[On the prospects of using the DNA repair inhibitors in radiotherapy of tumors].

The cellular DNA repair systems sufficiently provide the resistance of tumors to ionizing radiation, and thus contribute to reducing the effectiveness of their radiotherapy. Therefore, suppression of the activity of critical DNA repair enzymes in tumor cells is considered one of the promising directions to overcome this resistance. As can be seen from the literature analysis, the use of many inhibitors of DNA repair enzymes have not yet yielded the results, which can be extrapolated to preclinical models or clinical trials. However, experimental studies show that the inhibitors of the enzyme family of poly(ADP-ribose) polymerases (PARP) are able to inhibit the growth of various human tumor cells. Pre-clinical and clinical trials of the PARP inhibitors also show promising results in terms of the possibility of their wide practical application. The effect of the PARP inhibitors consists in the blockage of the most important DNA repair systems. This leads to the accumulation of DNA single-strand breaks, the collapse of replication forks and to generation of lethal double-strand breaks (DSB). The PARP inhibitors can be used to suppress breast cancer and ovarian cancer with mutations in BRCA-1/2 genes ("BRCAness" cancer) without combination with radiotherapy or chemotherapy. In the tumor cells deficient in BRCA-1/2 genes DSB repair by homologous recombination pathway does not function. Therefore, the process of DNA DSB repair is switched to the non-homologous end-joining pathway, which operates with formation of the chromosomal rearrangements leading to cell death. Thus, the analysis results show that DNA repair inhibitors have the potential to improve the efficiency of cancer radiotherapy. Further research in this direction is very promising.

[Pathways for maintenance of mitochondrial DNA integrity and mitochondrial functions in cells exposed to ionizing radiation].

The analytical review deals with the results of studies devoted to mitochondrial DNA (mtDNA) disorders, the development of oxidative stress and possible pathways for the maintenance of mitochondrial functions in cells exposed to ionizing radiation (IR). Mitochondrial functions, which are closely related to the integrity of mtDNA, play a key role in many cellular processes. A wide range of degenerative diseases, carcinogenesis, and aging is associated with disturbances in mtDNA. MtDNA and the mitochondrion as a whole are increasingly considered as sensitive targets for cancer radio-chemotherapy. Knowledge of post-radiation processes in the mitochondria also facilitates creation of possible additional ways to reduce the radiation reaction of the organism. Injuries and mutations in mtDNA occur with a greater frequency than in the nuclear DNA (nDNA) in cells exposed to IR and other genotoxicants. On the other hand, functionally active copies of mtDNA can persist and survive in the cells exposed to clinically relevant doses of radiation. This safety is ensured by numerous copies of mtDNA in the cell, and due to their shielding from the effects of reactive oxygen (and nitrogen) species (ROS) by nucleoid proteins and by the operation of base excision repair in mitochondria. However, the generation of ROS increases in the mitochondria of cells exposed to IR. The increased generation of ROS in mitochondria can sometimes persist up to several days after the exposure of cells. The prolonged increased generation of ROS may be due to the involvement in the electron transport chain of the complexes of aberrant proteins expressed by the genes of mutated mtDNA copies. This may lead to the additional DNA damage, mitochondrial dysfunction, and instability of the nuclear genome. However, the development of oxidative stress can be restrained by antioxidant systems in the mitochondria. The key role here is played by activation of Mn-SOD2 and the protein p53. In addition, activation of mitochondrial biogenesis with the mtDNA synthesis, mitochondrial dynamics and mitophagy may be conjugated with the development of oxidative stress in mitochondria and mitochondrial dysfunction in irradiated cells. Thus, we can assume, that, although damage to mtDNA occurs at a high frequency and its repair is less efficient in mitochondria, the presence of multiple copies of mtDNA associated with proteins, the induction of antioxidant systems, the biogenesis of mitochondria with mtDNA synthesis, the activation of mitochondrial dynamics and mitophagy may contribute to the maintenance of mtDNA and functionally active mitochondria in the irradiated cells.

[Variability of DNA simple sequence repeats in peripheral blood of humans subjected to prolonged exposures of ionizing radiation].

