Paper
EFFECT OF DIFFERENT ANTIOXIDANTS ON X-RAY INDUCED
DNA DOUBLE-STRAND BREAKS USING g-H2AX IN HUMAN
BLOOD LYMPHOCYTES
Nicoleta Simona Bicheru,1 Cerasela Haidoiu,1 Octavian Călborean,1,2 Adrian Popa,1
Ioana Porosnicu,3 and Radu Hertzog1
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Abstract—Ionizing radiation exposure produces direct or indirect
biological effects on genomic DNA. The latter are ionizing radiation
mediated by induction of free radicals and oxygen species (ROS).
The study was conducted to evaluate the dose-effect/time-effect of
antioxidant treatments in reducing the induction of double-strand
breaks in human blood lymphocytes. Human peripheral blood
samples of 2 mL each from healthy donors were irradiated
with 10 mGy after pre-incubation with different antioxidants
(b-carotene, vitamin E, vitamin C, N-acetyl L-cysteine). In order
to assess their efficiency as prophylactic therapy for irradiation,
various concentrations and combinations of antioxidants, as well
as different incubation times, have been evaluated. To assess
double-strand breaks induced by ionizing radiation, the phos-
phorylated histone g-H2AX has been used. A significant reduc-
tion (p < 0.001) in double-strand breaks studied with a g-H2AX
assay was observed with N-acetyl L-cysteine with a 1-h incubation
time, followed by vitamin C, vitamin E, and b-carotene. The use
of antioxidants, especially N-acetyl L-cysteine before irradiation,
significantly decreased the occurrence of double-strand breaks,
demonstrating the potential radiological protection for exposure
to ionizing radiation.
Health Phys. 119(1):101–108; 2020
Key words: DNA; health effects; radiation damage; radiation,
ionizing
INTRODUCTION
RADIATION GENERATES damage in all animal and human cells
mainly through the generation of aqueous free radicals, such
as OH, H, eaq−, HO2, and H3O+. Antioxidants can reduce
1
Military Medical Research Center, Bucharest, Romania; 2Faculty of
Biology, University of Bucharest, Bucharest, Romania; 3National Institute
for Laser Plasma and Radiation Physics, Bucharest, Romania.
The authors declare no conflicts of interest.
For correspondence contact: Octavian Călborean, Military Medical
Research Center, Bucharest, Romania, Military Medical Research Center, 3-
5 Institutul Medico-Militar Street, Bucharest, 010919 Romania or email at
ocalborean2@yahoo.com.
(Manuscript accepted 19 December 2019)
0017-9078/20/0
Copyright © 2020 Health Physics Society
DOI: 10.1097/HP.0000000000001267
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the deleterious effects of some of these radicals. Thus,
sulfhydril groups such as N-acetyl L-cysteine (NAC) can
form mixed disulfides with DNA and proteins that prevent
radiation damage by stabilizing the target. Vitamin C and
vitamin E reduce the formation of lipid hydroperoxide de-
rivatives (Nair et al. 2001). The human body’s response to
radiation is a complex process that depends on external fac-
tors such as radiation type, dosage, and duration, as well as
its intrinsic genetic and epigenetic characteristics. The main
types of radiation exposure include the background dose,
computer tomography, and other investigation and radio-
therapy tools, as well as environmental exposure due to nu-
clear incidents and disasters (Reisz et al. 2014) or due to the
increased use of depleted uranium on modern battlefields.
The changes induced by radiation are mainly mediated
through reactive oxygen species (Valko et al. 2004) that
generate different types of DNA damages, such as double-
strand breaks (DSB). These damages can be assessed using
fluorescence microscopy by counting the foci caused by
DNA breaks (Reliene et al. 2009) that generate the early
