Objective: To investigate an effect of astaxanthin on global DNA methylation in human peripheral blood lympho-cytes 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 °Cfor 3 h. Samples were exposed to γ-ray (emitter IBL-237C, dose-rate 2.34 Gy/min) in dose 1.0 Gy. Astaxanthin wasadded into culture medium in final concentrations 20.0 μg/ml before irradiation. The analysis of the global DNAmethylation 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 treatmentof non-irradiated and irradiated human peripheral blood lymphocytes with astaxanthin resulted to a statisticallysignificant (p < 0.05) shift of comet distribution with tendency to increasing level of DNA migration which indicatesincrease 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 DNAmethylation both in irradiated in vitro and native human lymphocytes. It suggests that astaxanthin has an effect onmechanisms of epigenetic regulation of gene expression.
Meta. Vyznachyty vplyv astaksantynu na riven' global'nogo metyliuvannia DNK v oprominenykh in vitro limfotsy-takh peryferychnoï krovi liudynyMetody. Tsil'nu krov dodavaly u kul'tural'ne seredovyshche RPMI-1640 u spivvidnoshenni 1 : 10 i vytrymuvaly 3godyny pry 37 °S. Oprominiuvannia γ-kvantamy zdiĭsniuvaly z vykorystanniam vyprominiuvacha IBL-237C (doza 1,0Gr, potuzhnist' 2,34 Gr/khv). Astaksantyn u kintseviĭ kontsentratsiï 20,0 mkg/ml dodavaly pered oprominenniam.Analiz statusu global'nogo metyliuvannia DNK v limfotsytakh peryferychnoï krovi liudyny provodyly za dopomo-goiu modyfikovanogo metodu kometnogo elektroforezu (Somet assay) v neĭtral'nykh umovakh z vykorystanniamchutlyvogo do metyliuvannia fermentu restryktsiï HpaII.Rezul'taty. Dlia kozhnogo variantu eksperymentu bulo provedeno doslidzhennia chastotnogo rozpodilu «komet».Dodavannia astaksantynu do neoprominenykh i oprominenykh limfotsytiv liudyny pryzvelo do statystychno znachu-shchogo (p -#060; 0,05) zsuvu rozpodiliv «komet» u napriamku zbil'shennia vykhodu DNK, shcho vkazuie na zrostannia pulu klitynzi znyzhenym rivnem metyliuvannia DNK.Vysnovky. Vstanovleno, shcho astaksantyn v kintseviĭ kontsentratsiï 20,0 mkg/ml pryzvodyt' do znyzhennia rivniaglobal'nogo metyliuvannia DNK v oprominenykh in vitro ta neoprominenykh limfotsytakh liudyny, shcho svidchyt' pro ĭo-go zdatnist' vplyvaty na epigenetychni mekhanizmy kontroliu gennoï ekspresiï.
Keywords: DNA methylation; astaxanthin; human peripheral blood lymphocytes; γ-irradiation.
D. A. Кurinnyi, O. M. Demchenko, M. G. Romanenko, S. R. Rushkovsky.