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Fullerene nano particles and their anti-oxidative effects: A comparison to other radio protective agents

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  • Faculty of Medicine in Hradec Králové, Charles University in Prague

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Radiation therapy occupies an important position in the treatment of malignant diseases in spite of the existence of radiation side effects on normal tissues. Thus, substances are being developed which are designed to reduce both the acute and long term radiation effects on healthy tissues. Currently a sulphur-containing compound amifostine (WR2721, ethyol) is used in clinical practice as a radioprotectant. However, it itself has considerable side effects including hypotension (found in 62% of patients), hypocalcaemia, diarrhoea, nausea, and vomiting. Carbon nanospheres, known as fullerenes, and their water soluble derivatives (e.g. C 60 (OH) 24 , dendrofullerene DF-1) exert anti-oxidative properties and reduce damage to the DNA in irradiated cells. Water soluble fullerenes are low-toxic substances and thus, are attractive in terms of their use as radioprotectants.
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Journal of
APPLIED
BIOMEDICINE
J Appl Biomed. 10: 1–8, 2012
DOI 10.2478/v10136-012-0002-2
ISSN 1214-0287
REVIEW
Fullerene nanoparticles and their anti-oxidative effects:
a comparison to other radioprotective agents
Jiřina Vávrová1, Martina Řezáčová2, Jaroslav Pejchal1
1Department of Radiobiology, University of Defence, Faculty of Military Health Sciences, Hradec Králové,
Czech Republic
2Department of Medical Biochemistry, Faculty of Medicine in Hradec Králové, Charles University in Prague,
Czech Republic
Received 17th August 2011.
Revised 31st October 2011.
Published online 2nd November 2011.
Summary
Radiation therapy occupies an important position in the treatment of malignant diseases in spite of the
existence of radiation side effects on normal tissues. Thus, substances are being developed which are designed
to reduce both the acute and long term radiation effects on healthy tissues. Currently a sulphur-containing
compound amifostine (WR2721, ethyol) is used in clinical practice as a radioprotectant. However, it itself
has considerable side effects including hypotension (found in 62% of patients), hypocalcaemia, diarrhoea,
nausea, and vomiting. Carbon nanospheres, known as fullerenes, and their water soluble derivatives (e.g.
C60(OH)24, dendrofullerene DF-1) exert anti-oxidative properties and reduce damage to the DNA in irradiated
cells. Water soluble fullerenes are low-toxic substances and thus, are attractive in terms of their use as
radioprotectants.
Key words: ionizing radiation; fullerenes; antioxidant; radioprotection; nanoparticles
INTRODUCTION
Radiotherapy is one of the major treatment modalities
in the management of human cancer. However, it can
lead to a number of side effects in the human body as
a consequence of a series of events over different
time periods varying from less than 10–12 s to many
weeks. The energy transfer from a photon and/or a
particle to atoms and molecules results in a direct
change, i.e. a chemical conversion of a macro-
molecule, which could be of importance for the
biological function. The critical event is damage to
the DNA in the nucleus and formation of DNA
double-strand breaks (DSB). This initial event can be
caused by two mechanisms: either by direct damage
to the DNA by the radiation energy or indirect
damage mediated through radicals, peroxides and
superoxides produced during the water radiolysis. In
the case of radiation with low LET (Linear Energetic
Transfer) including gamma radiation and X-rays, a
prevalent proportion of the radiation damage results
from the indirect mechanism. In this review the effect
of classical radioprotectants is compared to the effect
of water-soluble C60 fullerenes.
Jiřina Vávrová, Department of Radio-
biology, University of Defence, Faculty of
Military Health Sciences, Třebešská 1575,
500 01 Hradec Králové, Czech Republic
vavrova@pmfhk.cz
+420 973 253 214
+420 495 513 018
© Journal of Applied Biomedicine
Vávrová et al.: Fullerene nanoparticles and their anti-oxidative effects
2
MECHANISM OF RADIOPROTECTION
The radioprotection of living organisms by
pharmacological substances particularly depends on
their ability to reduce intracellular concentrations of
free radicals and peroxides produced over the first
milliseconds after irradiation. Substances, which
could be used in the protection of healthy tissues from
ionizing radiation effects in radiation therapy, should
adhere to the following two basic conditions: 1) they
must selectively protect normal tissues (without
affecting tumour cells) and 2) they must exert
minimum toxicity.
Radioprotection due to hypoxia
The degree of radiation damage to tissues directly
correlates with their oxygenation. Substances able to
reduce oxygenation can therefore have protective
effects. A reduction of oxygen levels to 3–10% in the
air inhaled during the course of irradiation can exert
protective effects in mice and rats comparable to
those achieved with traditional radioprotectants
(Vacek et al. 1971). Radioprotectants taking
advantage of hypoxia as the main mechanism of the
effect include indolylalkylamines (serotonin and
mexamine). The mechanism of their protective effect
is explained by the post-vasoconstriction hypoxia
(Zherebchenko and Suvorov 1963). However, given
their side effects, such as a decrease in arterial blood
pressure, a decrease in body temperature, and the
teratogenic effect or degenerative changes in testicles
(Kuna 1985), these substances did not find any use in
clinical practice.
