[Frequency of Lymphocytes with Mutations at the Locus of T-Cell Receptor in Residents of Radiation Polluted Bryansk Region 28 Years after the Chernobyl Nuclear Power Plant Accident].

The aim of this study is to assess the level of somatic mutagenesis according to the frequency of lymphocytes bearing mutations at the locus of T-cell receptor (TCR) in the residents of the Bryansk region contaminated with radionuclides as a result of the Chernobyl accident. The study was :conducted in 2014 in two regional centers - Klintsy and Novozybkov (average¹³7Cs pollution density of 322 and 708 kBq/m²,.respectively). The average frequency of the TCR-mutant cells in the total group of examined residents of the Bryansk region (n = 237) was not significantly different from that in the group of agematched control persons living in un- contaminated areas (n = 146): 3.8 x 10⁻4 vs 3.5 x 10⁻4, respectively (p = 0.84). However, after separation of examinees into 3 groups depending on age at the start of irradiation (at.the moment of the Chernobyl acci- dent) it was found that the average frequency of the TCR-mutant cells in the persons exposed in utero was 1.6 higher than that in the control group (p = 0.04). Proportion of persons with an increased frequency of the mutant cells (more than the age norm of this indicator) among prenatally exposed population reached 23.8%; which was about.4 times higher than in the control group (p = 0.04). Proportion of persons with an increased frequency of the TCR-mutant cells in group "0-17 years at the start of irradiation", was about 2 times higher than in controls, but this difference was not statistically significant (8.0% vs 4.3%, respectively, p = 0.33). In the third group "18 or more years old at the start of irradiation" we could not register the difference in the average frequency of theTCR mutant cells or the proportion of persons with an increased frequency of these cells in comparison with the age-matched control group. In general, comparison with earlier data shows that age-related regularities of somatic mutagenesis established 15-18 years after the Chernobyl accident persist at a later date (after 28 years in this study).

Formation of potential radiation risk groups to render timely targeted medical care: lessons of Chernobyl.

The paper discusses technology for establishing potential cancer risk groups, based on methods of molecular and radiation epidemiology. Assay of gene mutations at the T-cell receptor (TCR) locus as the method of molecular epidemiology was used for measuring the frequency of TCR-mutations in 320 nuclear workers of the Institute of Physics and Power Engineering (IPPE). The method of radiation epidemiology was applied to the estimation of attributable risk fraction (ARF) for solid cancers in these groups. The main estimates of radiation risk after the Chernobyl accident are in close agreement with the International Commission on Radiological Protection (ICRP) Publication, 103 models published in 2007. In nuclear workers of the IPPE with ARF ≥ 10%, the increased level of TCR-mutations occurs more often (risk ratio=9.7; 95% CI: 2.9; 32.1).

[Sensitivity of melanoma B16 side population to low- and high-LET radiation].

Cancer stem cells (CSC) found in multiple tumor types and cancer cell lines were shown to be more resistant to low-LET radiation in comparison to other cancer cells. Therefore, CSC are supposed to determine the long-term effect of cancer therapy. Research into the CSC sensitivity to high-LET radiation is of great interest because of the advances in hadron therapy. The aim of this investigation is to compare CSC and other cancer cell sensitivity to the low- (60Co gamma-rays) and high-LET (neutron) radiation. To identify CSC, we used the low cytometry-based side population (SP) technique based on the CSC capacity to produce the efflux of the vital dye Hoechst 33342. SP and non SP cells were sorted and exposed to gamma and neutron radiation at doses of 1-10 Gy and 0.1-4.7 Gy, correspondingly. We applied the colony-formation test to examine the SP and non SP survival rate after irradiation. It was shown that the sensitivity of SP to gamma-irradiation was lower than that of other cells: D0 average values (+/- SE) made up 2.3 +/- 0.3 Gy and 1.4 +/- 0.2 Gy, correspondingly (p = 0.047). The survival rate of SP and non SP did not differ after neutron irradiation. The values of relative biological effectiveness of neutron radiation relative to gamma-radiation at the D10 level were 2.6 for SP and 2.1 for other cells. The obtained results justify for the first time a high efficiency of application of neutrons in radiotherapy from the point of view of CSC elimination.

