**3.1. The impact of astaxanthin on the level of radiation-induced chromosomal aberrations in human lymphocytes**

levels was selected as an indicator of radioprotective effect of astaxanthin. The studies were

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 chromosomal apparatus of the cell (frequency of different 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 effect 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 effectiveness of the reparation systems and to assess the correctness of the operation of control mechanisms

feature of the comet assay is the identification of cells in which the apoptosis program has

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

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

observed (**Figure 1**), and "comets" have the typical elongated "tail" part.


–S, S–G<sup>2</sup>

, G<sup>2</sup>

–M). Moreover, an important


conducted using a combination of the methods of classical cytogenetic analysis (G<sup>0</sup>

sensitivity assay and G<sup>2</sup>

124 Progress in Carotenoid Research

resis (comet assay) [25–29].

at checkpoints between all stages of the cell cycle (G<sup>1</sup>

begun or has already been implemented [32–34].

DNA breaks and (D) "atypical comet" (apoptotic cell) [28].

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 chromosomal aberrations under the astaxanthin exposure in vitro in all tested concentrations did not differ 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 G<sup>0</sup> phase of the first mitotic cycle (**Figure 2**).

It is established that astaxanthin in all tested concentrations significantly (p < 0.01) reduced the frequencies of radiation-induced chromosome aberrations, but the effectiveness of its modifying action depended on its concentration in the irradiated culture.

The maximum radioprotective effect of astaxanthin (the most effective 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 affect the mitotic activity of the lymphocyte culture, had no mutagenic effect on non-irradiated cells, and effectively (to ~ 70%) reduced the level of aberrant metaphases and the frequency of cytogenetic markers of radiation exposure. So long as significant difference 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 effect on the γ-irradiated culture of human blood lymphocytes.

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 G<sup>0</sup> , S, and G<sup>2</sup> stages of the first 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 G<sup>0</sup> stage of the cell cycle, the effect of astaxanthin resulted in a significant reduction of the radiation-induced cytogenetic effect, 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 significant (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 irradiated on the G<sup>0</sup> phase of the cell cycle, the addition of carotenoid on the G<sup>2</sup> 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 differ 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 differ 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 effect on radiation-induced cytogenetic effects 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 astaxanthin modifying action were established:

**1.** The effectiveness of astaxanthin has a dependence on the stage of the cell cycle on which lymphocytes were irradiated.

**Frequency of** 

**Chromosome** 

**Frequency of chromosome aberrations**

**aberrations** 

**Chromatid type**

**Single** 

**Chromatid** 

**Total**

**Double** 

**Dysenteric**

**Centric** 

**Abnormal** 

**Acentric** 

**Total**

**monocentric**

**rings**

**rings**

**fragments**

**fragments**

**exchanges**

**Chromosome type**

**(per 100 cell)**

**the aberrant** 

**Metaphases**

Unirradiated

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 0.49 ± 0.20

23.02 ± 1.20

0.97 ± 0.22

culture

G<sup>0</sup>

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

> (1.0 Gy)

G<sup>0</sup>

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

> (1.0 Gy + А)

S

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

> (1.0 Gy)

S

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

> (1.0 Gy + А)

G2 (1.0 Gy)

G<sup>2</sup>

46.72 ± 1.48

71.54 ± 1.34

56.47 ± 1.47 1.95 ± 0.41

> (1.0 Gy + А)

**Table 1.**

Comparison of the mean group values of cytogenetic parameters in irradiated in

cell cycle and under the joint action of γ-radiation and astaxanthin in concentration 20.0

 μg/ml.

vitro in dose 1.0

Gy human lymphocyte cultures on G0, S, and G2 stages of the

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127

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

58.42 ± 1.47 12.94 ± 1.0

0.18 ± 0.41

0.00

0.00

0.00

13.12 ± 1.0

0.00

0.00

0.00

14.03 ± 0.91

**2.** The radioprotective effect of astaxanthin is realized in cells exposed only on G<sup>0</sup> 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.