Inactivation of oxidative radicals in water
Strongly reactive oxygen radicals produced during
irradiation by water radiolysis have harmful effects on
the cell. Radical scavenging is the basic mechanism
of many chemical substances and enzymes protecting
biological targets against ionising radiation. It is
essentially a competition for a radical between the
protective substance and the biological molecule. In
aqueous solutions, protective substances and e nzymes
react with free radicals and peroxides, produce stable
non-toxic products and thus reduce the amounts of
these species. Many of these radioprotectants are very
good extinguishers of oxygen radicals. Compounds
containing sulphur are well known radioprotectants
exerting the highest protective effects, but they are
also very toxic. In contrast, antioxidants of natural
origin can be characterized by a relatively low
toxicity, but also lower radioprotective properties.
RADIOPROTECTIVE AGENTS
Sulphur containing compounds
Radioprotectants containing sulphur are chemical
analogues of cysteine (a thiol group containing amino
acid). Similar to cysteine, these substances have their
SH group separated by two to three carbon atoms
from the basic amino group. To provide effective
radioprotection, these substances must be present in
the organism prior to irradiation. The optimum
radioprotection is achieved in the case of an
intravenous administration 15–30 min before
irradiation. Sulphur containing compounds act
through the mediation of three main mechanisms: as
extinguishers of free radicals, as carriers of oxygen
and last, but not least, they act as substances inducing
hypoxic effects. The most well known
radioprotectants of this group are cysteamine,
cystamine AET and WR2721 (Bacq 1954, Dostál
1967, Kuna 1985).
The Walter Reed Military Institute in the USA has
produced and tested 4000 compounds. In 1969, they
synthesized a substance marked WR-2721 (Piper et
al. 1969) (amifostine, ethyol) which is an organic
thiophosphate prodrug (2-(3-aminopropyla-
mino)ethylsulphanyl phosphonic acid) hydrolysed in
vivo by alkaline phosphatase to the active
cytoprotective thiol metabolite, WR-1065
(2-((aminopropyl)amino)ethanethiol). The selective
protection of non-malignant tissues is believed to be
related to higher alkaline phosphatase activity, higher
pH, and vascular permeation in normal tissues. The
combination of hypovascularity, low pH, and reduced
enzyme levels results in a low accumulation of the
active drug in tumour tissues (Kouvaris et al. 2007).
Mean lethal doses were established for accurate
determination of the radioprotective effects. These
doses are typically related to the 30th day after
irradiation. LD50/30 is a lethal dose after which 50%
of animals survive up to the 30th day after irradiation.
The DRF (Dose Reducing Factor) is a ratio of the
mean lethal dose in protected animals to that in
non-protected animals. Table 1 presents DRF values
of different radioprotectants used in mice. In the case
of whole-body γ-ray irradiation, WR-2721
administered at a dose of 300 mg/kg is obviously the
most effective radioprotectant (Kuna et al. 1983,
Kuna 1985). No radioprotective effect of WR-2721
was found when it was administered at a dose of
160 mg/kg (intravenously or intramuscularly) to rats
15–20 min before their whole-body exposure to
fission neutrons (Kuna et al. 2004). This is probably
due to the fact that neutrons primarily damage
biological molecules directly. WR-2721 also
Vávrová et al.: Fullerene nanoparticles and their anti-oxidative effects
3
Table 1. Comparison of the radioprotective effect of DF-1 dendrofullerene with other known radioprotectants.
Drug Dose Irradiation Animals DRF* Author
WR-2721 300 mg/kg i.m.
15–20 min before irrad. gamma mice 2.39 Kuna 1985
WR-2721 100 mg/kg i.m.
15–20 min before irrad. gamma mice 1.3 Kuna 1985
Cystamine 175 mg/kg i.m.
5–15 min before irrad. gamma mice 1.83 Kuna 1985
Superoxide-dismutase i.v. 2 h before irrad. (200 mg/kg)
and 1 h after irrad. (35 mg/kg) X-rays mice 1.56 Petkau 1978
Hypoxia – 8% O2in the course of irrad. gamma mice 1.5 Vacek et al.
1971
DF-1 dendrofullerene
nanoparticle
300 mg/kg
15 min before irrad. X-rays mice 1.22 Brown 2010
* The DRF (the Dose Reducing Factor) is a ratio of the mean lethal dose (LD50/30) in protected animals to that in non-protected
ones.
considerably reduces the toxicity of c hemotherapeutic
agents, particularly of cisplatin (Yuhas 1980).