phosphorylation of g-H2AX in the nucleii of affected cells
(Rothkamm and Löbrich 2003; Reliene et al. 2009). The
rate of DSB is positively correlated in a very linear way with
the radiation dose for lower doses, which makes it an excel-
lent biomarker for quantifying the radiation-induced DSBs
(Rogakou et al. 1998; Rothkamm and Löbrich 2003;
Löbrich et al. 2005; Rübe et al. 2008; Beels et al. 2009;
Kuefner et al. 2009, 2010b; Reliene et al. 2009). After an in-
terval of between 5 and 10 min after irradiation, the number
of g-H2AX foci is the highest, and after that the repair pro-
cess lowers it (Löbrich et al. 2005; Jeggo and Löbrich
2007; Copp et al. 2013).
In humans, there is a wide range of response to radiation,
which is determined by parameters including the radiation
source, radiation dosage (amount of radiation energy re-
ceived), length of exposure, and, importantly, the genetic
and epigenetic makeup of the exposed individual. These
101
102 Health Physics
parameters can range widely, and humans may be exposed
to low-dose radiation from commonly used diagnostic tools
in medicine, such as computer tomography (CT) scanning
or high doses of radiation used for radiotherapy or gener-
ated by radiation accidents (Reisz et al. 2014).
Various dietary antioxidants, such as vitamin C and vi-
tamin E, have been studied due to their proven capacity to
protect human cells and organs from radiation-induced
damages (Weiss and Landauer 2000) and thus reduce the ra-
diation tissue damage and the risk of different cancer types
(Borek 2004). However, the extension of radiation protec-
tion for doses present in radiological examinations and
workplace exposure in high-risk environments is an impor-
tant but less researched topic.
Our objective is to determine whether several natural
antioxidant compounds, when present in concentrations
lower, equal, and higher than their normal physiological
values, present in vitro protective effects against x-ray ioniz-
ing radiation at acute doses equal to the maximum allowed
annual dose for radiation workers and typical for multiple
CT imaging. We used the g-H2AX test, a well-established
method to assess the double strand DNA breaks at 24 h after
50 mGy irradiation doses (Brand et al. 2015).
MATERIALS AND METHODS
Chemicals
Phospho-Histone H2A.X (Ser139) Monoclonal An-
tibody (CR55T33), Alexa Fluor 488, was purchased
from ThermoFisher Scientific (Waltham, MA USA).
VECTASHIELD DAPI mounting medium was purchased
from Vector (Burlingame, CA USA). The Histopaque 1077,
phosphate buffered saline solution, albumin from bovine
serum >96% and DAPI (4´,6-Diamidine-2´-Phenylindole
dihydrochloride) were purchased from Merck KgaA
(Darmstadt, Germany). The Formaldehyde solution was
purchased from Bio-Optica Milano S.p.A. (Milan, Italy),
the Fluorescent Mounting Medium was purchased from
Agilent Technologies, Inc. (Santa Clara, CA USA), while
Triton
100 was purchased from Honeywell International,
Inc. (Charlotte, NC USA).
Table 1. Amount of antioxidant substances added to the blood samples adapted to the recommended daily intake (RDI) and
their average concentrations in plasma; aConcentration of substance used in the experiments per 1 mL blood; bCurrent RDI
(FDA, WHO); PI: recommendation on package insert; SC: Standard concentration.
Antioxidants 1 x SC/1 mL blooda
ß-carotene (hard capsules 12,000 U.I. 0.48 mg/mL
Rotta Natura, Romania)
Vitamin E (soft capsules, 400 U.I, 12.8 mg/mL
d-alpha-tocopherolacetal),
Cosmo Pharma, Romania)
Vitamin C (tablets 500 mgL, BioLand, Romania) 8.8 mg/mL
NAC (capsules, 300 mg, Zenyth, Romania) 10.7 mg/mL
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July 2020, Volume 119, Number 1
Antioxidants
We tested four antioxidant compounds: b-carotene, vi-
