Giulia Sciandrello

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

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.

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

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 1h 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 3h of irradiation.

Long-lasting genomic instability following arsenite exposure in mammalian 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. Environ. Mol. Mutagen. 2011.

Acrylamide catalytically inhibits topoisomerase II in V79 cells

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.

Biological effects and photodegradation by TiO2 of terpenes present in industrial wastewater

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.

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.

Mid-region parathyroid hormone-related protein (PTHrP) binds chromatin of MDA-MB231 breast cancer cells and isolated oligonucleotides in vitro

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 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.