Predicting Fetal Perchlorate Dose and Inhibition of Iodide Kinetics during Gestation: A Physiologically-Based Pharmacokinetic Analysis of Perchlorate and Iodide Kinetics in the Rat

Rebecca A. Clewell*,1, Elaine A. Merrill{dagger}, Kyung O. Yu{ddagger}, Deirdre A. Mahle§, Teresa R. Sterner{dagger}, David R. Mattie{ddagger}, Peter J. Robinson§, Jeffrey W. Fisher{ddagger},2 and Jeffery M. Gearhart§

* Geo-Centers, Inc., Wright-Patterson AFB, Ohio 45433; {dagger} Operational Technologies Corp., Dayton, Ohio 45432; {ddagger} AFRL/HEST, Wright-Patterson AFB, Ohio 45433; and § Mantech Environmental Technology, Inc., Dayton, Ohio 45437

Received November 20, 2002; accepted March 3, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Perchlorate (ClO4-) disrupts endocrine homeostasis by competitively inhibiting the transport of iodide (I-) into the thyroid. The potential for health effects from human exposure to ClO4- in drinking water is not known, but experimental animal studies are suggestive of developmental effects from ClO4- induced iodide deficiency during gestation. Normal hormone-dependent development relies, in part, on synthesis of hormones in the fetal thyroid from maternally supplied iodide. Although ClO4- crosses the placenta, the extent of inhibition in the fetal thyroid is unknown. A physiologically-based pharmacokinetic (PBPK) model was developed to simulate ClO4- exposure and the resulting effect on iodide kinetics in rat gestation. Similar to concurrent model development for the adult male rat, this model includes compartments for thyroid, stomach, skin, kidney, liver, and plasma in both mother and fetus, with additional compartments for the maternal mammary gland, fat, and placenta. Tissues with active uptake are described with multiple compartments and Michaelis-Menten (M-M) kinetics. Physiological and kinetic parameters were obtained from literature and experiment. Systemic clearance, placental-fetal transport, and M-M uptake parameters were estimated by fitting model simulations to experimental data. The PBPK model is able to reproduce maternal and fetal iodide data over five orders of magnitude (0.36 to 33,000 ng/kg 131I-), ClO4- distribution over three orders of magnitude (0.01 to 10 mg/kg-day ClO4-) and inhibition of maternal thyroid and total fetal I- uptake. The model suggests a significant fetal ClO4- dose in late gestation (up to 82% of maternal dose). A comparison of model-predicted internal dosimetrics in the adult male, pregnant, and fetal rat indicates that the fetal thyroid is more sensitive to inhibition than that of the adult.

Key Words: PBPK model; pregnancy; perchlorate; iodide; thyroid; inhibition; fetal dose.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent findings of perchlorate (ClO4-) contamination in groundwater and drinking water sources have resulted in widespread concern over the potential health effects from long-term ingestion of low-level perchlorate via drinking water (Motzer, 2001Go; Urbansky, 1998Go; Urbansky and Shock, 1999Go). Most drinking water sources reported to contain perchlorate appear to have concentrations less than 20 ppb, although concentrations as high as 3700 ppm have been reported in some Las Vegas ground water samples (Motzer, 2001Go). Perchlorate interferes with thyroidal iodide uptake by binding to the sodium iodide symporter (NIS) at the basolateral membrane of the thyroid follicular endothelial cell, resulting in decreased iodide uptake and decreased synthesis of thyroid hormones. However, hormone homeostasis is usually regained through upregulation of NIS via the hypothalamus-pituitary-thyroid (H-P-T) feedback mechanism (Wolff, 1998Go). Nevertheless, due to the identification of contaminated water sources, and the potential for interaction with thyroid iodide uptake in sensitive human subpopulations, the Environmental Protection Agency is currently in the process of evaluating human risk in order to recommend a safe water concentration of ClO4- (USEPA, 2002Go).

The endocrine system maintains a delicate balance of hormonal and inorganic iodide through the H-P-T feedback system, wherein the hypothalamus responds to diminished serum hormone levels by increasing production of thyrotropin releasing hormone (TRH). TRH then signals the pituitary to release thyroid stimulating hormone (TSH), which in turn increases NIS levels in the thyroid and downregulates hormone deiodination by decreasing synthesis of thyroid peroxidase (TPO). The NIS is responsible for transporting inorganic iodide (I-) into the thyroid follicular cell for the synthesis of thyroid hormones, particularly thyroxine (T4) and triiodothyronine (T3; Wolff, 1998Go).

