[Tissue-specific Changes in the Polymorphism of Simple Repeats in DNA of the Offspring of Different Sex Born from Irradiated Male or Female Mice].

Evidence is presented indicating the differences in the polymorphism of microsatellite (MCS) repeats in DNA of somatic tissues in the offspring of BALB/c mice of different sex born from preconceptionally irradiated males or females. Brother-sister groups of the offspring born by non-irradiated parental pairs were compared with the offspring obtained after the irradiation of one parent in the same pairs. The number of MCS repeats in DNA of somatic tissues of the offspring from irradiated males or females was compared by a polymerase chain reaction using an arbitrary primer. It was found that changes in the polymorphism of the number of MCS repeats in the offspring from the males irradiated at a dose of 2 Gy was insignificant as compared with the offspring from control animals. In the offspring born by the females irradiated at a dose of 2 Gy (which does not impair the reproductive capacity), a statistically significant increase in the polymorphism was observed. Changes in the polymorphism were different in the offspring of different sex. A higher level of polymorphism was revealed in the female offspring born from the females of the F0 generation after their irradiation at a dose of 2 Gy. The increase in the polymorphism of the number of MCS repeats in DNA was more pronounced in postmitotic tissues compared with proliferating tissues.

[Specialized software product for comparative analysis of multicomponent DNA fingerprints].

"GelAnalyzer" software, which is used to identify and correctly compare DNA fingerprints consisting of a large number of discrete bands, has been developed by the project to study the quantitative changes in DNA polymorphism patterns in animals and humans exposed to gamma radiation. The actual capabilities of this program are much broader and include the possibility to analyze the images of any multicomponent gels containing fragments of DNA, RNA, and proteins. This software product runs on Windows. "GelAnalyzer" allows one to analyze gel images obtained by a scanner, camera, or digital camera and ensures the visual control of the identification and comparative analysis of bands; it also makes it possible to take into account the bands that are poorly identified automatically and exclude the artifacts (incidental marks) on images. The operation of "GelAnalyzer" software is based on the determination of the values of normalized coordinates of bands with allowance for the relative electrophoretic mobility (Rf) of PCR products and comparison of their spectra (set of bands in gel lanes) to reveal the similarities or differences in their components with subsequent statistical data processing and display the results of the analysis.

[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.

Effects of X-ray irradiation of female mice in preconceptive period on polymorphism of simple repeats in DNA of offspring of different sex.

Sibs groups of F1-offspring born by non-irradiated mice and by female mice exposed to X-ray radiation in preconceptive period (50-200 cGy) were compared. Arbitrary primed PCR revealed significantly increased polymorphism of simple DNA repeats in somatic tissues of the offspring from female mice irradiated in a dose of 200 cGy. The increase in DNA polymorphism in postmitotic brain tissues and in peripheral blood was more pronounced than in proliferating spleen tissues and in the epithelium of tail tip. In the tissues of female offspring from irradiated mothers, higher increase in DNA polymorphism was observed in comparison with the tissues of male offspring from the same mothers.

[Detection of large deletions of mitochondrial DNA in tissues of mice exposed to X-rays].

Large mtDNA deletions in mouse brain and spleen cells, induced by X-radiation at doses of 2 and 5 Gy were studied within four weeks after the exposure of animals to X-rays. Variations in the content of extracellular (deletion) mtDNA were examined in the blood plasma of mice irradiated with 5 Gy in the same postirradiation times. Ionizing radiation was shown to effectively induce large mtDNA deletions at the doses chosen. The level of deletion mtDNA was dependent on dose and postirradiation time.

[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).

[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.

[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.

[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.

[Genome instability in the F1-progeny of mice irradiated by ionizing radiation as determined by micronucleus assay].

