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.

The Diagnostic, Prognostic, and Therapeutic Potential of Cell-Free DNA with a Special Focus on COVID-19 and Other Viral Infections.

Cell-free DNA (cfDNA) in human blood serum, urine, and other body fluids recently became a commonly used diagnostic marker associated with various pathologies. This is because cfDNA enables a much higher sensitivity than standard biochemical parameters. The presence of and/or increased level of cfDNA has been reported for various diseases, including viral infections, including COVID-19. Here, we review cfDNA in general, how it has been identified, where it can derive from, its molecular features, and mechanisms of release and clearance. General suitability of cfDNA for diagnostic questions, possible shortcomings and future directions are discussed, with a special focus on coronavirus infection.

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.

DNA Copy Number Variations as Markers of Mutagenic Impact.

DNA copy number variation (CNV) occurs due to deletion or duplication of DNA segments resulting in a different number of copies of a specific DNA-stretch on homologous chromosomes. Implications of CNVs in evolution and development of different diseases have been demonstrated although contribution of environmental factors, such as mutagens, in the origin of CNVs, is poorly understood. In this review, we summarize current knowledge about mutagen-induced CNVs in human, animal and plant cells. Differences in CNV frequencies induced by radiation and chemical mutagens, distribution of CNVs in the genome, as well as adaptive effects in plants, are discussed. Currently available information concerning impact of mutagens in induction of CNVs in germ cells is presented. Moreover, the potential of CNVs as a new endpoint in mutagenicity test-systems is discussed.

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.

Genotoxicity of Water Contaminants from the Basin of Lake Sevan, Armenia Evaluated by the Comet Assay in Gibel Carp (Carassius auratus gibelio) and Tradescantia Bioassays.

Combination of bioassays and chemical analysis was applied to determine the genotoxic/mutagenic contamination in four different sites of the basin of Lake Sevan in Armenia. Water genotoxicity was evaluated using the single cell gel electrophoresis technique (comet assay) in erythrocytes of gibel carp (Carassius auratus gibelio), Tradescantia micronucleus (Trad-MCN) and Tradescantia stamen hair mutation (Trad-SHM) assays. Significant inter-site differences in the levels of water genotoxicity according to fish and Trad-MCN bioassays have been revealed. Two groups of locations with lower (south-southwest of the village Shorzha and Peninsula of Lake Sevan) and higher (estuaries of Gavaraget and Dzknaget rivers) levels of water genotoxicity were distinguished. Correlation analysis support the hypothesis that the observed genetic alterations in fish and plant may be a manifestation of the effects of water contamination by nitrate ions, Si, Al, Fe, Mn and Cu. Increase of DNA damage in fish also correlated with content of total phosphorus.

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.

Use of Comet-FISH in the study of DNA damage and repair: review.

The Comet-FISH technique is a useful tool to detect overall and region-specific DNA damage and repair in individual cells. It combines two well-established methods, the Comet assay (single cell gel electrophoresis) and the technique of fluorescence in situ hybridization (FISH). Whereas the Comet assay allows separating fragmented from non-fragmented DNA, FISH helps to detect specifically labelled DNA sequences of interest, including whole chromosomes. Thus the combination of both techniques has been applied in particular for detection of site-specific breaks in DNA regions which are relevant for development of different diseases. This paper reviews the relevant literature and presents three examples on how Comet-FISH was used for studying the induction of DNA damage by genotoxic compounds related to oxidative stress in colon cancer-relevant genes (TP53, APC, KRAS) of a colon adenoma cell line. The accumulated evidence on relative sensitivity of these genes in comparison to global damage allows a more definite conclusion on the possible contribution of the genotoxic factors during colorectal carcinogenesis. Telomere fragility was compared in different cell lines treated with cytostatic agents, and revealed new patterns of biological activities through the drugs and different sensitivities of the cell lines that were found to be associated with their tumour origin. A third example relates to measuring repair of specific gene regions using Comet-FISH, a method that can be developed to biomarker application. Taken together, available data suggests that Comet-FISH helps to get further insights into sensitivity of specific DNA regions and consequently in mechanisms of carcinogenesis. Although the nature of the measured Comet-FISH endpoint precludes us from stating basically that damage and repair are occurring within the specific gene, it is at least possible to evaluate whether the damage and repair are occurring within the vicinity of the gene of interest.

