Abstract
The existence of thyroid stunning (i.e., inhibited thyroidal iodide uptake after administration of diagnostic amounts of 131I) is controversial and is currently a subject of debate. To our knowledge, the stunning phenomenon has not been investigated previously in vitro. Methods: Growth-arrested porcine thyroid cells that formed a tight and polarized monolayer in a bicameral chamber were irradiated with 3–80 Gy 131I present in the surrounding culture medium for 48 h. The iodide transport capacity after irradiation was evaluated 3 d later by measuring the transepithelial (basal to apical) flux of trace amounts of 125I. Results: The basal-to-apical 125I transport decreased with increasing absorbed dose acquired from 131I; a nearly 50% reduction was observed already at 3 Gy. Stable iodide at the same molarity as 131I (10−8 mol/L) had no effect on the 125I transport. Cell number and epithelial integrity were not affected by irradiation. Conclusion: Stunning of iodide transport is detected after 131I irradiation of cultured thyroid cells. The degree of inhibition of transport is dependent on the absorbed dose.
As stated in a recent review, “Evidence that stunning is a real phenomenon is now strong, albeit not yet conclusive” (1). This quotation summarizes the current view on the potential side effects of diagnostic amounts of 131I on subsequent radiation therapy due to inhibited iodide accumulation in thyroid tissue. Radioiodide has been used in therapy for >50 y. Thyroid stunning was first reported in 1951 by Rawson et al. (2), who found that the 131I uptake, as determined by scintigraphy, was reduced after administration of diagnostic amounts of 131I. However, the question of whether preexposure to diagnostic amounts of 131I might influence the outcome of 131I radiation therapy is still being debated (3– 10).
Efforts to identify and determine the magnitude of thyroid stunning in response to 131I radiation are based solely on in vivo studies. A measured reduction in thyroid uptake might be caused by stunning or dead-time losses related to high counting rates if not compensated for. McDougall (8) concluded that no stunning was observed in his study when the administered activity was <74 MBq 131I. However, other studies reported stunning when activities of >111 MBq were used (9,10). The use of 123I for diagnostics to eliminate the stunning effect has been recommended in some reports (11–13).
The mechanism by which thyroid stunning, if existing, occurs is unknown. Leger et al. (9), who described a 50% decrease in the uptake of 123I 5 wk after administration of 185 MBq 131I, suggested 2 possible mechanisms. One is simply that the β-emission of 131I given for diagnostic purposes is enough to cause cell damage, perhaps by induction of apoptosis, thereby reducing the number of iodide-transporting cells. The alternative explanation is that low-dose 131I radiation in some way affects iodide transport mechanisms, possibly also involving a change in the intrathyroidal iodide turnover, without being deleterious to cell viability.
This study was performed to determine if absorbed doses of 131I that are comparable with those used in clinical diagnostic investigations with 131I could induce stunning of iodide transport in thyroid cells in vitro. For this purpose, we used a bicameral culture system that mimics the structural and functional properties of the follicular epithelium in intact thyroid tissue.
MATERIALS AND METHODS
Cell Culture
Porcine thyroids obtained from the local abattoir were prepared according to Nilsson et al. (14) to obtain a pure fraction of follicular cells. Briefly, glands sterilized by flaming for 5 s in 70% ethanol were decapsulated and freed from macroscopic connective tissue, after which the parenchyme was subjected to enzymatic disintegration by collagenase. Segments of thyroid follicles were separated from large tissue fragments and blood cells by repeated centrifugation and filtration and then suspended in minimal essential medium supplemented with 5% fetal calf serum, penicillin (200 U/mL), streptomycin (200 μg/mL), and fungizone (2.5 μg/mL) (all culture medium constituents were purchased from GIBCO, Paisley, U.K.). The cells were cultured on the collagen-coated microporous filters (pore size, 0.4 μm) of bicameral chambers (Transwell 3413; Corning Costar, Cambridge, MA) at 37°C in an incubator with 5% CO2; the culture medium was exchanged every 2–3 d. During culture the growing cells reorganized spontaneously from follicular aggregates to form a continuous monolayer on the filter. Thus, the experimental system provides a model consisting of 3 compartments (Fig. 1) corresponding to the in vivo counterparts: the follicular epithelium (cell monolayer), the extrafollicular space (basal volume), and the follicular lumen (apical volume).
