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.
The chapter is devoted to study the effects 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 specificity of the modifying effect of astaxanthin on radiation-induced genomic injuries depending on the stage of the cell cycle had been determined. Significant weakening of the negative effect of ionizing radiation on the G0 stage and the absence of a radioprotective effect 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 testify about strong radioprotective effect 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 effect of mutagens on genome stability.
- DNA breaks
- chromosomal aberrations
The ecological situation that arose from nuclear accidents in Chornobyl and Fukushima, constant 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 for new safe and effective radioprotectors, preferably of natural origin, for prevention and treatment of radiation-induced damages in humans, especially which cause genome alterations and cancer. For that purpose, carotenoids, due to its chemical and biological properties, are the most promising substances .
Astaxanthin is a carotenoid of xanthophyll group, and it is one of the most common red pigments of algae, yeasts, krill, shrimps, crayfish, trout, and salmon . 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 , and has anti-inflammatory , immunomodulating , and anticarcinogenic [5, 6, 7] effects.
Since 2015, we have started the investigation of the radioprotective effects 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 effects 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 .
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 astaxanthin, 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].
Because the astaxanthin molecule contains conjugated double bonds, hydroxyl and keto groups, it has both lipophilic and hydrophilic properties . Astaxanthin has two chiral centers and can exist in three different 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
As shown in experiments
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 confirmed the absence of histopathological changes and the dose-effect dependence upon oral administration of astaxanthin in doses ranging from 4.161–17.076 to 465.0–557.0 mg/kg per day .
The accumulated published data have shown the multifaceted positive effect of astaxanthin in mammals by reducing the manifestations of oxidative stress, including during inflammation processes; it can prevent the development of atherosclerotic cardiovascular diseases and participate in the regulation of lipid and glucose metabolism [19, 20, 21, 22, 23].
These properties of astaxanthin primarily attributed to its ability to exhibit activity both at the level of the cell membrane and in the area of the cytoplasm, thus affecting the flow of intracellular processes . Due to these unique properties, astaxanthin exhibits significantly higher biological activity in comparison with other antioxidants .
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 effect on the human genome.
3. Investigation of radioprotective properties of astaxanthin
Since 2015, we examined the possibility of modification by astaxanthin and the negative effects 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 levels was selected as an indicator of radioprotective effect 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 electrophoresis (comet assay) [25, 26, 27, 28, 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 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 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 identification of cells in which the apoptosis program has begun or has already been implemented [32, 33, 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 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 chromosomal aberrations under the astaxanthin exposure in vitro in all tested concentrations did not differ from the corresponding background cytogenetic parameters (p > 0.05) .
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 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.
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 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.
|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|
|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|
(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|
|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 + 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|
(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|
After irradiation of lymphocyte culture in a dose of 1.0 Gy on the G0 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 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 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:
The effectiveness of astaxanthin has a dependence on the stage of the cell cycle on which lymphocytes were irradiated.
The radioprotective effect 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.
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. 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 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.
|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|
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, significant increasing in TM was detected (from 2.80 ± 0.54 to 6.55 ± 1.82, p
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 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, 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
|Control + astaxanthin||5.93||0.93|
|Irradiation at S phase||7.45||0.36|
|Irradiation at S phase + astaxanthin||4.79||0.23|
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
|Control + astaxanthin||5.93||0.93|
|Irradiation at G2 phase||12.06||1.88|
|Irradiation at G2 phase + astaxanthin||8.96||2.39|
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 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 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 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/G2 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
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 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 , while intensive DNA fragmentation occurs exactly during apoptosis .
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
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 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.
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 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 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 . Astaxanthin is a power antioxidant and capable to scavenge and quench free radicals and ipso facto to reduce the overall level of DNA damages . 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-/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 effects of astaxanthin on the proliferation of tumor cells [7, 17, 41].
The obtained results enable us to resume the following astaxanthin effects on irradiated cells that may be clearly observed depending on the phase of the cell cycle and the duration of cells cultivation after irradiation:
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)
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)
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)
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 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 apoptosis in the first place . 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 repair processes).
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