CYTOGENETIC STUDY OF MANIFESTATIONS OF THE UNIVERSAL PHENOMENON OF THE BYSTANDER RESPONSE. / TsYTOGENETYChNE DOSLIDZhENNIa PROIaVIV UNIVERSAL'NOGO IaVYShchA EFEKTU SVIDKA.

OBJECTIVE: to establish the level of chromosomal instability in peripheral blood lymphocytes (PBL) from healthyindividuals and cells from patients with B-cell chronic lymphocytic leukemia (CLL) under co-cultivation conditions and to determine the possibility of inducing manifestations of the universal phenomenon of bystander response in them. MATERIALS AND METHODS: Cytogenetic analysis of uniformly stained chromosomes from human PBL, which differed by cytogenetic markers of sex; the presence of oncological transformation and in vitro irradiation of 137Cs in a dose of 0.50 Gy under the conditions of their joint cultivation was performed. RESULTS: The frequency of chromosome aberrations in PBL from healthy individuals when co-cultured with the blood from CLL patients was 3.35 per 100 cells, exceeded the control (1.48 per 100 cells, р < 0.01), did not significantly differ from the rate in non-irradiated blood T-lymphocytes from patients with B-cell CLL (3.18 per 100 cells, р > 0.05) and was lower than when co-cultivated with irradiated blood from CLL patients (5.00 per 100 cells, р < 0.01). In irradiated in vitro blood lymphocytes from CLL patients, the mean group level of chromosome aberrations under separate cultivation was 12.36 per 100 cells and exceeded the indicator during their co-cultivation with lymphocytes from healthy individuals (8.35 per 100 cells, р < 0.05). CONCLUSIONS: A tumor-induced bystander effect (TIBE) develops in PBL from healthy individuals when co-cultured with the blood from CLL patients and in T lymphocytes of B-cell CLL patients, the cytogenetic manifestation of which is an increase in the frequency of single chromatid fragments. The interaction of irradiated blood cells from CLL patients with lymphocytes from healthy individuals causes an increase in chromosomal instability in the latter due to the development of a radiation-induced bystander effect (RIBE) and leads to a decrease in the level of chromosomal instability in irradiated lymphocytes from CLL patients (rescue effect). An increase in the level of chromatid-type aberrations in T-lymphocytes of CLL patients during in vitro irradiation is a consequence of the development of RIBE against the background of TIBE.

MODIFICATION OF THE TUMOR/INDUCED BYSTANDER EFFECT BY IRRADIATION UNDER COCULTIVATION OF LYMPHOCYTES FROM PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA AND LYMPHOCYTES FROM HEALTHY DONORS. / MODYFIKATsIIa OPROMINENNIaM PUKhLYNNO/INDUKOVANOGO EFEKTU SVIDKA PRY SUMISNO/ROZDIL'NOMU KUL'TYVUVANNI LIMFOTsYTIV KhVORYKh NA KhRONIChNU LIMFOTsYTARNU LEIKEMIIu Z LIMFOTsYTAMY ZDOROVYKh DONORIV.

OBJECTIVE: Study the tumor-induced bystander effect of blood cells from chronic lymphocytic leukemia (CLL)patients on non-transformed bystander cells (peripheral blood lymphocytes (PBL) of conditionally healthy individ-uals) and the possibility of its modification after the impact of ionizing radiation. MATERIALS AND METHODS: We carried out cocultivation and separate cultivation of blood samples from conditionallyhealthy volunteers and patients with CLL according to our technique. Using the Comet assay, the relative level ofDNA damage was evaluated. RESULTS: A statistically significant increase (р < 0.001) in the level of DNA damage in PBL culture of conditionallyhealthy individuals after co-cultivation with malignant cells of CLL patients was observed. After irradiation, a drop in the level of cells with a high degree of DNA damage was noted, which was connected with an increase in the frequency of cells that were delayed in division at the S stage of the cell cycle. An increase in apoptotic activity in cultures of bystander cells was observed in all variants of the experiment (р < 0.001). CONCLUSION: The influence of irradiated blood cells of patients with CLL results in an enhancement of the tumor-induced bystander effect manifestation in the PBL of conditionally healthy individuals.

Study of modifying effects of astaxanthin on cytogenetic manifestations of bystander response in human peripheral blood lymphocytes in vitro.

