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.

DEVELOPMENT OF RADIATION-INDUCED BYSTANDER EFFECT IN THE SOMATIC CELLS OF PERSONS FROM DIFFERENT AGE GROUPS. / ROZVYTOK RADIATsIINO-INDUKOVANOGO EFEKTU SVIDKA V SOMATYChNYKh KLITYNAKh OSIB RIZNOGO VIKU.

OBJECTIVE: To investigate the development of chromosomal instability as a result of the radiation-induced bystandereffect in blood lymphocytes of persons from different age groups. MATERIALS AND METHODS: Materials of research were blood lymphocytes from 42 persons of different age (from 12 to102 years), divided into four age groups - teenagers, middle-aged, elderly and centenarians. Bystander effect wasstudied by modeling its induction in human lymphocytes, at which 0.3 ml of non-irradiated blood (served asbystander cells) and 0.3 ml of blood from persons of another sex exposed in vitro to X-ray in a dose of 0.25 Gy wereadded to the incubation mixture, followed by cultivation according to generally accepted semi-micro-method. Slidesof metaphase chromosomes were GTG-stained and analyzed under light microscopes with magnification x 1000. RESULTS: The average level of chromosomal aberrations in bystander cells of teenagers (6.08 ± 0.67 per 100metaphases), middle-aged people (4.56 ± 0.61 per 100 metaphases) and elderly persons (6.34 ± 0.76 per 100metaphases) significantly exceeded those of the corresponding age-related controls (p <0.01) due to the aberra-tions of chromatid type. The level of chromosome aberrations in centenarians' bystander cells (2.84 ± 0.51 per 100metaphases) was not significantly different from the control (p >0.05). The bystander effect was registered in 83%of teenagers, 90 % middle-aged persons and 50 % of the elderly persons. CONCLUSIONS: In the non-irradiated blood lymphocytes of teenagers, middle-aged and elderly persons under condi-tion of co-cultivation with cells X-irradiated in vitro in a dose of 0.25 Gy the bystander effect was induced. In thenon-irradiated centenarians' blood lymphocytes the bystander effect was not revealed. Interindividual variability inthe induction of bystander effect was registered. The development of bystander effect was independent on the levelof chromosomal instability in control cultures.

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.

Comparative analysis the frequency and spectrum of chromosome aberrations in irradiated in vitro peripheral blood lymphocytes of the elderly and centenarians from Kyiv. / PORIVNIaL'NYY̆ ANALIZ RIVNIa TA SPEKTRU ABERATsIY̆ KhROMOSOM V OPROMINENYKh IN VITRO LIMFOTsYTAKh PERYFERYChNOÏ KROVI OSIB LITN'OGO VIKU TA DOVGOZhYTELIV m. KYIeVA.

OBJECTIVE: To establish and compare the frequency and spectrum of chromosome aberrations under X radiation exposure in vitro in dose 0.25 Gy peripheral blood lymphocytes of the elderly and centenarians. MATERIAL AND METHODS: Material of cytogenetic research were peripheral blood lymphocytes from 11 elderly and 10 centenarians, which were irradiated in vitro in dose 0.25 Gy and cultured by generally accepted semi micromethod; slides of metaphase chromosomes were GTG stained and analyzed under the microscope with magnification x 1000. RESULTS: Under irradiation of blood in vitro the mean group frequencies of chromosome aberrations exceeded such without irradiation (р < 0.001) and were 11.60 ± 0.95 аnd 6.82 ± 0.63 per 100 cells in the elderly and the centenar ians, accordingly. Radiation induced increase in the frequency of chromosomal injuries occurred due to chromo some type aberrations which are markers of radiation exposure. In the elderly the elevated frequency of chromatid type aberrations also was registered what is considered a sign of chromosome instability. CONCLUSIONS: The results indicate increased sensitivity the blood lymphocytes from the elderly to radiation expo sure in low doses and allow to assume the advantage of persons with hereditary determined chromosomal stability in achieving longevity.

[Effects of mono-, poly- and composite probiotics on the ulceration caused by restraint stress].

It was studied the effect of probiotic strains of Bifidobacterium animalis VKL, Bifidobacterium animalis VKB and Lactobacillus casei IMVB-7280, and their mixtures on erosive and ulcerative lesions of the gastric mucosa (GM) of rats. GM was induced by water-immersion restraint stress. It was found that investigated probiotics did not have gastroprotective properties under a single and seven-day prophylactic administration. However, multiprobiotics (polyprobiotic Bifidobacterium animalis VKL and Bifidobacterium animalis VKB and composite probiotic Bifidobacterium animalis VKL, Bifidobacterium animalis VKB and Lactobacillus casei IMVB-7280) reduced the erosive and ulcerative lesions and the intensity of bleeding in rat GM when given within 14 days. It was shown that one of the mechanisms of antiulcer preventive effect of the multistrain probiotics is the restoration of pro/antioxidant balance in the GM under the stress action. The obtained results show the effectiveness of poly- and composite probiotics in the gastric ulcer prevention.

