Chapter 7 RADIOPROTECTIVE PROPERTIES OF ASTAXANTHIN: THE IMPACT ON RADIATION INDUCED CHROMOSOMAL ABERRATIONS AND DNA BREAKS IN HUMAN LYMPHOCYTES IN VITRO

Abstract

ABSTRACT

Purpose: Astaxanthin (a carotenoid of xanthophyll group) satisfies all requirements demanded to potential radioprotectors. The purpose of our study was to investigate a modifying effect of astaxanthin on radiation induced genome damages in cultivating human peripheral blood lymphocytes (PBL).
Materials and methods: To establish astaxanthin radioprotective activity, we used method for scoring of structural chromosomal aberrations (ChA) in PBL and neutral single-cell gel electrophoresis (comet assay) for estimation of the level of DNA breaks in cells (expressed as tail moment (TM) for “classical comets” and rate of “atypical comets” (АС) for cells with greatly fragmented DNA). Blood samples were obtained by venepuncture from five conditionally healthy volunteers. Astaxanthin in final concentrations of 2.0, 10.0, 20.0 and 40.0 µg/ml had been added into the culture medium before cultivation began, prior to γ- irradiation of PBL (dose 1.0 Gy). Cultures were incubated at 37°C for 48-50 h. Chromosome slides were prepared after 50 h of cultivation. Comet assay was carried out before cultivation and on 48 h of culturing.
Results: Astaxanthin in all studied concentrations per se had no mutagenic activity and obviously reduced the level of radiation induced chromosomal aberrations. The most efficient concentration of astaxanthin was 20.0 µg/ml. The effect of astaxanthin in this concentration on exposed cells manifested in significant decreasing of the radiation-induced ChA level: from 26.06 ± 1.81 per 100 metaphases (phm) to
9.03 ± 0.73 phm (p<0,001). Comparative analysis of the ChA spectra indicated the most substantial reduction of classic unstable cytogenetic markers of radiation exposure (dicentric and ring chromosomes). Likewise, statistically significant decrease in TM in irradiated lymphocytes treated by astaxanthin compared with untreated irradiated samples was observed both before cultivation (3.21 ± 0.48 and 6.55 ± 1.82 respectively, p<0,05) and after 48 h of cultivation (5.27 ± 1.77 and 12.86 ± 0.74 respectively, p<0,05). The level of AC, which evidently arose from apoptotic cells, in irradiated cultures treated by astaxanthin was approximately two times higher as compared with irradiated samples (7.15 ± 1.13% and 3.57 ± 0,81% respectively, p<0,05).
Conclusions: Our study indicated that astaxanthin in concentration of 20.0 µg/ml demonstrated evident radioprotective properties by reducing the level of ChA, decreasing DNA damage and increasing the apoptotic rate.

Keywords Astaxanthin, human peripheral blood lymphocytes, chromosomal aberrations, DNA breaks, apoptosis, radioprotective effect

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Chapter 7
Astaxanthin as a Modifier of Genome Instability after
γ-Radiation
Denys Kurinnyi, Stanislav Rushkovsky,
Olena Demchenko and Mariya Pilinska
Additional information is available at the end of the chapter
http://dx.doi.org/10.5772/intechopen.79341
© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
Astaxanthin as a Modier of Genome Instability after
DenysKurinnyi, StanislavRushkovsky,
OlenaDemchenko and MariyaPilinska
Additional information is available at the end of the chapter
Abstract
The chapter is devoted to study the eects of astaxanthin on the frequency of chromosomal
aberrations and the level of DNA damages in human peripheral blood lymphocytes under
ionizing radiation exposure in vitro. To achieve the purpose of the research, a combination
of classical cytogenetic methods (G0- and G2-radiation sensitivity assays) and method of
single-cell electrophoresis (comet assay) was used. The specicity of the modifying eect
of astaxanthin on radiation-induced genomic injuries depending on the stage of the cell
cycle had been determined. Signicant weakening of the negative eect of ionizing radia-
tion on the G0 stage and the absence of a radioprotective eect on the S and G2 stages of
the cell cycle may be associated with activation by astaxanthin of apoptosis in irradiated
cells with a critically high level of the genome damages. The research results not only tes-
tify about strong radioprotective eect of astaxanthin but also demonstrate the feasibility
of the parallel use of cytogenetic and molecular genetic methods to assess the impact as
mutagens as well as factors that modify the eect of mutagens on genome stability.
Keywords: astaxanthin, lymphocytes, γ-radiation, DNA breaks, chromosomal
aberrations
1. Introduction
The ecological situation that arose from nuclear accidents in Chornobyl and Fukushima, con-
stant expansion of usage of the ionizing radiation in industry and medicine, and the threats
of nuclear terrorism especially aggravated in the last decade are risk factors for the growth of
radiation burden on human populations. The abovementioned conditions require the search
© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