tamin E, vitamin C, and N-acetyl L-cysteine (NAC) that
were sold as natural supplements by local producers in the
form of either hard capsules or powder (Table 1).
For the standard concentrations (SC) of antioxidants,
we followed the protocol described by Brand et al. (2015).
Briefly, we prepared pre-incubation media with antioxidant
concentrations based on the recommended daily dose values
(RDI) on FDA and OMS and assuming a 6,000 mL total
blood volume (see Table 1). Given these standard concen-
trations (SC), we tested the effect of three concentrations
1
SC, 10
SC, and 1/10
SC values (see Table 1).
Irradiation setup
The irradiation system is based on a transmission x-ray
source from YXLON International (Hamburg, Germany)
that is normally a part of non-destructive testing equipment.
It can be operated with a submicron focal spot at tube high
voltage up to 225 kV and a maximum power of 10–20 W.
The main advantages of this type of x-ray source are the
cone beam opening angle of around 170o, which ensures ir-
radiation of large volumes and the tunability of the energy
and dose debit, which can provide high precision for low-
dose irradiation setup. The dose rate was measured with a
Standard UNIDOS dosimeter and a Farmer Chamber type
30010 (for absolute dose measurement in radiation therapy)
calibrated in air kerma at the primary standards of the Ger-
man Federal Institute of Physics and Metrology (PTB-
Braunschweig). The human peripheral blood samples were
placed at 13 cm distance from the x-ray source focus set to
90 keVand 100 mA and exposed to it through a 1-mm alumi-
num filter. The measured dose rate was 1.34 mGy s−1, and
the radiation dose of 50 mGy was delivered in a 37-s expo-
sure time.
Patient blood samples
We took 2 mL of peripheral blood in Vacutainer blood
collection tubes from each of the 10 patients using sampling
and informed consent procedures approved by our internal
Bioethics Committee (authorization number: 1/20.02.2019).
All subjects were healthy males, non-smokers, without any
1 x SC initial data RDI/PI per dayb
0.9 mmoL/L in plasma 6 mg PI (12,000 U.I.)
27 mmol/L in plasma 10–15 mg PI (400 U.I.)
50 mmol/L in plasma 100–150 mg PI (500 mg)
300 mg daily dose for a 70 kg adult, PI (300 mg)
bio-availability = 0.4
 Effect of antioxidants on x-ray-induced DNA double-strand breaks c N. S. BICHERU ET AL.
acute or chronic disease. The subjects were aged between 30
and 35 and had an average weight of 65 kg.
In vitro radioprotection of cell DNA
The human peripheral blood samples were treated with
antioxidant and then were exposed the following to a
50 mGy dose of x-ray radiation as follows:
• Group 1: 2 mL Blood + 0 mGy;
• Group 2: 2 mL Blood + 50 mGy;
• Group 3: 4–2 mL Blood + NAC (1
SC) (1/2 h; 1 h) in-
cubation + 50 mGy;
• Group 5: 6–2 mL Blood + NAC (10
SC) (1/2 h; 1 h) in-
cubation + 50 mGy;
• Group 7: 8–2 mL Blood + NAC (1/10
SC) (1/2 h; 1 h) in-
cubation + 50 mGy;
• Group 9: 10–2 mL Blood + vitamin C (1
SC) (1/2 h;
1 h) incubation + 50 mGy;
• Group 11: 12–2 mL Blood + vitamin C (10
SC) (1/2 h;
1 h) incubation + 50 mGy;
• Group 13: 14–2 mL Blood + vitamin C (1/10
SC) (1/2 h;
1 h) incubation + 50 mGy;
• Group 15: 16–2 mL Blood + vitamin E (1
SC) (1/2 h.;
1 h) incubation + 50 mGy;
• Group 17: 18–2 mL Blood + vitamin E (10
SC) (1/2 h.;
1 h) incubation + 50 mGy;
• Group 19: 20–2 mL Blood + vitamin E (1/10
SC) (1/2 h.;
1 h) incubation + 50 mGy;
• Group 21: 22–2 mL Blood + b-carotene (1
SC) (1/2 h.;
1 h) incubation + 50 mGy;
• Group 23: 24–2 mL Blood + b-carotene (10
SC) (1/2 h.;
1 h) incubation + 50 mGy;
• Group 25: 26–2 mL Blood + b-carotene (1/10
SC)
(1/2 h.; 1 h) incubation + 50 mGy;
• Group 27: 2 mL Blood + NAC (1 x SC) + vitamin C (1