In an effort to quantitatively predict ClO4-, I-, and inhibition kinetics in response to both acute and subchronic ClO4- exposure, we have developed a physiologically-based pharmacokinetic (PBPK) model in the adult male rat (Merrill et al., 2003Go). Because the PBPK model is based on physiological, biochemical, and mechanistic data, it allows us to determine target site dosimetry and in vivo chemical interactions (inhibition) while improving confidence in extrapolation between dose levels, exposure durations and routes. However, with ClO4-, as with other potential endocrine disruptors, the primary concern is not necessarily potential risk to the adult, but rather the possible developmental effects resulting from perinatal exposure. During gestation and early infancy, a critical window exists in which thyroid hormones are needed for normal physical and mental development (Bakke et al., 1976Go; Howdeshell, 2002Go; Myant, 1971Go; Porterfield, 1994Go). The most susceptible developmental periods to thyroid perturbations are not known. However, even short-term iodide deficiency in the perinatal period can result in lifelong consequences. In the human, gestational iodide deficiency has been linked to increased incidence of stillbirth, congenital abnormalities, lowered IQ, mental retardation, and impaired hearing (Delange, 2000Go; Haddow et al., 1999Go; Klein et al., 1972Go; Porterfield, 1994Go). In the rat, gestational and neonatal hypothyroidism can result in brain cell disorganization and delayed onset of puberty and estrus (Bakke et al., 1976Go; Clos et al., 1974Go).

Although the roles of maternal versus fetal hormone in fetal development are currently not well-defined, it appears that both maternal and fetal hormone production is necessary to carry out normal development (Howdeshell, 2002Go). As early as gestation day (GD) 9, before the onset of fetal thyroid function, maternal T3 and T4 have been detected in the rat fetus (Morreale de Escobar et al., 1985Go). The fetal thyroid begins to sequester iodide and synthesize and secrete hormones by gestation week 12 in the human (Roti et al., 1983Go) or GD 17–20 in the rat (Eguchi et al., 1980Go; Geloso, 1961Go; Nataf and Sfez, 1961Go), while the necessary iodide is obtained from the mother via placental transport (Brown-Grant, 1961Go; Roti et al., 1983Go). Maturation of the fetal thyroid results in a significant increase in total thyroid hormone levels in the fetus between GD 18 and birth (Howdeshell, 2002Go), indicating that fetal thyroid hormone production may play a significant role in maintaining total serum hormone levels in late gestation. Since thyroid hormones regulate various aspects of brain development throughout gestation (Howdeshell, 2002Go), it is important to determine possible perturbation of thyroid activity, be it maternal or fetal. Because ClO4- affects maternal hormone production, placental iodide transfer, and fetal hormone production, it could present a potential concern for fetal development at any time in gestation.

In order to determine the sensitivity of the developing fetus to perchlorate, it is necessary to associate an observed effect with a measured fetal ClO4- dose. However, the lack of data in both rats and humans makes the calculation of a human fetal ClO4- dose from maternal drinking water measurements nearly impossible by classical methods. In this article, we propose the use of a PBPK model to predict both fetal ClO4- dose and subsequent iodide kinetic changes in the dam and fetus after maternal exposure. The developed PBPK model accounts for changing physiology and maternal-fetal transfer during gestation and is able to predict both ClO4- and radioiodide kinetics in the pregnant and fetal rat, as well as ClO4- induced inhibition of iodide kinetics. Together with the model predictions for male rat kinetics (Merrill et al., 2003Go), this model allows for a more quantitative comparison of life stage differences (Clewell et al., 2001Go). Additionally, further extrapolation of this model to human ClO4- exposure could allow the fetal risk to be estimated in the absence of direct human data (Clewell et al., 2001Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Supporting Experiments
All in-house experiments were performed with timed-pregnant dams of the Sprague-Dawley strain (Crl: CD, Charles River Laboratory, Raleigh, NC). The animals used in in-house studies were handled in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 1996, and the Animal Welfare Act of 1966, as amended. GD 1 was verified by the presence of a vaginal plug. In all studies pregnant rats were housed individually in light, heat, and humidity controlled cages. Rats were kept on a 12 h light/dark cycle with access to food and water ad libitum. All rats from in-house studies (control and ClO4-/I- dosed groups) were given the same diet (Purina #5000) containing 0.8 ppm iodine. Maternal drinking water consumption was measured daily and perchlorate drinking water levels were adjusted when necessary to ensure the accuracy of dosing levels in the drinking water studies. On GD 20, dams and fetuses were euthanized by CO2 asphyxiation.

