Accepted Manuscript

Unmodified hydrated С60 fullerene molecules exhibit antioxidant

properties, prevent damage to DNA and proteins induced by
reactive oxygen species and protect mice against injuries caused
by radiation-induced oxidative stress

Sergey V. Gudkov, Evgenii L. Guryev, Andrei B. Gapeyev, Mars

G. Sharapov, Nikolai F. Bunkin, Alexey V. Shkirin, Tatiana S.
Zabelina, Alexey P. Glinushkin, Mikhail A. Sevost'yanov,
Konstantin N. Belosludtsev, Anatoly V. Chernikov, Vadim I.
Bruskov, Andrey V. Zvyagin

PII: S1549-9634(18)30518-5
DOI: doi:10.1016/j.nano.2018.09.001
Reference: NANO 1865
To appear in: Nanomedicine: Nanotechnology, Biology, and Medicine
Received date: 8 March 2018
Revised date: 19 May 2018
Accepted date: 4 September 2018

Please cite this article as: Sergey V. Gudkov, Evgenii L. Guryev, Andrei B. Gapeyev,
Mars G. Sharapov, Nikolai F. Bunkin, Alexey V. Shkirin, Tatiana S. Zabelina, Alexey P.
Glinushkin, Mikhail A. Sevost'yanov, Konstantin N. Belosludtsev, Anatoly V. Chernikov,
Vadim I. Bruskov, Andrey V. Zvyagin , Unmodified hydrated С60 fullerene molecules
exhibit antioxidant properties, prevent damage to DNA and proteins induced by reactive
oxygen species and protect mice against injuries caused by radiation-induced oxidative
stress. Nano (2018), doi:10.1016/j.nano.2018.09.001

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 ACCEPTED MANUSCRIPT
UNMODIFIED HYDRATED С60 FULLERENE MOLECULES EXHIBIT
ANTIOXIDANT PROPERTIES, PREVENT DAMAGE TO DNA AND
PROTEINS INDUCED BY REACTIVE OXYGEN SPECIES AND PROTECT
MICE AGAINST INJURIES CAUSED BY RADIATION-INDUCED
OXIDATIVE STRESS

a, b, c
Sergey V. Gudkov *, Evgenii L. Guryev a, Andrei B. Gapeyev d, Mars G. Sharapov d,
Nikolai F. Bunkin e, Alexey V. Shkirin b, f, Tatiana S. Zabelina c, Alexey P. Glinushkin g, Mikhail
A. Sevost’yanov h, Konstantin N. Belosludtsev i, j, Anatoly V. Chernikov j, Vadim I. Bruskov j,

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Andrey V. Zvyagin a, k, l

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a
Lobachevsky State University of Nizhni Novgorod, Nizhni Novgorod, 23 Gagarin Ave., Nizhny
Novgorod, 603950, Russian Federation

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b
Prokhorov General Physics Institute of the Russian Academy of Sciences, 38 Vavilova St.,
Moscow, 119991, Russian Federation
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c
Moscow Regional Research and Clinical Institute (MONIKI), 61/2 Shchepkina St., Moscow,
129110, Russian Federation
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d
Institute of Cell Biophysics of the Russian Academy of Sciences, 3 Institutskaya St., Pushchino,
Moscow Region, 142290, Russian Federation
e
Bauman Moscow State Technical University, 5 Vtoraya Baumanskaya St., Moscow, 105005,
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Russian Federation
E

f
National Research Nuclear University MEPhI, 31 Kashirskoe Ave., Moscow, 115409, Russian
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Federation
g
All-Russian Research Institute of Phytopathology, Institute St., 5, Bolshie Viazemy, Moscow
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Region, 143050, Russian Federation

h
Baikov Institute of Metallurgy and Materials Science of the Russian Academy of Sciences, 49
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Leninskii Ave., Moscow, 119334, Russian Federation

i
Mari State University, 1 Lenina pl., Yoshkar-Ola, Mari El, 424001, Russian Federation
j
Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences, 3.
Institutskaya St., Pushchino, Moscow Region, 142290, Russian Federation
k
Sechenov University, 8 Malaya Trubetskaya, Moscow, Russian Federation
l
ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Department of Physics &
Astronomy, Macquarie University, Sydney, NSW 2109, Australia.
* Corresponding authors. E-mail address: s_makariy@rambler.ru (S.V. Gudkov)
This work was supported by the Ministry of Education and Science of the Russian Federation
(project No.14.Z50.31.0022).
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ABSTRACT
Unmodified hydrated С60 fullerene molecules (C60UHFM) were shown to reduce the
formation ROS in water and 8-oxoguanine in DNA upon ionizing radiation impact. C60UHFM
efficiently eliminates long-lived protein radicals arising after irradiation. In irradiated mice
C60UHFM reducing the rate of single/double-strand DNA breaks and amount of chromosomal
breaks. The radioprotective activity of C60UHFM was estimated by the survival rate of animals,
the dose modification factor for animal survival was 1.3. Hematological tests showed that
C60UHFM injection in mice prior to irradiation results in a decrement of irradiation-induced

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leucopenia and thrombocytopenia. Histological analysis testified that C60UHFM provides
significant protection of small intestine tissues in mice against irradiation-induced damage. The

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obtained data assume that the radioprotective properties of C60UHFM are determined by their
antioxidant, antiradical and DNA-protective qualities. Thus, it was demonstrated that C60UHFM

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is a novel antioxidant and radioprotective agent capable of substantial reduction of the harmful
effects of ionizing radiation.
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Key words: fullerene C60, nanostuctures, X-rays, reactive oxygen species, DNA damage,
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radioprotectors

(Word count for abstract: 150; Word count for manuscript: 4564; Number of references: 59;
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Number of figures: 8; Number of tables: 4)

