Free Radical Biology & Medicine 47 (2009) 786–793

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Free Radical Biology & Medicine

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Original Contribution

Peculiarities of the antioxidant and radioprotective effects of hydrated C60 fullerene

nanostuctures in vitro and in vivo
Grigory V. Andrievsky a,b, Vadim I. Bruskov c, Artem A. Tykhomyrov d,⁎, Sergey V. Gudkov c
a
Institute of Physiologically Active Compounds LLC, Kharkiv, Ukraine
b
Institute of Scintillating Materials (ISMA), STC “Institute of Single Crystals,” National Academy of Sciences, Kharkiv, Ukraine
c
Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Russian Federation
d
Department of Biophysics and Biochemistry, National University of Dniepropetrovsk, Gagarin ave., 72, Dniepropetrovsk, Ukraine, 49010

a r t i c l e i n f o a b s t r a c t

Article history: Aqueous solutions of highly stable supramolecular donor–acceptor complexes of chemically nonmodified
Received 31 December 2008 pristine C60 fullerene molecules with H2O molecules (hydrated C60 fullerene–C60HyFn) and their labile nano-
Revised 30 April 2009 U
sized clusters were examined for their antioxidant effects on removal of hydroxyl radicals ( OH) and
Accepted 12 June 2009
protecting DNA against oxidative damage induced by ionizing radiation in vitro. The suppressing influence of
Available online 17 June 2009
C60HyFn on the formation of OH-radicals in water exposed to X-rays at doses of 1–7 Gy was assessed by
Keywords:
determination of oxidation levels of coumarin-3-carboxylic acid. C60HyFn demonstrates apparent antiradical
U
Hydrated C60 fullerene activity in vitro in the range of concentrations of 10−11–10−6 M. Paradoxically, the OH-removing efficacy of
Hydroxyl radicals C60HyFn was in reverse correlation with fullerene concentration. It was hypothesized that the antiradical
Ionizing radiation action of C60HyFn in water medium generally is due to a “nonstoichiometric” mechanism, supposedly to a
DNA damage hydrated free radical recombination (self-neutralization), which is catalyzed by specific water structures
Antiradical activity ordered by C60HyFn. With the use of 8-oxoguanine as a marker of oxidative damage to DNA, it has been
Antioxidant demonstrated that C60HyFn in concentrations of 10−7–10−6 M protects nucleic acids against radical-induced
Radioprotector
damage. The second part of the present study was aimed to evaluate the overall radioprotective efficacy of
Free radicals
C60HyFn in doses of 0.1 or 1 mg/kg b.w. injected intraperitoneally to mice either 1 h before or 15 min after
lethal dose exposure of the X-ray (7 Gy) irradiation. Survival rate of the mice was observed at 30 day intervals
after irradiation, while the weight gains of experimental animals were monitored as well. The most
significant protective effect was demonstrated when 1 mg/kg dosage of C60HyFn was administered before
irradiation. The outcome of the substance testing is 15% survival rate of irradiated animals at 30 days of
observation, and prevention of noticeable weight loss characteristic for radiation impact, versus unprotected
control animals. In conclusion, results of the study obviate that the apparent protective action of C60HyFn in
vivo is determined by its considerable ability to decrease X-ray-generated reactive oxygen species. Based on
the results and that neat C60 is nontoxic, actually in the hydrated form, without side effects and with
sufficient radioprotective effects in low doses, C60HyFn may be considered as a novel antioxidant agent,
which substantially diminishes the harmful effects of ionizing radiation.
© 2009 Elsevier Inc. All rights reserved.

Introduction fullerene family. Molecules of C60 consist of 60 carbon atoms

Fullerenes are the third natural allotropic variation of carbon [1]. In
the past years fullerenes have attracted considerable interest in many
fields of research, including in biomedical applications [2–4]. Full-
erene С60 is considered to be the most investigated member of the
Abbreviations: C60HyFn, hydrated C60 fullerene; C60FWS, C60 fullerene water
solution; ROS, reactive oxygen species; SOD, superoxide dismutase; ABTS, 3-
ethybenzthiazoline-6-sulfonic acid; CCA, coumarin-3-carboxylic acid; 7-OH-CCA, 7-
OH-coumarin-3-carboxylic acid; 8-oxoG, 8-oxoguanine; b.w., body weight; DRF, dose
reduction factor.
⁎ Corresponding author.
E-mail addresses: yard@kharkov.ua (G.V. Andrievsky), artfullerene@gmail.com
(A.A. Tykhomyrov), s_makariy@rambler.ru (S.V. Gudkov).
connected by sp2,5-bonds, which determine its pseudo-aromatic
structure due to delocalization of π-electrons over its carbon core.
