Nitric oxide coordinates development of genomic instability in realization of combined effect with ionizing radiation
The aim of this study was to investigate the ability of environmental nitrogen oxides or natural nitric oxide (NO) donors to modify free radicals balance and development of genomic instability alone or in combination with ionizing radiation. Methods: Genotoxicity and cytogenetic abnormalities were...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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irk-123456789-1391342018-06-20T03:04:45Z Nitric oxide coordinates development of genomic instability in realization of combined effect with ionizing radiation Mikhailenko, V.M. Diomina, E.A. Muzalov, I.I. Gerashchenko, B.I. Original contributions The aim of this study was to investigate the ability of environmental nitrogen oxides or natural nitric oxide (NO) donors to modify free radicals balance and development of genomic instability alone or in combination with ionizing radiation. Methods: Genotoxicity and cytogenetic abnormalities were assessed in vitro in peripheral blood lymphocytes (PBL) isolated from healthy humans or in vivo in rats PBL. Human PBL were treated with physiologically relevant NO donor — S-Nitrosoglutathione and X-ray irradiation. The inhalation treatment of animals with NO was carried out in chamber with purified gaseous NO mixed inside with air. Levels of S-Nitrosohemoglobin and methemoglobin in the blood were assessed with electron paramagnetic resonance. The total level of reactive oxygen and nitrogen species in PBL was determined fluorometrically, and serum levels of reactive oxygen species was determined by spectrophotometric assay. DNA damages were assessed by alkaline single-cell gel electrophoresis. The frequency of chromosomal aberrations in human PBL measured with the conventional cytogenetic assay in metaphase cells on short-term (52 h) and long-term (72 h) cultures. Results: Environmental nitrogen oxides or release of NO from stable complexes with biomolecules (such as S-Nitrosothiols) intensified generation of free radicals, DNA damage and development of genomic instability alone or in combination with ionizing radiation. Treatment of PBL by S-Nitrosoglutathione caused prevalent induction of chromatid type but irradiation — chromosome aberrations. The dose dependence of chromatid-type aberrations observed in human PBL after combined influence of S-Nitrosoglutathione and ionizing radiation indicates a crucial role of NO in the formation of chromosomal instability. Conclusion: NO can deregulate free radicals balance resulted in genotoxic effect, posttranslational modification of repair enzymes and thus coordinated development of genomic instability and increase of cancer risk. 2013 Article Nitric oxide coordinates development of genomic instability in realization of combined effect with ionizing radiation / V.M. Mikhailenko, E.A. Diomina, I.I. Muzalov, B.I. Gerashchenko // Experimental Oncology. — 2013. — Т. 35, № 1. — С. 58-64. — Бібліогр.: 42 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/139134 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions |
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Original contributions Original contributions Mikhailenko, V.M. Diomina, E.A. Muzalov, I.I. Gerashchenko, B.I. Nitric oxide coordinates development of genomic instability in realization of combined effect with ionizing radiation Experimental Oncology |
| description |
The aim of this study was to investigate the ability of environmental nitrogen oxides or natural nitric oxide (NO) donors to modify free radicals balance and development of genomic instability alone or in combination with ionizing radiation. Methods: Genotoxicity and cytogenetic abnormalities were assessed in vitro in peripheral blood lymphocytes (PBL) isolated from healthy humans or in vivo in rats PBL. Human PBL were treated with physiologically relevant NO donor — S-Nitrosoglutathione and X-ray irradiation. The inhalation treatment of animals with NO was carried out in chamber with purified gaseous NO mixed inside with air. Levels of S-Nitrosohemoglobin and methemoglobin in the blood were assessed with electron paramagnetic resonance. The total level of reactive oxygen and nitrogen species in PBL was determined fluorometrically, and serum levels of reactive oxygen species was determined by spectrophotometric assay. DNA damages were assessed by alkaline single-cell gel electrophoresis. The frequency of chromosomal aberrations in human PBL measured with the conventional cytogenetic assay in metaphase cells on short-term (52 h) and long-term (72 h) cultures. Results: Environmental nitrogen oxides or release of NO from stable complexes with biomolecules (such as S-Nitrosothiols) intensified generation of free radicals, DNA damage and development of genomic instability alone or in combination with ionizing radiation. Treatment of PBL by S-Nitrosoglutathione caused prevalent induction of chromatid type but irradiation — chromosome aberrations. The dose dependence of chromatid-type aberrations observed in human PBL after combined influence of S-Nitrosoglutathione and ionizing radiation indicates a crucial role of NO in the formation of chromosomal instability. Conclusion: NO can deregulate free radicals balance resulted in genotoxic effect, posttranslational modification of repair enzymes and thus coordinated development of genomic instability and increase of cancer risk. |
