Changes in Telomere Length in Leukocytes and Leukemic Cells after Ultrashort Electron Beam Radiation.

Application of laser-generated electron beams in radiotherapy is a recent development. Accordingly, mechanisms of biological response to radiation damage need to be investigated. In this study, telomere length (TL) as endpoint of genetic damage was analyzed in human blood cells (leukocytes) and K562 leukemic cells irradiated with laser-generated ultrashort electron beam. Metaphases and interphases were analyzed in quantitative fluorescence in situ hybridization (Q-FISH) to assess TL. TLs were shortened compared to non-irradiated controls in both settings (metaphase and interphase) after irradiation with 0.5, 1.5, and 3.0 Gy in blood leukocytes. Radiation also caused a significant TL shortening detectable in the interphase of K562 cells. Overall, a negative correlation between TL and radiation doses was observed in normal and leukemic cells in a dose-dependent manner. K562 cells were more sensitive than normal blood cells to increasing doses of ultrashort electron beam radiation. As telomere shortening leads to genome instability and cell death, the results obtained confirm the suitability of this biomarker for assessing genotoxic effects of accelerated electrons for their further use in radiation therapy. Observed differences in TL shortening between normal and K562 cells provide an opportunity for further development of optimal radiation parameters to reduce side effects in normal cells during radiotherapy.

Application of land snail Helix lucorum for evaluation of genotoxicity of soil pollution.

Application of native species as sentinels allows environmental scientists to determine real genotoxic impact of environmental pollutants. The present study aims at investigating the DNA damage in the land snail Helix lucorum as a biomarker of soil pollution. For this reason, the genotoxic impact of contaminated soil on H. lucorum, collected from different polluted areas, was investigated using the comet assay in haemocytes and digestive gland cells. An increase in DNA damage was found in the snails sampled from polluted sites compared with the reference one. Strong correlations between DNA damage in haemocytes and digestive gland cells with the level of contamination indicate pollution-induced genotoxic effects in both tissues. At the same time, the digestive gland was more sensitive towards pollutants compared with haemolymph. A direct relationship between concentrations of Cu, As and Mo in soil and the number of damaged cells for hаemolymph and digestive gland tissue was found. However, the data obtained reflect the total genotoxicity of all pollutants in the studied areas. Significant correlations between the DNA damage measured by the comet assay and metal contents in soil indicate that it is a suitable biomarker in ecotoxicological studies. Our results indicate the effectiveness of H. lucorum in biomonitoring of environmental pollution.

Doxorubicin-Induced Translocation of mtDNA into the Nuclear Genome of Human Lymphocytes Detected Using a Molecular-Cytogenetic Approach.

Translocation of mtDNA in the nuclear genome is an ongoing process that contributes to the development of pathological conditions in humans. However, the causal factors of this biological phenomenon in human cells are poorly studied. Here we analyzed mtDNA insertions in the nuclear genome of human lymphocytes after in vitro treatment with doxorubicin (DOX) using a fluorescence in situ hybridization (FISH) technique. The number of mtDNA insertions positively correlated with the number of DOX-induced micronuclei, suggesting that DOX-induced chromosome breaks contribute to insertion events. Analysis of the odds ratios (OR) revealed that DOX at concentrations of 0.025 and 0.035 µg/mL significantly increases the rate of mtDNA insertions (OR: 3.53 (95% CI: 1.42-8.76, p < 0.05) and 3.02 (95% CI: 1.19-7.62, p < 0.05), respectively). Analysis of the distribution of mtDNA insertions in the genome revealed that DOX-induced mtDNA insertions are more frequent in larger chromosomes, which are more prone to the damaging action of DOX. Overall, our data suggest that DOX-induced chromosome damage can be a causal factor for insertions of mtDNA in the nuclear genome of human lymphocytes. It can be assumed that the impact of a large number of external and internal mutagenic factors contributes significantly to the origin and amount of mtDNA in nuclear genomes.

Application of the comet assay, micronucleus test and global DNA methylation analysis in Darevskia lizards as a sentinel organism for genotoxic monitoring of soil pollution.

