Prevention of DNA Double-Strand Breaks Induced by Radioiodide-¹³¹I in FRTL-5 Thyroid Cells

¹³¹I causes DNA double-strand breaks (DSB) in FRTL-5 thyroid cells but not in cells that lack sodium/iodide symporter expression; perchlorate, iodide, and thiocyanate protect against the ¹³¹I-induced DSB.

Abstract

Radioiodine-131 released from nuclear reactor accidents has dramatically increased the incidence of papillary thyroid cancer in exposed individuals. The deposition of ionizing radiation in cells results in double-strand DNA breaks (DSB) at fragile sites, and this early event can generate oncogenic rearrangements that eventually cause cancer. The aims of this study were to develop a method to show DNA DSBs induced by ¹³¹I in thyroid cells; to test monovalent anions that are transported by the sodium/iodide symporter to determine whether they prevent ¹³¹I-induced DSB; and to test other radioprotective agents for their effect on irradiated thyroid cells. Rat FRTL-5 thyroid cells were incubated with ¹³¹I. DSBs were measured by nuclear immunofluorescence using antibodies to p53-binding protein 1 or γH2AX. Incubation with 1–10 μCi ¹³¹I per milliliter for 90 min resulted in a dose-related increase of DSBs; the number of DSBs increased from a baseline of 4–15% before radiation to 65–90% after radiation. GH3 or CHO cells that do not transport iodide did not develop DSBs when incubated with ¹³¹I. Incubation with 20–100 μm iodide or thiocyanate markedly attenuated DSBs. Perchlorate was about 6 times more potent than iodide or thiocyanate^. The effects of the anions were much greater when each was added 30–120 min before the ¹³¹I. Two natural organic compounds recently shown to provide radiation protection partially prevented DSBs caused by ¹³¹I and had an additive effect with perchlorate. In conclusion, we developed a thyroid cell model to quantify the mitogenic effect of ¹³¹I. ¹³¹I causes DNA DSBs in FRTL-5 cells and had no effect on cells that do not transport iodide. Perchlorate, iodide, and thiocyanate protect against DSBs induced by ¹³¹I.

Radioiodine-131 released from nuclear reactor accidents has dramatically increased the incidence of papillary thyroid cancer in exposed individuals, especially young children who were exposed in the Marshall Islands or in areas affected by the Chernobyl catastrophe (1–3). For prevention of radiation-induced thyroid cancer, the Food and Drug Administration in 2001 recommended that potentially exposed people take potassium iodide tablets that contain 100 mg iodide per day to block thyroid uptake of the ¹³¹I (http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm080542.pdf). based on the work of Braverman and colleagues (4). The deposition of ionizing radiation in cells results in double-strand DNA breaks (DSBs) at fragile sites, and this early event can generate oncogenic rearrangements that eventually cause the cancer (5, 6).

Ionizing radiation causes double-strand breaks in DNA that lead to downstream activation of repair processes within cells (7). The two main pathways for repair of DSBs are nonhomologous end-joining and homologous recombination. Nonhomologous end joining is the main pathway by which cells repair damage from ionizing radiation because it does not require a template for repair and involves limited processing of the damaged ends before religation of the DSBs (7). This process is more likely to result in rearrangements leading to oncogenic mutations than repair by homologous recombination. The presence of γH2AX (histone H2AX, which is phosphorylated at serine 139 located in the carboxy terminal tail) is accepted as a specific indicator for the presence of DSBs (8). P53-binding protein-1 (53BP1) is another component of the DNA repair system for nonhomologous end joining of DSBs that accumulates in the nucleus after DSBs caused by ionizing radiation (9, 10).

The goals of this study were: 1) to develop a method to show DSBs induced by ¹³¹I in thyroid cells; 2) to test monovalent anions that are transported by the sodium/iodide symporter (NIS) to determine whether they prevent ¹³¹I-induced DSB; and 3) to test other radioprotective or mitigating agents for their effect on irradiated thyroid cells.

Materials and Methods

FRTL-5 rat thyroid cells were cultured in Coon's modified F-12 medium (Sigma, St. Louis, MO) supplemented with six hormones (TSH, 1 U/liter; insulin, 246 mU/liter; somatostatin, 10 μg/liter; hydrocortisone, 10 nm; transferrin, 5 mg/liter; glycyl-histidyl-lysine, 2.5 μg/liter), 5% calf serum and antibiotics (6H medium) as previously described (11). Cells were maintained in a 5% CO₂-95% air atmosphere at 37 C with a change of medium every second day and passed every 7 d. For the experiments, cells were then transferred to LabTek chamber slides that were ionized for cell adherence (Thermo Fisher Scientific, Los Angeles, CA).