| format |
Article |
| author |
Mikhailenko, V.M. Diomina, E.A. Muzalov, I.I. Gerashchenko, B.I. |
| author_facet |
Mikhailenko, V.M. Diomina, E.A. Muzalov, I.I. Gerashchenko, B.I. |
| author_sort |
Mikhailenko, V.M. |
| title |
Nitric oxide coordinates development of genomic instability in realization of combined effect with ionizing radiation |
| title_short |
Nitric oxide coordinates development of genomic instability in realization of combined effect with ionizing radiation |
| title_full |
Nitric oxide coordinates development of genomic instability in realization of combined effect with ionizing radiation |
| title_fullStr |
Nitric oxide coordinates development of genomic instability in realization of combined effect with ionizing radiation |
| title_full_unstemmed |
Nitric oxide coordinates development of genomic instability in realization of combined effect with ionizing radiation |
| title_sort |
nitric oxide coordinates development of genomic instability in realization of combined effect with ionizing radiation |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2013 |
| topic_facet |
Original contributions |
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http://dspace.nbuv.gov.ua/handle/123456789/139134 |
| citation_txt |
Nitric oxide coordinates development of genomic instability in realization of combined effect with ionizing radiation / V.M. Mikhailenko, E.A. Diomina, I.I. Muzalov, B.I. Gerashchenko // Experimental Oncology. — 2013. — Т. 35, № 1. — С. 58-64. — Бібліогр.: 42 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
AT mikhailenkovm nitricoxidecoordinatesdevelopmentofgenomicinstabilityinrealizationofcombinedeffectwithionizingradiation AT diominaea nitricoxidecoordinatesdevelopmentofgenomicinstabilityinrealizationofcombinedeffectwithionizingradiation AT muzalovii nitricoxidecoordinatesdevelopmentofgenomicinstabilityinrealizationofcombinedeffectwithionizingradiation AT gerashchenkobi nitricoxidecoordinatesdevelopmentofgenomicinstabilityinrealizationofcombinedeffectwithionizingradiation |
| first_indexed |
2025-07-10T07:38:38Z |
| last_indexed |
2025-07-10T07:38:38Z |
| _version_ |
1837244741698191360 |
| fulltext |
58 Experimental Oncology 35, 58–64, 2013 (March)
NITRIC OXIDE COORDINATES DEVELOPMENT OF GENOMIC
INSTABILITY IN REALIZATION OF COMBINED EFFECT
WITH IONIZING RADIATION
V.M. Mikhailenko*, E.A. Diomina, I.I. Muzalov, B.I. Gerashchenko
R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, National Academy
of Sciences of Ukraine, Kyiv 03022, Ukraine
The aim of this study was to investigate the ability of environmental nitrogen oxides or natural nitric oxide (NO) donors to modify free
radicals ba lance and development of genomic instability alone or in combination with ionizing radiation. Methods: Genotoxicity and
cytogenetic abnormalities were assessed in vitro in peripheral blood lymphocytes (PBL) isolated from healthy humans or in vivo in rats
PBL. Human PBL were treated with physiologically relevant NO donor — S-Nitrosoglutathione and X-ray irradiation. The inhalation
treatment of animals with NO was carried out in chamber with purified gaseous NO mixed inside with air. Levels of S-Nitrosohemo-
globin and methemoglobin in the blood were assessed with electron paramagnetic resonance. The total level of reactive oxygen and
nitrogen species in PBL was determined fluorometrically, and serum levels of reactive oxygen species was determined by spectropho-
tometric assay. DNA damages were assessed by alkaline single-cell gel electrophoresis. The frequency of chromosomal aberrations
in human PBL measured with the conventional cytogenetic assay in metaphase cells on short-term (52 h) and long-term (72 h) cultures.
Results: Environmental nitrogen oxides or release of NO from stable complexes with biomolecules (such as S-Nitrosothiols) intensified
generation of free radicals, DNA damage and development of genomic instability alone or in combination with ionizing radiation.
Treatment of PBL by S-Nitrosoglutathione caused prevalent induction of chromatid type but irradiation — chromosome aberrations.
The dose dependence of chromatid-type aberrations observed in human PBL after combined influence of S-Nitrosoglutathione and
ionizing radiation indicates a crucial role of NO in the formation of chromosomal instability. Conclusion: NO can deregulate free
radicals balance resulted in genotoxic effect, posttranslational modification of repair enzymes and thus coordinated development
of genomic instability and increase of cancer risk.
Key Words: nitric oxide, S-Nitrosothiols, nitrosative stress, ionizing radiation, DNA damage, genomic instability.
Intensification of anthropogenic activities leads
to the growth of environmental pollution by chemical
and physical factors that affect both the state of eco
systems and human health. Combinations of chemical
and physical agents are dangerous in terms of muta
genic and carcinogenic effects. Nitrogen oxides (NOx)
are one of the main air pollutants. The possible role
of NOx in the formation of genomic instability is deba
ting due to their ability to inhibit DNA repair enzymes [1,
2]. Ionizing radiation (IR) is a powerful physical geno
toxic factor that affects three related systems: redox
homeostasis, cell cycle control and DNA repair [3].