Transwell bicameral culture chamber system. Porcine thyroid cells form continuous monolayer epithelium on microporous filter, which constitutes bottom of Transwell insert. Monolayer is continuous and thus separates chamber into lower (basal) and upper (apical) compartment. Insert that holds filter is placed at fixed distance of 1 mm from base of basal well. Filter area, 0.33 cm2; filter height, 10 μm; filter pore size, 0.4 μm.
To evaluate the barrier function of the cell layer before and during experiments, the transepithelial resistance was measured with a Millicell ERS ohmmeter (Millipore Corp., Bedford, MA). As described earlier (15), the monolayered cells we used for the radioiodide experiments were largely nondividing because of contact-dependent inhibition of growth. Cell number was estimated by determination of the total DNA content (16).
Irradiation Procedures
In the standard experimental protocol (Fig. 2), cultures were pretreated with 1 mU/mL thyroid-stimulating hormone ([TSH] Sigma Chemical Co., St. Louis, MO) for 48 h before irradiation to accelerate the iodide transport capacity. TSH stimulation was also maintained throughout irradiation and subsequent steps of the experiment. Irradiation was started by replacing the culture medium with medium containing 131I (Nycomed Amersham Plc., Little Chalfont, U.K.) and, to prevent radioiodide-protein binding and possible confounding side effects of iodinated compounds, 1 mmol/L methimazole (Sigma) was added. The addition of alkaline 131I stock solution, finally diluted 1:500 or more, to minimal essential medium did not change the pH. The medium with radioactivity was distributed, 500 μL in the basal compartment and 100 μL in the apical compartment. The activity concentration was the same basally and apically at the start of incubation. Assuming that the ratio of apical and basal radioactivity concentrations did not change over time, different amounts of 131I estimated to give absorbed doses of 1, 5, 10, and 30 Gy were added to the cultures. To more accurately determine the actual absorbed dose, taking into account changes in the distribution of radioactivity likely to occur because of ongoing transport, the 131I activity was determined in consecutive samples of the basal and apical media during irradiation. In such experiments, the starting volumes were 400 and 200 μL and the sampling volumes were 10 and 5 μL, respectively, to maintain a constant volume ratio between the compartments on either side of the cell monolayer. In some experiments, 1 mmol/L perchlorate (Sigma) was present in the culture medium throughout irradiation to block the accumulation of 131I in the apical compartment. To investigate the possible effects of iodide per se on 125I transport, irradiated cultures were compared with those that were exposed to stable iodide, 127I, at similar molarity (10− 8 mol/L). Untreated control cultures were included in all experiments. In general, each type of experiment was repeated 3 times on cultures from different platings. The 48-h irradiation period was terminated by multiple, gentle washings in culture medium to remove all radioactivity, after which the culture was continued until transport studies were performed.
Protocol for thyroid cell culture treatments (open arrows) and radioiodide transport analysis (filled arrows). Time scale indicates number of days after starting primary culture.
Measurement of Iodide Transport
The ability of the cells to transport iodide was evaluated 3 d after terminating the irradiation period. The chambers were transferred from the incubator to a 37°C water bath, and the culture medium was replaced by Tyrode’s salt solution, pH 7.2, containing 1 mmol/L methimazole to prevent iodination. To study the transport of iodide in the basal-to-apical direction, 125I (Nycomed Amersham) was added to the basal medium (150 kBq/mL; 400 μL) and the radioactivity accumulated in the apical medium (200 μL) was analyzed in 150-μL samples. The samples were taken at 10-min intervals for 30 min, and the collected volume was replaced immediately with nonradioactive medium. The remaining 50 μL of apical medium at each interval was left in to minimize the risk of damaging the cell layer with the tip of the pipette. Finally, the filters and cells were washed twice and cut out of the holders. The 125I activities present in the apical medium, the basal medium, and the filters with adherent cells were measured using a Wallac 1480 γ-counter (Wallac Oy, Turku, Finland). Corrections were made for background, radioactive decay, and, when appropriate, spillover in the 125I energy window from photons emitted by the minimal amounts of 131I remaining in filters and cells after the wash.
