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Am J Physiol Endocrinol Metab 294: E802–E806, 2008.
First published February 26, 2008; doi:10.1152/ajpendo.00013.2008.
Thyroid-stimulating hormone increases active transport of perchlorate
into thyroid cells
Neil Tran, Liza Valentı́n-Blasini, Benjamin C. Blount, Caroline Gibbs McCuistion, Mike S. Fenton,
Eric Gin, Andrew Salem, and Jerome M. Hershman
Endocrine Research Laboratory, Veterans Affairs Medical Center West Los Angeles, University of California Los Angeles
School of Medicine, Los Angeles, California; and National Center for Environmental Health, Centers for Disease Control
and Prevention, Atlanta, Georgia
Submitted 7 January 2008; accepted in final form 24 February 2008
by which iodide is concentrated in
the thyroid has been well studied and characterized over the
past 50 years, it was not until 1996 that the Na⫹/I⫺ symporter
(NIS) was cloned (12). NIS functions as the transport system
for transferring iodide from the blood against an electrochemical gradient into thyroid follicular cells. This is the initial step
in the biosynthesis of the iodine-containing hormones thyroxine and triiodothyronine, which are essential for growth, development, and many metabolic reactions (22). NIS has also
been shown to have a high affinity for many ions of similar size
and charge to iodide, such as perchlorate, thiocyanate, nitrate,
bromide, perrhenate, and pertechnetate (31, 34). Many of these
anions, including perchlorate, have been shown to be competitive inhibitors of iodide transport (34).
Perchlorate blocks iodide transport in human thyroid follicular cells in a dose-dependent manner (17, 27) and has been
used pharmacologically to treat hyperthyroidism (33). In cells
transfected with human NIS, NIS had a 30-fold higher affinity
for perchlorate than for iodide (29). NIS is expressed in human
placenta and lactating breast (20, 26) and thus may transport
perchlorate across these tissues, resulting in exposure of the
developing fetus and neonate to perchlorate (4, 19). This has
led to public health concerns because low-level perchlorate
exposure is common in the US population (5) and was associated with increased serum TSH and decreased thyroxine
levels in women with low iodine status in one epidemiological
study (3).
Perchlorate both inhibits iodide uptake by NIS and stimulates iodide efflux from thyroid follicular cells. These processes also are likely to occur in vivo on the basis of the
historical use of the perchlorate discharge test to diagnose
defects in iodine organification. Unlike iodide, however, perchlorate is not metabolized by the thyroid gland (1), and the
mechanism of action of perchlorate on the NIS is not fully clear
(31, 32). There are conflicting results from the electrophysiological research on NIS and anion transport studies in thyroid
tissue (2, 16, 34, 35, 36) in regard to whether or not perchlorate
is transported by NIS.
NIS transports two Na⫹ ions for every one I⫺ ion, generating an inward current; this is the basis for electrophysiological
studies of NIS using Xenopus oocytes (13, 16) and patch
clamping in FRTL-5 cells (36). These methods define a selectivity series for anion transport as follows: I⫺ ⬎ ClO⫺
3 ⬎
⫺
SCN⫺ ⬎ NO⫺
3 ⬎ Br . No measurable current was produced
by the tetrahedral anions perchlorate and perrhenate in the two
studies (16, 36), which led some researchers to the conclusion
that these anions are not transported by NIS (13). This is in
contradiction to previous studies showing that radiolabeled
perchlorate was concentrated in thyroid tissue in vivo (9, 10,
32) and contrasts with the selectivity series based on physio⫺
logical anion transport in thyroid tissue: ClO⫺
4 ⬎ ReO4 ⬎
⫺
⫺
⫺
⫺
SCN ⱖ I ⬎ NO3 ⬎ Br (2, 34, 35). Recently, Dohan et al.
(14) published additional evidence of NIS-mediated active
transport of perchlorate both in vivo and in vitro. Their conclusions are based on indirect techniques for measuring perchlorate and thus need to be confirmed using direct perchlorate
detection methods.
