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Mutagenesis Advance Access originally published online on September 5, 2008
Mutagenesis 2009 24(1):25-33; doi:10.1093/mutage/gen048
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© The Author 2008. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

Effects of hTERT on genomic instability caused by either metal or radiation or combined exposure

A. Glaviano1,2,*, C. Mothersill3, C. P. Case2, M. A. Rubio4, R. Newson5 and F. Lyng1

1Radiation and Environmental Science Centre, Focas Institute, Dublin Institute of Technology, Dublin, Ireland 2Bristol Implant Research Centre, University of Bristol, Bristol, UK 3Department of Medical Physics and Applied Radiation Sciences, McMaster University, Hamilton, Canada 4The Netherlands Cancer Institute, Amsterdam, The Netherlands 5National Heart and Lung Institute, Imperial College London, London, UK

Genomic instability is considered to be an important component in carcinogenesis. It can be caused by low-dose exposure to agents, which appear to act through induction of stress–response pathways related to oxidative stress. These agents have been studied mostly in the radiation field but evidence is accumulating that chemicals, especially heavy metals such as Cr (VI), can also act in the same manner. Previous work showed that metal ions could initiate long-term genomic instability in human primary fibroblasts and this phenomenon was regulated by telomerase. The aim of this study was to examine the difference in clonogenic survival and cytogenetic damage after exposure to Cr (VI) and radiation both singly and in combination in normal human fibroblasts (hTERT– cells) and engineered human fibroblasts, infected with a retrovirus carrying a cDNA encoding hTERT, which rendered these cells telomerase positive and replicatively immortal (hTERT+ cells). Cr (VI) induced genomic instability in hTERT– cells but not in hTERT+ cells, whereas radiation induced genomic instability in hTERT+ cells and to a lesser extent in hTERT– cells. Combined exposure caused genomic instability in both types of cells. However, this genomic instability was more pronounced in hTERT– cells after radiation followed by Cr (VI) and more pronounced in hTERT+ cells after Cr (VI) followed by radiation. Moreover, the biological effects provoked by combined exposure of Cr (VI) and radiation also led to a synergistic action in both types of cells, compared to either Cr (VI) treatment only or radiation exposure only. This study suggests that telomerase can prevent genomic instability caused by Cr (VI), but not by radiation. Furthermore, genomic instability may be prevented by telomerase when cells are exposed to radiation and then Cr (VI) but not after exposure to Cr (VI) and then radiation.


   Introduction
Top
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Human beings may be exposed to low doses of mutagenic agents from a wide variety of sources. Many cancers show a multivariate pattern of genetic mutations suggesting that several aetiological agents are involved. Therefore, it is relevant to look at the actions of mutagenic agents, such as radiation and heavy metals, both singly and in combination with each other. Both radiation (1Go–6Go) and heavy metals (7Go–9Go) have been shown to provoke genomic instability and there is a need to determine the combined effects of these two toxic agents.

Studies have shown that combined exposure of Cr (VI) and gamma rays can have enhancing effects on the outcome, compared to the single agent (either metal or gamma rays) (10Go,11Go). In fact, chromium (VI) ions enhanced mutagenic effects of gamma rays in both acute and chronic experiments, assessed by a micronucleus test in mouse bone marrow polychromatocytes (10Go). Moreover, Anan'eva et al. (11Go) showed that combined exposure of rats to low-dose irradiation and heavy metal (Cu2+) ions caused significant accumulation of free-radical products in various organs and tissues, compared to either radiation or metal only. Other authors have shown similar effects when metals, different from Cr (VI), were combined with radiation (12Go–18Go).

Like the radiation-induced genomic instability (1Go,2Go), cadmium and nickel ions, as well as orthopaedic wear debris from a titanium aluminium vanadium alloy hip replacement, also cause genomic instability (7Go,8Go). There was a persistent reduction in clonogenic survival and an increase in chromosome aberrations after exposure in the distant progeny of cells, which appeared to have thoroughly recovered from the exposure (7Go,8Go). Genomic instability induced by agents such as metals (9Go) and radiation (19Go) is influenced by telomerase including hTERT and/or telomeres in a number of species including yeast, plants, mice and cultured human cells (20Go–22Go).

The specific mechanisms involved in the induction and particularly in the transmission of the genomic instability phenotype to progeny are still unknown. Oxidative stress responses are known to be involved and subtle checkpoint control of sectoring cellular ‘decisions’ on how to manage damage can be linked to genotype (23Go). Furthermore, the actual genes involved have not been identified. It has been shown that double-strand breaks (DSBs) are the main initiators for genomic instability (24Go). Therefore, in the present study, it was interesting to see whether the long-term effects of radiation and Cr (VI), which are both known to induce DSBs, would or would not enhance the genomic instability outcome.