Thanks to such a structure, C60 can readily react with oxygen free
radicals. Neat fullerenes and their water-soluble derivates are shown
to be a potent free radical scavenger that makes this class of
compounds attractive tools for regulation of free radical processes
and for reducing the severity of oxidative stress in biological systems
[5,6]. Since the first studies of C60's antioxidant abilities it is supposed
that an extended electron-conjugation system only determines the
high reactivity of fullerene molecules toward reactive oxygen species
(ROS). Until recently fullerene was considered to be a novel
“structural” antioxidant and characterized as a “radical sponge” by
Krusic et al. [7]. Nevertheless, based on the available data, it is obvious

0891-5849/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.freeradbiomed.2009.06.016
 G.V. Andrievsky et al. / Free Radical Biology & Medicine 47 (2009) 786–793
that C60's antiradical properties are not limited only by direct reaction
of the fullerene carbon cage with ROS. The experimental results [8]
demonstrate that water-soluble fullerene derivates can deactivate ROS
through a nonstoichiometric mechanism; thus, the current antiox-
idant concept of fullerene activity must be revised. A more recent
report [9] introduced evidence about the superoxide dismutase (SOD)
mimetic properties of fullerene derivates. Tris-malonyl C60 derivates
U
appear to be able to remove superoxide radical (O2 −) at a rate
comparable to that shown for SOD. Besides, it was also found that
U
dismutation of O2 − by fullerene derivates was a result of an enzyme-
like catalytic antioxidant activity of С3 malonic acid C60 derivates
U
against O2 −. Moreover, С3 was shown to decrease oxidative stress in
mice brain, improve cognitive performance, and extend the life span
of rodents [9,10]. The underlying evidence of quenching singlet
oxygen (1O2) in the presence of fullerenes, which occurs in a more
accelerated rate in water medium compared to all other tested
solvents, observed by Bensasson et al. [11], leads to the suggestion of a
possible role of water structures conjoined on the fullerene surface in
free radical neutralization.
During the last decade the biological effects of the water-soluble
form of C60 that is denoted as hydrated C60 fullerene (C60HyFn) are
being studied extensively [12–15]. Stable aqueous solutions of
chemically nonmodified C60 fullerene in water (C60 fullerene water
solution—C60FWS) contain single hydrated C60 fullerene molecules as
well as their labile clusters (secondary associates) with the size of 3–
36 nm. C60HyFn is highly hydrophilic and highly stable donor–
acceptor complexes of C60 with water molecules—C60@{H2O}n, n =
22–24 [16–19]. A great deal of information has accumulated concern-
ing the beneficial effects of C60HyFn, its neuroprotective, anticancer,
anti-inflammatory, antiatherogenic action, mainly determined by the
antioxidative capacity of C60HyFn, which is revealed unexpectedly at
extremely low concentrations [13]. Nevertheless, the intrinsic
mechanisms of C60HyFn antioxidant and tissue-protective activities
are not yet completely elucidated. Despite substantial experimental
results, highlighting the positive influence of C60HyFn on biological
systems in vivo, few attempts were made at direct measurement to
evaluate the radical scavenging profile of C60HyFn in order to clarify
the molecular mechanism of its antioxidant action. It is well known
that ionizing radiation causes radiolysis of H2O and ROS production
U U
(UOH, HO2 , O2 −). Thus, X-irradiation impact is proposed as a relevant
model of acute oxidative stress [20–22]. The present work was
inspired by numerous obtained data from recent studies, which
demonstrated the radioprotective activity of some water-soluble C60
derivates, dendrofullerenes and polyhydroxyfullerenes (fullerenols)
[23–25]. Nevertheless, Andrievsky and co-workers were the first
investigators who reported a 3-year duration clinical observation
about the beneficial effects of C60HyFn application in small doses
administered to a volunteer with malignancy and detected a positive
therapeutic effect of C60HyFn during the course of radiotherapy, which
was referred initially to its high antioxidant activity [26,27]. Afterward
we explored whether C60HyFn could exert significant antioxidant
effects in different concentrations to neutralize hydroxyl radicals, the
most reactive ones among all ROS species, and protect DNA against
oxidative damage induced by X-irradiation in vitro. The aim of the
second part of the present study was to evaluate the general
radioprotective activity of C60HyFn in mice irradiated at lethal doses
of X-rays.
Materials and methods
Chemicals
Аnti-mouse IgG–horseradish peroxidase conjugates, 3-ethy-
benzthiazoline-6-sulfonic acid (ABTS), Tris-HCl, and casein were
purchased from Sigma (USA); coumarin-3-carboxylic acid and 7-
OH-coumarin-3-carboxylic acid were obtained from Aldrich (USA);
787
Na2HPO4·7H2O and NaH2PO4·H2O were from Amresco (USA). Highly
polymerized DNA from salmon sperm (ICN, USA) was used. All
solutions were prepared in bidistilled water with specific conductance
of 200 μS/m. We also used Triton X-100, sodium chloride (AppliChem,
Germany), citric acid, trisodium citrate, glucose, and hydrogen
peroxide (ReaChim, Russia). Monoclonal antibodies specific for 8-
oxoguanine were obtained using methods previously described in
detail [28]. The affinity constant determined by enzyme-linked
immunosorbent assay (ELISA) was 1.3 × 106 M−1 and exceeded the
binding constants of possible cross-structural analogs by more than
three orders of magnitude. The initial concentration of monoclonal
antibodies determined by the Lowry method was 2.85 × 10−4 M.
