Multicolour FISH detection of radioactive iodine-induced 17cen–p53 chromosomal breakage in buccal cells from therapeutically exposed patients

M.J. Ramírez, S. Puerto, P. Galofré1, E.M. Parry2, J.M. Parry2, A. Creus, R. Marcos and J. Surrallés3

Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Edifici Cn, Universitat Autònoma de Barcelona, 08193 Bellaterra,
1 Servei de Medicina Nuclear, Ciutat Sanitària i Universitària Vall d'Hebron, Pg. Vall d'Hebron 119, 08035 Barcelona, Spain and
2 School of Biological Sciences, University of Wales, Swansea, Singleton Park, Swansea, UK


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Simultaneous labelling of 17cen and the p53 locus by multicolour FISH was used to monitor radioactive iodine-induced structural and numerical chromosome abnormalities in buccal cells from 29 hyperthyroidism and thyroid cancer patients sampled before and after therapeutic treatment. This novel methodology allowed the efficient detection of 17p deletions leading to p53 allelic deletions, 17p gains and whole chromosome 17 numerical abnormalities in epithelial cells. Highly significant increases in the frequency of cells with (i) 17p abnormalities (1.8-fold; P < 0.001), including p53 monoallelic deletions (2.1-fold; P < 0.001) and 17p gains (3.5-fold; P < 0.001); (ii) chromosome 17 numerical abnormalities (2-fold; P < 0.001); and (iii) simultaneous 17p breakage and chromosome 17 numerical abnormalities (2.3-fold; P < 0.001), were observed after radioactive iodine treatment. As expected, the major contribution to these increases was detected in hyperthyroidism patients compared with thyroid cancer patients who suffered thyroidectomy before radioactive iodine exposure and, therefore, experienced a rapid elimination of the radioisotope. Considering that both the genetic endpoints and the target tissue are extremely relevant in carcinogenesis, it is suggested that the observed genetic damage could contribute to the reported increase in cancer risk of people therapeutically or accidentally exposed to radioactive iodine.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Genetic alterations and development of cancer have been reported to be consequences of exposure to several physical or chemical agents. Amongst them, radioactive iodine (131I) has been associated with a high increase in the incidence of thyroid cancer in children after the Chernobyl nuclear power plant accident (13). Radioactive iodine is commonly used in various diagnostic and therapeutic applications such as the treatment of patients with hyperthyroidism or thyroid cancer (4,5). These patients provide a good opportunity to study cytogenetic damage induced by known doses of 131I in exposed people and, therefore, to carry out risk estimations in people environmentally exposed to such a radionuclide. At present, the great majority of such studies, including ours, have been performed in peripheral blood lymphocytes from exposed patients (622).

More recently, interphase molecular cytogenetics by fluorescence in situ hybridization (FISH) in epithelial tissues has been proposed to measure 131I-induced chromosome damage (23). Exfoliated epithelial cells represent an alternative cellular system for biomonitoring studies since they are commonly the target for carcinogens, and >90% of cancers arise from epithelial tissues (24). In addition, exfoliated cells can be easily collected and studied to detect abnormal morphology, premalignant changes or cancer (25,26). This is extremely relevant when dealing with patients therapeutically exposed to radioactive iodine, considering the reported data suggesting that they have a low but significantly increased risk for subsequent cancers after radioactive iodine treatment. This is especially true in the most exposed tissues including salivary glands (27,28), stomach (29) and bladder (30).

The application of emerging molecular cytogenetic methods in biologically based risk assessment may help to clarify the uncertainties of low risk exposures such as radioactive iodine therapy. In this context, it is important to develop new biomarkers of genetic damage with special relevance in terms of cancer induction. FISH with chromosome- or locus-specific DNA probes allows cytogenetic information to be obtained rapidly from interphase cells. A highly relevant chromosomal region in carcinogenesis is the short arm of chromosome 17, where the p53 gene is located (band 17p13.1). The wild-type p53 gene is a tumour suppressor gene coding for a nuclear phosphoprotein that plays a crucial role in the cellular response to DNA damage by inducing cells to arrest in G1 or enter into apoptotic cell death (31,32). Thus, failure to express functional p53 protein, after loss or mutation of the p53 gene, leads to an increased incidence of spontaneous tumours, as a result of an inability to undergo cell-cycle arrest or to induce the apoptotic pathway of the cell death in response to a damage induced by an environmental agent. Inactivation of p53, with resulting inappropriate entry into S phase, leads to genetic instability (33) and aneuploidy in vitro (3436) and in vivo (37,38).