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BACKGROUND
Fullerene is the third allotropic form of carbon, with molecules forming a closed cage
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structure and consisting of an even number of carbon atoms in the sp2 hybridized state [1]. The
known fullerene forms consist of 60 and 70 carbon atoms, as well as other “higher” fullerenes
with a larger number of atoms. The physicochemical properties of the С60 fullerene and its
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derivatives and their effects on the biological processes are the most well-studied due to their
abundance [2]. From the biological point of view, unmodified С60 fullerene possesses the most
attractive characteristics, including non-toxicity upon intraperitoneal injection in animals in a
wide concentration up to 2.5 mg/g of body weight [3], non-immunogenicity [4], as well as
capability to neutralize [5] and generate [6] reactive oxygen species (ROS) under various
physical impacts. In biological media the С60 fullerene exhibits significant antioxidant properties,
serving as a “sponge for radicals” [5]. At present attempts are made to create antimicrobial
agents [2], preparations for treatment of AIDS [7] and Alzheimer disease [8] based on
chemically modified С60 fullerene. C60 has a great potential in bio related applications, such as
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photosensitizer for PDT, drug delivery carrier for targeted drug delivery etc. [9-12]. The role of
fullerenes in radiobiology is poorly investigated [review article 13,14]. It was found that in vitro
dendrofullerene (DF-1) prevent irradiation-induced DNA damage and death of mammalian cell
culture [15], protect Danio rerio embryos from toxic effects of irradiation [16], and demonstrate
radioprotective action in mice [17]. It was demonstrated that fullerenol С60(OH)12–26 protects
Stylonychia mytilus from the damaging effect of γ-irradiation [18], enhances the activity of
antioxidant enzymes in irradiated cells К562 [19], improving the survival rate of irradiated rats
[20]. Earlier, with the participation of members of our team, it was found that labile aggregates

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of C60 fullerenes containing a small number of single molecules of fullerenes partially prevent
irradiation-induced death of mice, 15 percent of mice irradiated at a dose of 7 Gy and received

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fullerene remain alive by 30 days [21]. In general, the studies aimed at investigation of the
radioprotective properties of fullerenes and their involvement in different reactions in irradiated

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organism and water solutions are not large in number. The optimal modes and schedules of
fullerene administration in the animal organism have not been determined, and the mechanisms
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of their radioprotective effect remain unclear. The knowledge on the impact of fullerene on the
hydrolysis of water solutions and its radioprotective action on DNA, proteins and other
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macromolecules also lacks experimental data. The choice of a specific type of fullerene for
studying presents a separate issue. The majority of studies are focused on only highly water-
soluble chemically modified fullerenes (dendrofullerenes and fullerenols), though it is known
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that chemical modification, and particularly polyhydroxylation, reduces the capability of

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fullerenes to interact with free radicals [22, 23].

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The objective of our research was to fill the gaps mentioned above. The work included
investigation of the effect of unmodified hydrated С60 fullerene molecules (C60UHFM) on the
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generation of water radiolysis products, such as H2O2 and OH-radicals, as well as the capability
of C60UHFM to prevent DNA and protein damage under ionizing radiation, and its
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radioprotective properties and the mechanisms of antiradiation activity in mice.

METHODS
C60UHFM production and characterization.
Highly stable C60UHFM water solution was synthesized using a special technology for
transfer of fullerene molecules (MER Corp., USA, purity >99.5%) from their solution in an
organic solvent into an aqueous phase by sonication without addition of any solubilizers and
stabilizers [24]. With the help of this technology could be obtained labile aggregates of
C60UHFM containing a small number of single molecules [24]. The use of short-time laser

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fragmentation and a number of a special modification of this [25] technology allowed to increase
the maximum concentration of fullerenes to 3.0×10−4 M (0.22 mg/ml) and to achieve an almost
complete absence of aggregation and the formation of fullerene associates. Stable aqueous
solutions of chemically unmodified C60 fullerene in water contain single C60UHFM (~99%) as
well as their labile clusters (secondary associates) (~1%). C60UHFM was examined by dynamic
light scattering (ZetaSizer Nano ZS (Malvern Instruments Ltd., GB) (Fig.1). Labile C60UHFM
clusters were sized from 3 to 300 nm, falling into several representative groups: I – 3-4 nm, II –
7-10 nm, III – 40-70 nm, IV – 70-300 nm (Fig.1). The ratio of particles belonging to the

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aggregates of different classes can be evaluated by the dependence of light diffusion intensity I
from particle size r. Rayleigh scattering assuming I ~ r6, is applicable for spherical particles with

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far smaller sizes than the wavelength of scattered light [26]. Accordingly, one aggregate of group
IV is accompanied by 55 aggregates of group III, 1.4106 small aggregates belonging to groups

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II and 5.0108 molecules to groups I. The stability of the aqueous and buffer colloids of
C60UHFM changed negligibly, and insignificant aggregation of C60UHFM was observed in
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biological fluids. Fullerol C60(OH)24 was used in several experiments for comparison with
C60UHFM.
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Animals
Males of randomly bred white Kv:SHK mice aged 5-6 weeks and weighing 18–22 g
(nursery Kryukovo) were used in the experiments. The animals were housed in polypropylene
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cages with sawdust as bedding material. They were maintained under controlled temperature (22
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± 3°C) and were given standard commercial mouse feed (Arno, Russia) and drinking water ad
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libitum. Radiobiological and physiological parameters of Kv:SHK mice were previously

described in detail [27]. The institutional regulations for the care and use of laboratory animals
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were carefully followed. All the experimental protocols received approval from the Bioethics
Committee ITEB RAS.
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Irradiation
Solutions and animals were irradiated at room temperature using an RUT-15
therapeutical X-ray device (Mosrentgen,Russia) with dose power 0.1 Gy/min (7 mA, 200 kV,
focal length 105 cm, data on insert to Fig.2), 4.5 Gy/min (17 mA, 200 kV, focal length 19.5 cm,
data on Fig.4) and 1.0 Gy/min (15 mA, 200 kV, focal length 37.5 cm, data on Fig.3,5-8, Tables
1-4). Filters (Cu-1mm + Al-l mm) of a half value thickness of 1.6 mm Cu were used in the
system.
In vitro experiments
The concentration of H2O2 was determined by the method of enhanced
chemiluminescence in the “luminol/4-iodophenol/peroxidase” system [28]. Chemiluminescence
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intensity was measured by using a Beta-1 liquid scintillation counter (Medapparatura,Russia) in
the photon-counting operation mode; calibration was carried out with H2O2 samples of known
concentration [28].
The concentration of OH-radicals was determined using coumarin-3-carboxylic acid [29].
The fluorescence intensity was measured on a Cary Eclipse spectrofluorimeter (Varian,Australia)
with ex=400 nm and em=450 nm. Before measurement, the pH was adjusted to 8.5 using Tris-
HCl buffer.
Measurement of 8-oxoguanine (8-oxoG) in DNA was carried out with the method of

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immunoenzyme analysis. The details of procedure have been published elsewhere [30].
Long-lived protein radicals were studied by measuring the X-ray-induced

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chemiluminescence of protein solutions using a specially elaborated highly sensitive
chemiluminometer Biotoks-7 AM (Econ,Russia) [31]. Measurements were carried out in the

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dark at room temperature in 20-ml plastic polypropylene vials for liquid scintillation counting
[32].
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In vivo experiments
To assess DNA damage in mouse blood leukocytes, we used the alkaline comet assay
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with some modifications [33]. Routinely, a total of about 50 cells per slide were registered and
DNA damage was assessed by the percentage of DNA in a comet tail.
The cytogenetic damage to the red bone marrow cells of mice and rats was determined
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from the formation of polychromatophilic erythrocytes (PCE) with micronucleus/micronuclei

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(MN). Mice were sacrificed by cervical dislocation 28 hours after the treatment. Histological
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samples were prepared by a standard method with some modifications [27,31].

