Mutagenesis, Vol. 14, No. 1, 121-127,
January 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
FISH analysis of 1cen–1q12 breakage, chromosome 1 numerical abnormalities and centromeric content of micronuclei in buccal cells from thyroid cancer and hyperthyroidism patients treated with radioactive iodine
1 Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Edifici Cn, Universitat Autònoma de Barcelona, 08193 Bellaterra and 2 Servei de Medicina Nuclear, Ciutat Sanitària i Universitària Vall d'Hebron, Pg. Vall d'Hebron 119, 08035 Barcelona, Spain
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One of the health consequences of the Chernobyl nuclear power plant accident was a radioactive iodine-related increase in the incidence of thyroid cancer in exposed children. This radioisotope is used in the treatment of thyroid cancer and hyperthyroidism patients providing a convenient opportunity to study cytogenetic damage induced by known doses of radioactive iodine in treated patients. We used pancentromeric FISH on micronuclei and chromosome 1 tandem labelling FISH to monitor overall chromosome breakage and loss, 1q12 breakage and decondensation and chromosome 1 numerical abnormalities in buccal cells from 31 radioactive iodine-exposed hyperthyroidism and thyroid cancer patients. The overall outcome of the study, with 250 000 buccal cells analysed, is that there was no radioactive iodine-related increase in the frequency of micronuclei, 1q12 breakage, 1q12 decondensation or chromosome 1 numerical abnormalities. In addition, neither age nor gender, health status nor radioactive iodine dose modulated the frequency of the above cytogenetic end points. Although several uncertainties of these emerging molecular cytogenetic methodologies will require further experimentation, we conclude that, at the reported exposure levels, radioactive iodine did not induce detectable chromosome damage in buccal cells from treated patients.
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Introduction |
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One of the consequences of the Chernobyl nuclear power plant accident was a high increase in the incidence of thyroid cancer in exposed children from both Belarus (Kazakov et al., 1992
) and Ukraine (Likhtarev et al., 1995) which has been directly linked to high exposure to radioactive iodine, mainly 131I (Likhtarev et al., 1995). This radioisotope is currently used in the treatment of thyroid cancer and hyperthyroidism patients. Around 90% of the 131I radiation effects are the result of ß-radiation, with a short length in soft tissues (Searle, 1983) and with extrathyroidal radiation aparently minimal and restricted to 
-emissions.
The existence of patients treated with radioactive iodine provides a good opportunity to study the cytogenetic effects of this radioisotope. The great majority of such studies have been done in peripheral blood lymphocytes from exposed patients, giving contradictory results (Gutiérrez et al., 1995, 1997; Ramírez et al., 1997a). However, since the advent of interphase molecular cytogenetics, epithelial tissues such as buccal mucosa are an alternative cell target to measure environmentally-induced chromosome damage. The cells in the basal layer of the stratified squamous epithelium of the oral mucosa are in continuous division producing exfoliation of the superficial cells. There exists a continuous cell turnover in the different strata because the cells produced by mitosis in the basal layer migrate to the surface to replace those that are shed. A given cell needs ~25 days to cover the different strata of the epithelium (Ten Cate, 1985). Exfoliated cells are a very appropriate cell system for biomonitoring studies, since they can be easily collected and studied to detect abnormal morphology, premalignant changes or cancer (Bryan and Cohen, 1983; Brawn, 1984). In addition, epithelial tissues are commonly the target for chemical agents and >90% of cancers arise from epithelial tissues (Cairns, 1975; Rosin and Gilbert, 1990).
Since the pioneering work of Stich and co-workers (Stich et al., 1982a), the micronucleus (MN) assay in exfoliated cells has been extensively used. Thus, induction of MN in exfoliated cells was detected after different exposures, including ionizing radiation (Stich and Rosin, 1983a; Sarto et al., 1987; Moore et al., 1996), smoking and other tobacco products (Stich et al., 1982b; Fontham et al., 1986; Sarto et al., 1987; Livingston et al., 1990, Rupa and Eastmond, 1997), alcohol (Stich and Rosin, 1983b), antiblastic chemotherapy (Sarto et al., 1990), formaldehyde (Titenko-Holland et al., 1996), arsenic (Moore et al., 1996) and others.
