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 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.

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.

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.

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. 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. 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.