Problems of Radiation Medicine and Radiobiology

D. A. Кurinnyi¹, O. M. Demchenko¹, M. G. Romanenko², S. R. Rushkovsky²

¹State institution «National Research Center for Radiation Medicine of the National Academy of Medical Sciences of Ukraine», 53 Melnykova str., Kyiv, 04050, Ukraine
²Educational and Research Center «Institute of Biology and Medicine» Taras Shevchenko National University of Kyiv, 64/13, Volodymyrska str., Kyiv, 01601, Ukraine

THE IMPACT OF ASTAXANTHIN ON THE LEVEL OF DNA METHYLATION IN IRRADIATED IN VITRO HUMAN LYMPHOCYTES

Objective. To investigate an effect of astaxanthin on global DNA methylation in human peripheral blood lymphcytes exposed to γ-radiation in vitro.
Methods. Samples of whole blood were diluted with RPMI-1640 medium in proportion 1 : 10 and incubated at 37 °C for 3 h. Samples were exposed to γ-ray (emitter IBL-237C, dose-rate 2.34 Gy/min) in dose 1.0 Gy. Astaxanthin was added into culture medium in final concentrations 20.0 μg/ml before irradiation. The analysis of the global DNA methylation state was carried out using modified method of neutral single-cell gel electrophoresis (Comet assay) after digestion with the methylation-sensitive endonuclease HpaII.
Results. For every experiment the analysis of the frequency distribution of «comets» was carried out. The treatment of non-irradiated and irradiated human peripheral blood lymphocytes with astaxanthin resulted to a statistically significant (p < 0.05) shift of comet distribution with tendency to increasing level of DNA migration which indicates increase in the pool of cells with a reduced level of DNA methylation.
Conclusions. It has been found that astaxanthin in concentration of 20.0 μg/ml reduced the levels of global DNA methylation both in irradiated in vitro and native human lymphocytes. It suggests that astaxanthin has an effect on mechanisms of epigenetic regulation of gene expression.
Key words: astaxanthin, human peripheral blood lymphocytes, γ-irradiation, DNA methylation.

Problems of radiation medicine and radiobiology.
2018;23:235_245. doi: 10.33145/2304-8336-2018-23-235-245.

full text

1. Weinhold B. Epigenetics: The Science of Change Environ Health Perspect. 2006;114:160-7.

2. Jin B, Li Ya, Robertson KD. DNA methylation. Superior or subordinate in the epigenetic hierarchy? Genes Cancer. 2011;2(6): 607-17. doi: 10.1177/1947601910393957

3. Moore LD, Le T, Fan G. DNA methylation and its basic function. Neuropsychopharmacology. 2013;38:23-38. doi: 10.1038/npp.2012.112.

4. Brenet F, Moh M, Funk P, Feierstein E, Viale AJ, Socci ND, Scandura JM. DNA methylation of the first exon is tightly linked to transcriptional silencing. PLoS One. 2011;6(1):e14524. doi: 10.1371/journal.pone.0014524.

5. Sharma S, Kelly TK, Jones PA. Epigenetics in cancer. Carcinogenesis 2010;31:27-36.

6. Li E, Beard C, Jaenisch R. Role for DNA methylation in genomic imprinting. Nature. 1993;366:362-5.

7. Zoghbi HY, Beaudet AL. Epigenetics and human disease. Cold Spring Harb Perspect Biol. 2016;8(2):a019497. doi: 10.1101/cshperspect.a019497.

8. Banno K, Kisu I, Yanokura M, Tsuji K, Masuda K, Ueki A, et al. Epimutation and cancer: a new carcinogenic mechanism of Lynch syndrome (Review). Int J Oncol. 2012;41(3):793-7. doi: 10.3892/ijo.2012.1528.

9. Novak K. Epigenetics changes in cancer cells: highlights of the American Association for Cancer Research Special Conference on Chromatin, Chromosomes, and Cancer Epigenetics. Medscape General Medicine. 2004;6(4):17. doi: 10.3892/ijo.2012.1528.

10. Simmons D. Epigenetic influences and disease. Nature Education. 2008;1(1):6.

11. Obe G, Durante M. DNA double strand breaks and chromosomal aberrations. Cytogenet Genome Res. 2010;28:8-16. doi: 10.1159/000303328.

12. Turgeon M, Perry N, Poulogiannis G. DNA damage, repair, and cancer metabolism. Front Oncol. 2018;8:15. doi: 10.3389/fonc.2018.00015.

13. Lloyd DC, Dolphin G. W. Radiation-induced chromosome damage in human lymphocytes. Br J Ind Med. 1977;34(4):261-73.

14. Merrifield M., Kovalchuk O. Epigenetics in radiation biology: a new research frontier. Front Genet. 2013;4:a 40. doi: 10.3389/fgene.2013.00040.

15. Shah DJ, Sachs RK, Wilson DJ. Radiation-induced cancer: a modern view. Br J Radiol. 2012;1020:1166-73. doi: 10.1259/bjr/25026140.

16. Lee Y, Kim YJ, Choi YJ, Lee JW, Lee S, Cho YH, et al. Radiation-induced changes in DNA methylation and their relationship to chromosome aberrations in nuclear power plant workers. Int J Radiat Biol. 2015;91:142-9. doi: 10.3389/fgene.2013.00040.

17. Pilinska MA, Kurinnyi DA, Rushkovsky SR, Dybska OB. Genoprotective properties of astaxanthin revealed by ionizing radiation exposure in vitro on human peripheral blood lymphocytes. Probl Radiac Med Radiobiol. 2016;21:141-8.