An advantage of the MN assay in exfoliated cells is that it is simple, fast, free of cell culture-related artifacts and can be applied directly to the first target organs of the mutagenic pollutants. It is well known that MN arise from chromosomal fragments or whole chromosomes that are left behind during mitotic division. The dual origin of the MN can be distinguished using fluorescence in situ hybridization (FISH) with probes labelling the centromeric region of all human chromosomes, so MN derived from acentric chromosomal fragments will not be labelled by the probe and MN harbouring whole chromosomes will be positively labelled. This approach was first introduced by Becker and co-workers in human lymphocytes (Becker et al., 1990) and then applied to buccal cells (Moore et al., 1993a,b, 1996; Titenko-Holland et al., 1994, 1996; Surrallés et al., 1997a). Thus, Moore et al. (1996) reported a 16.6-fold increase in the frequency of MN after radiotherapy treatment for oral cancer, virtually all MN detected being centromere negative (C–MN). Thus, the FISH/MN assay potentially allows a quick and reliable identification of both aneugenic and clastogenic effects in buccal cells. This is particularly relevant when dealing with agents that produce both kinds of cytogenetic effects in vivo, such as ionizing radiation (reviewed by Natarajan et al., 1996; Ramírez et al., 1997a).
In spite of the above advantages, the MN assay has the potential limitation that it only detects missegregating chromosomal fragments or whole chromosomes, but is unable to detect stable chromosome aberrations such us translocations. This limitation can be overcome by using chromosome 1 tandem labelling FISH, allowing the detection of chromosome aberrations involving the band 1q12 as well as chromosome 1 numerical abnormalities (Eastmond et al., 1994; Rupa et al., 1995, 1997a, Rupa et al., b; Surrallés et al., 1997b). Tandem labelling FISH is performed with an 
satellite probe hybridizing with 1cen and visualized in green together with a classical satellite probe detecting 1q12 in red. As reported by Rupa et al. (1995), the presence of a nucleus with three satellite signals adjacent to three classical satellite signals is indicative of three copies of chromosome 1 (trisomy or triploidy). However, a cell with two satellite signals and three classical satellite signals, one of them non-adjacent, indicates a nucleus with a break within 1q12. Similarly, a wide separation between 1cen and 1q12 indicates a break between both regions.
The 1q12 constitutive heterochromatin band is a breakage-prone region involved in a number of chromosome abnormalities encountered in human cancers. This fragile site has been reported to be sensitive to a variety of clastogenic agents, including ionizing radiation, melphalan, busulfan, mitomycin C, triethylenemelamine, nifurtimox and hydroquinone (Bourgeois, 1974; Brogger, 1977; Honeycombe, 1978; Yunis et al., 1987; Sabatier et al., 1989; Gorla et al., 1989; Aurias, 1993; Eastmond et al., 1994; Rupa et al., 1995, 1997a, Rupa et al., b; Surrallés et al., 1997b). The tandem labelling methodology has been successfully used to detect chromosome damage in lymphocytes of human populations exposed to pesticides (Rupa et al., 1995) and in buccal cells of betel quid chewers (Rupa and Eastmond, 1997).
In previous studies we analysed radioactive iodine-induced cytogenetic damage in human lymphocytes from hyperthyroidism and thyroid cancer patients using several methodologies, including the MN (Gutiérrez et al., 1995, 1997), FISH (Ramírez et al., 1997a) and COMET assays (Gutiérrez et al., 1998a,b). The aim of the present study was to investigate the possible aneugenic or clastogenic effect of 131I treatment in buccal cells of similarly exposed thyroid cancer and hyperthyroidism patients, using the MN assay complemented with pancentromeric FISH as well as chromosome 1 tandem labelling FISH.
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Materials and methods |
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Patients
The study was performed with a total of 31 patients of the Nuclear Medicine Service of the University Hospital of Vall d'Hebron (Barcelona), 16 patients (five male and 11 female) with hyperthyroidism and 15 (two male and 13 female) with papillar or follicular thyroid cancer. The age of the patients ranged between 23 and 80 years.
The therapeutic treatment consisted of sodium iodide (131I) given orally as an adjuvant radiation dose to eliminate excess 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). 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 obtained by brushing the inside of the cheeks with a toothbrush. The cells were rinsed in a 50 ml conical tube containing 20 ml buffer solution (0.1 M EDTA, 0.01 M Tris–HCl and 0.02 M NaCl, pH 7; Moore et al., 1993a). The cells were washed three times by centrifugation at 1500 r.p.m. for 10 min in the same buffer. Then, 50 µl cell suspension was dropped onto preheated (45–55°C) slides and allowed to air dry for 15 min at the same temperature. Cell density was checked with a phase contrast microscope and the cell suspension was diluted or concentrated when necessary. After 1 h at room temperature, the slides were fixed in 80% cold methanol (0°C) for 30 min, air dried at room temperature and stored at –20°C until use.