The purpose of this study was to investigate whether the short- and long-term response of cells exposed to sublethal concentrations of either metal or radiation or combined exposure would induce genomic instability, in order to further understand the process of cancer formation from multiple causes. In addition, we have examined the role of hTERT to test whether it would protect against the genomic instability induced by both agents, as it had previously been shown to do so against metal exposure alone (9Go).


   Materials and methods
Top
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Cell lines
Two types of BJ human foreskin fibroblasts were used for all the experiments: hTERT– human fibroblasts (normal cells) and hTERT+ human fibroblasts (immortalized cells). BJ normal human foreskin fibroblasts were infected at population doubling (PD) 50 with pLXSN-hTERT retroviruses, as in Rubio et al. (25Go). These cells were reselected from the bulk of the cell population as in our previous study (9Go). The plasmid was created by introducing hTERT from pBABE-PURO-hTERT (25Go) in the pLXSN retroviral vector (Clontech Berkeley, CA, USA). Cells were selected with G418, and the population (hTERT+) expanded before being frozen prior to use after PD 55. BJ hTERT– cells were also used after PD 50. These cells became senescent after PD 80. Both types of cells were grown at 70% confluency in Dulbecco`s modified Eagle's medium (Sigma London UK), supplemented with 20% of Medium 199 (Sigma), 10% foetal bovine serum (Gibco), 20 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer (Sigma), 4 mM L-glutamine (Gibco), 1 mM sodium pyruvate (Sigma) and 1x antibiotics antimycotic (Sigma). During each harvesting, cells were detached from the bottom surface of the flasks using Versene (Gibco) supplemented with 0.6x trypsin (Sigma). The cells were trypsinized every 3 days. The range of telomerase expression, as well as the range of telomere sizes in the control cell population, was shown in our previous work (9Go). Both types of cells were cultured at 37°C, either in 25- or 75-cm2 flasks, depending on the experiment carried out. All the experiments were started with cells at approximately PD 60. All experiments were performed in duplicate.

Experimental procedure
For all the experiments, 5 x 105 cells, plated in 75-cm2 flasks were allowed to attach for 24 h. Exactly a day later, these cells followed a different experimental procedure, depending on the sequence of the different insults.

(i) Cells were first treated for 24 h with a single dose of metal, 0.4 µM Cr (VI), they were then exposed to a single dose of radiation (0.05 or 0.5 Gy) and the next day they were harvested.
(ii) Cells were first exposed to a single dose of radiation (0.05 or 0.5 Gy), the next day they were treated for 24 h with a single dose of metal, 0.4 µM Cr (VI), and the next day they were harvested.

Therefore, in both experimental procedures, the cells were kept in culture for 3 days.

For each type of cells, there were 11 dose points, four of which (8, 9, 10 and 11) were combined exposure:

(1) Control
(2) Metal + Sham Irradiation (M + SI)
(3) Sham Irradiation + Metal (SI + M)
(4) Radiation 0.05 Gy + Vehicle Control (0.05 Gy + VC)
(5) Vehicle Control + Radiation 0.05 Gy (VC + 0.05 Gy)
(6) Radiation 0.5 Gy + Vehicle Control (0.5 Gy + VC)
(7) Vehicle Control + Radiation 0.5 Gy (VC + 0.5 Gy)
(8) Metal + Radiation 0.05 Gy (M + 0.05 Gy)
(9) Radiation 0.05 Gy + Metal (0.05 Gy + M)
(10) Metal + Radiation 0.5 Gy (M + 0.5 Gy)
(11) Radiation 0.5 Gy + Metal (0.5 Gy + M)

Control flasks were exposed to neither metal nor radiation. Phosphate-buffered saline (PBS) was used as a control for the metal treatment using the same volume (vehicle control), whereas the flasks were treated identically to the irradiated cells without the actual irradiation (sham irradiation).

The effects of the insults on the cells were studied immediately (Day 0) and 30 days (Day 30) after either a single metal treatment, or a single radiation exposure, or a combined exposure (metal + radiation or vice versa). These cells were trypsinized every 3 days, and the plating density during post-treatment passaging was 5 x 105 cells (in 75-cm2 flasks). These cells were counted with the haemocytometer and the trypan blue staining (Sigma), and the initial cell inoculum was 100% viable. Twenty-four hours after the metal treatment, the Day 0 cells were washed twice with 1x PBS to remove thoroughly any traces of metal ions. The other cells, used for delayed effects (Day 30) of either only metal exposure or combined exposure, were also washed twice with 1x PBS and fresh medium was added into the flasks.