In some cases, 17p allelic losses are not correlated with p53 gene loss, for instance in some breast cancers (38). For this reason, the existence of another gene(s) in the short arm of chromosome 17 that might play a critical role in carcinogenesis has been hypothesized. In lung, breast and ovarian cancers, and leukaemias, this candidate tumour suppressor gene is located in the band 17p13.3 (3942). Thus, in terms of developing new cytogenetic biomarkers for cancer risk, it would be interesting to focus not only on p53 allelic losses but also on overall 17p deletions involving the p53 gene and other 17p genes implicated in carcinogenesis.

The aim of the present study was to investigate 131I-induced structural and numerical abnormalities involving the chromosome 17 and the 17p region in epithelial cells of therapeutically exposed thyroid cancer and hyperthyroidism patients. To do so we have developed a novel FISH methodology that detects simultaneously, and in different colours, the centromeric region of chromosome 17 and the p53 locus (Figure 1Go). This technique was applied to buccal cells from thyroid cancer and hyperthyroidism patients sampled before and after radioactive iodine treatment.



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Fig. 1. Schematic representation of the 17cen–p53 locus bicolour FISH methodology to detect structural and numerical abnormalities involving chromosome 17p and chromosome 17, respectively. Normal cells would harbour two 17cen signals and two p53 locus signals (a). Cells with two 17cen signals but three p53 signals are interpreted as 17p gains with breakpoints in 17cen–p53 (b). p53 monoallelic deletions resulting from chromosome 17p deletions with breakpoints in the 17cen–p53 regions will lead to cells with two 17cen signals but only one p53 signal (c). Cells with one copy of chromosome 17 (haploid or monosomic cells) will show one 17cen signal and one p53 signal (d). Similarly, cells with three copies of chromosome 17 (triploid or trisomic cells) will harbour three 17cen signals and three p53 signals (e).

 

    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
The study was performed with a total of 29 patients of the Nuclear Medicine Service of the University Hospital of Vall d'Hebron (Barcelona): 14 patients (five males and nine females) with hyperthyroidism and 15 (two males and thirteen females) with papillary or follicular thyroid cancer. The age of the patients ranged between 23 and 79 years. The therapeutic treatment consisted of [131I]NaI given orally as an adjuvant radiation dose to eliminate any excess of cells for hyperthyroidism patients or to eliminate the remaining tumour cells after total thyroidectomy for cancer patients. The final treatment dose received was for hyperthyroidism patients 155.40–1110.00 MBq (4.2–30 mCi) and for cancer patients 3700–4440 MBq (100–120 mCi). All the patients responded to a detailed questionnaire on lifestyle and other potentially confounding factors. Before proceeding with the study we obtained clearance from the ethical committee of our institutions and all patients gave informed consent.

Buccal cell sampling and slide preparation
Two buccal cell samples were collected from each patient, one before 131I treatment and a second sample 3–4 weeks (mean 27.2 ± 2.6 days) after treatment. The cell samples were processed and the slides were prepared as described elsewhere (23,43).