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

cycle. Astaxanthin was added to cultures of lymphocytes at least an hour before irradiation.

effect of astaxanthin resulted in a significant reduction of the radiation-induced cytogenetic effect, namely, a decrease of almost in 3.5 times both the mean frequency of the aberrant

in the frequency of classical unstable cytogenetic markers of radiation exposure—dysenteric

as well as the total level of simple acentrics—free double fragments, and acentric rings (up to

In contrast to the modifying activity shown by astaxanthin in lymphocyte cultures irra

not change as the total average frequency of radiation-induced chromosomal damages

of chromatid type represented by single fragments and chromatid exchanges with the total

per 100 metaphases) in exposed cultures without adding astaxanthin. Aberrations of the chromosome type were mainly represented by free double fragments; the average group

In much the same way, astaxanthin did not exhibit modifying effect on radiation-induced cytogenetic effects in lymphocyte cultures irradiated on the S stage of the cell cycle. The total

Thus, due to the use of cytogenetic methods, the following important aspects of the astaxan

**1.** The effectiveness of astaxanthin has a dependence on the stage of the cell cycle on which

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

± 1.15 per 100 metaphases in exposed cultures without and with the previous addition of astaxanthin, respectively. Among the chromosomal damages, simple aberrations prevailed

mean group frequencies of radiation-induced chromosomal damages were 19.57

**2.** The radioprotective effect of astaxanthin is realized in cells exposed only on G

(single and double fragments) in both variants of the experiment (**Table**

± 1.34 per 100 metaphases, respectively, p

**3**).

phase of the cell cycle, the addition of carotenoid on the G

100 cells, respectively—and exclusively due to aberrations of chromosome type (**Table**

± 0.41 and 0.43

**1**, **Figure**

**1** . 0

, S, and G

0

**4**). Among chromosomal damages, dominated aberrations

± 1.00 and 14.03

± 1.47 per 100 metaphases did not differ from such (58.32

2

± 0.72% and 8.48

± 0.18 per 100 metaphases, respectively),

stages of the first cell

± 0.75 per

< 0.001) decrease

2

> 0.05) as the spectrum of

**1**, **Figure**

**4**).

0

**1**). The


phase did

± 1.34

± 0.91 per 100

± 1.11 and

stage of the


stage of the cell cycle, the

cultures were irradiated at 0, 40, and 46 h, corresponding to G

After irradiation of lymphocyte culture in a dose of 1.0 Gy on the G

metaphases and the level of chromosome aberrations—up to 7.82

frequencies of it did not differ between themselves (13.12

antimutagenic activity of astaxanthin was characterized by significant (p

The obtained data are presented in the **Table**

and ring chromosomes (up to 2.37

0

chromosome aberrations (**Figure**

± 1.17 and 71.54

average frequency 58.42

metaphases, respectively).

thin modifying action were established:

on cells with the high level of genomic instability.

lymphocytes were irradiated.

± 0.62 per 100 metaphases) (**Table**

4.74

(72.35

18.46

diated on the G

126 Progress in Carotenoid Research

**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.

culturing (0 h) and from 48 human-PBL cultures. Some cultures were exposed to γ-ray (emitter IBL-237C, dose rate 2.34 Gy/min) in dose 1.0 Gy at 0, 40, and 46 h of cultivation. Nonirradiated 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

20.0 μg/ml, which was defined during our cytogenetic study, was added to the cultures of

Similarly to our cytogenetic data, no significant changes in DNA breakage were detected in non-irradiated samples supplemented with astaxanthin compared with untreated lymphocytes both after 0 and 48 h of cultivation (**Table 2**). This confirms our suggestion that astaxan-

As can be seen from the **Table 2** and **Figure 6**, after γ-irradiation of lymphocytes in dose

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-

The effect of astaxanthin on irradiated cells manifested in significant (р < 0.001) decrease in the

of non-irradiated control both after 0 and 48 h of cultivation (TM = 3.74 ± 0.82 and 5.27 ± 1.77,