Counting of blood cells was carried out as follows [26,34]. Groups of 10 mice were
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originally used. Five mice from each group were randomly taken for blood cell count, and then
the results were averaged for these five animals. At the end of the experiment, if the number of
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survived mice in each group was less than five, all the mice were taken for blood draws. To
provide morphopathological examination dead mice were necropsied within 24 h. Postmortem
examination was performed both in irradiated unprotected control and in C60UHFM pre-treated
irradiated animals. The organs were taken and thoroughly washed with isotonic solution for
several times. Each organ was placed on a slide and revised from both sides with binocular
magnifier with 20 magnification.
Survival of mice. Concentrated C60UHFM was diluted with sterile 5% glucose solution
immediately before the experiment. Control mice, or unprotected control group, were injected
i.p. with 5% glucose solution. The numbers of surviving mice in all groups and their body weight
(gain) were monitored for 30 days after irradiation and fixed once a day at the same time.
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Statistical analysis
Mean and standard error of the mean (SEM) were calculated for most variables.
Correlation coefficient was calculated, and the means from the different treatment groups were
compared by the Mann-Whitney U test or Student’s unpaired t-test when appropriate. An 95%-
confidence interval was calculated for the dose modification factor (DRF) and given in
parentheses in the text. In the survival experiments, the survival curves of different groups were
compared by Fisher’s exact test. Statistical significance was assigned to P<0.05.

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RESULTS
The effect of C60UHFM on the generation of Н2О2 and ОН-radicals upon water radiolysis

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The influence of C60UHFM on the generation of Н2О2 in water under X-ray irradiation is
shown in Fig.2A. The concentration of originating H2O2 linearly depends from the absorbed X-

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ray radiation dose, both in the presence and in the absence of C60UHFM. Reduction of Н2О2
generation by 20-35% was observed at C60UHFM concentration range of 0.1-1 µM. The DMF
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values for Н2О2 generation were 1.22(1.19-1,28) and 1.55(1,45-1,67) at 0.1 μM and 1 μM of
C60UHFM, respectively. The radiation chemical yield (G) of Н2О2 was 0.80±0.04
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molecules/100eV (80nM/Gy) in the absence of C60UHFM, while at 0.1μM C60UHFM the G-

value was 0.64±0.03 molecules/100 eV (64 nM/Gy), and G = 0.52±0.03 molecules/100 eV
(52nM/Gy) in the presence of 1μM C60UHFM. These values also persisted in a small dose range
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(Fig. 2A, inset). C60(OH)24 was used in control experiments, with a concentration of 1 μM G of
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Н2О2 equivalent to 0.59±0.03 molecules/100eV (59nM/Gy).

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The effect of C60UHFM on the generation of OH-radicals in X-ray irradiated water is

shown in Fig.2B. The amount of originating OH-radicals, both with and without C60UHFM
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linearly depends from the dose of X-ray radiation. Reduction on 70-90% in generation of OH-
radicals was observed in the presence of C60UHFM in the concentration range of 0.1-1 µM. The
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DMF values were determined to be 3.1(2.8-3.4) at 0.1μM of C60UHFM and 14.5(13.2-16.0) at a

1 μM. The G-value of OH-radicals in the absence of C60UHFM was 2.4 molecules/100 eV (0.24
μM/Gy), and in the presence of C60UHFM G=0.8 molecules/100 eV (0.08μM/Gy) at a
concentration of 0.1 μM and approximately 0.1-0.2 molecules/100 eV (0.01-0.02μM/Gy) at 1
μM. These patterns were also observed in the small dose (Fig.2B, inset). C60(OH)24 was used in
control experiments, with a concentration of 1 μM G of Н2О2 equivalent to 0.7 molecules/100eV
(0.07μM/Gy).
The effect of C60UHFM on the formation of long-lived protein radicals induced by X-ray
radiation

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The inset in Fig.3 demonstrates dose dependence for the formation of long-lived bovine
serum albumin (BSA) radicals upon X-ray irradiation. The intensity of chemiluminescence in
protein solutions linearly depends from the dosage of X-ray radiation up to 15 Gy, in the
presence (data not shown), as well as in the absence of C60UHFM. Fig.3 exemplifies the variance
of chemiluminescence intensity for BSA solution over time. The mean half-life of long-lived
BSA radicals in control solution is about 5 h. The addition of 0.1 and 1.0 µM C60UHFM
increases the rate of elimination for long-lived X-ray-induced BSA radicals by 1.5-2.5 times, the
observed half-life is 2-3 hours.

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The effect of C60UHFM on DNA damage occurrence upon X-ray irradiation in vitro and in
vivo

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The dose dependence of C60UHFM at concentrations of 0.1 and 1 µM, affecting the
generation of 8-oxoG in a DNA solution under X-ray radiation, was studied (Fig.4). The amount

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of 8-oxoG in the presence and absence of C60UHFM linearly depends from the dose of X-
radiation. Reduction of 8-oxoG formation in the DNA by 35-50% was demonstrated at
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C60UHFM concentration range of 0.1-1 µM. The DMF values for C60UHFM at concentrations
0.1 and 1 μM were 1.6(1.4-1.7) and 2.0(1.9-2.1), respectively. The radiation chemical yield
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values for 8-oxoG in conditions 0.0/ 0.1/1.0 µM C60UHFM were 0.37, 0.23 and 0.19
molecules/100 eV, respective.
Table 1 represents data on the influence of C60UHFM on the formation of strand breaks
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and alkali labile sites in DNA of mouse leukocytes upon X-ray irradiation. C60UHFM (0.1-1
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µM) does not affect the DNA damage rate in intact leukocytes. Following X-ray irradiation of a
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blood cell suspension at a dose of 7 Gy, the amount of DNA in the comet tail increased
approximately 11-fold. The addition of C60UHFM to the suspension 60 minutes before
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irradiation ensured a reduction of the DNA damage by 30-50% in terms of the amount of DNA
in the comet tail and by 40-50% in terms of the tail moment. Additional analysis was aimed to
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investigate the dependence of the effect of 1 µM C60UHFM on the degree of DNA damage from
time of its addition to cell suspension prior to irradiation.
The addition of C60UHFM into a suspension of cells 15 minutes before irradiation
provided a minimal protective action in terms of the amount of DNA in the comet tail
(14.21.2% against 16.01.0% in the control), which improved along with the increase of time
period to 30 (10.51.3%), 60 (8.30.9%) and 120 min (7.91.0%). Therefore, upon X-ray
irradiation C60UHFM exhibits antioxidant and radioprotective properties in vitro over the
concentration range of 0.1-1 µM, hence in further in vivo experiments we used similar
concentrations of 0.1 and 1.0 mg/kg of mouse body weight.
The effect of C60UHFM on the survival of irradiated mice
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The influence of intraperitoneal and intravenous administration of C60UHFM on the
survival of mice irradiated with X-rays at a lethal dose of 7 Gy was studied (Table 2).
Intravenous administration of C60UHFM provided a more expressed radioprotective effect.
Injection of C60UHFM into the bloodstream 60 min before irradiation demonstrated the highest
efficiency.
The survival rate was investigated in detail for mice after single intravenous injection of
C60UHFM 1 h before X-ray irradiation at a lethal dose of 7 Gy (Fig.5). Given the 100% lethality
in the control group of irradiated animals, about 30 and 80% of mice injected with 0.1 and 1

