[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.

[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.

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.

[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).

[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.

[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.

[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.

[Study of combined chronic effects of gamma radiation and lead on the occurrence and repair of DNA injury in mice]. / Issledovanie sochetannogo khronicheskogo vozdeistviia gamma-radiatsii i svintsa na obrazovanie i reparatsiiu povrezhdenii DNK u myshei.

It has been shown that neither exposure of mice to lead in the drinking water (20 mg/l) for 50 days nor chronic gamma-irradiation of animals (1.5 Gy, 1.3 mGy/h, 50 days) induces single-stranded DNA breaks in thymocytes. Acute gamma-irradiation (1 and 4 Gy) of lead-pretreated mice resulted in an inhibiting of repair of radiation-degraded DNA in thymocytes and in an increasing of the level of DNA lesions detected in erythroblasts of bone marrow by the micronuclear test method. Inhibition of ssDNA break repair in thymocytes caused by lead was not seen upon exposure of mice to combined chronic action of gamma-irradiation and lead. Chronic irradiation did not affect the micronuclei rate increase revealing after acute irradiation of lead-treated mice.

[A vitamin-antioxidant diet decreases the level of chromosomal damages and the frequency of gene mutations in irradiated mice]. / Vitaminno-antioksidantnaia dieta snizhaet uroven' khromosomnykh povrezhdenii i chastotu gennykh mutatsii u obluchennykh myshei.

Two different methods were used to study the effect of the antioxidant vitamins mixture (AVM) containing beta-carotene, alpha-tocopherol, ascorbic acid, rutin, and microelements zinc and selenium on mouse resistance to acute and chronic irradiation. Micronucleus test demonstrated that a daily dietary supplement of AVM reliably decreased the rate of chromosomal damage, namely, X-ray-induced micronuclei in the bone marrow polychromatophilic erythrocytes of aging mice, but did not induce micronuclei formation in the young mice. Assay for somatic mutations in the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus showed that AVM significantly decreased the rate of genic mutations in mouse splenocytes after chronic irradiation.

Effect of vitamin-antioxidant micronutrients on the frequency of spontaneous and in vitro gamma-ray-induced micronuclei in lymphocytes of donors: the age factor.

The effect of prolonged consumption of a vitamin-antioxidant mixture (VAM) on the frequency of spontaneous and in vitro gamma-radiation-induced micronuclei (MN) in peripheral blood lymphocytes in donors of various ages was investigated. Three groups of donors were recruited: (i) 56-83 years old (35 subjects), (ii) 23-30 years old (13 subjects), and (iii) 63-82 years old (12 subjects). Blood was sampled every 4 months for one year in all donors of the three groups. After the first sampling of blood, the donors of groups (i) and (ii) took VAM containing the vitamins A, C, E, as well as beta-carotene, folic acid, and rutin daily for 4 months. After the second blood sampling, the intake of VAM was terminated. The third blood sample was taken 4 months after termination of VAM intake. A part of the blood was exposed to gamma-radiation and the frequency of spontaneous and induced MN in lymphocytes was assayed. The analyses showed that the frequency of spontaneous and in vitro gamma-ray-induced MN in aged donors was significantly higher than that in young donors. No seasonal variations in MN frequency were observed in human lymphocytes during one year. Aged donors showed a statistically significant decrease in spontaneous MN in lymphocytes after a 4 month period of consumption of VAM. The intake of VAM by both aged and young donors promoted a decrease in MN induced lymphocytes in vitro by gamma-radiation. The results of our observations enable the suggestion that consumption of VAM favours a decrease in the chromosome damage produced by endogenous and exogenous factors in human lymphocytes.

[Micronucleus formation in the bone marrow cells of chronically irradiated mice with subsequent acute gamma irradiation]. / Issledovanie obrazovaniia mikroiader v kletkakh kostnogo mozga khronicheski obluchennykh myshei pri ikh posleduiushchem ostrom gamma-obluchenii.

With the use of the micronuclear test method it has been shown that mice preirradiated with gamma rays at a low dose rate exhibit a decreased frequency of chromosome aberrations induced in bone marrow cells by subsequent acute exposure to gamma radiation as compared to mice not subjected to preliminary irradiation. Such animals have a higher radioresistance with respect to the survival rate. The results obtained suggest the possibility of induction by ionizing radiation, at a low dose rate, of adaptive repair response at the organism level.

[Increase in the activity of nuclear DNA polymerases in the tissues of mice subjected to long-term gamma irradiation]. / Povyshenie aktivnosti iadernykh DNK-polimeraz v tkaniakh myshei, podvergnutykh dlitel'nomu gamma-oblucheniiu.