Widely distributed in Armenia bisexual and parthenogenetic species of Darevskia rock lizards have the potential to be considered as bioindicators of the effects of environmental pollutants. Juvenile and adult bisexual D. raddei and parthenogenetic D. armeniaca lizards were sampled from four locations with different levels of soil contamination. The comet assay, micronucleus (MN) test and global DNA methylation detection were applied in peripheral blood erythrocytes to assess genotoxic and epigenetic effects of pollutants. The concentrations of heavy metals were analyzed in the corresponding soil samples. In polluted areas levels of DNA damage were significantly higher and levels of global DNA methylation were significantly lower in both species than in reference sites. The levels of comets and global DNA methylation decreased with age but did not depend on sex. MN test did not show significant differences among localities or between sexes and age groups. The positive correlation between DNA damage and contents of Cr, Cu, As and Mo and negative correlation between global DNA methylation and contents of Cr, Cu, Zn, Mo and Pb were shown for D. raddei. The positive correlations between DNA damage and contents of Cr, Zn and Pb and negative correlations between global DNA methylation and contents of Cr, Zn, Mo, Cd and Pb were revealed for D. armeniaca. The correlation results for some metals are different in juveniles and adults. D. armeniaca showed higher sensitivity toward environmental pollution than D. raddei in their common habitat. Genetic homogeneity of parthenogenetic lizards permits to evaluate the effects of environmental pollutants independently from inter-individual genetic variation. In conclusion, the current study suggests that DNA damage and global DNA methylation may be applied to Darevskia lizards as a sentinel organism for assessing the effects of environmental pollutants.

Analysis of copy number variations induced by ultrashort electron beam radiation in human leukocytes in vitro.

BACKGROUND: Environmental risk factors have been shown to alter DNA copy number variations (CNVs). Recently, CNVs have been described to arise after low-dose ionizing radiation in vitro and in vivo. Development of cost- and size-effective laser-driven electron accelerators (LDEAs), capable to deliver high energy beams in pico- or femtosecond durations requires examination of their biological effects. Here we studied in vitro impact of LDEAs radiation on known CNV hotspots in human peripheral blood lymphocytes on single cell level. RESULTS: Here CNVs in chromosomal regions 1p31.1, 7q11.22, 9q21.3, 10q21.1 and 16q23.1 earlier reported to be sensitive to ionizing radiation were analyzed using molecular cytogenetics. Irradiation of cells with 0.5, 1.5 and 3.0 Gy significantly increased signal intensities in all analyzed chromosomal regions compared to controls. The latter is suggested to be due to radiation-induced duplication or amplification of CNV stretches. As significantly lower gains in mean fluorescence intensities were observed only for chromosomal locus 1p31.1 (after irradiation with 3.0 Gy variant sensitivites of different loci to LDEA is suggested. Negative correlation was found between fluorescence intensities and chromosome size (r = - 0.783, p < 0.001) in cells exposed to 3.0 Gy irradiation and between fluorescence intensities and gene density (r = - 0.475, p < 0.05) in cells exposed to 0.5 Gy irradiation. CONCLUSIONS: In this study we demonstrated that irradiation with laser-driven electron bunches can induce molecular-cytogenetically visible CNVs in human blood leukocytes in vitro. These CNVs occur most likely due to duplications or amplification and tend to inversely correlate with chromosome size and gene density. CNVs can last in cell population as stable chromosomal changes for several days after radiation exposure; therefore this endpoint can be used for characterization of genetic effects of accelerated electrons. These findings should be complemented with other studies and implementation of more sophisticated approaches for CNVs analysis.

DNA damage and micronuclei in parthenogenetic and bisexual Darevskia rock lizards from the areas with different levels of soil pollution.

Natural species are widely used as indicator organisms to estimate of the impact of environmental pollution. Here we present the results of first study of a reliability of parthenogenetic Darevskia аrmeniaca and bisexual Darevskia raddei rock lizards as sentinels for monitoring of environmental genotoxicity. The comet assay and micronucleus test were applied to the lizards sampled in six areas in Armenia and Artsakh with different levels of soil contamination. The results obtained showed a clear relationship between the pollution level of lizards' habitats and the frequency of DNA damage in the comet assay. Low baseline frequency of micronuclei in D. аrmeniaca and D. raddei, however, makes this parameter ineffective for environmental genotoxicity evaluation. The parthenogenetic lizards D. аrmeniaca showed higher sensitivity toward genotoxic pollutions compared with bisexual D. raddei living in the same environment. The correlations between soil content of heavy metals Cr, Cu, Zn, Mo, Pb and DNA damage in D. аrmeniaca and between Cu, As, Mo, Pb and DNA damage in D. raddei were revealed. Overall, the lizards D. raddei and D. аrmeniaca appeared to be sensitive species in detecting soil pollution in natural environment. The application of the comet assay in Darevskia lizard species can be considered as a more appropriate method than a micronucleus test. The use of parthenogenetic lizards D. аrmeniaca as bioindicator will permit to assess the environmental genotoxicity independent of the genetic polymorphism of bisexual species.