for new safe and eective radioprotectors, preferably of natural origin, for prevention and
treatment of radiation-induced damages in humans, especially which cause genome altera-
tions and cancer. For that purpose, carotenoids, due to its chemical and biological properties,
are the most promising substances [1].
Astaxanthin is a carotenoid of xanthophyll group, and it is one of the most common red
pigments of algae, yeasts, krill, shrimps, craysh, trout, and salmon [2]. It is known that
astaxanthin is the most powerful antioxidant, which has the ability to scavenge free radicals
in tens of times higher than α-tocopherol or β-carotene [3], and has anti-inammatory [2],
immunomodulating [4], and anticarcinogenic [5–7] eects.
Since 2015, we have started the investigation of the radioprotective eects of astaxanthin
studying parameters of genome damages in human somatic cells. In this chapter, we have
concentrated on physicochemical properties of astaxanthin and its biological eects with
the main focus on the data from our investigations concerning the impact of astaxanthin on
radiation-induced genome damages in human somatic cells and have discussed eventual
mechanisms of its action.
2. Physicochemical properties and peculiarities of biological action
of astaxanthin
Astaxanthin is a secondary carotenoid, which belongs to the group of xanthophylls and has
two additional oxygen atoms on each benzene ring in comparison with β-carotene. This gives
astaxanthin a rich red color and greatly increases its antioxidant properties. Unlike β-carotene,
astaxanthin is not a vitamin A precursor [8].
Empirical formula: C40H52O4
Molar weight: 596.84 g/M
In contrast to primary carotenoids, which are associated with the structural and functional
components of the photosynthetic apparatus, secondary carotenoids, which include astax-
anthin, are in the cell in oil droplets, and their main function is to form a protective layer to
prevent the damages, which are provoked by stress conditions [9, 10].
Progress in Carotenoid Research122

Because the astaxanthin molecule contains conjugated double bonds, hydroxyl and keto
groups, it has both lipophilic and hydrophilic properties [11]. Astaxanthin has two chiral cen-
ters and can exist in three dierent stereoisomers—3S, 3′S; 3R, 3′S; and 3R, 3′R. The probability
of obtaining these isomers of astaxanthin in the process of chemical synthesis is 1:2:1 [12, 13].
Nowadays natural astaxanthin mainly derived from microalgae (hyperproducer Haema-
tococcus pluvialis), yeast (Phaa rhodozyma) and animal-consumers included a number of
small marine crustaceans (Euphausiacea) and the salmon family (Salmonidae) [2]. Microalgae
Haematococcus pluvialis produces astaxanthin mainly 3S, 3′S stereoisomeric form; precisely,
such molecular structure is considered the most valuable [14].
As shown in experiments in vitro, astaxanthin eectively protects cells from nonspecic oxida-
tion by quenching singlet oxygen, eectively inhibits lipid peroxidation in biological samples,
and owing to the capture of free radical prevents or stops the chain reaction of oxidation [2,
15, 16]. In addition to direct protective eect, astaxanthin inhibits the activation of the H2O2-
mediated transcription of the factor NF-kB (the nuclear factor “kappa-b”—a universal tran-
scription factor ) that controls the expression of heme oxygenase 1 (HMOX1), one of the markers
of oxidative stress, and nitric oxide synthase (iNOS) [17, 18]. Astaxanthin blocks the cytokine
production declined by modulating the expression of protein tyrosine phosphatase 1 [18].
Experiments on the determination of astaxanthin toxicity showed a high level of safety—LD50
was not established after single administration of substance to rats. The studies conrmed the
absence of histopathological changes and the dose-eect dependence upon oral administra-
tion of astaxanthin in doses ranging from 4.161–17.076 to 465.0–557.0 mg/kg per day [19].
The accumulated published data have shown the multifaceted positive eect of astaxanthin
in mammals by reducing the manifestations of oxidative stress, including during inamma-
tion processes; it can prevent the development of atherosclerotic cardiovascular diseases and
participate in the regulation of lipid and glucose metabolism [19–23].
These properties of astaxanthin primarily aributed to its ability to exhibit activity both at the
level of the cell membrane and in the area of the cytoplasm, thus aecting the ow of intracel-
lular processes [2]. Due to these unique properties, astaxanthin exhibits signicantly higher
biological activity in comparison with other antioxidants [24].
Thus, the above data indicate that astaxanthin complies with all the requirements that apply
to radioprotectors (low toxicity, high antiradical and antioxidant activity, the ability to act
both at the membrane level and in the intracellular space). These properties of astaxanthin
suggest that it may have antimutagenic activity and, as consequence, radioprotective eect
on the human genome.
3. Investigation of radioprotective properties of astaxanthin
Since 2015, we examined the possibility of modication by astaxanthin and the negative
eects of ionizing radiation on the human blood lymphocyte genome in vitro. The decrease
in the intensity of radiation-induced genome damages on the chromosomal and molecular
Astaxanthin as a Modifier of Genome Instability after γ-Radiation
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123