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.

Dose-rate effect of ultrashort electron beam radiation on DNA damage and repair in vitro.

Laser-generated electron beams are distinguished from conventional accelerated particles by ultrashort beam pulses in the femtoseconds to picoseconds duration range, and their application may elucidate primary radiobiological effects. The aim of the present study was to determine the dose-rate effect of laser-generated ultrashort pulses of 4 MeV electron beam radiation on DNA damage and repair in human cells. The dose rate was increased via changing the pulse repetition frequency, without increasing the electron energy. The human chronic myeloid leukemia K-562 cell line was used to estimate the DNA damage and repair after irradiation, via the comet assay. A distribution analysis of the DNA damage was performed. The same mean level of initial DNA damages was observed at low (3.6 Gy/min) and high (36 Gy/min) dose-rate irradiation. In the case of low-dose-rate irradiation, the detected DNA damages were completely repairable, whereas the high-dose-rate irradiation demonstrated a lower level of reparability. The distribution analysis of initial DNA damages after high-dose-rate irradiation revealed a shift towards higher amounts of damage and a broadening in distribution. Thus, increasing the dose rate via changing the pulse frequency of ultrafast electrons leads to an increase in the complexity of DNA damages, with a consequent decrease in their reparability. Since the application of an ultrashort pulsed electron beam permits us to describe the primary radiobiological effects, it can be assumed that the observed dose-rate effect on DNA damage/repair is mainly caused by primary lesions appearing at the moment of irradiation.

Comparative analysis of individual chromosome involvement in micronuclei induced by mitomycin C and bleomycin in human leukocytes.

BACKGROUND: Micronucleus (MN) assay is a well standardized approach for evaluation of clastogenic/aneugenic effects of mutagens. Fluorescence in situ hybridization (FISH) is successfully used to characterize the chromosomal content of MN. However, the relationships between nuclear positioning, length, and gene density of individual chromosomes and their involvement in MN induced by different mutagens have not been clearly defined. RESULTS: Chromosomal content of MN was characterized in human leukocytes treated with mitomycin C (MMC) and bleomycin (BLM) by FISH using centromeric (cep) and whole-chromosome painting (wcp) probes. Involvement of chromosomes 8, 15 and 20 in MMC-induced and chromosomes 1, 9 and 16 in BLM-induced MN was studied, and correlated with chromosome size, gene density and interphase position. The results obtained were analyzed together with previous own data on the frequencies of inclusion of chromosomes 3, 4, 6, 7, 9, 16, 17, 18, and X in MMC-induced MN. It could be shown that MMC- and BLM-induced MN could contain material derived from all chromosomes investigated. Involvement of whole chromosomes 8, 15 and 20 in MMC-induced MN negatively correlated with gene density; however, analysis together with earlier studied chromosomes did not confirm this correlation. Inclusion of chromosomes 8, 15 and 20 in MMC-induced MN does not depend on their size and interphase position; the same result was found for the twelve overall analyzed chromosomes. In BLM-treated cells significant correlation between frequencies of involvement of chromosomes 1, 9 and 16 in MN and their size was found. CONCLUSIONS: Our results clearly revealed that BLM differs from MMC with respect to the distribution of induced chromosome damage and MN formation. Thus, DNA-damaging agents with diverse mechanism of action induce qualitatively different MN with regard to their chromosomal composition. Also this study demonstrates the utility of combined sequential application of cep and wcp probes for efficient detection of MN chromosomal content in terms of centric and acentric fragments.

A New Case of a Complex Small Supernumerary Marker Chromosome: A Der(9)t(7;9)(p22;q22) due to a Maternal Balanced Rearrangement.

Complex small supernumerary marker chromosomes (sSMCs) constitute one of the smallest subsets within the patients with an sSMC. Complex sSMCs consist of chromosomal material derived from more than one chromosome, for example, the derivative der(22)t(11;22)(q23;q11.2) in Emanuel syndrome. Here, a yet unreported case of a complex sSMC formed due to a t(7;9)(p22;q22)mat is presented.

