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The phenomenon of atypical DNA comets in experiments using DNA comet assay is described and illustrated. The current hypotheses explaining the nature of atypical DNA comets and own vision of the issue are considered. The practical importance of the registration of atypical DNA comets in assessing the genotoxicity is discussed.

Modern methods of genetic homeostasis assessment in animals are described in the present article. The single gel-electrophoresis test (Comet Assay), micronuclei test, chromosome aberration frequency, and sister chromatid exchanges are reviewed in detail. The questions of test-sensitivity of given methods and principles or their application for genetic homeostasis assessment in wild populations of animals are considered.

The Comet Assay and micronucleus assays have been used to evaluate the condition of the nuclear DNA in erythrocytes of peripheral blood of roach (Rutilus rutilus L.) from water-storage of low-level radioactive waste. The Rutilus rutilus L. from the Shershny reservoir, Chelyabinsk, was used as a control population. Radionuclide maintenance in water, sediments and roach in those reservoirs and Shershny reservoir was defined. The dose rate for Rutilus rutilus L. was calculated using program complex ERICA Assessment Tool 1.0 May 2009. Our investigation has shown that a chronic radiation of population (dose rate - 5.2 mGy/day and 19.5 mGy/day) leads to a significantly higher level of the DNA damage in erythrocytes of peripheral blood and increases the speed of nuclear DNA reparation after irradiation of erythrocytes in vitro. We suppose that it may be a result of the increased quantity of active form of oxygen in cells of the fish in water-storage of low-level radioactive waste.

The comet assay is a current highly sensitive method to evaluate primary DNA damages and repair. The paper considers the principle and procedure of the DNA comet assay and its modification for the detection of different types of DNA damages. It also discusses the prospects of its use as an indicator test during epidemiological and a variety of experimental and clinical studies. Whether the DNA comet assay is of importance for the expert evaluation of genotoxicity and for the prediction of mutagenicity and carcinogenicity is considered.

DNA breaks and their repair efficiency were analyzed in irradiated in vitro lymphocytes (at doses 1 Gy, gamma-radiation of 60Co, dose rate 1 Gy/min) isolated from peripheral blood of 41 untreated patients with breast cancer and 25 healthy donors using the DNA comet assay under non-denaturing conditions (mainly double-strand DNA breaks (DSB), as well as apoptotic cell death using the DNA halo assay. To estimate the expression of bystander effect, the cells were incubated in a culture medium obtained from lymphocytes irradiated in vitro at doses 1 Gy. The average DSB level in blood lymphocytes of breast cancer patients was shown to be significantly higher (p < 0.05) compared with that in control donors. In general, the following effects were observed in irradiated in vitro lymphocytes of cancer patients: (1) increased sensitivity to y-radiation-induced DNA DSBs compared with lymphocytes from healthy donors, (2) reduced repair efficiency of these damages. Incubation of irradiated blood lymphocytes in a medium from irradiated cells led to an increased relative number of DNA DSBs and an elevated fraction of cells dying through apoptotic pathway both in blood lymphocytes from cancer patients and control donors. However, these non-targeted effects were more expressed for the blood lymphocytes of breast cancer patients.