levels was selected as an indicator of radioprotective eect of astaxanthin. The studies were
conducted using a combination of the methods of classical cytogenetic analysis (G0-radiation
sensitivity assay and G2-radiation sensitivity assay) and the method of single-cell electropho-
resis (comet assay) [25–29].
The parallel application of two methodological approaches for such a study greatly expanded
the experimental possibilities. Thus, due to cytogenetic methods, the state of the chromo-
somal apparatus of the cell (frequency of dierent types of chromosome aberrations) is clearly
visualized starting from the 48 h of cultivation. The comet electrophoresis is highly sensitive
and provides the ability to determine the relative levels of single- and double-strand DNA
breaks in individual cell. When conducting cell electrophoresis, the DNA migrates into the
agarose gel, forming a structure that resembles a comet (Figure 1), and the use of the comet
assay can simultaneously estimate the eect of both mutagenic and antimutagenic factors
on the stability of the human somatic cell genome, starting from 0 h of cultivation [30, 31]. In
addition, the use of single-cell electrophoresis makes it possible to determine the eectiveness
of the reparation systems and to assess the correctness of the operation of control mechanisms
at checkpoints between all stages of the cell cycle (G1–S, S–G2, G2–M). Moreover, an important
feature of the comet assay is the identication of cells in which the apoptosis program has
begun or has already been implemented [32–34].
In cells with a lack or a low level of damages, the “tail” is formed also by the release of DNA
loops into the gel. Because in the cell during realization of the apoptotic process genomic
fragmentation of the high level occurs, a massive yield of DNA fragments into agarose gel is
observed (Figure 1), and “comets” have the typical elongated “tail” part.
To quantify the migration of DNA into the agarose gel, two indices are used: the percentage of
DNA in the “tails” and tail moment (TM). TM simultaneously which takes into account both the
Figure 1. Examples of “comets” obtained in the experiment: (А, B, C) The “comets” arisen from cells with a low level of
DNA breaks and (D) “atypical comet” (apoptotic cell) [28].
Progress in Carotenoid Research124

