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ISSN: 0005-2523
Volume 62, Issue 06, August, 2022
Effect of metformin on gamma radiation -Induced DNA
oxidation damage in Human Peripheral Blood
Lymphocytes in vitro.
Ali Niama Salman1, Farha A.Ali Shafi2, Abdual Sahib Kadhim Al-Zayadi3
Ministry of Environment, Baghdad, Iraq1
Department OF Biology, College of science, University of Mustansiryah, Baghdad Iraq2
Ministry of Science and Technology, Baghdad, Iraq3
8-Hydroxydeoxyguanosine (8OHdG), gamma ray, DNA oxidative
damage, metformin
The aim of the study was to assess the effect of metformin (an antihyperglycemic drug in type 2 diabetes (T2D) patient) on DNA oxidative
damage in vitro in human peripheral blood lymphocyte from 10 donors;
evaluation of the oxidative damage induced by 1 and 2 Gy gamma rays;
and assessed the role of metformin in protecting lymphocytes against the
8Hydroxydeoxyguanosine (8-OHdG) ELISA Kit was used to detect the
DNA oxidative damage in blood lymphocytes. Metformin (10 and
50µM) in vitro increased the rate of (8-OHdG) significantly (P ≤ 0.001)
than control samples value. Irradiated blood lymphocyte with 1 Gy
gamma rays elevated the DNA oxidative damage in rate (95%) and
significantly (P ≤ 0.001) than control values, and the rise of irradiation
into 2 Gy, caused increase the DNA oxidative damage (8-OHdG) 2.3
folds and significantly (P ≤ 0.001) than 1 Gy irradiated. Blood treated
with metformin prior irradiation enhance the reduction of (8-OHdG)
specially in blood irradiated by 2 Gy gamma ray, the reduction
significantly (P ≤ 0.05 and P ≤ 0.001) respectively for (10 and 50 µM)
metformin treated compared with irradiated group only.
This work is licensed under a Creative Commons Attribution Non-Commercial 4.0
International License.
1. Introduction
Ionizing radiation (IR), including gamma rays, associated with many aspects of people lives and causes
harmful genetic effects on the cells due to the influence of its high energy on cellular molecules, and ionize
the cellular water producing free radicals that reacts aggressively with cellular components such nucleic
acids, proteins, and lipids [1], [2].
Water radiolysis generate reactive oxygen species (ROS) (including: Hydrogen peroxide (H2O2) [3],
Superoxide ion (O2•) and hydroxyl radical (OH•)), ionized water (H2O+), hydrogen radical (H) and hydrated
electrons (eaq-)[4]. In the presence of (O2, eaq- and H) atoms immediately transformed to superoxide
/perhydroxyl O2•–/HO2• radicals [5].
A. N. Salman, F. A.A. Shafi and A. S. K. Al-Zayadi, 2022
Azerbaijan Medical Journal
Hydroxyl radicals (HO•) capable to attack and modify DNA, specially guanine (G) base and generate C8hydroxyguanine (8-OHGua) or its nucleoside counterpart deoxyguanosine (8-hydroxy-2-deoxyguanosine)
convert it to 8-hydroxy-2'-deoxyguanosine (8-OHdG) or to structural isomer 8-oxo-7-hydro-2'deoxyguanosine (8-oxodG)) [6], [7].
Super peroxide (O2•-) attacks readily oxidized guanine to generate (8-oxodG) in DNA reactions [8]. During
the DNA correcting operation, (8-OHdG) within the DNA strand is corrected and discharged from cells.
Which known as markers for oxidative DNA damage often utilized as an oxidative stress biomarker [9], [5],
Metformin classified as an anti-hyperglycaemic drug for T2D patient, [11]. Metformin confirmed to reduce
cellular endogenous (ROS) and disrupt the energy in mitochondria by inhibiting complex I and OXPHOS
enzymes [12- 14], lead in activates AMP-Activated Kinase (AMPK); [15]. Metformin lower intracellular
(ROS) levels by increasing the production of the antioxidant protein thioredoxin via the AMPK-FOXO3
pathway, an essential endogenous antioxidant mechanism and plays a key role in cellular redox equilibrium,
[16], [17]. Metformin connected to operate on the ataxia telangiectasia mutated-checkpoint kinase 2 ATM
/Chk2 pathway, which can stop the cell cycle and allow for DNA repair [18], and involved in response to
oxidative stress [19].
2. Material Methods
This study was approved by the Ethics Committee of the College of Science, Al-Mustansiriya University.
Fresh peripheral venous blood samples were obtained from 10 healthy donors (five male and five female),
ranged in age from 25 to 35 years old, from Baghdad. All participants were non-smokers, did not consume
alcohol and had not been exposed to IR for least 3 months.
Soon after blood sample collection were heparinized and distributed in sterile (2ml) Eppendorf tubes, for
metformin treatment or irradiation or both. metformin solvent (in sterile doubled distilled water) was added
into tubes were reached to final concentrations (10 and 50 µM/mL), then incubated at 37◦C. Subsequently
(after 2 hours of incubation) samples were irradiated with 1or 2 Gy of gamma radiation (Gamma chamber
900 Birt India) and were incubated again at 37◦C for 1 h following irradiation to enable for DNA repair.
Blood samples were distributed into groups; as describe bellow: Group one control: Blood sample was treated with sterile doubled distilled water.
