Multicolour FISH in an analysis of chromosome aberrations induced by N-nitroso-N-methylurea and maleic hydrazide in barley cells.

The present study is a rare example of a detailed characterization of chromosomal aberrations by identification of individual chromosomes (or chromosome arms) involved in their formation in plant cells by using fluorescent in situ hybridization (FISH). In addition, the first application of more than 2 DNA probes in FISH experiments in order to analyse chromosomal aberrations in plant cells is presented. Simultaneous FISH with 5S and 25S rDNA and, after reprobing of preparations, telomeric and centromeric DNA sequences as probes, were used to compare the cytogenetic effects of 2 chemical mutagens: N-nitroso-N-methylurea (MNU) and maleic hydrazide (MH) on root tip meristem cells of Hordeum vulgare (2n=14). The micronucleus (MN) test combined with FISH allowed the quantitative analysis of the involvement of specific chromosome fragments in micronuclei formation and thus enabled the possible origin of mutagen-induced micronuclei to be explained. Terminal deletions were most frequently caused by MH and MNU. The analysis of the frequency of micronuclei with signals of the investigated DNA probes showed differences between the frequency of MH- and MNU-induced micronuclei with specific signals. The micronuclei with 2 signals, telomeric DNA and rDNA (5S and/or 25S rDNA), were the most frequently observed in the case of both mutagens, but with a higher frequency after treatment with MH (46%) than MNU (37%). Also, 10% of MH-induced micronuclei were characterized by the presence of only telomere DNA sequences, whereas there were almost 3-fold more in the case of MNU-induced micronuclei (28%). Additionally, by using FISH with the same probes, an attempt was made to identify the origin of chromosome fragments in mitotic anaphase.

Zinc-induced DNA damage and the distribution of metals in the brain of grasshoppers by the comet assay and micro-PIXE.

The distribution and concentration of selected elements by PIXE method and DNA damage using comet assay in brains of 1st instars of grasshoppers Chorthippus brunneus from unpolluted (Pilica) and polluted (Olkusz) site, additionally exposed to various doses of zinc during diapause or after hatching, were measured. We tried to assess the degree of possible pre-adaptation of the insects to heavy metals and evaluate the utility of these parameters in estimation of insect exposure to industrial pollutants. Additionally, the mechanism of zinc toxicity for grasshopper brains was discussed. We observed the correlation between experimental zinc dose, zinc contents in the brain and DNA damage in neuroblasts, but only in groups exposed to lower zinc concentration. For higher zinc concentration the amount of the metal in brain and DNA damage remained at the control level. Some site-related differences in DNA damage between grasshoppers from Pilica and Olkusz were observed during short-term exposure (after hatching). Significant increase in the calcium contents in the brain, proportional to zinc concentration in sand, was also observed, especially in the offsprings from Olkusz. The results may be the basis for further searching for molecular mechanisms of defense against heavy metals in insects living in polluted habitats.

DNA damage induced by mutagens in plant and human cell nuclei in acellular comet assay.

Higher plant cells have a long tradition of use in the studies on environmental mutagenesis in situ, especially in relation to human health risk determination. The studies on the response of plant and human cells to physical and chemical mutagens showed differences in their sensitivity. The differences in the presence of cell components in plants and humans could influence such response. Additionally, the level of the organization of the employed material could influence DNA-damaging effect: leukocytes are isolated cells and plant--an intact organism. To preclude these obstacles, the effects of direct treatment of isolated nuclei with genotoxic agents were determined to compare the sensitivity of plant and human cells. In the present study, we have determined the DNA-damaging effects of two chemical mutagens: maleic acid hydrazide (MH) and N-methyl-N-nitroso-urea (MNU) applied to isolated nuclei of both plant and human cells. In order to compare the sensitivity of the nuclei of Nicotiana tabacum var. xanthi and the nuclei of leukocytes, the acellular Comet assay was carried out. The results showed higher sensitivity of the nuclei of leukocytes as compared to the nuclei of plant cells to mutagenic treatment with the applied doses of MH and MNU.

Plant genotoxicity: a molecular cytogenetic approach in plant bioassays.

It is important for the prevention of DNA changes caused by environment to understand the biological consequences of DNA damages and their molecular modes of action that lead to repair or alterations of the genetic material. Numerous genotoxicity assay systems have been developed to identify DNA reactive compounds. The available data show that plant bioassays are important tests in the detection of genotoxic contamination in the environment and the establishment of controlling systems. Plant system can detect a wide range of genetic damage, including gene mutations and chromosome aberrations. Recently introduced molecular cytogenetic methods allow analysis of genotoxicity, both at the chromosomal and DNA level. FISH gives a new possibility of the detection and analysis of chromosomal rearrangements in a great detail. DNA fragmentation can be estimated using the TUNEL test and the single cell gel electrophoresis (Comet assay).

Transformed roots of Crepis capillaris--a [corrected] sensitive system for the evaluation of the clastogenicity of abiotic agents.

The presence of a large number of pollutants, including mutagenic agents in the environment is a problem of a major concern. Rapid progress in plant biotechnology, especially in the development of cell transformation methods, including the production of transformed roots -- 'hairy roots' -- has opened new possibilities to use transformed root cultures in plant bioassays for the evaluation mutagenic effects of different agents. We have used Crepis capillaris hairy roots for evaluation of cytogenetic effects of mutagenic treatment. Effects of maleic acid hydrazide (MH) and X-ray treatment were analysed in chromosomal aberration, sister chromatid exchange (SCE) and TUNEL tests. Comparison of cytogenetic effects in hairy roots and roots of seedlings showed a much higher sensitivity of hairy roots, which makes them convenient material for monitoring DNA damage after mutagenic treatment.

Chromosomal aberrations in Crepis capillaris cells detected by FISH.

Crepis capillaris (2n=6) is an excellent plant for the assay of chromosome aberrations after mutagenic treatment. It has simple karyotype: three pairs of morphologically distinct and relatively large chromosomes. The frequency of structural chromosome aberrations and micronuclei in root meristem cells has been used for evaluation of the genotoxicity of chemicals and environmental pollutants. The introduction of fluorescence in situ hybridization method allows more detailed detection and localization of chromosomal rearrangements not only in mitotic but also in interphase nuclei. We demonstrate a few examples of the detection of chromosomal aberrations using rDNA and telomeric sequences as probes for in situ hybridization to C. capillaris chromosomes.