The undesirable side effects of WR-2721 are
related to the application of high doses. The LD50/48
value for mouse strain H after an intra peritoneal (i.p.)
administration was 764–1054 mg/kg. The best
radioprotective effect was achieved by an i.p.
application of 300 mg/kg, when DRF was 2.11–2.39.
A decrease in the dose to 100 mg/kg caused a
significant decrease in the protective effect (DRF =
1.3) (Kuna 1985). The side effects of WR-2721
include hypotension, hypocalcaemia, diarrhoea and
nausea (France et al. 1986). Hwang et al. (2004)
applied WR-2721 to patients during myeloablative
conditioning therapy for allogenic bone marrow
transplantation. WR-2721 was administered at a dose
of 1000 mg/day of conditioning and was well
tolerated if attention was paid to the serum calcium
level, blood pressure and antiemetics.
Antioxidants of natural origin
There are a few substances of natural origin that are
able to protect cells from the negative effects of free
radicals and reactive oxygen species. These
substances can be divided into two groups: 1)
low-molecular substances acting as scavengers of
oxygen radicals and 2) enzymes detoxifying reactive
oxygen radicals and peroxides.
The low-molecular compounds acting as oxygen
radical scavengers include vitamins A and E, which
are lipophilic, and vitamin C, which is hydrophilic.
Several studies have established the radioprotective
values of vitamins A, C and E and carotenoids in
normal cells (Malick et al. 1978, Konopacka et al.
1998, Prasad et al. 2002). In these compounds, the
DRF values range between 1.1 and 1.2. Lipophilic
vitamins A and E administrated i.p. and hydrophilic
vitamin C administered i.m. to mice for 14 days
(3 days before immunoradiotherapy and 11 days after
immunoradiotherapy) reduced the body weight loss
and myelosupression associated with radio-
immunotherapy (Blumenthal et al. 2000).
The group of enzymes with antioxidant properties
includes superoxide-dismutase (SOD), catalase,
glutathione peroxidase and glutathione reductase
(Citrin et al. 2010) and these are described below.
Superoxide-dismutase (SOD)
Superoxide-dismutase is an enzyme important for the
detoxification of reactive oxygen radicals catalyzing
the superoxide radical conversion to hydrogen
peroxide (H2O2) and hydrogen. H2O2 is subsequently
removed by a reaction with the participation of two
enzymes (catalase and glutathione peroxidase). The
administration of superoxide-dismutase 2 h prior to
irradiation (200 mg/kg) and 1 h after irradiation
(35 mg/kg) provides a relatively high radioprotective
Vávrová et al.: Fullerene nanoparticles and their anti-oxidative effects
4
effect – DRF = 1.58 (Petkau 1978). In contrast to
radioprotectants containing sulphur, these enzymes
exert only a minimum toxicity.
In terms of radiation damage, not only DNA
impairment is important, but also the damage to
mitochondria mediated through the production of
superoxide and other reactive oxygen species (ROS)
derived from superoxide. An increased ROS
production can be observed in the irradiated tissues 6
months after the exposure (Epperly et al. 2008). The
damage to the mitochondria is manifested by
induction of apoptosis. Manganese superoxide
dismutase (MnSOD), which is an enzyme present in
human cells, is actually the first line of defense
against an increased superoxide production in the
mitochondria (Belikova et al. 2009). Thus,
antioxidant gene therapy studies have utilised
manganese superoxide dismutase-plasmid liposomes
(MnSOD-PL). Overexpression of the mitochondria
localized MnSOD gene product have been shown to
decrease the ionizing radiation-induced apoptosis of
cells in vitro (Epperly et al. 2002). In the case of
intravenous application of MnSOD-PL to mice before
their exposure to 9.5 Gy (antioxidant gene therapy –
the mice received intravenously 100 μl of liposomes
containing 100 μg of human MnSOD-transgene
plasmid 24 hours prior to irradiation), increased
animal survival was observed 30 days as well as
340 days after irradiation (Epperly et al. 2008).
Fullerene – derivatives
Compounds developed based on nanotechnology,
such as for example, carbon nanospheres named
fullerenes (C60, C70, C80–C200) also represent an
important group of antioxidants due to the possible
absorption of many oxygen radicals in a single
fullerene molecule (Bosi et al. 2003). The most
abundant form of fullerenes is buckminsterfullerene
C60 (Fig. 1) with 60 carbon atoms arranged in a
spherical structure (Marković and Trajković 2008).
Fig. 1. Structure of fullerene C60.
C60 is soluble in aromatic solvents and carbon
disulfide but essentially insoluble in water and
alcohol (Jensen et al. 1996). For their use in biology,
it is necessary to obtain fullerene derivatives, which
are soluble in polar solvents. Chemical modification
of the fullerene carbon cage by the attachment of
various functional groups (e.g.-OH, NH2, -COOH)
enables the fullerene molecule to establish bonds with
water via hydrophilic functional adducts (Marković
and Trajković 2008). Johnston et al. (2010) reviewed
analyses of the toxicity of fullerenes in detail.