Biological effects of nitric oxide (NO) are broad
ranging and significantly depend on the concentration.
Signal properties of NO molecules occur at physiologi
cal concentrations as low as 1–30 nM. An increase
of NO concentration in 10 times activates onco
genic signaling cascades, and at NO concentrations
of above 500 nM results in nitrosative stress [4]. NOx
participate in all stages of neoplasia, demonstrating
cytotoxic and cytostatic activities, and under certain
conditions, are able to increase the effectiveness
of anticancer therapy [5, 6]. Manifestation of NO cyto
toxicity depends on both concentration and cumulative
dose. Significant cytotoxic effects occur at concentra
tions exceeding 150–300 mkM/min of comparable
cumulative dose [7].
Relatively high NO concentrations lead to the
failure of antioxidant defense and DNA repair sys
tems accompanied by formation and accumulation
of DNA strand breaks that significantly enhance the
level of somatic mutations [8]. NO can cause direct
or/and indirect mutagenic effects. Direct effects im
ply interaction of NO hydrated derivatives with DNA
amino groups. Nitrosation of primary amines lead
to formation of alkylating agents. Indirect mutagenic
effects involve formation of Nnitrosocompounds
such as Nalkylnitrosamines in reactions of NO with
secondary amines and amides.
Exogenous NOx, after being inhaled are transferred
to the blood stream. In the blood, NO binds to hemoglo
bin, albumin and other iron or SHcontaining proteins
and compounds, that are transported through vessels
to different tissues and organs [9], where NO oxidized
to nitrite and/or nitrates prior removal from the body.
Some part of NO reversibly binds to biological mo
lecules thus forming SNitrosothiols (RSNO) and ni
trosated complexes of nonheme iron ((RS─)2Fe+(NO+)2)
that provide NO stabilization and transfer from donor
to target cells [10, 11]. Recent data suggest a vital role
of RSNO in posttranslational modification of signaling
cascades in cells and modulation of biological pro
cesses and pathological states [12, 13].
Received: November 2, 2012.
*Correspondence: Fax: (380 44) 258 1656
E-mail: mvmik@yahoo.com
Abbreviations used: DSB — double strand break; GSNO — S-
Nitrosoglutathione; HbNO — S-Nitrosohemoglobin; IR — ionizing
radiation; metHB — methemoglobin; NO — nitric oxide; RNS — re-
active nitrogen species; ROS − reactive oxygen species; RSNO —
S-Nitrosothiols; SSB — single strand break.
Exp Oncol 2013
35, 1, 58–64
Experimental Oncology 35, 58–64, 2013 (March)35, 58–64, 2013 (March) (March) 59
Gaseous NO can induce DNA single (SSB) and
doublestrand breaks (DSB), DNA SOSrepair, forma
tion of micronuclei, chromatidtype aberrations, and
exchanges between sister chromatids [7, 14]. SSB
formation is characteristic for NO effects, detection
of which may serve as biomarker of NO genotoxicity [1].
NO plays a significant role in formation of mammali
an cells sensitivity to IR both in vivo and in vitro [15], but
at the genetic level the NO doseeffect dependence
has not been determined yet. Many NOdependent
biological functions are directly linked with the RSNO
synthesis. RSNO is a primary form of NO transport,
which released under physiological conditions and
is able to establish the background for appearance
and accumulation of aberrant cells [6, 16].
The common mechanism of NO and IR genoto
xicity is the formation of reactive oxygen (ROS) and
nitrogen (RNS) species. About 80% of IR–induced
damages are due to the influence of free radicals [8].
DNAfree radicals interactions lead to DNA SSB and
DSB, sister chromatids exchanges, point mutations,
microdeletions [17]. The reaction of NO with super
oxide radicals leads to peroxynitrite formation, that
causes DNA DSB [18].
Chemical agents alone or in combination with
IR can cause the formation of DSB, that if unrepaired
or inefficiently repaired can give rise to chromatid
breaks subsequently converted into chromosomal
aberrations [19].
To date, the question about duration of induced
chromosomal instability and its ability to be transmit
ted to future generations of cells remains unanswered.
This issue needed for prediction of longterm health
effects as a result of combined action of mutagenic
and carcinogenic factors [20]. It has been shown
that radiationinduced signaling, which leads to the
development of genomic instability, is inherited epi
genetically [21]. Therefore, investigation of heritability
of chromosomal instability under combined action
of IR and NO in somatic cells is of importance.
Taking into account the multiple impact of NO and
its derivatives on human health together with radio
logical situation after the Chernobyl accident in some
regions of Ukraine, the study of the combined action
of NO and IR on DNA and development of chromo
somal instability is very important.
