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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.

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.

Here, we report the effects of exposure of mammalian cells to α-pinene, a bicyclic monoterpene used in insecticides, solvents and perfumes. Morphological analysis, performed in V79-Cl3 cells exposed for 1 h to increasing concentrations (25 up to 50 μM) of α-pinene, indicated a statistically significant increase in micronucleated and multinucleated cell frequencies; apoptotic cells were seen at 40 and 50 μM. 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 demonstrated 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-H(2)DCFDA and then analysed by flow cytometry. Results demonstrated an increase in fluorescence intensity after α-pinene treatment indicating increased oxidative stress. On the whole, these findings strongly suggest that α-pinene is able to compromise genome stability preferentially through mitotic alterations and to damage DNA through ROS production.

The presence of (±)α-pinene, (+)β-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 Micro-extraction (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.

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. Environ. Mol. Mutagen. 2011. © 2011 Wiley-Liss, Inc.

It is well established that inorganic arsenic induces neurotoxic effects and neurological defects in humans and laboratory animals. The cellular and molecular mechanisms of its actions, however, remain elusive. Herein we report the effects of arsenite (NaAsO2) on primary cultures of rat astrocytes. Cells underwent induction of heat shock protein 70 only at the highest doses of inorganic arsenic (30 and 60 microM), suggesting a high threshold to respond to stress. We also investigated arsenic genotoxicity with the comet assay. Interestingly, although cells treated with 10 microM arsenite for 24 h maintained >70% viability, with respect to untreated cells, high DNA damage was already observed. Since arsenic is not known to be a direct-acting genotoxic agent, we investigated the possibility that its effects are due, in astrocytes as well, to ROS formation, as already described for other cell types. However, FACS analysis after CM-H2DCFDA staining did not evidence any significant increase of ROS production while, on the contrary, at the highest arsenite concentrations used, ROS production decreased. Concordantly, we found that, if most cells in the culture are still alive (i.e. up to 10 microM arsenite), they show a treatment-dependent increase in the concentration of SOD1. On the other hand, SOD2 concentration did not change. Finally, we found that astrocytes also synthesize PIPPin, an RNA-binding protein, the concentration of which was recently reported to change in response to stress induced by cadmium. Here we also report that, in cells exposed to high doses of arsenite, an anti-PIPPin antibody-positive faster migrating protein appears.

The aim of this work was to study the biological effects of four monoterpenes, i.e. α-pinene, β-pinene, 3-carene and d -limonene present in the wastewater of a citrus transformation factory. The study was carried out by exposing V79 Chinese hamster cells to single terpene or to the mixture of four terpenes at concentrations corresponding to those in the wastewater evaluated by head space solid phase micro extraction and gas chromatography (HS-SPME-GC) analyses. Treatments with single or combined terpenes similarly affected cell vitality, but only the combined treatments induced the 6-thioguanine resistant mutants. Moreover the photocatalytic degradation of the four terpenes was successfully achieved with the photocatalyst TiO 2 Degussa P25 in both the actual effluent and in synthetic solutions.