Long-term post-radiation changes in the level of microsatellite DNA polymorphism in peripheral blood of the male "Mayak" employees (Ozyorsk, Russia), who had been exposed to prolonged gamma-irradiation during professional activities, were studied. DNA samples were obtained from the Radiobiology Repository of Human Tissue (Southern-Urals Biophysics Institute FMBA) and used as templates for arbitrarily primed PCR. Comparative analysis of the obtained samples of DNA fragments showed a significant increase in the number of high-molecular fragments and reduction in the number of amplified low molecular weight DNA fragments in comparison with the control. However, a direct correlation of the level of DNA polymorphism with the accumulated total dose of radiation was not found. The study of the polymorphism of microsatellite DNA repeats can be used for qualitative assessment of the levels of genetic variability.

[Experimental detection of integration of mTDNA in the nuclear genome induced by ionizing radiation].

Transfer of mtDNA in the nuclear genome is usually regarded as a continued and dynamic process of forming numt-pseudogenes or numt-insertions. They can be regarded not only as a neutral polymorphism, but may be involved in oncogenesis, aging and genetic diseases. Experimental identification of numt-insertions arising de novo is limited due to the presence of numerous homology mtDNA constitutively existing in the nuclear genomes of eukaryotes. It is known that the chick nuclear DNA (nDNA) constitutively contains 12 numt-pseudogenes. We attempted to experimentally detect the formation of numt-insertions de novo in the nDNA of chick embryos (Gallus gallus) from the eggs exposed to X-rays. Free mtDNAs were removed from preparations of nDNA of liver embryos through double gel electrophoresis. Numt-inserts in nDNA of control and survival embryos (from irradiated eggs) were revealed by PCR using 11 pairs of primers flanking the region of mtDNA of about 300-400 bp. PCR analysis with nDNA of control group showed no presence of homology mtDNA amplified with selected primers. PCR assays of nDNA of eight embryos from irradiated eggs showed that nDNA of two embryos contained new sites of mtDNA. PCR amplification of 3 loci of mtDNA is stably detected in nDNA from one embryo and 4 loci of mtDNA in nDNA from another embryo. Sequencing of PCR amplicons synthesized on templates of these nDNA showed that their sequences are identical to mtDNA and accurately cover the sites of several genes and the site of mtDNA D-loop. Thus, the experimental results indicate that ionizing radiation can induce integration of mtDNA fragments in the nuclear genome, apparently, through the mechanism of nonhomologous end-joining repair of double-strand breaks of nDNA.

[Changes of mitochondrial DNA/nuclear DNA ratio in the blood serum following X-ray irradiation of mice at various doses].

Using quantitative real-time PCR, the copy number of mitochondrial and nuclear DNA fragments in mouse blood serum was estimated at different time points following X-ray irradiation at various doses (from 0.5 to 10 Gy). The changes in the correlation between mtDNA and nuclear DNA (mtDNA/nucDNA) in blood serum reflect the degree of radiation injury depending on the dose of irradiation. Exposure to radiation at 10 Gy and massive cell death caused by this lethal dose result in a sharp decrease by an order of magnitude of the mtDNA/nucDNA ratio in the mouse serum; the value of this parameter did not recover within the next 3 days. The opposite effect was revealed when mice were exposed to irradiation at the dose of I Gy, which is not followed by massive cell death, but leads to a higher level of the mtDNA damage as compared with the nuclear DNA protected by histones. Defective mtDNA molecules enter the bloodstream, which results in an increase of the mtDNA/nucDNA ratio in serum. Under irradiation of mice at the intermediate dose of 3 Gy the two processes described above are exhibited at once. During the first hours after irradiation an apoptotic death of radiosensitive cells and release of a large number of nuclear DNA fragments in the serum are initiated, which reduces the mtDNA/nucDNA ratio. However, at later times after irradiation, starting from 5 days, an increase of the mtDNA/nucDNA ratio is observed in the serum, presumably as a result of reparation and elimination of defective mtDNA. Thus, the mtDNA/nucDNA ratio in the serum of irradiated mice reflects the degree of the radiation damage to cells and may be considered as a biological marker of radiation injury in the future.

[Delayed and transgenerational molecular and genetic effects of prolonged influence of ionizing radiation in nuclear plant workers].

