Document Type

Theses, Ph.D

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This item is available under a Creative Commons License for non-commercial use only

Publication Details

Successfully submitted for the award of Doctor of Philosophy (Ph.D) to the Dublin Institute of Technology 2007.

Abstract

There is currently a great interest in delayed chromosomal damage and other damaging effects of low-dose exposure to a variety of agents, which appear collectively to act through induction of stress-response pathways related to oxidative stress (and aging). These agents have been studied mostly in the radiation field but evidence is accumulating that chemicals, especially heavy metals, can also act in the same manner. Therefore, this work investigated the effects of metals and/or radiation in human fibroblasts in vitro. Humans are exposed to metals, including chromium (CR) VI) and vanadium (V) (V) from the environment, industry and surgical implants. Thus the impact of low-dose stress responses may be greater than expected from individual toxicity projections. In this study, a short (24 hours) exposure of human fibroblasts to low doses of Cr (VI) and V (V) caused both acute chromosome damage and genomic instability in the progeny of exposed cells for a t least 30 days after exposure. Acutely, Cr (VI) caused chromatid/ breaks without aneuploidy while V (V) caused aneuploidy without chromatid breaks. The long-term genomic instability was similar but depended on hTERT positivity. In telomerase-negative hTERT- cells, CR (VI) and V (V) caused a long lasting and transmissible induction of dicentric chromosome, nucleoplasmic bridges, micronuclei and aneuploidy. There was also a long term and transmissible reduction of clonogenic survival, with and increased b-galactosidase staining and apoptosis. This instability was not present in telomerase positive hTERT + cells. In contrast, in HTERT + cells the metals caused a persistent induction of tetraploidy, which was not noted in hTERT-cells. Interestingly, the clonogenic assay demonstrated that radiation induced genomic instability in hTERT + cells and to a lesser extent, in hTERT-cells. This showed that the telomerase activity in hTERT+cells did not provide protection against genomic instability caused by the radiation insult. Furthermore, neither 0.05 Gy nor 0.5 Gy doses of radiation induced chromosomal instability in either types of cells used (hTERT-and hTERT + cells). However, hTERT + cells had a slight higher incidence of micronuclei, immediately after radiation exposure of 0.5 Gy compared to hTERT- cells. Similarly to the metal only experiments, there was a higher level of tetraploidy in the hTERT+cells compared to the hTERT-cells, although it only reached a level of statistical significance immediately after the radiation exposure of the 0.05 Gy dose. This finding was different to what was seen for the metal only treated [CR (VI)] cells, where hTERT-cells showed significant cell damage and this damage was less compared to hTERT+cells. Combined exposure caused loss of clonogenic survival and therefore genomic instability in both types of cells (hTERT – and hTERT + cells). This genomic instability was more pronounced in hTERT + cells after Metal Followed by Radiation, and it was more pronounced in hTERT- cells after Radiation Followed by Metal. Similarly, cytogenetic damage was higher in hTERT+ cells after Metal Followed by Radiation, and higher in hTERT- cells after Radiation Followed by Metal. Similar to the metal only experiments, there was a higher level of tetraploidy in the hTERT +cells compared to the hTERT-cells, although it did not reach a level of statistical significance. It appears that the biological effects provoked by combined exposure of metal and radiation has led to a synergistic action in both types of cells, compared to metal treatment only or radiation exposure only. In fact, in most of the significant results, the damage caused by the combination of metal and radiation was higher than the damage induced by either metal itself or radiation itself. Similarly to the radiation only experiments, it was interesting to observe that ectopic hTERT expression had no effect in preventing the lost of clonogenic survival, as well as the formation of cell damage after combined exposure. This was in contrast to metal only treated [CR (VI)] cells, where hTERT – cells showed cell damage which was less compared to that observed in hTERT+cells. This study suggests that the type of genomic instability caused by metals in human cells may depend critically on whether they are telomerase-positive or – negative. However, the type of genomic instability caused by either radiation or combined exposure to metals and radiation in human cells appears to be not prevented by telomerase activity.

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