Changes in the Activity of Genes Involved in the Regulation of Hematopoiesis during Tumorigenesis in Irradiated Mice.

We used 183 F1 CBA×C57Bl hybrid mice to study the delayed effects of low-power long-term γ-irradiation at a dose of 12.6 Gy (10 mGy/min) 8 and 10 months after the treatment. Eight months after the treatment we found the increased expression of the transcription factor NFκB and its target genes iNOS and G-SCF in the bone marrow (BM). Ten months after the treatment malignant lymphomas were revealed in 14 of 94 mice in the liver, abdominal cavity, and subcutaneously. In the BM of these mice, the transcription of the PTEN, NFκB, and iNOS genes was inhibited and the contents of long non-coding RNAs (lncRNAs) lnc p21, NEAT1, and microRNA miR-125b were decreased. The expression of the NFκB(p65) gene and miR-125b was inhibited in the BM of irradiated mice without tumors ten months after the treatment. These data show the deregulation of the P53 system supporting the genome stability in the BM of irradiated mice. These indices will be studied as potential markers of risk of development of irradiation-induced tumors.

Investigation of Thionins from Black Cumin (Nigella sativa L.) Seeds Showing Cytotoxic, Regulatory and Antifungal Activity.

Thionins (NsW1 and NsW2), earlier isolated from the seeds of endemic Middle-Asian black cumin (Aligella sativa L.), showing signilicant inhibitory action on some bacterial and yeast pathogens were investigated for cytotoxic properties against several tumor cell lines (AsPC-1, Colo357, RD and Jukart) in vitro within nano- and micromolar ranges of the active concentrations and as modulators of expression of the genes controlling conversion of normal cells to malignant ones. Suppression of the expression of the genes from MMP, RhoA, miR21 families in human rhabdomyosarcoma (RD) cells was observed, whereas the influence of the molecules on the genes in normal blood cells was not identified. It was shown that the thionins from black cumin induced almost 90% of the cell death in RD and Jukart lines. Moreover, the polypeptides inhibited clinical isolates of Aspergillus ochraceus and A.fimigatus at the level comparable with that of amphotericin B. The data demonstrated that the peptides could be considered as perspective antitumor and antimycotic agents.

[Searching Radiation Countermeasures using the Model of Prolonged Irradiation of Mice with Low Dose Rate and Evaluation of Their Influence on Heat Shock Protein Genes Expression].

Different radiomodificators (cytokine betaleukine, antioxidant phenoxan, antigipoksant limontar and nucleoside riboxin) were investigated on mice for evaluating their radiation protective capacity against prolonged (21 h) exposure at a dose of 12.6 Gy at a low dose rate of 10 mGy/min. Bone marrow cellularity and endogenic CFUs were used as evaluation criteria 9 days after exposure. Simultaneously, expression of the heat shock proteins of 25, 70 and 90 kDa in unexposed mice bone marrow was studied 2, 24 and 48 h after injections. Betaleukine only had a positive significant effect in both tests in the variants of 50 mcg/kg and 3 mcg/kg when administered 2 h and 22 h before exposure, correspondingly. Effects of betaleukine HSPs on expression were both stimulating and inhibiting, that was in contradiction with a constant positive effect in 5 experiments on exposed mice for each betaleukine variant. It argues against the vital role of HSPs in the betaleukine antiradiation effect. In 2 experiments with high temperatures betaleukine administered at a dose of 50 mcg/kg evoked a very high HSP-70 gene expression after 24 h, and mice exposed to irradiation at that time in a parallel experiment showed an increased radiation effect. It corresponds to the idea that HSPs serve a stress indicator.

[Comparative analysis of natural and synthetic antimutagens as regulators of gene expression in human cells under exposure to ionizing radiation].

