STATUS OF THE CELLULAR IMMUNITY IN CLEAN-UP WORKERS OF THE CHORNOBYL ACCIDENT AND PATIENTS WITH CANCER OF THE MOUTH CAVITY, ORAL AND LARYNGEAL PART OF THE PHARYNX. / STAN KLITYNNOGO IMUNITETU V UChASNYKIV LIKVIDATsIÏ NASLIDKIV AVARIÏ NA ChAES TA KhVORYKh NA ZLOIaKISNI NOVOUTVORENNIa POROZhNYNY ROTA, ROTOVOÏ TA GORTANNOÏ ChASTYN GLOTKY.

OBJECTIVE: to establish cellular immunity in clean-up workers of the Chornobyl accident compared to patients with malignant neoplasms of the oral cavity, oral and laryngeal parts of the pharynx according to the subpopulation organization of peripheral blood leukocytes. MATERIALS AND METHODS: We examined 112 males, age (56,92 ± 6,17) years (M ± SD), including 26 male clean-up workers exposed at the radiation dose 10-500 mSv; 20 male clean-up workers exposed at the dose range 504-990mSv; 33 patients with malignant neoplasms of the oral cavity, oral and laryngeal parts of the pharynx and 33 non-exposed subjects of the control group. Immune cell subsets analysis was performed by the expression of differential and activation antigens of peripheral blood leukocytes using flow cytometry. RESULTS: In assessing each group's cellular immunity, there was a decrease in the number of CD19+ B-lymphocytes, CD3+ HLA-DR+ T- and CD3- HLA-DR+ B-lymphocytes, CD3- 16+ 56+ natural killer cells, combined with an increase in the number of CD8+ T-lymphocytes. An increase in the relative number of CD4+ CD8+ T-lymphocytes was determined in clean-up workers (D < 500 mSv) and cancer patients. An increase in the number of CD8+ T-lymphocytes and a decrease in the number of CD4+ T-lymphocytes were observed in clean-up workers (D > 500 mSv), as well as in cancer patients. In addition, a decrease of monocytes, CD3+ 16+ 56+ , and CD3+ TCRαß+ T-lymphocytes was determined in patients with oncological pathology. CONCLUSIONS: The obtained results show the unidirectionality of changes in cellular immunity in the participants of the liquidation of the consequences of the accident at the Chornobyl nuclear power plant and patients with the investigated oncological pathology, which indicates the formation of persistent violations of antitumor protection in the participants of the liquidation of the consequences of the accident as the basis of oncogenesis. Determining changes in the number of lymphocytes, monocytes, granulocytes, CD4+ T-lymphocytes, CD4+ CD8+ immature T-lymphocytes, the immunoregulatory ratio CD4+ / CD8+, CD3+TCRαß+ T-lymphocytes and CD3+ 16+ 56+ CTL can have an additional effect on the effectiveness antitumor protection and the probability of oncogenesis risk in immunocompromised individuals.

СHARACTERISTICS OF CYCLIN D1-MEDIATED REGULATION OF CELL CYCLE OF PERIPHERAL BLOOD LIMPHOCYTES OF CHORNOBYL CLEAN-UP WORKERS AND PERSONS WITH MALIGNANT NEOPLASMS OF THE ORAL CAVITY, OROPHARYNX AND LARYNGOPHARYNX. / KhARAKTERYSTYKA TsYKLIN D1-OPOSEREDKOVANOÏ REGULIaTsIÏ KLITYNNOGO TsYKLU LIMFOTsYTIV PERYFERYChNOÏ KROVI UChASNYKIV LIKVIDATsIÏ NASLIDKIV AVARIÏ NA ChAES TA OSIB, KhVORYKh NA ZLOIaKISNI NOVOUTVORENNIa POROZhNYNY ROTA, ROTOVOÏ TA GORTANNOÏ ChASTYN GLOTKY.