In situ hybridization
Slides were treated and denatured essentially as described previously (23,43). The probes used were an {alpha}-satellite, biotin-labelled DNA probe that hybridizes specifically with the centromeric region of chromosome 17 (D17Z1; ONCOR, Gaithersburg, MD) and a p53 locus specific probe, directly labelled with Cy3 (LSI p53; VYSIS, Downers Grove, IL). The hybridization mixture, consisting of 0.25 µl of 17cen probe, 0.3 µl of p53 locus probe, 3.5 µl of hybridization buffer (VYSIS) and 1 µl of distilled water per slide, was denatured for 5 min at 73 ± 1°C. Five microlitres of denatured hybridization mixture were immediately pipetted onto the denatured and dried slides, which were then incubated overnight at 37°C in a moist chamber. The next day, three washes in 50% formamide/2x SSC, for 10 min at 46 ± 1°C followed by a wash in 2x SSC for 10 min at the same temperature, were carried out. Then, the slides were rinsed in 2x SSC, 0.1% Tween-20, at the same temperature, and incubated with blocking reagent in 0.1 M phosphate buffer for 5 min at room temperature. The biotin-labelled probe was detected using FITC-conjugated avidin and biotin-conjugated anti-avidin antibodies. All the incubations were performed at 37°C for 20 min in a moist chamber. After each incubation, three washes were carried out at room temperature, for 2 min each in phosphate buffer supplemented with 0.1% Tween-20. Slides were finally rinsed in phosphate buffer and dried with sequential washes of 70%, 90% and absolute ethanol. The nuclear material was counterstained with 0.01 µg/ml DAPI in antifading solution (Vectashield, Vector). All the slides were stored at 4°C until required for microscopy.

Microscopic analysis and scoring
Microscopic analysis was performed on an Olympus BX-50 microscope equipped with a 100 W mercury lamp and a 1000x magnification objective with iris aperture. A total of 2000 cells was examined for each patient both before and after treatment. Cells were classified according to the number of green (17cen) and red (p53 locus) signals. All the slides were coded prior to scoring.

Statistical analysis
The data did not fulfil the requirements of parametric texts (normality distribution and homogeneity of variances). Therefore, the Wilcoxon matched pairs non-parametric test for dependent variables was used to evaluate the genotoxic effect of 131I by comparing the incidence of (i) cells with chromosome breaks in the region 17cen–p53, (ii) cells with chromosome 17 numerical abnormalities and (iii) cells with both numerical and structural alterations, before and after 131I treatment. The Mann–Whitney test was applied to assess the influence of confounding factors. The dose–effect within each diagnostic group was analysed by a regression analysis.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Chromosome 17p breakage
In this group, only cells with structural abnormalities involving one or two breaks in the region 17cen–p53 were considered (Figure 1Go): cells with one or two 17p deletions (two 17cen signals and only one p53 signal or two 17cen signals and no p53 signal, respectively) and cells with a 17p gain (two 17cen signals and more than two p53 signals). The frequencies of these three types of aberrant cells are pooled in Figure 2aGo. The actual results are summarized in Table IGo. After scoring 2000 buccal cells in each of the 29 patients, before and after radioactive treatment, a 1.8-fold increase in the frequency of 17cen–p53 breaks was observed (P < 0.001) after exposure (Figure 2aGo). This increased breakage was mainly observed in hyperthyroidism patients, with an increment of 2.6-fold after treatment (P < 0.01), whereas the thyroid cancer patients showed a statistically significant increment of 17p gains (Table IGo) and a non-significant increase in overall 17p breakage. In the pooled population, a clear increment in the frequency of cells with one 17p deletion (2.1-fold; P < 0.001) or with one or more 17p gains (3.5-fold; P < 0.001) was observed. The frequency of cells with two deletions involving the p53 locus was not increased after treatment. This behaviour was consistently observed both in thyroid cancer and in hyperthyroidism patients. The radiation-induced increase in chromosome 17p breakage was not modulated by gender, age or dose of radiation administered.



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Fig. 2. Incidence in 2000 buccal cells of 17p breakage (a), chromosome 17 numerical abnormalities (b) and simultaneous 17p breakage and chromosome 17 numerical abnormalities (c), before (open box) and after (shaded box) 131I treatment. Means, standard errors and standard deviations are represented by middle points, boxes and bars, respectively. The statistical significance of the comparison of values obtained before and after radioactive treatment is also shown (***P < 0.001).

