mean
age 55.4 5.3 years) who were hospitalized at the Lviv
State Regional oncology treatment and diagnostic
Center in the period 2016 -2019 and underwent a full
clinical and laboratory examination. The study
included p atients diagnosed with ovarian cancer
without comorbi dities at the start of the research.
Clinical diagnoses were established on the basis of
a wide range of general clinical, laboratory,
instrumental and special oncological research methods.
In addition, the level of tumor marker glycoprotein CA -
125 in seru m was determined to differentiate almost
healthy women and diagnose ovarian cancer [8]. All
ovarian cancer patients and practically healthy persons
were informed in detail about the purpose, objectives
and terms of research and gave written informed
consen t to participate in research on blood samples.
Any violations of moral and ethical norms were not
revealed by the Bioethics Commission of Danylo
Halytsky Lviv National Medical University.
Peripheral bloo d lymphocytes were isolated by the
method of A. Boyum (1968). Blood diluted 1:1 with
physiological saline was layered in a density gradient
of phycol -triumbrast ( =1.08 g/cm 3) and centrifuged
for 20 minutes at 500 g. Removed interphase rings of
mononuclear cells were washed twice for 10 minutes
with physiolo gical saline. After the last centrifugation
a small amount of physiological saline was added to the
pellet, resuspended, and the number of living and dead
cells was counted in the Goryaev chamber using t rypan
blue. The integrity and viability of blood lymp hocytes
in all experiments was at least 95%. Saponin was added
to the suspension to permeabilize blood lymphocyte
membranes and reveal latent enzymatic activities [6].
Plasma lipid peroxidation was asses sed by the
concentration of malonic dialdehyde (MDA) in the
reaction with 2 -thiobarbituric acid (Timirbulatov,
1981). Glutathione peroxidase (GP) activity was
determined by the rate of GSH oxidation in the
presence of tert -Butyl hydroperoxide (Moin, 1986).
The determination of glutathione reductase (GR)
act ivity was performed by decreasing the NADPH
content (Carlberg, 1985). Glutathione S -transferase
(GsT) activity was determined by the rate of enzymatic
formation of glutathione S -conjugate in the GSH
redu ction reaction with 1 -chloro -2,4 dinitrobenzene
(Karp ishenko, 2002). The content of reduced
glutathione (GSH) was calculated as the difference
between the concentrations of total and oxidized
glutathione using Elman's reagent (Anderson, 1985).
The signific ance of changes between the statistical
characteristi cs of two alternative data sets was
established by Student's t -test. The difference in
probability value p<0.05 was considered significant.
Results and discussion . We conducted a
comparative study of li pid peroxidation (LP) and
glutathione system in practic ally healthy women and
women with ovarian cancer (OC). Activation of LP
processes in blood lymphocytes of women with ovarian
cancer wa s shown by determining the co ncentration of
malonic dialdehyde (MDA ) which is a secondary
product of lipid peroxidation. Thus, in the control group
the concentration of MDA in blood lymphocytes in
calcium -free medium was (62 .1±4 .5) μmol/mg protein
(Fig. 1). At OC this value increases statistically and
significantly up to (96 .9±7 .4) μmol/mg protein
(p<0 .001).

Fig. 1. The concentration of malonic dialdehyde in the lymphocytes of practically healthy women (C) and
women with ovarian cancer (OC)
Comment: *p<0,00 1 compared to practically healthy women

It should be noted that the intensity of lipid
peroxidation in lymphocytes depends on the presence
of calcium ions in the medium (Fig. 2).

0
50
100
150
C OC
MDA,
μmol /mg protein ,

14 American Scientific Journal № ( 42 ) / 2020
Fig. 2. The dependence of the level of malonic dialdehyde in blood lymphocytes on the concentration of Ca 2+.