In order to evaluate the combined genotoxic poten
tial of chemical and radiation factors and estimate the
efficiency of DNA repair processes were used alkaline
gel electrophoresis of isolated cells (DNA comet assay)
[22, 23] and chromosomal aberrations [24]. Chromo
somal aberrations are considered as integral index,
which takes into account both the implementation
of primary DNA damage and repair processes. An in
crease of chromosomal aberrations in peripheral blood
lymphocytes (PBL) is a valuable biological marker for
the assessment of stochastic effects, including can
cer [25]. High sensitivity of chromosomal apparatus
in PBL, low and quite stable level of spontaneous
chromosomal aberrations in these cells, their ability
to accumulate cytogenetic abnormalities, together
with the natural cells synchronization led to the wide
spread use of PBL culture in assessing mutagenicity
of physical and chemical environmental factors [26].
The aim of investigation was to study ability of en
vironmental NO or natural NO donors to modify free
radicals balance and cause development of genomic
instability alone or in combination with IR.
MATERIALS AND METHODS
Adult randombred male rats (120−150 g) were
obtained from the vivarium of R.E. Kavetsky Institute
of Experimental Pathology, Oncology and Radiobio
logy, NAS of Ukraine (Kyiv, Ukraine) and kept at steady
state conditions with a constant temperature and
natural light. The work with animals was performed ac
cording to the rules of local Ethic Committee [27]. The
animals were divided into four groups: 1) intact control
(IC); 2) animals that inhaled NOx for 1 month (16 h per
day); 3) animals that were fractionally irradiated with
Xrays at a frequency of exposure 0.1 Gy per 3 days
giving the total absorbed dose 1 Gy; 4) animals that
received combined treatment of NO and IR.
Genotoxicity and cytogenetic abnormalities were
assessed in PBL isolated from healthy humans for
an in vitro study or rats treated with NO and/or IR for
in vivo study. An informed consent of donors for taking
blood samples and conducting cytogenetic studies
was obtained.
NO inhalation. The inhalation treatment of animals
with NO was carried out in 0.1 m3 chamber equipped
with device for input of purified gaseous NO mixed in
side with air. Air circulation inside the chamber allowing
triple total replacement of air per 1 h. NOx concentra
tion at the chamber’s output was 150 mg/m3 of air,
40% corresponds to NO and 60% — NO2 of their
total content. Concentration of NOx was expressed
in mg of NO per m3 of air. The control of NO content
in the inhalation chamber was performed as described
previously [28].
X-ray irradiation. Xray irradiation of rat and
human PBL was performed at dose rate of 0.89 Gy/
min with doses range 0.1–2 Gy. Measurements
of absorbed dose were performed using an ionization
chamber and ferrosulfate dosimeter.
Isolation of PBL. Whole blood was diluted
in an equal volume of PBS and stratified on His
topaque1077 (“Sigma”, St Louis, MO) for lymphocyte
separation according to the manufacturer’s instruc
tion. After isolation, lymphocytes were washed in PBS,
diluted in 1 ml culture medium and counted after trypan
blue (“Euroclone”, Pero, IT) exclusion staining. PBL
were suspended in PBS and kept at 4−6 °C before use.
Electron paramagnetic resonance (EPR) stu-
dies. The levels of SNitrosohemoglobin (HbNO) and
methemoglobin (metHB) were measured by EPR in the
blood of rats. EPR spectra were obtained using spec
trometer RE1307 (USSR) and Е109 (Varian, USA)
in the centimeter wavelength range at T = 77K. Results
60 Experimental Oncology 35, 58–64, 2013 (March)
ware expressed in arbitrary units (a.u.), characterizing
the intensity of the EPR signal.
RSNO concentration in the blood serum of rats
was determined according to [29] and expressed
in nM. The total level of ROS and RNS in PBL deter
mined fluorometrically [30]. The serum levels of ROS
determined by spectrophotometric assay [31].
Alkaline Comet Assay. The singlecell gel elec
trophoresis was used for visualizing and measuring
SSB and DSB DNA damage in individual cells. The
method is based on detection of various mobil
ity of damaged DNA [32]. PBL were washed in PBS
and suspended in agarose gel at concentration
of 0.5–0.7*106 cells/ml then processed as previously
described [33]. Slides were stained with SYBR Green
(Sigma, 15 μg/ml). The images of comets were ob
served at 40x–100x magnification with a fluorescence
microscope equipped with video camera (CCD,
Webbers, USA). One hundred images were randomly
selected from each sample and analyzed by an image
analysis program “CometScore” (TriTek Corp, Sum
erduck, VA, USA). The degree of DNA damage was
estimated by the DNA percentage in the tail (%DNAT).