AIM: To study the possible impact of astaxanthin on the cytogenetic manifestations of the simultaneous development of radiation-induced (RIBE) and tumor-induced bystander effect (TIBE) in human peripheral blood lymphocytes. MATERIALS AND METHODS: Peripheral blood lymphocytes from healthy persons and patients with chronic lymphocytic leukemia were cultured separately or cocultured with or without previous irradiation in vitro by 137Cs at a dose of 0.5 Gy. The cells were cultured with and without 20 µg/ml astaxanthin. RESULTS: In the presence of astaxanthin, the decrease of chromosomal instability both in the variant with separate TIBE and with simultaneous development of TIBE and RIBE was observed as the reduction in the frequency of simple chromosome-type aberrations, namely, double fragments. The average level of chromatid-type aberrations did not change under the action of astaxanthin. Although the total chromosome instability in bystander cells diminished, this did not lead to the elimination of the RIBE and TIBE development in the presence of astaxanthin. CONCLUSION: In the setting of experiment, astaxanthin did not reduce the frequency of chromatid-type aberrations in bystander cells due to RIBE and TIBE but reduced the frequency of simple aberrations of chromosomal type, not associated with the development of bystander response phenomenon.

STUDY THE EFFECTS OF IONIZING RADIATION ON THE LEVEL OF CHROMOSOME INSTABILITY IN HUMAN SOMATIC CELLS DURING THE DEVELOPMENT OF TUMOR-INDUCED BYSTANDER EFFECT. / DOSLIDZhENNIa VPLYVU IONIZUIuChOÏ RADIATsIÏ NA RIVEN' KhROMOSOMNOÏ NESTABIL'NOSTI V SOMATYChNYKh KLITYNAKh LIuDYNY PRY ROZVYTKU PUKhLYNO-INDUKOVANOGO EFEKTU SVIDKA.

OBJECTIVE: to determine the impact of the irradiated in vitro blood cells from patients with B-cell chronic lymphocytic leukemia (CLL) on the level of chromosomal instability in peripheral blood lymphocytes (PBL) from healthy persons during the development of tumor-induced bystander effect. MATERIALS AND METHODS: Separate and joint cultivation of PBL from healthy persons (cells-bystanders) together withblood cells from CLL patients irradiated in vitro at the G0 stage of the mitotic cycle by γ-quanta 137Cs in a dose of0.5 Gy 137Cs (cells-inductors) was used. For joint cultivation our own model system for co-cultivation of PBL fromindividuals of different sex, designed by us to investigate the bystander effects at the cytogenetic level was used.Traditional cytogenetic analysis of uniformly painted chromosomes with group karyotyping was performed. The frequency of chromosome aberrations in cells-inductors and cells-bystanders as the markers of chromosome instability were determined. RESULTS: Found that at co-cultivation of PBL from healthy individuals with irradiated blood cells from CLL patientsthe middle group frequency of chromosome aberrations in the bystander cells (5.18 ± 0.51 per 100 metaphases,p < 0.001) was statistically significant higher than its background level determined at a separate cultivaton (1.52± 0.30 per 100 metaphases), and at co-cultivation with non-irradiated blood cells from CLL patients (3.31 ± 0.50 per100 metaphases, p < 0.01). CONCLUSIONS: Co-cultivation of in vitro irradiated blood cells from CLL patients with PBL from healthy persons leadsto an increase in the level of chromosome instability in the bystander cells due to synergism between tumor-inducedand radiation-induced bystander effects.

RADIATION-INDUCED BYSTANDER EFFECT - MODELING, MANIFESTATION, MECHANISMS, PERSISTENCE, CANCER RISKS (literature review). / RADIATsIY̆NO-INDUKOVANYY̆ EFEKT SVIDKA ­ MODELIuVANNIa, PROIaVY, MEKhANIZMY ROZVYTKU, PERSYSTENTsIIa, ONKOLOGIChNI RYZYKY (ogliad literatury).

The review summarizes and analyzes the data of world scientific literature and the results of the own research con- cerning one of the main non-targeted effects of ionizing radiation - the radiation induced bystander effect (RIBE) - the ability of irradiated target cells to induce secondary biological changes in non-irradiated receptor cells. The his- tory of studies of this phenomenon is presented - it described under various names since 1905, began to study from the end of the twentieth century when named as RIBE and caused particular interest in the scientific community during recent decades. It is shown that the development of biological science and the improvement of research methods allowed to get new in-depth data on the development of RIBE not only at the level of the whole organism, but even at the genome level. The review highlights the key points of numerous RIBE investigations including mod- eling; methodological approaches to studying; classification; features of interaction between irradiated and intact cells; the role of the immune system, oxidative stress, cytogenetic disorders, changes in gene expression in the mechanism of development of RIBE; rescue effect, abscopal effect, persistence, modification, medical effects. It is emphasized that despite the considerable amount of research concerning the bystander response as the universal phenomenon and RIBE as one of its manifestations, there are still enough «white spots¼ in determining the mech- anisms of the RIBE formation and assessing the possible consequences of its development for human health.

