Various published data sets that investigate the potential effect of exogenous perchlorate (
) on the uptake of iodide in the thyroid and subsequent changes in thyroid hormone levels are available. In order to best use the data towards the prediction of human health effects resulting from
exposure, the available literature data must be integrated into a self-consistent, coherent, and parsimonious quantitative model based on the most likely mode of action of perchlorate effect on thyroid function. We submit that the simplest mode of action for
in the thyroid that remains consistent with all available data involves competitive inhibition of iodide transport into the thyroid follicle, transport of perchlorate into the thyroid follicle against a concentration gradient, further transport into the thyroid lumen (where it may again interfere with iodide transport), and, finally, passive diffusion back into the blood. We believe this description of perchlorate’s kinetic behavior should serve as the foundation for predictive physiologically based pharmacokinetic (PBPK) models and as a working hypothesis for further experimental exploration.
Perchlorate (
) is the dissociation product of the solid rocket fuel ingredient and oxidizer, ammonium perchlorate. The high solubility and the stability of
in water, together with past disposal and handling procedures, have resulted in widespread contamination of drinking water sources (Urban-sky and Shock 1999). Ground and drinking water reserves have been found across the country to range from less than 4 ppb to up to 3700 ppm
in some Las Vegas water samples, though most were less than 20 ppb (Motzer 2001).
blocks the sodium iodide symporter (NIS) from transporting iodide into the thyroid follicle, potentially inhibiting the production of thyroid hormones responsible for control of metabolism and governing several aspects of perinatal development (Wolff 1998). Thus, the risk of long-term health effects from environmental exposures, especially in the case of perinatal development, must be thoroughly explored. The United States Environmental Protection Agency (USEPA) is currently in the process of evaluating the potential human health risk associated with environmental perchlorate exposure in order to recommend safe water concentrations (USEPA 2002).
In order to develop reasonable estimates of risk based on actual target tissue dosimetry, our laboratory has developed a series of physiologically based pharmacokinetic (PBPK) models in the male, pregnant, lactating, fetal, and neonatal rats and the adult human (Clewell et al. 2003a, 2003b, Merrill et al. 2001, 2003). They are mechanistically based, and incorporate changes in physiology and chemical-specific parameters resulting from life stage and species differences. The models are consistent with existing experimental data and can be extrapolated across life stage and species to allow quantitation of
levels in the target tissues (e.g., thyroid, milk) for the population of interest, the perinatal human (Clewell, Merrill, and Robinson 2001).
In order for mathematical models such as these to be useful for predictive purposes, the number of estimated model parameters must be limited. In theory, an unlimited number of models could be constructed to fit a particular set of data if they are allowed to be arbitrarily complex. However, such models are not useful for predictive purposes. Therefore, in accordance with the principle of parsimony, an effort is made to keep the working hypothesis for the model as simple as possible while explaining the available data. The chosen mechanism should therefore limit the number of adjustable parameters and ad hoc assumptions. In this case, we believe the simplest explanation that is consistent with the entire body of data involves competitive inhibition of
and iodide transport into the thyroid follicle by NIS. Thus,
competes with I− for binding sites on the NIS and is then transported into the thyroid follicle against a concentration gradient. Intrathyroidal
is able to interfere with the transport of I− into the lumen, but as
is not bound within the thyroid, the anion eventually diffuses back through the follicle to the blood and is later eliminated through the urine. This three-compartment model for the thyroid perchlorate uptake is shown in Figure 1.
Historically,
has been classified as a competitive inhibitor based on its ability to block thyroid iodide uptake (Wolff and Maurey 1963). Recently, however, some concerns have been expressed over the assumption that
was transferred into the thyroid cell via the NIS (Riedel et al. 2001a, 2001b; Soldin 2002). This concern is primarily based on published electrogenicity studies that showed a bathing medium containing I−caused an inward current in an oocyte with NIS, but when 500 μM
was added to the bathing medium, the inward current disappeared (Eskandari et al. 1997). The authors of this paper presented two theories to explain the disappearance of the current: (1) that
blocks I− transport, but is not transferred into the cell; and (2)
competes with I− and is transferred into the cell by NIS at a 1:1 ratio with Na+.
