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

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

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"

Telomere dysfunction in cells with arsenic-induced genomic instability

It is well known that the occurrence of dicentric chromosomes represent signature of telomere dysfunction and is a clear symptom of genomic instability. V79 Chinese hamster cells, treated with 10µM sodium arsenite for 24h and allowed to grow in drug-free medium (ASO cells), showed genomic instability with aneuploidy and nuclear abnormalities as well as the appearance of dicentric chromosomes since the 90th cell generation. TRAP assay was performed on growing ASO cells and on clones isolated during the course of the expanded growth. As expected, some clones with dicentric chromosomes and severely reduced telomerase activity went to death; surprisingly, other clones also bearing chromosomal end-to-end fusions maintained telomerase activity and were capable of proliferating accumulating genomic instability. These findings indicate that arsenic induces continuing instability by telomere dysfunction and suggest that this abnormal phenotype could arise from deregulation of the protein complex that protect chromosome ends from degradation and end-to-end fusion. Preliminary results by FISH analysis, performed on the unstable clones with active telomerase, seem to be in favour of a possible involvement of DNA damage response proteins. In fact, the most part of the dicentric rearrangements showed telomeric sequences clearly detectable at fusion point, in agreement with some data reported for mice Ku-deficient cells in which genomic instability is related to the impairment of DNA damage repair mechanism

In vivo and in vitro inhibitory effects of acrylamide on DNA topoisomerase II

Acrylamide (AA), a chemical produced in several foodstuffs when cooked at a high temperature, is considered a probable human carcinogen, but the molecular mechanism underlying its genotoxicity has not fully known. Numerous authors have reported the induction by AA of DNA double strand breaks and sister chromatid exchange (SCE); we here confirmed the acrylamide capability of damaging DNA by utilizing Comet assay, which showed a dose-dependent increase of tail lenght, in metabolically non competent V79 Chinese hamster cells. Moreover, we observed that Acrylamide (AA) was able to antagonize in vivo the citotoxicity of well know poison etoposide; this suggested that topoisomerase II activity was affected by AA. The hypothesis was confirmed in in vitro tests: we observed, in fact, a strong inhibitory effect of AA against topoisomerase II in the kDNA assay and in topoisomerase II-mediated supercoiled DNA relaxation assay. In particular, this latter inhibition was not accompanied by stabilization of a covalent topoisomerase II-DNA intermediate. In order to characterize AA target(s), we pretreated pBR322 with AA and observed that this substrate became quickly incompetent in the topoisomerase II catalytic assay. These results suggest that DNA could be a target for Acrylamide-induced inhibition of topoisomerase II. Furthermore, preliminary results seem to indicate the possibility that another mode of action of acrylamide is related to its affinity for topoisomerase II sulphydryl groups, according to recent evidences reporting that thiol-reactive compounds can induce DNA damage through a nongenotoxic mechanism, such as the thiolation of the nuclear protein topoisomerase II

Persistent genomic instability by arsenic exposure in V79 Chinese hamster cells

Previously, we demonstrated that acute treatment with arsenic leads mammalian cells to exhibit persistent chromosomal instability and DNA hypomethylation, by performing investigations after about 2 months of subculturing. In order to evaluate quantitatively the continuing instability during the expanded growth, we carried out cytogenetic, morphologic and molecular analyses immediately after exposure and every week up to 112 cell generations. Briefly, V79 Chinese hamster cells were treated with 10 µM sodium arsenite (SA) for 24h; at the end of exposure, mitotic rounded-up cells were harvested by shake-off and allowed to grow in drug-free medium. The instability markers, micronucleated and multinucleated cells as well as aneuploid cells, seen just after treatment, reappeared since 60th cell generation. Metaphases with dicentric chromosomes or telomeric associations characterized cell population since 90th generation. To gain insight into the mechanism involved in perpetuating the unstable phenotype, groups of clones, stable and unstable, were analysed also for telomerase activity by TRAP assay and for levels of reactive oxygen species (ROS) measured by ability to oxidize fluorogenic dye. Some of the isolated unstable clones, also bearing chromosomal end-to-end fusions, maintained telomerase activity and were capable to proliferate accumulating genomic instability as well as transformed phenotype and spontaneous increased gene mutation oxidative stress associated. Furthermore these clones showed altered DNA methylation pattern. On the whole, these results raise the possibility that cell variants induced by the short-term exposure to arsenic probably gain a selective advantage when they are able to epigenetically reprogram their genome and proliferate in an error-prone mode

Antagonist effects of Acrylamide on clastogenity of VP16

We investigated on the Acrylamide (AA) capability of influencing the clastogenic effects of VP16, the topoisomerase II targeting drug, by performing sequential treatments in V79 Chinese hamster cells. The VP16 cytotoxicity resulted almost completely antagonized by preincubating cells with nontoxic concentrations of AA, as inferred by statistically significant differences versus response with VP16 alone. Moreover, the severe clastogenic effect of VP16, evidenced by the presence of complex structural chromosome aberrations and by high frequencies of micronulei and sister chromatid exchanges, was reduced by AA in a dose-dependent manner. For example, the frequency of micronucleated cells induced by VP16 was 24.9% , and it became 11.2% or 6.3% when cells were pretreated with 1mM or 5mM AA, respectively. These findings let us to suppose that AA interacts with topoisomerase II. To address this question, we performed the kDNA decatenation assay and found that nuclear extracts from cells treated with either VP16 or AA had reduced topoisomerase II activity, as demonstrated by an inability to convert kDNA from the catenated to the decatenated form; on the contrary, nuclear extracts from cells pretreated with AA and then with VP16 were able to convert kDNa to the decatenated form. Taken together, these results show that acrylamide can efficiently minimize the clastogenicity of VP16 and suggest that it plays a role in the inhibition of topoisomerase II