Monoclonal antibodies were dissolved in 0.05 M phosphate buffer, pH
7.4, mixed with an equal volume of glycerol, and then stored at −20°C
until used.
C60HyFn production and characterization
Highly stable fullerene water solution (C60FWS) was synthesized
using the special technology for transfer of fullerene molecules (MER
Corp., USA, purity N99.5%) from their solution in an organic solvent
into an aqueous phase by sonication without addition of any
solubilizers and stabilizers [16–19]. The C60HyFn concentration in
concentrated C60FWS was 2.6 × 10−4 M (0.19 mg/ml).
For confirmation of our data obtained previously, fullerene
nanoparticles in C60FWS were examined by high-resolution transmis-
sion electron microscopy (microscope PEM-125K, Selmi, Ukraine). For
HR-TEM analysis, specimens were prepared by drying of a 2 μl drop of
C60FWS (with 30 μM C60) on a copper grid with a transparent carbon
film. The typical TEM image of C60HyFn nanoparticles with sizes of
1.6–1.8 nm (single C60HyFn) and 3.4 nm (the first spherical clusters
consisting of 13 hydrated C60) is shown on the Fig. 1 [17,18].
Animals
Males of random bred white Kv:SHK mice aged 5 to 6 weeks and
weighing 18–22 g (nursery Kryukovo, Russian Academy of Medical
Sciences) were used in the experiments. The animals were housed in
polypropylene cages with sawdust as bedding material. They were
maintained under controlled conditions of temperature (22 ± 3°C)
and were given standard commercial mouse feed (Arno, Russia) and
drinking water ad libitum. Radiobiological and physiological para-
meters of Kv:SHK mice were previously described in detail [29]. The
institutional guide for the care and use of laboratory animals was
carefully followed. All the experimental protocols received approval
from the Bioethics Committee of the Institute of Theoretical and
Experimental Biophysics, Russian Academy of Sciences.
Fig. 1. TEM image of coagulated C60HyFn nanoparticles received after C60FWS drying.
788 G.V. Andrievsky et al. / Free Radical Biology & Medicine 47 (2009) 786–793
Fig. 2. The influence of hydrated fullerene (10−13–10−6 M) on formation of hydroxyl
radicals in double distilled water exposed to X-rays at doses of 1, 3, 5, or 7 Gy. Inset: the
U buffer, pH 4.3. The optical density of the samples was measured with
influence of C60HyFn (10−12–10−6 M) on formation of OH in double distilled water
exposed to X-rays at a dose of 7 Gy. ⁎P b 0.05 vs control level (irradiated water without
adding of C60HyFn); ⁎⁎P b 0.01 vs control level; ⁎⁎⁎P b 0.001 vs control level.
Irradiation
Solutions and animals were irradiated at room temperature using
an RUT-15 therapeutical X-ray device (Mosrentgen, Russia) with doses
from 1 to 7 Gy at a dose power 1 Gy/min (current 20 mA, voltage
200 kV, focal distance 37.5 cm) and 4.5 Gy/min (current 20 mA,
voltage 200 kV, focal distance 19.5 cm).
Determination of hydroxyl radical generation
Quantitative analysis of hydroxyl radicals was performed with use
of a highly specific fluorescent probe coumarin-3-carboxylic acid
(CCA) whose hydroxylation product, 7-OH-coumarin-3-carboxylic
acid (7-OH-CCA), fluoresces intensely. Experimental conditions have
been described in detail elsewhere [30]. CCA was dissolved in a plastic
bottle without heating by shaking for 5 h in air-saturated water to a
concentration of 0.5 mM. Concentrated C60FWS was added to such
solution just before the experiment. Thus, a series of the solutions
containing constant concentration of CCA has been obtained, and
C60HyFn concentration varied from 10−6 to 10−13 M. These solutions
were irradiated at room temperature with doses of 1, 3, 5, or 7 Gy at a
dose rate of 1.0 Gy/min in polypropylene flasks for a liquid
scintillation counter (Beckman, USA). The fluorescence of 7-OH-CCA,
U
а product of reaction between CCA and OH, was measured with а
Kontron SFM 25А spectrofluorimeter (Kontron Instruments S.р.а.,
Italy) at λex = 400 nm and λem = 450 nm in a mirror quartz cell of
size 10 × 10 mm. Solutions of an authentic preparation of 7-OH-CCA
(Aldrich, USA) of known concentration were used to calibrate the
results.
Determination of 8-oxoguanine (8-oxoG) in DNA
The method of immunoenzyme assay was described in detail
previously [28]. DNA from salmon sperm in 10−3 M phosphate buffer,
pH 6.8, was used at a concentration of 350 μg/ml. The concentration of
DNA solutions was determined with a Specord UV-VIS spectro-
photometer (Carl Zeiss Jena, Germany). A quantity of 50 μg/ml was
taken as one unit of optical absorption of double-helical DNA at
260 nm with an optical path of 1 cm. Concentrated C60FWS was added
to DNA solution immediately before the experiment. DNA solutions
without and with C60HyFn were irradiated at room temperature with
doses of 1, 3, 5, and 7 Gy at a dose rate of 4.5 Gy/min.