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mg/kg C60UHFM, respectively, survived irradiation for 30 days. Only 10% of animals survived
(2 animal out of 20) after 7-Gy irradiation, when C60(OH)24 at a concentration of 1 mg/kg was

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used as a control.
Weighing of animals was done throughout the whole span of experiment. Irradiated mice

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lost nearly 33% of body weight by the 10th day. Animals treated by 0.1 mg/kg C60UHFM lost
approximately 25% of weight, and subsequently the mass showed a marginal increase. The
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group of animals treated by 1 mg/kg C60UHFM exhibited weight loss of about 18% by the 10th
day, followed by a significant increase of mass, so that by day 30 their weight did not differ
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statistically from that measured before irradiation. A similar trend was observed regarding feed
and water consumption by mice.
To estimate the radioprotective efficiency of C60UHFM in a 30-day survival trial in mice
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we calculated the dose modification factor (DMF) from LD50/30 upon injection of C60UHFM 60
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min before X-ray irradiation at a dose range of 5-10 Gy. It was demonstrated that in control mice
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LD50/30 was 6.0 Gy, while for animals treated by 0.1 and 1 mg/kg C60UHFM this parameter was
6.7 and 7.9 Gy, respectively (Fig.6). Thus, the DMF value for C60UHFM injection at
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concentrations of 0.1 and 1 mg/kg was 1.1(1.0-1.2) and 1.3(1.2-1.4), respectively.

The effect of C60UHFM on the formation of micronuclei in mouse bone marrow
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polychromatophilic erythrocytes
The efficiency of radioprotectors against cytogenetic damage in bone marrow
polychromatophilic erythrocytes (PCE) can be conveniently estimated by the micronucleus
(MN) assay. The results of studying the effect of C60UHFM on the yield of PCE with MN in
mouse bone marrow upon X-ray irradiation at a dose of 1.5 Gy are presented in Table 3.
Notably, irradiation of animals with a dose of 1.5 Gy led to increase in the number of PCE
with MN (~13 times). Injection of 0.1/1.0 mg/kg C60UHFM in mice resulted in an average
decrease of MN number by 38±9% and 58±10%, respectively. Thus, the C60UHFM is effective
in protection of bone marrow cells against cytogenetic damage induced by ionizing radiation.
The effect of C60UHFM on the blood cell counts of irradiated mice
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We studied the effect of single intravenous administration of 0.1 and 1 mg/kg C60UHFM on
the leukocyte count in the peripheral blood of mice before exposure to X-rays at a dose of 7 Gy
(Fig.7). The leukocyte count showed no significant altering during the course of the experiment
in the group of unirradiated animals. C60UHFM injection in intact mice did not provide any
marked changes in the leukocyte count. All groups of animals exposed to radiation demonstrated
a reduction in the number of leukocytes through day 8 after X-ray exposure. The leukocyte count
of animals from the irradiated control group reduced by ~98% relative to the intact mice, and did
not change prominently till their death. The group of irradiated mice treated by 0.1 mg/kg

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C60UHFM showed an increase of the leukocyte count starting from day 12, making
approximately 50% of their initial number by day 30. The leukocyte count in irradiated mice

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injected with 1 mg/kg C60UHFM decreased by 85% by day 8 compared to the unirradiated
control, but their number started to raise after day 12, reaching nearly the norm by day 30. The

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influence of intravenous administration of C60UHFM on the granulocyte count in the peripheral
blood of irradiated mice was investigated. The change in the number of granulocytes in the
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bloodstream of all the groups studied is similar to the changes that were observed for leukocytes.
It is known that radiation-induced thrombocytopenia plays a key role in the capillary
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structural failure and emergence of bleeding, promoting bacteremia and radiation sepsis [36].
The effect of C60UHFM injected intravenously prior to irradiation on the platelet count in the
peripheral blood of mice is demonstrated in Fig.8. The platelet count showed practically no
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alteration in the group of unirradiated animals throughout the experiment. C60UHFM injection in
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intact animals resulted in a marginal increase in the number of blood platelets by day 2, with
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virtually no further changes. In the control group of irradiated animals the number of platelets
showed a 90% reduction by day 8, and no increase was registered further on till the death of
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animals. The platelet count in mice injected with C60UHFM 1 h before irradiation was 60-85%
by day 30, as compared to the control without radiation exposure.
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Mice irradiated at a dose of 7 Gy showed no signs of diarrhea or rectal bleedings. Necropsy

of animals exposed to X-rays revealed lung and pericardium hemorrhages and no visible
distention and obstruction of the gastrointestinal tract. Morphometric analysis identified
significantly larger size and density of lung hemorrhages in the control group of irradiated mice
in comparison to the group of animals injected intravenously with C60UHFM prior to X-
irradiation (Table 4).

DISCUSSION
In general, the present study clearly demonstrated that C60UHFM exhibited significant
antioxidant properties, reducing the radiation chemical yield of ROS after X-irradiation of water

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(Fig. 2A-B). At the same time, it is understood that the antioxidant properties of C60UHFM
measured in aqueous solutions and in vivo differ quantitatively. Despite this, our data well
correspond to the previously obtained results on the antioxidant properties of fullerenes in other
ROS-producing systems [37-40]. Previously it has been recognized that the marked antiradical
activity of fullerene is determined solely by the electron-deficient properties of the system
composed of 30 conjugated double bonds in its molecule, which makes С60 a very efficient
radical acceptor. Due to the notion that, unlike other known antioxidants, a С60 molecule can
theoretically bind up to 60 radicals [5]. However, the results of recent studies testify that the

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mechanism of the antioxidant effect of some water-soluble fullerene derivatives cannot be
explained only by this conception. Other possible hypothetical mechanisms (catalytic,

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recombination and proton adsorption mechanisms) have been proposed to explain the antioxidant
characteristics of fullerenes [4, 41-43].