Absorbed Dose Calculations
The absorbed dose of 131I applied to the cells was initially estimated by assuming that the activity was distributed evenly between the culture chamber compartments. The distance from the cell layer to the wall of the chamber and medium surfaces was greater than the continuous slowing-down approximation range of the emitted particles. Therefore, the absorbed dose affecting the cells was assumed to be close to the average absorbed dose in the chamber and was calculated according to:
where à is the cumulated activity, Δ is the energy emitted per transition, φ is the absorbed fraction, and m is the mass of cells (17). The absorbed fraction was set to 1 for electrons and calculated to be 0.0125 for photons (364 keV), using the method from the International Commission on Radiation Units and Measurements (17):
where μen is the mass energy-absorption coefficient and x is the radius of the compartment. This approximation can be used assuming that the product of μenx ≪ 1, the volume is approximated by a sphere, and the radioactivity is distributed homogeneously within the volume. However, during the time course of irradiation a net transport of 131I through the cell layer occurred in the apical direction, resulting in a gradual change in the apical-to-basal radioiodide concentration ratio (Fig. 3). In most experiments, the concentration of 131I in the apical medium was 100–300 times higher than that in the basal medium when irradiation was stopped after 48 h. Taking into account the different 131I concentrations and geometries of the apical and basal compartments, the corrected absorbed dose, Dc, was calculated according to:
where D0 is the uncorrected absorbed dose as determined by the equations above, CAB is the apical-to-basal 131I concentration ratio, and VA and VB are the apical and basal volumes, respectively (Fig. 1). Theoretically, if the entire radioactivity were transferred instantly to the apical compartment, the absorbed dose would be 3 times higher than if the applied radioactivity were to remain at a ratio of 1:1 between the 2 compartments. On the basis of the kinetics of the gradual accumulation of 131I in the apical medium, as illustrated in Figure 3, the absorbed doses were adjusted by introducing an average correction factor of 2.7.
Redistribution of 131I between basal and apical media of filter-cultured porcine thyrocytes during irradiation period (Dc = 15 Gy). Equal concentrations of 131I were present initially at both sides of cell monolayer. Results are presented as mean ± SEM (n = 6).
Statistical Analysis
The Student t test was used to determine the statistical significance of differences between the groups. Results are given as mean ± SEM.
RESULTS
The ability of the cells to unidirectionally transfer 125I from the basal to the apical medium, corresponding to the natural route of transepithelial iodide transport in vivo, was studied 3 d after terminating the 131I irradiation. Before each transport experiment, the tightness of the cell layer was confirmed by measurements of the transepithelial electrical resistance. No signs of paracellular leakage were found, regardless of whether the cells had been exposed to 131I. 125I transport was almost completely blocked in cultures irradiated with 80 Gy (Fig. 4). In contrast, no significant change in the 125I transport capacity of the cells was found in the presence of 10−8 mol/L stable iodide (Fig. 4), which is equivalent to the highest 131I molarity used. Lower absorbed doses also reduced the basal-to-apical transport of 125I (Fig. 5). The 125I transport was decreased by nearly 50% already at 3 Gy compared with the control group. Perchlorate present during irradiation to prevent accumulation of 131I in the apical medium partially blocked the reduction of 125I transport appearing at the corresponding absorbed dose (Fig. 6). Determination of the DNA content revealed that 131I radiation at doses that caused pronounced inhibition of the 125I transport did not change significantly the total number of cells (Table 1).