Group two (10 µM) Metformin: blood samples were subjected to (10 µM) metformin
Group three (50 µM) Metformin: blood samples were subjected to (50 µM) metformin
Group four Radiation only: Blood sample irradiated with 1 or 2 Gy gamma ray.
Group five (10 µM) Metformin + Radiation: blood samples were subjected to (10 µM) metformin for 2 h
formerly exposure to gamma radiation 2 or 1 Gy
Group six (50 µM) Metformin + Radiation: blood samples were subjected to (50 µM) metformin for 2 h
before exposure to gamma radiation 2 or 1 Gy.
2.1 Sample culturing
Approximately one-half milliliter of each blood sample was mixed with (4.5 ml) medium RPMI 1640
(Capricorn) supplemented with L-Glutamine and antibiotics (gentamicin), 15% fetal bovine serum
(Capricorn) and 5% (0.25 ml) of Phytohaemagglutinin (PHA) (Capricorn). The samples were kept in
incubator at 37◦C for 72 h. After that the samples were centrifuged at 1500rpm for 10 min, 2 ml of
suspension was withdrawn for the purpose of ELISA examination.
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Volume 62, Issue 06, August, 2022
8-Hydroxydeoxyguanosine (8-OHdG) concentration in cell culture supernatant was detected by used 8Hydroxydeoxyguanosine (8-OHdG) ELISA Kit, measurements procedure applied according to the kit
Catalogue: MBS764814; mybiosource.com
3. Statistical analysis
The statistical analysis program IBM SPSS version 28.0 was used, the mean and SE of mean, the
probability and the significantly compare between the means of groups were calculated by the least
significant difference (LSD), and analysis of variation (ANOVA) to detect the effects of different factors in
the study parameters [20].
4. Results and discussion
4.1 8-Hydroxydeoxyguanosine (8-OHdG) as biomarkers of oxidative DNA damage
Table (1 and 2) summarized the data of (8-OHdG) obtained from samples in this study
The means value of (8-OHdG ng/ml) in control samples and samples treated with two concentration of
metformin (10 and 50 µM) were (7.03 ± 0.16; 12.03 ± 0.15 and 13.92 ± 1.03) respectively. The mean value
of 8-OHdG in sample treated with (10 and 50 µM) of metformin substantially higher and significantly
differences (P ≤ 0.001) when compared with control samples (table 1).
The blood samples were treated with metformin clearly contained more 8-OHdG value but it is noteworthy
that untreated samples (control) similarly contained (8-OHdG). It is important to mention that some of
(ROS) result from normal cellular metabolism. With absence exposed to exogenous DNA damage material,
and through oxidative metabolism in mitochondria some oxygen converted to (ROS). Approximately (180)
guanines are oxidized to (8-oxoG) for each mammalian cell each day [21]. [22] detect small amount of (8OHdG) approximately (0.6 - 1.4 residue/ l05 dG) in DNA samples isolated from mouse liver; HeLa cells
and S. typhmirium that had not been treated DNA oxidation agents. According to results increased
concentration of (8-OHdG) in metformin treated sample indicate DNA damage in lymphocytes cells in
vitro. [23] reported the mean value of (8-OHdG) in metformin treated sample were (114.4 μg/ml) was
higher than detected in control samples. [24] suggest that the genotoxicity impacts of metformin in
mammalian result from activation of AMPK that led to increases nitric oxide production, as well as
mitochondria-produce reactive-nitrogen species.
Numerous of the DNA lesions are generated from exposed to (ROS) that repaired via base excision repair
system, some of (ROS) result from metabolic process in cell with absence exposed to exogenous DNAdamaging material for example through oxidative metabolism in mitochondria some oxygen is converted to
(ROS) [25].
Table (1) The mean± SE of (8-OHdG) induced in vitro by 10 and 50 µM of metformin in cultured blood
lymphocytes from human volunteers.
10µM met
7.03 ± 0.16
12.03***a ± 0.15
***. P ≤ 0.001; a. compares to control
50µM met
13.92***a ± 1.03
Samples exposed to 1 and 2 Gy gamma ray show increased in 8-OHdG, the mean value of the 8-OHdG ±
SE were (13.69 ± 1.18 and 31.98 ± 2.9) respectively that were significant higher (P ≤ 0.001) when parallel
to the control value (figure 1), the increase rates were (1.95 and 4.54) folds respectively than control, and
the doubling of the radiation dose from 1 to 2Gy led rising the DNA oxidation damage by (2.34) folds.
A. N. Salman, F. A.A. Shafi and A. S. K. Al-Zayadi, 2022
Azerbaijan Medical Journal
Figure (1) The mean± SE of 8-OHdG induced in vitro by 1 and 2 Gy of gamma radiation in cultured
blood lymphocytes from human volunteers
The first target of ionizing radiation is the aqueous material of the cells as a consequence of radiolysis of
water the creation of reactive oxygen species (ROS). Among the (ROS) the mainly significant are hydrogen
peroxide (H2O2), as the longest-lived and hydroxyl radical (OH•) most reactive (ROS) produced by IR,
capable to attack and modify chromosomal and mitochondrial DNA [1].