SC) 1 h incubation + 50 mGy;
• Group 28: 2 mL Blood + NAC (1 x SC) + vitamin E (1

SC) 1 h incubation + 50 mGy; and
• Group 29: 2 mL Blood + NAC (1 x SC) + vitamin C (1

SC) + vitamin E (1
SC) 1 h incubation + 50 mGy.
The H2AX assay was performed only after the irradiation.
The experiment was done in duplicate and repeated twice.
g-H2AX foci identification using
fluorescence microscopy
We used a widely used method (Rothkamm and Löbrich
2003; Löbrich et al. 2005; Beels et al. 2009; Kuefner et al.
2009, 2010a and b; Brand et al. 2012) with a few changes.
Briefly, the staining intensity of the assay is associated with
the degree of early phosphorylation of H2AX- type histones
caused by the presence of DNA double stranded breaks.
We mixed the peripheral blood with an equal volume of
Histopaque 1077 lymphocyte separation medium (Biochrom,
Berlin, Germany) and centrifuged it to 1,200 g in a density
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103
gradient medium for 15 min at 37 °C. After separation,
the lymphocytes were cultured in a RPMI-1640 medium
supplemented with 10% fetal bovine serum, 4 mM gluta-
mine, 1% penicillin/streptomycin and 5% CO2. The cells
were maintained for 24 h in a humidified atmosphere with
5% CO2 at 37 °C before the radiation exposure.
The lymphocytes were then fixed with a 3.7% formalde-
hyde solution for 15 min, made permeable by treating it with
a 0.2% Triton
100 solution for 15 min and blocked with a
Triton and 2% BSA solution for 1 h. For detection, we incu-
bated the lymphocytes with a 1:50 diluted solution of
Phospho-Histone H2A.X (Ser139) Monoclonal Antibody
(CR55T33), Alexa Fluor 488 for 1 h at room temperature.
For mounting, we used a VECTASHIELD mounting
medium with 4′,6-diamidino-2-phenylindole (DAPI). For the
fluorescence analysis, we used a Zeiss LSM510 (Rossdorf,
Germany) fluorescence microscope with x63 and x100 magni-
fying lenses. The double strand DNA breaks (“g-H2AX
foci”) appear as green points. We analyzed 40 cells, and
we counted the detected g-H2AX foci. We calculated the
excess foci/cell by subtracting the background g-H2AX
foci before irradiation from the g-H2AX foci after irradia-
tion and dividing the difference by the number of cells ana-
lyzed. Each microscope slide was counted by at least two
independent observers. We used the raw data to calculate
the mean for each analyzed blood sample.
Statistical analysis
In order to reduce the error due to a larger cell number,
we chose to pool the data from the two independent experi-
ments of 10 patients each, which resulted in a n = 20. We then
performed the Shapiro-Wilk test to test whether the data are
normally distributed, then calculated the standard deviation
for each sample. We used the paired t-test and Dunnett’s test
to compare the excess foci pre-treated with antioxidants to
the excess foci without antioxidants. A p-value <0.05 was
considered statistically significant. Statistical analysis was
performed using the software R Core Team (2017).
RESULTS
Experimental scheme
Most of the test populations did not pass the Shapiro-Wilk
test and therefore cannot be considered as normally distrib-
uted (see the underlined values in Table 2).
The excess foci/cell for the samples with irradiation
and without antioxidant is 0.79±0.0636 (n = 10). We calcu-
lated the percentage of protection level for every antioxidant
and antioxidant combination tested as
100
ðEcontrol −EwithAO Þ=Econtrol ; ð1Þ
where Econtrol is the average of excess foci/cell for the ir-
radiated samples without antioxidant (0.79); Ewith AO is
104 Health Physics July 2020, Volume 119, Number 1

Table 2. The number of foci/cell and radiation protection percentage corresponding to four anti-oxidants (at 1/10
SC, 1