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Water radiolysis can be generally represented stated as follows: Н2ООН+Н+Н++е-
aq+Н2+Н2О2 [44]. When non-ionizing radiation is applied to aqueous solutions, ROS generation
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is mainly associated not with water, but with dissolved oxygen in it [45]. It is known that the
ratio of originating OH-radicals and H2O2 in radiolysis of pure water is 3:1 [46]. This ratio does
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not change significantly upon addition of various low-molecular antioxidants. The present
investigation demonstrated that addition of the C60UHFM (1 µM) leads to a 1.5-fold reduction of
the radiation chemical yield of H2O2 (Fig.2A), whereas the G-value of OH-radicals is reduced
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15-fold (Fig.2B), i.e. by an order of magnitude greater. Thus, the ratio relation between the
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amount of registered OH-radicals and H2O2 in the presence of C60UHFM is approximately 3:10.
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The obtained data allow assuming that only about 10% of the antiradical effect of C60UHFM is
realized through attachment of ОН-radicals by its double bond, while the rest of the antioxidant
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potential is ensured by other mechanisms. Hence, though in the presence of C60UHFM the
predominant process is neutralization of OH-radicals, reactions involving oxygen, atomic
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hydrogen, hydrated electron and hydroperoxide radicals, and resulting in Н2О2 formation
through recombination of the abovementioned radical products of water radiolysis [4] and,
presumably, proton sorption by fullerene [41], largely compensate for the reduction of H2O2
production.
The capability of C60UHFM to efficiently neutralize OH-radicals with a slight impact
on the formation of H2O2 may underlie their radioprotective properties. For instance, it is known
that OH-radical is the most active type of ROS responsible for most damage of biomolecules
upon exposure to ionizing radiation [47]. Along with its damaging effect at high concentrations,
H2O2 plays an important signaling and regulatory role, resulting in activation of the protective
systems of cells and promoting their survival under oxidative stress, which is in our case induced
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by ionizing radiation. H2O2 mediates diverse responses as a secondary messenger. Recent data
on the role of H2O2 in the intra- and intercellular signaling in mammalian cells, including its
influence on cellular growth, death, and aging, have been reported in a number of reviews [48-
50]. Thus, the results obtained in our study on the C60UHFM-mediated neutralization of
oxidative stress induced by ionizing radiation may also to a certain degree explain the
pronounced geno-, cyto- and tissue-protective properties of fullerenes upon the development of
oxidative stress induced by various physical and chemical factors.
A considerable percentage of biopolymer damage caused in a cell by ionizing radiation

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is due to the short-lived radicals originating upon water radiolysis and having half-lives of
fractions of a second [44]. It has been found that long-lived protein radicals are capable of

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damaging the DNA structure in vitro and in viro, similarly to short-lived radicals [32,51]. Some
low-molecular bioantioxidants can effectively eliminate long-lived protein radicals [52-55]. We

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demonstrated (Fig.3) that the addition of 1 µM C60UMFM accelerates the elimination of long-
lived protein radicals 2.5-fold, which is the most impressive result among all studied
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antioxidants. Moreover, the C60UMFM largely prevents the formation of oxidative damage of
the DNA (Table 1). It can be suggested that owing to the expressed antioxidant and antiradical
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potential, the capability of the C60UHFM to prevent the DNA damage under ionizing radiation
underlies its radioprotective activity in vivo.
In the present study we found that the radioprotective properties of C60UHFM (Fig.5,6)
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remarkably surpass the previously reported effects of fullerenol C60(OH)24 and dendrofullerene
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DF-1. For instance, injection of C60(OH)24 before irradiation provided the survival of 60% of
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animals by day 30 [18]. The maximum DMF value for DF-1 was 1.22 [15]. Our findings
confirmed the superior radioprotective properties of C60UHFM, even when used at
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concentrations 1-2 orders of magnitude lower than that in the above-mentioned studies. It is
believed that the radioprotective efficacy of C60UHFM can be enhanced by including positively
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charged functional groups to the surface of C60UHFM, in analogy with positively charged
aminothiols demonstrated in Ref. [56].
We showed (Table 2), that C60UHFM exhibits a far greater efficiency in intravenous
injections compared to intraperitoneal administration. Additionally, it was established that the
common practice of C60UHFM injection in the organism 15-30 min prior to irradiation is
ineffective, possibly due to longer time required for C60UHFM distribution in the animal
organism [57]. It is known that neutropenia is a serious complication in radiation and chemical
therapy of cancer [58], and thrombocytopenia-induced hemorrhage provides significant
complications in many diseases [59]. The current investigation demonstrated for the first time
that intravenous C60UHFM injection 1h before exposure to ionizing radiation significantly
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diminishes the severity of radiation-induced leucopenia and thrombocytopenia, reduces the
amount of hemorrhages in the viscera and protects the small intestine of mice from damages
caused by radiation (Fig.7,8, Table4). Thus, C60UHFM is an efficient antiradical agent,
promising radioprotector and an active remedy for the prevention of radiation-induced leuco-
and thrombocytopenia.

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REFERENCES
1. Kroto HW, Heath JP, O'Brien SC, Curl RF, Smalley RE. (1985). "C60:Buckminsterfullerene."
Nature 318:162-163.
2. Yang X, Ebrahimi A, Li J, Cui Q. (2014). "Fullerene-biomolecule conjugates and their
biomedicinal applications." Int J Nanomedicine 9:77-92.
3. Moussa F, Trivin F, Ceolin R, Hadchouel M, Sizaret PY, Greugny V, Fabre C, Rassat A,
Szwarc H. (1996). "Early effects of C60 Administration in Swiss Mice: A Preliminary
Account for In Vivo C60 Toxicity." Full Sci Thenol 4:21-29.

PT
4. Andrievsky GV, Klochkov VK, Derevyanchenk LI. (2005). "Is C60 fullerene molecule
toxic?!" Fullerenes, Nanotubes Carbon Nanostruct 13:363-376.

RI
5. Krustic PJ, Wassermann PN, Keizer PN, Morton JR, Preston KF. (1991). "Radical reactions of
C60." Science 254:1183-1185.