The DNA samples were denatured in a water bath for 5 min and
cooled on ice for 3–4 min. Aliquots of 40 μl (350 μg/ml) were
transferred to the wells of immunoassay plates (Costar, USA). DNA
was immobilized by a simple dry adsorption procedure with incu-
bation for 3 h at 80°C until the solution had evaporated [31]. The
blocking of nonspecific sites was performed using 300 μl of solution
containing 1% casein in 0.15 M Tris-HCl buffer, pH 8.7, and 0.15 M NaCl.
Then the plates were incubated for 3 h at 37°C. Formation of antigen
complexes with 8-oxoguanine-specific antibodies (100 μl/well,
1:2000) was carried out in the blocking solution by incubation with
stirring for 2 h at 30°C. The samples were washed twice (300 μl/well)
with a washing solution (50 mM Tris-HCl, pH 8.7, 0.15 M NaCl, 0.1%
Triton X-100). Then a secondary complex of antibodies with IgG–
horseradish peroxidase conjugate (80 μl/well, 1:1000) was formed for
2 h at 30°C in blocking solution. This complex was washed three times
as described above. After that a chromogenic substrate (18.2 mM) and
2.6 mM hydrogen peroxide in 75 mM citrate buffer, pH 4.2, were
added. After achievement of green staining, the reaction was stopped
by the addition of an equal volume of 1.5 mM NaN3 in 0.1 M citrate
a microwell plate reader at 405 nm (Titertek Multiscan, Finland). The
linear dose–yield relationship for the formation of 8-oxoG in DNA on
irradiation with a G value (radiation-chemical yield) of 0.37 molecules/
100 eV absorbed radiation energy was used as the standard for the
determination of the amount 8-oxoG in DNA in every experiment. The
details of the calibration procedure have been published elsewhere [32].
C60HyFn testing in vivo and survival of mice after irradiation
Concentrated C60FWS was diluted by sterile 5% glucose solution
immediately before the experiment and two solutions of C60HyFn
containing 0.004 and 0.04 mg/ml, that corresponds to concentrations
of C60 5.6 and 56 μM, respectively, were prepared. Single C60HyFn
intraperitoneal (ip) administration to mice was in the amount of
0.5 ml that corresponds to doses of the tested compound at 0.1 or
1.0 mg/kg of b.w. Experimental animals were injected 1 h before or
15 min after irradiation with a lethal dose of X-rays (7 Gy). Control
mice, or unprotected control group, were injected ip with 0.5 ml of 5%
glucose solution. The numbers of surviving mice in all groups and
body weight gain were monitored for 30 days after irradiation and
fixed once a day at the same time.
Statistical analysis
Data in Figs. 2 and 3 are presented as the mean ± SEM. These data
were analyzed using the Student t test, and P values of 0.05 or less
were considered statistically significant. In the survival experiments,
Fig. 3. The influence of C60HyFn (10−9–10−6 M) on formation of 8-oxoguanine (8-oxoG)
in DNA in vitro under irradiation with doses of 1, 3, 5, or 7 Gy. Inset: the influence of
hydrated fullerene (10−9–10−6 M) on formation of 8-oxoG in DNA in vitro under
irradiation at dose of 7 Gy. (⁎P b 0.05 vs control level).
 G.V. Andrievsky et al. / Free Radical Biology & Medicine 47 (2009) 786–793
the survival curves of different groups were compared by the Fisher
exact test.
Results
Influence of C60HyFn on formation of hydroxyl radical in vitro under
irradiation
The effects of C60HyFn on hydroxyl radical generation induced by
X-irradiation in various doses were assessed by determination of
U
7-OH-CCA, the end product of reaction between OH and CCA. As
shown in Fig. 2, hydrated fullerene decreased the radiation-induced
formation of hydroxyl radicals in a concentration-dependent manner.
U
Statistically significant OH-removing effects of C60HyFn were
observed in the range of C60 concentration of 10−11–10−6 M. The
quantity of hydroxyl radicals formed on irradiation in the presence of
C60HyFn depended almost linearly on dose of X-rays. Therefore, the G
value (radiation-chemical yield of hydroxyl radicals in water) is 2.4
molecules/100 eV absorbed radiation energy; our G value agrees with
earlier published data [20]. Hydrated fullerenes in all concentrations
decreased to various extents the yield of 7-OH-CCA and, therefore,
OH-radicals (G value of 0.2–2.2 molecules/100 eV).
The influence of C60HyFn at the same concentrations on produc-
tion of ОН-radicals caused by X-rays at a dose of 7 Gy is seen in the
inset to Fig. 2. No statistically significant reduction of 7-OH-CCA levels
(P N 0.05) was revealed when hydrated fullerene in concentrations of
10−13–10−12 M was added to the water before irradiation. C60HyFn at
a concentration of 10−11 M exhibits weak antiradical effects (P b
0.05) and less diluted C60FWS displays more significant effects (P b
0.01). The most prominent decrease of radiation-induced hydroxyl
radical yield is observed in the presence of C60HyFn in concentrations
no less than 10−8 M (P b 0.001). The dose reduction factors (DRF) for
various concentrations of fullerene were estimated to be 1.1 (10−11 M),
1.47 (10−10 M), 1.72 (10−9 M), 2.43 (10−8 M), 3.13 (10−7 M), and
14.87 (10−6 M). These results indicate that the tested agent represents
a DRF value exceeding 3.0 only at concentrations of more than 10−7 M.