OBJECTIVE: to explore proliferative potential of peripheral blood lymphocytes of Chornobyl clean-up workers and persons with malignant neoplasms of the oral cavity, oropharynx and laryngopharynx by level of expression of cyclin D1 and quantitative parameters of cell cycle. MATERIALS AND METHODS: A total of 294 men aged (58.47 ± 7.32) were surveyed, 215 of them were Chornobyl clean"up workers (1986-1987), exposed at the dose range 10.43-3623.31 mSv; 49 persons of the control group and 30persons with malignant neoplasms of the oral cavity, oropharynx and laryngopharynx at III, IVА and IVВ stages ofthe disease. The analysis of parameters of cell cycle and proliferative activity of peripheral blood (PB) lymphocyteswas performed using the flow cytometry. The evaluation of distribution of cells by G0/G1, S, G2/M cell cycle phaseswas done in vivo and in in vitro. Proliferative potential was analyzed by level of expression of cytoplasmic protein ofcyclin D1. RESULTS: Proliferative potential of PB lymphocytes of Chornobyl clean"up workers and persons with malignant neo"plasms of the oral cavity, oropharynx and laryngopharynx was assessed. An increase in the level of spontaneousсyclin D1 expression and disturbance of сyclin D1-dependent regulation of cell cycle of PB lymphocytes after mito"gen activation were determined in the Chornobyl clean-up workers. An increase in pool of cells in the S" and G2/M"phases of cell cycle was detected, which characterizes high proliferative potential of PB lymphocytes. These changesare most pronounced in the subgroup of persons with a radiation dose of D > 500 mSv, and in persons with oncolo"gical pathology. CONCLUSIONS: A positive linear dependence has been established between the radiation dose and the number of cellsin the S"phase of cell cycle in the subgroup of Chornobyl clean"up workers with a radiation dose of D > 500 mSv. The detected changes of cyclin D1-dependent regulation of cell cycle and proliferative status of lymphocytes depend on the radiation dose, can be a manifestation of genome instability and be a cause for risks of oncogenesis, in a remote period after radiation exposure.

CHANGES IN GENE EXPRESSION ASSOCIATED WITH NON-CANCER EFFECTS OF THE CHORNOBYL CLEAN-UP WORKERS IN THE REMOTE PERIOD AFTER EXPOSURE. / ZMINY GENNOÏ EKSPRESIÏ, ASOTsIIOVANI Z NEPUKhLYNNYMY EFEKTAMY VIDDALENOGO PERIODU PISLIa OPROMINENNIa V UChASNYKIV LIKVIDATsIÏ NASLIDKIV AVARIÏ NA ChAES.

OBJECTIVE: to establish the connection of radiation-induced changes in gene expression with the realized pathology of the broncho-pulmonary and cardiovascular systems in Chornobyl clean-up workers. MATERIALS AND METHODS: We examined 314 male Chornobyl clean-up workers (main group; age (58.94 ± 6.82) years(M ± SD); min 33, max 79 years; radiation dose (411.82 ± 625.41) mSv (M ± SD); min 1.74, max 3600 mSv) with various nosological forms of cardiovascular and broncho-pulmonary pathology (BPP) and 50 subjects of the controlgroup: age (50.50 ± 5.73) years (M ± SD); min 41, max 67 years. The relative level of BCL2, CDKN2A, CLSTN2, GSTM1,IFNG, IL1B, MCF2L, SERPINB9, STAT3, TERF1, TERF2, TERT, TNF, TP53, CCND1, CSF2, VEGFA genes expression was determined inperipheral blood leukocytes by real-time PCR (7900 HT Fast Real-Time PCR System (Applied Biosystems, USA)). The«gene-disease¼ association was determined on statistical models stratified separately for each disease and gene.Logistic regression was used to calculate the odds ratio. RESULTS: Increased GSTM1 gene expression and no changes in angiogenesis-related VEGFA gene expression werefound in the main group of patients with coronary heart disease (CHD). It was established overexpression of TP53,VEGF and IFNG genes in the group of patients with arterial hypertension (AH). At combination of these diseases anincrease of expression of СSF2, TERF1, TERF2 genes was established. The detected changes demonstrate an activationof the antioxidative defense system in patients with CHD, while AH is associated with the expression of genes ofangiogenesis and immune inflammation. It was shown an increase in the expression of genes associated with apoptosis and kinase activity (BCL2, CLSTN2, CDKN2), immune inflammation (CSF2, IL1B, TNF) in Chornobyl clean-upworkers with BPP. Expression of TP53 and GSTM1 (gene, associated with the glutathione system) was significantlyupregulated in the group of individuals with chronic bronchitis, whereas in patients with chronic obstructive pulmonary disease, no increase was detected; the expression of SERPINB9 and MCF2L genes was downregulated. CONCLUSIONS: Changes in the expression of genes, associated with the development of somatic pathology in theremote period after irradiation, in particular the genes of the immune response and inflammatory reactions CSF2,IFNG, IL1B, TNF; expression of genes that regulate cell proliferation, aging and apoptosis TP53, BCL2, MCF2L, CDKN2A,SERPINB9, TERF1, TERF2, TERT; genes that regulate cell adhesion and angiogenesis CLSTN2, VEGF.