In the present work we used a very sensitive perchlorate
measurement by ion chromatography-electrospray ionizationtandem mass spectrometry to directly measure perchlorate
Address for reprint requests and other correspondence: J. M. Hershman,
Endocrinology-111D, VA Medical Center West Los Angeles, 11301 Wilshire
Blvd., Los Angeles, CA 90073 (e-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
iodide; Na⫹/I⫺ symporter
ALTHOUGH THE MECHANISM
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Tran N, Valentı́n-Blasini L, Blount BC, McCuistion CG, Fenton
MS, Gin E, Salem A, Hershman JM. Thyroid-stimulating hormone
increases active transport of perchlorate into thyroid cells. Am J
Physiol Endocrinol Metab 294: E802–E806, 2008. First published
February 26, 2008; doi:10.1152/ajpendo.00013.2008.—Perchlorate
blocks thyroidal iodide transport in a dose-dependent manner. The
human sodium/iodide symporter (NIS) has a 30-fold higher affinity
for perchlorate than for iodide. However, active transport of perchlorate into thyroid cells has not previously been demonstrated by direct
measurement techniques. To demonstrate intracellular perchlorate
accumulation, we incubated NIS-expressing FRTL-5 rat thyroid cells
in various concentrations of perchlorate, and we used a sensitive ion
chromatography tandem mass spectrometry method to measure perchlorate accumulation in the cells. Perchlorate caused a dose-related inhibition of 125-iodide uptake at 1–10 ␮M. The perchlorate content from
cell lysate was analyzed, showing a higher amount of perchlorate in
cells that were incubated in medium with higher perchlorate concentration. Thyroid-stimulating hormone increased perchlorate uptake in
a dose-related manner, thus supporting the hypothesis that perchlorate
is actively transported into thyroid cells. Incubation with nonradiolabeled iodide led to a dose-related reduction of intracellular accumulation of perchlorate. To determine potential toxicity of perchlorate,
the cells were incubated in 1 nM to 100 ␮M perchlorate and cell
proliferation was measured. Even the highest concentration of perchlorate (100 ␮M) did not inhibit cell proliferation after 72 h of
incubation. In conclusion, perchlorate is actively transported into
thyroid cells and does not inhibit cell proliferation.
ACTIVE THYROID PERCHLORATE UPTAKE
accumulation in FRTL-5 rat cells to test the hypotheses that
perchlorate is actively transported into thyroid cells, that the
process is TSH dependent, and that iodide can block perchlorate uptake by thyroid cells.
MATERIALS AND METHODS
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Perchlorate was analyzed using a modified version of the method of
Valentı́n-Blasini et al. (30). Briefly, lysed cells (⬃100,000 cells) were
spiked with 2 ng of 18O4-perchlorate internal standard. Following a
30-min equilibration at room temperature, the solution was transferred
to a microcentrifuge tube containing a 0.2-␮m nylon filter. The
sample was centrifuged at 16,060 g for 35 min. An aliquot of the
filtrate was transferred to an autosampler vial and subsequently
analyzed using ion chromatography-electrospray ionization-tandem
mass spectrometry. Perchlorate was quantified on the basis of the peak
area ratio of analyte to stable isotope-labeled internal standard. Two
quality control pools were analyzed in each analytical batch with
unknown samples. Reported results met the accuracy and precision
specifications of the quality control/quality assurance program of the
Division of Laboratory Sciences, National Center for Environmental
Health, Center for Disease Control [similar to rules outlined by
Westgard et al. (31a)]. We assessed perchlorate contamination by lot
screening all reagents and analyzing blanks with each batch of
unknowns and identified a background of trace levels of perchlorate in
the medium and reagents. If background perchlorate levels exceeded
the detection limit of 0.05 ng/ml, then the data were corrected for this
small perchlorate background in the medium and NaOH (⬍0.17
ng/ml).
The results presented are representative of at least three replicate
experiments. Iodide and perchlorate uptake data were fit to a curve
using a single-site competitive ligand binding macro in Sigma Plot
v10 (Systat Software, San Jose, CA). TSH response and washout data
were curve fit using the Sigma Plot simple exponential rise to
maximum curve fit.
Measurement of cell growth. FRTL-5 cells were grown to confluence in 6H medium and then maintained in 5H medium for 3 days.
The medium was aspirated, and 200 ␮l of 6H medium containing
various concentrations of ClO⫺
4 was added to quadruplicate wells of
the 96-well plate. After 1–5 days, the perchlorate-containing medium
was aspirated from each well, and growth inhibition due to possible
perchlorate toxicity was tested using Promega’s MTS-CellTiter 96
AQueous One Solution Cell Proliferation Assay (Madison, WI).