Manipulating fullerene water solubility has included
the use of surface modifications, solvents, extended
stirring, and mechanical processes. However, the
ability of these processes to have an impact on
fullerene toxicity requires further assessment,
especially when considering the use of solvents,
which particularly enhance the toxicity of fullerene
derivates (Johnston et al. (2010).
Inhibition of HIV replication
These substances were also shown to possess
considerable antiviral effects. In 1993, the
water-soluble derivative of C60 [bis(monosuccini-
mide) derivative of bis(2-aminoethyl)diphenyl-C60]
was found to be a substance inhibiting HIV-1
protease (Schinazi et al. 1993). A derivative of
tris-hydroxymethyl methano-fullerene was later
discovered to exert an even higher antiviral activity
(Jensen et al. 1996). The antiviral activity seems to be
characteristic for many non-toxic derivatives of the
C60 fullerene (Friedmann et al. 1998, Cheng et al.
2010).
Photodynamic therapy of tumours
Mroz et al. (2007) showed that the C60 molecule
mono-substituted with a single pyrollidinium group is
a remarkably efficient photosensibilizer and can
mediate the killing of a panel of mouse cancer after
exposure to white light. Following intravenous
injection of C60-PEG (polyethylene glycol – PEG
conjugated with C60) to tumour-bearing mice, coupled
with exposure of the tumour site to visible light, the
volume increase of tumour mass was suppressed and
C60-PEG conjugate exhibited a stronger suppressive
effect than Photofrin (Tabata et al. 1997, Liu et al.
2007). These data demonstrate the potential use of
these compounds as photo-sensibilizers for
photodynamic therapy of tumours.
Antioxidant activity
Oxidative stress and associated oxidative damage are
mediators of cellular injury in many pathological
conditions, including autoimmunity, atherosclerosis,
diabetes, and neurodegenerative disorders (Marković
Vávrová et al.: Fullerene nanoparticles and their anti-oxidative effects
5
and Trajković 2008). In many studies, it has been
shown that water-soluble fullerene derivates can act
as antioxidant substances scavenging oxygen radicals
(including ROS generated by ionising radiation) and
protecting cells and/or tissues against ROS damage.
The known antioxidant activity of water soluble C60
derivatives is summarized in Table 2.
Table 2. Water soluble C60 derivates and their antioxidative effects.
Fullerene type Biological effects Author
C60(OH)24
radioprotection
protection from doxorubicin toxicity
Trajković et al. 2007, Cai et al. 2010
Injac et al. 2008
C60(OH)22
protection from H2O2 induced
oxidative injury Yin et al. 2008
Malonic acid C60 derivates
carboxyfullerenes neuroprotection Dugan et al. 1996, 2001,
Ali et al. 2008
C60 dendrofullerene radioprotection Brown et al. 2010, Theriot et al. 2010
Polyvinylpyrrolidone wrapped C60 reduces synovitis Yudoh et al. 2009
For instance, polyhydroxylated fullerenes –
C60(OH)x, also referred to as fullerenols, were studied
by Trajković et al. (2007) and Cai et al. (2010).
Trajković et al. (2007) demonstrated the radio-
protective effect of fullerenol (C60(OH)24)
administered to rats intraperitoneally in a dose of
100 mg/kg 30 min prior to 8 Gy irradiation. The
fullerenol protected the rats’ haemopoietic and
lymphoid tissues. Cai et al. (2010) studied the
radioprotective effects of repeated fullerenol
administrations (for a period of 14 days) at a dose of
40 mg/kg on the mouse immune system. It was found
that 2-week C60(OH)24 pretreatment effectively
reduced whole body irradiation-induced mortality
without apparent toxicity. C60(OH)24 pretreatment also
showed significant protective effects against ionizing-
radiation-induced decreases in immune and
mitochondrial function and antioxidant defense in the
liver and spleen. This suggests that the
polyhydroxylated fullerene derivative C60(OH)24
protects against ionizing-radiation-induced mortality,
possibly by enhancing the immune function,
decreasing oxidative damage and improving the
mitochondrial function.
The antioxidant and protective properties of
carboxy-fullerenes have been described by Dugan et
al. (1996) and Ali et al. (2008). Both studies showed
that carboxy-fullerenes are able to protect neurons
against the oxidative stress associated with
Parkinson’s disease and ischaemic brain injury.
Moreover, Ali et al. (2008) compared the structure of
6 carboxy-fullerenes and found the best antioxidative
effect in the tris–adduct malonic acid derivate of
fullerene – C60(C(COOH)2)3. Ali et al. (2008)
described carboxy-fullerenes as three-dimensional
carbon carriers with the antioxidative properties
depending not only on the number of bound
carboxylic groups but also on their distribution on the
fullerene ball.
