THE IMPACT OF ASTAXANTHIN ON THE LEVEL OF DNA METHYLATION IN IRRADIATED IN VITRO HUMAN LYMPHOCYTES. / VPLYV ASTAKSANTYNU NA RIVEN' METYLIuVANNIa DNK V OPROMINENYKh IN VITRO LIMFOTsYTAKh LIuDYNY.

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 «comets¼ 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.

Study the impact of astaxanthin on developing of genomic instability in human peripheral blood lymphocytes irradiated in vitro on G2 phase of cell cycle. / DOSLIDZhENNIa VPLYVU ASTAKSANTYNU NA ROZVYTOK GENOMNOÏ NESTABIL'NOSTI V LIMFOTsYTAKh PERYFERYChNOÏ KROVI LIuDYNY, OPROMINENYKh IN VITRO NA G2 STADIÏ KLITYNNOGO TsYKLU.

OBJECTIVE: 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. MATERIALS AND METHODS: 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. RESULTS: 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). CONCLUSIONS: Noimpactofastaxanthinongenomicinstabilityinducedbygammairradiation invitroinculturesof human peripheral blood lymphocytes on postsynthetic (G2) phase of first mitotic cycle had been established.

Genoprotective properties of astaxanthin revealed by ionizing radiation exposure in vitro on human peripheral blood lymphocytes. / GENOPROTEKTORNI VLASTYVOSTY ASTAKSANTYNU, VYIaVLENI PRY DIÏ IONIZUIuChOGO VYPROMINIuVANNIa IN VITRO NA LIMFOTsYTY PERYFERYChNOÏ KROVI LIuDYNY.

OBJECTIVE: to identify possible radioprotective properties of astaxanthin by means of cytogenetic criteria. METHODS: Cultivation of peripheral blood lymphocytes from five apparently healthy volunteers; treatment of lym phocytes' cultures by astaxanthin in final concentrations 20 µg/ml in Go phase of mitotic cycle, prior to ? irradia tion in vitro in a dose 1 Gy; cytogenetic analysis the uniformly stained slides of metaphase chromosomes. The elec trophoresis of individual cells (Comet assay); visualization of results under fluorescent microscope; accounting the number of nucleoid the fourth grade that correspond to apoptosis of the cells. RESULTS: Established that astaxanthin in final concentration 20.0 µg/ml exposed to the culture of human peripher al blood lymphocytes in the early G0 phase of mitotic cycle leads to significant reduction of cytogenetic effects induced by gamma irradiation in vitro in dose 1.0 Gy (from 26.05 ± 1.81 to 9.08 ± 0.78 per 100 cells, respectively) and to significant increase the frequency of apoptotic cells at the 48 hour of cultivation (from (3.78 ± 0.24) to (8.26 ± 0.91) %, respectively). CONCLUSIONS: The results obtained show the ability of astaxanthin to considerable weakening of radioinduced muta genic effect in human peripheral blood lymphocytes, which testify its powerful radioprotective potential.

Astaxanthin modifies clastogenic effects of ionizing radiation in vitro in peripheral blood lymphocytes of the persons recovered from acute radiation sickness.

AIM: To assess radioprotective activity of astaxanthin toward radiation-induced in vitro cytogenetic effects in human peripheral blood lymphocytes (PBL). MATERIALS AND METHODS: PBL from the cleanup workers exposed to ionizing radiation at high doses in 1986 during accident on Chornobyl nuclear power plant and who were diagnosed with acute radiation sickness of the first and second degrees, were cultured in vitro. Astaxanthin was added into the culture medium at a final concentration of 20.0 µg/ml, prior to γ-irradiation of PBL in vitro at a dose of 1 Gy. The slides of metaphase chromosomes were analyzed. RESULTS: Astaxanthin demonstrated considerable radioprotective effect in irradiated PBL manifested in significantly decreased levels of unstable cytogenetic markers of radiation exposure (dicentrics and centric rings). CONCLUSION: The data evidence on radioprotective capacity of astaxanthin toward radiation-induced cytogenetic effects in vitro in PBL of liquidators irradiated during Chornobyl nuclear power plant accident. This article is a part of a Special Issue entitled "The Chornobyl Nuclear Accident: Thirty Years After".

Redistribution of DNA loop domains in human lymphocytes under blast transformation with interleukin 2.

At higher order levels chromatin fibers in interphase nuclei are organized into loop domains. Gene regulatory elements (promoters and enhancers) are often located near the sites of loop attachments. Therefore, loop domains play a key role in regulation of cell transcriptional activity. We investigated the kinetics of DNA loop exit during single cell gel electrophoresis (the comet assay) of nucleoids obtained from two cell types that differ in their synthetic activity ­ human lymphocytes and lymphoblasts. Lymphocyte activation and transformation into lymphoblasts (blast transformation) was performed with interleukin 2. The results obtained suggest that a rearrangement of the loops occurs after lymphocyte activation. After blast transformation we observed an increase of the amount of loop domains on the surface of nucleoids against a decrease of the inner loop fraction. Therefore, the comet assay can be used for detection of large-scale changes in the cell nucleus that follow changes in cell functional state.