Perchlorate drinking water study.
Dams (n = 6 per dose group) were exposed to drinking water treated with perchlorate from GD 2 through 20, at doses of 0.0, 0.01, 0.1, 1.0, and 10.0 mg ClO4-/kg-day. Two hours prior to euthanization, dams were given an iv dose of 33 µg/kg 125I- with carrier (trace level radioiodide mixed with nonradiolabeld iodide). Maternal and fetal serum samples were analyzed for free and total thyroxine (fT4 and tT4), T3, and TSH. ClO4- was also analyzed in the maternal and fetal serum, skin and gastrointestinal (GI) tract, as well as maternal thyroid, GI contents, and placenta following the methods described by Fisher et al.(2000)Go. The same tissues were also analyzed for 125I- with a gamma counter.

Radioiodide and inhibition kinetic studies.
Timed-pregnant Sprague-Dawley dams were exposed via tail vein injection to a tracer dose of 125I- (average dose = 2.19 ng/kg body weight [BW]) on GD 20. Dams (n = 6) were euthanized at 0.5, 1, 2, 4, 8, 12, and 24 h postdosing. Maternal and fetal serum, skin, and GI tract, as well as maternal thyroid, GI content, placenta, and mammary gland tissue were collected and analyzed for 125I- with a gamma counter. Fetal serum was pooled by litter due to small sample volume while fetal skin and GI tract were analyzed individually. Inhibition kinetics were examined in this same manner; however, 2 h prior to the administration of 125I- (average dose = 1.87 ng/kg BW), dams from the inhibition group were dosed with 1.0 mg/kg BW ClO4- via tail vein injection. This particular ClO4- dose was chosen to be large enough dose to significantly affect iodide uptake, based on inhibition in the male rat (Merrill et al., 2003Go), while being lower than the dose required to saturate the symporter, based on the drinking water study results (see Results section below). Euthanization was performed at 0.5, 1, 2, 4, 8, 12, and 24 h post-125I- dosing.

Model structure.
All model code was written in ACSL (Advanced Continuous Simulation Language, Aegis Technologies Group, Inc., Huntsville, AL). Several classical mathematical models exist for iodine kinetics in both humans and rats (Berman et al., 1968Go; DiStefano et al., 1982Go; Hays and Wegner, 1965Go). However, since these models are not physiologically based, they are limited in their usefulness for extrapolation across species and life stage. The PBPK model proposed here supports these types of extrapolations by accounting for physiological differences. The maternal models for both I- and ClO4- consist of compartments for plasma, thyroid, skin, gut, kidney, liver, fat, mammary gland, and placenta, plus two lumped compartments for the remaining slowly and richly perfused tissues. The thyroid and gut are described with three subcompartments representing the stroma, follicle, and colloid in the thyroid, and the capillary bed, GI tract, and GI contents in the gut. Skin, placenta, and mammary gland are described with two subcompartments, representing the capillary bed and tissue. Active uptake into the thyroid follicle and colloid, as well as the skin tissue, mammary gland, GI contents, and placenta, was described with Michaelis-Menten (M-M) terms for saturable processes (bold arrows in Fig. 1Go). Permeability area cross-products (PA) and partition coefficients were used to describe the passive movement of the anions (I- and ClO4-) between the capillary bed, tissue, and inner compartments (small arrows in Fig. 1Go), which results from the inherent electrochemical gradients within these tissues (Chow et al., 1969Go). The flow-limited kidney, liver, and fat compartments were described using partition coefficients and blood flows. Urinary clearance and transfer of anions between the placenta and fetal serum were represented by first order clearance rates. Binding of ClO4- to plasma proteins was described with a saturable term for association of the ClO4- anions to binding sites in the plasma and a first order clearance rate for dissociation from plasma binding sites. However, unlike ClO4- (Merrill et al., 2003Go), the majority of iodide in the serum is not bound to proteins (Yu et al., 2002aGo,bGo). Thus, binding of I- to plasma proteins was not included in the model.