The ability of fullerenes to modulate cytokine
production and cellular damage was shown in
bis-adduct malonic acid derivate and water-soluble
C60 fullerene (polyvinylpyrrolidone wrapped C60).
Bis-adduct malonic acid derivate inhibited the
TNF-alpha initiated apoptosis in HeLa cells (Li et al.
2011). On the other hand, findings by Yudoh et al.
(2009) indicate that polyvinylpyrrolidone wrapped
C60 reduces pro-inflammatory cytokine production
from synovial inflammation-related cells and
mitigates the resultant synovitis in vitro. Intra-
articular treatment with this compound significantly
attenuates synovitis and joint destruction in the rat
model of arthritis.
Another promising fullerene derivate is dendro
(60) fullerene-1 (DF-1). The derivate is characterised
by a single branched dendromer architecture
containing 18 terminal carboxylic groups attached to
the fullerene ball. DF-1 is readily soluble in water, is
non-toxic and has radioprotective effects (Brown et
al. 2010, Theriot et al. 2010). Theriot et al. (2010) has
shown that DF-1 protects lymphocytes as well as cells
in the intestinal crypts from radiation-induced cell
Vávrová et al.: Fullerene nanoparticles and their anti-oxidative effects
6
death. In this study, human lymphocytes and rat
intestinal crypt cells (IEC-6) were incubated with
100 μM DF-1 one hour prior to irradiation, rinsed and
immediately exposed to a single dose of 4 Gy in the
fresh medium. The study shows that 1-hour
incubation with DF-1 reduces the number of
micronuclei (an indicator of DNA damage) in both
types of cells compared to the irradiated
non-protected groups. Brown et al. (2010) evaluated
the DF-1 DNA protective effects via the expression of
phosphorylated histone H2AX (γ-H2AX). Γ-H2AX
serves as an indicator of DNA double strand brakes.
In the DU145 cell culture, a 30-min pre-treatment
with 100 μM DF-1 resulted in a significant decrease
in the number of γ- H2AX foci 1 and 6 h after 4 Gy
irradiation. Both studies demonstrate that there is a
reduction in DNA damage after DF-1 incubation and
that DF-1 acts not only against the oxidative stress
but also against the DNA damage generated by
ionizing radiation.
CONCLUSION
Polyamino- and polyhydroxy-fullerenes show that
water-solubility increases with the number of groups
introduced into the molecule. It is possible to state
conclusively that water-soluble fullerene derivatives
exert considerable protective effects against the
oxidative stress as scavengers of free radicals in vitro
as well as in vivo (Dugan et al. 2001, Ali et al. 2004,
Bakry et al. 2007, Injac et al. 2008). The radio-
protective effects were demonstrated in fullerenols,
carboxy-fullerenes, polyvinylpyrrolidone wrapped
fullerene, and DF-1. Table 1 summarizes a
comparison of the DRFs after a single water-soluble
dendrofullerene DF-1 application 30 min before
irradiation (DRF = 1.22) with the effects of other
radioprotectants. Given the fact that these substances
(fullerenol, DF-1) have no or only slight side effects,
they offer a great potential to become radioprotectants
with the possibility of repeated administration, which
is necessary in standard fractionated radiotherapy.
ACKNOWLEDGEMENTS
The authors would like to thank the Ministry of
Defence of Czech Republic (project
MO0FVZ0000501 and project OVUOFVZ200806)
for financial support.
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... Moreover, treatment with C 60 was more effective than with VOO in improving the CP-induced genetic toxicity because the percentages of DNA polymorphism in hepatic and cardiac tissues were 8.9% and 9.8% and 12 and 12.7% in CP + C 60 and CP + VOO groups, respectively. Fullerene C 60 causes no damage in DNA strands and had no impact on the level of aberrant chromosomes in vivo and in vitro [15,37,38]. In the present study, C 60 and VOO induced an improved DNA level and decreased the chromosomal aberration in CP + C 60 or CP + VOO compared to that in the CP group. ...
... The chromosomal aberration is an important parameter for investigating the protective effects of antigenotoxic agents on chemical and drug-induced toxicity. C 60 had no genotoxic effects, and it induced antigenotoxic effects at subcytotoxic concentrations on human lymphocytes, presented by the decreased in micronuclei and chromosomal aberration frequency [37,38]. Moreover, C 60 prevents the toxic effect of doxorubicin (chemotherapeutic agent) on normal cells and possesses no genotoxic effect on human lymphocytes [18]. ...