Genome variability and changes in immune homeostasis, induced in man in the course of long-term industrial contact with ionizing radiation (IR) sources were studied by using unique biomaterials stored in the Radiobiological Repository for Human Tissues at the Southern Urals Biophysics Institute, FMBA. The biomaterials, peripheral blood samples and blood DNA were obtained from the "Mayak" PA employers occupationally exposed to prolonged external gamma-radiation and/or internal alpha-radiation from incorporated 239Pu in a wide range of accumulated doses. A significant increase in the polymorphism of microsatellite-associated peripheral blood DNA repeats was revealed in a group of persons with accumulated doses of external gamma-radiation above 2.0 Gy, as well as in the descendants of parents with preconceptive doses of higher than 2.0 Gy. In persons whose parents had a preconceptive dose above 2.0 Gy, an increase in the gene p53 mutation rate was observed, and descendants of persons with dose of 3.0 Gy and higher showed mtDNA heteroplasmy, regardless of the sex of an exposed parent. Changes in the expression of membrane markers for the effector and regulatory T-lymphocytes depending on radiation type and dose load were determined. The growth factor level variations (TGF-beta1, EGF, HGF, FGF) in peripheral blood serum in persons exposed to radiation from gamma- or alpha-sources, allow us to consider them as biomarkers of radiation-induced disturbances in immune homeostasis. The concentration changes of TGF-beta1, apoptosis proteins (p53, TPA-cyk, sAPO-1/Fas), and the adhesion molecule sCD27 in the case of cardiovascular diseases in the serum of both irradiated and non-irradiated "Mayak" PA employers point to the information value of these immune response characteristics as specific biomarkers of cardiac disorders. It is proposed that the revealed changes in immune homeostasis and in the variability of somatic cell genome may provoke development of tumors and cardiovascular diseases in man in delayed periods after prolonged exposure to IR.

[Increased genomic instability in somatic cells of the progeny of female mice exposed to acute X-radiation in the preconceptional period].

The level of genome instability (GI) was studied in the progeny of female mice exposed in the preconceptional period to radiation doses of 0.5, 1, and 2 Gy in comparison to that in the progeny of the same parent pairs born before irradiation of the females. To assess the level of genome instability, we analyzed polymorphism of DNA fragments from postmitotic (blood and brain) and proliferating (spleen and tail tip) tissues amplified by AP-PCR (PCR amplification with an arbitrary primer). It was found that polymorphism of the spectrum of AP-PCR products, which is a multilocus genetic marker (MGM), in the genome of somatic cells in the progeny of female mice exposed to 2 Gy was higher than in the progeny of male mice exposed to the same doses. In the progenies of female mice born before and after irradiation, tissue-specific variations in the level of DNA polymorphism were detected. The maximum value of this polymorphism (with respect to the frequency of "nonparental bands") was determined for peripheral blood DNA in comparison with the other tissues. Estimations of the MGM polymorphism with the AP-PCR method demonstrate an increased level of genome instability in somatic cells of offsprings from female mice exposed to a single acute dose of X-rays (0.5, 1, and 2 Gy) in the pre-conceptional period. Radiation-induced transgenerational genome instability with an increase in the dose of preconceptional irradiation of female mice was more pronounced in DNA of the postmitotic tissues (blood and brain DNA) than in DNA of the proliferating tissues (spleen and tail tip epithelium).

[Low efficiency of repair of critical DNA damage induced by low doses of radiation].

This study provides an analysis of the development of cellular response to the critical DNA damage and the mechanisms for limiting the efficiency of repairing such damages induced by low doses of ionizing radiation exposure. Based on the data of many studies, one can conclude that the majority of damages occurring in the DNA of the cells after exposure to ionizing radiation significantly differ in their chemical nature from the endogenous ones. The most important characteristic of radiation-induced DNA damages is their complexity and clustering. Double strand breaks, interstrand crosslinks or destruction of the replication fork and formation of long single-stranded gaps in DNA are considered to be critical damages for the fate of cells. The occurrence of such lesions in DNA may be a key event in the etiology and the therapy of cancer. The appearance in the cells of the critical DNA damage induces a rapid development of a complex and ramified network of molecular and biochemical reactions which are called the cellular response to DNA damage. Induction of the cellular response to DNA damage involves the activation of the systems of cell cycle checkpoints, DNA repair, changes in the expression of many genes, reconstruction of the chromatin or apoptosis. However, the efficiency of repair of the complex DNA damage in cells after exposure to low doses of radiation remains at low levels. The development of the cell response to DNA damages after exposure to low doses of radiation does not reach the desired result due to a small amount of damage, with the progression of the phase cell cycle being ahead of the processes of DNA repair. This is primarily due to the failure of signalization to activate the checkpoint of the cell cycle for its arrest in the case of a small number of critical DNA lesions. In the absence of the arrest of the phase cell cycle progression, especially during the G2/M transition, the reparation mechanisms fail to completely restore DNA, and cells pass into mitosis with a damaged DNA. It is assumed that another reason for the low efficiency of DNA repair in the cells after exposure to low doses of radiation is the existence of a restricted access for the repair system components to the complex damages at the DNA sites of highly compacted chromatin.