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.

BIOLOGICAL MARKERS OF EXTERNAL AND INTERNAL EXPOSURE IN SHELTER CONSTRUCTION WORKERS: A 13-YEAR EXPERIENCE.

OBJECTIVE: The aim of this study is to investigate the cellular, molecular, genetic and non-invasive functional biomarkers of occupational irradiation in workers exposed to a combination of external gamma-radiation and incorporation of transuranium elements. RESULTS: A study was performed in 688 radiation workers of Shelter object conversion into a safe system with mean shift dose of external exposure of 26.06 mSv (range: 0.1-113.35) and risk of internal exposure with transuranium elements. Several biological parameters could serve as biomarkers of exposure at radiation doses below 100 mSv and even in 20-50 mSv interval. The parallel changes were shown in decline of brain electric activity, telomere length, differences in CCND1, CDKN1A, CDKN2A, VEGFA, TP53, DDB2 genes expression. An increase in counts of dicentrics, pair fragments and TCR-variant lymphocytes at doses over professional limits shows the need of biological dosimetry. The most sensitive markers include TCR-CD4+, γ-H2AX+ and CyclinD1+ cell counts. Implementation of flow cytometry approach for these markers enables quick obtaining of quantitative data. Confounding factors included respiratory function and smoking. The study of the radiation workers with the history of chronic exposure in radiation area during 3-5 years demonstrates changes of compensatory origin, i.e. absence of telomere shortening, increased number of NK-cells in combination with lower expression of intracellular γ-H2AX. CONCLUSION: This study confirms the presence of radiation-induced changes in gene regulation of cell proliferation, telomere function and apoptosis in radiation workers exposed to external and internal exposure at doses above professional limits and increase of compensatory changes.

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.

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".

Chromosomal mutagenesis in human somatic cells: 30-year cytogenetic monitoring after Chornobyl accident.

In the lecture we have generalized and analyzed the data of cytogenetic laboratory of National Research Center for Radiation Medicine of the National Academy of Medical Sciences of Ukraine on 30-year selective cytogenetic monitoring among the priority contingents of different ages exposed to radiation after Chornobyl accident in Ukraine. It is highlighted that not only targeted but also untargeted radiation-induced cytogenetic effects should be explored, especially in delayed terms following radiation exposure. The new methodical approaches for studying "bystander effect", individual radiosensitivity, and various forms of radiation-induced chromosomal instability (delayed, hidden, transmissible) have been proposed. These approaches proved to be advantageous for analyzing cytogenetic patterns of induction and persistence of chromosomal instability in human somatic cells because of "bystander effect" and "bystander type effect". The phenomenon of positive "reverse" bystander effect has been found. The possibility of modifying the inherited individual human susceptibility to mutagenic exposure by ionizing radiation has been estimated. Finally, the association between hypersensitivity to radiation exposure and realization of oncopathology in exposed individuals has been revealed. The increased intensity of human somatic chromosomal mutagenesis was confirmed not only in the nearest but in the delayed terms following Chornobyl accident as a result of radiation-induced both targeted and untargeted cytogenetic effects. Such effects can be considered as risk factors for malignant transformation of cells, hereditary diseases, birth defects, and multifactorial somatic pathology. This article is a part of a Special Issue entitled "The Chornobyl Nuclear Accident: Thirty Years After".

Peculiarities of induction and persistence of hidden chromosome instability in peripheral blood lymphocytes of persons occupationally exposed to ionizing radiation. / Osoblyvosti indukcii' ta persystencii' pryhovanoi' hromosomnoi' nestabil''nosti v limfocytah peryferychnoi' krovi osib, jaki profesijno kontaktuvaly z ionizujuchoju radiacijeju.

Objective - to investigate the induction of hidden chromosome instability in persons occupationally exposed to ionizing radiation and its persistence in vitro in successive mitoses. Materials and methods. Using two tests ("G2-bleomycin sensitivity assay" and two-term cultivation of human peripheral blood lymphocytes) voluntary cytogenetic examination of 15 individuals participated in the conversion of the "Shelter" ("Chornobyl NPP") into ecologically safe system had been carried out. Total 24 034 metaphase had been analyzed, of which 12 243 - without additional mutagenic exposure, 11 791 - exposed to bleomycin in vitro at concentration of 0.05 µg/ml. Results. The magnitude and dynamics of background as well as bleomycin-induced cytogenetic effects in both terms of lymphocytes' cultivation in occupational group differed significantly from the group of comparison towards increasing of chromosome instability indices with significant interindividual fluctuations. Conclusion. Interindividual differences in persistence of radiation-induced hidden chromosome instability in successive generations of human somatic cells had been found.