RESULTS
Perchlorate is taken up by FRTL-5 cells. Figure 1A shows
that, when cells were incubated with 1 ␮M iodide, the maximal
uptake of radioactive iodide was 42,250 counts䡠min⫺1 䡠well⫺1
when there was no perchlorate present. Addition of perchlorate
caused a dose-related inhibition of iodide uptake. Figure 1B
shows data on perchlorate uptake from a concurrent experiment with identical conditions, except that no 125I was used
because this would contaminate the instrumentation for perchlorate measurement. The amount of perchlorate taken up by
the cells increased with increasing perchlorate in the incubation
medium. The percentage of perchlorate that was taken up by
cells was calculated using the measured amount (in ng) divided
into the expected amount present in the incubation medium
(based on concentration of perchlorate used for each particular
point). The percentage of perchlorate taken up by cells decreased as the concentration of perchlorate in the incubating
medium increased. The perchlorate uptake was 8, 5, and 2% at
perchlorate concentrations of 1, 2, and 10 ␮M, respectively.
Perchlorate uptake is TSH dependent. We investigated the
effect of TSH on iodide and perchlorate uptake. FRTL-5 cells
were grown in 6H medium at usual concentration of 2,000
␮U/ml until ⬃95% confluent. They were then transferred to
12-well plates and grown in the absence of TSH (5H medium)
for 7 days. The cells were then incubated in various concentrations of TSH ranging from 0 to 80 ␮U/ml for 96 h to
stimulate various amounts of NIS expression. Subsequent in-
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Cell culture and reagents. FRTL-5 rat thyroid cells used in all
experiments were generously provided by Dr. Leonard Kohn. FRTL-5
cells were used because no human thyroid cell line has functional NIS.
The only human cell lines that can be maintained in cell culture are
thyroid cancer cells that do not express NIS and do not transport
iodide. Rat and human NIS cDNA share 93% similarity and are
functionally interchangeable. FRTL-5 cells were cultured in Coon’s
modified Ham’s F-12 medium supplemented with six hormones
(bovine TSH, 2 U/l; insulin, 246 U/l; somatostatin, 10 ␮g/l; hydrocortisone, 10 nM; transferrin, 5 mg/l; glycyl-histidyl-lysine, 2.5 ␮g/l),
5% calf serum, and antibiotics (6H medium) as described previously
(23, 25). Cells were maintained in a 5% CO2-95% air atmosphere at
37°C with a change of medium every 2nd or 3rd day and passed every
7 days. TSH is a pituitary hormone that induces NIS expression in
thyrocytes. Cells to be studied in TSH-free (5H) medium were rinsed
once with sterile saline, and the TSH-free medium was changed 3
days before and on the day of test substance addition. Three days was
the minimum time required for TSH-stimulated effects on NIS expression to dissipate.
For experiments measuring uptake of 125I and perchlorate, FRTL-5
cells were transferred to 12-well plates and grown to confluence.
FRTL-5 cells are contact inhibited and thus will not grow upon each
other to physically block access to the medium. In experiments
examining the effect of TSH on iodide and perchlorate uptake,
FRTL-5 cells were grown in 12-well plates using 6H medium until
⬃50% confluence. They were then cultured in 5H medium for ⱖ3
days. When the confluence reached ⬃75%, TSH was reintroduced in
concentrations designed for the particular experiment.
Chemicals, hormones, and reagents were purchased from Sigma.
Iodide uptake assay. Na125I was purchased from Amersham and
diluted for experiments in HBSS to 0.2 ␮Ci/ml. During 1-h incubation, 0.5 ml of this assay buffer was placed in each well so that each
well contained 0.1 ␮Ci of Na125I. The concentrations of KClO4 and
NaI were modified depending on the particular objective of each
experiment.
The 6H medium was removed from each well in 12-well plates, and
the cells were rinsed once with 1 ml of HBSS (previously heated
to 37°C). After removal of HBSS rinse, 1 ml of the assay buffer
containing 125I, perchlorate, and/or sodium iodide, as specified in each
experiment, was added to each well. Cells were then incubated in
37°C water bath for 60 min.