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The potential effects of the fullerene C 60 nanoparticle (C 60 ) as well as virgin olive oil (VOO) against the cyclophosphamide- (CP-) induced cytotoxic and mutagenic effects were evaluated by two main methods: molecular intersimple sequence repeat (ISSR) assay and cytogenetic biomarkers. Thirty adult male rats were divided to five groups (control, CP, C 60 , CP + C 60 , and CP + VOO). CP was i.p. injected with a single dose of 200 mg/kg; C 60 and VOO were given orally (4 mg/kg dissolved in VOO and 1 ml, resp.) in alternative days for 20 days. The ISSR analysis revealed an increased in the DNA fragmentation level for liver and heart tissues represented by 21.2% and 32.6%, respectively, in the CP group. The DNA polymorphism levels were modulated and improved in CP + C 60 (8.9% and 12%) and CP + VOO (9.8% and 12.7%) for hepatic and cardiac tissues, respectively. The bone marrow cytogenetic analysis revealed that C 60 and VOO had significantly decreased the frequency of CP-induced chromosomal aberrations (chromosomal ring, deletion, dicentric chromosome, fragmentation, and polyploidy). Fullerene C 60 and VOO have ability to reduce DNA damage and decrease chromosomal aberrations. In conclusion, fullerene C 60 and VOO have protective effects against the CP-induced mutagenicity and genotoxicity. Fullerene C 60 and VOO open an interesting field concerning their potential antigenotoxic agents against deleterious side effects of chemotherapeutics.
... Cell death is related to damage to nuclear DNA and formation of DNA double-strand breaks (DSBs). Damage to the DNA can be caused directly by radiation or indirectly, through mediation of radicals, peroxides, and superoxides [29]. In order to reduce adverse radiation effects on biological systems, radioprotective compounds are researched [27]. ...
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The aim of this study was to investigate the uptake and accumulation of fullerenol C60(OH)36 into peripheral blood mononuclear cells (PBMCs). Some additional studies were also performed: measurement of fullerenol nanoparticle size, zeta potential, and the influence of fullerenol on the ionizing radiation-induced damage to PMBCs. Fullerenol C60(OH)36 demonstrated an ability to accumulate in PBMCs. The accumulation of fullerenol in those cells did not have a significant effect on cell survival, nor on the distribution of phosphatidylserine in the plasma membrane. However, fullerenol-induced depolarization of the mitochondrial membrane proportional to the compound level in the medium was observed. Results also indicated that increased fullerenol level in the medium was associated with its enhanced transport into cells, corresponding to its influence on the mitochondrial membrane. The obtained results clearly showed the ability of C60(OH)36 to enter cells and its effect on PBMC mitochondrial membrane potential. However, we did not observe radioprotective properties of fullerenol under the conditions used in our study.
... In addition, AFM 1 was detected in both mycelia and medium, and the sum of the concentrations revealed the strongest aflatoxigenic effect at an average rate of 93% until 144 hours of growth at the lowest FNP concentration (Fig. 5). AFM 1 is primarily found in animal tissues and fluids (milk and urine) as a metabolic product of AFB 1 39 but is also found as a product of secondary metabolism of Aspergillus species [40][41][42][43] . There was no observed statistically significant difference between the ratio of aflatoxins B 1 and B 2 for the applied FNP concentrations. ...
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Abstract The water soluble fullerene C60 daughter product - fullerols C60(OH)24 (FNP) possesses a great potential of modifying secondary metabolites biosynthesis. In order to clarify the extent of interaction, the impact of FNP (10, 100 and 1000 ng mL−1) on aflatoxin production and the available precursors of biosynthesis pathway from Aspergillus flavus NRRL 3251 was determined, in both the mycelia and yeast extract sucrose (YES) medium, during a 168-hour growth period at 29 °C in the dark. The FNP of 8 nm in diameter, and with a zeta potential of −33 mV affected mycelial growth at 1000 ng mL−1 while conidia production was slightly affected at 10 ng mL−1. The FNP effect on aflatoxin and it biosynthetic precursors was concentration dependent and alteration of the sterigmatocystin (ST) export from the cell was observed. Most of the monitored aflatoxin precursors, except norsolorinic acid, were detected in both mycelia and YES medium. However, observed precursor concentrations were much higher in mycelia, with exception of ST. The study shows the loss of FNP antioxidative effect after 120 hours of growth, and strong concentration dependent aflatoxigenic effect after that time. Thus, this data is relevant to guide future considerations on FNP-fungal interactions in the environments and on risk assessment.
... The scavenging of free radicals by nanoparticle performance is the inhibition of reactive oxygen species (ROS). These species are highly unstable and reactive, as they capture cellular macromolecule electrons that results to their inefficiencies [9][10][11][12]. ...