[Share of extracellular mutated mitochondrial DNA increases in plasma of lung cancer patients following radiotherapy].

Quantitative and qualitative changes in circulating extracellular DNA (ec-DNA) of blood plasma are considered as markers for diagnosis and prognostic of tumor pathology. We investigated the content of mutant copies of the circulating extracellular mitochondrial DNA (ec-mtDNA) in blood plasma (using the enzymatic method, based on the cleavage of DNA with unpaired bases by CEL-I endonuclease) in 8 patients with lung cancer before and after radiotherapy, as well as in healthy young and elderly donors. It was found that in the plasma of healthy elderly donors share of ec-mtDNA with mutations (consisting of total circulating DNA) is much greater, than that of young donors. On the other hand, in the plasma of lung cancer patients (aged 70-76 years) before radiotherapy a substantial increase in the share of ec-mtDNA with mutations, compared with that of healthy elderly donors. Following radiotherapy, patients with lung cancer found a twofold increase of the proportion of ec-mtDNA with mutations in the total circulating plasma DNA. This increase is largely, perhaps due to the release of ec-mtDNA with mutations from dying tumor cells and cells damaged by normal tissues.

[Mutant copies of mitochondrial DNA in tissues and plasma of X-rays exposed mice].

With violations of the mitochondrial genome associated wide range of degenerative diseases, the development of tumor pathology, aging and the processes of cell death. We investigated the levels of mitochondrial DNA (mtDNA) with mutations and their total content in the tissues of the brain, and spleen of mice exposed to X-rays at doses of 1-5 Gy, and depending on the post-radiation time (8-28 days). These same mice were analyzed for the level of mutant copies of the extra cellular mtDNA (ec-mtDNA) and its total content in blood plasma. Mutations were determined by means of CEL-I endonuclease (mismatch-specific enzyme) cleavage of heteroduplexes, obtained by hybridization of PCR amplicons of mtDNA (D-loop region) of irradiated and control mice. The changes of total amount of mtDNA (ND4 gene) copies vs. nuclear DNA (GAPDH gene) measured by real-time PCR method. The results showed that in the tissues of the brain, and of the spleen of irradiated mice (with a maximum at 8 days after exposure) the level of mutant mtDNA copies, with a subsequent decline to a 28-day post-radiation time dramatically increased. Was shown that mutagenesis of mtDNA in the brain and spleen tissues of irradiated mice, well as mutagenesis of nuclear genes, has a linear dependence on the dose X-rays. In contrast, mutant nuclear genes, the majority of mutant mtDNA copies is eliminated from the tissues of the brain and of the spleen, while maintaining them at the same level of total content of mtDNA within 28 days after irradiation in mice. The results show that during this post-radiation time in the plasma of irradiated mice high levels of ec-mtDNA with mutations, with a maximum at the 14th day in the total circulating DNA maintained. These data suggest that the mutant copies of mtDNA eliminated from tissues cells of irradiated animals in the post-radiation period. Elevated levels of ec-mtDNA with mutations in the plasma can be considered as a potential marker for the assessment of radiation injury of organism.

[Nuclear mitochondrial pseudogenes].

