Karyotype abnormalities in a variant Chinese hamster cell line resistant to methyl methanesulphonate

A variant cell population, isolated from V79-CI 3 Chinese hamster cells after two consecutive treatments with methyl methanesulphonate (MMS), was found to be highly resistant to killing by this alkylating agent. The resistant cell line was cytogenetically characterized both by the presence of a stable translocation involving metacentric chromosome 2 and acrocentric chromosome 6 and by a supernumerary chromosome originated by the duplication of a small telocentric chromosome. This cell population also showed a transient transformed phenotype, seen as formation of transformed foci containing cells with high chromosomes counts and multiple chromosomal aberrations. As MMS-resistance and karyotype changes are permanent and heritable traits, we suppose that they are related events

Abnormal mitotic spindle assembly and cytokinesis induced by D-Limonene in cultured mammalian cells

d-Limonene is found widely in citrus and many other plant species; it is a major constituent of many essential oils and is used as a solvent for commercial purposes. With the discovery of its chemotherapeutic properties against cancer, it is important to investigate the biological effects of the exposure to d-Limonene and elucidate its, as yet unknown, mechanism of action. We reported here that d-Limonene is toxic in V79 Chinese hamster cells in a dose-dependent manner. Moreover, to determine the cellular target of d-Limonene, we performed morphological observations and immunocytochemical analysis and we showed that this drug has a direct effect on dividing cells preventing assembly of mitotic spindle microtubules. This affects both chromosome segregation and cytokinesis, resulting in aneuploidy that in turn can lead to cell death or genomic instability

Genomic instability induced by a-pinene in Chinese hamster cell line

Here, we report the effects of exposure of mammalian cells to a-pinene, a bicyclic monoterpene used in insecticides, solvents and perfums. Morphological analysis, performed in V79-Cl3 cells exposed for 1 h to increasing concentrations (25 up to 50 mM) of a-pinene, indicated a statistically significant increase in micronucleated and multinucleated cell frequencies; apoptotic cells were seen at 40 and 50 mM. This monoterpene caused genomic instability by interfering with mitotic process; in fact, 50% of cells (versus 19% of control cells) showed irregular mitosis with multipolar or incorrectly localised spindles. Cytogenetic analysis demonstrated high-frequency hypodiploid metaphases as well as endoreduplicated cells and chromosome breaks. Clastogenic damage was prevalent over aneuploidogenic damage as demonstred by the higher proportion of kinetochore-negative micronuclei. Alkaline comet confirmed that monoterpene exposure caused DNA lesions in a concentration-dependent manner. This damage probably arose by increased reactive oxygen species (ROS) production. In order to assess the generation of ROS, the cells were incubated with CM-H2DCFDA and then analysed by flow cytometry. Results demonstrated an increase in fluorescence intensity after a-pinene treatment indicating increased oxidative stress. On the whole, these findings strongly suggest that a-pinene is able to compromise genome stability preferentially through mitotic alterations and to damage DNA through ROS production

Long-Lasting Genomic Instability Following Arsenite Exposure inMammalian Cells: The Role of Reactive Oxygen Species

Previously, we reported that the progeny of mammalian cells, which has been exposed to sodium arsenite for two cell cycles, exhibited chromosomal instability and concurrent DNA hypomethylation, when they were subsequently investigated after two months of subculturing (about 120 cell generations) in arsenite-free medium. In this work, we continued our investigations of the long-lasting arsenite-induced genomic instability by analyzing additional endpoints at several time points during the cell expanded growth. In addition to the progressive increase of aneuploid cells, we also noted micronucleated and multinucleated cells that continued to accumulate up to the 50th cell generation, as well as dicentric chromosomes and/or telomeric associations and other complex chromosome rearrangements that began to appear much later, at the 90th cell generation following arsenite exposure. The increasing genomic instability was further characterized by an increased frequency of spontaneous mutations. Furthermore, the long-lasting genomic instability was related to elevated levels of reactive oxygen species (ROS), which at the 50th cell generation appeared higher than in stable parental cells. To gain additional insight into the continuing genomic instability, we examined several individual clones isolated at different time points from the growing cell population. Chromosomally and morphologically unstable cell clones, the number of which increased with the expanded growth,were also present at early phases of growth without arsenite. All genomically unstable clones exhibited higher ROS levels than untreated cells suggesting that oxidative stress is an important factor for the progression of genomic instability induced by arsenite

Genotoxicity of citrus wastewater in prokaryotic and eukaryotic cells and efficiency of heterogeneous photocatalysis by TiO2

The presence of (±)a-pinene, (+)b-pinene, (+)3-carene, and R-(+)limonene terpenes in wastewater of a citrus transformation factory was detected and analyzed, in a previous study, by using Solid Phase Microextraction (SPME) followed by GC analyses. Purpose of that research was to compare the genotoxic responses of mixtures of terpenes with the genotoxicity of the individual compounds, and the biological effects of actual wastewater. Genotoxicity was evaluated in the Salmonella reversion assay (Ames test) and in V79 cells by Comet assay. Ames tests indicated that the four single terpenes did not induce an increase of revertants frequency. On the contrary, the mixtures of terpenes caused, in the presence of metabolic activation, a highly significant increase of the revertants in TA100 strain in comparison to the control. The Comet assay showed a significant increase in DNA damage in V79 cells treated for 1 h with single or mixed terpenes. Moreover, the actual wastewater was found highly genotoxic in bacterial and mammalian cells. Photocatalytic tests completely photodegraded the pollutants present in aqueous wastewater and the initial high genotoxicity of samples of wastewater collected during the photocatalytic run, was completely lose in 3 h of irradiation