Metal
Both types of cells were exposed to a single dose of Cr (VI) (0.4 µM), using potassium dichromate (K2Cr2O7) (Sigma) for 24 h. The metal salt was diluted in PBS in order to reach the concentration used.

Radiation
Both types of cells were exposed to two different doses of gamma radiation (either 0.05 or 0.5 Gy). The cells were irradiated at room temperature using a Cobalt 60Co teletherapy unit (St Luke's Hospital, Rathgar, Dublin, Ireland). The dose administered from this unit is a function of the distance of the source from the culture flask/object, dose rate and size of radiation field. The standard distance from source to skin/flask (SSD) is 80 cm, but in this study, flasks were irradiated at a distance of 100 and 170 cm from the source. A correction factor was applied for the change in distance. The dose rate was ~1.8 Gy/min during these experiments. TLDs were used to confirm that the appropriate dose was delivered.

Cell survival
In experiments of clonogenic survival, the cells were treated with 0.4 µM Cr (VI) for 24 h and/or exposed to either 0.05 or 0.5 Gy doses of ionizing radiation. The metal dose of 0.4 µM was chosen because it had given a significant reduction of cell survival in our previous study (9Go). These radiation doses were selected because higher doses of radiation had induced high toxicity, resulting in a low plating efficiency (PE) [PE (%) = (no. of colonies counted/no. of cells plated) x 100]. After each time interval (Day 0 or Day 30), the cells were harvested and then plated (2000 cells) in 25-cm2 flasks at concentrations adjusted to yield ~100 colonies according to the method established by Puck and Marcus (26Go). Six flasks for each dose point were used for this experiment. The cells were cultured for 2 weeks and then stained with carbol fuchsin (20%, Ziehl Niehlson) to assess the colony formation.

Cell damage
In the experiments using the micronucleus assay, the cells were treated with 0.4 µM Cr (VI) for 24 h and/or exposed to either 0.05 or 0.5 Gy doses of ionizing radiation. The metal dose of 0.4 µM was chosen because it had given significant results in previous cytogenetic studies (9Go). The radiation doses of 0.05 or 0.5 Gy were selected because higher doses of radiation were supposed to induce high cytotoxicity, resulting in a low yield of binucleated cells in the micronucleus assay. Immediately after the combined exposure, and therefore 24 h before the harvesting, the cells were exposed to cytochalasin-B (Sigma) at concentration of 6 µg/ml for 24 h. After 24 h of exposure, cells were harvested for the detection of micronuclei (MNi) formation to assess the initial effects (Day 0) of metal only, radiation only or combined exposure insult. The cells were then centrifuged (1200 r.p.m. for 6 min) and re-suspended in 1 ml of fresh media. Flasks containing cells used for the detection of damage at Day 30 were grown 30 days more. These cells were subcultured every 3 days. The micronucleus assay (in binucleated cells) was performed according to the method of Fenech and Morley (27Go–29Go).

Statistical methods
Analyses used generalized linear models with quasi-likelihood standard errors (30Go) and hypergeometric confidence intervals and Fisher exact tests for odds ratios. For the assays of proportions of cells that were clonogenic or micronucleate, a generalized linear model was used, with a log link function, a binomial variance function with total equal to the number of cells in the sample assayed and a common overdispersion parameter based on Pearson's chi squared to calculate confidence intervals for population proportions of cells assayed as positive (positivity rates) and their treated/control and hTERT+/hTERT– ratios (rate ratios).


   Results
Top
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Cell survival
Combined exposure of M + 0.05 Gy M + SI and M + 0.05 Gy caused a significant decrease in clonogenic survival (respectively, P < 0.01 and P < 0.05) in hTERT– cells at Day 0 (Figure 1a). The hTERT+ cells followed a different pattern since M + 0.05 Gy caused a statistically significant reduction of clonogenic survival (P < 0.05) immediately after the combined exposure, which increased up to 30 days after the combined exposure (P < 0.01) (Figure 1b). Furthermore, there was a significant increase of clonogenic survival (P < 0.01) 30 days after the radiation exposure (VC + 0.05 Gy). A direct comparison of hTERT– and hTERT+ cells showed that at both survival times, with either metal treatment or radiation exposure or combined exposure, there were more colonies in the hTERT+ cells compared to hTERT– cells (with the only exception of M + 0.05 Gy at Day 0). The clonogenicity rate ratios of hTERT+/hTERT– cells were plotted with their confidence intervals in Figure 3. These values were expressed as a percentage of control (PBS) and control was set to 100%.