SC and 10
SC) and three anti-oxidant combinations (only at 1
SC). The two pre-incubation periods used for the four anti-
oxidants were 1 h and ½ h, respectively. SD = standard deviation. a = Statistically significant (p < 0.001, Dunnet’s test) com-
pared to control. The values corresponding to a test populations that are from normally distributed populations (p > 0.05,
Shapiro-Wilk test) are underlined.
1/10 x SC 1 x SC 10 x SC
Excess Radiation Excess Radiation Excess Radiation
Experiment foci/cell SD protection (%) foci/cell SD protection (%) foci/cell SD protection (%)

without antioxidant 0.790 0.0636 0

NAC ½ h 0.524a 0.0676 33.7 0.333a 0.0245 57.9 0.288a 0.0263 63.6
Vitamin C ½ h 0.566a 0.0317 28.3 0.399a 0.0190 49.5 0.359a 0.0203 54.6
Vitamin E ½ h 0.669 0.0379 15.3 0.470a 0.0310 40.5 0.411a 0.0151 47.9
ß-caroten ½ h 0.741 0.0400 6.2 0.531a 0.0242 32.8 0.470a 0.0377 40.5
NAC 1 h 0.413a 0.0462 47.8 0.251a 0.0222 68.2 0.204a 0.0203 74.2
Vitamin C 1 h 0.515a 0.0328 34.8 0.328a 0.0255 58.5 0.278a 0.0242 64.9
Vitamin E 1 h 0.611 0.0559 22.6 0.389a 0.0128 50.8 0.339a 0.0190 57.1
ß-caroten 1 h 0.681 0.0323 13.8 0.460a 0.0205 41.8 0.416a 0.0272 47.3
NAC + vitamin C 1 h 0.238a 0.0309 69.9
NAC + vitamin E 1 h 0.329a 0.0233 58.4
NAC + vitamin C + 0.284a 0.0233 64.1
vitamin E 1 h

the average of excess foci/cell for the irradiated samples
with an antioxidant or antioxidant combination.
All treatments except those with 1/10
Vitamin C
(both 1 h and ½ h) and 1/10X ß-carotene (both 1 h and 30
min) show statistically significant reductions (p < 0.001,
Dunnett’s test) of the excess foci/cell compared with the
negative control without any antioxidant treatment.
For the pre-incubation with 1
SC (both 1 h and 30 min)
of antioxidants, the four antioxidants show statistically signif-
icant (p < 0.001, Student’s t-test) differences between them.
The efficiency order is NAC (0.251 foci/cell for 1 h) > vi-
tamin C (0.328 foci/cell) > vitamin E (0.389 foci/cell) >
ß-carotene (0.46 foci/cell) (Figs. 1 and 2 and Table 2).
The protection against the effects of 50 mGy radiation
doses (the DSB reduction effect) is up to 68.2% for
NAC (1 h) and to as low as 41.8% for ß-carotene.
The pre-incubation with antioxidants for 30 min shows
the order of effectiveness as that of 1 h but with a slightly
lower effectiveness, with vitamin E and ß-carotene being
less effective (Fig. 2 and Table 2). The lower effectiveness
of the reduced exposure time (30 min) is statistically signif-
icant for all four antioxidants and for every concentration
(p < 0.01, Student’s t-test).
The differences between the effects of the three
concentrations for the same antioxidant are statistically signif-
icant between 1/10
SC and 1
SC (p < 0.001, Student’s
t-test) and are not statistically significant between 1
SC
and 10
SC. Thus, antioxidant concentrations 10 times
higher than the physiological values do not significantly in-
crease their radiation protection effects.
After 1 h at 1/10
SC, the H2AX excess foci/cell
varies from 0.413 for NAC to 0.681 for ß-carotene

Fig. 1. Microscopic image of g-H2AX foci in human blood lymphocytes irradiation 50 mGy: without antioxidant (left); with 1
SC NAC (right).
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 Effect of antioxidants on x-ray-induced DNA double-strand breaks c N. S. BICHERU ET AL. 105

Fig. 2. The effect of 30 min and 1 h pre-incubation with 1
standard concentration (SC) of four different antioxidants on the excess foci/cell of
human lymphocytes following a 50 mGy irradiation dose. The columns and error bars represent the average ± SD for the samples (n = 10). All
antioxidant-treated samples present significant (*) reductions in excess foci/cell (p < 0.001, Dunnett’s test) compared to the negative control.