To prepare cells for irradiation with ¹³¹I, when the cells in the 75-cm² flask were approximately 75% confluent, they were resuspended into 1 ml 6H, and 25 μl containing approximately 10⁵ cells was added into each well with 475 μl 6H. Cells were allowed approximately 45 min to adhere. They were then incubated with ¹³¹I-iodide (Mallinkrodt, Commerce, CA), usually for 90 min. The radioactive medium was removed, and the cells were rinsed three times with 500 μl of PBS and then incubated in 500 μl 4% paraformaldehyde for 15 min. The cells were then rinsed three times more with PBS, once with 0.5% Triton X-100, and again three times with PBS. Then 500 μl of 10% fetal bovine serum (FBS) was added to the wells to block nonspecific binding, and the cells were incubated overnight at 4 C (or, alternatively, 1 h at room temperature). Chemicals were obtained from Sigma unless stated otherwise.

The primary antibody (53BP1 rabbit antibody) (Santa Cruz Biotechnology, Santa Cruz, CA), used to indicate DSBs, was prepared in 10% FBS at a 1:800 dilution. In some experiments γH2AX was used as the primary antibody (Santa Cruz Biotechnology). The cells were incubated in the primary antibody (200 μl/well) for 1 h at room temperature. The primary antibody was removed and the cells were rinsed once with 0.1% Triton X-100 and twice with PBS and then incubated in 10% FBS to block nonspecific binding for 1 h at room temperature. The secondary antibody (Alexa Fluor 488 goat antirabbit IgG antibody; Invitrogen Molecular Probes, Eugene OR), used to produce immunofluorescence, was prepared in 10% FBS at a 1:500 dilution. The cells were incubated with this antibody (200 μl/well) for 45 min at room temperature. They were then washed three times with 500 μl of PBS and mounted with coverslips using 10 μl of 4′,6′-diamino-2-phenylindole/well.

Nuclear immunofluorescence was viewed using a Zeiss Axioskop 2 plus fluorescent microscope (New York, NY). Approximately 100–150 cells were counted in each well; when the number of positive nuclear foci was five or more, the cells were considered to have significant DNA damage indicative of a DSB. Each condition was studied in duplicate wells and the mean number of DSB ± sd was calculated. Each experiment was repeated, usually three times with close agreement of results.

Statistical significance between groups was determined by an unpaired two-tailed Student's t test, Tukey-Kramer multiple comparison test, or Dunnett multiple comparison test, as appropriate, using InStat3 for the Mac (GraphPad Software, Inc., La Jolla, CA).

The radioactive uptakes by the cells were determined by counting an aliquot of the supernatant in triplicate after the 90-min incubation in a scintillation counter. The mean uptake was 30.7 ± 12.4% (sd).

Results

Induction of DSBs by ¹³¹I

Figure 1 shows that incubation of FRTL-5 cells with ¹³¹I for 90 min resulted in a dose-related increase in DSBs detected by γH2AX immunostaining at doses of 1–20 μCi ¹³¹I per milliliter. There was a dose-related increase in the uptake of ¹³¹I by the FRTL-5 cells (Fig. 1). Figure 2 shows that incubation of cells with 10 μCi ¹³¹I per milliliter for 90 min caused 91% of the cells to be positive for DSBs, compared with only 4.3% of cells being positive for DSB when they were not incubated with ¹³¹I (P < 0.01). We found similar effects of ¹³¹I using the PCCL3 rat thyroid cell line kindly provided by Jacques Dumont (Universite Libre de Bruxelles, Bruxelles, Belgium) (data not shown).

Fig. 1.

¹³¹I-induced DSBs in FRTL-5 cells detected by γH2AX immunostaining, 90 min incubation. The solid line shows the relationship of DSBs with concentration of ¹³¹I in the incubation medium; there was no increase of DSB at ¹³¹I concentrations greater than 10 μCi/ml. The dashed line shows the ¹³¹I taken up by cells (microcuries per 10⁵ cells) at each concentration of ¹³¹I.

Fig. 2.

FRTL cells were incubated with 10 μCi/ml for 90 min. Left panel shows that 91% had positive 53BP1 nuclear immunostaining indicating DSBs. Right panel shows that 4.3% of control FRTL-5 cells not incubated with ¹³¹I had positive 53BP1 nuclear immunostaining.

To determine whether the DSBs induced by ¹³¹I might be caused by a bystander effect that was not due to transport of the ¹³¹I into the cell, studies were performed with two cell lines that do not transport iodide, CHO cells and GH3 rat pituitary cells. Figure 3 shows that there was no induction of DSBs by ¹³¹I in these cell lines in contrast with the effect of ¹³¹I on FRTL-5 cells.