Influence of fullerene on formation of 8-oxoG in DNA in vitro under
irradiation
The influence of C60HyFn in various concentrations on formation
of 8-oxoG in salmon sperm DNA exposed to X-rays at doses of 1, 3, 5,
Fig. 4. Influence of C60HyFn pretreatment on the survival rate and body mass gain of
mice irradiated with a dose of 7 Gy. Inset: results of body weight monitoring of intact
control mice, irradiated control mice, irradiated animals injected with C60HyFn in a dose
of 0.1 or 1.0 mg/kg b.w.
789
Table 1
Influence of pretreatment of mice with fullerene on vascular damage in different organs
after total body X-irradiation (7 Gy)
Grade of vascular damage Control C60 C60
0.1 mg/kg 1 mg/kg
SI P L SI P L SI P L
Normal finding 4 7 1
Dilatation of small blood vessels 9 6 2 1 3 6 3
Isolated hemorrhages 1 4 2 8 6 3 7
Diffuse hemorrhages 6 8 3
SI, small intestine; P, pericardium; L, lung. Each experimental group consisted of 10
animals. The table shows the number of animals with the different degrees of vascular
damage.
or 7 Gy was studied (Fig. 3). It was demonstrated that hydrated
fullerene decreased significantly (P b 0.05) the radiation-induced
formation of 8-oxoG in DNA in vitro in C60 concentrations of 10−6 and
10−7 M. We did not perform the experiment using C60FWS with
concentrations of C60HyFn less than 10−9 M due to the lack of sig-
nificant DNA-protective effects observed in all ranges of studied X-ray
doses. The radiation-chemical yield (G value) for 8-oxoG formation in
DNA was 3.7 × 10−2 μmol J−1 (G = 0.37 molecules/100 eV absorbed
radiation energy). Decrease of the 8-oxoG G value has been produced
by C60HyFn in concentrations of 10−9–10−6 M up to 0.19–0.30
molecules/100 eV. The influence of C60HyFn at the same concentrations
on formation of 8-oxoG induced by X-rays in a dose of 7 Gy is illustrated
in the inset to Fig. 3. C60HyFn in concentrations of 10−9–10−8 M has not
provided significant reduction of radiation-induced DNA damage in
comparison with control (P N 0.05). Notable inhibition of radiation-
induced yield of 8-oxoG is seen for fullerenes in concentrations at least of
10−7 M. The DRF for various concentrations of fullerenes appears to be
1.58 (10−7 M) and 1.97 (10−6 M). Collectively, in our in vitro experiments
C60HyFn displays the most significant antiradical/antioxidant potency in
concentrations of 10−7–10−6 M; therefore, the dosage adequate to these
values, 0.1 and 1.0 mg/kg b.w., has been applied to study the general
radioprotective effects of fullerene in vivo.
Influence of C60HyFn on survival of mice irradiated with a lethal dose
The effects of C60HyFn on survival and body weight gain of mice
irradiated with a lethal dose of 7 Gy were investigated in experiments
with intraperitoneal injections in a dose of 0.1 or 1.0 mg/kg b.w. (Fig. 4).
The median survival time of irradiated control mice was about 5 days
and the maximum was 12 days. When hydrated fullerene (0.1 mg/kg)
was injected before irradiation, the mean survival times was approxi-
mately 11 days, while the maximum survival time increased up to
23 days. The most pronounced radioprotective effect of C60HyFn was
observed when fullerene was injected in a dose of 1.0 mg/kg. In this case,
15% of animals were alive at 30 days after irradiation. Survival of animals
injected with C60HyFn (0.1 or 1.0 mg/kg b.w.) after irradiation was not
increased compared to control irradiated mice (data not shown).
Results of mice weights indicate that radiation caused the loss of
body weight in all groups of irradiated animals (see inset to Fig. 4). It
should be noted that body weight decrease declined at a slower pace
in mice pretreated with C60HyFn. Fullerene treatment at a dose of
0.1 mg/kg has provided normalization of body weight after 10 days
postirradiation. C60HyFn at a dosage of 1.0 mg/kg has completely
prevented loss of weight by 30 days after irradiation, and this
parameter did not statistically differ from that observed at the
beginning of the study. No signs of diarrhea or obvious rectal bleeding
were observed throughout the experiment in mice irradiated with 7
Gy of X-rays. To provide morphopathological examination dead mice
were necropsied within 24 h. A postmortem examination was
performed both in irradiated unprotected control and in fullerene-
pretreated irradiated animals (Table 1). Necropsies revealed the absence
of distensions, obstructions, intussusceptions, or hemorrhages in the
790 G.V. Andrievsky et al. / Free Radical Biology & Medicine 47 (2009) 786–793

gastrointestinal tract in all groups of irradiated animals. Dilatation of

intestinal blood vessels was revealed in unprotected irradiated mice. In
contrast, in mice injected by fullerenes this injury was less apparent.