Transfer of genetic material from mitochondria to the nucleus and their integration into the nuclear genome is a continuous and dynamic process. Fragments of mitochondrial DNA (mtDNA) in the nuclear genome are incorporated as non-encoded sequences, which are called nuclear mitochondrial pseudogenes (NUMT-pseudogenes). At present, the formation NUMT-pseudogenes in the nuclear genome is shown in many eukaryotes. They are distributed on different chromosomes, form a "library" of mtDNA fragments, migrated into the nuclear genome and provide important information on the history of the evolution of genomes. Escape of mtDNA from the mitochondria most is associated with damage and mitophagy these organelles. The integration of mtDNA fragments into the nuclear genome may occur during repair of double strand breaks of nuclear DNA (nDNA) arising under the action of endogenous and exogenous agents. Reparation of nDNA double strand breaks with "capture" fragments of mtDNA, occurs by non-homologous end joining and a similar mechanism, but with the involvement microhomology, located on the terminal sequences. Analysis of data allows us to suppose that the rate of formation NUMT-pseudogenes will depend on the rate of double strand breaks in nDNA, activity systems, their repair, as well--the number of mtDNA fragments that have emerged from the organelles, with their further migration into the nucleus. Such situations can be expected, most often after exposure to the damaging agents, in the first place--ionizing radiation. The emergence of new NUMT-pseudogenes, obviously, is changing not only the structure of the genome in the areas of their implementation, but may have a significant impact on the realization of genetic information. Integration NUMT-pseudogenes in the nuclear genome de novo may play a role in the development of various pathologies and aging. NUMT-pseudogenes can make serious errors in analyzing free mtDNA of total cellular DNA (using PCR), as a result of their co-amplification.

[Reduction of the number of mutant copies of mitochondrial DNA in tissues of irradiated mice in the postradiation period].

Changes in the number of mutant copies of mitochondrial DNA (mtDNA) were studied in the brain and spleen tissues of mice after their X-irradiation at a dose of 5 Gy. For this purpose, heteroduplexes obtained via hybridization of the products of PCR amplification of mtDNA (ND3 gene and two D-loop regions) from irradiated and control mice were digested with the CelI nuclease capable of specific mismatch cleavage. Heteroduplexes obtained via hybridization of the products of PCR amplification of mtDNA from irradiated and control mice were digested by the CelI nuclease to a greater degree than heteroduplexes of the PCR products of mtDNA of mice from the control group. This suggests the presence of mutations in mtDNA regions in irradiated mice. Digestion by the CelI nuclease of heteroduplexes obtained via hybridization of the PCR products of mtDNA (ND3 gene and D-loop regions) on day 8 after irradiation is essentially more efficient than digestion of heteroduplexes obtained via hybridization of the PCR products of mtDNA isolated from mouse tissues on days 14 and 28 of the postradiation period. These results indicate a reduction in the number of mtDNA copies with mutations in tissues of irradiated mice by day 28 of the postradiation period. The reduction in the level of mutant mtDNA copies by this term is especially significant in the spleen. The total number of mtDNA copies in the mouse brain and spleen tissues estimated by real-time PCR, relative to the nuclear beta-actin gene, is also decreased by 30-50% as compared to the control on days 8 to 28 after irradiation. The results of the study suggest that mutant mtDNA copies are eliminated from tissues of irradiated animals in the postradiation period. This elimination can be regarded either as a result of selective degradation of mitochondria carrying mutant DNA copies or as a result of cell death being continued in tissues of irradiated animals.

[The comparative determination mutations in mtDNA of irradiated mice using mismatch-specific endonuclease and TTGE-technique].

We defined the mutations in mtDNA of X-irradiated mice brair using mismatch-specific endonuclease (CEL I-nuclease method) and by temporal temperature gradient gel electrophoresis (TTGE-technique). The comparison of the received by both methods, allows to conclude, that CEL I-nuclease method gives more qualitative results, than TTGE-technique. Moreover, CEL I-nuclease method is more sensitive, in contrast with TTGE-technique. The CEL I-nuclease method allows simultaneously to conduct the analysis of big amount of sample DNA, to get the reproducible results. It does not require complex equipment and economical. The analysis of mutations in mtDNA of brain of X-irradiated mice by CEL I-nuclease method has shown, that the amount of mutant copies mtDNA is essentially reduced (in 2-3 times) with 8 up to 28 days of the post-radiation period. However the amount mtDNA copies in brain tissue of the irradiated animals is remains during all post radiation time without change though lower, concerning given control group. The results permit the suggestion that mutant mtDNA copies are eliminated from the tissues of irradiated animals in the post-radiation period.

[Extracellular mutant mitochondrial DNA content is sharply elevated in the blood plasma of irradiated mice].