Cytogenetic analysis. The frequency of chromo
somal aberrations in human PBL measured with the
conventional cytogenetic assay in metaphase cells
on shortterm (52 h) and longterm (72 h) cultures. The
frequency of chromosomal aberrations was expressed
as a number of damaged chromosomes per 100 ana
lyzed metaphases. Total chromosomal aberrations
were sub classified as chromosometype aberrations
(including chromosometype breaks, ring chromo
somes, marker chromosomes, and dicentrics) and
chromatidtype aberrations (including chromatidtype
breaks and chromatid exchanges) [34]. As a transport
form of NO, SNitrosoglutathione (GSNO) was used,
which was added to the cell culture in the concentra
tion range of 0.5–1.5 μM.
Statistical analysis. Statistical analysis was per
formed using Student’s ttest. Values are reported
as mean ± standard error. Significance level was set
at P ≤ 0.05 [35].
RESULTS AND DISCUSSION
Prolonged inhalation of exogenous NOx was ac
companied by significant increase of RSNO level in the
blood serum of rats and by formation of a large amount
of Snitrosyl complexes of hemoglobin in erythro
cytes. The reaction between NOx and hemoglobin
resulted in formation of HbNO complex registered
at g = 2.03 on EPR spectrum (Fig. 1). The blood
of experimental animals was sampled in less than
1 hour after termination of NO treatment and showed
a 3.6fold increase in HbNO content (Fig. 2).
The moderate concentration of RSNO was present
in the blood serum of intact rats, but after NO inhalation
the level of RSNO increased in a 9.5fold, indicating
the development of nitrosative stress in organism
of animals (Fig. 2). The most prevalent form of RSNO
in organism is nitrosated form of glutathione —
GSNO. Previous work has shown that transnitrosation
between oxygenated hemoglobin and GSNO is a slow,
reversible process. However, GSNO reacts with de
oxygenated hemoglobin to form glutathione, NO and
ferric hemoglobin (metHb). NO formed from this reac
tion is immediately autocaptured to form HbNO [36].
Fig. 1. EPR spectrum of blood of intact rats (1) and rats exposed
to NOx (2)
0
1000
2000
3000
4000
5000
0
1000
2000
3000
4000
5000
HbNO RSNO
*
*
A.
U. nM
IC
the influence
of exogenous NO
* — differences
from IC; P ≤ 0.05
Fig. 2. The level of HbNO (a.u.) and RSNO concentration (nM)
in the whole blood of rats exposed to exogenous NO. Data shown
are the mean ± standard error of the mean.
The mechanism of RSNO formation involves direct
reaction of NO with reduced thiol and is accompanied
by production of a radical intermediates (RSN·OH)
including superoxide (O
2), peroxynitrite (ONOO¯) that
in the presence of Cu,Znsuperoxide dismutase form
hydrogen peroxide (H2O2) [37].
The intensity of free radical processes in the blood
of experimental animals after prolonged influence
of NO and/or low doses of IR was assessed by measur
ing the total levels of oxygen radicals and peroxynitrite
(ROS+RNS) in PBL and serum of rats. The total level
of ROS + RNS in PBL of rats exposed to NO was 3.1
fold higher than their level in PBL of intact animals.
However, fractionated irradiation of rats resulted in mi
nor reduction of free radicals level (1.2fold compared
with control). In animals exposed to NO combined with
IR, the production of ROS + RNS raised in 1.6fold
compared to their initial level (Fig. 3).
The tendency of ROS formation in serum and PBL
was similar. Maximum ROS generation (1.8fold higher
than in control) was observed after prolonged inhalation
intake of exogenous NO, while IR did not affect the ROS
level. Combined action of NO and IR resulted in an in
significant increase (1.2fold) of radicals level (Fig. 3).
Experimental Oncology 35, 58–64, 2013 (March)35, 58–64, 2013 (March) (March) 61
0
500
1000
1500
2000
2500
3000
3500
4000
IC NO IR NO + IR
0
20
40
60
80
100
120
140
160
180
RO
S
in
P
BL
(u
ni
ts
)
RO
S
in
s
er
um
(u
ni
ts
)
* — differences
from IC; P ≤ 0.05.
*
*
ROS in PBL
ROS in blood serum
Fig. 3. The total level of ROS in PBL and blood serum of rats
exposed to exogenous NO and/or IR. Data shown are the mean
± standard error of the mean.
Free radicals react with various molecules in the
organism, resulting in the production of stable ROS
metabolites (e.g., lipid peroxide, DNA adduct). They
oxidize purines and pyrimidines, causing breaks
in DNA and involved in nitrosation of primary hetero
cyclic amines.
The study of DNA fragmentation in the rat PBL
showed that the spontaneous level of fragmented DNA
in control animals reached 4.85%, that corresponds
to literature data [38]. Prolonged inhalation intake
of exogenous NO resulted in 2.4fold increase of DNA
breaks compared with control animals (Fig. 4).