The radioactive assay buffer was removed, and cells in each well
were rinsed twice, each with 1 ml of ice-cold HBSS, as quickly as
possible. The duration of cell contact to ice-cold HBSS was ⬍30 s to
avoid release of trapped iodide from cells. After the last rinse, 0.5 ml
of 0.5 M NaOH was added to each well and cells were incubated at
room temperature for 30 min. The NaOH acts as a cell lysate,
breaking cell membranes and releasing trapped radioiodide into the
NaOH solution. The entire volume (0.5 ml) was placed in a sample
tube, and the 125I was counted in a ␥-well counter. Total radioactivity
of cells in each well was expressed as counts per minute per well.
Each concentration was run in triplicate, and the uptake was calculated as an average of those three wells.
Perchlorate assay. For the purpose of analysis of perchlorate
content in each well, an additional set of triplicate wells underwent
exactly the same procedure as described above, but the Na125I was
omitted. The cell pellets were lysed with NaOH and stored frozen at
⫺20°C in plastic-stoppered tubes until shipment. Samples were
shipped on dry ice by overnight courier to the Center for Disease
Control’s National Center for Environmental Health in Atlanta, GA.
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ACTIVE THYROID PERCHLORATE UPTAKE
DISCUSSION
Fig. 1. A: iodide uptake as a function of perchlorate concentration in incubation
medium with constant 1 ␮M sodium iodide. All cells were grown in 6H medium.
Error bars represent ⫾1 SD. B: perchlorate content in cell lysate as a function of
perchlorate concentration in incubation medium. Error bars represent ⫾1 SD.
cubation of these cells with 125I-labeled iodide, no nonlabeled
iodide, and 1 ␮M perchlorate indicated a dose-related increase
in iodide uptake (Fig. 2A) and in perchlorate uptake (Fig. 2B),
respectively, consistent with NIS-mediated transport of these
anions.
Perchlorate is displaced by nonradioactive iodide in a
dose-dependent fashion. We examined the effect of various
concentrations of nonradioactive (“cold”) iodide on the uptake
of radioactive iodide and perchlorate by FRTL-5 cells. Cells
were grown in 6H medium until near confluence and then in
medium with 500 ␮U TSH/ml, 1 ␮M perchlorate, and various
amounts of iodide for 48 h. As iodide concentration of the
incubation medium increased, there was a reduction in radioiodide uptake (Fig. 3A) as well as in the amount of perchlorate
taken up by FRTL-5 cells (Fig. 3B).
Perchlorate effect on cell proliferation. To evaluate the
toxicity of perchlorate on thyroid cells, the effect of perchlorate
on cell proliferation was studied using the MTS-CellTiter 96
AQueous One Solution Cell Proliferation Assay. Incubation of
the FRTL-5 cells with 10 nM to 100 ␮M perchlorate for 24 –72
h did not reduce cell proliferation (Fig. 4). In another study,
incubation with similar amounts of perchlorate for 5 days did
not significantly reduce cell proliferation (data not shown).
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Fig. 2. A: iodide uptake as a function of TSH concentration. B: perchlorate
content in cell lysate as a function of TSH concentration. Error bars
represent ⫾1 SD.
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The results show that perchlorate is actively transported into
FRTL-5 thyroid cells by a TSH-dependent process, that the
amount of perchlorate taken up increases with the concentration of perchlorate in the medium and is saturated at high
perchlorate concentration, and that the perchlorate uptake is
blocked by iodide in a dose-related manner. These results
support the hypothesis that perchlorate is actively transported
into the thyroid cell; this active transport is most likely mediated by NIS. However, our data do not exclude the possibility
that another transporter besides NIS may contribute to the
transport of perchlorate. Based on the data shown in Fig. 2, a
mean cell count of 5.0 ⫻ 105/well and a mean thyroid cell
volume of 3.73 pl (8), the calculated cell/medium ratio was 62
at the TSH-induced maximum uptake of perchlorate. Our
results are consistent with studies of perchlorate uptake using
36
ClO⫺
4 in rats and guinea pigs showing that the isotope was
concentrated in the thyroid tissue and that the concentration
was TSH dependent (9, 21).