Slides pretreatment and denaturation
Before hybridization, slides were treated with pepsin (300 µg/ml) (Sigma) in 0.01 M HCl, pH 2.7, for 7 min at 37°C to remove excess keratin and cytoplasm from the buccal cells (Surrallés et al., 1997a). The pepsin treatment was stopped by washing in phosphate-buffered saline (PBS) and PBS supplemented with 50 mM MgCl2. The slides were finally dehydrated in washes of 70, 90 and 100% ethanol for 3 min each at –20°C.
Slides were denatured in 70% formamide, 2x SSC for 4 min at 74°C, chilled with ice-cold 70% ethanol and dehydrated with sequential washes in 90 and 100% ethanol.
Pancentromeric FISH
A 30 nt digoxigenin-labelled pancetromeric synthetic oligomer probe (SO-All Cen) with the sequence 5'-GTTTTGAAAC10ACTCTTTTTG20TAGAATCTGC-3' (MWG-Biotech, Germany) was used. This probe hybridizes with the centromeric region of all human chromosomes (Meyne et al., 1989; Norppa et al., 1993; Surrallés et al., 1995, 1996a, 1997a; Ramírez et al., 1997a,b) and was used to distinguish MN harbouring whole chromosomes from those containing acentric fragments.
The probe was denatured at 70°C for 5 min in a mixture containing 0.5 µg/ml herring sperm DNA and 2.5 µg/ml probe in 2x SSC, then placed immediately on ice and finally dropped onto the denatured slides under coverslips. Slides were sealed with glue and in situ hybridization with target DNA occurred overnight at 37°C in a moist chamber.
After overnight hybridization, four washes in 6x SSC were done, three at room temperature for 10 min each and a final one at 42°C for 5 min. Then the slides were rinsed in 4x SSC, 0.1% Tween 20 (5 min at room temperature) and incubated with 1% blocking reagent (Boehringer Mannheim, Germany) in 4x SSC for 15 min at 37°C. After a wash in 4x SSC, 0.1% Tween 20 (5 min at room temperature), the digoxigenin-labelled probe was immunodetected using a mouse monoclonal anti-digoxigenin antibody (Sigma Chemical Co., St Louis, MO), followed by a sheep FITC-conjugated anti-mouse (Boehringer Mannheim) and a donkey FITC-conjugated anti-sheep (Sigma) secondary antibodies as described previously (Surrallés et al., 1995; Ramírez et al., 1997a,b). After each antibody incubation, three washes in 4x SSC, 0.1% Tween 20 for 5 min each were done. Slides were finally rinsed with PBS and the nuclear material was counterstained with 4',6'-diamidino-2-phenyloindole (DAPI) and propidium iodide in antifade solution (Vectashield; Vector Laboratories Inc., Burlingame, CA). All slides were stored at –20°C until microscopic analysis.
Tandem labelling FISH
Tandem labelling FISH was performed essentially as described previously (Surrallés et al., 1997b) with some modifications. The probes used were an satellite, biotin-labelled DNA probe that hybridizes specifically with the centromeric region of chromosome 1 (D1Z5; ONCOR, Gaithersburg, MD) and a classical satellite, digoxigenin-labelled DNA probe detecting the adjacent heterochromatic band 1q12 (PUC1.77; Boehringer Mannheim).
Denatured slides were incubated overnight with a 5 µl hybridization mixture (previously denatured as described before) made of 60% formamide in 2x SSC, 1 mg/ml herring sperm DNA, 6.25% dextran sulphate, 0.250 µl satellite probe and 0.125 µl classical satellite probe.