amount of DNA and the length of the “tail” (TM = “tail” length multiplied by the percentage of
DNA in the “tail”) is more informative and calculated automatically during the computer analysis.
3.1. The impact of astaxanthin on the level of radiation-induced chromosomal
aberrations in human lymphocytes
To evaluate the possible mutagenic activity of astaxanthin, it was tested at concentrations of
2.0, 10.0, 20.0, and 40.0 μg/ml in the culture of human peripheral blood lymphocytes. In the
cytogenetic assay, it was found that the frequencies of aberrant cells and the levels of chromo-
somal aberrations under the astaxanthin exposure in vitro in all tested concentrations did not
dier from the corresponding background cytogenetic parameters (p > 0.05) [25].
To determine the optimal working concentration of astaxanthin for further research of its
radiomodifying capacity, a pilot study of its impact on the culture of human peripheral blood
lymphocytes is exposed in vitro to gamma quanta in a dose of 1.0 Gy on G0 phase of the rst
mitotic cycle (Figure 2).
It is established that astaxanthin in all tested concentrations signicantly (p < 0.01) reduced
the frequencies of radiation-induced chromosome aberrations, but the eectiveness of its
modifying action depended on its concentration in the irradiated culture.
The maximum radioprotective eect of astaxanthin (the most eective drop in the frequency
of cytogenetic markers of radiation exposure) was observed after administration of astaxanthin
before irradiation of cultures at concentrations of 20.0 and 40.0 μg/ml (7.69 ± 1.74 and 7.72 ± 1.80
per 100 cells, respectively). These concentrations did not aect the mitotic activity of the lympho-
cyte culture, had no mutagenic eect on non-irradiated cells, and eectively (to ~ 70%) reduced
the level of aberrant metaphases and the frequency of cytogenetic markers of radiation expo-
sure. So long as signicant dierence between the values that characterize carotenoid activity in
these concentrations (p > 0.05) was not observed, for the further studies of the radiomodifying
capacity of astaxanthin, the concentrations of 20.0 μg/ml were chosen.
Figure 2. Selection of the optimal concentration of astaxanthin to study its modifying eect on the γ-irradiated culture
of human blood lymphocytes.
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To analyze the possible dependence of radioprotective properties of astaxanthin from the
stage of the mitotic cycle on which the cells were exposed to ionizing radiation, lymphocyte
cultures were irradiated at 0, 40, and 46 h, corresponding to G0, S, and G2 stages of the rst cell
cycle. Astaxanthin was added to cultures of lymphocytes at least an hour before irradiation.
The obtained data are presented in the Table 1.
After irradiation of lymphocyte culture in a dose of 1.0 Gy on the G0 stage of the cell cycle, the
eect of astaxanthin resulted in a signicant reduction of the radiation-induced cytogenetic
eect, namely, a decrease of almost in 3.5 times both the mean frequency of the aberrant
metaphases and the level of chromosome aberrations—up to 7.82 ± 0.72% and 8.48 ± 0.75 per
100 cells, respectively—and exclusively due to aberrations of chromosome type (Table 1). The
antimutagenic activity of astaxanthin was characterized by signicant (p < 0.001) decrease
in the frequency of classical unstable cytogenetic markers of radiation exposure—dysenteric
and ring chromosomes (up to 2.37 ± 0.41 and 0.43 ± 0.18 per 100 metaphases, respectively),
as well as the total level of simple acentrics—free double fragments, and acentric rings (up to
4.74 ± 0.62 per 100 metaphases) (Table 1, Figure 3).
In contrast to the modifying activity shown by astaxanthin in lymphocyte cultures irra-
diated on the G0 phase of the cell cycle, the addition of carotenoid on the G2 phase did
not change as the total average frequency of radiation-induced chromosomal damages
(72.35 ± 1.17 and 71.54 ± 1.34 per 100 metaphases, respectively, p > 0.05) as the spectrum of
chromosome aberrations (Figure 4). Among chromosomal damages, dominated aberrations
of chromatid type represented by single fragments and chromatid exchanges with the total
average frequency 58.42 ± 1.47 per 100 metaphases did not dier from such (58.32 ± 1.34
per 100 metaphases) in exposed cultures without adding astaxanthin. Aberrations of the
chromosome type were mainly represented by free double fragments; the average group
frequencies of it did not dier between themselves (13.12 ± 1.00 and 14.03 ± 0.91 per 100
metaphases, respectively).
In much the same way, astaxanthin did not exhibit modifying eect on radiation-induced
cytogenetic eects in lymphocyte cultures irradiated on the S stage of the cell cycle. The total
mean group frequencies of radiation-induced chromosomal damages were 19.57 ± 1.11 and
18.46 ± 1.15 per 100 metaphases in exposed cultures without and with the previous addition
of astaxanthin, respectively. Among the chromosomal damages, simple aberrations prevailed
(single and double fragments) in both variants of the experiment (Table 1, Figure 4).
Thus, due to the use of cytogenetic methods, the following important aspects of the astaxan-
thin modifying action were established:
1. The eectiveness of astaxanthin has a dependence on the stage of the cell cycle on which
lymphocytes were irradiated.
2. The radioprotective eect of astaxanthin is realized in cells exposed only on G0 stage of the
mitotic cycle which manifests in lowering the frequency of chromosome-type aberrations
for the induction of which a large number of double-stranded DNA breaks as the error of
repairing of such damages are needed, which permit to suggest the impact of carotenoid
on cells with the high level of genomic instability.
Progress in Carotenoid Research126