LUNG FUNCTION AND TELOMERE RELATIVE LENGTH IN CLEAN-UP WORKERS OF CHORNOBYL NPP ACCIDENT IN A REMOTE POST-ACCIDENT PERIOD. / FUNKTsIONAL'NYY̆ STAN BRONKhOLEGENEVOÏ SYSTEMY TA VIDNOSNA DOVZhYNA TELOMER V UChASNYKIV LIKVIDATsIÏ NASLIDKIV AVARIÏ NA ChAES U VIDDALENOMU PISLIaAVARIY̆NOMU PERIODI.

OBJECTIVE: The objective was to study the relationship between functional status of bronchopulmonary system and telomere length in clean-up workers of Chornobyl NPP accident in a remote post-accident period. MATERIALS AND METHODS: A study was performed in 113 clean-up workers of Chornobyl NPP accident. Individual do- cumented doses of irradiation in clean-up workers ranged from 1,0 to 880 mSv (330.4 ± 317.7 (M ± SD)). The aver- age age of the Chornobyl NPP participants was (62.21 ± 6.99) years. A complex of functional pulmonary tests (spirometry, body plethysmography, examination of lung diffusion capacity) was performed. Relative telomere length (RTL) was analysed by flow-FISH. RESULTS: There was a tendency to decrease the relative telomere length in clean-up workers with COPD I-II stage and COPD III-IV, compared with patients with the absence of bronchopulmonary diseases (RTL 15,2 ± 2,7). Significantly shorter telomeres were observed in patients with COPD who were exposed to radiation at a dose of more than 500 mSv (13.6 ± 2.5) compared with COPD patients who were exposed at a dose <10 mSv (RTL 15.3 ± 2.3). When analyzing the correlation relationships of the studied indicators, no significant associations were found with the relative telomere length. At this stage of the study no association of relative telomere length with age, body mass index, and functional criteria (FEV1 (l), intrathoracic pressure (ITGV), total lung capacity (TLC), diffusion lung capac- ity (DLCO)) was detected. CONCLUSIONS: The analyzed telomere length relationship from liquidators of the Chernobyl found no direct associa- tion with indicators of lung function tests, however, showed a trend towards reducing the relative telomere length in clean-up workers who suffer from COPD and exposed to doses from 100 to 500 mSv and above 500 mSv.

EXPRESSION OF BIOLOGICAL MARKERS INDUCED BY IONIZING RADIATION AT THE LATE PERIOD AFTER EXPOSURE IN A WIDE RANGE OF DOSES. / EKSPRESIIa RADIATsIINO-INDUKOVANYKh BIOLOGIChNYKh MARKERIV U VIDDALENOMU PERIODI PISLIa OPROMINENNIa U ShYROKOMU INTERVALI DOZ.