View larger version (36K):
[in this window]
[in a new window]
 
FIG. 1. Schematic of PBPK model for pregnant dam (left) and fetus (right). Note: plasma compartment includes protein binding of ClO4-, not I-.

 
Given that the majority of thyroid iodide is organified shortly after transport into the follicle, it was necessary include a simplified description of the incorporation of iodide into thyroid hormones and hormone precursors (monoiodothyronine, diiodothyronine), as well as the secretion of these hormones from the maternal thyroid. However, since the purpose of this present model is primarily to determine perchlorate distribution and resulting inhibition of free iodide transfer by NIS, it was possible to simplify the model by assuming that the radioiodine behaved as inorganic iodide in all compartments other than the thyroid, including serum. In the thyroid compartment, the partitioning and active transport mechanisms account only for the movement of free iodide. Hormone production was modeled as a first order production rate from the thyroid follicle to an "incorporated iodine" compartment (ClProdi). Clearance of this incorporated iodine from the thyroid was then described with a first order clearance rate from the incorporated iodide compartment into the serum (ClSecri). Due to the dynamic nature of the fetal thyroid, the uncertainty in hormone production kinetics, and the lack of supporting data, a description of hormone production was not included in the fetal rat on GD 17–20. Rather, total iodine in the fetal thyroid was modeled using active uptake and diffusion from the serum.

The perchlorate anion was modeled in the same manner as inorganic iodide, based on the similar size and charge of the ions and their shared affinity for NIS. The thyroid, skin, GI, placenta, and mammary glands contain active NIS and were therefore defined separately in the structure of the model (Kotani et al., 1998Go; Spitzweg et al., 1998Go). The thyroid, skin, and GI contents maintain higher concentrations of ClO4- and I- than the plasma (Brown-Grant and Pethes, 1959Go; Chow et al., 1969Go; Halmi and Stuelke, 1959Go; Wolff, 1998Go; Zeghal et al., 1995Go), requiring active transport mechanisms to work against the concentration gradient. Although other tissues, such as the salivary gland, ovary, and choroid plexus, are also known to sequester iodide and perchlorate in the rat and human (Brown-Grant, 1961Go; Honour et al., 1952Go; Spitzweg et al., 1998Go), small amounts of the anions in these tissues do not affect plasma concentrations. Therefore, these tissues were lumped together with the richly and slowly perfused compartments.

In addition to the reported presence of NIS, studies in our laboratory have shown the uptake of iodide in placental tissue to be inhibited by ClO4- (Mahle et al., 2002Go). Thus, it was necessary to include a description of NIS in the placental compartment to account for ClO4- and I- concentrations and the measured ClO4- induced inhibition of placental iodide uptake. The mammary gland has also been shown to concentrate both anions during lactation and is known to contain NIS, which is regulated by hormones produced during lactation (Tazebay et al., 2000Go). Intralaboratory studies found mammary gland:plasma ratios of less than one for ClO4- and I- during gestation. However, time-course data indicate that mammary gland levels remain elevated well into the clearance phase of the serum. In order to maintain these elevated tissue concentrations, it was necessary to include symporter activity in the mammary gland of the pregnant rat.

The kidney and liver were also separately defined in the structure of the model to describe the rapid urinary clearance of the anions and to allow future elaboration of the model to address hormone metabolism in the liver. A compartment was also included to account for the effect of changing fat volume on the kinetics of hydrophilic anions during gestation. Since kidney, liver, and fat do not maintain tissue:plasma ratios greater than one for either anion, these tissues were described as single, flow-limited compartments and do not contain terms for active uptake. Effective partitioning into these compartments is thought to result from the electrochemical gradient that moves ClO4- from serum to tissue, as in the thyroid (Chow and Woodbury, 1970Go).

Plasma binding of perchlorate was included in the model, in order to simulate the relatively high serum ClO4- concentrations observed in our laboratory at low administered doses (Fig. 2Go). This binding has been measured in human, bovine (Carr, 1952Go; Scatchard and Black, 1949Go), and rat serum (Merrill et al., 2003Go). At low serum concentrations (<=100 µg/l), Fisher reported approximately 99% of the anion is bound to plasma proteins and at higher concentrations (>=500 µg/l), 50% is bound (Fisher, 2002, as cited in Merrill et al., 2003Go). This plasma binding of perchlorate is also evidenced by the ability of the anion to interfere with T4 binding to serum albumin and pre-albumin rats as it does in humans (Shishiba et al., 1970Go; Yamada, 1967Go). These studies suggest that perchlorate inhibits binding of T4 to albumin by reversibly binding to the albumin via weak covalent interactions.