Electrophysiological studies showed that perchlorate did not
elicit any current and that perrhenate elicited only a minor
current in Xenopus laevis oocytes transfected with rat NIS,
suggesting that these anions were not transported (16). In an
ACTIVE THYROID PERCHLORATE UPTAKE
elegant study using both FRTL-5 cells and COS-7 cells transfected with human NIS, Van Sande et al. (31) showed that
there was active transport of perrhenate. Their studies of the
inhibition of transport by various anions suggested that perchlorate and perrhenate were similar in potency. They suggested that the “explanation for the discrepancy between the
electrophysiological and tracer uptake measurement of tetrahedral oxyanion transport would be that the symporter would
have equal stoichiometry for Na⫹ and these anions, i.e., that
this transport would be electrically neutral” (31). NIS-mediated
active vectorial transport of perchlorate was recently demonstrated by Dohan et al. (14) using polarized NIS-expressing
Madin-Darby canine kidney cells in a bicameral setup. Studies
of 186ReO⫺
4 in this elegant model showed that its transport
stochiometry was electoneutral, suggesting that Na⫹/ClO⫺
4
transport is also electroneutral (14). By directly measuring
perchlorate accumulation in thyroid cells for the first time, we
confirm physiological studies that inferred active perchlorate
transport into thyroid cells rather than perchlorate’s being
bound to NIS and functioning only as an inhibitor of iodide
transport. A possible basis for the electrical neutrality is that
AJP-Endocrinol Metab • VOL
each of the two sodium ions cotransported with iodide are
bound by different regions of the symporter and that the
symporter functions even when only one sodium ion is cotransported. Additionally, our findings agree with the conclusions of
Clewell et al. (11), who believed that the weight of evidence
favors active transport of perchlorate into the thyroid follicle
against a concentration gradient.
Although perchlorate is an excellent oxidizer under some
conditions, the activation energy required to initiate these
chemical reactions is high. This high activation energy may
explain why humans do not metabolize perchlorate; instead,
humans rapidly excrete perchlorate in urine with a t1/2 of ⬃8 h
(17). Our data show that even a concentration of 100 ␮M,
50-fold greater than that which will markedly inhibit iodide
uptake, is not cytotoxic in cultured FRTL-5 cells.
Although a rat cell line was used for the present study, we
believe the results are applicable to humans. The cDNA that
encodes human NIS exhibits an 84% identity and 93% similarity to rat NIS (26). The transcriptional regulation of human
and rat NIS in thyroid cells is also very similar (20). Accordingly, our results are likely to be entirely applicable to human
thyroid cells in regard to perchlorate uptake.
The principal concern regarding perchlorate exposure of
humans is inhibition of thyroidal iodide uptake, possibly resulting in reduced synthesis of thyroid hormone. Because
trace-level perchlorate exposure is common in the US population, there is considerable public and governmental interest in
this issue (18). An epidemiological analysis of the National
Health and Nutrition Examination Survey (NHANES) data has
reported that background perchlorate exposure levels are associated with changes in thyroxine and TSH concentrations in
women with low iodine status (3). Further analysis of the
NHANES data found that the combination of exposure to
perchlorate and cigarette smoke is associated with decreased
thyroxine and increased TSH levels, consistent with inhibition
of NIS-mediated iodide uptake (28). These published findings
are consistent with our results; active transport of perchlorate is
modulated by the presence of other anions, such as iodide.
Additionally, our data suggest that the NIS expressed in pla-
Fig. 4. Effect of various concentrations of perchlorate on FRTL-5 cell proliferation.
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Fig. 3. A: iodide uptake as a function of nonradioactive iodide concentrations
all in 1 ␮M of perchlorate. Error bars represent ⫾1 SD. B: perchlorate content
in cell lysate as a function of nonradioactive iodide concentrations. Error bars
represent ⫾1 SD.
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ACTIVE THYROID PERCHLORATE UPTAKE
cental tissue could transport perchlorate across the placental
barrier and lead to increased exposure to the developing fetus.
Further research is needed to assess the toxicological implications of NIS expression in both placental and mammary tissue
(11a) and the modulation of perchlorate transport by iodide.
Active transport of perchlorate across membranes is also consistent with the active secretion of perchlorate in human and
bovine milk (7, 15, 19, 24).
In conclusion, our data show that perchlorate is actively
transported into FRTL-5 cells by a TSH-stimulated process and
that iodide in high concentrations is a competitive inhibitor of
perchlorate uptake.
ACKNOWLEDGMENTS
GRANTS
This study was upported by a research grant from the University of
California Toxic Substances Research and Training Program and Veterans
Affairs Merit Research Funds.
The findings and conclusions in this report are those of the authors and do
not necessarily represent the views of the Centers for Disease Control and
Prevention.
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