 

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Table I. Results on the induction of 17p monoallelic and diallelic deletions, 17p gains, chromosome 17 numerical abnormalities and cells with simultaneous 17cen–p53 breakage and chromosome 17 numerical abnormalities as detected by multicolour FISH
 
Chromosome 17 numerical abnormalities
Cells with only numerical abnormalities are included in this group (Figure 1Go): monosomic or haploid cells (cells with only one 17cen signal and only one p53 signal) and polysomic or polyploid cells (cells with three 17cen signals and three p53 signals or cells with four 17cen signals and four p53 signals). Thus, cells with three chromosome 17 centromeric signals are assumed to carry a numerical abnormality (trisomy or triploidy). The fact that the same cell harbours three p53 signals is only a further confirmation that this cell is numerically abnormal. All numerically aberrant cells were pooled together in Figure 2bGo. As detailed in Table IGo, a 2-fold increment in the frequency of chromosome 17 numerical abnormalities was observed in the pooled patients after treatment (P < 0.001), as a result of a clear and significant increment (3.3-fold; P < 0.01) in the hyperthyroidism patients. In thyroid cancer patients, only a non-significant increase was detected (Table IGo). Most of this increment was attributed to monosomic or haploid cells, with a 2.1-fold increment (P < 0.001) and, above all, in hyperthyroidism patients (3.7-fold increase; P < 0.01). The radiation-induced increase in chromosome 17 numerical abnormalities was not modulated by gender, age or dose of radiation administered.

Simultaneous 17p breakage and chromosome 17 numerical abnormalities
Cells with both numerical and structural abnormalities as a result of a simultaneous clastogenic and aneugenic event are included in this group. For example, monosomic, haploid, polysomic or polyploid cells with one or more 17p deletions or 17p gains. All these multiple aberrant cells are pooled together in Figure 2cGo. However, only monosomic or haploid cells with one or more 17p gains are indicated in Table IGo since they were the most frequently observed. The rest of cells simultaneously presenting 17p breakage and chromosome 17 numerical abnormalities were pooled together in Table IGo under the heading `other'. A clear and statistically significant 2.3-fold increase in the frequency of cells simultaneously presenting 17p breakage and chromosome 17 numerical abnormalities was observed after treatment in the data pooled from all the patients (P < 0.001). This clear increment was statistically significant both in hyperthyroidism patients (2.9-fold increase; P < 0.01) and in thyroid cancer patients (1.8-fold increase; P < 0.05). The radiation-induced increase in the frequency of cells with simultaneous 17p breakage and chromosome 17 numerical abnormalities was not modulated by gender, age or dose of radiation administered.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Radioactive iodine has been proven to be a low risk and effective treatment for thyroid disease. Nonetheless, there are persisting concerns regarding carcinogenic risk to therapeutically exposed patients. Given the widespread interest in developing new biomarkers for cancer risk, we have introduced a novel multicolour FISH approach especially designed to detect environmentally induced 17p alterations and chromosome 17 aneuploidy in epithelial tissues. Considering that 17p allelic deletions are the hallmark of many human tumours, including colon, breast, bladder, lung and liver cancer (4449), and that >90% of human tumours arise from epithelial tissues, the proposed methodology offers the possibility of detecting DNA lesions with special relevance in carcinogenesis. From a mechanistic point of view, 17p gains (Figure 1bGo) probably arise from radiation-induced chromatid translocations formed in the G2 phase of the continuously proliferating buccal stem cells. Unpublished results from our laboratory indicate that this novel methodology is equally effective in detecting ionizing-radiation-induced chromosome damage in cultured human cells exposed in vitro.

In the present study, cells were sampled from the buccal mucosa of thyroid cancer and hyperthyroidism patients therapeutically exposed to radioactive iodine. Our results clearly indicate that radioactive iodine, at the level of therapeutic exposure, was able to induce chromosome 17 numerical abnormalities and breakage within the 17cen–p53 region. Radiation-induced chromosome breakage resulted in a significant increase of 17p gains and 17p deletions involving the p53 gene in the exposed individuals, especially those suffering hyperthyroidism. The observed increase in both 17p deletions and 17p gains argues against a technical artefact as an explanation to our findings. In addition, the preparation method did not include hypotonic treatment or fixation in suspension, thus maintaining the integrity of the nucleus and precluding the likelihood of whole chromosome or chromosome fragment losses due to technical artefact.