Effect of 131I irradiation (Dc = 80 Gy) and equimolar concentrations of stable iodide (10−8 mol/L) on subsequent (3 d later) basal-to-apical 125I transport. Results are given as 125I activity transported into apical medium at indicated times relative to activity added to basal medium at time = 0. Values obtained are expressed as percentage of total activity present basally at start of experiment. Results are expressed as mean ± SEM (n = 3).
Effect of different absorbed doses on iodide transport in 131I irradiated cultures. Results are presented as relative amount of 125I activity transported from basal to apical medium during 30 min. Values are given as mean ± SEM (n = 4).
Effect of perchlorate (ClO4−) on iodide transport in 131I-irradiated cultures. Perchlorate (1 mmol/L) was present in apical and basal media during exposure to 131I (corresponding to Dc = 30 Gy without perchlorate) for 48 h. 125I transport was evaluated 3 d later. 131I-induced stunning of 125I transport was 90% when perchlorate was not present during irradiation. Results are expressed as mean ± SEM (n = 3).
Effect of 131I Radiation on DNA Content in Confluent, Growth-Arrested Porcine Thyrocytes Cultured on Filter
DISCUSSION
In this study, we examined whether 131I at absorbed doses commonly obtained from diagnostic investigations are able to induce stunning of thyroid iodide transport in vitro. Porcine thyroid cells were cultured in Transwell bicameral chambers to provide an experimental system in which parameters affecting the viability and function of the cells as a polarized epithelium could be controlled and monitored (14). The cells were prestimulated with TSH to maintain the iodide-transporting capacity close to its maximal level (14) and then irradiated with 131I for 2 d. To rule out side effects related to iodination, such as reduced expression of the sodium/iodide symporter (18), the thyroid-blocking agent methimazole was present throughout 131I exposure. Iodide transport was evaluated 3 d after washing out the radioactivity by measurements of the transepithelial transport of trace amounts of 125I. Clinical studies have not elucidated whether the reduction in radioiodide uptake observed after administration of diagnostic amounts of 131I is caused by a decrease in activity of the iodide-concentrating mechanism or merely reflects a therapeutic effect related to radiation-induced cell death. Our results show that 131I radiation for 48 h reduces the vectorial (basal to apical) 125I transport across filter-cultured porcine thyrocytes in a dose-dependent way. A 50% inhibition of transport was noted at an absorbed dose of approximately 3 Gy (Fig. 5). The lack of effect of stable iodide present for the same period of time at a concentration (10−8 mol/L) similar to that of the highest 131I concentration proves that the stunning effect is radiation dependent and is not related to iodide exposure per se (Fig. 4). This notion is supported further by the results of the perchlorate experiment (Fig. 6). Iodide uptake mediated by the sodium/iodide symporter is known to be blocked by perchlorate (19). Perchlorate was found to counteract stunning, and a likely explanation is that a lower absorbed dose was obtained because of the reduced apical 131I accumulation.
To eliminate misleading results due to changes in cell number, this study was designed to examine the effect of 131I on iodide transport in confluent, nondividing cells. On the basis of 3H-thymidine incorporation experiments (data not shown), it is evident that irradiation equivalent to the applied absorbed doses used in this study inhibits the proliferation of subconfluent, rapidly growing thyroid cells. Therefore, irradiation might very well influence cell renewal in the monolayer. However, because this is known to occur at a very low rate in contact-inhibited, high-density cultures, it is likely that any small loss in cell number due to cell cycle arrest contributes only marginally to the pronounced reduction in iodide transport observed 3 d after irradiation was terminated. The similar levels of total DNA obtained in the irradiated and nonirradiated cultures (Table 1) confirmed this assumption. Cell density, as revealed by light microscopic examination after nuclear staining (data not shown), was also the same with and without pretreatment with 131I. Preliminary observations on caspase-3 activity and nuclear morphology (data not shown) suggest that apoptosis was not induced by 131I radiation under the current experimental conditions.