[The association of TCR-mutant cells with DNA polymorphism in women residing in radiation polluted regions of the Russian Federation].

Using flow-cytometric method the frequency of lymphocytes beaming mutations at T-cell receptor (TCR) locus was assessed in women residing in radiation polluted regions of Bryansk and Tula Districts. Simultaneously genotyping of the 8 polymorph loci for genes involved in detoxication of xenobiotics and oestrogen metabolism was carried out. The increased TCR-mutant cell frequency was found to be characteristic of homozygotes of the low activity appropriated enzymes for 3 loci (HFE187, GSTM1 and MTHFR) at least. This tendency was statistically significant in case of deletion polymorphism of the GSTM1 gene: TCR-mutant cell frequency of the homozygous carriers of a deletion at the GSTM1 locus was (4.63 +/- 0.18) x 10(-4) while it was (4.05 +/- 0.15) x 10(-4) in other groups of persons. The greatest mutant cell frequency was observed in carriers of the minor allele 4889G of the locus CYP1A. More often the increased values of the TCR-mutant cells (outside range "3sigma") were determined in women with genotypes A/G or G/G of the locus CYP1A1 (25%) than in carries of the normal genotype A/A (1.6%) (OR = 20.6; p = 0.0002). The comparison of the groups of women with reproductive system diseases reveals significant elevation in the mean TCR-mutant cell frequency in inhabitants of the most radiation polluted region among others.

[The changing of cell-free DNA properties of peripheral blood and TCR-mutant cell frequency in individuals exposed to ionizing radiation].

Some properties of the cell-free DNA (cfDNA) of peripheral blood plasma were assessed in 153 employees of atomic industry enterprises. The contents of ribosomal repeat (rDNA) and its concentration in plasma increased in cfDNA of the group of persons in comparison with non-irradiated individuals. The contents of satellite III in cfDNA of donors and of irradiated persons do not differ and less than in DNA nucleus. The correlation between cumulative dose of radiation, contents of rDNA in cfDNA and the frequency of lymphocytes bearing mutations at T-cell receptor (TCR) locus was obtained. The definition of three indications in irradiated persons: the contents of ribosomal genes in cfDNA, TCR-mutant cell frequency and concentration of ribosomal genes in blood plasma--may be useful for revealing individuals in organism of which an intensive cell apoptosis takes place and there is an increased probability of carcinogenesis and of progress of disease of immune system.

[The correlation between intracellular level of nitric oxide and frequency of gene somatic mutations after low dose radiation exposure].

The purpose of this work was the study of possible relationship between intracellular NO level and somatic mutagenesis after irradiation with low doses. The level of NO in peripheral blood lymphocytes and frequency of the TCR-mutant cells were assessed by flow cytometry in 64 workers of atomic industry with mean dose (+/- SE) 114.9 +/- 10.8 MSV, accumulated within 21.4 +/- 1.1 years, and 66 age- matched control donors. The mean frequency of the TCR-mutant cells in this groups was (6.1 +/- 1.0) x 10(-4) and (4.1 +/- 0.2) x 10(-4) respectively (p = 0.06). 14% of workers of atomic industry had the TCR-mutant cell frequencies exceeding the 95% confidence interval in control donors. It was found the positive correlation between the intracellular NO level and the TCR-mutant frequency (R = 0.36, p < 0.01). The mean level of NO in individuals with the elevated TCR-mutant frequency was significantly higher than in others: 1619 +/- 57 vs 1340 +/- 40 relative units (p = 0.01). The results suggest that nitric oxide may come into elevating frequency of the mutant cells in some proportion of individuals exposed to low doses of ionizing radiation not excepting formation of genome instability.