As expected, the genotoxic effect in cancer patients was weaker than the effect observed in hyperthyroidism patients because, after total thyroidectomy, the effective half-life of the radioiodine in cancer patients is very low. Thus, the overall exposure in cancer patients is lower than in hyperthyroidism patients in spite of the higher activity administered (50). In addition, the localized effect of irradiation from the thyroid to the buccal mucosa is probably higher in hyperthyroid patients than in cancer patients, as the former had an overdeveloped thyroid gland and the later suffered thyroidectomy before treatment.

It is known that, in addition to its major clastogenic effect, ionizing radiation can also induce aneuploidy (21,5156). The high increment in the frequency of 17p breakage and chromosome 17 numerical abnormalities confirms the dual effect induced by ionizing radiation in general, and by radioactive iodine in particular. The increase in cells with both types of damage (simultaneous breakage and aneuploidy) is much higher than might be expected if the two events had occurred independently. This association was actually expected considering that the cells have been cycling and, therefore, irradiated during all the phases of the cell cycle and that p53 plays an important role in chromosome segregation (5760). Thus, cells irradiated after the G1/S checkpoint, e.g. during the S or G2 phase, would lead to p53 deletions in the daughter cells which would be prone to aneuploidy. Our observation is consistent with a number of reports confirming that the inactivation of the p53 gene produces an inappropriate entry into S phase, leading to genetic instability and aneuploidy in vitro (3436) and in vivo (37,38). Our results are also in agreement with the suggestion that 17p allelic losses occur as early events in a pathway of genetic instability that leads to aneuploidy and other allelic losses and cancer, as suggested by Blount et al. (37).

In a recently published study, we used replicate slides from the same individuals to study chromosome breakage in the 1q12 region (23). Breaks in 1q12 were detected by tandem labelling multicolour FISH (61,62). Considering the frequent implication of this band in cancer chromosomal aberrations, the tandem labelling methodology was proposed as a useful tool in the detection of early chromosomal changes involved in neoplastic development (61). However, we did not detect any increase in the frequency of 1q12 breakage after treatment using the same slides where we now report an increase in 17p breakage. This apparent contradiction is probably explained by the high instability and low persistence of 1q12 translocations through cell divisions, as suggested by our recent results in a highly proliferative in vitro cell system (M.J.Ramírez, S.Puerto, A.Creus, R.Marcos and J.Surrallés, submitted for publication). We therefore suggest that the tandem labelling method is a good biomarker for radiation-induced chromosome breakage in non-proliferative tissues such as G0 lymphocytes, but not in highly proliferative cell systems. We are currently investigating whether the 17cen–p53 region is more radiosensitive than the 1q12 region as an alternative or additional explanation to our observations.

Considering the implication of 17p alterations and epithelial tissues in cancer, it is suggested that the reported methodology could be useful to assess the genotoxic potential of environmental exposures and, therefore, could be valid as a biomarker of cancer risk in people therapeutically, environmentally or accidentally exposed to carcinogens.


    Notes
 
3 To whom correspondence should be addressed Email: jsurralles{at}einstein.uab.es Back


    Acknowledgments
 
We would like to thank Lancer, S.A. (Barcelona) for the gift of the toothbrushes. This work was partially funded by the Spanish Ministry of Education and Culture (MEC, CICYT, PM98-0179), the Generalitat de Catalunya (SGR95-00512, CIRIT), the Commission of the European Union (Euratom Programme, FIGH-CT99-00011), the Fanconi Anemia Research Fund, Inc. (USA) and the Consejo de Seguridad Nuclear (394/98/GTP/481.00). The work in Swansea was supported by a grant from the Cancer Research, Wales. M.J.R.'s visit to Swansea was supported by a travel grant awarded by the MEC. M.J.R. and S.P. were supported during this work by a fellowship awarded by the MEC and the Universitat Autònoma de Barcelona, respectively.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 17, 2000; revised April 27, 2000; accepted April 27, 2000.