SC
6. Arbogast JW, Darmanyan AP, Foote CS, Rubin Y, Diederich FN, Albarez MM, Anz SJ,
Whetten RL. (1991). "Photophysical properties of sixty atom carbon molecule (C60)." J Phys
NU
Chem 95:11-12.
7. Luo Z, Xu X, Zhang X, Hu L. (2013). "Development of calixarenes, cyclodextrins and
MA

fullerenes as new platforms for anti-HIV drug design: an overview." Mini Rev Med Chem
13:1160-1165.
8. Gordon R, Podolski I, Makarova E, Deev A, Mugantseva E, Khutsyan S, Sengpiel F,
D

Murashev A, Vorobyov V. (2017). "Intrahippocampal pathways involved in learning/memory

E

mechanisms are affected by intracerebral infusions of amyloid-β25-35 peptide and hydrated

PT

fullerene C60 in rats." J Alzheimers Dis 58(3):711-724.

9. Shi J, Wang L, Gao J, Liu Y, Zhang J, Ma R, Liu R, Zhang Z. (2014). "A fullerene-based
CE

multi-functional nanoplatform for cancer theranostic applications." Biomaterials 35(22):5771-

5784.
AC

10. Shi J, Yu X, Wang L, Liu Y, Gao J, Zhang J, Ma R, Liu R, Zhang Z. (2013). "PEGylated
fullerene/iron oxide nanocomposites for photodynamic therapy, targeted drug delivery and
MR imaging." Biomaterials 34(37):9666-9677.
11. Shi J, Liu Y, Wang L, Gao J, Zhang J, Yu X, Ma R, Liu R, Zhang Z. (2014). "A tumoral
acidic pH-responsive drug delivery system based on a novel photosensitizer (fullerene) for in
vitro and in vivo chemo-photodynamic therapy." Acta Biomater 10(3):1280-1291.
12. Shi J, Wang B, Wang L, Lu T, Fu Y, Zhang H, Zhang Z. (2016). "Fullerene (C 60)-based
tumor-targeting nanoparticles with "off-on" state for enhanced treatment of cancer. " J Control
Release 235:245-258.

13
 ACCEPTED MANUSCRIPT
13. Krokosz A, Lichota A, Nowak KE, Grebowski J. (2016). "Carbon nanoparticles as possible
radioprotectors in biological systems." Radiation Phys and Chem 128:143-150.
14. Gudkov SV, Popova NR, Bruskov VI. (2015). "Radioprotectors: History, Trends and
Prospects." Biofizika 60(4):801-811.
15. Theriot CA, Casey RC, Moore VC, Mitchell L, Reynolds JO, Burgoyne M, Partha R, Huff
JL, Conyers JL, Jeevarajan A, Wu H. (2010). "Dendro[C(60)]fullerene DF-1 provides
radioprotection to radiosensitive mammalian cells." Radiat Environ Biophys 49:437-445.
16. Daroczi B, Kari G, McAleer MF, Wolf JC, Rodeck U, Dicker A. (2006). "In vivo

PT
radioprotection by the fullerene nanoparticle DF-1 as assessed in a Zebrafish model." Clin
Cancer Res 12:7086-7091.

RI
17. Brown AP, Chung EJ, Urick ME, Shield WP, Sowers AL, Thetford A, Shankavaram UT,
Mitchell JB, Citrin DE. (2010). "Evaluation of the fullerene compound DF-1 as a radiation

SC
protector." Radiat Oncol 5:34-43.
18. Zhao Q, Li Y, Xu J, Liu R, Li W. (2005). "Radioprotection by fullerenols of Stylonychia
NU
mytilus exposed to γ-rays." Int J Radiat Biol 81:169-175.
19. Bogdanovic V, Stankov K, Icevic I, Zikic D, Nikolic A, Solajic S, Djordjevic A, Bogdanovic
MA

G. (2008). "Fullerenol C60(OH)24 effects on antioxidative enzymes activity in irradiated

human erythroleukemia cell line." J Radiat Res 49:321-327.
20. Trajkovic S, Dobric S, Jacevic V, Dragojevic-Simic V, Milovanovic Z, Dordevic A. (2007).
D

"Tissue-protective effects of fullerenol C60(OH)24 and amifostine in irradiated rats." Colloids

E

Surf B Biointerfaces 58(1):39-43.

PT

21. Andrievsky GV, Bruskov VI, Tykhomyrov AA, Gudkov SV. (2009). "Peculiarities of the
antioxidant and radioprotective effects of hydrated C60 fullerene nanostuctures in vitro and in
CE

vivo." Free Radic Biol Med 47(6):786-793.

22. Chiang LY, Swirczewski JW, Hsu CS, Chowdhury SK, Cameron S, Creegan K. (1992).
AC

"Multi-hydroxy additions onto C60 fullerene molecules." J Chem Soc Chem Commun
24:1791-1793.
23. Guldi DM, Asmus KD. (1999). "Activity of water-soluble fullerenes towards OH-radicals
and molecular oxygen." Radiat Phys Chem 56:449-456.
24. Andrievsky GV, Kosevich MV, Vovk OM, Shelkovsky VS, Vashchenko LA. (1995). "On
the production of an aqueous colloidal solution of fullerenes." J Chem Soc Chem Commun
12:1281-1282.
25. Barmina E.V., Gudkov S.V., Simakin A.V., Shafeev G.A. (2017). "Stable Products of Laser-
Induced Breakdown of Aqueous Colloidal Solutions of Nanoparticles." J of Laser
Micro/Nanoengineering 12(3):254-257.
14
 ACCEPTED MANUSCRIPT
26. Bunkin NF, Shkirin AV, Suyazov NV, Babenko VA, Sychev AA, Penkov NV, Belosludtsev
KN, Gudkov SV. (2016). "Formation and Dynamics of Ion-Stabilized Gas Nanobubble Phase
in the Bulk of Aqueous NaCl Solutions." J Phys Chem B 120:1291-1303.
27. Sharapov MG, Novoselov VI, Fesenko EE, Bruskov VI, Gudkov SV. (2017). "The role of
peroxiredoxin 6 in neutralization of X-ray mediated oxidative stress: effects on gene
expression, preservation of radiosensitive tissues and postradiation survival of animals." Free
Radical Research 51(2):148-166.
28. Shtarkman IN, Gudkov SV, Chernikov AV, Bruskov VI. (2008). "Effect of amino acids on

PT
X-ray-induced hydrogen peroxide and hydroxyl radical formation in water and 8-oxoguanine
in DNA." Biochemistry-Moscow 73:470-478.

RI
29. Chernikov AV, Gudkov SV, Shtarkman IN, Bruskov VI. (2007). "Oxygen effect in heat-
mediated damage to DNA." Biofizika 52(2):244-251.