Obvious hemorrhages in the lungs and pericardium in irradiated mice of
all groups were detected by necropsy examination. Incidence and
severity of pulmonary hemorrhages were distinctly higher in control
mice irradiated with 7 Gy compared to irradiated mice receiving
C60HyFn ip injection.

Discussion

The present study demonstrates for the first time that the hydrated
form of chemically nonmodified C60 fullerene can act simultaneously
as an antiradical, antioxidant, and radioprotective agent. The bene-
ficial effects of C60HyFn in the case of X-ray impact are determined by
hydroxyl radical removing action, protection of DNA against oxidative
modification, resulting in prolongation of survival rate of irradiated
mice even after a single injection of the tested drug in low doses.
Ionizing radiation produces ROS during water radiolysis. It is well
known that hydroxyl radical is a powerful oxidant and the most
harmful among all species of the ROS family. OH-radicals are capable
of damaging cellular macromolecules and organelles, hence initiating
the propagation and further aggravation of radiation-induced oxida-
tive stress, total injury of an irradiated organism with resulting
shortening of life span. Numerous experimental works were con-
ducted to study the application of various antioxidants to protect
macromolecules and cells against oxidative damage caused by
irradiation [33–35]. Special attention is paid to investigation of the
compounds of the fullerene family, due to their unique physical and Fig. 6. Model of the hydrated C60 fullerene (C60HyFn= stable donor–acceptor complex
of C60@{H2O}n) surrounded by long-range shells of ordered water [13].
chemical properties and biological activity [36]. Until recently, it was
considered that high antioxidative potential of fullerenes is deter-
mined by the ability of the C60 cage to bind readily free radicals [37].
With 30 double bonds, C60 can theoretically scavenge up to 60 radicals the radical removing efficacy of C60HyFn is in reverse correlation with
according to the following scheme: C60 concentration in solution. This means that in highly diluted
U
solution (10−11–10−9 M) each molecule of C60 quenches OH in quan-
NC = C b + ·OHY NCðOHÞ −·Cb + ·OH Y N CðOHÞ− CðOHÞb: tities which exceeds the theoretically accessible binding capacity of
carbon molecules. While evaluating the antiradical efficacy of hydrated
Nevertheless, results obtained in our study indicate that an fullerene we discovered a dependence of the amount of neutralized
U
alternative mechanism of antiradical action of C60HyFn may occur in OH-radicals per one C60HyFn ([ OH]/[C60]) from C60 concentration
aqueous solutions contrary to a custom recognized mechanism of ([C60]) that is calculated as logarithmic graph and demonstrated in
direct binding of free radicals to double bonds of the fullerene core. Fig. 5. It is obvious that one hydrated C60 in highly diluted C60FWS is
Analysis of data shown in the inset to Figs. 2 and 3 has revealed that able to neutralize 103–104 UOH at concentrations 10−9–10−11 M, the
greatly prevailing number of hydroxyl radicals, which exceeds the
theoretical possible binding capacity of fullerenes (60 OH-radicals per
one molecule of C60). Moreover, actual antiradical activity of C60HyFn
must probably be higher if we study utilization of the other short-
lived radical species produced during water radiolysis.
Therefore, results of our investigations, unexpected to some extent,
reveal an inversely proportional and nonstoichiometric character of
antiradical efficacy of C60HyFn in the concentration range of 10−11–10−6
M. This phenomenon is difficult to explain on the basis of the common
conception of direct radical trapping by C60 molecules. We suppose that,
in the simple system “neat C60–water,” radiation-induced formation of
OH-radicals as well as their further annihilation occurs in the ordered
heterogeneous water shells, which are shaped and stabilized due to
hydrophobic hydration of electron-deficient C60 core by H2O dipoles
[13,18,38] (Fig. 6). This model corresponds entirely with Pollack's
conception about water of “exclusion zones” [39], which emphasizes a
key role of mobility-limited superficial water layers in various biological
events, including long-range recognition of complementary entities
such as enzyme–substrate and antigen–antibody. In accordance with
this conception, water molecules near hydrophilic surfaces can form
Fig. 5. Inversely proportional and nonstoichiometric dependence of antiradical efficacy long-range stable and ordered water layers, extending to a distance of
of C60HyFn in the range of fullerene concentration 10−11–10−6 M (measurement of
U
OH-induced formation of 7-OH-CCA in water irradiated at the dose of 7 Gy was taken as
more than 10–100 μm (that is equal to many thousands layers of H2O
U molecules). Similarly, we suggest that C60HyFn—hydrophilic and highly
an example). Actual values of [ OH]/[C60] ratio are presented near the points;
percentage of removed OH-radicals is given in brackets. stable donor–acceptor complex “C60–H2O molecules”—is able to induce
 G.V. Andrievsky et al. / Free Radical Biology & Medicine 47 (2009) 786–793
in aqueous media the long-range and ordered water shells. In such case,
heterogeneous ordered water medium promotes localization and
concentration of hydrated free radicals (FR) in those of its regions, in
which the structures of water are similar to structures of its own
hydrated shells that cover certain radicals (Fig. 7). A similar accumula-
tion of free radicals increases essentially the probability of their
encounter in aqueous medium with their following recombination
(self-neutralization), resulting in the formation of stable (nonradical)
molecules [13,40]. In other words, the unusual nature of C60HyFn
antiradical activity, in contrast to other natural antioxidants (tocopher-
ols, carotinoids, bioflavonoids, ascorbic acid etc.), is based on its ability
to “catalyze” accumulation and deactivation of free radicals by means of
long-range and ordered water shells, which are induced by C60HyFn and
its nano-sized clusters. The second probable mechanism of C60HyFn
indirect antiradical action considers that generation of primary
products of H2O radiolysis occurs in ordered and “viscous” water layers
surrounding C60. This sufficiently limits diffusion of free radicals from
the loci of their formation. According to this tentative mechanism of
action, free radicals may not be separated spatially in the fullerene-
ordered aqueous medium. Because of this, immediately after the
formation of free radicals, they are able to recombine fast yielding
molecular products [see explanation in legend of Fig. 7]. According to
current observations, it is noteworthy that in highly ordered aqueous
media free radicals exist for a shorter period versus in nonclustered bulk
water.