Figure 1
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  		Fig. 1. Clonogenic survival. The figures show the clonogenic survival in hTERT– cells (a and c) and hTERT+ cells (b and d) after a single exposure to M + SI, either VC + 0.05 Gy or VC + 0.5 Gy and either a combined exposure to M + 0.05 Gy or M + 0.5 Gy and at different times after the exposure. Metal (M) is Cr (VI) (0.4 µM), sham irradiation (SI) is room temperature and vehicle control (VC) is PBS. These values were expressed as a percentage of control (PBS) and control was set to 100%. Error bars represent standard deviations. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the control (PBS).

 
Combined exposure of M + 0.5 Gy M + SI and M + 0.5 Gy caused a significant decrease of clonogenic survival (respectively, P < 0.01 and P < 0.05) in hTERT– cells at Day 0. This clonogenic reduction persisted only with M + 0.5 Gy (P < 0.01) up to 30 days after the combined exposure (Figure 1c). The hTERT+ cells followed a different pattern since M + 0.5 Gy caused a significant reduction in clonogenic survival only 30 days after the combined exposure (P < 0.001). Furthermore, there was a 10% reduction in clonogenic survival 30 days after radiation exposure only (VC + 0.5 Gy) (P < 0.01) (Figure 1d). A direct comparison of hTERT– and hTERT+ cells showed that at both survival times, with either metal treatment or radiation exposure or combined exposure, there were more colonies in the hTERT+ cells compared to hTERT– cells. The clonogenicity rate ratios of hTERT+/hTERT– cells were plotted with their confidence intervals in Figure 3. These values were expressed as a percentage of control (PBS) and control was set to 100%.

Combined exposure of 0.05 Gy + M The 0.05 Gy + VC, SI + M and 0.05 Gy + M resulted in a significant decrease (~13%) in clonogenic survival in hTERT– cells at Day 0 (P < 0.05). SI + M and 0.05 Gy + M caused a persistent decrease in clonogenic survival (respectively, P < 0.01 and P < 0.001) up to 30 days after either the metal or the combined exposure (Figure 2a). The hTERT+ cells showed a reduction in clonogenic survival following radiation exposure (0.05 Gy + VC) at Day 0 (P < 0.01), which persisted up to 30 days after the metal exposure (P < 0.05). The 0.05 + M caused a significant reduction in clonogenic survival at Day 0 (P < 0.05), which persisted and became more significant up to 30 days after the combined exposure (P < 0.01) (Figure 2b). A direct comparison of hTERT– and hTERT+ cells showed that at both survival times, with either metal treatment or radiation exposure or combined exposure, there were more colonies in the hTERT+ cells compared to hTERT– cells (with the only exception of 0.05 Gy + VC at Day 0). The clonogenicity rate ratios of hTERT+/hTERT– cells were plotted with their confidence intervals in Figure 3. These values were expressed as a percentage of control (PBS) and control was set to 100%.


Figure 2
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  		Fig. 2. Clonogenic survival. The figures show the clonogenic survival in hTERT– cells (a and c) and hTERT+ cells (b and d) after a single exposure to either 0.05 Gy + VC or 0.5 Gy + VC, SI + M and either a combined exposure to 0.05 Gy + M or 0.5 Gy + M and at different times after the exposure. Metal (M) is Cr (VI) (0.4 µM), sham irradiation (SI) is at room temperature and vehicle control (VC) is PBS. These values were expressed as a percentage of control (PBS) and control was set to 100%. Error bars represent standard deviations. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the control (PBS).

 


Figure 3
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  		Fig. 3. Clonogenic survival. The figure illustrates the rate ratio of hTERT+/hTERT– for clonogenic survival, with 95% confidence intervals, showing combinations of exposure (metal only, radiation only and combined) and days (Day 0 and Day 30) post exposure. ***P < 0.001 compared to the control (PBS).

 
Combined exposure of 0.5 Gy + M SI + M and 0.5 Gy + M caused a significant decrease (~18%) in clonogenic survival in hTERT– cells (P < 0.05) at Day 0. There was a further reduction in clonogenic survival with SI + M (P < 0.01) and 0.5 Gy + M (P < 0.001) up to 30 days after either the metal or the combined exposure (Figure 2c). Furthermore, there was a loss of clonogenic survival 30 days after the radiation exposure (0.5 Gy + VC) (P < 0.05). The hTERT+ followed a different pattern since only the radiation exposure (0.5 Gy + VC), but not the combined exposure (0.5 Gy + M), resulted in a decrease (~14.5%) in clonogenic survival immediately after the radiation exposure (Day 0) (P < 0.001), which persisted up to 30 days after the radiation exposure (P < 0.001) (Figure 2d). A direct comparison of hTERT– and hTERT+ cells showed that at both survival times, with either metal treatment or radiation exposure or combined exposure, there were more colonies in the hTERT+ cells compared to hTERT– cells (with the only exception of 0.5 Gy + VC at Day 0). The clonogenicity rate ratios of hTERT+/hTERT– cells are plotted with their confidence intervals in Figure 3. These values were expressed as a percentage of control (PBS) and control was set to 100%.