Influence of aflatoxin B1 on copy number variants in human leukocytes in vitro.

BACKGROUND: Aflatoxin B1 (AFB1) is a mycotoxin produced by Aspergillus spec. The latter are worldwide contaminants of food with mutagenic and carcinogenic activities in animals and humans. AFB1 was shown to have deleterious effects on metabolism of eukaryotes in many model systems, including the ability to inhibit DNA replication. An agent that disturbs DNA replication may also have the potential to induce de novo DNA copy number variations (CNVs). RESULTS: Blood samples of three clinically healthy carriers were treated in vitro with AFB1 and chromosome preparations were subjected to parental origin determination fluorescence in situ hybridization (pod-FISH). Probes able to visualize CNVs in 8p21.2 and 15q11.2 were applied. In this setting here for the first time an influence of AFB1 on molecular-cytogenetically detectable CNVs could be shown. CONCLUSIONS: The obtained results indicate that: (i) pod-FISH is a single cell directed, sensitive and suitable method for the analysis of mutagen induced CNVs, (ii) AFB1 has the potential to induce in vitro instability of known CNVs in human leukocytes.

Chromosomal composition of micronuclei in human leukocytes exposed to mitomycin C.

Micronuclei (MN) can be induced by different mutagenic substances. Even though this has been known for decades, it is still not clear which genetic content, especially which chromosomes, these MN are constituted of and if there are any influences on this content by the MN-inducing substance. Also, the interphase position, size, and gene density of a chromosome could influence its involvement in MN formation. To study some of these questions, fluorescence in situ hybridization using centromeric and whole-chromosome painting probes for chromosomes 3, 4, 6, 7, 9, 16, 17, 18, and X was applied in mitomycin C (MMC)-induced MN in human leukocytes. The obtained results showed that material from all studied chromosomes was present in MN. Also, there was no correlation between interphase position, size, and gene density of the studied chromosomes and their migration in MN. Interestingly, material derived from chromosomes 9 and 16 was overrepresented in MMC-induced MN. Finally, further studies using substances other than MMC are necessary to clarify if the MN-inducing mutagen has an influence on the chromosomal content of the MN.

Fluorescence in situ hybridization in combination with the comet assay and micronucleus test in genetic toxicology.

Comet assay and micronucleus (MN) test are widely applied in genotoxicity testing and biomonitoring. While comet assay permits to measure direct DNA-strand breaking capacity of a tested agent MN test allows estimating the induced amount of chromosome and/or genome mutations. The potential of these two methods can be enhanced by the combination with fluorescence in situ hybridization (FISH) techniques. FISH plus comet assay allows the recognition of targets of DNA damage and repairing directly. FISH combined with MN test is able to characterize the occurrence of different chromosomes in MN and to identify potential chromosomal targets of mutagenic substances. Thus, combination of FISH with the comet assay or MN test proved to be promising techniques for evaluation of the distribution of DNA and chromosome damage in the entire genome of individual cells. FISH technique also permits to study comet and MN formation, necessary for correct application of these methods. This paper reviews the relevant literature on advantages and limitations of Comet-FISH and MN-FISH assays application in genetic toxicology.

Butyrate reduces the frequency of micronuclei in human colon carcinoma cells in vitro.

Butyrate, formed by bacterial fermentation of plant foods, has been shown to protect human colon cells from selected genotoxic substances. The mechanism for this effect could be the enhancement of toxicological defence leading to an increased detoxification of genotoxic risk factors and thus to a reduction of DNA and chromosome damage. Previous protective properties of butyrate against DNA damage induction in colon cells were demonstrated using the comet assay. In the present study the effect of butyrate on chromosome damage induced by ferric nitrilotriacetate (Fe-NTA) and hydrogen peroxide (H(2)O(2)) (suggested to be putative risk factors of colorectal carcinogenesis) was investigated using the cytokinesis-block micronucleus (CBMN) test. It was possible to reveal that pre-treatment of HT29 colon carcinoma cells with butyrate (2 and 4mM) for 15 min caused a reduction of micronuclei induced with H(2)O(2) (75 microM; p<0.01) and Fe-NTA (500 and 1000 microM; p<0.05). The decrease in the level of Fe-NTA- and H(2)O(2)-induced micronuclei was also confirmed in most of the corresponding variants of 24h pre-treatment of cells with butyrate. The results obtained demonstrate for the first time protective properties of butyrate against chromosome damage induced by H(2)O(2) and Fe-NTA in human colon carcinoma cells.

