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

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

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

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

Assessment of aquatic genotoxicity of the Lake Sevan basin, Armenia using natural bioindicators

Water pollution can cause genomic instability, thereby threatening aquatic wildlife and human health․ For a correct assessment of the impact of the mixture of pollutants, chemical analysis of environmental contaminants should be simultaneously applied with genotoxicity assays. The сomet assay is a sensitive, versatile and extensively used method for genotoxicity assessment in a water environment and was successfully applied in aquatic wild species, including fish and crayfish. Earlier we investigated the genotoxicity of waters of the Lake Sevan basin (Simonyan et al. 2016, Simonyan et al. 2019)․ Lake Sevan is the largest lake of the Caucasus Region, situated in the Republic of Armenia, in the Gegharkunik Province. Water pollution in the lake is a result of both anthropogenic and natural pressures. A combination of comet assay (Tice et al. 2000) and chemical analysis was applied for evaluation of water genotoxicity using gibel carp Carassius auratus gibelio and crayfish Astacus leptodactylus as sentinel organisms. Fish and water samples were collected from the south–southwest of the village Shorzha (40028′33″N and 45014′22″E), Peninsula of Lake Sevan (40033′46″N and 45001′37″E), the estuaries of the Rivers Gavaraget (40025′12″N and 45009′53″E) and Dzknaget (40036′55″N and 44058′13″E). Сrayfish and water samples were collected from the estuary of the River Masrik (40013′25″N and 45038′21″E) and near the villages of Artanish (40027′19″N and 45025′12″E) and Tsapatakh (40024′34″N and 45028′22″E). According to the chemical analysis of waters, the south–southwest of the village Shorzha and the estuary of the River Masrik were chosen as reference sites․ The content of contaminants in water was analyzed according to the standard methods in the Environmental Impact Monitoring Center, Ministry of Environment of the Republic of Armenia. The obtained results showed that the levels of DNA damage were significantly higher in both species in polluted areas than in reference sites. A significant positive correlation between DNA damage in C. auratus gibelio and contents of Al, Fe, Cu and Mn in water was shown. DNA damage in A. leptodactylus correlated with Al, Fe, Cu and Mo. C. auratus gibelio and A. leptodactylus were found to be sensitive mostly to the same pollutants, which indicate that these metals may be of primary concern as contaminants of the studied aquatic environment. Thus, the current study indicates that C. auratus gibelio and A. leptodactylus are sensitive bioindicators for monitoring of water pollution in the Lake Sevan basin

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

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