Nucleic acids circulating in blood plasma and other biological fluids may be of interest as potential markers for diagnosis of various pathologies and monitoring of stress influences. For many genotoxic agents, mitochondrial DNA (mtDNA) is a more vulnerable target than nuclear DNA, and mutations of mitochondrial genome may be markers of many diseases. In the present study extracellular mtDNA with mutations was determined in the blood plasma of mice exposed to X-radiation at a dose of 5 Gy. Mutations were assessed from cleavage by CEL-endonuclease (mismatch specific cleavage enzyme) of heteroduplexes obtained by hybridization of mtDNA PCR amplikons (gene ND3 and D-loop region) from the blood plasma of irradiated and control mice. The total number of copies of mtDNA (gene ND4) against nuclear DNA (gene GAPDH) was measured by the Real-Time PCR method. A content of mtDNA with mutations in the blood plasma of mice was elevated during one month of the post-radiation period, however, the levels were not the same at different time periods (1, 4, 8, 14, 28 days), the highest one being detected on the 14 day after irradiation. The increased content of extracellular mutant mtDNA in blood plasma of X-irradiated mice can be considered as a sensitive biomarker for assessing radiation injury and effects of other genotoxic agents.

[Lesions of the mitochondrial genome and ways of its preservation].

The results of studies on damage, repair, mutation induction, and compensatory amplification of mitochondrial DNA (mtDNA) in mammalian cells are analyzed. The analysis have shown that mtDNA is a more susceptible target than nuclear DNA for endogenous and exogenous genotoxic agents, whereas DNA repair systems in mitochondria function less efficiently then in the nucleus. The rate of mutation accumulation is higher in mtDNA than in the nuclear DNA. In contrast to nuclear DNA, replication of damaged mtDNA is not blocked. MtDNA with multiple or complex lesions may be removed from mitochondria. An important mechanism of mitochondrial genome preservation is compensatory induction of new mtDNA copies. The cascade of events leading to the activation of the expression of nuclear genes controlling DNA repair in response to energy crisis is analyzed. As the key regulators of the activation of mtDNA replication and mitochondrial biogenesis, transcription coactivators PGC-1alpha and PGC-1beta are considered. These coactivators induce the expression of genes for nuclear respiratory factors NRF-1 and NRF-2. In their turn, NRF-1 and NRF-2 control the expression of mitochondrial transcription factors mtTFA, mtTFB-1, and mtTFB-2. MtTFA plays a key role not only in the mtDNA transcription regulation, but also in its stabilization and replication initiation. Thus, induction of the expression of many genes, resulting in activating mtDNA replication and increasing the number of its copies, is an important mechanism of preserving the mitochondrial genome upon the action of endogenous and exogenous damaging agents.

[The use of temporal temperature gradient gel electrophoresis to reveal mutations in peripheral blood mitochondrial DNA].

The mutations in mitochondrial DNA (mtDNA) arise at a higher frequency than in nuclear DNA, and their appearance in peripheral blood can be considered as a sensitive marker to estimate the level of genotoxic load. For revealing the presence of mutations in mtDNA of peripheral blood, we used the method of temporal temperature gradient gel electrophoresis (TTGE). The samples of whole blood DNA from four donor groups were used. Group I contained 10 young (23-26 years) donors and Group II 12 elderly (65-74 years) donors. Group III was formed from patients with breast cancer (12 women) past sessions of radio-chemotherapies (RCHT). Group IV was made of professionals of a nucleus plant occupationally exposed to chronic gamma-irradiation. PCR was carried out on four coding sequences and on one hypervariable sequence of the D-loop (DloopI) of mtDNA. PCR products were tested with TTGE. Most mutations were revealed in the DloopI. Heteroplasmy in the region of DloopI was registered in the blood of each donor of Group III 7 days after the RCHT session. Also, mutations in mtDNA Dloop1 were found in 6 of 13 individuals of Group IV. The blood of this donor group was taken 16 to 28 years after prolonged irradiations in a dose range of 250-350 cGy. In the elderly donor group, the same results were observed in 3 of 12 individuals. The results show that the method of TTGE can be used in mass analyses to assess the effects of radiation and other genotoxic agents in man by detection of unknown mutations in peripheral blood mtDNA.