(Fig. 3). The radiation protection percentage is much
smaller for 1/10
SC, for NAC being 47.8%. The
protection effect of vitamin C and NAC is higher at both
incubation times with about 20% that for vitamin E and ß-
carotene (1/10
SC, ½ h, 1 h).
At a 10
SC and a 1 h incubation time, there is the
highest reduction of excess g-H2AX foci/cell: 0.204 for
NAC and 0.278 for vitamin C, with protection percentages
of 74.2% and 64.9%, respectively (Fig. 4).
The effect of three combinations of several antioxi-
dants was determined only at 1
SC concentrations and
1-h pre-incubation times. The combined effect of NAC
and vitamin C is not significantly better than NAC alone
(0.238 foci/cell against 0.251 foci/cell), while the other
two combinations have slightly weaker effects than NAC
alone (Fig. 5).
DISCUSSION
In every instance, the order of effectiveness among
anti-oxidants decreased in the following order: NAC, vita-
min C, vitamin E, b-carotene. Moreover, the increase in an-
tioxidant pre-incubation times and concentrations constantly
and significantly increased their effectiveness. The protection
percentage of NAC for 1
SC was close to 70%. Using
different combinations of NAC and other vitamins, there
was no synergistic protection effect in one case, and there
was a decrease of radiation protection in the other two

Fig. 3. The effect of 30 min and 1 h pre-incubation with 1/10
SC of four different antioxidants on the excess foci/cell of human lymphocytes
following a 50-mGy irradiation dose. The columns and error bars represent the average ± SD for the samples (n = 10). Only samples treated with
NAC and Vitamin C present significant (*) reductions in excess foci/cell (p < 0.001, Dunnett’s test) compared to the negative control.
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106 Health Physics July 2020, Volume 119, Number 1

Fig. 4. The effect of 30 min and 1 h pre-incubation with 10
SC of four different anti-oxidants on the excess foci/cell of human lymphocytes
following a 50 mGy irradiation dose. The columns and error bars represent the average ± SD for the samples (n = 10). All antioxidant-treated sam-
ples present significant (*) reductions in excess foci/cell (p < 0.001, Dunnett’s test) compared to the negative control.

cases (when vitamin E was added), which is in accord
with a few previous studies (Kuefner et al. 2012; Brand
et al. 2015).
There are several studies on the protective in vitro and/or
in vivo effect of antioxidants after irradiation. A previous
study on the protective effects of several antioxidants (zinc,
trolox, lipoic acid, ß-carotene, selenium, vitamin E, vitamin C,
NAC, and Q10 co-enzyme) showed that some of them have a
protecting effect of up to 43% against irradiation-induced
DNA damages at 10 mGy doses on human lymphocytes,
which is equivalent to procedures used in interventional and
diagnostic radiology (Brand et al. 2015). As in our study, the
most effective antioxidants were NAC and NAC combinations
with vitamin C and vitamin E, with higher pre-incubation
times and doses having a stronger effect.
The study of Wan et al. (2006) assessed the effect of
anti-oxidants (vitamin C, NAC, Selenium, Q10 co-enzyme
and vitamin E succinate) on human breast epithelial cells
irradiated with x-rays (0.5–2 Gy) and found radiation pro-
tection of up to 87% for a single antioxidant (Vitamin E
succinate) and up to 94% for the combination of them.
In our studies on human blood lymphocytes pre-incubated
with antioxidants and irradiated with 50 mGy and analyzed af-
ter 24 h, we found a reduction from 0.79 excess foci/cell for un-
treated cells to 0.20 excess foci/cell for 1
SC NAC, which is
in accord with previous studies (Rothkamm and Löbrich 2003;
Löbrich et al. 2005; Rothkamm et al. 2007; Beels et al. 2009;
Kuefner et al. 2012). For instance, our result of 0.79 excess
foci/cell for untreated cells is about five times higher than the
value found in a previous study (Brand et al. 2015) that used