SC
30. Garmash SA, Smirnova VS, Karp OE, Usacheva AM, Berezhnov AV, Ivanov VE,
Chernikov AV, Bruskov VI, Gudkov SV. (2014). "Pro-oxidative, genotoxic and cytotoxic
NU
properties of uranyl ions." J Environ Radioact 127(1):163-170.
31. Gudkov SV, Garmash SA, Shtarkman IN, Chernikov AV, Karp OE, Bruskov VI. (2010).
MA

"Long-lived protein radicals induced by X-ray irradiation are the source of reactive oxygen
species in aqueous medium." Dokl Biochem Biophys 430:1-4.
32. Bruskov VI, Popova NR, Ivanov VE, Karp OE, Chernikov AV, Gudkov SV. (2014).
D

"Formation of long-lived reactive species of blood serum proteins by the action of heat."
E

Biochem Biophys Res Commun 443:957-961.

PT

33. Gapeyev AB, Lukyanova NA, Gudkov SV (2014). "Hydrogen peroxide induced by
modulated electromagnetic radiation protects the cells from DNA damage." Cent Eur J of Biol
CE

9(10):915-921.
34. Asadullina NR, Usacheva AM, Smirnova VS, Gudkov SV. (2010). "Antioxidative and
AC

radiation modulating properties of guanosine-5’-monophosphate." Nucleosides Nucleotides &

Nucleic Acids 29:786-799.
35. Landes RD, Lensing SY, Kodell RL (2013). "Hauer-Jensen M. Practical advice on
calculating confidence intervals for radioprotection effects and reducing animal numbers in
radiation countermeasure experiments." Radiat Res 180(6):567-574.
36. Donnelly EH, Nemhauser JB, Smith JM, Kazzi ZN, Farfan EB, Chang AS, Naeem SF.
(2010). "Acute radiation syndrome: assessment and management." South Med J 103(6):541-
546.
37. Kong L, Zepp RG. (2012). "Production and consumption of reactive oxygen species by
fullerenes." Environ Toxicol Chem 31(1):136-143.
15
 ACCEPTED MANUSCRIPT
38. Cai X, Hao J, Zhang X, Yu B, Ren J, Luo C, Li Q, Huang Q, Shi X, Li W, Liu J. (2010).
"The polyhydroxylated fullerene derivative C60(OH)24 protects mice from ionizing-
radiation-induced immune and mitochondrial dysfunction." Toxicol Appl Pharmacol
243(1):27-34.
39. Lao F, Li W, Han D, Qu Y, Liu Y, Zhao Y, Chen C. (2009). "Fullerene derivatives protect
endothelial cells against NO-induced damage." Nanotechnol 20(22):225103.
40. Yin JJ, Lao F, Fu PP, Wamer WG, Zhao Y, Wang PC, Qiu Y, Sun B, Xing G, Dong J, Liang
XJ, Chen C. (2009). "The scavenging of reactive oxygen species and the potential for cell

PT
protection by functionalized fullerene materials." Biomaterials 30(4):611-621.
41. Chistyakov VA, Smirnova YuO, Prazdnova EV, Soldatov AV. (2013). "Possible

RI
Mechanisms of Fullerene C60 Antioxidant Action." BioMed Res Int Article ID 821498, 4 P.
42. Ali SS, Hardt JI, Quick KL, Kim-Han JS, Erlanger BF, Huang TT, Epstein CJ, Dugan L.

SC
(2004). "A biologically effective fullerene (C60) derivate with superoxide dismutase mimetic
properties." Free Rad Biol Med 37:1191-1202.
NU
43. Quick KL, Ali SS, Arch R, Xiong C, Wozniak D, Dugan LL. (2008). "A carboxyfullerene
SOD mimetic improves cognition and extends the lifespan of mice." Neurobiol Aging 29:117-
MA

128.
44. Gudkov SV, Shilyagina NY, Vodeneev VA, Zvyagin AV. (2016). "Targeted Radionuclide
Therapy of Human Tumors." Int J Mol Sci 17(1):33.
D

45. Gudkov SV, Andreev SN, Barmina EV, Bunkin NF, Kartabaeva BB, Nesvat AP, Stepanov
E

EV, Taranda NI, Khramov RN, Glinushkin AP. (2017). "Effect of Visible Light on Biological
PT

Objects: Physiological and Pathophysiological Aspects." Physics of Wave Phenomena

25(3):1-7.
CE

46. Ward JF. (1988). "DNA damage produced by ionizing radiation in mammalian cells:
Identities, mechanisms of formation and reparability." Prog Nucl Acid Res Mol Biol 35:95-
AC

125.
47. Gudkov SV, Chernikov AV, Bruskov VI. (2016). "Chemical and radiological toxicity of
uranium compounds." Rus J of General Chem 86(6):1531-1538.
48. Burgoyne JR, Oka S, Ale-Agha N, Eaton P. (2013). "Hydrogen peroxide sensing and
signaling by protein kinases in the cardiovascular system." Antioxid Redox Signal 18:1042-
1052.
49. Antunes F, Brito PM. (2017). "Quantitative biology of hydrogen peroxide signaling." Redox
Biol 13:1-7.
50. Sies H. (2017). "Hydrogen peroxide as a central redox signaling molecule in physiological
oxidative stress: Oxidative eustress." Redox Biol 11:613-619.
16
 ACCEPTED MANUSCRIPT
51. Ostdal H, Davies MJ, Andersen HJ. (2002). "Reaction between protein radicals and other
biomolecules." Free Rad Biol Med 33:201-209.
52. Kumagai J, Kawaura T, Miyazaki T, Prost M, Prost E, Watanabe M, Quetin-Leclerq J.
(2003). "Test for antioxidant ability by scavenging long-lived mutagenic radicals in
mammalian cells and by blood test with intentional radicals: an application of gallic acid."
Radiat Phys Chem 66:17-25.
53. Pietraforte D, Minetti M. (1997). "Direct ESR detection or peroxynitrite-induced tyrosine-
centred protein radicals in human blood plasma." Biochem J 325:675-684.

PT
54. Koyama S, Kodama S, Suzuki K, Matsumoto T, Miyazaki T, Watanabe M. (1998).
"Radiation-induced long-lived radicals which cause mutation and transformation." Mutat Res

RI
421:45-54.
55. Gudkov SV, Shtarkman IN, Chernikov AV, Usacheva AM, Bruskov VI (2007). "Guanosine

SC
and inosine (riboxin) eliminate the long-lived protein radicals induced X-ray radiation." Dokl
Biochem Biophys 413:50-53.
NU
56. Weiss JF, Landauer MR (2009). "History and development of radiation-protective agents."
Int J Radiat Biol 85(7):539-573.
MA

57. Hendrickson OD, Morozova OV, Zherdev AV, Yaropolov AI, Klochkov SG, Bachurin SO,
Dzantiev BB. (2014). "Study of Distribution and Biological Effects of Fullerene C60 after
Single and Multiple Intragastrical Administrations to Rats." Fullerenes, Nanotubes and
D

Carbon Nanostruct 23:658-668.