View larger version (42K):
[in this window]
[in a new window]
 
FIG. 2. Perchlorate concentration in maternal (a) serum (Clewell et al., 2001Go), (b) thyroid (upregulated), (c) GI contents, and (d) skin as well as fetal (e) serum (Clewell et al., 2001Go) and (f) skin at the 0.01, 0.1, 1.0, and 10.0 mg/kg-day dose on GD 20.

 
The structure of the fetal perchlorate model is similar to that of the pregnant rat, with the exception of the mammary gland and placenta compartments. In order to simplify the model, all fetuses from a single litter were lumped together within the model structure, essentially viewing all individual fetuses as one entity. Although there is evidence that delivered fetal dose of some toxins may vary due to position within the uterine horn, the difficulty of chemical analysis and the small sample volume obtained from the GD 20 fetus preclude the possibility of analyzing serum for each individual fetus. Thus, the model attempts to predict the available data, which consists of pooled fetal samples. Though this may present a crude estimate of a particular individual fetus, the model is able to provide a reasonably accurate estimate of the average fetal dose, thereby enabling risk estimates to be made from dosimetry in the subject of interest, the fetus, rather than the less comparable but more available adult rat. The model description of fetal dose is based on transfer of the anions between the placenta and fetal serum. Although a kidney compartment is included in the fetal model, urinary excretion is not used to identify the loss of perchlorate for the fetus, as the ability to produce urine is not well developed until after parturition. Loss of ClO4- and I- from the fetus was described as clearance from the fetal serum to the placenta. The anions are then able to diffuse into the maternal serum and are, therefore, available for redistribution in the dam.

Perchlorate inhibition of iodide uptake was included in the maternal thyroid follicle and colloid, GI contents, skin and placenta, as well as the fetal GI contents and skin throughout gestation. Inhibition in the fetal thyroid follicle and colloid was also included from the onset of fetal thyroid iodide accumulation (GD 17) through parturition, based on the observation that perchlorate was present in the fetal blood and could, therefore, inhibit fetal NIS transport of iodide into the thyroid. Placental inhibition was observed in studies in our laboratory (Mahle et al., 2002Go) and was included in the model in order to predict the effect on iodide transfer to the fetus. Literature sources have reported inhibition in gastric juice of the male rat (Halmi and Stuelke, 1959Go) and intralaboratory studies showed consistent evidence of significant (>60%) inhibition of iodide uptake in both the fetal GI and skin, and slight inhibition in the maternal skin (Mahle et al., 2002Go). Since the release of iodide from these extrathyroidal tissues could affect the serum levels and the amount of stored iodide in the fetus, it was necessary to include iodide inhibition in the tissues with active uptake.

An additional description of thyroid perchlorate inhibition by iodide was determined to be unnecessary, as it would not affect perchlorate thyroid levels. Although both anions are transported with NIS and would therefore inhibit the other’s ability to bind to the symporter, the fact that the Km for ClO4- is an order of magnitude lower than that of I- indicates that ClO4- has a much greater affinity for NIS. Additionally, even when considering dietary iodide, the relative intake of perchlorate in the drinking water dosing scenarios (on the order of mg/kg-day) is much greater than that of the trace element iodide (on the order of µg/kg-day). Competitive inhibition of ClO4- by I- would be modeled by adjusting the Kmp by one plus the ratio of serum iodide concentration to the Km for iodide. Because the Kmi is large (4 x 106 ng/l) and the endogenous serum iodide concentration (roughly 4000 ng/l) is approximately 1000 times smaller than Km, it is apparent that the effect of iodide on thyroid ClO4- will be negligible.

Dosing procedures.
In order to simulate the daily dosing regimen of the drinking water experiment, rats were modeled as drinking at a constant rate for 12 of the 24 h per day (1800 to 0600 hours). A pulse function in ACSL was used to introduce drinking water to the GI contents of the dam for the first 12 h of each 24-h period. Intravenous dosing was introduced into the venous blood compartment of the maternal model.