Fig. 5. The effect of 1 h pre-incubation with 1 SC of three antioxidant combinations on the excess foci/cell of human lymphocytes following a
50 mGy irradiation dose. The columns and error bars represent the average ± SD for the samples (n = 10). All samples treated with antioxidant
combinations present significant (*) reductions in excess foci/cell (p < 0.001, Dunnett’s test) compared to the negative control.
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 Effect of antioxidants on x-ray-induced DNA double-strand breaks c N. S. BICHERU ET AL.
a much lower dose (10 mGy), which indicates that our re-
sults are valid.
Another study showed that a reduction of excess foci/
cell in human peripheral blood lymphocytes for pre-
incubation for 1 h with a combination of antioxidants was
about 58% at a 10 mGy dose (Kuefner et al. 2012). In con-
trast, we studied the effects of four antioxidants and three
combinations of them after 30 min and 1 h on human lym-
phocytes irradiated with 50 mGy.
A previous study on rat lymphocytes (Reliene et al.
2009) with 1–4 Gy doses and NAC administered for 10 d
showed 80% reduction of excess foci/cell, which is close
to our maximum protection value of ~70%. A new study
(Brand et al. 2018) on primary lymphocytes irradiated with
10–100 mGy showed a 50%–80% reduction of excess foci/
cell by using PrC-210, a synthetic aminothiol radioprotector.
Even at a 20 mGy dose, the radiation protection was 60%
after a 2–3 h pre-incubation time.
Our study agrees with previous studies indicating that,
among various anti-oxidants, NAC offers the best protec-
tion. The increase of incubation time from 30 min to 1 h in-
creased the radiation protection of all antioxidants (p < 0.01,
Student’s t-test), probably due to the increase in the time the
antioxidants can permeate the cell through active transport
and diffusion. Another possible explanation could be an indi-
rect protection mechanism, which involves erythroid 2–related
factor 2 (Nrf2) activation, followed by DNA radiation protec-
tion through phase II anti-oxidant enzymes (Kuefner et al.
2012; Brand et al. 2015).
In conclusion, our study proves the significant and
major in vitro protective effects of two antioxidant food sup-
plements (NAC and vitamin C) on human cells against x-ray
radiation doses of 50 mGy, which is the maximum annual ac-
cepted dose for US nuclear plant workers and corresponds to
3–7 CT scans a year.
LIMITATIONS
The number of samples from patients was low, but it
was enough to observe the effects of antioxidants on DSBs.
The standard concentrations (SC) used are not based on
physiology or toxicological equivalence but follow the cur-
rent FDA or OMS recommendations or, in their absence, the
prospect indications (PI).
Irradiation damage to cell components other than DNA
can also lead to decreases in cell viability; therefore, a holistic
approach to radiation protection of antioxidants must take
into account their protective effects on all cell components,
including proteins and lipid membranes. For example, the
general protective effect of Vitamin C and Vitamin E, known
to protect cell lipids against irradiation damage (Nair et al.
2001), may be understated by only taking into account their
protective effects against DNA DSBs. Therefore, future
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107
holistic studies on protective cell effects of antioxidants
against irradiation damage are needed.
The previous studies (Löbrich et al. 2005; Rothkamm
et al. 2007; Kuefner et al. 2009, 2012) clearly prove that
the irradiation dose is proportional with the number of excess
H2AX foci/cell after exposure. We used a dose of 50 mGy
and determined the excess foci/cell at 24 h after irradiation.
We only worked in vitro on the more easily accessible periph-
eral lymphocytes. Therefore, the in vivo validation of our re-
sults is needed. Moreover, various issues such as the variation
of gH2AX foci numbers, effective dose, and DNA repair ki-
netics may crop up in the analysis of DSBs in response to
irradiation of different tissues of irradiated patients. Even
though our study and others show that some antioxidants
decrease the gH2AX foci numbers after irradiation, their
protective effect against radiation-induced cancers has not
yet been proven, while lymphocytes are differentiated cells
that cannot become malignant.
In conclusion, the administration of antioxidants before
radiation exposure protects the cells by reducing the number
of DSBs in DNA.
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