NIS typically transports two sodium ions (Na+) for every one iodide (I−) and is driven by an inwardly directed Na+ gradient (Eskandari et al. 1997; Kosugi et al. 1996; Riedel et al. 2001a, 2001b; Yoshida et al. 1997). Thus, it follows that a measurable current would be established as the NIS transfers a net positive charge into the thyroid.
, however, though similar in “size” to I−, is sterically quite different; iodide is a single atom, whereas
is a tetrahedral molecule with the negative charge located toward the center. Therefore it is quite possible that the binding of
to NIS would be different than I−, and factors such as steric hindrance, polarizability, and ionic interactions would affect the nature of transport relative to Na+. Although the authors of these studies (Dohan, De la Vieja, and Carrasco 2000; Nilsson 1999; Riedel et al. 2001a, 2001b) apparently favor the explanation that
is not translocated into the cell, they also concluded that
transport by NIS at 1:1 stoichiometric ratio with Na+ remains a viable explanation. Such a stoichiometry would abolish the iodide-driven inward current but maintain the competitive inhibition of iodide uptake. Because these studies do not attempt to quantitatively measure
in the thyroid, they cannot disprove the translocation of
across the follicular membrane. In fact, when the electrogenicity studies are taken in context with the many other studies in literature (Anbar, Guttmann, and Lewitus 1959; Bagchi and Fawcett 1973; Chow and Woodbury 1970; Golstein et al. 1992; Hildebrandt and Halmi 1981; Lewitus, Guttmann, and Anbar 1962; Wolff 1998; Van Sande et al. 2003) and recent experiments (Yu et al. 2001, 2002), the weight of evidence suggests that
is a competitive inhibitor of thyroid iodide uptake, both replacing iodide as a substrate of NIS and being transported by that carrier system into the cell itself.
EVIDENCE FOR PERCHLORATE BINDING TO NIS
Experimental data are consistent with the assumption that
is transported by NIS. The mutual inhibition of
and I− indicates that
inhibits I− transfer by binding to the symporter. Published studies illustrate the competitive nature of this binding by showing that I− and other iodide inhibitors (e.g.,
) also inhibit thyroidal uptake of
(Anbar, Guttmann, and Lewitus 1959; Lewitus, Guttmann, and Anbar 1962; Wolff 1998). Additionally,
is consistently concentrated in tissues with active NIS, including gastrointestinal (GI) contents, skin, mammary gland and milk, in both drinking water and intravenous (IV) studies (Yu et al. 2001, 2002), regardless of age or gender. The presence of NIS in these tissues has been confirmed by the studies of Brown-Grant (1961), Kotani et al. (1998), and Spitzweg et al. (1998). Table 1 presents data collected in our laboratory from drinking water studies in the lactating rat (Yu et al. 2001). When exposed to
in drinking water from gestation day (GD) 2 to postnatal day (PND) 5, maternal tissue:serum ratios are consistently greater than 1, suggesting that a mechanism for active transfer of
is present in each of these tissues. In addition to the data of Table 1, milk:plasma ratios on PND 10 under similar experimental conditions were 2:1 for all doses (Yu et al. 2001). Tissues lacking NIS did not show any accumulation of
. Tissue:serum ratios measured in muscle, fat, and liver were less than 1 (Yu et al. 2002). The fact that
is consistently sequestered only in tissues with NIS suggests that the symporter, and not another anion channel, is responsible for its uptake.
Thyroid uptake of
, like that of I, is subject to up-regulation by thyroid-stimulating hormone (TSH), suggesting that they share the same entry pathway: NIS. Chow, Chang, and Yen (1969) pretreated rats with propylthiouracil (PTU, to block organification of iodide) and TSH (to increase NIS number and activity), prior to dosing with radiolabeled
I−, or a combination of
and I−. The authors found that both
thyroid:blood ratios and percent inhibition of thyroid iodide uptake were increased in rats pretreated with TSH. Similarly, Lewitus, Guttmann, and Anbar (1962) found that thyroid
uptake was increased in the presence of TSH. The fact that thyroid
concentrations are dependent upon changes in symporter activity suggests that
must indeed be transported by NIS.