Model parameters.
Whenever possible, physiological and kinetic parameters were obtained from literature or experiments. Allometric scaling was employed when needed to account for differences in parameters due to variations in body weight of male, female, and fetal rats. Tissue volumes were scaled linearly by body weight. Blood flows, maximum velocities, permeability area cross products (PA), and clearance values were scaled by BW0.75. Fetal values were scaled in a similar manner to the maternal parameters; however, it was necessary to scale the individual fetal values first and then adjust for the total number of fetuses in the litter.

Physiological parameters.
The physiological description of maternal and fetal rats during gestation is based on O’Flaherty et al.(1992)Go, Gentry et al. (2002)Go, and Fisher et al.(1989)Go. The model is able to incorporate differences in gestation time, pup birth weight, and litter size between experiments and strains of rats. Physiological parameters and the sources from which they were obtained are listed in Table 1Go.


View this table:
[in this window]
[in a new window]
 
TABLE 1 Physiological Parameters
 
The maternal body weight (BW) increases significantly during the relatively short gestation time in the rat (21 days), with the majority of weight resulting from increasing volumes of the placenta, mammary gland, fat, and total fetal volume. Therefore, the total change in maternal body weight was described as the sum of the changes in these four tissue volumes and the initial (prepregnancy) body weight (BWinit). All other maternal organs were assumed to remain constant throughout gestation.

Mammary tissue (VM) growth was described as a linear process, reaching a maximum volume of 4.6% BW at GD 21 (Hanwell and Linzell, 1973Go; Knight and Peaker, 1982Go). Maternal fat (VF) was also modeled with a linear increase during gestation (Naismith et al., 1982Go), with a 40% increase from the nonpregnant value (7.0% BW) for Sprague-Dawley rats (Brown et al., 1997Go). Placental volume (VPl) was described in the model as a sum of three stages of growth, involving changes in both the yolk sac and chorioallantoic placenta, based on the model of O’Flaherty et al.(1992)Go and data from Buelke-Sam et al.(1982)Go and Sikov and Thomas (1970)Go.

A three-stage description of fetal growth was also described in O’Flaherty et al.(1992)Go to mathematically reproduce several data sets (Beaton et al., 1954Go; Buelke-Sam et al., 1982Go; Goedbloed, 1972Go; Sikov and Thomas, 1970Go). In the absence of data for fetal volumes before GD 11, an exponential growth curve was used to approximate early embryonic growth, starting at zero liters and intercepting the first available data points at GD 11. The final equation for fetal growth is dependent on the weight of the pup at the time of birth (PupBW), allowing the model to be adjusted for variations in birth weight of different rat strains.

Fetal GI, liver, kidney, and thyroid weights given for the Wistar rat (Schneidereit, 1985Go) on days 17 through 21 of gestation and skin volumes given for days GD 19 through PND 1 (Palou et al., 1983Go) were fit to exponential functions. These equations were then used in the model to describe organ growth versus fetal body weight over the course of gestation. Since organ weights are not available for earlier time points in gestation, the best-fit exponential curve to the above data was used to extrapolate to earlier time points in fetal development.

In accordance with the reported values of Schneidereit (1985)Go, the fetal GI and kidney increased from 2.0 and 0.3 to 3.0 and 0.44% BW, respectively, from GD 17–21. In the same time period, the relative volumes of the fetal liver and thyroid decreased (with respect to body weight) from 8.5 and 0.058 to 7.15 and 0.038% BW. Fetal skin relative volume increased more than threefold in the last three days of gestation, comprising 8.8, 13.6, and 19.3% of the body weight on GD 19, 21, and PND 1 (Palou et al., 1983Go). Volume fractions of fetal stroma (VTSfet), follicle (VTFfet), and colloid (VTLfet) were also significantly different than those of the dam (see Table 1Go) and were given values reported for the rat at birth (Conde et al., 1991Go). Fetal body fat (VFfet) was assumed to be negligible for the purpose of the model, based on the work of Naismith et al.(1982)Go, who found that two-day-old rats contained only 0.16% fat and that body fat quickly increased in the neonatal period.