As expected, significant 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 concentration 20 μg/ml significantly (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 significant decrease of radiation-induced DNA damages (TM = 3.21 ± 0.48, p < 0.05

Similar results were obtained after treatment at 46 h of incubation (**Table 4**). Irradiation of

outcome, to material increase in ТМ value (12.06 ± 1.88, p *<* 0.001). The effect of astaxanthin

phase of the cell cycle led to a large amount of DNA breaks and, as

**Tail moment (X ± Se) Tail moment (X ± Se)**

phase and astaxanthin supplementation on DNA damages in human lym-

average level of DNA damages in lymphocytes from cultures irradiated at G<sup>0</sup>

compared with irradiated samples) likewise after irradiation of lymphocytes at G<sup>0</sup>

**Treatment 0 h 48 h**

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

phase of the cell cycle, significant increasing in TM was detected (from 2.80 ± 0.54

(46 h) phases of the cell cycle. Astaxanthin in the final concentration

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129

nearly to the value

phase of the

G0

1.0 Gy at G<sup>0</sup>

cell cycle.

tion, respectively).

respectively) (**Figure 5**).

lymphocyte cultures at G<sup>2</sup>

**Table 2.** The impact of γ-irradiation at G<sup>0</sup>

phocytes after 0 and 48 h of cultivation.

(0 h), S (40 h), and G<sup>2</sup>

lymphocytes before irradiation.

thin in chosen concentration has no mutagenic activity.

**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 G<sup>0</sup> stage of the cell cycle.

**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 different stages of the cell cycle. G<sup>0</sup> , S, and G<sup>2</sup> (cell cultures irradiated without astaxanthin) and G<sup>0</sup> (A), S(A), and G<sup>2</sup> (A) (cell cultures irradiated with supplemented 20.0 μg/ ml astaxanthin).

#### **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 culturing (0 h) and from 48 human-PBL cultures. Some cultures were exposed to γ-ray (emitter IBL-237C, dose rate 2.34 Gy/min) in dose 1.0 Gy at 0, 40, and 46 h of cultivation. Nonirradiated 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 G<sup>2</sup> (46 h) phases of the cell cycle. Astaxanthin in the final concentration 20.0 μg/ml, which was defined during our cytogenetic study, was added to the cultures of lymphocytes before irradiation.

Similarly to our cytogenetic data, no significant changes in DNA breakage were detected in non-irradiated samples supplemented with astaxanthin compared with untreated lymphocytes both after 0 and 48 h of cultivation (**Table 2**). This confirms our suggestion that astaxanthin 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 G<sup>0</sup> phase of the cell cycle, significant 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 cultivation, respectively).

The effect of astaxanthin on irradiated cells manifested in significant (р < 0.001) decrease in the average level of DNA damages in lymphocytes from cultures irradiated at G<sup>0</sup> 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, significant 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 concentration 20 μg/ml significantly (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 significant decrease of radiation-induced DNA damages (TM = 3.21 ± 0.48, p < 0.05 compared with irradiated samples) likewise after irradiation of lymphocytes at G<sup>0</sup> phase of the cell cycle.

Similar results were obtained after treatment at 46 h of incubation (**Table 4**). Irradiation of lymphocyte cultures at G<sup>2</sup> 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 effect of astaxanthin


**3.2. The impact of astaxanthin on the level of DNA damages in human lymphocytes**

(A), S(A), and G<sup>2</sup>

of 1.0 Gy in vitro and astaxanthin in concentration of 20.0 μg/ml on different stages of the cell cycle. G<sup>0</sup>

cultures irradiated without astaxanthin) and G<sup>0</sup>

ml astaxanthin).

128 Progress in Carotenoid Research

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

**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 G<sup>0</sup>

, S, and G<sup>2</sup>

(A) (cell cultures irradiated with supplemented 20.0 μg/

stage of the cell cycle.