Finally, the work of Bagchi and Fawcett (1973) showed the effect of
on intrathyroidal iodide to be dependent on extracellular Na+. The authors utilized thyroid cells that were preloaded with 131I− and placed in either a Na+-sufficient or Na+-deficient medium. The addition of
to the medium caused increased 131I− efflux from the thyroid cells within the Na+-sufficient medium, whereas in the Na+-deficient medium,
had no effect on 131I− efflux. Thus, this study is consistent with the hypothesis that Na+-dependent transporter (NIS) is required for the transfer of
to the cell. The observed effect of
on intrathyroidal iodide efflux also provides additional support for the hypothesis that
is actually translocated into the thyroid cell (see below). If perchlorate were irreversibly bound to NIS, not transported into the thyrocyte, there would be no explanation for the increased efflux of 131I− found in this study.
EVIDENCE FOR PERCHLORATE UPTAKE FROM THE SERUM INTO THE THYROID FOLLICLE
Despite recent suggestions to the contrary (Reidel 2001a, Reidel 2001b), most evidence suggests
does not remain bound to NIS and trapped at the follicular membrane, but rather, is transported into the thyroid cell. Although autoradiography has not been used to determine the regions in which radiolabeled
is concentrated in the thyroid, several studies lend support to the hypothesis that the anion is translocated into the follicle. In order to distinguish between alternative explanations for the results of the aforementioned electrogenicity studies, Van Sande et al. (2003) measured the uptake of iodide, pertechnetate, and perrhenate in rat thyroid (FTRL5) cells and COS-7 cells stably transfected with human NIS. Like perchlorate, perrhenate was shown to abolish the inward iodide current reported by Eskandari et al. (1997), which, according to the hypothesis presented by the authors, would suggest that this anion too is not transported into the cell. However, in the in vitro studies of Van Sande et al. (2003), perrhenate was taken up into the cell by both rat and human NIS. Thus, it is apparent that the electrogenicity studies alone do not provide enough information to determine which anions are transferred into the cell via NIS. Despite the lack of direct perchlorate uptake measurements, the work of Van Sande and coauthors (2003) with similar anions (perrhenate and pertechnetate) supports the feasibility of the transfer of
into the cell via NIS. According to the authors, “the evidence that it [
] behaves as its chemical analog perrhenate and pertechnetate is overwhelming. The great chemical similarity among the tetrahedral oxyanions, and the even greater biological similarity detailed in virtually all published work, suggests that perchlorate is handled by the NIS and the thyroid as are perrhenate and pertechnetate.”
Perchlorate has been directly measured in homogenized thyroids at concentrations as much as 30 times that of the serum in rats given
in drinking water (Yu et al. 2002). Rats subjected to acute
exposures also showed thyroid:blood ratios greater than one. Table 2 shows thyroid and serum levels at various time points after acute intravenous exposures at 0.1, 1.0, and 3.0 mg
/kg from the experiments described in Yu et al. (2002).
The relationship of thyroid
concentration to inhibition of iodide uptake from the data of Yu et al. (2002) is shown in Figure 2. If perchlorate were irreversibly bound to the symporter at the follicular membrane, rather than transferred into the thyroid cell, a linear relationship would be expected between the measured iodide inhibition and thyroid perchlorate concentration. Additionally, from the equation of this line, the maximum perchlorate concentration could be calculated from the dose at which 100% inhibition occurs. The nonlinearity of this relationship demonstrates the ability of the thyroid to continue to accumulate
even at levels where iodide uptake is almost completely blocked. Indeed, Chow and Woodbury (1970) measured a thyroid
concentration of 270 mg/kg thyroid in the male rat after an IV dose of 14 mg
/kg body weight (BW), which is nearly an order of magnitude higher than concentrations sufficient to block uptake of iodide by NIS (Figure 2). In contrast, this hyperbolic relationship is completely consistent with expectations for competitive transport.