[Primary prevention of cardiovascular diseases: long term results of five year long preventive intervention in 12-year old boys (ten year prospective study)].

In a longitudinal cohort (prevention group, n=213, comparison group, n=163) of 10-year prospective follow-up we addressed efficacy of 5-year-long multifactor preventive intervention, conducted in a sample of population of 12 year old boys. Preventive intervention was carried out both at populational level and among persons with risk factors of development of cardiovascular diseases with the use of group, individual, and partly family approaches, and was directed at rationalization of nutrition, elevation of physical activity and prevention of harmful habits. During first 3 years of prevention we succeeded to achieve stable statistically significant lowering of mean levels of total cholesterol, low density lipoprotein cholesterol, triglycerides, and atherogeneity index, as well as to affect fatty component of body mass (skinfold thickness). Long term effect of 5-year long preventive intervention manifested as significantly lower level of systolic blood pressure, lower prevalence of low levels of high density lipoprotein cholesterol, smaller increment of low density lipoprotein cholesterol and index of atherogeneity in the prevention group. These results evidence that prevention of main factors of risk of development of cardiovascular diseases (obesity, arterial hypertension, disorders of lipid composition of the blood, and low physical activity) in child and adolescent age in the period of active growth and development is feasible, effective, safe and is able to lead to decrease of levels of these factors in adults, but should last uninterruptedly until formation of stable habits of healthy life style.

[Somatic mutagenesis at T-cell receptor locus in inhabitants of radiation polluted regions as a result of the Chernobyl disaster].

In the period of 2001-2004, frequency of cells bearing mutations at T-cell receptor (TCR) locus was assessed in 553 inhabitants of radiation polluted regions of the Russian Federation and 154 unexposed control persons. The inhabitants were divided into three groups according to age at the moment of the Chernobyl disaster and 137Cs pollution density: 1) in utero, 37-555 kBq/m2; 2) 0-14 years old, 20-555 kBq/m2; 3) 18 and more years old, highest 137Cs density (185 more than 555 kBq/m2). The most intense changes of the TCR-mutant cell frequency were observed in the group of persons exposed to ionizing radiation in utero. The mean frequency of the mutant cells was higher in the first group than in age-matched control group by about 1.5-fold: 4.0 x 10(-4) vs 2.7 x 10(-4) accordingly (p < 0.0001). Elevation in the mean TCR-mutant cell frequency was less expressed in group of inhabitants aged 0-14 years at the moment of irradiation start: 1.3-fold increase in comparison to age-matched control (3.8 x 10(-4) vs 2.9 x 10(-4), p = 0.0002). It was not found significant differences in mutant cell frequencies between control group and adults consisting in the third group (18 and more years old at the moment of the Chernobyl accident). The changes of the TCR-mutant cell frequency in persons exposed in pre- and postnatal periods differ not only quantitatively, but qualitatively. In the fist case all persons react to irradiation by increasing number of the TCR-mutant cells in some degree. In the second case - only a part of population. Proportion of reacting persons depends on age at the start of irradiation and, perhaps, on dose absorbed. The TCR-mutant frequency was significantly higher in persons with benign tumors of different localizations and nodules in thyroid gland than in persons without this pathology.

[The comparative analysis of gene and structural somatic mutations in inhabitants of Orel district areas contaminated with radionuclides as a result of Chernobyl accident].

The results of comparative analysis of gene and structural mutations found in peripheral blood lymphocytes of inhabitants of Orel district areas contaminated with radionuclides as a result of Chernobyl accident are presented. The average level of 137Cs contamination in those areas ranged about 22-113 kBq/m2. In the study group was found the enhanced frequency of somatic cells with gene and structural mutations compared with laboratory control level by synchronous applying a T-cell receptor (TCR) loci mutation assay and cytogenetic analysis of unstable aberrations. The case-control comparison was carried out using the measured mutation frequencies and cases of various thyroid gland sickness recognized by ultrasonic examination. The cytogenetic assay did not show the statistical difference between healthy group and subjects with thyroid gland sickness. The average frequency of TCR loci mutation cells in the subjects with thyroid gland sickness was found to be statistically higher comparing with healthy persons. This finding was true for each study region and for Orel district in total. The subgroup of subject exposed in utero in 1986, soon after accident was analyzed. Both cytogenetic and TCR loci mutation assays shown enhancement of average mutation frequency in somatic cells in the subjects of this subgroup with thyroid gland sickness comparing with healthy persons.