0
4
8
12
16
IC NO IR NO + IR
DN
A
da
m
ag
e,
%
*
*
** — differences from IC; P ≤ 0.05.
Fig. 4. The level of DNA damage (%) in PBL of rats exposed
to NO and/or IR. Data shown are the mean ± standard error
of the mean.
Fractionated irradiation of rats resulted in 2.7fold
increase of DNA damage in PBL (13.3 ± 0.2%). The
highest level of DNA damage was observed under the
combined action of NO and IR (15.4 ± 0.1%) and it was
3.1fold greater than in control.
The combined effect of investigated factors, which
is not reducible to the sum of their individual impacts,
can be explained by common mechanism of genotoxic
effects for both factors (formation of ROS and RNS),
and individual contribution of NO to the overall effect
due to the nitrosation of primary amines by interac
tion with aminogroups of DNA and Nnitrosamines
synthesis. In addition, interaction of NO and ligases
lead to their nitrosation and formation of DNA hyper
sensitivity to other factors, such as IR.
HbNO and GSNO had been suggested to act
as an endogenous NO donors [6]. In order to investi
gate the combined effect of NO donors and IR, we per
formed parallel in vitro studies on human PBL. Spon
taneous level of DNA fragmentation in human PBL was
3.8 ± 0.06%. Irradiation of PBL led to dosedependent
increase (1.3fold at dose of 0.5 Gy and 6.3–fold
at dose of 2 Gy) of DNA breaks formation (Fig. 5).
0
5
10
15
20
25
30
0 0,5 1 1,5 2 2,5
Gy
DN
A
da
m
ag
e,
%
1
2
GSNO + IR
IR
Fig. 5 The level of DNA damage (%) in human PBL treated with
GSNO and IR (in vitro). Data shown are the mean ± standard
error of the mean.
The differences in genotoxic effect of IR in human
and rat PBL can be explained by variations in species
specific radiosensitivity [39] and hypersensitivity
of DNA exposed to fractionated lowdose IR [40].
The results of DNA damage study after combined
treatment of PBL with IR and GSNO (1 μM) in vitro shown
on Fig. 5. The treatment of PBL with GSNO alone led
to 1.4fold increase in DNA fragmentation compared
with control cells. Combined treatment of PBL with
GSNO and IR at dose of 0.5 Gy caused 2.2fold in
crease of DNA breaks. This effect was 1.8fold greater
than the effect of IR alone, and in 1.6fold exceeded
DNA damage by GSNO alone. Increase of IR dose
up to 1 Gy together with 1 μM of GSNO resulted
in 3.1fold rise of genotoxic effect (Fig. 5). But fur
ther elevation of IR dose up to 1.5 Gy combined with
GSNO did not cause increase of DNA damage. On the
contrary, their level dropped 1.6fold compared with
level of DNA damage induced by 1 Gy, also it remained
1.9fold higher compared to control PBL. Thus, the
genotoxic effect after combined treatment with GSNO
and IR was 1.2fold weaker than after individual action
of 1.5 Gy of IR, but this effect was in a 1.4fold stronger
than individual effect of GSNO (data not shown) (Fig. 5).
The interaction of NO and DNA repair enzymes may
lead to the failure of DNA repair and increase of the
genotoxic effect. It is known that during the DNA exci
sion repair SSB are formed. The replication of DNA with
unrepaired SSB may cause formation of DSB, associ
ated with development of genetic instability. Existence
of DNA DSB accompanied by impaired DNA repair
resulted in formation of chromosomal aberrations.
The cytogenetic effect of GSNO and IR on human PBL
in vitro (Table) was evaluated under short time cultivation
condition in the first cell generation. Results suggest
that upon an increase of GSNO concentration from
0.5 to 1.0 μM the number of PBL with chromosome ab
errations and the overall frequency of induced chromo
somal aberrations are also increased (from 6.0 ± 0.2 and
7.0 ± 0.2 to 12.0 ± 1.0 and 18.3 ± 1.4, respectively).