In general, guanine is the most vulnerable nucleic acid component to oxidative processes mediated by free
radical induced by IR specially (•OH), that modified to 8-hydroxydeoxyguanosine (8-OHdG) when reacts at
C8 guanine nucleotide. The existence of 8-OHdG in DNA templates may induce the miscoded insertion of
nucleotides in the duplicated strand, such damage must be corrected to avoid mutations by recognizing then
removing the (8-OHdG) and repair the damaged base [26], the base excision repair (BER) is a major
pathway for DNA base repair damaged by (ROS) [27].
The findings of the current study are consistent with results of [22] who Found that (8-OHdG) increased
with increase the dose of ionizing radiation. Also, he detects that there is positive correlation between
concentration of (H2O2) and percentage of (8-OHdG) in the cells exposed to gamma radiations. While
numerous studies have identified increased value of (8-OHdG) in oxidative-stress-associated diseases, the
precise biological role of (8-OHdG) has not been discovered. Oxidized deoxyguanosine has been related
with an increased risk of genomic damage, thus, most researchers have assumed that oxidized
deoxyguanosine could have mutagenic or at least critical effects in cells, therefore mammalian physiology
tries to eliminate it [28]. According to the latest hypothesis cells create of (8-OHdG) be one of the
protection mechanisms of cells against oxidative-stress-induced inflammation, oxidized guanosine can react
with the GTPase family (Rho; Rac ;Ras and cdc42), which is generally involved in cytoskeleton
modification, triggering inflammation controlling programmed cell death, as well as carcinogenesis [29],
[30]. For example Rac1 activation result in growing NADPH oxidase (NOX) complex and later ROS
generation [31]. So, inhibition of Rac1 via 8-OHdG, can meaningfully block ROS-mediated inflammation.
Treatment of blood samples with metformin (10 and 50µM) before Exposure to gamma radiation 1 or 2 Gy
resulted in decrease in the concentration of (8-OHdG) when parallel to sample irradiated with gamma
radiation only table (2).
Table (2) The changes of 8-OHdG induced in vitro by different treatment in (1and 2 Gy gamma-ray and
metformin + 1 or 2 Gy radiation groups in cultured blood lymphocytes from human volunteers. data
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Volume 62, Issue 06, August, 2022
represent as mean ± S.E.
10µm met+ 1Gy
13.69 ±1.18
***. P ≤ 0.001,
50µm met+ 1Gy
13.34 ±1.16
12.44 ± 1.05
31.98 ± 2.9
*. P ≤ 0.01, a. compares to 2Gy irradiated group
22.55*a ± 3.16
50µm met+ 2Gy
19.19*** a ± 2.84
The mean value of the 8-OHdG ± SE of sample treated with (10 and 50µM) Metformin prior irradiation
with 2 Gy were (22.55 ± 3.16 and 19.19± 2.84) respectively (Table 2). These values were significant
difference (p<0.05) when compared with sample exposed to 2 Gy of gamma radiation only. The reduction
rates were ( -29.49% and -39.99%) respectively figure (1).
The present findings consistent with other research which found that metformin have many mechanisms to
reduce the oxidative stress. First, metformin might engage directly with (ROS) in vitro. BonnefontRousselot demonstrated that metformin was able to scavenge hydroxyl free radicals (but not hydrogen
peroxides or superoxides) [32]. Second, metformin is capable of reduction (ROS) via arresting its creation
in the cells through reducing NAD(P)H, or via reduce respiratory chain reactions in mitochondria [33].
Third, metformin possibly will exert its protective role in part through alteration concentrations of serum
insulin. Moreover earlier studies proven that metformin has a significant influence on DNA repair pathways
through up-regulating of AMP-activated protein kinase (AMPK) [34].
DNA base excision repair (BER) system is efficient repair mechanism for correcting (8-OHdG) base,
which depend on several enzymes to recognize, hydrolyses the damage and fill the gaps, 8-oxodeoxyguanosine DNA glycosylase 1 (OGG1) is essential enzyme in recognize the damage and two major
proteins XRCC1 and DNA POL BETA contribution in fill the gaps,mXRCC1 has been proved to increased
significantly in diabetes patients using metformin which effect on modulate the oxidative stress, also
metformin is up-regulate P53 expression which activates a range of DNA repair genes, such as DNA POL
beta, and a cellular responses to oxidative stresses [35], [27].
5. Conclusion
Exposed blood samples to gamma radiation 1 and 2 Gy induced cellular damage via ionizing the cellular
water molecules producing high reactive free radicals result in increased DNA oxidation damage biomarker
(8-OHdG). Reflecting genotoxic and cytotoxicity effects of gamma radiation treated blood samples with
metformin prior exposed to gamma radiation enhanced cell’s ability to resist damage and decreases the
genotoxic and cytotoxic effects of gamma radiation on peripheral blood lymphocytes revealing antioxidant,
genoprotective and radio mitigating properties of metformin drug.
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