18. Kurinnyi DA, Rushkovsky SR, Dybska OB, Dubrovina GV, Pilinska MA. Astaxanthin modifies clastogenic effects of ionizing radiation in vitro in peripheral blood lymphocytes of the persons recovered from acute radiation sickness. Exp Onc. 2016;38(4):280-2.

19. Kurinnyi DA, Rushkovsky SR, Demchenko OM, Pilinska MA. Study the impact of astaxanthin on developing of genomic instability in human peripheral blood lymphocytes irradiated in vitro on G2 phase of cell cycle. Probl Radiac Med Radiobiol. 2017;22:208-16.

20. Kurinnyi DA, Rushkovsky SR, Demchenko OM, Pilinska MA. Peculiarities of modification by astaxanthin the radiation-induced damages in the genome of human blood lymphocytes exposed in vitro on different stages of the mitotic cycle. Cytol Genet. 2018;52(1):40-5.

21. Ambati RR, Phang SM, Ravi S, Aswathanarayana RG. Astaxanthin: sources, extraction, stability, biological activities and its commercial applications. Mar Drugs. 2014;12(1):128-52. doi: 10.3390/md12010128.

22. Hama S, Uenishi S, Yamada A, Ohgita T, Tsuchiya H, Yamashita E, et al. Scavenging of hydroxyl radicals in aqueous solution by astaxanthin encapsulated in liposomes. Biol Pharm Bull. 2012;35:2238-42.

23. Stewart J, Lignell A, Pettersson A, Elfving E, Soni G. Safety assessment of astaxanthin - rich microalgae biomass: Acute and subchronic toxicity studies in rats. Food Chem Toxicol. 2008;46(9):3030-6. doi: 10.1016/j.fct.2008.05.038.

24. Tago Y, Fujii T, Wada J, Kato M, Wei M, Wanibuchi H, et al. Genotoxicity and subacute toxicity studies of a new astaxanthin- containing Phaffia rhodozyma extract. J Toxicol Sci. 2014;39(3):373-82.

25. Kurinnyi DA, Kostikov IYu. Co-cultivation of unicellular green algae (Chlorophyta, Chlorophyceae) and human peripheral blood lymphocytes as a test system for radiobiological studies. Int J Algae. 2017, Vol. 19,no. 2, P. 163-172. https://doi.org/10.15407/alg27.02.215.

26. Кurinnyi DA, Rushkovsky SR, Demchenko OM, Pilinska MA. Radioprotective properties of astaxanthin: The Impact on radiation induced chromosomal aberrations and DNA breaks in human lymphocytes in vitro. In: Reeve T., editor. Ionizing radiation. Advances in research and applications. NY: Nova science publishers; 2018. p. 221-40.

27. Lewies A, Van Dyk E, Wentzel JF, Pretorius PJ. Using a medium-throughput comet assay to evaluate the global DNA methylation status of single cells. Front Genet. 2014;5(215):1-6. doi: 10.3389/fgene.2014.00215.

28. Olive PL, Banath JP. Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the «comet» assay. Radiat Res. 2012;178:35-42.

29. Gyori BM, Venkatachalam G, Thiagarajan PS, Hsu D, Clement M. OpenComet: An automated tool for comet assay image analysis. Redox Biology. 2014;2:457-65.

30. Lee E, Oh E, Lee J, Sul D, Lee J. Use of the tail moment of the lymphocytes to evaluate DNA damage in human biomonitoring studies. Toxicol Sci. 2004;81:121-32.

31. Azqueta A, Langie S, Collins A, editors. 30 years of the Comet Assay: an overview with some new insights. Frontiers; 2007. 560 p.

32. Rosner B. Fundamentals of biostatistics. 8th ed. Cengage Learning; 2015. 962 p.

33. Ehrlich M, Gama Sosa MA, Huang L-H, Midgett RM, Kuo KC, McCune RA, Gehrke C. Amount and distribution of 5-methylcytosine in human DNA from different types of tissues or cells. Nucleic Acids Res. 1982;10:2709-21. doi:10.1093/nar/10.8.2709.PMC 320645.

34. Lister R, Pelizzola M, Dowen R, Hawkins RD, Hon Gary, Tonti-Filippini J, et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature. 2009;462(7271):315-22. doi:10.1038/nature08514.

35. Ahnstrom G, Erixon K. Measurement of strand breaks by alkaline denaturation and hydroxyapatite chromatography, in DNA Repair. In: Friedberg EC, Hanawalt PC, editors. A laboratory manual of research procedures. New York, NY: Marcel Dekker; 1981. p. 403-18.

36. Kulis M, Esteller M. DNA methylation and cancer. Adv Genet. 2010;70:27-56. doi: 10.1016/B978-0-12-380866-0.60002-2.

37. Zhang L, Wang H. Multiple mechanisms of anti-cancer effects exerted by astaxanthin. Mar Drugs. 2015;13:4310-30. doi:10.3390/md13074310.

38. Glasauer A, Chandel NS. Targeting antioxidants for cancer therapy. Biochem Pharmacol. 2014;92:90-101.

39. Zhang X, Zhao WE, Hu L, Zhao L, Huang J. Carotenoids inhibit proliferation and regulate expression of peroxisome proliferators-activated receptor gamma (PPARgamma) in K562 cancer cells. Arch Biochem Biophys. 2011;512:96-106.

40. Kowshik J, Baba AB, Giri H, Deepak RG, Dixit M, Nagini S. Astaxanthin inhibits JAK/STAT-3 signaling to abrogate cell proliferation, invasion and angiogenesis in a hamster model of oral cancer. PLoS One. 2014;9:e109114. doi: 10.1371/journal.pone. 0109114.