Temporal changes in maternal cardiac output during gestation are described in the model as the sum of initial cardiac output (Brown et al., 1997Go) and the change in blood flow to the placenta, mammary and fat tissues, per the approach of O’Flaherty et al.(1992)Go, which employs changing blood flows in the placenta, mammary gland, and fat. The fraction of maternal cardiac output to the mammary gland, fat, and yolk sac change proportionally to the change in tissue volumes. Blood flow to the chorioallantoic placenta increases more rapidly than the tissue volume.

Fetal blood flow was assumed to operate independently from the mother, increasing with fetal volume. Since regional blood flow and cardiac output data were not available for the rat fetus, the measured values could not be extrapolated back through gestation with certainty. Therefore, the values for regional blood flow to the fetal tissues were scaled allometrically (tissue weight0.75) from the earliest available time point in the pup and cardiac output was scaled by BW0.75 from the value given by Gotshall et al.(1987)Go at PND 1. Fractional blood flows to fetal GI, skin, liver, and kidney (as % cardiac output) were given for the PND 1 rat in Rakusan and Marcinek (1973)Go and were significantly different from those of the adult rat (4.6, 10.4, 4.5, and 3.6% vs. 13.6, 5.8, 18.3, and 14.0% of the cardiac output, respectively). These changing fractions are in agreement with the time-line for maturation of organ function in the developing rat. For example, the liver and kidney are not fully functional during gestation. In fact, the kidneys do not reach full function until well after birth (glomeruli levels increase up to day 100; Bengele and Solomon, 1974Go). Thus, the increasing trend seen in the fractional blood flows to the fetal liver and kidney with age may, at least in part, be due to their increasing functional capability.

Chemical-specific parameters.
Chemical-specific parameters for perchlorate and iodide are listed in Table 2Go. Binding of I- to human NIS was determined to have an average Km of 4.0 x 106 ng/l (Gluzman and Niepomniszcze, 1983Go). A similar value (4.4 x 106 ng/l) was measured by Kosugi and coauthors (1996)Go. This value for the follicular Km (KmTFi) remained constant across species (Gluzman and Niepomniszcze, 1983Go) and tissues (Wolff, 1998Go) and was therefore applied to all compartments in the model with active uptake by NIS. Golstein et al.(1992)Go measured a Km value of approximately 4.0 x 109 ng/l for iodide for the second thyroid transport mechanism at the apical membrane (KmTLi) in bovine thyroid. In the model, a slightly lower value than that measured by Golstein et al. of 1.0 x 109 was used for KmTLi, based on the ability of the model to fit the later (>8 h) time-points. Like the NIS, this apical channel is also inhibited by ClO4-.


View this table:
[in this window]
[in a new window]
 
TABLE 2 Chemical-Specific Parameters
 
The affinity of ClO4- for NIS was not available in literature nor was it determined by experiment in our laboratory. However, since this anion competitively inhibits iodide uptake by NIS, the value for Km should be equal to its Ki. Wolff and Maurey (1963)Go and Kosugi et al.(1996)Go measured the Ki for ClO4- at 0.4 x 105 and 1.5 x 105 ng/l, respectively. Thus the Km value for binding of ClO4- to NIS (KmTFp) was set between these two values (1.0 x 105 ng/l) in the model. This value is further supported by various literature sources suggesting that ClO4- actually has as much as an order of magnitude greater affinity for NIS than I- itself (Chow et al., 1969Go; Halmi and Stuelke, 1959Go; Harden et al., 1968Go; Lazarus et al., 1974Go). The Km for the second transport mechanism in the thyroid colloid (KmTLp) was set to 1.0 x 108 ng/l, approximately a factor of 10 lower than the Km values used for iodide. This value for the colloid Vmax is supported by the data of Chow and Woodbury (1970)Go, who reported saturation of this transport mechanism at 1.0 x 108 ng/l ClO4-.

Unlike Km, values for Vmax vary significantly between different species and tissues with NIS (Wolff, 1998Go). Therefore, values for Vmax in the thyroid, GI, and skin were determined by the fit of the model to available data in the pregnant rat on GD 20. The nonlinear behavior of the ClO4- uptake in tissues with NIS suggested that the symporter was saturated between the 1.0 and 10.0 mg/kg-day doses in the perchlorate drinking water study. The model fit to data from the lower dose groups (0.01 through 1.0 mg/kg-day) in the drinking water study were used to set Vmax values for ClO4- uptake, since nonsaturable processes dominate at higher doses (10 mg ClO4-/kg-day). Kinetic data were available for radioiodide in the maternal and fetal rat; the experiments were performed at tracer doses so as to stay well below saturation of NIS. Thus, the uptake and clearance of radioiodide as measured in the kinetic study were used to determine values for Vmax in tissues with active uptake.