Cell damage
Combined exposure of M + 0.05 Gy M + SI, VC + 0.05 and M + 0.05 Gy did not induce significant MNi in hTERT– cells (Figure 4a). The hTERT+ cells followed the same pattern (Figure 4b). However, in all the experiments at both survival times, with or without either metal treatment or radiation exposure or combined exposure, there was a lower level of MNi in the hTERT+ cells compared to the hTERT– cells. This was statistically significant in the control (P < 0.05) and after metal exposure to M + SI (P < 0.01) at Day 30 only (Figure 6). These results were expressed as a percentage of MNi.


Figure 4
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  		Fig. 4. Micronuclei (MNi). The figures show the percentage of MNi in hTERT– cells (a and c) and hTERT+ cells (b and d) after a single exposure to M + SI, either VC + 0.05 Gy or VC + 0.5 Gy and either a combined exposure to M + 0.05 Gy or M + 0.5 Gy and at different times after the exposure. Metal (M) is Cr (VI) (0.4 µM), sham irradiation (SI) is room temperature and vehicle control (VC) is PBS. Error bars represent standard deviations. *P < 0.05 compared to the control (PBS).

 
Combined exposure of M + 0.5 Gy M + 0.5 Gy caused a statistically significant increase in MNi immediately after the combined exposure (Day 0) in hTERT– cells, which did not persist up to Day 30 (Figure 4c). The hTERT+ cells followed a similar pattern, but M + 0.5 Gy induced a significant persistence of MNi since they were present at both survival times. Furthermore, VC + 0.5 Gy caused significant increase in MNi immediately after the radiation exposure (Day 0), which did not persist up to Day 30 (Figure 4d). In all the experiments, at both survival times, with or without either metal treatment or radiation exposure or combined exposure, there was a lower level of MNi in the hTERT+ cells compared to the hTERT– cells. This was particularly true at Day 30, where control (P < 0.05), exposure to metal M + SI (P < 0.01) and radiation VC + 0.5 (P < 0.05) reached level of statistical significance (Figure 6). These results were expressed as a percentage of MNi.

Combined exposure of 0.05 Gy + M The 0.05 + VC, SI + M and 0.05 Gy + M did not induce significant MNi in hTERT– cells, although SI + M and 0.05 Gy + M were higher than control (Figure 5a). The hTERT+ cells followed the same pattern (Figures 5b). In all the experiments at both survival times, with or without either metal treatment or radiation exposure or combined exposure, there was a lower level of MNi in the hTERT+ cells compared to the hTERT– cells. This was statistically significant after exposure to SI + M at Day 0 (P < 0.05) and Day 30 (P < 0.01), and in the control (P < 0.05), after radiation exposure to 0.05 + VC (P < 0.05) and combined exposure to 0.05 Gy + M (P < 0.05) at Day 30 only (Figure 6). These results were expressed as a percentage of MNi.


Figure 5
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  		Fig. 5. Micronuclei (MNi). The figures show the percentage of MNi in hTERT– cells (a and c) and hTERT+ cells (b and d) after a single exposure to either 0.05 Gy + VC or 0.5 Gy + VC, SI + M and either a combined exposure to 0.05 Gy + M or 0.5 Gy + M and at different times after the exposure. Metal (M) is Cr (VI) (0.4 µM), sham irradiation (SI) is at room temperature and vehicle control (VC) is PBS. Error bars represent standard deviations. *P < 0.05 compared to the control (PBS).

 


Figure 6
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  		Fig. 6. Micronuclei (MNi). The figure illustrates the rate ratio of hTERT+/hTERT– for MNi, with 95% confidence intervals, showing combinations of exposure (metal only, radiation only and combined) and days (Day 0 and Day 30) post-exposure. *P < 0.05, **P < 0.01 compared to the control (PBS).

 
Combined exposure of 0.5 Gy + M The 0.5 Gy + VC and 0.5 Gy + M caused a statistically significant increase in MNi immediately after the combined exposure (Day 0) in hTERT– cells, which had no persistence up to Day 30 (Figure 5c). The response of hTERT+ cells was different since there were no MNi. Only the combined exposure to 0.5 Gy + M induced some MNi at Day 0, but this was not significant (Figure 5d). In all the experiments at both survival times, with or without either metal treatment or radiation exposure or combined exposure, there was a lower level of MNi in the hTERT+ cells compared to the hTERT– cells. This was statistically significant in the control at Day 30 (P < 0.05), after exposure to SI + M at Day 0 (P < 0.05) and Day 30 (P < 0.01) and after radiation exposure to 0.5 + VC at Day 0 only (P < 0.05) (Figure 6). These results were expressed as a percentage of MNi.