Notulae to the Italian flora of algae, bryophytes, fungi and lichens: 14

In this contribution, new data concerning bryophytes, fungi and lichens of the Italian flora are presented. It includes new records and confirmations for the algal genus Chara, for the bryophyte genera Bryum, Grimmia, Cephaloziella, Hypnum, Nogopterium, Physcomitrium, Polytrichastrum, Rhynchostegiella, Saelania, and Schistostega, the fungal genera Cortinarius, Lentinellus, Omphalina, and Xerophorus, and the lichen genera Acarospora, Agonimia, Candelariella, Cladonia, Graphis, Gyalolechia, Hypogymnia, Lichinella, Megalaria, Nephroma, Ochrolechia, Opegrapha, Peltigera, Placidium, Ramalina, Rhizoplaca, Ropalospora, Strangospora, Toniniopsis, Usnea, and Zahlbrucknerell

The DNA methylation inhibitor 5-azacytidine modulates 6-thioguanine toxicity in mammalian cells

In order to assess the effects of combining two antimetabolites used separately to treat human leukemias, we carried out an experimental study by exposing V79 Chinese hamster cells, a 6-thioguanine (6-tG)-sensitive cell line, to sequential and concurrent treatments with 5-azacytidine (5-azaC) and 6-tG. In this paper, we demonstrate that there is a clear dependency for the way in which this combination was tested. Pre-treatment with 5-azaC made V79 cells more resistant to 6-tG by a substantial reduction in 6-tG incorporation into DNA; this effect could still be detected for several cell divisions after the removal of 5-azaC, and was achieved neither by reduced cell growth nor by the induction of hypoxanthine-guanine-phosphoribosyltransferase (HGPRT(-)) mutants. The reverse order of treatment produced a higher toxic effect than exposure to each prodrug alone. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved

Genomewide hypomethylation and PTHrP gene hypermethylation as a model for the prediction of cancer risk in rheumatoid arthritis

We have previously shown that PTHrP(38-94)-amide restrains growth and invasion "in vitro", causes striking toxicity and accelerates death of some breast cancer cell lines, the most responsive being MDA-MB231 whose tumorigenesis was also attenuated "in vivo". PTHrP(38-94)-amide contains the domain implicated in the nuclear import of PTHrP. Although the nucleus was identified as a destination for mid-region PTHrP, evidence for direct DNA-binding capability is lacking to date. Here, we examined the localization of PTHrP(38-94)-amide within MDA-MB231 cells and within metaphase spread preparations and characterized its DNA-binding properties, employing a combination of immunocytochemical, cytogenetic, "whole genome"/conventional PCR, EMSA and DNase footprinting techniques. The results obtained: (i) show that PTHrP(38-94)-amide gains access to the nuclear compartment of MDA-MB231 cell; (ii) demonstrate that PTHrP(38-94)-amide is a DNA-binding peptide; and, (iii) represent the first data to date on the potential molecular targets in both cellular chromatin and isolated oligonucleotides "in vitro"

The High-Altitude Volcanic Caves of Mount Etna (Eastern Sicily): A Relevant Refuge for Some Ptero-Bryophyte Communities

A phytosociological analysis of the plant communities in high-altitude volcanic caves of Mount Etna (Sicily), based on literature data and unpublished relevés, is presented. A total of 147 phytosociological relevés were processed and analyzed using classification and ordination methods. Classification of the relevés, supported by ordination, showed two main vegetation groups: the first one includes communities of the Pohlion crudae alliance, and the other includes the vegetation from the Pohlio crudae–Asplenion septentrionalis alliance. Furthermore, two new communities, Pohlio crudae–Cystopteridetum dickieanae subass. amphidietosum mougeotii and Pohlio crudae–Cystopteridetum dickieanae subass. polystichetosum lonchitis, are proposed for Etna. The high-altitude caves can be considered a refuge for these ptero-bryophytic rare communities

Early induction of genetic instability and apoptosis by arsenic in cultured Chinese hamster cells

In order to assess at what time from the beginning of exposure inorganic arsenic can give rise to genetic instability and trigger apoptosis, V79-C13 Chinese hamster cells were treated with 10 muM sodium arsenite for 24 h. Under these conditions, cell survival was >70% and cells showed neither an increase in chromosome aberration frequency nor a delay in cell cycle progression. Investigations, which were carried out every 6 h during the treatment, revealed an early appearance of genetically unstable cells, namely micronucleated, multinucleated and mononucleated 'giant' cells, as well as apoptotic cells. Indirect immunostaining using anti-beta-tubulin antibody showed severe alterations in spindle morphology after only 6 h treatment, when cells with small spindles whose poles were inside the metaphase plate appeared, and after 12 h treatment, when cells in which spindle assembly had completely failed were observed. These cells, unable to complete mitosis, underwent apoptosis. In fact, cells which turned out to be positive in the TdT-FragEL test had condensed chromatin arranged in metaphase-like plates; their maximum frequency was reached after 24 h treatment. A cytogenetic study was conducted at the end of the period of exposure to arsenic and after post-treatment incubation in fresh medium for up to 5 days. It showed that the percentage of cells with 21 chromosomes (modal number of the cell line) decreased, making way for aneuploid cells. Arsenic, therefore, induced early genetic instability or apoptosis in dividing cells. However, while apoptosis tended to cease when arsenic was removed from the culture medium, the acquired instability remained and propagated within the cell population