In vitro testing of cyto- and genotoxicity of new porphyrin water-soluble metal derivatives.

Porphyrins and porphyrin derivatives have an outstanding potential for discovery of novel pharmacological agents due to their ability for numerous chemical modifications and a variety of mechanisms of biological effects. New water-soluble Ag and Zn derivatives of tetrachloride meso-tetra (4-N-oxiethylpyridyl) porphyne were synthesized. Cyto- and genotoxicity of these substances were tested in vitro by the vital dye (trypan blue) exclusion and the micronucleus tests, respectively. Both metalloporphyrins were shown to be cytotoxic for Cos-7 (fibroblast-like African green monkey kidney cells transformed by simian virus 40 [SV40]), DU 145 (epithelial-like cells of human prostate carcinoma), and K-562 (human chronic myeloid leukemia cells) cell lines. At the same time they did not cause chromosome fragmentation in K-562 cell line at as high concentrations as IC(50) (20 micromol/L for Ag and 70 micromol/L for Zn derivative). Thus, the metalloporphyrins tested meet at least two important demands to potential anticancer drugs as they combine the cytotoxicity with low genotoxicity. The three in vitro tumor models used are relevant to further in vitro and in vivo preclinical investigation of the studied metalloporphyrins as potential chemotherapeutics.

Fragility of telomeres after bleomycin and cisplatin combined treatment measured in human leukocytes with the Comet-FISH technique.

UNLABELLED: THE AIM of the present study was the comparative investigation of action of widely applied anticancer preparations: cisplatin (cis-DDP) and bleomycin (BLM) on total DNA and telomeres damage in human blood cells. METHODS: The "Comet-FISH technique" -- single cell gel electrophoresis ("comet assay") in combination with fluorescent in situ hybridization (FISH) was used for this purpose. This newly applied combined approach permits to detect on the same specimen the total DNA damage in individual cells and evaluate specific DNA sequences as well. Telomere -- specific -- PNA (peptide nucleic acid) probes were used for the localization of telomeres in the comet's head and their migration to the tail. THE RESULTS obtained indicate that in control variants, due to DNA metabolism and handling, approximately 7% of the DNA and 17% of the telomeres were found in the tail. In cells treated with BLM alone, telomeres leak out with equal probability as total DNA. In turn, the combination of cis-DDP with BLM reduces telomere migration more than the migration of total DNA due to cis-DDP crosslinking effect. Thus, preferentially telomeric action of the cis-DDP can be concluded. CONCLUSION: The Comet-FISH approach permitted us to reveal the induction of DNA breaks with BLM and its modification due to platinum-crosslink formation, using telomeric PNA probes.

Evaluation of cisplatin-DNA crosslinks formation with UV-C application by the alkaline Comet-assay.

UNLABELLED: The AIM of the present study was to investigate DNA damage induced by widely applied anticancer preparation cisplatin (cis-DDP) at doses comparable with therapeutic ones in leukocytes of healthy donors. METHODS: DNA damage and repair was estimated by the single cell gel electrophoresis or Comet-assay. For estimation of cis-DDP-induced DNA crosslinks the combined treatment by cis-DDP with the DNA damaging agent UV-C was used. The RESULTS obtained indicate that cis-DDP is forming crosslinks with DNA in human leukocytes and significantly reduce UV-C-induced DNA migration. CONCLUSION: The Comet-assay with UV-C application is a useful tool to detect cis-DDP- induced DNA crosslinks. Data on the cis-DDP-induced DNA damage in vitro may be important for their extrapolation on in vivo level.