   Discussion
Top
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
This work has provided direct evidence that combined exposure to Cr (VI) and radiation can induce characteristics in progeny cells that are associated with genomic instability. Both types of cells in this study (hTERT– and hTERT+ cells) showed genomic instability. Previous work confirmed that a wide range of chemicals could reduce the reproductive integrity of progeny cells for several generations (7Go,9Go,31Go). However, there had been no evidence yet that a combination of metal and radiation could induce genomic instability in human fibroblasts. In this work, evidence was provided of persistent loss of clonogenic survival (in both type of cells) and persistent cell damage such as MNi in hTERT+ cells.

The clonogenic assay demonstrated that Cr (VI) induced aberrations in surviving progeny that were lethal and therefore could not have been carried by the surviving cells. Non-clonality is a feature of this type of instability. The fact that this instability is non-clonal and lethal suggests that it is not due to a defect sustained by the cells at the time of the initial insult, as the defect should manifest in all clonal progenies of the affected cells and not in any of the progeny of cells that did not sustain the damage (32Go). According to other studies, Cr (VI) exposure caused genomic instability in hTERT– cells, but not in hTERT+ cells, and therefore ectopic hTERT expression had significant effects in preventing the loss of clonogenic survival after Cr (VI) exposure (9Go). This genomic instability, in Cr (VI)-treated hTERT– cells, might be partly explained by a persistent induction of apoptosis and a delayed induction of senescent-like β-galactosidase staining (33Go) within the colonies, as in our previous study (9Go). Rubio et al. (34Go) have suggested that hTERT is only protective for clonogenic survival in cells with short telomeres. This might be the reason why hTERT+ cells showed no reduction of clonogenic survival. Furthermore, it could be possible that the reduced growth in the colonies of Cr (VI)-treated hTERT– cells was related to a higher level of genomic instability in the colonies caused by the microenvironment (35Go) or an increased concentration of bystander effects (36Go). If true, hTERT might provide one genetic influence for the control of the bystander effect (23Go).

Surprisingly, the clonogenic assay showed that radiation of 0.5 Gy alone induced genomic instability in hTERT+ cells, but not in hTERT– cells. Porter et al. (37Go) showed that telomerase-immortalized human fibroblasts retain UV-induced mutagenesis and p53-mediated DNA damage responses using the clonogenic assay. This is in line with the present work since telomerase expression in hTERT+ cells did not protect against radiation exposure, in term of clonogenic survival. Other studies have shown that ectopic expression of hTERT does not rescue the radiosensitivity, in term of chromosome damage, in Ataxia-Telangiectasia (A-T) engineered fibroblasts. However, these cells can rescue the premature senescence phenotype (38Go). Anyway, in our work, it is unclear why radiation of 0.5 Gy alone induced genomic instability in hTERT+ cells and not in hTERT– cells. In addition, it was surprising that radiation of 0.05 Gy alone increased the clonogenic survival at Day 30 in the hTERT+ cells. This phenomenon has never been seen before and currently, there is no literature for an explanation.