The irradiated cells were also evaluated by measuring the transepithelial resistance before the 125I transport was studied. A high resistance indicates that leaky tight junctions or ruptured plasma membranes due to cell damage cannot be present. We found that the barrier function of the cell layer did not change, even after the highest absorbed dose (80 Gy). That the integrity of the cultured epithelium was maintained is supported further by the observation of a high apical-to-basal 131I ratio in the bicameral culture chamber at the end of irradiation. Also, the pronounced reduction in iodide transport observed 3 d later is compatible only with a continuous and tight cell layer attached to the filter. Indeed, an impaired epithelial barrier would result in the unrestricted diffusion of solutes, including iodide, between the basal and apical compartments (i.e., the reverse of the actual findings in the irradiated cultures) (20).
When 131I was added at the start of the experiment, the apical and basal iodide molarities were equal. However, during the course of irradiation the ability of the cells to transport iodide generated a gradual accumulation of the radionuclide in the apical medium, resulting in a very high apical-to-basal 131I ratio after 48 h. The enrichment of most of the 131I in the smaller apical volume requires the absorbed dose, initially calculated on the basis of the assumption of a constant apical-to-basal ratio of 1, to be corrected. The estimated correction factor was approximately 2.7 taking into account the kinetics of the 131I accumulation in the apical medium (Fig. 3). The maximum error in Dc was estimated to be ±10%. That 131I transport prevailing during irradiation indeed caused an increased absorbed dose to the cells was shown in experiments in which the generation of a high apical-to-basal 131I gradient was inhibited by perchlorate.
The estimated absorbed doses reported to induce thyroid stunning in previous in vivo studies vary. Muratet et al. (21) showed stunning in a group of patients who received an absorbed dose of >17.5 Gy to thyroid remnants after 131I scanning. Kao and Yen (10) observed a 74% reduction in radioiodide uptake when the diagnostic absorbed dose was >35 Gy. Sabri et al. (22) reported stunning at similar absorbed doses. Our findings of inhibited 125I transport by approximately 90% at 30 Gy and almost 50% at 3 Gy (corrected absorbed doses) in the 131I-irradiated thyroid cell cultures suggest that thyroid stunning may occur also at lower absorbed doses than those considered on the basis of clinical observations. Another matter of concern, which has been discussed only briefly in previous in vivo studies, is the time of onset and the duration of thyroid stunning. Indeed, the variation in time between the administration of diagnostic and therapeutic amounts of 131I is considerable. Recently, McDougall (8) and Cholewinski et al. (3) found no signs of inhibited uptake when therapy was performed 72 h or earlier after diagnostic examination using 74 MBq and 185 MBq 131I, respectively. This suggests that stunning might be manifest after a delay of a few days. To reproduce a time interval of relevance in clinical applications, the iodide transport was evaluated 3 d after ending the 48-h irradiation period. The fact that the inhibition of iodide transport in cultured thyrocytes was pronounced only 5 d after the start of irradiation indicates that stunning must be initiated earlier. In future studies, the in vitro model will be useful to determine more precisely the kinetics of radioiodide-induced thyroid stunning and its dependence on the absorbed dose for different iodine isotopes.
CONCLUSION
This study shows that incubation with 131I inhibits the transepithelial transport of 125I in cultured porcine thyroid cells. The reduction in transport is not related to possible side effects of stable iodide or loss of damaged cells but is interpreted as a direct effect of radiation on thyroid function. Thus, the results provide in vitro evidence favoring the hypothesis that thyroid stunning is a real phenomenon that must be considered in radiation therapy.
Acknowledgments
The valuable work of Bertil Arvidsson, MSc, and the technical assistance of Therese Carlsson are greatly appreciated. This study was supported by the Swedish National Cancer Society (grants 3682 and 3427) and the Swedish Medical Research Council (grant 537).
Footnotes
Received Jun. 12, 2001; revision accepted Dec. 3, 2001.
For correspondence or reprints contact: Per Postgård, MSc, Department of Radiation Physics, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden.
E-mail: per.postg\|[aring]\|rd{at}radfys.gu.se
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