Fig. 7. Probable scheme of free radicals (FR) absorption, concentration, and
recombination governed by long-range and ordered structures of interfacial water
U U U
formed around the hydrated C60 fullerene [13]. Radical species, such as OH, H , HO2 ,
U U−
e−
hyd (hydrated electron), CO2, O2 , could be considered on the role of hypothetical
hydrated radicals R1, R2, R3, and R4, which are formed at radiolysis of water under
ambient conditions. Reactions of recombination between them yield molecular
products such as H2O, H2O2, H2, O2, H2CO3, etc. The simplest examples of such reactions
U U U U U U
are as follows: OH + H → H2O; OH + OH → H2O2; H + H → H2.
791
It is important to note that our idea about an indirect, catalytic-like
mechanism of free radical elimination in the presence of C60HyFn is
supported by experimental data, demonstrating that native C60
fullerene exhibits extremely high stability against ROS-induced
oxidative modification. For example, it has been shown that prolonged
high-dose (6 MGy) irradiation of C60 water suspension prepared by
ultrasonic treatment caused destruction of only 5% of C60 molecules
[41]. This observation suggests that general pathways of C60 radiolysis
in the aqueous phase are polymerization, breaking the carbon
backbone, C–H-group formation, and incorporation of oxygen atoms
as carbonyl and OH-groups. In our experiment, the chemical stability of
the C60 carbon cage toward radiation-induced oxidation of hydrated
fullerene was verified using UV-Vis-spectroscopy as described pre-
viously [18]. In this case, absorption spectra of C60FWS (10−7–10−5 M
solutions of C60), irradiated at doses up to 24 Gy, appeared to be the
same as those obtained for nonirradiated hydrated fullerenes (data not
shown). Postirradiation constant spectral characteristics of C60HyFn did
not show any direct action of X-rays or highly reactive products of water
radiolysis on the oxidative modification of C60 molecules without
formation of their oxy-and/or polyhydroxylated derivates.
C60HyFn was shown to prevent formation of 8-oxoG in DNA in vitro
under the influence of ionizing radiation (Fig. 3). In contrast to results
obtained for hydroxyl radical removal (Fig. 2), hydrated fullerene did
not display notable protective effects at C60 concentrations lower than
10−7 M. This observation may be attributed to relatively low
sensitivity of ELISA compared to this parameter for the 7-OH-CCA
determination procedure. Nevertheless, we suppose that antiradical
and antioxidative, in particular, DNA-protective effects of C60HyFn
may also underlie its radioprotective activity in vivo.
It was previously shown that water-soluble nanoparticles of
dendrofullerenes (DF-1), containing carboxylic groups, protect zebra-
fish embryos against ionizing radiation [24]. DF-1, as expected for an
agent with potential antioxidant properties, reduced ROS level in
irradiated embryos, mitigated radiation-induced lethality, alleviated
developmental defects caused by radiation, attenuated disorders of
excretory function, and, finally, protected against neurotoxicity. In
another study, fullerenols C60(OH)18–22 were demonstrated to protect
protozoa Stylonychia mytilus exposed to γ-rays [23]. At the same time,
little is known about potential radioprotective effects of fullerenes in
mammals. In the study of Trajković et al. [25] it was revealed that C60
(OH)12–26 could serve as antioxidative agents and radioprotectors in
rats irradiated at a dose of 8 Gy. Our results match the data obtained by
corresponding authors. But it should be noted, that in our experiment
irradiated animals were treated with fullerenes at doses that are 1–2
orders lower than in the cited work [25], though efficacy was
comparable for both radioprotectors.
Reviewing all above-cited reports, high effectiveness of water-
soluble functionalized fullerenes in radiation-induced injury is
referred to their potent ROS scavenging activity in vivo. Nevertheless,
underlying mechanisms by virtue of which C60 derivates can trap free
radicals and act as powerful ROS scavengers or “radical sponges” in
such conditions remain unresolved, while adding only 6 radicals
substantially decreases the ability of the C60 carbon cage to interact
with UOH in aqueous media, as it has been experimentally proved [11].