The work of Hildebrandt and Halmi (1981) also supports the hypothesis that
does, in fact, enter the cell. Similar to Bagchi and Fawcett (1973), who demonstrated the effect of
on the efflux of intrathyroidal iodide, Hildebrandt and Halmi (1981) reported that
affects the utilization of iodine after being taken up into the thyroid cell. The authors administered radioiodide to rats 72 hours prior to
dosing and measured the effect on internal iodide measurements. In the presence of propylthiouracil (PTU) and TSH, significant discharge of the internal iodide was observed as a result of
exposure. The fact that perchlorate is able to affect iodide transfer between the follicle and follicular lumen (Bagchi and Fawcett 1973; Golstein et al. 1992; Hildebrant and Halmi 1981) suggests that
must be present in the thyrocytes.
EVIDENCE FOR PERCHLORATE TRANSPORT FROM THYROCYTES INTO THE FOLLICULAR LUMEN
Several perchlorate studies indicate that perchlorate is not only transferred into the thyroid follicle, but that once in the thyroid cell, the anion is further transported into the follicular lumen. For example, Chow and Woodbury (1970) examined thyroid
time course data taken after an IV dose and observed that there were two apparent phases in the uptake of
into the thyroid; the first being a rapid transport (t
1
/
2 =2 minutes) and the second being much slower (t
1
/
2 =33 minutes). The authors suggest that this first phase represents the rapid transport into the interstitial and cellular compartments and the second phase represents the transport and slow equilibration of
in the luminal fluid. These data, along with histological measurements of the stroma, cell, and luminal volumes, were used to calculate relative
concentrations. This process was repeated for Cl−, which did not show the same accumulation seen with
. From the time course behavior and measured potentials in the cellular compartments, the authors conclude, “
is concentrated in the lumen of the thyroid by a two-step process. Firstly,
is actively transported across the basal cell membrane from the stromal fluid into the cellular fluid. In the cellular fluid,
then passes across the apical cell membrane into the lumen and it is further concentrated during this process” (Chow and Woodbury 1970).
Similar to the uptake data of Chow and Woodbury (1970), newly available data from our laboratory (Yu et al. 2002) showed two distinct phases in the clearance of
from the male rat thyroid after an IV dose. The first phase shows quick elimination, presumably diffusion from the follicle; the second phase is slower, suggesting slow release from a deep compartment (the lumen). When constructing a pharmacokinetic model for
, a three-compartmental thyroid, consistent with the physiological structure (i.e., stroma, follicle and lumen), was required to capture the kinetic behavior of thyroid
(Clewell et al. 2001; Merrill et al. 2003). A two-compartment structure failed to simulate the behavior of
. By adding a third compartment to the thyroid, it was possible to describe the entire time course. The kinetic behavior is well described, assuming rapid transfer between the stroma and follicle, and a slower diffusion between the lumen and follicle (Figure 3).
OTHER INFORMATION PERTINENT TO PERCHLORATE DISPOSITION
Ruling out Analytical Interference in Perchlorate Measurements
Perchlorate has been measured in thyroid and other biological tissues using both radiolabled (
) and cold perchlorate analytical methods. Both methods have confirmed the ability of the thyroid to concentrate perchlorate both in vivo and in vitro (Anbar, Guttmann, and Lewitus 1959; Chow and Woodbury 1970; Chow et al. 1969; Yu et al. 2002). However, in order to ensure the integrity of the measurements, the potential for interference by in vivo metabolism and analytical interference must be addressed. Anbar, Guttmann, and Lewitus (1959) explored the possibility of in vivo metabolism in the rat using double-labeled
and found greater than 99.9% of the
dose unchanged in the urine. Less than 1/1000th of the dose constituted total possible metabolites and radiolabeled impurities, including
and Cl− (Anbar, Guttmann, and Lewitus 1959). Studies in our laboratory show similar findings. Measured cold perchlorate in the urine of adult male rats after an IV
dose showed 90% recovery of the
in the urine within 24 hours of dosing (Yu et al. 2002). Human subjects administered
in drinking water excreted 88% of the ingested dose over 24 hours (Merrill et al. 2001). No metabolites were found in either the rat or human urine samples.