62 Experimental Oncology 35, 58–64, 2013 (March)
Table. The frequency and spectrum of chromosome aberrations in human
PBL treated with GSNO and IR
№ Groups
Cytogenetic parameters (per 100 analyzed
metaphase)
The fre-
quency
of aberrant
cells, %
The total fre-
quency of chro-
mosome aber-
rations
Chro-
mosome
type ab-
errations
Chroma-
tide type
aberra-
tions
1 Intact control 1.1 ± 0.3 1.1 ± 0.3 0.3 0.8
2 IR, 0.5 Gy 11.0 ± 1.3 11.0 ± 1.3 5.8 5.2
3 IR, 1.0 Gy 18.0 ± 1.4 18.0 ± 1.4 12.0 6.0
4 IR, 1.5 Gy 26.0 ± 1.8 34.0 ± 1.8 26.0 8.0
5 GSNO, 0.5 μM 6.0 ± 0.2 7.0 ± 0.2 - 6.0
6 GSNO, 1.0 μM 12.0 ± 1.0 18.3 ± 1.4 0.3 18.0
7 IR, 0.5 Gy + GSNO, 0.5 μM 20.0 ± 1.4 21.0 ± 1.7 8.0 13.0
8 IR, 0.5 Gy + GSNO, 1.0 μM 24.0 ± 1.6 26.0 ± 1.6 7.0 19.0
9 IR, 1.0 Gy + GSNO, 0.5 μM 27.0 ± 1.5 30.0 ± 1.9 10.0 20.0
10 IR, 1.0 Gy + GSNO, 1.0 μM 37.0 ± 1.8 46.0 ± 1.6 13.0 33.0
11 IR, 1.5 Gy + GSNO, 0.5 μM 35.0 ± 2.0 48.0 ± 1.9 23.0 25.0
12 IR, 1.5 Gy + GSNO, 1.0 μM 23.0 ± 1.4 25.0 ± 1.4 17.0 8.0
The aberrations of chromatid type, such as dele
tions and exchanges, were prevalent in the spectrum
of GSNO induced damage. On the contrary, irradiation
of PBL mainly caused chromosomal type aberrations.
The combined effect of low dose of IR (0.5 Gy) and
smaller GSNO concentrations (0.5 μM and 1.0 μM)
caused an increase in the number of chromosome
aberrations in human PBL (Table), as well as an in
crease of DNA damage frequency in rat PBL (Fig. 5).
The total effect does not exceed the sum of individual
effects of these factors, while it was larger than the
effect of a single factor. Increasing the dose of GSNO
(1.0 μM) and IR (1 Gy) caused the additive effect
of all studied cytogenetic parameters. The obtained
data indirectly indicates inhibition of DNA repair due
to combined effect of IR and GSNO, possibly because
of induction of slow or unrepairable DNA breaks,
as evidenced by formation of structural chromosomes
rearrangements.
However, further increase of IR dose up to 1.5 Gy com
bined with GSNO treatment (1.0 μM) led to considerable
decrease of cytogenetic effects, in particular, the num
ber of cells with chromosome aberrations and the total
frequency of chromosomal aberrations (Table). The
overall aberrations rate decreased 1.8fold compared
to cells treated with IR (1 Gy) and GSNO (1 μM). Chro
mosometype aberrations were the prevalent damage
type, while the number of chromatidtype aberrations
dropped to the level of individual impact of 1.5 Gy of IR,
and decreased 2fold compared to 1.0 μM of GSNO.
As shown on Fig. 5, the number of DNA breaks
increased with increasing of IR dose up to 1.5 Gy. How
ever, the number of DNA breaks in PBL treated with
IR at the dose of 1.5 Gy and GSNO at the concentration
of 1.0 μM decreased compared with effect of IR and
GSNO lower dose as well as with effects of individual
factors. The common trend in results of molecular and
cytogenetic studies suggests the direct link between
the frequency of chromosomal aberrations and the
level of DNA damage. Reduction of induced cytoge
netic effect can be attributed to elimination of cells
with heavily damaged DNA, in addition experiencing
deficiency in DNA repair and, as a result, more loaded
with chromosomal rearrangements.
In order to confirm this assumption we have stud
ied the qualitative and quantitative characteristics
of chromosomal instability of PBL at longterm culti
vation (72 h, Fig. 6–8). Decrease of overall frequency
of chromosome aberrations (1.5fold) in irradiated
PBL treated with GSNO was observed mainly in com
bination with low doses of IR, but the changes of the
spectrum of chromosome aberrations were found
throughout whole dose range of IR (Fig. 6).
0
8
16
24
32
0,5 1,0 1,5
Ab
er
ra
tio
ns
fr
eq
ue
nc
y
Gy
Control GSNO
IC
GSNO
IR
GSNO + IR
Fig. 6. The total frequency of chromosome aberrations (per
100 analyzed metaphase) in human PBL treated with GSNO
and IR. Data shown are the mean ± standard error of the mean.
The radiation effects in human PBL were character
ized by linear dose dependence of the chromosome
type aberrations frequency, which is a good marker
of the IR impact, but additional influence of GSNO
caused marked decrease of cytogenetic effect without
dependence on the IR dose (Fig. 7).
0,5 1,0 1,5
0
7
14
21
28
Ab
er
ra
tio
ns
fr
eq
ue
nc
y
Gy
IR
IR+GSNO
Fig. 7. The frequency of chromosome type aberrations (per
100 analyzed metaphase) in the human PBL treated with GSNO
and IR. Data shown are the mean ± standard error of the mean.