(cell

**Table 2.** The impact of γ-irradiation at G<sup>0</sup> phase and astaxanthin supplementation on DNA damages in human lymphocytes after 0 and 48 h of cultivation.

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

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 estimate 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

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

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 reflects not so much on the existence 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 significant decrease of DNA exit under the neutral conditions of electrophoresis is observed [35, 36]. This opinion is confirmed 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,

The supplementation with astaxanthin resulted in significant reduction in the levels of "comets" 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

It is known that astaxanthin reveals apoptotic activity in experiments with different 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

increment of last three groups of "comets" (TM > 6.48) was observed.

and cells with the very high level of DNA damages cannot pass S/G<sup>2</sup>

and from 8.60 ± 1.30 to 3.25 ± 0.75%, p *<* 0.01, respectively).

phase of the cell cycle.

phase of the cell cycle, after 48 h of incubation,

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131

checkpoint, and, as a

aberration only if cells were irradiated at G<sup>0</sup>

When lymphocytes were irradiated at the G<sup>0</sup>

result, they are delayed on this phase.

are shown on **Figure 6**.

**Figure 5.** The relative levels of DNA damages irradiated at G<sup>0</sup> -phase human lymphocytes non-supplemented or supplemented with astaxanthin cultures.


**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.


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 G<sup>2</sup> phase of the cell cycle.

supplementation was somewhat not only weaker than in previous experiments but also significant compared to cultures irradiated on G<sup>2</sup> stage (TM = 8.96 ± 2.39 and 12.06 ± 1.88, respectively, p *<* 0.05).

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 G<sup>0</sup> 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 estimate 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 G<sup>0</sup> 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 reflects not so much on the existence 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 significant decrease of DNA exit under the neutral conditions of electrophoresis is observed [35, 36]. This opinion is confirmed 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/G<sup>2</sup> checkpoint, and, as a result, they are delayed on this phase.

The supplementation with astaxanthin resulted in significant reduction in the levels of "comets" 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 different 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

supplementation was somewhat not only weaker than in previous experiments but also

**Table 4.** The tail moment values in human blood lymphocytes after γ-radiation exposure and under combined action of

phase 12.06 1.88

phase + astaxanthin 8.96 2.39

**Table 3.** The tail moment values in human blood lymphocytes after γ-radiation exposure and under combined action of

**Treatment Tail moment**

**Figure 5.** The relative levels of DNA damages irradiated at G<sup>0</sup>

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

**Treatment Tail moment**

Control 4.07 0.60 Control + astaxanthin 5.93 0.93

stage (TM = 8.96 ± 2.39 and 12.06 ± 1.88,

**X ±Se**

**X ±Se**


significant compared to cultures irradiated on G<sup>2</sup>

phase of the cell cycle.

respectively, p *<* 0.05).

radiation and astaxanthin at G<sup>2</sup>

Irradiation at G<sup>2</sup>

Irradiation at G<sup>2</sup>

Notes: X, mean value; Se, standard error.

supplemented with astaxanthin cultures.

130 Progress in Carotenoid Research

Notes: X, mean value; Se, standard error.

radiation and astaxanthin at S phase of the cell cycle.

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

In control cultures after 48 h of cultivation, the AC level was low and did not exceed

further cultivation led to an increase in the frequency of AC from to 3.11 ± 0.71% (p *<* 0.05), but such effect was not observed after radiation exposure neither at the 40th hour nor at the

Astaxanthin in concentration of 20 μg/ml per se did not affect the amount of apoptotic cells in non-irradiated cultures of lymphocytes, but the AC level irradiated and treated by astaxanthin 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

stage of the cell cycle and not observed after irradiation in other terms of cultivation.