Changes of DNA structural condition, the level of membrane Fas-receptor expression, caspase-3 functional activity, concentrations of Ca2+, p53 and cytochrome c proteins of human lymphocytes in dynamics of apoptosis development induced by UV-light (240-390 nm) at doses 151, 1510, 3020 J/m2 and reactive oxygen species (superoxide anion-radical, hydroxyl radicals, hydrogen peroxide, singlet oxygen) have been studied. UV-light and reactive oxygen species have been established to induce fragmentation of lymphocyte DNA after 20 h incubation of the modified cells. It has been shown, that the increase in the expression level of membrane death Fas-receptors is observed during 1-5 h after exposure oflymphocytes to UV-light and ROS compared with intact cells. Also revealed is augmentation of lymphocyte caspase-3 functional activity 4 h after generation of singlet oxygen, hydroxyl radical and hydrogen peroxide addition, as well as 8 and 24 and 6 and 8 h after UV-irradiation of the cells at doses 151 and 1510 J/m2, correspondingly. Using DNA-comet method made it possible to tape that DNA damages (single-strand breaks) appear 15-20 min after lymphocyte UV-irradiation at doses 1510 and 3020 J/m and addition of hydrogen peroxide in concentration 10(-6) mol/l (C1 type comet) and reach their maximum 6 h after modification of the cells (C2 and C3 type comets). It has been observed, that 6 h after exposure oflymphocytes to hydrogen peroxide and UV-light at doses 1510 and 3020 J/m2, the p53 level of investigated cells raises. It has also been shown that the higher level of calcium in lymphocyte cytosol in conditions of UV-light exposure (1510 J/m2) and exogenous generation of reactive oxygen species is caused by Ca2+ exit from intracellular depots as a result of activating the components of the phosphoinositide mechanism for transferring information into a cell. Ideas about correlation between alterations of the calcium level and initiation of programmed cellular destruction of human lymphocytes after exposure to UV-irradiation and ROS is proposed. The authors come to the conclusion about the leading role of receptor-mediated (Fas-dependent) caspase- and p53-dependent ways of realizing apoptosis oflymphocytes induced by UV-light at doses 151 and 1510 J/m2 and active oxygen metabolites. The pattern of the possible intracellular events leading to apoptotic destruction of lymphocytes after their UV-irradiation is offered.

The main hybrid techniques of molecular cytogenetics are described. Methodological aspects of combination of fluorescence in situ hybridization (FISH) with the comet assay and micronuclei (MN) test are discussed along with results of their application to evaluate and locate DNA and chromosome damage in the genome. The experience of the authors with the application of FISH in combination with the comet assay and MN test are reported.

The analysis of the literature data on application of the gel electrophoresis of individual cells ("comet assay") in radiobiological investigations was carried out. The descriptions of various variants of the method are presented; its alkaline version is in more detail considered. The works concerning to induction and DNA damage repair of single stranded and double stranded DNA breaks, DNA alkali labile sites, crosslinks, DNA bases damage, cellular radiosensitivity and revealing of apoptotic cells were analyzed. The application of the method at biomonitoring of DNA damage level in cells of the person and the animals exposed to genotoxic agents, including ionizing radiation is described. The analysis of the literary data testifies to perceptivity of development and further uses of this method in radiobiological researches.

The results are presented on comparison of the kinetics of DNA exit during neutral and alkaline variants of single cell gel electrophoresis. It has been shown that pre-incubation of samples in alkaline buffer makes impossible DNA exit during the neutral electrophoresis, in contrast to the alkaline conditions where DNA exit is very efficient. The conclusion has been made that the alkaline conditions induce a disruption of DNA matrix interactions. The hypothetical nature of these interactions is discussed. The results obtained suggest that the mechanisms of DNA exit in the course of neutral and alkaline electrophoresis are essentially different. In the case of the neutral electrophoresis the comet tails are formed by the relaxed loop domains while during the alkaline electrophoresis they are formed by single stranded DNA fragments, which are pulled out by one and from the coil that is not bound to the matrix.

Using comet assay, a statistically significant increase (p < 0.05) in the level of DNA breaks in spleen cells was revealed in male CBA/lac mice exposed to gamma-radiation (1.7 cGy/day) or 90Sr (150-250 Bq/day) for 210 days. The level of DNA breaks also increased under combined exposure to both gamma-radiation and 90Sr (p < 0.05), but to a lesser degree than under exposure to each of these factors alone. Upon additional in vitro treatment of spleen cells with hydrogen peroxide, the relative increase in the level of DNA breaks was smaller in cells of irradiated mice than in the control. The ratio of the level of DNA breaks after hydrogen peroxide treatment to that before this treatment in control mice was 4.2 +/- 0.9, compared to 1.4 +/- 0.6 in gamma-irradiated mice, 1.9 +/- 0.8 in 90Sr-irradiated mice, and 2.3 +/- 0.8 in mice exposed to both gamma- and 90Sr-irradiation.