[Frequency of mutant lymphocytes in T-cell receptor locus as a possible predictor of thyroid cancer development in irradiated and unirradiated persons].

Was compared frequency of lymphocytes mutant at loci of T-cell receptor (TCR) from samples of peripheral blood taken from 186 healthy donors and 46 untreated thyroid cancer patients, including the persons exposed to ionizing radiation as a result of inhabitation in radioactive polluted region of Russian Federation. Was shown that the cell mutation rate within thyroid cancer group was significantly higher than the same parameter for the healthy person with similar age distribution (p < 0.01). It could be a result of such factors as genotoxic influence, different sensitivity or possible genome instability (including radiation-induced). It was found that 37% of patients have the increased frequency of somatic mutation cells, i.e. it exceeded 95% confidence interval for the screening group. The presented results cause to anticipate that TCR-test could be used as one of criteria for formation groups of high cancer risk development.

[Comparative investigation of structural and gene somatic mutations in workers of nuclear chemical plants. II. Frequency of lymphocytes mutant in T-cell receptor loci].

Frequency of lymphocytes mutant at T-cell receptor (TCR) loci was defined in 42 workers of nuclear chemical plants. In 11 persons mainly exposed to external radiation the mean frequency of TCR-mutant lymphocytes was statistically significant by higher compared with control group of unexposed donors: 9.1 x 10(-4) vs 3.5 x 10(-4) correspondently (p < 0.01). Frequency of TCR-mutant lymphocytes did not correlate neither the frequency of structural mutations non doses of external exposure. In group of workers exposed to combined external and internal radiation (n = 31) the average frequency of TCR-mutant lymphocytes was higher compared with control level: 8.9 x 10(-4) vs 3.5 x 10(-4) correspondently (p < 0.01). Correlations between the frequency of TCR-mutant cells and Pu content in organism (r = 0.5; p = 0.005) and between the frequency of chromosome aberration of unstable and stable types (r = 0.5; p = 0.002 and r = 0.6; p = 0.036, correspondently) were set. Comparison of results of analysis of structural and gene mutations allows us to supose that in case of external exposure the observed disturbances can result from genome instability in remote period after irradiation. In case of combined exposure the genetic changes were possibly caused by the constant action of alpha-radiation from Pu containing in the body.

[Use of thymodepressin for treating autoimmune cytopenia]. / Primenenie timodepressinoa dlia lecheniia autoimmunnykh tsitopenii.

AIM: To study influence of thymodepressin on the course of autoimmune cytopenia. MATERIAL AND METHODS: Thymodepressin is a new synthetic hemoregulatory dipeptide (gamma-D-Glu-D-Trp). It was used for the treatment of 22 patients with autoimmune cytopenia. RESULTS: Hemoglobin levels were elevated in autoimmune hemolytic anemia and platelet levels were high in idiopathic thrombocytopenic purpura. A thymodepressin course resulted in a fall of total lymphocyte count and activated CD3+CD69+ lymphocytes. CONCLUSION: The above results, safety, absence of toxicity and allergenicity, parenteral and intranasal useability open perspectives for further studies of therapeutic action of thymodepressin as an immunodepressant in autoimmune processes.

[Mutation at the T-cell receptor locus in people a long time after acute and prolonged irradiation]. / Mutatsii po lokusu T-kletochnogo retseptora u liudei v otdalennye sroki posle ostrogo i prolongirovannogo oblucheniia.