Concerns of whether the measurements of cold perchlorate (Yu et al. 2002) could represent analytical interference by other metabolites rather than the injected anion (
) are ruled out by the selectivity of the analytical method used in these studies (Narayanan et al. 2003). The ion chromatography (IC) method used to measure the anion in the thyroid, serum, urine, and other tissues of the rat easily distinguishes between
and other possible reduction products, such as chlorate (
) and chloride (Cl−), based on both size and charge density. Some anions, such as
, are tightly bound to the exchange medium and remain in the column for longer periods of time. Other anions, such as
, Cl−, F−, and I−, move quickly through the column. Thus, it is possible to distinguish between similar anions with confidence, based on their retention times. The following samples were analyzed for validation of the IC method: water standards spiked with
and
and thyroid samples obtained from rats dosed in vivo with
. One of the thyroid samples was run as a perchlorate-only control; the other was spiked with
ex vivo. The resulting chromatograms are shown in Figure 4. The chromatograms demonstrate that the perchlorate peak is free of interference using this method.
Possible Effect of Perchlorate on Thyroid Iodide Organification
In 1966, Greer, Stott, and Milne published a study in which they exposed rat thyroid lobes to varying concentrations of
in vitro. After allowing the lobes to incubate in the medium for 3 hours, they were rinsed and total 131I− uptake was measured in a well scintillation counter. Paper chromatography was used to determine the relative proportions of the inorganic and organically bound intrathyroidal iodine. As expected,
significantly reduced the total uptake of 131I−. However,
also caused a greater dose-dependent decrease in the relative proportions of organic iodine (iodotyrosines) than the inorganic iodide within the thyroid. Thus, the authors conclude, “organic binding was actually being inhibited rather than being reduced due to a smaller quantity of iodide entering the gland.”
CONCLUSION
When evaluated in light of recently reported pharmacokinetic data (Yu et al. 2001, 2002), the large amount of literature data (Chow and Woodbury 1970; Hildebrandt and Halmi 1981; Wolff 1998) provides substantial information about the effect of
on thyroid iodide uptake and subsequent hormone changes. Such an extensive body of data, though not exhaustive, is useful in forming hypotheses about the mode of action of perchlorate in the thyroid. This mode of action will be the foundation for extrapolation to human exposure scenarios with predictive physiologically based pharmacokinetic models. As such, it is important to ensure that all the essential observations of chemical kinetics are accurately described, with the simplest possible description of the mechanism. In the case of perchlorate, the most parsimonious explanation that remains consistent with the database for perchlorate involves competitive inhibition of
and iodide transport into the thyroid follicle by NIS, transport of
into the thyroid follicle against its concentration gradient, further transport into the thyroid lumen (where
may again interfere with iodide transport) and passive diffusion back into the blood (Figure 1).
The above mode of action explains the following experimental observations:
Short-term perchlorate-induced inhibition of iodide uptake into the thyroid
Mutual inhibition of I− and
uptake into the thyroid and inhibition of
by other competitive inhibitors, such as
Accumulation of
in the thyroid and other NIS-containing tissues against prevailing concentration gradients
Up-regulation of
uptake in the thyroid, paralleling iodide up-regulation, as a result of increases in serum TSH
Increased efflux of intrathyroidal 131I− in the presence of extracellular
inhibition of intrathyroidal iodide transfer from the thyroid follicle to the lumen
Biphasic nature of
uptake and clearance curves into and out of the thyroid (suggesting distribution of
into two compartments of the thyroid: follicle and lumen)
Nonlinearity of iodide inhibition with respect to thyroid
concentration, and the ability of the thyroid to continue sequestering
at serum levels sufficient to inhibit nearly all thyroid iodide uptake
Thus, although the precise mechanism of action for
is not known, the weight of evidence suggests that inhibition of thyroid iodide uptake by
is truly competitive. We believe this description of perchlorate’s mode of action should serve as a working hypothesis for further experimental exploration and for preliminary predictive purposes until such time as it may be validated or contradicted by new experimental evidence.