The clonogenic assay showed that ‘metal + radiation’ and ‘radiation + metal’ did not result in the same amount of genomic instability in both types of cells. In fact, metal + radiation caused more genomic instability in hTERT+ cells (P < 0.01 with M + 0.05 Gy and P < 0.001 with M + 0.5 Gy at Day 30), while radiation + metal causes more genomic instability in hTERT– cells (P < 0.001 with 0.05 Gy + M and 0.5 Gy + M at Day 30). Similarly, the micronucleus assay showed that metal + radiation caused cell damage in hTERT+ cells (P < 0.05 with M + 0.5 Gy at Day 0 and Day 30), while radiation + metal caused cell damage in hTERT– cells (P < 0.05 with 0.5 Gy + M at Day 0). However, the genomic instability was present in hTERT+ cells only, where combined exposure of metal + radiation (M + 0.5 Gy) determined a persistence of MNi (chromosomal breakage) (39Go) up to Day 30 (P < 0.05). In order to understand the type of persistent damage (MNi) caused by the combined exposure of metal + radiation (M + 0.5 Gy) in hTERT+ cells, it is important to remember the type of damage that metal only [Cr (VI)] and radiation only (0.5 Gy) normally induces. It is known that genomic instability after metal exposure might be provoked by a mechanism such as single-strand break (SSB) (31Go). The production of SSBs by Cr (VI) reduction, either directly as a consequence of Cr–DNA interactions or as a result of oxygen/carbon radical generation or by the replication past/repair of DNA lesions, represents one of the most commonly reported lesions arising from chromium (VI) treatment. Besides, it has been suggested that DNA is the main target for the biological effects of radiation (40Go), since cells irradiated with {gamma} rays, show susceptibility to several breaks in either of the single strands in the DNA molecule. It may be possible that the persistent incidence of MNi observed in hTERT+ cells after combined exposure of metal + radiation (M + 0.5 Gy) have been provoked by a mechanism such as SSBs (31Go). But it has been shown that the most lethal damage caused by ionizing radiation is DSB. Therefore, it is possible that the cell damage (MNi) observed in this work, after combined exposure of metal + radiation (M + 0.5 Gy), was induced by DSBs since conclusive experimental data linking Cr (VI) reduction to the development of DSBs, as observed after ionizing radiation (like the 0.5 Gy radiation used in this project) or radiomimetic drug exposure, are still lacking (41Go–43Go). However, MNi formation is not always the consequences of DSBs since chromosomal aberrations at metaphase or in interphase must be examined to determine whether the different cell types show a difference in the induction or residual amounts of DSBs. Damage to the DNA caused by oxidative stress can be detected in the bases and sugar–phospate backbone in the structure of DNA, as well as SSBs and DSBs. In fact, SSBs and DSBs can be induced from direct ionizing radiation. However, indirect damage can be caused by radicals generated from radiation and lead to base damage. Therefore, combined exposure of metal + radiation (M + 0.5 Gy) in this work may have had the same effects, through direct interaction with nucleic acid and production of free radical species or dysfunction of cellular organelles, in the hTERT+ cells, leading to genomic instability. Furthermore, ectopic hTERT expression had no effect in preventing the formation of MNi (chromosome breakage) after combined exposure of metal + radiation (M + 0.5 Gy) since this combination caused significant MNi, which persisted up to Day 30 in immortalized hTERT+ cells, but surprisingly not in normal hTERT– cells. In this study, the difference in effects, depending on the order of treatments is an unexpected finding. It is possible that epigenetic effects of chromium could be linked to this phenomenon, where the modification of histone deacetylase and DNA methyltransferase might be responsible for altering the response of cells to subsequent irradiation (44Go). However, this does not explain why there was different damage in the two types of cell used, depending on the order of the treatments.

The results obtained in the combined exposure experiment suggested that telomerase (in hTERT+ cells) could have conferred a protection to these cells in radiation + metal, but not in metal + radiation. This was reflected by the normal clonogenic survival and low incidence of MNi, when hTERT+ cells were exposed to the combination of radiation + metal. This phenomenon can be explained by the findings that ectopic expression of hTERT leads to transcriptional alterations of a subset of genes that may lead to increased genomic stability and enhanced repair of genetic damage. This is therefore linked with reduction of spontaneous chromosome damage in G1 cells, enhancement of the kinetics of DNA repair and an increase in NTP levels (45Go). However, it is unknown why this protection did not occur when hTERT+ cells were exposed to the combination of Metal + Radiation, and to date there is no literature which offers any suggestions about this phenomenon. It may be possible that metal + radiation resulted in severe DNA damage, which could not be prevented by the telomerase enzyme of the hTERT+ cells. Besides, it was interesting to observe that combined exposure in hTERT– cells induced loss of clonogenic survival, as well as increase in MNi, in radiation + metal, but not in metal + radiation. There is no literature either which can offer a suggestion about this phenomenon, but it may be possible that radiation + metal resulted in severe DNA damage, which could not be repaired by the DNA repair machinery of the hTERT– cells. Moreover, the combined exposure of metal + radiation, which was not as toxic as radiation + metal, may not have induced such severe damage or this damage may have been repaired by the DNA repair machinery of the hTERT– cells.

It is likely that the biological effects provoked by combined exposure of metal and radiation has led to an additive action, 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.

In this work, it was interesting to have observed the following characteristics of the combined exposure:

(i) Additive effects were observed in both types of cells.
In hTERT– cells, M + 0.5 Gy, and to a lesser extent 0.05 Gy + M and 0.5 Gy + M, caused a loss of clonogenic survival 30 days after the combined exposure, and MNi were induced by M + 0.5 Gy immediately after the combined exposure (Day 0).
In hTERT+ cells, M + 0.05 Gy and 0.05 Gy + M caused a loss of clonogenic survival immediately after the combined exposure (Day 0), M + 0.5 Gy resulted in a loss of clonogenic survival 30 days after the combined exposure (Day 30), and MNi were induced by M + 0.5 Gy 30 days after the combined exposure (Day 30).
(ii) Additive effects were observed in both types of combinations.
The combined exposure of metal + radiation showed additive effects in the clonogenic assay since there was loss of clonogenic survival with M + 0.5 Gy at Day 30 in hTERT– cells, with M + 0.05 Gy at Day 0 in hTERT+ cells and with M + 0.5 Gy at Day 30 in hTERT+ cells. In terms of MNi, synergism occurred in hTERT– cells immediately after the combined exposure (Day 0) and in hTERT+ cells 30 days after the combined exposure (Day 30).
The combined exposure of radiation + metal showed additive effects in the clonogenic assay since there was loss of clonogenic survival with 0.05 Gy + M and 0.5 Gy + M in hTERT– cells 30 days after the combined exposure (Day 30), and with 0.05 Gy + M in hTERT+ cells 30 days after the combined exposure (Day 30).
(iii) Additive effects may be related to genomic instability.
The evidence that additive effects were present as delayed damage, and therefore likely to be linked with genomic instability, was provided by the loss of clonogenic survival with M + 0.5 Gy, 0.05 Gy + M and 0.5 Gy + M in hTERT– cells 30 days after the combined exposure (Day 30) and with M + 0.5 Gy and 0.05 Gy + M in hTERT+ cells 30 days after the combined exposure (Day 30). The evidence that synergism was also present as delayed damage was provided by the increase of MNi in hTERT+ cells 30 days after the combined exposure (Day 30) of metal + radiation (M + 0.5 Gy). This demonstrated that genomic instability (delayed loss of clonogenic survival and delayed damage) occurred, in most of the cases, when metal treatment was in combination with radiation exposure.

Similar to the present work, Vitvitskii et al. (10Go) demonstrated that combined exposure of gamma rays and Cr (VI) can have enhancing effects on the outcome, compared to the single agent (either gamma rays or metal alone). In their studies, the effects of chromium ions (VI) on the mutagenic activity of gamma rays were assessed by a micronucleus test in mouse bone marrow polychromatocytes. They observed that Cr (VI) ions enhanced mutagenic effects of gamma rays in both acute and chronic experiments (10Go).

Muller and Streffer, who analysed the risk to pre-implantation mouse embryos of combined exposure of heavy metals (arsenic, cadmium or lead) and radiation, used morphological development to evaluate the risk after combined exposure to these metals and X-rays. Either metal combined with radiation showed additive effects since morphological development was extra-damaged than expected from the addition of the single effects (46Go). Sahu et al. observed that either nickel sulphate or lead sulphate or sodium arsenite combined with UV light give an additive sister chromatid exchanges response in cultured human lymphocytes, compared to the single exposure only (47Go).

Anan'eva et al. showed that combined exposure of rats to low-dose irradiation and heavy metal (Cu2+) ions caused significant accumulation of the free radical products proportional to exhausting antioxidant and oxidizing–reduction potential in various organs and tissues, compared to either radiation or metal only (11Go). Therefore, the additive effects observed after combined exposure of metal + radiation (and vice versa) could be due to the accumulation of free-radical products, which induced DNA damage.

In conclusion, the effects of hTERT expression detected in this study can be summarized as listed below.

(i) Metal induced genomic instability in hTERT– cells (cell survival and cell damage), radiation induced genomic instability only in hTERT+ cells (cell survival only) and combined exposure caused genomic instability in hTERT– cells (cell survival only) and hTERT+ cells (cell survival only).
(ii) Ectopic hTERT expression had a dramatic effect in preventing genomic instability after metal exposure, whereas telomerase activity in hTERT+ cells did not provide protection against genomic instability (loss of clonogenic survival) caused by the radiation insult.
(iii) Combined exposure of metal + radiation caused more genomic instability (loss of clonogenic survival) in hTERT+ cells, while combined exposure of radiation + metal caused more genomic instability (loss of clonogenic survival) in hTERT– cells. Therefore, it might be possible that ectopic hTERT expression could prevent genomic instability in hTERT+ cells only in the combined exposure of radiation + metal.

This study investigated the short- and long-term response of cells exposed to sublethal concentrations of either metal or radiation or combined exposure and showed in some cases that genomic instability was enhanced by the combined exposure of the two toxic agents. Furthermore, combined exposure of metal and radiation showed in some cases additive effects, compared to that found after exposure to either single agent. This work may provide helpful information to understand the process of cancer formation from multiple causes.


   Funding
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Ireland Technological Sector Research Strand 1 Post-Graduate Research & Development Skills Programme.


   Acknowledgments
 
Conflict of interest statement: None declared.


   Notes
 
* To whom correspondence should be addressed. Tel: +35318968475; Email: a.glaviano{at}libero.it or a.glaviano{at}tcd.ie


   References
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received on April 15, 2008; revised on July 10, 2008; accepted on August 4, 2008.


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