Activity of nuclear DNA-polymerase in the liver, lung and spleen tissues of mice subjected to long-term chronic gamma-irradiation (1.3 mGy/h) has been investigated. Chronic gamma-irradiation with a cumulative dose of 1.7 Gy during 55 days raises DNA polymerase activity in the irradiated tissue nuclei. Analysis of DNA-polymerase activity in the liver nuclei have demonstrated that this increase is connected with activation of DNA-polymerase beta.

[Activation of the DNA repair system in tissues of mice subjected to chronic gamma irradiation]. / Aktivatsiia sistemy reparatsii DNK v tkaniakh myshei, podvergnutykh khronicheskomy gamma-oblucheniiu.

Mice exposed to gamma-quanta during 47 and 82 days at a dose-rate of 1.3 mGy/h and cumulative doses of 1.45 and 2.54 Gy, respectively, were subsequently subjected to a single acute irradiation with a dose of 20 Gy. Repair of DNA damages induced by the acute exposure was shown to proceed in the brain, pulmonary and splenic tissues of chronically exposed mice more readily than in the tissues of mice not subjected to chronic irradiation. The data obtained indicate that the induced adaptive response activates DNA repair in tissues of mice exposed to long-term low-level radiation.

[DNA-synthesizing activity of the nucleoid protein core in Escherichia coli]. / DNK-sinteziruiushchaia aktivnost' belkovogo ostova nukleoida Escherichia coli.

Nucleoids obtained from E. coli cells by extraction with 1 M NaCl and detergents containing solution were further extracted with 2 M NaCl. From these samples, that contain only tightly bound proteins, fractions of protein core and peripheral nucleoprotein were obtained. It is shown that DNA synthesis proceeds mainly in the core structures. We have found that DNA polymerase I, which is bound with DNA nucleoid loops and with the above mentioned core structures, is not dissociating in 2M NaCl solution.

Proteins tightly bound to DNA and DNA-synthesizing activity of nucleoids from Escherichia coli.

Membrane-attached nucleoids were isolated from E. coli and separated from proteins by 2 M NaCl. Disintegration of such nucleoids by ultrasound and subsequent centrifugation resulted in the formation of two fractions: a sediment (fraction I) and a supernatant (fraction II). The protein:DNA ratio of fraction I was equal to 27 and was different from that to fraction II (2.6). More than 70% of the proteins not dissociating at 2 M NaCl and bound to DNA of both fractions were polypeptides with molecular weights (Mw) of 31,000-23,000 daltons (31-23 Kdal). After pulse labelling of the cells with [3H]-thymidine, the specific radioactivity of newly synthesized DNA associated with fraction I was shown to be considerably higher than that of fraction II. The analysis of DNA-synthesizing activities in fractions I and II showed that both nucleoid fractions contained DNA polymerase I. After dissolving the two fractions in 8 M urea - 0.15% sodium dodecylsulphate (SDS) they were chromatographed on hydroxyapatite. DNA-protein complexes were obtained that did not dissociate at 4 M guanidine X HCl - 5 M urea and 1% SDS. The main protein of the complexes was a 31 Kdal polypeptide tightly bound to DNA.

[Proteins firmly bound to DNA in the E. coli nucleoid]. / Prochno sviazannye s DNK belki v sostave nukleoida E. coli.

The nucleoid isolated from E. coli cells was subjected to further deletion by treatment with 2 M NaCl. After disintegration of this nucleoid by ultrasonication, two fractions were obtained, i. e., a rapidly (RS) and slowly sedimenting (SS) ones. The protein, RNA and DNA patterns in the RS fraction are similar to that of the eukaryotic cell nuclear matrix. Electrophoretic analysis of total non-dissociating by 2 M NaCl proteins revealed that the RS and SS fractions predominantly contain proteins with Mr 31,27 and 23 kD. The protein with Mr = 31 kD is firmly bound to DNA, does not dissociate in the guanidine hydrochloride (4 M)-urea (5 M) mixture as well as in solution of 1% sodium-dodecyl sulphate and may be responsible for the chromosome binding to the E. coli membrane.

Effect of mercaptoethylamine on DNA degradation in thermophilic bacteria bac. Stearothermophilus exposed to gamma-, UV-radiation or methylnitrosourea.

The effect of mercaptoethylamine (MEA) on degradation of DNA in thermophilic bacteria Bac. stear. exposed to gamma-, UV-rays or methylnitrosourea (MNU) was studied. Using centrifugation on alkaline and neutral sucrose gradients, it was shown that MEA inhibits the accumulation of breaks in the DNA of Bac. stear. It also lowers the level of DNA degradation in toluene-treated cells of Bac. stear. under the action of the intrinsic nuclease, reduces the activity of the endonuclease specific for apurinic DNA, as well as that of S1-nuclease and DNase-I in vitro. The inhibition in the accumulation of DNA breaks is assumed to be due to a decrease of the endonuclease activity in the cells of thermophilic bacteria.