Fig. 3.

¹³¹I (10 μCi/ml, 90 min) caused a marked increase of DSBs in FRTL-5 cells detected by 53BP1 immunostaining (P < 0.01) but did not induce DSBs in Chinese hamster ovary (CHO) cells or GH3 cells that do not transport iodide.

To determine whether the FRTL-5 cells recovered from the DSBs induced by ¹³¹I, cells were incubated with ¹³¹I for 90 min, the ¹³¹I was removed, the cells were washed twice with medium, and the incubation was continued for 24 h. There was only partial recovery to the baseline state of DSBs at 24 h (Fig. 4). To determine whether incubation with 10 μCi ¹³¹I per milliliter compromised cell viability, FRTL-5 cells were incubated in flasks for 90 min with ¹³¹I rather than in the LabTek chamber slides (Thermo Fisher Scientific, Los Angeles, CA). The ¹³¹I was removed, the cells were washed twice, and the incubation was continued with passage of the cells two times over 7 d. Study of cell viability by flow cytometry using 7-amino actinomycin D showed no impairment of viability in comparison with control cells not incubated with ¹³¹I (12).

Fig. 4.

Recovery from DSBs induced by exposure to ¹³¹I. FRTL-5 cells were incubated in 10 μCi ¹³¹I per milliliter. The DSB of cells not incubated with ¹³¹I was 10% (basal DSB). The DSB at 0 time shown here is the maximum DSB after the 90-min incubation. After the removal of the ¹³¹I, the incubation was continued for 24 h and immunostaining for 53BP1 was performed at various times (P < 0.05 at 6 h and P < 0.01 at 24 h compared with 0 time).

Prevention of DSBs by monovalent anions and other compounds

Studies were performed to assess the relative effect of monovalent anions for prevention of DSBs induced by ¹³¹I. We compared iodide, perchlorate, and thiocyanate, monovalent anions that are transported by the NIS. Figure 5A shows the dose-related reduction of DSBs caused by perchlorate and iodide. Numerous comparisons were performed to determine the relative efficacy of perchlorate, iodide, and thiocyanate as inhibitors of ¹³¹I-induced DSBs. The concentrations of these anions chosen for the experiment in Fig. 5B were selected to produce similar effects. The results show that iodide and thiocyanate are similar in blocking potency and perchlorate is about 6-fold more potent than the other anions. In various experiments, the relative effect of perchlorate compared with iodide for blocking DSBs varied from 4- to 20-fold, although the 6-fold relative potency, perchlorate/iodide, shown in Fig. 5B was repeatable in several experiments.

Fig. 5.

A, Inhibition of DSBs caused by perchlorate (2, 4, or 6 μm) or iodide (10, 20, or 30 μm). FRTL-5 cells were incubated with 10 μCi per milliliter ¹³¹I for 90 min; anions were added at 0 time; basal DSB was 11.7%. Perchlorate and iodide caused a dose-related reduction of DSBs by a multiple comparison test (P < 0.01). B, Inhibition of DSBs by anions (90 min incubation, 10 μCi/ml ¹³¹I). Basal DSB was 11%; maximum DSB with no anion was 84%. Anions added 60, 30, and 0 min before ¹³¹I were similarly effective in preventing DSBs (P < 0.01); anions added 30 min after ¹³¹I were less effective (P < 0.05) compared with no anion.

Figure 6A shows that preincubation with stable iodide or perchlorate was more effective in prevention of DSBs than addition of the inhibitor after a 30-min incubation with ¹³¹I. In this experiment, perchlorate was more than 10 times as potent as iodide for prevention of DSBs. Figure 6B shows that delayed addition of the inhibitory anion resulted in a time-dependent loss of the inhibitory effect.

Fig. 6.

A, Inhibition by KClO₄ and KI using 53BP1 (90 min incubation with ¹³¹I; basal DSB, 8.1%; maximum DSB, 86.5%). Perchlorate or iodide added after 30 min incubation was less protective than when added 120, 60, or 0 min before ¹³¹I (P < 0.05). B, Perchlorate or iodide was added at various times shown after incubation with ¹³¹I (basal DSB, 11%; maximum DSB, 76%). Addition of the anion at 40 min was less protective than when added at 0 time (P < 0.01).

γH2AX is a well-established indicator of radiation damage. Figure 7 shows that results obtained with γH2AX were very similar to those found using 53BP1 as the indicator of DSBs. Several experiments with γH2AX gave results similar to those using 53BP1 as the indicator of DSBs.

Fig. 7.

Comparison of 53BP1 and γH2AX (90 min incubation with ¹³¹I). Results are qualitatively similar in basal state and at maximum DSBs but differ slightly during incubation with perchlorate that inhibits ¹³¹I-induced DSBs.