Thus, similar to cytogenetic effect, the apoptotic activity of astaxanthin was detected only

elimination of cells with a large number of DNA breaks and, as consequence, the reduction of

**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 radiation-induced level of chromosomal aberrations we observed earlier.

stage of the cell cycle with

phase of the cell cycle. This may be the cause of the

Astaxanthin as a Modifier of Genome Instability after γ-Radiation

http://dx.doi.org/10.5772/intechopen.79341

133

[39], while intensive DNA fragmentation occurs exactly during apoptosis [33].

1.45 ± 0.53%. The irradiation of lymphocyte cultures at the G<sup>0</sup>

46th hours of cultivation (**Figure 7**).

when the irradiated cells were on the G<sup>0</sup>

the 40th, and at the 46th hours of cultivation, respectively.

G0

**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].

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 G<sup>0</sup> 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 effect 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 affect the amount of apoptotic cells in non-irradiated cultures of lymphocytes, but the AC level irradiated and treated by astaxanthin 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 effect, the apoptotic activity of astaxanthin was detected only when the irradiated cells were on the G<sup>0</sup> 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.

**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].

132 Progress in Carotenoid Research

The absence of increase in the frequency of apoptosis after treatment in other terms of cultivation both under the influence of only ionizing radiation and under the combined action of γ-radiation and astaxanthin can be explained by either insufficient 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 G<sup>2</sup> phases of the cell cycle.

All of these effects are potentially radio- and genoprotective. However, we have previously shown that the protective action of astaxanthin concerning the radiation-induced cytogenetic effect similarly to its apoptotic effect was observed exclusively when irradiated cells

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 apoptosis in the first place [42]. So, the radioprotective effect 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

phase of the cell cycle. Moreover, analyzing the ChA spectra (**Figure 3**), we

, Olena Demchenko<sup>1</sup>

1 State Institution "National Research Center for Radiation Medicine of the National

2 Educational and Research Center "Institute of Biology and Medicine" Taras Shevchenko

[1] Azqueta A, Collins AR. Carotenoids and DNA damage. Mutation Research. 2012;**733**:4-13 [2] Ambati RR, Phang SM, Ravi S, Aswathanarayana RG. Astaxanthin: Sources, extraction, stability, biological activities and its commercial applications—A review. Marine Drugs.

[3] Nishida Y, Yamashita E, Miki W. Quenching activities of common hydrophilic and lipophilic antioxidants against singlet oxygen using chemiluminescence detection system.

[4] Lin KH, Lin KC, Lu WJ, Thomas PA, Jayakumar T, Sheu JR. Astaxanthin, a carotenoid, stimulates immune responses by enhancing IFN-γ and IL-2 secretion in primary cultured lymphocytes in vitro and ex vivo. International Journal of Molecular Sciences.

[5] Ohno T, Shimizu M, Shirakami Y, et al. Preventive effects of astaxanthin on diethylnitrosamine-induced liver tumorigenesis in C57/BL/KsJ-db/db obese mice. Hepatology

and Mariya Pilinska<sup>1</sup>

Astaxanthin as a Modifier of Genome Instability after γ-Radiation

http://dx.doi.org/10.5772/intechopen.79341

135

were on the G<sup>0</sup>

repair processes).

**Author details**

Denys Кurinnyi<sup>1</sup>

**References**

2014;**12**:128-152

\*, Stanislav Rushkovsky<sup>2</sup>

\*Address all correspondence to: kurinnyi.d@gmail.com

Academy of Medical Sciences of Ukraine", Kyiv, Ukraine

National University of Kyiv, Kyiv, Ukraine

Carotenoid Science. 2007;**11**:16-20

2015;**17**(1). DOI: 10.3390/ijms17010044

Research. 2016;**46**(3):201-209

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 influence after irradiation at 40 and 46 h of cultivation, the question remains: what is the cause of such effect 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 scavenge 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 increasing (although not always statistically significant) 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 "comets" 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 sufficiently large number of lesions enough to trigger mechanisms for the cell cycle arrest on the S phase. Probably, astaxanthin activates S-/G<sup>2</sup> -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 effects of astaxanthin on the proliferation of tumor cells [7, 17, 41].