The F1-progeny of BALB/c male mice chronically exposed to low-dose gamma-radiation (0.1; 0.25 and 0.5 Gy; dose rate 0.01 Gy/day) as well as the F1-progeny of females exposed to acute X-radiation (0.5; 1.0 and 2.0 Gy; dose rate 0.1 Gy/min) shown the significant elevated micronuclei frequencies in bone marrow erythrocytes, as compared to the F1-progeny of unirradiated males and females. The increase in the micronuclei frequency in the F1-progeny was determined by the dose of irradiation of parents. The values of elevated micronuclei frequency in the F1-progeny of chronically irradiated males and acutely irradiated females for a dose of 0.5 Gy were comparable. The micronuclei frequencies in the F1-progeny of irradiated females and males for this dose were in 1.5 and in 1.6 times higher than ones in the F1-progeny of unirradiated mice correspondingly. The results suggest the possibility of transfer of genome instability from irradiated parents to the somatic cells of the F1-progeny via non-lethally damaged germ cells of parents.

[Changes in copy number and deletion development of mitochondrial DNA in blood cells from breast cancer patients during radiochemotherapy].

Changes in mitochondrial DNA (mtDNA) copy number were compared versus the nuclear beta-globulin gene (internal standard), as well as occurrence of large mtDNA deletions in peripheral blood samples from 21 breast cancer patients following chemoradiotherapy. The study used polymerase chain reaction. Distinct variations were identified both in mtDNA copy number versus nuclear DNA and large mtDNA deletions occurrence in blood cells in response to genotoxic influence of chemoradiotherapy. Some mtDNA copies in breast cancer patients blood cells revealed large deletions whose frequency increased after chemoradiotherapy. Mitochondrial DNA lesions would induce synthesis of mtDNA copies in the course of chemoradiotherapy, which pointed to a compensatory reaction in peripheral blood cells as a consequence of a disturbed energy biogenesis.

[Increase of mitochondrial DNA copies with low level of DNA repair in tissue cells of gamma-irradiated mice].

The damage and the change in the number of mitochondrial DNA (mtDNA) copies in brain and spleen tissues of gamma-irradiated mice were studied. The changes in the number of mitochondrial DNA (mtDNA) copies were assayed by the comparative analysis of the density values of long-extension PCR products of the mtDNA fragments (16 kb) and the cluster nuclear gene of beta-globin (8.7 kb). PCRs of mtDNA fragments and the nuclear gene of beta-globin were carried out simultaneously in one test-tube within total DNA. Our results showed that in brain and in spleen cells of mice exposed to gamma-radiation an increase in copy number (polyploidization) of mtDNA with regard to the nuclear gene beta-globin took place. The induction of polyploidization of mtDNA observed in cells of gamma-irradiated animals is regarded as the development of a compensatory reaction because of the energy deficiency due to the increased ATP consumption and structural alteration of genes controlling OXPHOS. The data enabled the assumption that because of the low efficiency of repair systems in mitochondria the induction of synthesis of new mtDNA copies on intact or little affected mtDNA templates may be the major mechanism for the retention of the mitochondrial genome which is constantly damaged by the endogenous ROS and is affected by ionizing radiation and/or other exogenous factors.

[The increase in the variability of microsatellite-associated repeats in the genome of gamma-irradiated male mice is tissue specific]. / Tkanespetsificheskii kharakter povysheniia variabel'nosti mikrosatellit-assotsiirovannykh povtorov v genome potomstva gamma-obluchennykh samtsov myshei.

The arbitrarily primed polymerase chain reaction (AP-PCR) was used to measure the level of polymorphism of microsatellite (MCS)-associated repeating sequences of spleen, lung, and brain DNA in the F1 progeny of male BALB/c mice exposed to acute gamma-radiation at doses of 50 cGy and 200 cGy 15 days before mating with unirradiated females. The variability of MCS-associated sequences in the genome of brain and lung cells was higher as compared to the spleen cells of the progeny of unirradiated males. In the progeny of irradiated males, a 20% increase in MCS polymorphism of spleen DNA was found as an increase in the frequency of "non-parent" bands in DNA-fingerprints as against to the progeny of unirradiated males. Significant changes in this parameter were revealed for brain tissue and not for lung tissue only in the progeny of males exposed to 200 cGy. The results suggest a tissue-specific character of transmission of radiation-induced alterations in the genome of germ cells of male parents to the somatic cells of the progeny.

