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Study the impact of astaxanthin on developing of genomic instability in human peripheral blood lymphocytes irradiated in vitro on G2 phase of cell cycle

2017; Volume: 22; Linguagem: Inglês

10.33145/2304-8336-2017-22-208-215

2313-4607

D. A. Кurinnyi, S. R. Rushkovsky, O. M. Demchenko, M. A. Pilinska,

Anesthesia and Neurotoxicity Research

To identify the possibility of modification by astaxanthin the level of genome damages induced by gamma quanta in the culture of human peripheral blood lymphocytes exposed in vitro on postsynthetic (G2) phase of the first mitotic cycle.Peripheral blood lymphocytes from four apparently healthy volunteers 35-51 years old were cultivated using modified micromethod. To obtain genomic damages in G2 phase of the first mitotic cycle the part of cultures was irradiated by γ quanta in dose 1.0 Gy through 46 hours of cultivation. Astaxanthin in final con centration 20 μg/ml was exposed to lymphocytes' cultures before the irradiation. Cytogenetic analysis the uniform ly stained slides of metaphase chromosomes was carried out to determine the frequencies of chromosome and chro matid types of aberrations. Using the method of individual cells electrophoresis (Comet assay) the relative level of DNA damages (Tail Moment index) and the frequency of apoptotic cells with high level of DNA fragmentation were evaluated.Mean group frequencies of chromosome aberrations after gamma irradiation of lymphocytes in vitro exceed ed those without radiation exposure and were 72.35 ± 1.17 and 2.46 ± 0.30 per 100 metaphases, respectively (p < 0.001), mainly due to chromatid type of aberrations (58.32 ± 1.29 per 100 metaphases). Adding of astaxanthin into culture medium before the irradiation did not result in changes as in the frequency of chromosomal damages (71.54 ± 1.34 per 100 metaphases) as in the spectrum of aberrations - also prevailed chromatid type of aberrations (58.47 ± 1.47 per 100 metaphases). The increase of Tail Moment index after radiation exposure (from 3.84 ± 0.36 to 12.06 ± 1.88, respectively, p < 0.001) and lack of significant impact of astaxanthin on this index in the irradiated lym phocytes (8.96 ± 2.39, p > 0.05) was established, ie astaxanthin didn't change the relative level of radiation induced DNA damages. Also apoptogenic effect of astaxanthin was not found: frequency of apoptotic cells were (2.25 ± 1.49) % in cultures of intact lymphocytes, (2.08 ± 1.54) % in irradiated cultures and (1.78 ± 1.25) % under joint action of gamma radiation and astaxanthin (p > 0.05).Noimpactofastaxanthinongenomicinstabilityinducedbygammairradiation invitroinculturesof human peripheral blood lymphocytes on postsynthetic (G2) phase of first mitotic cycle had been established.Meta: vyznachennia mozhlyvosti modyfikatsiï astaksantynom rivnia indukovanykh gamma kvantamy poshkodzhen' ge nomu v kul'turi limfotsytiv peryferychnoï krovi liudyny, oprominenoï in vitro na postsyntetychniy̆ (G2) stadiï per shogo mitotychnogo tsyklu.Materyal i metody. Limfotsyty peryferychnoï krovi chotyr'okh umovno zdorovykh volonteriv vikom 35–51 rokiv kul'tyvuvaly za modyfikovanym mikrometodom. Dlia vyznachennia poshkodzhen' genomu na G2 stadiï mitotychnogo tsyklu chastynu kul'tur oprominiuvaly gamma kvantamy v dozi 1,0 Gr na 46 y̆ godyni kul'tyvuvannia. Neopromineni kul'tury sluguvaly kontrolem. Astaksantyn v kintseviy̆ kontsentratsiï 20,0 mkg/ml vvodyly v kul'tury limfotsytiv pered oprominenniam. Provodyly tsytogenetychnyy̆ analiz rivnomirno zabarvlenykh preparativ metafaznykh khro mosom, vyznachaly chastotu aberatsiy̆ khromatydnogo ta khromosomnogo typiv. Za dopomogoiu metodu elektroforezu okremykh klityn (Comet assay) otsiniuvaly vidnosnyy̆ riven' poshkodzhen' DNK (pokaznyk «Tail Moment») ta chasto tu apoptychnykh klityn (klityny z vysokym rivnem fragmentatsiï DNK).Rezul'taty. Seredn'ogrupovi chastoty aberatsiy̆ khromosom pry gamma oprominenni limfotsytiv in vitro perevyshchu valy taki bez oprominennia i stanovyly 72,35 ± 1,17 ta 2,46 ± 0,30 na 100 metafaz, vidpovidno (r < 0,001), pere vazhno, za rakhunok aberatsiy̆ khromatydnogo typu (58,32 ± 1,29 na 100 metafaz). Dodavannia astaksantynu pered oprominenniam limfotsytiv ne pryzvelo do zminy iak chastoty khromosomnykh porushen' (71,54 ± 1,34 na 100 meta faz), tak i spektru aberatsiy̆ – prevaliuvaly takozh aberatsiï khromatydnogo typu (58,47 ± 1,47 na 100 metafaz). Vstanovyly zrostannia pokaznyka «Tail Moment» pry oprominenni limfotsytiv (z 3,84 ± 0,36 do 12,06 ± 1,88, vidpovidno, r < 0,001) ta vidsutnist' statystychno znachushchogo vplyvu astaksantynu na tsey̆ pokaznyk v opromine nykh limfotsytakh (8,96 ± 2,39, p > 0,05), tobto astaksantyn ne zminiuvav vidnosnyy̆ riven' radiatsiy̆no indukovanykh poshkodzhen' DNK. Ne bulo vyiavleno takozh apoptogennoï diï astaksantynu: chastoty apoptychnykh klityn stanovy ly (2,25 ± 1,49) % v kul'turakh intaktnykh limfotsytiv, (2,08 ± 1,54) % v oprominenykh kul'turakh i (1,78 ± 1,25) % – pry sumisniy̆ diï gamma oprominennia ta astaksantynu (p > 0,05). Na vidminu vid vstanovlenoï namy ranishe genop rotektornoï diï astaksantynu na limfotsyty peryferychnoï krovi liudyny, shcho buly opromineni na stadiï spokoiu (G0), otrymani dani svidchat' pro vidsutnist' podibnogo efektu pislia oprominennia limfotsytiv na G2 stadiï klityn nogo tsyklu.Vysnovky. V ramkakh vykonanogo doslidzhennia ne vstanovleno modyfikuiuchogo (zakhysnogo) vplyvu astaksantynu na radiatsiy̆no indukovanu genomnu nestabil'nist' pry oprominenni kul'tury limfotsytiv peryferychnoï krovi liudyny gamma kvantamy v dozi 1,0 Gr na postsyntetychniy̆ (G2) stadiï pershogo mitotychnogo tsyklu.