This paper studies the effect of plant peptides of thionine Ns-W2 extracted from seeds of fennel flower (Nigella sativa) and ß-purothionine from wheat germs (Triticum kiharae), as well as a synthetic antimutagen (crown-compound), on the expression of several genes involved in the.control of cellular homeostasis, processes of carcinogenesis, and radiation response in human rhabdomyosarcoma cells (RD cells), T-lymphoblastoid cell line Jurkat, and blood cells. All of these agents acted as antimutagens-anticarcinogens, reducing the expression of genes involved in carcinogenesis (genes of families MMP, TIMP, and IAP and G-protein genes) in a tumor cell. A pronounced reduction in the mRNA level of these genes was caused by thionine Ns-W2, and the least effect was demonstrated by ß-purothionine. Antimutagens had very little effect on the mRNA levels of the several studied genes in normal blood cells.

[Expression of P53, NPM1, Kras, c-Myc, p14(ARF) genes in blood cells of cancer patients before and after radiation therapy].

The mRNA levels of P53gene, as well as NPM1, Kras, c-Myc, p14(ARF) genes, which, according to the published data, code for the proteins regulating the p53 activity, were studied using RT-PCR method in blood cells of patients with different localization of tumor process (prostate cancer, breast cancer and head and neck cancer) before and after application of radiation therapy. Changes in gene expression of cancer patients were compared with the control group of healthy donors. We have established that all patients had a decreased level of the Kras gene expression even before radiotherapy; moreover, the group of patients with prostate cancer had a low content of mRNA in NPM1 and p14(ARF), and the group of patients with head and neck cancerhad a reliably reduced mRNA in P53, NPM1 and p14(ARF). The radiation therapy did not cause essential changes in the expression of these genes of cancer patients, ecpect for the Kras gene, whose the mRNA level in the group of patients with head and neck cancer was reliably lower than the mRNA level prior to beginning of radiation therapy. The correlations of P53, NPM1, Kras, p14(ARF) gene expression were studied. We have shown that p14(ARF) mRNA level negatively correlates with Kras mRNA (R = -0.6, p = 0.002) and P53 mRNA levels (R = -0.49, p = 0.013) in the control group of healthy donors. A positive correlation was observed between P53 mRNA and NPM1 mRNA (R = 0.54, p = 0.006). Similar correlations between mRNA levels of these genes in blood cells were absent in the cancer patients before radiotherapy. After radiotherapy in patients with prostate cancer, p14(ARF) mRNA level positively correlated with NPM1 mRNA (R = 0.7, p = 0.001) and negatively with Kras mRNA (R = - 0.5, p = 0.03). Our results provide evidence that expression P53, NPM1, Kras and p14(ARF) genes may be coordinated in blood cells of healthy donors. The low expression levels of the studied genes in patients can contribute to the increase in the mutation changes in blood cells of the examined subjects after the action of genotoxic factors.

[The peculiarities of polymorphism of XPD and XRCC1 repair genes in cells of Down and Ehlers-Danlo syndrome patients characterized by increased radiosensitivity].

Codon 312 and 751 polymorphisms ofXPD gene and codon 399 polymorphism of XRCC1 gene of peripheral blood lymphocytes in patients with Down syndrome (DS) (46 individuals), Ehlers-Danlo syndrome (EDS) (47 individuals) and in a group of healthy donors (control) (40 individuals) have been studied. Frequency of XPD genotype (G312G) coding for the most effectively functioning form of XPD protein was lower in patients with DS (26%) than in a group of healthy donors (42.5%) (p = 0.035), whereas no significant differences with the control were revealed for this codon in patients with EDS. No patients with XPD genotype (C751C) (p = 0.036) were revealed in a group of EDS patients, while this genotype was found in 16% of a group of healthy donors and in 17% of patients with DS. The trend of XRCC1 genotype frequency reduction (A399A) (p = 0.085) in EDS patients (3.9%) compared with the group of healthy donors (13.5%) and DS patients (13.3%) has been obtained. These data show that polymorphisms of the excision repair genes under study are accompanied by an elevated individual radio sensitivity in patients with DS. Genes investigated (their polymorphic variants) did not participate in the mechanisms for radio sensitive phenotype formation in EDS patients.

[Investigation of detoxification polymorphisms genes, methylenetetrahydrofolate-reductase (MTHFR) and P53 in the radiosensitive human cells].