OBJECTIVE: To study radiation induced biological markers of the late period after exposure. SUBJECTS AND METHODS: A study was performed in 235 Chornobyl accident male clean-up workers exposed in 1986-1987 (doses of external exposure: (M ± SD: 419.48 ± 654.60; range 0.10-3,500 mSv); age 58,34 ± 6,57 years. Controlgroup included 45 non-exposed subjects (mean age: 50.60 ± 5.37 (M ± SD). Gene expression was performed by RT-PCR on 7900HT Analyzer using TLDA for BCL2, CDKN2A, CLSTN2, GSTM1, IFNG, IL1B, MCF2L, SERPINB9, STAT3, TERF1, TERF2,TERT, TNF, TP53, CCND1 genes. Relative telomere length (RTL) was analysed by flow-FISH; immune cell subsets,γ-H2AÐ¥ and CyclinD1 expression by flow cytometry. RESULTS: A statistically significant and dose-dependent decrease in expression of the BCL2, SERPINB9, CDKN2A, andSTAT3 genes was demonstrated in parallel to a dose-dependent overexpression of MCF2L and upregulation of TP53 (upto 100 mSv). IL1B expression was the highest in exposed to doses from 0.1 to 100 mSv with a negative correlationbetween at IL1B expression and CD19+3-, CD3-HLA-DR+, CD4+8- cell counts and CD4+/CD8+ ratio. Hyperexpression ofTNF gene in doses above 100 mSv to 1,000 mSv was shown, and in higher doses a combination of TNF downregula-tion with increase in IFNG gene expression were demonstrated with correlations with numbers of CD3+16+56+ andCD25+ lymphocytes and inhibition of expression CLSTN2. An increased expression of γ-H2AÐ¥ and Cyclin D1 corre-lated to radiation dose, telomere shortening to age and concommittant pathology. CONCLUSIONS: Cellular immunity, gene expression, telomere length, intracellular protein parameters are shown to beamong perspective biological markers at a late period after radiation exposure.

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.

Impact of chronic blood viral infection on lymphocyte telomere length in Chornobyl clean-up workers in a remote period after radiation exposure. / VPLYV KhRONIChNOÏ VIRUSNOÏ INFEKTsIÏ NA DOVZhYNU TELOMER LIMFOTsYTIV PERYFERYChNOÏ KROVI UChASNYKIV LIKVIDATsIÏ AVARIÏ NA ChAES U VIDDALENOMU PERIODI PISLIa OPROMINENNIa.

OBJECTIVE: To assess whether telomere length in lymphocytes of Chornobyl clean up workers at a late period 30 years after the exposure to ionizing radiation is influenced by a chronic blood viral infection and to determine role of viral carriage in cellular senescence. PATIENTS AND METHODS: Study group included 70 Chornobyl cleanup male workers 30 years after exposure {doses of external exposure (602.67 ± 114.19) mSv (M ± m); age (59.75 ± 0.82) yrs}. Relative telomere length (RTL) was analysed by fluorescence in situ hybridization and flow cytometry, immune cell subsets by standard combinations of monoclonal antibodies (CD45/14, CD3/19, CD4/8, CD3/HLADR, CD3/16/56, TCRγδ) and flow cytometry; antiviral immunity was performed determining the chronic phase antibodies to viruses: Hepatitis C (HCV), Cytomegalovirus (CMV), Toxoplasma gondii (TOX), Herpes simplex (HSV) and Epstein Barr virus (EBV VCA IgG and EBV NA IgG). The object of the study was peripheral blood (PB) of clean up workers. RESULTS: RTL changes were associated at the group level with the carrier state of the viral infection. RTL shortening was demonstrated as a significant difference between the groups (M ± SD) (HCV negative 15.27 ± 3.35, HCV posi tive 13.09 ± 3.05, p < 0.08, n = 12/52) or as a tendency (CMV negative 15.99 ± 5.41, CMV positive 14.86 ± 3.46 (M ± SD), p < 0.57, n = 11/53; HSV negative 17.01 ± 1.35, HSV positive 14.79 ± 3.80, p < 0.33, n = 13/51; TOX neg ative 15.94 ± 3.41, TOX positive 14.30 ± 3.81(M ± SD), p < 0.23, n = 27/37). These unidirectional changes can be associated with premature early cell aging of immune cells. To the contrary the significant RTL elongation was demonstrated in the group of EBV NA chronic carriers (EBV NA negative 11.25 ± 3.02 (M ± SD), EBV NA positive 16.15 ± 3.08 (M ± SD), p < 0.001, n = 15/49). CONCLUSION: The study confirmed the assumption on a relationship existing between the telomere length, chronic viral infection and late effects in immune cells. The changes of telomeres length on the background of immune dys function may be a sign of cellular aging, and concomitant chronic blood viral infection such as Hepatitis C, Epstein Barr viruses carriage could form a background for an error prone DNA reparation system as a factor of accumulation of pathological conditions, including malignant transformation.