Moreover, the capacity of carboxyfullerenes to trap free radicals is
weakened while simultaneously the degree of derivatization of the
fullerene core is increasing. For instance, C60 bearing 4 COOH-groups
does not display any UOH-scavenging activity [42]. Thus, it becomes
clear that fullerenes cannot act as radical “sponges” in physiological
condition and alternative antiradical mechanism is believed to be
realized.
It is noteworthy that actually every derivatized water-soluble C60
molecule, demonstrating antioxidative activity in aqueous solutions,
resides as nanoscale aggregated clusters (in the range of 20–200 nm
and more), which are highly hydrophilic and negatively charged
[43,44]. Therefore, according to Pollack's conception considering water
792 G.V. Andrievsky et al. / Free Radical Biology & Medicine 47 (2009) 786–793
as an “exclusion zone” [39], long-range water structures with various
degrees of ordering can form near such fullerene's aggregates. Finely,
compiling our findings, evidencing new concepts of distinct intrinsic
mechanisms of antiradical activity of С60HyFn, with similar data
concerning the characteristics of various water-soluble fullerenes, we
suggest that the mechanism, described above in the current report and
illustrated schematically in the Fig. 7, could underlie their capacity to
eliminate free radicals.
Also, we should note that the offered mechanism of antiradical
activity of hydrated fullerenes, probably, is not specific only to them.
Apparently, effects similar to those observed for С60HyFn can be
displayed by several other substances as well, which are characterized
by nanocluster, nonmolecular, solubility in water. For example, it has
been shown recently that nanoparticles of metal oxides (CeO2 and
Yt2O3!) increase the neuronal life span in culture, exhibiting a strong
and unusual antioxidant potency. At the same time, their micrometer-
sized counterparts do not have any effect on cell survival [45]. Other
authors [46] have revealed that 2-to 5-nm nanoparticles of cerium
oxide protect normal breast epithelial cells against apoptosis induced
by irradiation at a dose of 10 Gy. When pretreated with 10−8 M CeO2
nanoparticles 24 h prior to irradiation, these cells display almost 100%
level of survival. Moreover, nanoceria (with size of 60 nm) appeared to
be highly effective catalysts of superoxide neutralization acting as
SOD-mimetics [47].
As a whole, the results presented in this article suggest that physical,
chemical, and biological processes, including radiolysis, occurring in
highly ordered water environments, should differ essentially from the
similar processes investigated in the majority of in vitro conventional
experiments. Explanation of this phenomenon requires additional
investigations; though based on the observations of numerous experi-
ments, obtained data may be interpreted in such a way that the basics
for effective deactivation of free radicals is rather special clustered water
structures, which are ordered by C60 fullerene molecules (or other
nanoparticles), but not carbon molecule itself [48].
Collectively, our results demonstrates that nanostructures of
hydrated and chemically nonmodified fullerene C60 decrease radia-
tion-induced yields of hydroxyl radicals in aqueous solutions and
prevent oxidative modification of DNA caused by X-rays. It has been
estimated that C60HyFn significantly prolongs the life span of mice in
conditions of modeling total irradiation at full-spread lethal doses.
Newly discovered effects of unusually low doses of C60HyFn as potent
antioxidants and excellent radioprotectors are not explainable based
on commonly recognized mechanisms of action of C60 and its
derivates as free radical scavengers, i.e., absence of stoichiometry, in
U [17] Andrievsky, G. V.; Klochkov, V. K.; Karyakina, E. L.; Mchedlov-Petrossyan, N. O.
OH-removing profile and revealed a reverse correlation between
hydrated fullerenes' antiradical effects and its concentration in
aqueous solutions, is claimed as the “position statement” in our
study. According to obtained results, we hypothesize catalytic-like
mechanisms, which determine antiradical activity of C60HyFn. A
catalytic mechanism of action may be confirmed by similar results of
beneficial effects of super-small doses of C60HyFn in a number of
experimental pathological states and intoxication models [14,15].
We propose a tentative mechanism of action by means of the
existence of long-range and stable water layers ordered by C60HyFn,
capable of promoting local attraction, concentration, with subsequent
deactivation of free radicals. In C60HyFn the fullerene's core remains
stabile against oxidation by ROS; thus, C60HyFn will be used as a long-
lasting antioxidant administered even in super-small doses. Due to its
unique features, such as, postradiation maintenance of structural
stability, ability to withstand the influence of reactive products of
water radiolysis, long-lasting efficiency, and low toxicity, revealed by
the hydrated form of pristine C60 fullerene, it may be considered as a
novel highly effective antioxidant and radioprotective agent. To
explore the proposed current explanation of our hypothesis, we
suppose that further cross-disciplinary investigations may unveil
subtle catalytic mechanisms of hydrated fullerenes.
Acknowledgments
The authors gratefully acknowledge the advice and assistance of
Ms. Tamar Chachibaia (Georgian National Nanoinnovation Initiative).
We also thank Dr. Aleksander Kryshtal' (Karasin National University,
Kharkov, Ukraine) for help with TEM analysis.
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