**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 (G<sup>0</sup> -radiation sensitivity assay and G<sup>2</sup> -radiation sensitivity assay) and the method of single-cell electrophoresis (comet assay) [25–29].

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.

Astaxanthin as a Modifier of Genome Instability after γ-Radiation

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

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

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

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

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

**Figure 2.** Selection of the optimal concentration of astaxanthin to study its modifying effect on the γ-irradiated culture

phase of the first

125

**3.1. The impact of astaxanthin on the level of radiation-induced chromosomal** 

differ from the corresponding background cytogenetic parameters (p > 0.05) [25].

lymphocytes is exposed in vitro to gamma quanta in a dose of 1.0 Gy on G<sup>0</sup>

modifying action depended on its concentration in the irradiated culture.

capacity of astaxanthin, the concentrations of 20.0 μg/ml were chosen.

**aberrations in human lymphocytes**

mitotic cycle (**Figure 2**).

of human blood lymphocytes.

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 (G<sup>1</sup> –S, S–G<sup>2</sup> , G<sup>2</sup> –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 [32–34].

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].

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.