The genes of detoxication, MTHFR and p53 were studied in Down' and Ehlers-Danlos syndrome cells. The frequency GSTM1(0/0) genotype in Down syndrome patients was in 1.5 times higher than in control cells (p < 0.069). Opposite the frequency GSTM1(0/0) genotype in Ehlers-Danlos syndrome was 23.3% 2 times lower than in control cells (p < 0.034). This indication was in 2 times lower in women cells than in men cells and in 3 times lower than in control cells (p < 0.026). The mutations of p53 gene (7th exon) were detected in 4 from 11 Down patients (36.7%; in 2 cases af women and men), in Ehlers-Danlos patients--in 5 cases and only in men (29.4% among all the observed patients). The observations 24 healthy donors weren't revealed any mutations (p < 0.013-0.001). The hypothesis about the connection between gene polymorphisms which take a part in genome stability and radiosensitivity in Down and Ehlers-Danlos patients was developed.

[Research of mutations in oncogenes and tumour suppressor genes as the approach to research of ways of the individual prognosis of delayed effects of exposure to ionizing radiation].

The frequency of mutant forms p53 and N-Ras genes was investigated in DNA from peripheral blood of the patients by method Polymerase Chain Reaction - Single Strand Conformation Polymorphism Analysis. The patients were investigated in late period after radiation accidents exhibited acute radiation syndrome. It is established that the mutations among patients in areas of codons 246-250 exon 7 of p53 gene and codon 12 of N-Ras gene were meet more often than in control group. It is shown that these mutations possibly arise in insignificant number of the cells with the radiation-induced genomic instability. Possibility of use of mutations in protooncogenes and tumour suppressor genes as markers of risk of development of the main thing from delayed effects of exposure to ionizing radiation - malignant tumours is discussed.

[Autoimmune processes after long-term low-level exposure to electromagnetic fields (the results of an experiment). Part 4. Manifestation of oxidative intracellular stress-reaction after long-term non-thermal EMF exposure of rats].

This paper presents the results of the study of the effects of long-term low-level exposure of rats to microwaves. Rats were exposed in far field to 2450 MHz continuous wave fields providing an incident power density at the cages of 500 microW/cm2 for 7 hours daily for a total of 30 days resulting in a whole-body SAR of 0.16 +/- 0.04 W/kg. Three groups ("EMF-exposure", "sham-exposure" and cage-control) were formed, each consisting of 16 rats. Circulating antibodies (IgA, IgG and IgM) directed against 16 chemical substances were evaluated in coded serum from each group of rats by enzyme multiplied analysis (ELISA test). An increased amount of compounds resulting from interaction of amino acids with nitric oxide (NO) or its derivatives (NO2-Tyrosine, NO-Arginine, NO-Cysteine + NO-Bovine Serum Albumin, NJ-Methionine + NO-Asparagine + No-Histidine, NO-BTrypnohan + NJ-Tyrosin), fatty acids with small chains, hydroxylated fatty acids, palmitic/myristic/oleic acid, AZE (product of oxidation of fatty acids) was found in blood serum from EMF-exposed rats. As a rule, antibodies to conjugated antigens were seen for IgM, rarely seen for IgG and were completely absent for IgA. The levels of antibodies were higher on day 7 after the exposure compared to those on day 14 after the exposure.

[Gene polymorphisms in patients with Down's syndrome].

Polymorphisms of glutation-S-transferase (GSTM1, GSTT1 GSTP1) and methylentetrahydrofolate reductase (MTHFR) genes have been studied in DNA from blood lymphocytes of 18 patients with Down's syndrome and 61 controls. Frequencies of normal alleles of GST genotypes were lower in patients as compared to the controls. A DNA analysis of 11 patients and 17 controls revealed the presence of mutations in region 246-250 of exon 7 of the p53 gene in 4 patients. Mutations were not found in the control group. Due to the small sample size, the results of this study should be interpreted with caution and need replication in larger studies.

[The comparison of molecular-biochemical and cytogenetic analyses of blood cells of patients long after the acute radiation sickness].