Post-hybridization washes were performed in 60% formamide in 2x SSC for 15 min at 45°C followed by a wash in 2x SSC for 8 min at the same temperature. Then the slides were rinsed in 4x SSC, 0.1% Tween-20 (5 min at room temperature) and incubated with 5% non-fat dry milk in 4x SSC for 15 min at room temperature. After two washes in 4x SSC, 0.1% Tween-20 (5 min each at room temperature), the digoxigenin-labelled probe was immunodetected using monoclonal anti-digoxigenin (Sigma), anti-mouse digoxigenin-labelled (Boehringer Mannheim) and rhodamine-conjugated anti-digoxigenin (Boehriger Mannheim) antibodies. The biotin-labelled probe was detected using FITC-conjugated avidin and biotin-conjugated anti-avidin. All the incubations were performed at 37°C for 30 min in a moist chamber. After each incubation, washes were performed as described elsewhere (Surrallés et al., 1997c). The nuclear material was counterstained with DAPI in antifade solution (Vectashield) and stored at 4°C until microscopy.
Microscopic analysis and scoring
Microscopic analysis was performed on an Olympus BX-50 microscope equipped with a 100 W mercury lamp and a x1000 magnification objective with iris aperture. Two thousand cells were examined for each patient both before and after treatment for the presence or absence of MN. MN found were classified as centromere positive (C+MN) or negative (C–MN) only when the expected pattern of FISH centromeric signals was observed in the main nucleus. Otherwise, MN were classified as MN of unknown origin (UnMN).
Tandem labelling analysis was performed on 2000 buccal cells/individual before and after treatment. Cells were classified according to the number and localization of green (1cen) and red (1q12) signals.
Statistical analysis
The Student's t-test for dependent variables (paired t-test) was used to evaluate the genotoxic effect of 131I by comparing the frequencies of total MN, C+MN, C–MN, 1q12 breakage, chromosome 1 numerical abnormalities and 1q12 decondensations before and after 131I treatment. Comparisons between hyperthyroidism and thyroid cancer patients, between males and females, as well as between young (
45 years old) and older (>45 years old) patients were performed with a Student's t-test for independent variables (unpaired t-test). Both age groups were arbitrarily defined as giving two groups with a similar number of patients. Dose effects were assessed by performing multiple regression analysis.
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Results |
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MN and pancentromeric FISH
The results on MN induction and cytogenetic origin of MN after scoring 2000 buccal cells/individual before and after treatment are shown in detail in Table I
and summarized in Table II and Figure 1. Sex, health status and age of all donors are also indicated in Table I.
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The spontaneous frequency of MN in hyperthyroidism patients was 3.1 ± 3.1/2000 cells (0.156 ± 0.155%), similar to that found in thyroid cancer patients (2.3 ± 1.4/2000 cells, 0.113 ± 0.069%). Pooling the data from the 31 patients analysed, the spontaneous frequency of MN in buccal cells was 2.7 ± 2.4/2000 cells (0.135 ± 0.121%).
Eighty nine per cent of the MN found were classifiable as harbouring either acentric fragments or whole chromosomes. The remaining 11% of MN were classified as MN of unknown origin, since the nuclei of the cell where those MN were found did not present the expected pattern of FISH centromeric signals due to lack of hybridization. Of the total spontaneous MN 16.8% harboured centromeric signal and were therefore derived from whole chromosomes. As much as 83.2 % of spontaneous MN derived from acentric fragments.
Statistical analysis of the results indicated that none of the cytogenetic end points analysed (total MN, C+MN and C–MN) was increased after 131I treatment (P > 0.05, paired Student's t-test). In addition, neither age nor health status nor gender modulated the frequency of any of the cytogenetic variables (P > 0.05, unpaired Student's t-test). The only tendency observed was a slight increase (1.7-fold) in the frequency of total MN in thyroid cancer patients when compared with hyperthyroidism. Nevertheless, this tendency did not reach statistical significance (P = 0.071).
No 131I dose-related increase in the frequency of any of the cytogenetic end points analysed (total MN, C–MN and C+MN) was observed.
Tandem labelling FISH
Various cytogenetic abnormalities could be distinguished using the tandem labelling methodology, including breaks in the middle of the 1q12 band (M breaks), breaks between 1q12 and 1cen, i.e. in the proximal edge of 1q12 (E breaks), numerical abnormalities (NA) of chromosome 1 (cells with three or more FISH signals) and 1q12 decondensations (with one of the two red signals per nucleus being not compacted but spread along the nucleus). Decondensation might reflect dicentric chromosomes pulling in opposite directions, as their frequency increased after X-ray treatment in a dose-related fashion (Surrallés et al., 1997b).