Buccal mucosa micronuclei counts in relation to exposure to low dose-rate radiation from the Chornobyl nuclear accident and other medical and occupational radiation exposures.

BACKGROUND: Ionizing radiation is a well-known carcinogen. Chromosome aberrations, and in particular micronuclei represent an early biological predictor of cancer risk. There are well-documented associations of micronuclei with ionizing radiation dose in some radiation-exposed groups, although not all. That associations are not seen in all radiation-exposed groups may be because cells with micronuclei will not generally pass through mitosis, so that radiation-induced micronuclei decay, generally within a few years after exposure. METHODS: Buccal samples from a group of 111 male workers in Ukraine exposed to ionizing radiation during the cleanup activities at the Chornobyl nuclear power plant were studied. Samples were taken between 12 and 18 years after their last radiation exposure from the Chornobyl cleanup. The frequency of binucleated micronuclei was analyzed in relation to estimated bone marrow dose from the cleanup activities along with a number of environmental/occupational risk factors using Poisson regression adjusted for overdispersion. RESULTS: Among the 105 persons without a previous cancer diagnosis, the mean Chornobyl-related dose was 59.5 mSv (range 0-748.4 mSv). There was a borderline significant increase in micronuclei frequency among those reporting work as an industrial radiographer compared with all others, with a relative risk of 6.19 (95% CI 0.90, 31.08, 2-sided p = 0.0729), although this was based on a single person. There was a borderline significant positive radiation dose response for micronuclei frequency with increase in micronuclei per 1000 scored cells per Gy of 3.03 (95% CI -0.78, 7.65, 2-sided p = 0.1170), and a borderline significant reduction of excess relative MN prevalence with increasing time since last exposure (p = 0.0949). There was a significant (p = 0.0388) reduction in MN prevalence associated with bone X-ray exposure, but no significant trend (p = 0.3845) of MN prevalence with numbers of bone X-ray procedures. CONCLUSIONS: There are indications of increasing trends of micronuclei prevalence with Chornobyl-cleanup-associated dose, and indications of reduction in radiation-associated excess prevalence of micronuclei with time after exposure. There are also indications of substantially increased micronuclei associated with work as an industrial radiographer. This analysis adds to the understanding of the long-term effects of low-dose radiation exposures on relevant cellular structures and methods appropriate for long-term radiation biodosimetry.

Overexpression of TP53, TP53I3 and BIRC5, alterations of gene regulation of apoptosis and aging of human immune cells in a remote period after radiation exposure.