Frequency of
the aberrant
Metaphases
Chromosome
aberrations
(per 100 cell)
Frequency of chromosome aberrations
Chromatid type Chromosome type
Single
fragments
Chromatid
exchanges
Total Double
fragments
Dysenteric Centric
rings
Abnormal
monocentric
Acentric
rings
Total
Unirradiated
culture
2.52 ± 0.34 2.57 ± 0.35 1.60 ± 0.28 0.00 1.60 ± 0.28 0.96 ± 0.21 0.00 0.00 0.01 ± 0.01 0.00 0.97 ± 0.22
G0
(1.0 Gy)
22.93 ± 1.19 24.55 ± 1.22 1.54 ± 0.35 0.00 1.54 ± 0.35 6.47 ± 0.70 12.80 ± 0.95 2.76 ± 0.47 0.49 ± 0.20 0.49 ± 0.20 23.02 ± 1.20
G0
(1.0 Gy + А)
7.82 ± 0.72 8.48 ± 0.75 0.72 ± 0.23 0.00 0.72 ± 0.23 4.67 ± 0.57 2.37 ± 0.41 0.43 ± 0.18 0.22 ± 0.13 0.07 ± 0.07 7.76 ± 0.71
S
(1.0 Gy)
18.30 ± 0.97 19.57 ± 1.11 7.94 ± 0.67 2.27 ± 0.33 10.21 ± 0.37 9.36 ± 0.67 0.00 0.00 0.00 0.00 9.36 ± 0.67
S
(1.0 Gy + А)
16.92 ± 1.12 18.46 ± 1.15 6.15 ± 0.67 2.30 ± 0.33 8.45 ± 0.67 10.01 ± 1.0 0.00 0.00 0.00 0.00 10.01 ± 1.0
G2 (1.0 Gy) 47.06 ± 1.31 72.35 ± 1.17 56.04 ± 1.30 2.28 ± 0.39 58.32 ± 1.29 13.76 ± 0.90 0.27 ± 0.51 0.00 0.00 0.00 14.03 ± 0.91
G2
(1.0 Gy + А)
46.72 ± 1.48 71.54 ± 1.34 56.47 ± 1.47 1.95 ± 0.41 58.42 ± 1.47 12.94 ± 1.0 0.18 ± 0.41 0.00 0.00 0.00 13.12 ± 1.0
Table 1. Comparison of the mean group values of cytogenetic parameters in irradiated in vitro in dose 1.0 Gy human lymphocyte cultures on G0, S, and G2 stages of the
cell cycle and under the joint action of γ-radiation and astaxanthin in concentration 20.0 μg/ml.
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3.2. The impact of astaxanthin on the level of DNA damages in human lymphocytes
For evaluation of the relative level of DNA damages (single- and double-strand DNA breaks),
the method of single-cell gel electrophoresis (comet assay) in neutral condition was used. As a
parameter of DNA breakage, the TM computed as the %DNA in the comet tail multiplied by
the tail length was chosen. For comet assay, we used peripheral blood lymphocytes without
Figure 4. Frequencies and spectra of radiation-induced chromosome aberrations under γ-radiation exposure in dose
of 1.0 Gy in vitro and astaxanthin in concentration of 20.0 μg/ml on dierent stages of the cell cycle. G0, S, and G2 (cell
cultures irradiated without astaxanthin) and G0 (A), S(A), and G2(A) (cell cultures irradiated with supplemented 20.0 μg/
ml astaxanthin).
Figure 3. Change in the frequency and spectrum of chromosome-type aberrations under joint action of astaxanthin in the
concentration of 20.0 μg/ml and γ-radiation in a dose of 1.0 Gy on the G0 stage of the cell cycle.
Progress in Carotenoid Research128