The vinyl monomer acrylamide is characterized by the presence of an alpha,beta-unsaturated carbonyl group that makes it reactive towards thiol, hydroxyl or amino groups and towards the nucleophilic centers in DNA. The ability of acrylamide to chemically modify protein thiols has prompted us to consider topoisomerase II as one possible target of acrylamide, since agents targeting protein sulfhydryl groups act as either catalytic inhibitors or poisons of topoisomerase II. Nuclear extracts from V79 Chinese hamster cells incubated with acrylamide reduced topoisomerase II activity as inferred by an inability to convert kinetoplast DNA to the decatenated form. Nuclear extracts incubated with acrylamide pre-incubated with DTT converted kinetoplast DNA to the decatenated form, suggesting that acrylamide influences topoisomerase II activity through reaction with sulfhydryl groups on the enzyme. Furthermore, acrylamide did not induce the pBR322 DNA cleavage, as assessed by cleavage assay: thus, it cannot be regarded as a poison of topoisomerase II. As a catalytic inhibitor, acrylamide antagonizes the effect of etoposide, a topoisomerase II poison, as determined by clonogenic assay in V79 cells. This antagonism is confirmed by band depletion assay, from which it can be inferred that acrylamide reduces the level of catalytically active cellular topoisomerase II available for the action of etoposide. (C) 2009 Elsevier Ltd. All rights reserved. The vinyl monomer acrylamide is characterized by the presence of an a,b-unsaturated carbonyl group that makes it reactive towards thiol, hydroxyl or amino groups and towards the nucleophilic centers in DNA. The ability of acrylamide to chemically modify protein thiols has prompted us to consider topoisomerase II as one possible target of acrylamide, since agents targeting protein sulfhydryl groups act as either catalytic inhibitors or poisons of topoisomerase II. Nuclear extracts from V79 Chinese hamster cells incubated with acrylamide reduced topoisomerase II activity as inferred by an inability to convert kinetoplast DNA to the decatenated form. Nuclear extracts incubated with acrylamide pre-incubated with DTT converted kinetoplast DNA to the decatenated form, suggesting that acrylamide influences topoisomerase II activity through reaction with sulfhydryl groups on the enzyme. Furthermore, acrylamide did not induce the pBR322 DNA cleavage, as assessed by cleavage assay; thus, it cannot be regarded as a poison of topoisomerase II. As a catalytic inhibitor, acrylamide antagonizes the effect of etoposide, a topoisomerase II poison, as determined by clonogenic assay in V79 cells. This antagonism is confirmed by band depletion assay, from which it can be inferred that acrylamide reduces the level of catalytically active cellular topoisomerase II available for the action of etoposide.

The photocatalytic degradation of paraquat (1,1-dimethyl-4,4′-bipyridylium dichloride) aqueous solutions in the presence of polycrystalline TiO2 Degussa P25 irradiated by near-UV light was investigated. The substrate and total organic carbon concentrations were monitored by UV spectroscopy and TOC measurements, respectively: the complete photocatalytic mineralization of paraquat (20 ppm) was achieved after ca. 3 h of irradiation by using 0.4 g l−1 of catalyst amount at natural pH (ca 5.8). On the contrary no significant photodegradation of paraquat was observed in the absence of TiO2 under similar experimental conditions. To evaluate the genotoxicity of paraquat and its intermediates produced during heterogeneous photocatalytic treatment, in vitro tests such as Ames test, with and without rat liver microsomal fractions (S9 mix), and micronucleus test, were used. Results obtained with Salmonella typhimurium (strain TA100) showed that paraquat and photocatalytic products were unable to induce gene mutations when photocatalysis was used in the presence of the optimum amount of TiO2, i.e. 0.4 g l−1, whereas an increase of revertants his+ per plate was observed after 300 min irradiation in the presence of very low amount of TiO2 (0.04 g l−1). The negative results from micronucleus test suggest that mutagenic, but non-clastogenic, late intermediates of paraquat photo-oxidation were formed when the photocatalytic runs of paraquat degradation were carried out by using 0.04 g l−1 of photocatalyst.

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".

Early genetic instability induced in dividing V79-Cl3 Chinese hamster cells by inorganic arsenic, as demonstrated in our previous investigation, was evidenced by aneuploidy and nuclear abnormalities, but not by chromosomal rearrangements. Here we report the results of cytogenetic and morphological analyses performed on the progeny of cells dividing at the end of sodium arsenite treatment after they had been expanded through 120 generations (ASO cells) and then cloned. The acquired genetic instability persisted and was increased by highly unstable chromosomal rearrangements, namely dicentric chromosomes and telomeric associations, which were not seen following acute exposure. A peculiar finding was the preferential involvement of a particular chromosome in dicentric rearrangements observed in some isolated ASO clones. Interestingly, by immunostaining with anti-5-methylcytosine antibodies the genome-wide DNA hypomethylation, induced by arsenic immediately after the acute treatment, was found to affect those ASO clones characterized by aneuploidy and chromosomal rearrangements. These findings demonstrate that short-term exposure to arsenic has long-term effects and suggest that genome-wide DNA hypomethylation enhances genetic instability.

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.

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 microM 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.