Galina Hovhannisyan

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.

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.

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.

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 IC50 (20 μmol/L for Ag and 70 μmol/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 pre-clinical investigation of the studied metalloporphyrins as potential chemotherapeutics.

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

BackgroundMicronucleus (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.ResultsChromosomal 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.ConclusionsOur 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.

Study of the genotoxic effects of a proline-rich polypeptide using the comet assay

The genotoxicity of a proline-rich polypeptide was investigated in vitro in the human myeloid leukemia KCL-22 cell line. A comet assay, based on single-cell gel electrophoresis, was used for the analysis of the DNA damage and reparation rate. After electrophoresis, images resembling comets were formed; the intensity of the “tail” relative to the “head” reflected the number of DNA breaks. We demonstrated that the level of DNA damage depended on the polypeptide concentration and incubation time. Estimation of DNA damage levels by the comet assay is recommended for the evaluation of the genotoxicity of new pharmaceuticals.

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

BackgroundAflatoxin 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).ResultsBlood 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.ConclusionsThe 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.

Micronuclei and What They Can Tell Us in Cytogenetic Diagnostics

Purpose of ReviewThe micronucleus (MN) assay is a validated method of genetic toxicology, widely used for human biomonitoring studies. This review summarizes and discusses current data regarding involvement of MN in pathogenesis of different diseases, potential of MN assay to be used as cytogenetic diagnostic technique, as well as highlights current achievements in studies concerning clinically relevant chromosomal instability using MN assay.Recent FindingsRecent studies suggested that MN are indicator of pathological events in affected as well as not affected “target” tissues of an organism. They can be effectively used in risk assessment and to distinguish stage of pathological manifestations in diseases. Molecular-genetic studies revealed that MN are not only the markers of, but at the same time inducers of genomic instability.SummaryThe MN assay is an informative cytogenetic tool, alone and in combination with molecular genetic methods. Although it is not always clear if MN are a result or inducer of pathogenic effects, the vast number of clinical studies substantiated that they have high potential for clinical practice, as they are associated with diseases.

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

Abstract 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.