Molecular-biochemical and cytogenetic analyses were made on blood cells of 17 radiation accident victims who, from 1.7 to 43.8 years previously, had suffered acute radiation sickness (ARS) ranging from severity grades I to IV. Molecular-biochemical data were obtained with patients' leukocytes and with mononuclear cells on their oxidative status by a) the level of an anion-radical O2*- in the 3-(4,5-dimethyl-2-thiazol-2-yl)-2,5-diphenyl-2-tetrazolium bromide (MTT) test and b) the sum of reactive oxygen species in the 2,7-dichlorodihydrofluorescein diacetate (DCFDA) test together with a test for DNA strand unwinding in alkaline conditions by measurements of fluorescence intensity of ethidium bromide. Each parameter was measured in freshly sampled cells and during a 5-hour incubation as absolute means on 5 measurements and in % change from the initial values. Cytogenetic data were obtained from the standard metaphase preparations scored for routine unstable chromosomal aberrations (us-CA)--dicentrics; and stable aberrations (sCA)--translocations. The latter ones were assayed by the FISH method using whole chromosome 2, 4 and 12 fluorescent probes and scaled up to genome equivalence. For all patients reduced oxidative status of about 25-30% was obtained by the MTT-test (p < 0.005), and the DCFDA-test (p < 0.027). The yield of usCA depended on the time after irradiation with higher yields associated with the shorter postirradiation times and reducing almost to expected background frequencies. The yield of sCA was high for all patients, correlating with the severity of ARS whilst the molecular-biochemical parameters showed no relationship with ARS. A correlation was observed between parameters of oxidative status and % of cells with usCA: by the MTT-test r = from 0.50 up to 0.61 (p = from 0.06 up to 0.003), but by the DCFDA-test the strength of correlation was smaller: r = from 0.38 up to 0.48. An inverse correlation was found between initial oxidative state of mononuclear cells and the frequency of CA in lymphocytes. Similarly a marked inverse correlation between degree of DNA unwinding by the ethidium bromide assay on leukocytes and sCA in lymphocytes was also noted. The feasibility of radiation-induced delayed genomic instability in vivo for humans long time after irradiation is discussed.

[The individual prognosis of the gravity and of the outcome of acute radiation disease based on immunological indexes].

The information significance of the immunological indexes for the prognosis of gravity of course and of outcome of an acute radiation disease for the people after the exposure of ionizing radiation in clinically significant doses is studied. The value of indexes of the C-reactive protein contents, of the complement contents and of the titer of haemagglutinins in serum of a blood of 147 patients damaged at Chernobyl NPP accident as a result of external radiation gamma-exposure in combination with internal irradiation from the incorporation in an organism predominantly beta-emitting radionuclides were compared to the weight of acute radiation disease and its outcome (survival or loss). Was determined, that indexes of the contents of C-reactive protein in a peripheral blood during primary reactions on the irradiation (1-2 day after irradiation) and in latent period of disease (3-9 day after irradiation), and also titer of a complement on 3-9 day after irradiation can serve a source of information for the prognosis of probable gravity of a radiation injury and its outcome at irradiation of the man in clinically significant doses.

[Molecular manifestations of radiation-induced genome instability: the possibility of chemical modification].

The molecular manifestations of radiation-induced genome instability-changes of the DNA structure, the excision DNA repair and the contents of the reactive oxygen forms in bone marrow cells of the repair proficient mice (CBA) and of the repair-defective (101/H) lines in the dynamics up to 185 day after ionizing radiation exposure in the dose of 1.5 Gy were studied. Is was established, that after irradiation in bone marrow cells the descendants with the decreased activity of excision DNA repair and prone to increased changes of DNA structure DHK is arised. The injection of the phenozane in concentrations causing its receptor interaction with cells, did not defend DNA of the bone marrow cells from the radiation injury after the exposure in a sublethal dose, however it exerted influence on long-term changes. Due to the phenosane of the bone marrow cells of the irradiated mice of CBA line exhibited the larger activity in a DNA repair from damages and maintenance of vitality. The bone marrow cells of male mice of repair defective 101/H line, which phenozan was entered before the irradiation, remained unfit to the remuval of DNA damages by the repair, that probably resulted the activations of the program of the maintenance of genome constancy by the apoptosis in the cells--carriers of the structural defects and the cause of animal lethality.