E

58. Matikas A, Georgoulias V, Kotsakis A. (2016). "Emerging agents for the prevention of
PT

treatment induced neutropenia in adult cancer patients." Expert Opin Emerg Drugs 21(2):157-
166.
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59. Lisman T, Bernal W. (2017). "Hemostatic issues in pregnancy-induced liver disease."

Thromb Res 151:S78-S81.
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Figure captions
Fig. 1. Size distribution of secondary associates of C60UHFM (10-6 M) in aqueous phase.
Dynamic light scattering polymodal distribution plot. I-IV – dimensional groups.

Fig. 2. A The influence of C60UHFM on the formation of H2O2 in double distilled water exposed
to X-rays at doses of 0.1-10 Gy. Inset: The influence of C60UHFM on the formation of H2O2 in
double distilled water exposed to X-rays at doses of 0.1-0.5 Gy.
B The influence of C60UHFM on the formation of OH-radicals in double distilled water exposed
to X-rays at doses of 0.1-10 Gy. Inset: The influence of C60UHFM on the formation of •OH in

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double distilled water exposed to X-rays at doses of 0.1-0.5 Gy.

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Fig. 3. Time dependence of the luminescence intensity decay of BSA solutions (1 g/l) irradiated
with X-rays at a dose of 7 Gy. Background luminescence values were subtracted from the

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results. Inset: The luminescence intensity of BSA solutions (1 g/l) vs. X-ray radiation dose.

Fig. 4. The influence of C60UHFM on formation of 8-oxoG in DNA in vitro under irradiation (7
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Gy). The ordinate axis: The number of molecules of 8-oxoguanine per 105 molecules of guanine
in the DNA. Insert: Effect of C60UHFM on the formation of 8-oxoG in DNA in vitro exposed to
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X-rays at doses of 0-20 Gy.

Fig. 5. Kaplan-Meier estimate of 30-day survival of X-irradiated (7 Gy) mice injected i.v. with
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C60UHFM 60 min prior to exposure.

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Fig. 6. Radiation dose-response of C60UHFM injected i.v. to mice 60 min prior to irradiation
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with 5-10 Gy, plotted as probit mortality. For each experimental point, the data for 20 animals
were used.
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Fig. 7. Circulating leukocyte counts of whole-body 7 Gy X-irradiated mice injected i.v. with
C60UHFM 60 min prior to exposure. I - the lower and upper quartiles. Statistically significant
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differences between the irradiation control group and the other groups (Mann-Whitney U test, p
< 0.05) are marked by asterisks. For each experimental curve, the data for 10 animals were used.

Fig. 8. Circulating platelet counts of whole-body 7 Gy X-irradiated mice injected i.v. with
hydrated C60UHFM 60 min prior to exposure. I - the lower and upper quartiles. Statistically
significant differences between the irradiation control group and the other groups (Mann-
Whitney U test, p < 0.05) are marked by asterisks. For each experimental curve, the data for 10
animals were used.

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Table 1. The influence of C60UHFM on standard parameters of comets obtained from
image analysis of comets of mouse leucocytes exposed to X-rays (n=7).

Tail moment
Dose, C60UHFM*, Tail length,
Head DNA, % Tail DNA, % (arbitrary
Gy µM m
units)
- 98,60,4 1,40,4† 27,45,8 0,60,2†
0 0.1 98,50,5 1,50,5† 28,06,2 0,70,3†
1.0 98,70,6 1,30,6† 27,05,8 0,50,2†
- 84,01,0 16,01,0 96,63,6 19,71,2
7 0.1 89,01,1 11,01,1† 76,96,2 12,11,6†
1.0 8,30,9† 9,51,4†

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91,70,9 71,95,3

* - C60UHFM was added to the cell suspension, 1h prior to the irradiation.

†
– Values are significantly different from irradiated control values at P< 0.05 (Student’s

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unpaired t-test).

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Table 2. The influence of hydrated C60UHFM on 30-day survival of X-irradiated (7 Gy)
mice injected intraperitoneally (i.p.) or intravenously (i.v.) at different time intervals prior
exposure. In parentheses, the first digit indicates the number of alive animals after 30 days,
the second digit describes the initial number of animals in the group.

The percentage of surviving animals for 30 days

Time* after irradiation
i.p. i.v.
15 min 0 (0/10) 30 (3/10)
30 min 10 (2/20) 50 (10/20)
60 min 15 (3/20) 80 (16/20)
2h 25 (5/20) 65 (13/20)

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5h 20 (2/10) 30 (3/10)

* - The time elapsed between the injection of C60 and exposure to X-rays

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Table 3. Effect of hydrated C60UHFM intravenously to mice 60 min prior to their
irradiation with 1.5 Gy of X-rays on the formation of PCE with MN in the bone marrow of
the animals.

Number of
Dose, C60UHFM, Number of Number Percentage of
PCE with
Gy mg/kg animals of PCE PCE with MN
MN
- 5 13851 65 0.47±0.03*
0 0.1 4 10233 44 0.46±0.03*
1.0 4 10051 43 0.43±0.03*
- 5 12624 765 6.060.51*
1.5 0.1 4 10158 358 3.520.47*

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1.0 4 10078 235 2.330.32*

* – Values are significantly different from irradiated control values at P< 0.05 (Student’s

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Table 4. Influence of pretreatment of mice with C60UHFM on vascular damage in different
organs after total body X-irradiation (7 Gy).

Control C60UHFM C60UHFM

Degree of vascular damage 0.1 mg/kg 1.0 mg/kg
SI P L SI P L SI P L
Normal finding 5 8 4
Dilatation of small blood vessels 8 5 5 2 2 5 6
Isolated hemorrhages 2 5 1 4 5 1 4
Diffuse hemorrhages 5 9 1 3

Note: SI – small intestine; P – pericardium; L – lung. Each experimental group consisted of 10

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Graphical Abstract

The work included investigation of the effect of unmodified hydrated С60 fullerene molecules
(C60UHFM) on the generation of water radiolysis products, such as hydrogen peroxide and
hydroxyl radicals, as well as the capability of fullerene to prevent DNA and protein damage
under ionizing radiation, and its radioprotective properties and the mechanisms of antiradiation
activity in mice.

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Figure 1
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