Antistaphylococcal action of lacto- and bifidobacteria and interleukn-2.

Antistaphylococcal action of Lactobacillus delbrueckii subsp. bulgaricus IMV B-7281, L. acidophilus LMV B-7279 and Bifidobacterium animalis VKB as well as interleukin-2 (IL-2) has been determined on the model of experimental staphylococcal infection in mice. It has been established that peroral administration of certain probiotic strains of lacto- and bifidobacteria or composition L. acidophilus LMV B-7279--L. delbrueckii subsp. bulgaricus IMV B-7281 with IL-2 to mice favored the reducing of terms of staphylococcus persistence in the kidneys of experimental mice. The studied schemes are promising for treatment of patients with surgical infection.

Effect of cord blood cells on interferonogenesis and effector cells of immune system.

Human cord blood cells were shown to produce interferon-alpha- and -gamma in vitro, both spontaneously and following adequate induction. Transplantation of human blood cord cells to mice led to endogenous interferonogenesis activation, serum interferon levels as well as interferon-alpha and interferon-gamma synthesis by activated peripheral blood leucocytes becoming higher. The maximal interferon accumulation in murine blood sera following transplantation did not coincide with interferonogenesis dynamics observed due to the effect of standard early and late interferon inducers - poly I:C and ridostin, respectively. Interferonogenesis activation was accompanied by increased functional activty of phagocytic system cells.

[Age-related characteristics of glial fibrillary acidic protein level in the rat brain structures in pain syndrome].

In experiments in white Wistar rats of three-age groups (immature, mature and old rats) content of glial fibrous acidic protein (GFAP) was investigated in cerebral cortex, hippocampus, thalamus and brain stem after the carrying out of laparotomy. The correlation between emotional activity and the level of GFAP has been found in the given structures of a brain. In immature animals, the activation of emotional component of behaviour was accompanied by decrease in polypeptide's soluble and insoluble fractions in brain stem by 16% and 18%, respectively (p<0.05) in relation to intact animals. In mature rats, the operative trauma was accompanied by the lowering of grooming and lowering of the level of soluble fraction of GFAP in all structures. In old rats, laparotomy evoked accumulation of polymerized and depolymerized forms of GFAP in hippocampus by 34% and 48% (p<0.05), respectively.

[Effect of hypothyroidism on glial fibrillary acid protein in the structures of the rat brain].

The content and polypeptide composition of the glial fibrillary acid protein (GFAP) in the hemispheres' cortex, hippocamp, thalamus and brain stem in the condition of thyroid gland hypofunction in Wistar rats of different age (preadolescent rats five weeks old, pubertal rats five months old and old rats 18 - 20 months old) were investigated. Hypothyroidism was created by adding mercasolil (10 mg/kg) to the standard diet. At the end of experiment the level of thyrotropin and thyroxin in serum was defined with immunoenzyme method. It was established that experimental hypothyroidism caused significant increase 14-72% of the filament fraction GFAP content in the thalamus and brain stem (preadolescent rats) and in the thalamus and hemispheres' cortex (adult rats), Defragmented polypeptides with molecular weight 47 -37 kDa were revealed by immunoblotting. It is connected with the activation of cytosceletal changes of astroglia. There were changes in the group of old rats that is possibly a reason of reduced the flexibility of CNS.

[Fatty-acidic content in the brain structures of hypothyroid rats of different age groups].

The content of fatty acids in the cerebral cortex and hippocampus was studied in an experiment on Wistar rats of the three age groups (5-weeks, puberal and old- 20-22 weeks) with an induced hypothyroidism (simulation was achieved by a 2 week prior supplement of L-thyroxin and final monitoring of thyroxin levels in serum). It was determined that the major variations of free fatty acids were noticed in the cerebral cortex and hippocampus among puberal rats, and somewhat lower shifts in premature (5 weeks) and old animals. The increase of free fatty acids in premature rats was registered in spectrum C17-C22. The increase of linoleic and linolenic acids were registered five times higher in hippocampus. In old rats hyperthyroidism was characterized by more apparent shifts in fatty-acidic content of lipids of neocortex and less apparent in hippocampus.