[Sexual differences in a state systems of DNA structure maintenance and generation of reactive oxygen species in somatic cells of mice 101/H reparation-defective strain and manifestation of these differences after exposure to ionizing radiation]. / Polovye razlichiia sostoianiia sistem podderzhaniia struktury DNK i generatsii aktivnykh form kisloroda v somaticheskikh v somaticheskikh kletkakh u myshei repearatsionno-defektivnoi 101/H i ikh proiavleniia pri vozdeistvii ioniziruiushchei radiatsii.

To analyse a role of the factor of a genetic fundamentals of cells in formation of radiation-induced genome instability (RIGI) we investigated a condition of DNA pattern, content of superoxide anion-radical O2*- and a sum of reactive oxigen species (ROS) (O2*-, OH*, H2O2), and also catalase activity in bone marrow cells of male and female mice of 101/H strain in the norm and at once after chronic (10 day) exposure to 200 mGy gamma-radiation. Thus we based on conception about a significance of mechanisms of DNA repair and production of reactive oxygen species (ROS) in development of radiation-induced genome instability (RIGI), and also on the data on sex bound differences in efficiency of DNA repair in reply to impact of the genotoxic agents for male and female mice of 101/H strain. Sex connected differences in redox system of bone marrow cells were established. In males lower catalase activity was found in the norm, with considerable increase of the activity and the content of ROS after chronic irradiation with a low dose (200 mGy); at the same time a direct correlation between the ROS content and catalase activity occurred. In female, which have higher DNA repair potential, higher level of catalase activity was found in the norm, with reduction after irradiation and lower, than in male, level of O2*- content; no changes in the general ROS content, or direct correlation between the content of a superoxide anion-radical (O2*-) and the sum of ROS were observed. The detected differences between male and female the studied parameters in the norm and after irradiation indicate a connection of the studied characteristics and their changes with a sex, confirm the literature data about a significance of the factor of a genetic fundamentals of bioobject in formation of radiation-induced genome instability.

[Molecular epidemiology of the late radiation effects]. / Molekuliarnaia épidemiologiia otdalennykh radiatsionnykh éffektov.

The scientific bases, problems, methods and prospects for the development of a new scientific direction "Molecular epidemiology of late consequences of ionizing radiation influence on the man" are reviewed. It is marked, that for two decades on the basis of achievements in the field of molecular biology, biochemistry, genetics and genomics it has arisen and is developing the important direction in infectious and non-infectious epidemiology--molecular epidemiology. Molecular epidemiology is a new section of epidemiology on border with molecular biology and genetics, integrating efforts of epidemiologists, molecular biologists, genetics and researchers from many other areas of knowledge in a direction of an estimation of individual risk of development of chronic diseases at practically healthy people, development of recommendations on their preventive maintenance in cohorts of risk and, hence, in all population by studying by molecular methods of an etiology of illnesses, mechanisms of a susceptibility to them, and also biological mechanisms and frequency of their development in human cohorts. Special development in molecular epidemiology was noticed for the methods named biomarkers, subdivided on 4 categories: 1) markers of an internal dose; 2) markers of an effective dose; 3) markers of preclinical biological effects; 4) markers of a susceptibility. Use of biomarkers in molecular-epidemiological researches appeared especially productive in oncology. As radiogenic cancers are the main remote consequence of exposure to ionizing radiations, approaches and achievements of molecular epidemiology of nonradiation cancers are perspective at becoming of molecular epidemiology of radiation effects, with an object to solve problems concerning mechanisms and features of radiation carcinogenesis, risk assessment, distribution and preventive maintenance of radiogenic cancers in cohorts of professionals of the various branches having contact to sources of ionizing radiation, and the population, exposed to radiation medically.

[Signal function of the reactive oxygen species in regulatory networks of the cell reaction to damaging effects: contribution of radiosensitivity and genome instability]. / Signal'naia funktsiia aktivnykh form kisloroda v reguliatornykh setiakh otveta kletok na povrezhdaiushchie vozdeistviia: uchastie v realizatsii radiochuvstvitel'nosti i nestabil'nosti genoma.