The results obtained with the tandem labelling methodology after scoring 2000 buccal cells/individual before and after treatment are detailed in Table I
. The same data are summarized in Table II and Figure 1. M breaks and E breaks were pooled together in Table II and Figure 1. After tandem labelling analysis of 124 000 buccal cells, none of the cytogenetic abnormalities detected (1q12 breakage, 1q12 decondensation and chromosome 1 numerical abnormalities) was affected by 131I treatment, age, gender or health status.
The only tendency observed was a slight increase in the frequency of chromosome 1 numerical abnormalities in older patients (>45 years old) when compared with younger patients (<45 years old). However, this tendency did not reach statistical significance (P = 0.063).
No 131I dose-related increase in the frequency of any of the cytogenetic end points analysed (chromosome 1 NA, 1q12 breaks and 1q12 decondensations) was observed.
The spontaneous frequency of 1q12 breakage in hyperthyroidism patients was 5.6 ± 4.7/2000 cells, very similar to that found in thyroid cancer patients (5.7 ± 5.3/2000 cells). Pooling the data from all 31 patients analysed, the spontaneous frequency of 1q12 breakage in buccal cells was 5.7 ± 4.9/2000 cells. The distribution of cells according to the number of chromosomes 1 was ~1.50% of cells with one chromosome 1, ~98% of cells with two chromosomes 1 and ~0.5% of cells with more than two chromosomes 1.
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Discussion |
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In this study we used the most advanced interphase FISH methodologies to monitor chromosome damage in buccal cells from 131I-exposed patients with the overall outcome that no 131I treatment-related increase in the frequency of total MN, C–MN, C+MN, 1q12 breakage or decondensation and chromosome 1 numerical abnormalities was found.
Our results are apparently not in agreement with previous studies on radiation-induced MN in buccal cells. Tolbert and co-workers reported that radiotherapy of buccal tissues increased the frequency of MN in buccal cells from 0.16 to 1.00% (Tolbert et al., 1992). Sarto and co-workers reported an increase in the frequency of MN, especially chromosome fragments, in buccal cells after -ray treatment, reaching baseline levels 7–12 days after exposure (Sarto et al., 1987). Several studies detected an increase in the frequency of MN in buccal cells after head and neck radiotherapy, which declined 3 weeks after the last treatment (Stich and Rosin, 1983a; Tolbert et al., 1992). Similarly, Moore et al. (1996) reported 16.6- and 12.6-fold increases in the frequency of MN, specially C–MN, 3 and 6 weeks, respectively, after the initiation of radiotherapy for oral cancer, reaching baseline levels 3 weeks after the last treatment. Stich et al. (1983) found an increase in MN in buccal cells after radiotherapy, returning to background levels 1 or 2 months after the last irradiation.
In comparing our results with previously published data, one must take into account several biological and physical aspects, such as the type of treatment (acute versus chronic), the cell turnover and kinetics and the exposure level. Radioactive iodine has a half-life of ~8 days and accumulates in thyroid tissues, kidneys and bladder. Therefore, radiation exposure due to treatment cannot be considered as acute, although it is quickly excreted. The estimated turnover for buccal cells ranges between 1 week and 25 days (Stich et al., 1983; Hill, 1984), so we assume that a maximum peak of MN is expected 3–4 weeks after initiation of the treatment. In our study, the sample after treatment was obtained on average 27.2 ± 2.6 days after initiation of the treatment. We therefore consider that the schedule used in this study is valid for detecting induced chromosome damage in buccal cells. The fact that tandem labelling failed to detect radioactive iodine-induced chromosome aberrations gives support to our arguments in favour of an appropriate sampling schedule of our study, since no cell divisions are required to induced tandem labelling-detectable breaks (Rupa et al., 1997b). In addition, breaks detected by tandem labelling may reflect stable aberrations, such as chromosome translocations (Rupa et al., 1995; M.J.Ramírez, J.Surrallés, S.Puerto, A.Creus and R.Marcos, in preparation) which would be persistent in time and, therefore, would not decline to baseline levels after a few cell divisions (M.J.Ramírez, J.Surrallés, S.Puerto, A.Creus and R.Marcos, in preparation).
In the present study, the observed background frequency of MN in buccal cells after DAPI staining was 0.135 ± 0.121%. This frequency is in the range of the expected value, as previous studies reported a frequency of MN between 0.005 and 0.440 (Table III). The frequency of MN appears to be independent of the staining method used. Thus the DAPI staining method used in the present study is as sensitive as other staining methods, including Feulgen and propidium iodide.