culturing (0 h) and from 48 human-PBL cultures. Some cultures were exposed to γ-ray (emit-
ter IBL-237C, dose rate 2.34 Gy/min) in dose 1.0 Gy at 0, 40, and 46 h of cultivation. Non-
irradiated cultures were used as experimental control. Those times were chosen by the reason
that lymphocytes, which we can see after 48 h of cultivation on their metaphase stage, are at
G0 (0 h), S (40 h), and G2 (46 h) phases of the cell cycle. Astaxanthin in the nal concentration
20.0 μg/ml, which was dened during our cytogenetic study, was added to the cultures of
lymphocytes before irradiation.
Similarly to our cytogenetic data, no signicant changes in DNA breakage were detected in
non-irradiated samples supplemented with astaxanthin compared with untreated lympho-
cytes both after 0 and 48 h of cultivation (Table 2). This conrms our suggestion that astaxan-
thin in chosen concentration has no mutagenic activity.
As can be seen from the Table 2 and Figure 6, after γ-irradiation of lymphocytes in dose
1.0 Gy at G0 phase of the cell cycle, signicant increasing in TM was detected (from 2.80 ± 0.54
to 6.55 ± 1.82, p < 0.05 and from 4.07 ± 0.60 to 12.86 ± 0.74, p < 0.05, after 0 and 48 h of cultiva-
tion, respectively).
The eect of astaxanthin on irradiated cells manifested in signicant (р < 0.001) decrease in the
average level of DNA damages in lymphocytes from cultures irradiated at G0 nearly to the value
of non-irradiated control both after 0 and 48 h of cultivation (TM = 3.74 ± 0.82 and 5.27 ± 1.77,
respectively) (Figure 5).
As expected, signicant increase in the level of DNA breaks was detected in lymphocytes after
γ-irradiation at 40 h of cultivation (Table 3). The mean value of TM was equal to 7.45 ± 0.36 in
irradiated and 4.07 ± 0.60 in lymphocytes from intact cultures (p < 0.01). Astaxanthin in con-
centration 20 μg/ml signicantly (p < 0.01) decreased the DNA damages in lymphocytes from
cultures irradiated at 0 and 40 h of incubation nearly to the level of non-irradiated control
(TM = 5.27 ± 1.77 and 4.79 ± 0.23, respectively). The treatment of cells with astaxanthin resulted
in statistical signicant decrease of radiation-induced DNA damages (TM = 3.21 ± 0.48, p < 0.05
compared with irradiated samples) likewise after irradiation of lymphocytes at G0 phase of the
cell cycle.
Similar results were obtained after treatment at 46 h of incubation (Table 4). Irradiation of
lymphocyte cultures at G2 phase of the cell cycle led to a large amount of DNA breaks and, as
outcome, to material increase in ТМ value (12.06 ± 1.88, p < 0.001). The eect of astaxanthin
Treatment 0 h 48 h
Tail moment (X ± Se) Tail moment (X ± Se)
Control 2.80 ± 0.54 4.07 ± 0.60
Supplementation with astaxanthin 3.55 ± 1.37 5.93 ± 0.93
Irradiation 6.55 ± 1.82 12.86 ± 0.74
Table 2. The impact of γ-irradiation at G0 phase and astaxanthin supplementation on DNA damages in human lym-
phocytes after 0 and 48 h of cultivation.
Astaxanthin as a Modifier of Genome Instability after γ-Radiation
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supplementation was somewhat not only weaker than in previous experiments but also
signicant compared to cultures irradiated on G2 stage (TM = 8.96 ± 2.39 and 12.06 ± 1.88,
respectively, p < 0.05).
Treatment Tail moment
X ±Se
Control 4.07 0.60
Control + astaxanthin 5.93 0.93
Irradiation at S phase 7.45 0.36
Irradiation at S phase + astaxanthin 4.79 0.23
Notes: X, mean value; Se, standard error.
Table 3. The tail moment values in human blood lymphocytes after γ-radiation exposure and under combined action of
radiation and astaxanthin at S phase of the cell cycle.
Treatment Tail moment
X ±Se
Control 4.07 0.60
Control + astaxanthin 5.93 0.93
Irradiation at G2 phase 12.06 1.88
Irradiation at G2 phase + astaxanthin 8.96 2.39
Notes: X, mean value; Se, standard error.
Table 4. The tail moment values in human blood lymphocytes after γ-radiation exposure and under combined action of
radiation and astaxanthin at G2 phase of the cell cycle.
Figure 5. The relative levels of DNA damages irradiated at G0-phase human lymphocytes non-supplemented or
supplemented with astaxanthin cultures.
Progress in Carotenoid Research130

