[Induction of DNA damage in blood leucocytes and of cytogenetic injuries in bone marrow polychromatic erythrocytes in mice exposed to low-LET and high-LET radiation and in their progeny].

The present work was aimed at studying the molecular and cellular levels of the response of the hematopoietic system in mice and their progeny to the action of low-LET and high-LET radiation at different times after exposure. The damage to the genome at the molecular level was assessed by the comet assay in peripheral blood leucocytes, whereas at the cellular level it was estimated by means of the micronuclear test in the marrow cells, after exposure of mice to X-radiation of 1, 3 and 5 Gy and to a high-LET low-intensity radiation at thedoses of 0.14 and 0.35 Gy, as well as to a combined effect of these types of radiation. When accessing the level of the DNA damage to individual cells by the comet assay, we also used, apart from a commonly accepted parameter %TDNA, additional characteristics: the proportions of leucocytes with an intact and highly fragmented DNA. Using these parameters, we detected the changes characterizing the dynamics of the leukocyte population in mouse blood at different times after the action of X-ray and high-LET radiation. It was found that: (1) the DNA damage increases with the dose of high-LET radiation; (2) the level of damage in the progeny of the animals exposed to high-LET radiation does not differ from that in unirradiated animals both at the molecular and cytogenetic levels; and (3) a decrease in the radiosensitivity of the progeny of the mice exposed to high-LET radiation at a dose of 0.35 Gy makes itself evident only at the molecular level, which may point to the possible transgeneration transmission of genomic lesions.

[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.

[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.

[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.

[Diaphragmatic ruptures in combined injuries to the chest and abdomen]. / Razryvy diafragmy pri sochetannykh povrezhdeniiakh grudi i zhivota.

An experience with diagnosing and surgical treatment of 16 patients with rupture of the diaphragms is described. Operations were performed on 13 patients, 10 of them by the laparotomy access, 3 patients by the transpleural access. In patients with an extensive rupture of the diaphragm continuous respiratory insufficiency was observed in the postoperative period which required the artificial ventilation of the lungs. Eight patients died, 3 of them without an operative treatment, 3--from pulmonary complications and 2--from a cerebro-cranial trauma.