The frequency of chromatidtype aberrations,
which are cytogenetic markers of chemical mutagens
influence, in human PBL treated with GSNO and IR in
creased linearly with IR dose (Fig. 8). The treatment
with IR alone was characterized by doseindependent
decrease of number of chromatidtype aberrations.
These results suggest elimination of PBL with
chromosometype aberrations occurred due to DNA
DSB. The contribution of chromatidtype aberrations,
which are arising mainly due to DNA SSB, in the deve
lopment of genetic instability at longterm cultivation
of PBL was more significant. Accumulation of SSB
in a number of cell generations plays a crucial role
in the development of chromosomal instability under
the combined influence of IR and GSNO.
Experimental Oncology 35, 58–64, 2013 (March)35, 58–64, 2013 (March) (March) 63
0,5 1,5
0
2
4
6
8
10
12
Ab
er
ra
tio
ns
fr
eq
ue
nc
y
Gy
1,0
IR
IR+GSNO
Fig. 8. The frequency of chromatidtype aberrations (per
100 analyzed metaphase) in human PBL treated with GSNO
and IR. Data shown are the mean ± standard error of the mean.
Thus, inhalation intake of exogenous NO leads
to formation of complexes with hemoglobin, albumin
and other iron and SHcontaining proteins and bio
molecules. In addition, NO can be oxidized to nitrites
and nitrates, as well as reduced from these com
pounds. Hemoglobin plays a major role in the reco
very reaction of NO2
ions to NO, since O2 in the blood
primarily interacts with it. The interaction of NO2
ions
with deoxyhemoglobin caused the redox reaction,
in which deoxyhemoglobin was oxidized to metHB and
NO2
restored to NO:
Hb2+ + NO2
+ 2H+ → metHB + NO + H2O
Interacting with reduced hemoglobin, NO forms
a stable HbNO complex. Due to reverse binding
of NO with biological molecules and the formation
more stable RSNO and complexes of nonheme iron
((RS)2Fe+(NO+)2) NO can be transferred to various
tissues and organs. Under certain conditions, NO can
be released.
We showed that prolonged inhalation intake of ex
ogenous NO resulted in the development of nitrosa
tive stress in rats accompanied by formation of large
amount of metHb (68fold of control value, data not
shown), HbNO and increased level of RSNO in the
blood. The formation of HbNO complexes and RSNO
was of a specific interest due to possibility of revers
ible release of NO. As expected, the level of these
compounds in the blood was highest immediately after
exposure to NO, then their concentration gradually
decreased and reached the control level at 12th day.
Exogenous NO not only directly provide increase
of RSNO level, but also affect the processes associ
ated with production of endogenous NO. In our previ
ous study was shown that longterm exposure to ex
ogenous NOx negatively affected Tcells and caused
hyper activation of peritoneal macrophages [28].
Obtained data confirm that formation of chromo
somal aberrations is typical for radiationinduced
genotoxic effects. Effects associated with NO are
crucial in the formation of chromatidtype aberrations.
Analysis of literature showed that NO may mediate
radiationinduced effects, including development
of genomic instability in mammals. One of these ef
fects is the formation of DNA DSB and decreased
efficiency of repair system, which cumulatively lead
to formation of chromosome aberrations.
Thus, we showed that the combined influence
of NO and IR caused destabilization of genetic ap
paratus in PBL, the degree of manifestation of which
depended on the doses of these damaging factors.
Occurrence of chromosomal aberrations is con
sidered as a characteristic feature of neoplastic cells.
To date, more than 600 tumorassociated specific
chromosomal rearrangements are observed in most
cases of cancer. Carcinogenesis associated with
chromosomal aberrations can be induced by deregu
lation (usually overexpression) of the normal gene,
or by formation of hybrid gene from the fragments
of aberrated chromosomes [41]. There is a distinct
link between the total incidence of cancer and the
presence of chromosomal aberrations [42]. Since the
appearance of chromosomal changes is considered
to be potentially carcinogenic [43, 44], our data dem
onstrate the possibility of increasing carcinogenic risk
under the combined influence of NO and IR.
Thus, the combined effect of NO and IR caused
the formation of DNA SSB and DSB with the further
development of chromosomal instability in PBL, the
degree of manifestation and nature of which depends
on the doses of these factors. The dose dependence
of chromatidtype aberrations observed in human PBL
after combined influence of IR and GSNO indicates
a crucial role of NO in the formation of chromosomal
instability. Release of NO from stable complexes with
biomolecules (such as GSNO and GbNO) can deregu
late free radicals balance resulted in genotoxic effect,
posttranslational modification of repair enzymes and
thus coordinate development of genomic instability
and increase of cancer risk.
ACKNOWLEDGMENT
We thank Dr. Glavin A.A. for the assistance with
GSNO preparation.
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