[Sharp changes in the copy number of mtDNA and its transcription in the blood cells of X-ray irradiated mice are observed, and mtDNA fragments appear in the blood serum].

Changes in the number of mitochondrial DNA (mtDNA) copies and alterations in its transcription were followed in the blood cells of mice after their exposure to X-rays; also, extracellular mtDNA fragments were registered in the blood serum of these mice. By a real time polymerase chain reaction (RT-PCR), a sharp increase in mtDNA copy number one hour after the exposure was detected in the blood cells of 1 Gy X-ray irradiated mice. This increase in mtDNA copies is considered as a result of a compensatory reaction developed in mice in response to the radiation damage in a part of mtDNA molecules. Exposure to X-ray radiation at a dose of 10 Gy resulted in an arrest of mtDNA replication in mouse blood cells within three hours after irradiation. At the same time, RT-PCR assays showed that at the same radiation dose, an increase in the number of mtRNA of the mitochondrial nd6 gene relative to mRNA of the nuclear gene gapdh occurs. This indicates that, although mtDNA is more damaged in comparison with nuclear DNA (nDNA), the process of transcription continues in the blood cell mitochondria of mice after their exposure to 10 Gy, whereas the transcription of damaged nDNA is arrested. After the irradiation of mice with 10 Gy, mtDNA fragments of 1841 bp were registered in the blood serum. Thus, in the blood cells of mice exposed to X-rays, the copy number of mtDNA and its transcription are sharply changed, and large mtDNA fragments are observed in the blood serum.

[Ionizing radiation can activate the insertion of mitochondrial DNA fragments in the nuclear genome].

In analytical review is considered the possibility of the insertion of mitochondrial DNA (mtDNA) fragments into the nuclear genome of cells, exposed ionizing radiation (IR). Many studies show that integration fragment mtDNA in nuclear genome, as well as its fastening as NUMT-pseudogenes, proceed at ancient periods of the evolutions not only, but also at more late periods. The number of the investigations shows that under influence endogenous reactive oxygen species, chemical agent, UV-light and IR mtDNA is damaged with greater frequency, than nucleus DNA. Furthermore, the repair systems in mitochondria are low efficiency. In irradiated by IR cells mtDNA fragments can transition from the mitochondria to the cytoplasm. The binding of mtDNA fragment to a complex with proteins provides them the protection from nuclease destroying. Possibly, at such safe condition they and are carried to nucleus. At inductions of DNA double-strand breaks (under the action of IR and activated their reparation) mtDNA fragments may be inserted to nuclear genome. Such integration of mtDNA to nuclear genome, with shaping NUMT-pseudogenes de novo, may be proceed in irradiated cells in the course of the reparations DNA double-strand breaks by the nonhomologous end-joining pathway. These insertions of mtDNA can cardinally change the structure of nuclear genomes in area of their introduction and render the essential influence upon the realization of genetic information. Available information in literature also allows to suppose that integration mtDNA in nuclear genome can proceed and at raised genomic instability observed in cells at post radiation period. It in equal extent pertains and to malignant cells with raised by instability mitochondrial and nuclear genomes. As the most efficient agent, initiating insertion fragment mtDNA in nuclear genome, is considered ionizing radiation.

[Release of mtDNA from mitochondria and activation of its replication in tissues of irradiated mice].

A nessessary condition for normal functioning of mitochondria is the maintenance of certain numbers of intact mtDNA molecules. In the present study, we investigasted changes in the number of mtDNA copies in brain and spleen cells of mice subjected to irradiation. For the first time, we observed the irradiation-induced output of mtDNA fragments into brain and spleen cell cytosol. In the cytosol of these cells, examined in mice 5 h after 5 Gy irradiation, 1841 h.p. mtDNA fragments were detected able to persist for at 3 weeks. In addition, larger fragments of mtDNA (10,090 b.p.) were detected in the cytosol of brain cells of irradiated mice. The occurrence of mtDNA fragments in the cytosol of brain cells is accompanied with an increase in the number of mtDNA copies in the mitochondrial matrix. The induction of mtDNA replication in brain cells of irradiated animals may be considered as a compensatory reaction in response to mtDNA damage. A sharp decrease in the amount of mtDNA copies in the mitochondrial matrix of spleen cells on the first day after irradiation may be considered as apoptosis development. However, the compensatory reaction in brain cells was also noticed but in later terms.