Reactive oxygen species (ROS) have a few possible effects, such as metabolic (participation in regulation of protein functions), damaging (oxidative damage to proteins, lipids and nucleic acids) and signal; the latter is reviewed in the article. Superoxide anion-radical (O2-.), hydroperoxide (HO2) and nitroxide (NO) are capable to act as signal substances in the cell regulatory network, which determines a mode of cell response to disturbance: proliferation pace, a course of differentiation or a start of the apoptosis program. A role of ROS in the reaction network is reviewed: importance of their content in a cell; ROS-bound signal pathways, which trigger the programs of cell reactions to stimuli; initiations of the regulatory network, which determine a content of ROS in a cell; ROS reactions with network components, which influence its functioning. A significance of the ROS-bound segment of the network, which realizes regulatory signals of the damage, in formation of radiobiological effect is estimated. The data obtained by the authors are submitted; the prospects of studing substances (such as phenozan etc.), which can actively influence redox processes, as means of modification of radiation-induced genome instability and prevention of oncogenic transformation are considered.

[Interrelations between the content of reactive oxygen species and the state of DNA structure in bone marrow cells of mice after whole body gamma-irradiation]. / Vzaimosviaz' soderzhaniia aktivnykh form kisloroda i sostoianiia struktury DNK v kletkakh kostnogo mozga u myshei v dinamike posle obshchego vozdeistviia gamma-izlucheniia.

The aim of the research is a further analysis of a problem concerning two (regulatory and damaging) functions of reactive oxygen species (ROS) in viability of organism cells under acute exposure to ionizing radiation. For this purpose the ROS content and the state of DNA structure in bone marrow cells of male CBA and SHK mice have been studied in dynamics, from 15 minutes up to 185 day after acute exposure to a sublethal dose (1.5 Gy) of ionizing radiation. The analysis of dependencies between these parameters in the norm, immediately after irradiation and in later cell descendants showed the direct correlation between the ROS content and the DNA nativity in the norm; 185 days after irradiation the correlation disappeared. It was suggested that the correlation occurred in the norm indicates participation of the ROS (as a sensory link) in a system of reactions (under the control of the corresponding genetic program), that ensure the DNA structure and, ultimately, the genome stability. The loss of such connection after acute exposure to ionizing radiation indicates actuation of another module of reactions sustaining stability of cellular genome in new conditions, without regulatory participation of ROS, that can promote or demonstrate the development of radiation-induced genome instability.

[Dynamic component of maintaining genomic stability in murine bone marrow cells after chronic low-intensity irradiation lasting one year]. / Dinamicheskaii komponent podderzhaniia stabil'nosti genoma v kletkakh kostnogo mozga u myshei posle khronicheskogo oblucheniia nizkoi intensivnosti dlitel'nost'iu odin god.

For analysis of a dynamic component state of the system of maintenance of genome stability, which represent a condition of its expression (first of all, genes of the control of phases of cell cycle, the DNA repair and redox systems) after a long chronic exposure to a small dose, the activity of replicative, reparative DNA synthesis, DNA damage as well as oxyradical content in the bone marrow cells of mice (critical for radiation effects mammalian system) after 1 year radiation exposure to a dose 63.7 cGy (0.17 cGy/day) were studied. The considerable enhancement of replicative and reparative DNA synthesis activity by 67% (p = 0.0033) and 60% (p = 0.000004) accordingly in relation to the control and some, but statistically not significant (p = 0.149) tendency to increase (by 30%) the content of a superoxide anion-radical were established. Strong and highly significant correlation (r = 0.8681; P = 0.99975) between DNA damage and O2-. content in bone marrow cells of the irradiated mices, which indicate the large DNA damage by oxiradicals, probably, due to the loss of a part of structural proteins and conformation changes in expression sites of a chromatin, were detected. The obtained results interpreted as representing the change of a dynamic component state of a system of maintenance of genome stability, the epigenic transfer of that to descendants of the irradiated cells can be the cause for formation and maintenance of radiation-induced genome instability.