Our data suggested that astaxanthin decreased the rate of radiation-induced DNA breaks
in human lymphocytes regardless of the phase of the cell cycle when the irradiation was
performed. However, this conclusion is not consistent with our cytogenetic results: it was
observed that astaxanthin is able to decrease frequency of radiation-induced chromosome
aberration only if cells were irradiated at G0 phase of the cell cycle.
For more detailed analysis, we have studied the frequency distribution of individual cells
depending on their levels of DNA damages. According to TM, the sampling of “comets” from
control variants was divided into ten groups of 10% each. The established values of deciles
(TMs were 0.81, 1.28, 1.81, 2.69, 3.80, 5.07, 6.48, 10.19, 15.98) were chosen as boundary indices
to form ten groups of cells from irradiated cultures treated or not by astaxanthin and to esti-
mate percentage of “comets” that have TM within the appropriate range. If the value of TM
was equal to the boundary index, then “comet” was referred to the next group. The results
are shown on Figure 6.
When lymphocytes were irradiated at the G0 phase of the cell cycle, after 48 h of incubation,
the increase in the average TM level was caused exclusively by growth of the frequency of
the “comets” from the tenth group (TM > 15.98) (Figure 6A), which indicates accumulation
of cells with a large number of DNA damages with time. Irradiation at the 40th hours of
cultivation (Figure 6B) resulted in increased levels of the “comets” that belonged to groups 8
and 9 (TM from 6.48 to 15.98). After radiation exposure at 46 h of incubation (Figure 6C), the
increment of last three groups of “comets” (TM > 6.48) was observed.
It is noteworthy that γ-radiation exposure at 40 and 46 h of incubation did not cause decrease
in the frequency of group 1, which includes the “comets” with the smallest DNA release into
the “comet” tail (TM from 0 to 0.81). Probably, this situation reects not so much on the exis-
tence in lymphocyte cultures of the populations of radiation-resistant and/or fully recovered
cells, as the presence of heavily damaged cells in which the checkpoint has acted on the S
phase of the cell cycle, because if the cells are in this phase, then signicant decrease of DNA
exit under the neutral conditions of electrophoresis is observed [35, 36]. This opinion is con-
rmed by the lack of increase in frequency of “comets” from the tenth group after radiation
exposure at 40 h of cultivation: most of blast-transformed lymphocytes must be on S phase,
and cells with the very high level of DNA damages cannot pass S/G2 checkpoint, and, as a
result, they are delayed on this phase.
The supplementation with astaxanthin resulted in signicant reduction in the levels of “com-
ets” that belonged only to the ten groups after irradiation at 0 (from 25.07 ± 2.25 to 8.96 ± 1.74%,
p < 0.001) and at 46 h of cultivation (from 22.38 ± 1.77 to 10.45 ± 1.18%, p < 0.001) and groups 9
and 10 after radiation exposure at 40 h of incubation (from 16.56 ± 1.72 to 6.69 ± 1.06, p < 0.001
and from 8.60 ± 1.30 to 3.25 ± 0.75%, p < 0.01, respectively).
It is known that astaxanthin reveals apoptotic activity in experiments with dierent cultures
of cancer cells [6, 33]. In our studies, the decrease in the frequency of highly damaged cells
as a result of astaxanthin treatment may also be caused by activation of apoptotic processes.
The comet assay allowed not only estimating the relative level of DNA damages but also
determining the intensity of apoptotic processes [37, 38]. For this purpose simultaneously
Astaxanthin as a Modifier of Genome Instability after γ-Radiation
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131

Figure 6. The frequency distribution of “comets” according to the relative levels of DNA damages (see explanation in
the text) after irradiation at the 0th hour of cultivation (A), at the 40th hour of cultivation (B), and at the 46th hours of
cultivation (C). Irr, irradiated in dose 1.0 Gy cultures; Irr + A, irradiated and supplemented with 20.0 μg/ml astaxanthin
cultures. In one to ten groups of “comets,” 10% (bold line) is the control value for all groups [28].
Progress in Carotenoid Research132

with the evaluation of the levels of DNA damages, the count of “atypical comets” (AC)
(Figure 7) was carried out. It is obvious that AC were formed from the apoptotic cells, because
the radiation dose we applied is quite low and cannot induce DNA fragmentation like this
[39], while intensive DNA fragmentation occurs exactly during apoptosis [33].
In control cultures after 48 h of cultivation, the AC level was low and did not exceed
1.45 ± 0.53%. The irradiation of lymphocyte cultures at the G0 stage of the cell cycle with
further cultivation led to an increase in the frequency of AC from to 3.11 ± 0.71% (p < 0.05),
but such eect was not observed after radiation exposure neither at the 40th hour nor at the
46th hours of cultivation (Figure 7).
Astaxanthin in concentration of 20 μg/ml per se did not aect the amount of apoptotic cells
in non-irradiated cultures of lymphocytes, but the AC level irradiated and treated by astax-
anthin cultures was approximately in four times higher than with 48-hour control (7.15 ± 1.13
and 1.69 ± 0.56%, respectively, p < 0.01) and in two times higher than with irradiated samples
(7.15 ± 1.13 and 3.57 ± 0.81%, respectively, p < 0.05). The increase in the frequency of apoptotic
cells under the impact of astaxanthin was established exclusively in cultures irradiated at the
G0 stage of the cell cycle and not observed after irradiation in other terms of cultivation.
Thus, similar to cytogenetic eect, the apoptotic activity of astaxanthin was detected only
when the irradiated cells were on the G0 phase of the cell cycle. This may be the cause of the
elimination of cells with a large number of DNA breaks and, as consequence, the reduction of
the radiation-induced level of chromosomal aberrations we observed earlier.
Figure 7. The levels of “atypical comets” (AC%) in cultures of human lymphocytes after 48 hours of cultivation
depending on the irradiation terms and the addition of astaxanthin. C, control cultures; A, supplemented with 20.0 μg/
ml astaxanthin cultures; 0, 40, and 46 h, cultures irradiated at the 0th, at the 40th, and at the 46th hours of cultivation,
respectively; 0 h + A, 40 h + A, and 46 h + A, supplemented with 20.0 μg/ml astaxanthin cultures irradiated at the 0th, at
the 40th, and at the 46th hours of cultivation, respectively.
Astaxanthin as a Modifier of Genome Instability after γ-Radiation
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133