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Optimal distribution of cerium oxide nanoparticles (CONPs) or nanoceria can have a significant impact on their cytotoxicity, cellular uptake, and radioprotection effects. In this study, two different distribution plans of CONPs were investigated. A scanner electron microscope (SEM) was used for chemical analysis and recording of CONP images. Using MTT assay, the non-toxic concentrations of nanoceria with two different distribution plans were determined in MRC-5 and MCF-7 cell lines. Nanoceria cellular uptake at 50, 150, and 250 μM with two different dispersion plans was determined by using the UV/VIS absorbance of cell culture medium after 24 h of incubation. In order to quantify radioprotection effect, cells treated with non-toxic concentrations of nanoceria were exposed to 10, 40, and 100 cGy of 6 MV photon beams. The diameter of the spherical CONPs was 29 nm. Energy dispersive spectroscopy analysis showed that the cerium element has the highest weight percentage in CONPs (97.9%). Accumulation rate of filtered and non-filtered suspension were determined as 0.3608 and 14.2708 μg/ml/h, respectively. The 70 and 110 μM concentration of sustained nanoceria suspension did not have any toxicity for MRC-5 and MCF-7 cells, respectively. In both cell lines, 50, 150, and 250 μM of filtered nanoceria had a significant uptake than the non-filtered nanoceria. A total of results showed that the 70 μM of nanoceria have a significant radioprotection on normal cells in the radiation dose of 40 and 100 cGy, while the highest cellular uptake of nanoceria occurred in cancer cells. The results suggest that using of stable distribution of CONPs for radiation protection could be a good choice, knowing that these nanostructures will have selective protection in normal cells. Keywords Cerium oxide nanoparticles . Radioprotection effect . MTTassay . MRC-5 . MCF-7
... concentration in the medium, surface modifications, synthesis, processing conditions, size of aggregates, type of cells and duration of exposure Johnston et al., 2010;Su et al., 2010). Other numerous reports have shown that C 60 has a potential anti-oxidative stress effect with anti-genotoxic (Mrđanović et al., 2012;Rim et al., 2013;Vávrová et al., 2012), anti-cancer (Afanasieva et al., 2015;Didenko et al., 2013;Prylutska et al., 2011Prylutska et al., , 2017, hepatoprotective (Elshater et al., 2018;Halenova et al., 2016) and radioprotective (Theriot et al., 2010) properties due to the nano size. Under the current experimental settings and in agreement with these previous reports, C 60 shows hepato-and nephron-protective effects against CdCl 2 -induced genotoxicity. ...
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Currently, cadmium is considered to be one of the major environmental pollutants. Environmentally, cadmium is released in various forms e.g. oxide, chloride and sulphide. The aim of the present study was to examine the genotoxic impact of fullerene nanoparticles C60(C60) and virgin olive oil (VOO) on cadmium chloride (CdCl2)-induced genotoxicity in rats. To evaluate these effects on DNA damage and chromosomal frequency, 25 albino rats were randomly assigned to 5 groups (n=5 per group): Group 1 served as a control; Group 2 received a single intraperitoneal dose of CdCl2(3.5mg/kg); Group 3 animals were treated with C60(4mg/kg, orally) every other day for 20days; Group 4 received a single intraperitoneal dose of CdCl2(3.5mg/kg) and an oral dose of C60(4mg/kg); and Group 5 received a single intraperitoneal dose of CdCl2(3.5mg/kg) and oral doses of VOO every other day for 20 consecutive days. Genotoxic and anti-genotoxic effects of C60and VOO were evaluated in the liver, kidney and bone marrow using molecular and cytogenetic assays. As expected, CdCl2and C60administration was associated with band number alterations in both liver and kidney; however, C60pretreatment recovered to approximately basal number. Surprisingly, C60and VOO significantly attenuated the genotoxic effects caused by CdCl2in livers and kidneys. In bone marrow, in addition to a reduction in the chromosomal number, several chromosomal aberrations were caused by CdCl2. These chromosomal alterations were also reversed by C60and VOO. In conclusion, molecular and cytogenetic studies showed that C60and VOO exhibit anti-genotoxic agents against CdCl2-induced genotoxicity in rats. Further studies are needed to investigate the optimal conditions for potential biomedical applications of these anti-genotoxic agents.
... The functional impacts of dendron-C60-fullerene were similar to that of amifostineanother radioprotective agent and antioxidant. Dendro-C60-fullerene could internalize the experimental cells [70]. Fullerene upon interactions with polyamine and polyhydroxy compounds enhances water solubility. ...