OBJECTIVE: To identify a contributive role of changes in gene regulation of apoptosis and telomere length at tran scriptional and translational levels to the formation of radiation induced effects in immune system. PATIENTS AND METHODS: Study groups included 310 Chornobyl (Chornobyl) cleanup workers (dose of external expo sure (360.82 ± 32.3) mSv; age 58.9 ± 0.6 (M ± SD) years) and control (n = 77; age (52.9 ± 0.64) (M ± SD) years). Expression of CD95, phosphatidylserine receptors, bcl2 and p53 proteins was studied by flow cytometry; the relative expression (RQ) of BAX, BIRC5, FASLG, MADD, MAPK14, TP53, TP53I3, TERT, TERF1, TERF2 genes was performed using 7900 HT Fast RT PCR System and TagMan technology. Relative telomere length (RTL) was quantified by flow FISH assay. RESULTS: Dose dependent deregulation of apoptosis was shown at transcriptional level (TP53, TP53 I3, BAX, BIRC5, FASL genes) and translational level (bcl 2 and p53 proteins) with blocking entry to apoptosis, dose dependent activation of anti apoptotic proteins and TP53 mediated expression of genes inhibitors of apoptosis. After exposure below 100 mSv a decrease in TERT gene RQ was associated with shortened telomeres, after exposure to doses over 500 mSv the TERT RQ and RTL increase were associated with imbalance in TERF1 and TERF2 genes expression. CONCLUSION: Our study demonstrates a presence of subsequent changes in gene expression, regulatory proteins pres entation, telomere length and distribution of cells by the stages of apoptosis in a late period after radiation expo sure from low dose range to doses over 500 mSv. Results of the study contribute to the basic concepts on the late biological effects in immune system.

Incidence of multiple myeloma among cleanup workers of the Chornobyl accident and their survival.

The study presents the updated data on the multiple myeloma (MM) incidence in Ukrainian cleanup workers after the Chornobyl accident and their survival. The epidemiological analysis is based on the extended follow-up period to identify new MM cases for higher statistical power and to collect additional data on the disease course and outcome for the survival analysis. The objective of the study was to analyze the MM incidence and survival in Chornobyl cleanup workers in 1996-2013 in comparison with the national MM statistical data. MATERIALS AND METHODS: A study cohort consisted of 152,520 male cleanup workers resided in one of 6 regions of Ukraine or Kyiv city and registered in the Ukrainian State Chornobyl Registry (SChR). The Cohort Database was linked to the Ukrainian National Cancer Registry (NCR) Database to identify MM cases and to analyze MM incidence in 1996-2013. Standardized incidence ratios (SIR) for MM over the period 1996-2013 were calculated as compared to the general population of Ukraine. A 10-year lag period (when incident cases are not considered radiation-related) was applied. One-year and 5-year cause-specific survival of MM cases were calculated as percentage of those who were alive correspondingly for 1 or 5 years after diagnosis among overall diagnosed. By the moment, the 5-year survival rate either for the sub-period 2008-2013 or for the whole study period 1996-2013 cannot be determined. Vital status data were updated according to the recent SChR and NCR information. RESULTS: 75 MM cases diagnosed in 1986-2013 were identified in the studied cohort. 69 of them were included to the incidence analysis considering a 10-year lag period. While the incidence over 15 years after the accident did not differ significantly from the corresponding rate in general population of Ukraine, a tendency to increase was seen in the period from 16 to 21 years after the accident, and significantly increased SIR estimate was revealed for 2008-2013 (SIR 1.86, 95% confidence interval (CI) 1.27-2.44). The highest incidence was detected in 2008-2013 among cohort members diagnosed over the age of 50 years mostly due to the significant excess among those aged 60-69 (SIR 2.46, 95% CI 1.32-3.59). Survival rates in cleanup workers were shown to be higher than in the general population of Ukraine (73.9% and up to 65.0%, respectively). CONCLUSIONS: SIR for the 2008-2013 period, 22-27 years after the accident, demonstrated the significant excess of MM incidence among male cleanup workers. Survival of MM cases is higher in cleanup workers in comparison with that in general population. This article is a part of a Special Issue entitled "The Chornobyl Nuclear Accident: Thirty Years After".