V. G. Bezlepkin

The prolongation of survival in mice by dietary antioxidants depends on their age by the start of feeding this diet

The effect of daily dietary supplements of an antioxidant mixture (AM) consisting of beta carotene, alpha tocopherol, ascorbic acid, rutin, selenium, and zinc on the survival of male C57BL/6 mice starting at 2, 9, 16, and 23 months of age was investigated. The survival of mice given AM starting at 2 and 9 months of age was found to increase significantly (from 86 to 108 days) compared to the control. The times, of 50, 90, and 100% mortality in mice given AM starting at 2 and 9 months of age increased by 16-9.5% compared to the control, whereas in mice given AM, starting at 16 and 23 months of age, no effect was observed.

Modifying effect in vivo of interferon alpha on induction and repair of lesions of DNA of lymphoid cells of gamma-irradiated mice.

The induction of structural lesions and repair in DNA of lymphoid cells from the peripheral blood, spleen and thymus of mice treated with natural mouse interferon alpha (IFN-alpha) 24 and 48 h prior to gamma irradiation were studied using the comet assay and apurinic-apyrimidinic (AP) site radiolabeling. It was demonstrated that the radiation-induced damage assessed by the comet assay in the DNA of peripheral blood lymphocytes (PBLs), splenocytes and thymocytes of mice treated with IFN-alpha before irradiation was considerably less and was repaired more easily in the postirradiation period than that in untreated mice. The DNA of PBLs and splenocytes from interferon-treated mice showed a decrease in the spontaneously occurring and radiation-induced AP sites, as determined immediately and 90 min after irradiation, compared to the level of AP sites in the DNA of untreated mice. The results lead us to assume that IFN-alpha activates the DNA repair systems in the cells of lymphoid tissue.

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

Abstract By comparative analysis of fingerprints of arbitrarily primed polymerase chain reaction (AP-PCR) products, DNA alterations in somatic cells of the progeny (F 1 generation) of male mice chronically exposed to low-doses of γ-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 γ-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 F 1 generations.

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 extra-cellular deleted mtDNA were examined in the blood plasma of mice irradiated with 5 Gy in the same postir-radiation 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.

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.

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 preconceptional 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).

Mitochondrial DNA deletions in the peripheral blood of workers at the Mayak PA who were exposed to long-term combined effects of external γ- and internal α-radiation

The levels of large deletions in the mitochondrial DNA of workers at the Mayak Production Association (Mayak PA) who were exposed to external and combined occupational (external γ- and internal α-rays) radiation during the course of their duties were investigated. Peripheral blood-derived DNA samples were provided by the Radiobiological Human Tissue Repository of the Southern Urals Biophysics Institute (Russia). The samples were analyzed using long-extension PCR. The number of large-scale deletions in the mitochondrial DNA of workers who, in addition to external γ-radiation, were exposed to extra doses of irradiation due to incorporated 239Pu with a Pu body burden of 0.77–4.32 kBq, was 2.5-times lower compared to that of individuals who received only external γ-radiation. No significant gender-associated effects on the number of mitochondrial DNA deletions were detected among age-matched individuals.

Comparative assessment of mutations in mtDNA of x-irradiated mice using mismatch-specific endonuclease and temporal temperature gradient electrophoresis

Mutations in brain mtDNA of mice X-irradiated to 5 Gy were determined using a mismatch-specific endonuclease (CEL I) and by temporal temperature gradient gel electrophoresis (TTGE). The CEL I nuclease method allows simultaneous analysis of multiple DNA samples and proves to yield better results than TTGE; it is more sensitive, quite reproducible, and economical, requiring no complicated equipment. The CEL I nuclease assay for mtDNA mutations in the brain of x-irradiated mice has shown that the amount of mutant mtDNA copies is markedly reduced (2–3 times) from the 8th to the 28th day after irradiation, while the overall amount of mtDNA copies in the brain tissue remains fairly constant over this period, though lower than in the control. It can be suggested that mutant mtDNA copies are eliminated from the tissues of irradiated animals in the postirradiation period.