It can be seen that a t the presence 0 .5 mM Ca 2+in
incubation medium lipid peroxidation increases in 1 .6
times (p<0 .001). However, it should be noted that these
are not physiological concentrations of Ca 2+. They
exceed them by two orders of magnitude.
A similar situation as in lymphocytes is observed
when determining the concentration of MDA in serum.
In the blood serum of t he control group it reaches
(7.5±0.6) μmol/l (Fig. 3). At OC, LP processes
intensify in 1.6 times relative to the control group
(p<0 .001). Thus, at OC LP pr ocesses are significantly
intensified, in blood plasma in 1.6 times (p<0 .001) and
in blood lymphocyt es in 1.6 times (p<0 .001).

Fig. 3. The concentration of malonic dialdehyde in the serum of practically healthy women (C) and
women with ovarian cancer (OC).
Comment: *p<0.001 compared to practically healthy women

Simultaneously with the intensification of LP
processes the corresponding changes in the activity of
enzymes of the glutathione system were revealed
(Table 1).
Table 1
The state of the glu tathione antioxidant system and lipid peroxidation in practically healthy women and
patients with ovarian cancer, M±m, n=18 -22.
Groups

Indicators
Practically healthy women
Women with ovarian
cancer, stage III -IV

GSH , nmol /mg protein 17.8±1 .6 25.3±2 .1*
GP, nmol GSH/min∙mg of protein 161 .8±12 .7 96.1±8 .8***
GR, nmol NADPH/min∙mg of protein 51.9±5 .1 35.1±3 .1*
GsT, nmol GSH/min∙mg of protein 114 .9±9 .2 138 .8±11 .6
Comment: *р<0 .05,***р<0 .00 1 compared to practically healthy women

Thus, it is shown that in practically healthy women
the concentration of glutathione is (17.8±1.5) nmol/mg
protein. At OC, this value increases in 1.4 times and
reaches (25.3±2.2) nmol/mg protein (p<0.01). Also a
significant difference in glutathione perox idase
activities was reve aled. In the control group it was
(161.8±12.7) nmol GSH/min∙mg protein. At OC this
0
50
100
150
C 0,05 0,1 0,5 1 [Ca ²⁺],
мМ
MDA,
µmol/mg protein
0
2
4
6
8
10
12
14
C OC
MDA,
μmol /mg protein

American Scientific Journal № ( 42 ) / 2020 15

value decreases in 1.7 times up to (96.1±8.8) nmol
GSH/min∙mg protein (p<0.001).
Regarding the activity of glutathione reductase in
the lymphocytes of the control group it i s equal to
(51.9±5.1) nmol NADPH/min∙mg protein. At OC
enzyme activity is reduced up to (35.1±3.1) nmol
NADPH/min∙mg protein, that is lower in 1.4 times
(p<0.05) than in the control group.
The glutathione S-transferases activity of the
control group is (11 4.9±9.2) nmol GSH/min∙mg
protein. With the development of OC (stage III -IV)
GSTs activity increases up to (138.8±11.6) nmol
GSH/min∙mg protein, that is in 1.2 times more than in
the control group, however these changes are not
statistically significant (p> 0.05).
Thus, it is known from the literature that lipids,
especially polyunsaturated fatty acids are very sensitive
to free radicals that initiate lipid peroxidation [1]. One
of the final products of LPO is MDA, which due to its
high cytotoxic effect, in particular on the antioxidant
system, can act as a co -carcinogenic agent [1, 8, 9].
From the data obtained by us it is seen that at OC the
concentration of MDA is increased significantly,
whereas activitie s of two main antioxidant enzymes
glutathione perox idase and glutathione reductase are
decreased.
Conclusion. 1. At ovarian cancer the processes of
lipid peroxidation in lymphocytes and in blood plasma
are significantly intensified.
2. With the development of OC in blood
lymphocytes, the activities of two main antioxidant
enzymes glutathione peroxidase and glutathione
reductase are significantly reduced in comparison with
healthy donors.

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1Авдеева В. Г., 2Титова Е. М.
Россия, Пермь, образовательно -методический центр
«Пермская краевая шк ола медицины катастроф»
ГКУЗ «Пермский краевой территориальный центр медицины катастроф» 1Авдеева В. Г.,
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DOI: 10. 31618/asj.2707 -9864.2020.1.42.37
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