The absence of increase in the frequency of apoptosis after treatment in other terms of cul-
tivation both under the inuence of only ionizing radiation and under the combined action
of γ-radiation and astaxanthin can be explained by either insucient time for realization of
apoptosis or existence of contingent on the stage of the cell cycle apoptotic pathways, which
astaxanthin is unable to activate on the S and G2 phases of the cell cycle.
Since the increase in the level of apoptosis is not a reason for the decline of the pool of high
damaged cells under the astaxanthin inuence after irradiation at 40 and 46 h of cultivation,
the question remains: what is the cause of such eect of astaxanthin?
It is generally accepted that reactive oxygen species, which are formed by ionizing radiation
exposure, cause DNA breaks [40]. Astaxanthin is a power antioxidant and capable to scav-
enge and quench free radicals and ipso facto to reduce the overall level of DNA damages [3].
According to the data presented in Figure 7, the results obtained after irradiation of cells at
46 h of cultivation can be explained by the antioxidant properties of astaxanthin: reducing of
oxidative stress leads to a decrease in the number of “comets” of the ten groups and increas-
ing (although not always statistically signicant) in the frequencies of cells belonging to other
groups (except 3 and 9).
However, such impact of astaxanthin was not observed when cells were irradiated at 40 h of
cultivation. It is noteworthy that in this experiment the increase in the frequency of the “com-
ets” of group 1 (from 11.18 ± 1.46 to 17.54 ± 1.62%, p < 0.01) was detected. It can be explained by
the fact that this group may include cells having a suciently large number of lesions enough
to trigger mechanisms for the cell cycle arrest on the S phase. Probably, astaxanthin activates
S-/G2-phase checkpoint that leads to an increase in the frequency of the cells from which the
“comets” with low DNA are formed (by delay in S phase) and may cause decreasing in the
frequency of the “comets” of groups 9 and 10. The results are consistent with the literature
data concerning the eects of astaxanthin on the proliferation of tumor cells [7, 17, 41].
4. Conclusion
The obtained results enable us to resume the following astaxanthin eects on irradiated cells
that may be clearly observed depending on the phase of the cell cycle and the duration of cells
cultivation after irradiation:
1. Stimulation of apoptosis in the irradiated cells resulting in a decrease in the level of cells
with a large number of DNA damages (irradiation on the G0 phase of the cell cycle and
cultivation after irradiation for 48 h)
2. Stimulation of the processes that lead to the activation of the checkpoints on the S phase
and, accordingly, arrest the division of the most damaged cell population (irradiation on
the S phase of the cell cycle and cultivation after irradiation for 8 h)
3. Scavenge of reactive oxygen species resulting in reduction in the total level of DNA breaks
(irradiation on the G2 phase of the cell cycle and cultivation after irradiation for 2 h)
Progress in Carotenoid Research134

All of these eects are potentially radio- and genoprotective. However, we have previously
shown that the protective action of astaxanthin concerning the radiation-induced cytoge-
netic eect similarly to its apoptotic eect was observed exclusively when irradiated cells
were on the G0 phase of the cell cycle. Moreover, analyzing the ChA spectra (Figure 3), we
found that supplementation with astaxanthin reduces exactly the levels of classic unstable
cytogenetic markers of radiation exposure (dicentric and centric ring chromosomes), and it
is known that the cells bearing unstable chromosomal aberrations are eliminated by apop-
tosis in the rst place [42]. So, the radioprotective eect of astaxanthin rather may be due to
its ability to stimulate apoptosis in cells that carry a subcritical number of DNA breaks than
its potential genoprotective properties (defenses DNA from damages or activates of DNA
repair processes).
Author details
Denys Кurinnyi1*, Stanislav Rushkovsky2, Olena Demchenko1 and Mariya Pilinska1
*Address all correspondence to: kurinnyi.d@gmail.com
1 State Institution “National Research Center for Radiation Medicine of the National
Academy of Medical Sciences of Ukraine”, Kyiv, Ukraine
2 Educational and Research Center “Institute of Biology and Medicine” Taras Shevchenko
National University of Kyiv, Kyiv, Ukraine
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