[Induced germ line genomic instability at mini- and micro-satellites in animals]. / Indutsirovannaia nestabil'nost' genoma polovykh kletok zhivotnykh po mini- i mikrosatellitnym posledovatel'nostiam.

The recent data on the phenomenon of the induced germline genomic instability at mini- and microsatellites in animals were considered. Natural hypervariability of the minisatellites and microsatellites and their abundance in eukaryotic genome provide it's utility as the useful genetic markers for evaluation of the germline mutation frequency induced by treatment with different type of genotoxic factors at the low doses. High sensitivity of assays and possibility for direct determinations of the mutations, without the necessity to use extrapolation, are ensured. Some discussion is presented on the role of non-targeted mechanisms for the radiation-prone DNA lesions in the induction of germline genomic instability and also on the involving in this process the recombination events upon meiosis or during the early development stages of embryos. It is proposed that quantitative determination of germline genomic instability rate may be used as an acceptable variant for the genetic risk assessment and as indicator of increased probability for cancer and other pathologies at the offspring born to irradiated parents.

[Micronucleus frequency is increased in bone marrow erythrocytes from offspring of male mice exposed to chronic low-dose gamma irradiation]. / V éritrotsitakh kostnogo mozga potomstva samtsov myshei, podvergnutykh khronicheskomu gamma-oblucheniiu v malykh dozakh, povyshena chastota mikroiader.

The micronucleus frequency in bone marrow erythrocytes from the F1 progeny of male mice exposed to chronic low-dose gamma-irradiation was determined. Male BALB/c mice were irradiated with 10, 25 and 50 cGy at dose rates of 1, 5, and 15 cGy/day and mated with unirradiated females on day 15 after irradiation. The obtained offspring had an elevated micronucleus frequency in bone marrow erythrocytes at the age of 2 months. This suggests the transmission of genome instability from damaged germ-line cells of irradiated male parents to somatic cells of the progeny.

AP-PCR assay of DNA alterations in the progeny of male mice exposed to low-level gamma-radiation.

By comparative analysis of fingerprints of arbitrarily primed polymerase chain reaction (AP-PCR) products, DNA alterations in somatic cells of the progeny (F1 generation) of male mice chronically exposed to low-doses of gamma-radiation was investigated. Male BALB/c mice exposed to 10-50 cGy were mated with unirradiated females 15 days after irradiation. DNA was isolated from biopsies taken from tail tips of 2-month-old progeny. Preliminary AP-PCRs were carried out with 17 primers representing core sequences of micro- and/or minisatellites or their flanking oligonucleotides. Best quantitatively reproduced AP-PCR fingerprints of genomic DNA were obtained with one of these primers, a 20-mer oligonucleotide flanking the micro-satellite locus Atplb2 on mouse chromosome 11. Comparative analysis of individual fingerprints of AP-PCR products obtained on DNA templates from the progeny of irradiated and intact males revealed an increased variability of micro-satellite-associated sequences and an increased frequency of "non-parental bands" in DNA-fingerprints from the progeny of males chronically exposed to gamma-radiation 15 days before mating (at the postmeiotic stage of spermatogenesis). The results show that increased micro-satellite instability can be initiated by irradiation of the male parent to subsequently arise or be transmitted to the soma of the F1 generations.

[Computer analysis of band variability in DNA fingerprints]. / Komp'iuternyi analiz variabel'nosti polos na DNK-fingerprintakh.

Through the example of the distribution of PCR products DNA matrices of mouse tail tissue, a method of comparative analysis of DNA fingerprints is described. The PCR products were obtained using a 20-mer random primer flanking the Atp1b2 locus on mouse chromosome 11. A software program was designed that permits the simplification of comparison of DNA fragments variability or polymorphism detected on electrophoregrams from different individuals.