Yel-001 and Yel-002 are natural organic compounds that have been shown to protect against radiation-induced injury. Figure 8A shows that the addition of these compounds 30–60 min after exposure to ¹³¹I (in a 90 min incubation) reduced the percent of DSBs. Figure 8B shows that the combination of Yel-002 and 1 μm perchlorate was additive in prevention of DSBs. Ciprofloxacin, tetracycline, and tilorone, shown by others to confer radioprotection (13), did not prevent DSBs induced by ¹³¹I in FRTL-5 cells (data not shown).

Fig. 8.

A, Yel-001 and -002 are radioprotective when added before ¹³¹I (P < 0.05 for each comparison). B, Yel-002 blocked DSBs and had an additive effect with 1 μm KClO₄ (90 min incubation, 10 μCi/ml ¹³¹I; basal DSB was 4.1%; maximum DSB with no anion was 88%).

Discussion

Our results show that uptake of ¹³¹I causes DNA DSBs in FRTL-5 rat thyroid cells in a dose-related manner and that this does not occur in cell lines that do not actively transport iodide. Our work is the first demonstration in vitro that ¹³¹I causes DNA DSBs in thyroid cells. Others have shown that external radiation can cause DNA DSBs in primary cultures of human thyroid cells (14) and in the PCCL3 rat thyroid cell line (15). In contrast with the nearly complete loss of DSBs found by Galleani et al. (14) in human primary culture cells 24 h after external irradiation, we noted that there was a significant persistence of DSBs 24 h after removal of the ¹³¹I. This persistence of radiation-induced foci suggests the persistence of disorganized chromatin regions (16). The differences between results could be attributable to a higher radiation dose in our study or the fact that we used a rat cell line.

Perchlorate was about 6-fold more potent than iodide or thiocyanate in prevention of DSBs. A study of perchlorate transport into FRTL-5 cells indicated that the affinity of NIS for perchlorate is much greater than that for iodide (11). Van Sande et al. (17) calculated that the relative IC₅₀ of perchlorate/iodide for inhibition of radioiodide transport in FRTL-5 cells was 0.6:51. However, our data show that perchlorate is much less effective for blockade of DSBs than suggested by this ratio. Perhaps iodide has a special effect as a radioprotective agent in addition to prevention of radioiodide uptake. Iodide has been shown to reduce the mRNA and protein of the NIS in the thyroids of rats exposed to large amounts of iodide (18). Perhaps this effect of iodide plays a role in its radioprotective effect, although the rapid prevention of DSB by iodide in vitro makes this mechanism speculative.

Our data also show that maximum prevention of ¹³¹I-induced DSBs by competing monovalent anions requires addition of the anion to the incubation medium before the radioiodide exposure and that addition after incubation with the ¹³¹I has begun results in progressive loss of the radioprotective effect with the time of delay. Yel-001 and Yel-002 are examples of compounds under development for mitigation of the effects of ionizing radiation. Our study showed that the mitigating effects of these compounds are additive with those of anions that compete for NIS. The exact mechanisms of action of the Yel compounds are yet to be determined; however, in vivo and in vitro data (not reported yet) point to DNA repair up-regulation processes and rescue from cell death after genotoxic injuries.

The lack of induction of DSBs in cells that do not transport ¹³¹I or in whom the transport is blocked by competitive anions requires an explanation. The β-radiation of the ¹³¹I has an average energy of 190 kEv and penetration of about 1 mm in tissue or water (19). Yet there is an absence of significant bystander effect that could produce detectable DSBs from penetration of this radiation into the cells in this study. One possible explanation is the fact that the cells are grown in a monolayer, which exposes the cells to less of the ¹³¹I that is dispersed uniformly in the medium. Another factor is that the FRTL-5 cells transport a high proportion of the ¹³¹I into the cell, thereby increasing the radiation to the DNA in the nucleus.

In recent years, there has been an increase in the frequency of papillary thyroid cancer (20). The basis for the increase is unknown. The model developed for this study could provide a method for screening compounds that could be thyroid carcinogens by measuring their induction of DSBs as a precursor to neoplasia. However, proof of principle will require validation by suitable studies in experimental animals.

In summary, we have developed a cell model using FRTL-5 rat thyroid cells that concentrate ¹³¹I, which results in DSBs that are visualized with 53BP1 or γH2AX. Preincubation with perchlorate, iodide, or thiocyanate prevents the DSB. Addition of the anions after exposure to ¹³¹I is less effective in prevention of DSBs. These data provide a basis for studies of radioprotection against DSBs induced by ¹³¹I in animals and studies to refine the prevention of thyroid cancer resulting from nuclear fallout.