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One of the potential limitations of the MN assay in human buccal cells is the marked inter-individual difference as reflected by the standard deviation of the mean. Although in the present study the inter-individual difference is as high as 9-fold, it is normal or even low, considering that other authors showed inter-individual differences of 15-fold in urothelial cells from 24 individuals (Reali et al., 1987
) or 17-fold in urothelial and buccal cells (Stich and Rosin, 1983a).
It is interesting to note that the frequency of MN in buccal cells is one order of magnitude lower than in peripheral lymphocytes (Sarto et al., 1990; Surrallés et al., 1997a). However, the frequency of MN in different types of exfoliated cells seems to be in a similar range around ~0.3% (Stich and Rosin, 1984; Rosin and Anwar, 1992; Titenko-Holland et al., 1994). This effect is not due to the requirement of in vitro divisions to express MN in human lymphocytes, since the frequency of MN in uncultured human lymphocytes is still much higher than that observed in buccal cells (Surrallés et al., 1996a). Thus, for some unknown reason, buccal cells produce MN with low efficiency, at least when compared with circulating lymphocytes. The same phenomenon is not true for 1q12 breakage, as data obtained with buccal cells is quite similar to the results derived from blood cells. Thus, after pooling the data from the 31 patients analysed, the spontaneous frequency of 1q12 breakage in buccal cells was 2.8 ± 2.5
cells, very similar to the frequency reported in human lymphocytes, granulocytes and buccal cells (Eastmond et al., 1994; Rupa et al., 1995, 1997a, Rupa et al., b; Rupa and Eastmond, 1997; Surrallés et al., 1997b).
In contrast to the apparent concordance between different laboratories with respect to the spontaneous frequency of MN and 1q12 breaks in buccal cells, the centromeric content of MN is highly heterogeneous between laboratories. Thus, our results indicate that only 16.8% of the spontaneous MN in buccal cells derive from whole chromosomes, whereas other studies show percentages of C+MN of 34.2 (Surrallés et al., 1997a), 55.6 (Moore et al., 1993a), 56.0 (Titenko-Holland et al., 1994) and 66.7% (Moore et al., 1996) in the same cell type. This strong inter-laboratory variability is also observed in other cell types such as human peripheral lymphocytes, with percentages of C+MN ranging from 29.4 to 78 (Surrallés et al., 1995, 1996a, 1997a; Ramírez et al., 1997a). The low frequency of C+MN found in the present study cannot be attributed to the scoring criteria or the staining, as only those cells with the expected speckle pattern of FISH centromeric signals in the main nucleus were considered amendable for MN classification.
As previously reported (Sarto et al., 1987; Rupa and Eastmond, 1997), we did not find an age-related increase in the frequency of total MN, C–MN, C+MN or tandem labelling-detectable chromosome abnormalities. This result was unexpected, considering the well-documented ageassociated increase in the frequency of aneuploidy and MN, above all C+MN, in human lymphocytes (Fenech et al., 1994; Catalán et al., 1995; Surrallés et al., 1996a,b; Ramírez et al., 1997a). We recently reported that radioactive iodine-induced age-related aneugenic effects in lymphocytes of similarly exposed thyroid cancer patients, as well as dose-dependent clastogenic effects (Gutiérrez et al., 1997; Ramírez et al. 1997a).
In interpreting our negative results one must consider that the methodologies employed are new and their actual sensitivities have not been formally proven. The uncertainties, such as timing schedule, actual sensitivity and persistence of detectable chromosome damage, will require further experimentation. However, considering the arguments provided in the discussion, we conclude that, at the reported exposure levels, radioactive iodine did not induce detectable chromosome damage in buccal cells from exposed patients.
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Acknowledgments |
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This investigation was partially supported by grants from the Ministerio Español de Educación y Cultura (SAF95-0813, CICYT) and from the Generalitat de Catalunya (SGR95-00512, CIRIT). M.J.R and J.S. were supported during this work by a FPI fellowship and a Contrato de Incorporación de Doctores, respectively, by the Ministerio Español de Educación y Cultura. We thank M.McCarthy for her secretarial assistance.
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Notes |
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3 To whom correspondence should be addressed. Tel: 34 93 58120 52; Fax: 34 93 58123 87; Email: rmd{at}cc.uab.es
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References |
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Received on May 28, 1998; accepted on July 20, 1998.
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