The frequency of lymphocytes with mutations at genes of T-cell receptor (TCR) is determined in 165 persons exposed to ionizing radiation 16-40 years after the influence. Depending on a type of irradiation and time, elapsed from the moment of exposure, all inspected persons were divided into 3 groups: 1) the analysis performed 16-40 years after acute irradiation; 2) the analysis--9-13 years after acute irradiation; 3) the analysis--9-13 years after prolonged irradiation. The persons with elevated frequencies of TCR-mutant cells were detected in all three groups. In the first group they have compounded 36% of persons inspected, in the second--25%, in the third--15%. The percentage of persons with elevated frequencies of mutant cells in all groups was significantly higher than in control groups. Taking into account fast enough elimination of radiation-induced TCR-mutant cells it is possible to suspect, that the cause of elevated frequency of mutant cells in the part of the persons can be genome instability.

[Apoptosis of peripheral lymphocytes of healthy donors and patients with laryngeal neoplasms after gamma-irradiation in vitro]. / Apoptoz limfotsitov perifericheskoi krovi zdorovykh donorov i bol'nykh rakom gortani posle vozdeistviia gamma-izlucheniia in vitro.

Apoptosis in peripheral blood lymphocytes of healthy donors and cancer patients after gamma-irradiation with different doses was studied by the flow cytometry method. Wide intra- and interindividual variabilities of the lymphocyte radiosensitivity were observed. The radiosensitivity did not depend on the subpopulation composition of the lymphocyte pool. The persons with very low and high lymphocyte radiosensitivities were found significantly more often among the cancer patients than among the healthy donors. One can suggest that this method is useful as a biomarker of future cancer risk and prognosis of radiotherapy efficiency.

Determination of somatic mutant frequencies at glycophorin A and T-cell receptor loci for biodosimetry of acute and prolonged irradiation.

Somatic mutant frequencies at glycophorin A (GPA) and T-cell receptor (TCR) loci were assessed. The dependence of the GPA mutant frequency on doses of acute and prolonged irradiation was shown. In the case of acute irradiation the GPA mutant frequency displayed a three-fold greater dose-related increase as compared to prolonged irradiation. A dose-dependent increase in the TCR variant frequency was found only in a group of subjects with recent exposures. In Chernobyl clean-up workers the TCR mutant frequency was significantly higher than in control non-irradiated individuals.

[A noninvasive approach to studying fetal cells for prenatal diagnosis of chromosomal aneuploidies]. / Neinvazivnyi podkhod k issledovaniiu kletok ploda v tseliakh prenatal'noi diagnostiki khromosomnykh aneuploidii.

Fetal cells isolated from maternal peripheral blood during the second trimester of pregnancy were analyzed. Blood samples were centrifuged in a Ficoll-Paque gradient, the mononuclear cell fraction was isolated and stained with fluorescent monoclonal antibodies against glycophorine A (GPA + PE), transferrin (CD71 + FITC), and Hoechst 33342. Fluorescence-activated cell sorting (FACS) was conducted on a Vantage flow cytofluorimeter (Becton Dickinson). Fluorescence in situ hybridization (FISH) with Y chromosome-specific DNA probe revealed fetal cells that exhibited Y signal in all 20 blood samples obtained from women pregnant with healthy male fetuses. The concentration of these fetal cells averaged about 1.34% and ranged from 0.1 to 4.2% in different blood samples. In six cases, blood samples were obtained from pregnant women, in which prenatal cytogenetic analysis revealed various fetal aneuploidies. Using FISH with DNA probes specific for chromosomes X, 18, and 13/21, Fetal cells with chromosomal aberrations were detected in these six maternal blood samples at a concentration from 1.5 to 5.6% (on average 3.7%). These results indicate the possibility of a new noninvasive approach, which is safe for both mother and fetus when used for isolation of fetal cells from pregnant women's blood samples and prenatal diagnosis of a broad spectrum of fetal cell chromosomal aberrations.