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Polyhydroxylated fullerenes, named fullerenols (C60(OH)n; n = 12–26) are excellent antioxidants. Harmful effects of ionizing radiation on living organism are mainly mediated by free radical species and fullerenols attract an attention as a potential radioprotectors. Our preliminary investigations on mice and rats subjected to radiation injury show that fullerenol C60(OH)24 provides high survival rate of irradiated small rodents. Radioprotective effect was comparable to that of the standard radioprotector amifostine. The aim of this study was to compare the efficacy of fullerenol C60(OH)24 (10 and 100 mg/kg i.p.) and amifostine (300 mg/kg i.p.) in protection of rats against harmful effects of ionizing radiation. The animals were whole-body irradiated by X-rays (8 MV). Both compounds were given 30 min before irradiation. In order to evaluate the general radioprotective efficacy of fullerenol and amifostine rats were irradiated with an absolutely lethal dose of X-rays (8 Gy) and their survival and body mass gain were monitored during the period of 30 days after irradiation. The aim of the second part of the study is to investigate the tissue-protective effects of tested compounds (100 mg/kg i.p. of fullerenol and 300 mg/kg i.p. of amifostine, 30 min before irradiation). It was carried out on rats irradiated with a sublethal dose of X-rays (7 Gy). Influence of ionizing radiation on hematopoesis as well as the radioprotective efficiency of the compounds given were evaluated by determining blood cell count during 28 days after irradiation. For this purpose the blood was taken from tail vein before irradiation and on the 3rd, 7th, 14th, 21st and 28th day after irradiation. In order to estimate the radioprotective effects of fullerenol and amifostine on other rat tissue, the animals were sacrificed on the 7th and 28th day after irradiation and their main organs (lung, heart, liver, kidney, small intestine and spleen) were taken for histopathological analysis. In the experiment in which the general radioprotective efficacy of fullerenol and amifostinewas examined, fullerenol given in a dose of 100 mg/kg produced better protection than given in a dose of 10 mg/kg. This effect was comparable to that of amifostine. The results of hematological investigations showed that fullerenol better than amifostine prevented radiation-induced reduction in the white cell count (granulocytes and lymphocytes), particularly in the first 7 days after irradiation. Pathohistology examinations revealed better radioprotective effects of fullerenol compared to those of amifostine on the spleen, small intestine and lung, while amifostine had better radioprotective effects than fullerenol in protection of the heart, liver and kidney. These results confirm satisfactory radioprotective efficacy of fullerenol and encourage further investigations as a potential radioprotector.
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Nanoparticles such as fullerenes and carbon nanotubes have been extensively studied for biomedical applications. In this paper, we report the design of carbon nanotubes as HIV-1 protease inhibitors. Docking and molecular dynamics calculations are performed using an atomistic model to explore the optimal interaction structure and free energy between the nanotube and HIV-1 protease. A coarse-grained model is then developed based on the atomistic model, allowing us to investigate the dynamic behaviors of the protease in the bound and unbound states. The dynamic process reveals that the carbon nanotube is able to bind to the active site of the protease and prevent the active flaps from opening up, thus blocking the function of the protease. This process is strongly influenced by the size of the nanotube. The binding of carbon nanotubes to an alternative binding site other than the active site is also explored. Therefore, carbon nanotube-based inhibitors have great potential for application as HIV-1 protease inhibitors.
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We have developed and applied a computational strategy to increase the affinity of fullerene-based inhibitors of the HIV protease. The result is a 50-fold increase in affinity from previously tested fullerene compounds. The strategy is based on the design of derivatives which may potentially increase hydrophobic desolvation upon complex formation, followed by the docking of the hypothetical derivatives into the HIV protease active site and assessment of the model complexes so formed. The model complexes are generated by the program DOCK and then analyzed for desolvated hydrophobic surface. The amount of hydrophobic surface desolvated was compared with a previously tested compound, and if this amount was significantly greater, it was selected as a target. Using this approach, two targets were identified and synthesized, using two different synthetic approaches:  a diphenyl C60 alcohol (5) based on a cyclopropyl derivative of Bingel (Chem. Ber. 1993, 126, 1957−1959) and a diisopropyl cyclohexyl C60 alcohol (4a) as synthesized by Ganapathi et al. (J. Org. Chem. 1995, 60, 2954−2955). Both showed tighter binding than the originally tested compound (diphenethylaminosuccinate methano-C60, Ki = 5 μM) with Ki values of 103 and 150 nM, respectively. In addition to demonstrating the utility of this approach, it shows that simple modification of fullerenes can result in high-affinity ligands of the HIV protease, for which they are highly complementary in structure and chemical nature.
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Superoxide radical anion is a biologically important oxidant that has been linked to tissue injury and inflammation in several diseases. Here we carried out a structure-activity study on six different carboxyfullerene superoxide dismutase (SOD) mimetics with distinct electronic and biophysical characteristics. Neurotoxicity via N-methyl-D-aspartate receptors, which involves intracellular superoxide, was used as a model to evaluate structure-activity relationships between reactivity toward superoxide and neuronal rescue by these drugs. A significant correlation between neuroprotection by carboxyfullerenes and their ki toward superoxide radical was observed. Computer-assisted molecular modeling demonstrated that the reactivity toward superoxide is sensitive to changes in dipole moment, which are dictated not only by the number of carboxyl groups but also by their distribution on the fullerene ball. These results indicate that the SOD activity of these cell-permeable compounds predicts neuroprotection, and establishes a structure-activity relationship to aid in future studies on the biology of superoxide across disciplines.