Determination of somatic mutant frequencies at glycophorin A and T-cell receptor loci for biodosimetry of prolonged irradiation.

PURPOSE: To determine the variant frequencies (VF) at glycophorin A (GPA) and T-cell receptor (TCR) loci in persons exposed to prolonged ionizing radiation at different doses and to assess the significance of the GPA and TCR assays for biodosimetry of prolonged irradiation. MATERIALS AND METHODS: The VF values were determined by means of flow cytometry in 120 persons exposed between 1968-1996. Most exposures were in Chernobyl clean-up workers in 1986-1987. RESULTS: A significant correlation was shown between the NO GPA variant cell frequency and dose (r = 0.61, p < 0.0001). The slope of the linear regression was 6.3 x 10(-6) NO mutant cells/Gy. Dose-dependent increase in the TCR VF was found in the group with recent exposures (slope 2.1 x 10(-4) variant cells/Gy, r = 0.75, p = 0.0002). In the Chernobyl clean up workers who received doses less than 0.25 Gy the TCR VF unlike the GPA VF was significantly higher than in the control non-irradiated individuals (p < 0.01 and p > 0.05 respectively). CONCLUSIONS: The GPA assay has limited potential to be used as a biodosimeter of prolonged irradiation, at least in dose interval up to 2.0 Gy. The TCR assay is likely to have greater potential in estimation of recent radiation exposure than the GPA assay.

[Determination of mutation frequency at loci of glycophorin A and T-cell receptors: informativeness for biological dosimetry of acute and prolonged irradiation]. / Opredelenie chastoty mutatsii po lokusam glikoforina A i T-kletochnogo retseptora: informativnost' dlia biologicheskoi dozimetrii ostrogo i prolongirovannogo oblucheniia.

The frequencies of somatic mutations at loci of glycophorin A (GPA) and T-cell receptor (TCR) were determined in persons exposed professionally to ionizing radiation or a result of accidents at nuclear power plants and in control donors. Dependence of glycophorin A mutant (NO) cell frequency on doses of acute (up to 3.5 Gy) and prolonged (up to 15.0 Gy) irradiation was shown. The slope of linear regression corresponded to increase of NO-mutant frequency by 31.1 x 10(-6)/Gy (r = 0.87, p < 0.0001) for acute irradiation and by 6.3 x 10(-6)/Gy (r = 0.61, p < 0.0001) for prolonged one. The 5-fold decrease of the linear regression slope in the case of prolonged irradiation makes significantly worse permissive ability of the GPA test. Therefore its use for biological dosimetry of prolonged irradiation is not expedient in dose interval up to 1 Gy. The frequency of mutations in genes of T-cell receptor significantly correlated with dose of irradiation only in group of donors with recent radiation exposure (r = 0.75, p = 0.0002). Meanwhile, the TCR method is more sensitive and informative for biological dosimetry of recent radiation, than the GPA test.

[Determination of mutation frequency at loci of glycophorin A and T-cell receptor: study of Chernobyl AES accident cleanup workers]. / Opredelenie chastoty mutatsii po lokusam glikoforina A i T-kletochnogo retseptora: obsledovanie likvidatorov avarii na CHAES.

The frequencies of somatic mutations at loci of glycophorin A (GPA) and T-cell receptor (TCR) were determined in control unexposed donors and Chernobyl clean up workers, who received low doses of irradiation up to 0.25 Gy. High variability of mutant rates for two investigated genes was shown in the clean up workers. No significant difference in the GPA (NO) mutant frequencies was observed between the clean up workers and control donors (p > 0.05), though there is a tendency for increasing the GPA mutation rate in the clean up workers. Meanwhile, the TCR mutation rate was significantly increased the clean up workers (p < 0.01), perhaps because of acceleration of spontaneous mutagenesis and possible genome instability. Persons with elevated levels of mutations at two loci can present a group at high risk in respect to oncological diseases.