The ClO4- and I- anions are not expected to partition into tissues in the classical understanding of the process. Rather, these anions are thought to respond to the electrochemical potential present across tissue membranes. Chow and Woodbury (1970)Go explored the relationship of these electrochemical potentials to ClO4- concentrations in the stroma, follicle, and lumen in the male rat thyroid at three different doses of ClO4-. Theoretical effective partition coefficients can be calculated (Kotyk and Janacek, 1977Go) from the measured differences in electrical potentials between the thyroid stroma and follicle. The approximately equal and opposite potential from the follicle to the colloid enhances passage of negatively charged species into the colloid and indicates an effective partition coefficient of greater than one. From Chow and Woodbury (1970)Go, the potential difference for the stroma:follicle interface ranges from -58 to -51 mV; for a monovalent negatively charged ion, the resulting effective partition coefficient (PTFp) is between 0.114 and 0.149. Similarly, the follicle:lumen interface has a calculated effective partition coefficient (PTLp) between 6.48 and 8.74 (See Merrill et al., 2003Go, for calculation details). Thyroid effective partition coefficients for iodide were assigned within the range calculated for ClO4-, based on the fact that iodide and perchlorate have the same ionic charge and similar ionic radii.

Partition coefficients for ClO4- were estimated from the drinking water study, since it contained the most complete ClO4- data sets for pregnant and fetal rats. Whereas the values for Vmax were determined from the lower doses (prior to saturation of the symporter), partition parameters were determined from the fit of the model to the highest dose (10.0 mg/kg-day). At this level of perchlorate exposure, all measured tissues appeared to have saturated the mechanism of active uptake and would therefore primarily be influenced by partition coefficients and PA values. In the cases of muscle (slowly perfused), liver (richly perfused), kidney, and red blood cells, data were not available in the pregnant rat but were calculated in this laboratory for the adult male rat for use in the male rat PBPK model (Merrill et al., 2003Go). The partition coefficient for perchlorate in fat was estimated from measured tissue:blood ratios in the laying hen (Pena et al., 1976Go). Although measured in a different species, several other tissues were also found to have comparable values to the rat.

Iodide partition coefficients were calculated from the tissue:blood ratios measured during the clearance phase (24 h postdosing) of data for the tissue of interest either from literature or experimental data in the rat and PA values were visually optimized to measured data. The partitioning parameters for the muscle (slowly perfused), liver (richly perfused), kidney, and red blood cells were again taken from corresponding male rat parameters (Merrill et al., 2003Go) and the values for the fat and mammary gland tissues were given the same values as ClO4-, due to the lack of I- data and the similar polarity of the anions.

Binding of perchlorate in the serum was described as a saturable process with a first order release of perchlorate from serum proteins. Because data were not available for free versus bound serum ClO4- in the pregnant or fetal rat, parameters for binding were determined by fitting the model to data at 0.01 and 0.1 mg/kg-day ClO4- from the drinking water study, since serum binding was most relevant at these lower doses. Urinary clearance of perchlorate was determined from the 10.0 mg/kg-day dose group, where binding had little effect on serum concentrations.

Upregulation of thyroid NIS activity.
At the time of data collection in the drinking water study, rats had been exposed to ClO4- for 18 days. At this point, upregulation of the thyroid is evidenced by decreased T4 and elevated TSH levels at all doses, as well as a lack of noticeable thyroid inhibition (Yu et al., 2001Go). Increased TSH compensates for the competitive inhibition of I- uptake by increasing the number and activity of NIS at the basolateral membrane of the thyroid, while the affinity of iodide for the symporter remains constant (Wolff, 1998Go). Thus, the value for VmaxcTFi, which corresponds to the maximum capacity for active transport at the basolateral membrane, was increased to fit the measured radioiodide concentrations in the upregulated thyroids for each dose. The resulting values for VmaxcTFi were then plotted versus the corresponding concentrations of serum-free ClO4- and, since the ability of the thyroid to be upregulated is finite, the data were fit to a M-M equation. This equation was then used in the model to describe the induction of NIS upregulation with time and dose, in a similar manner to the description used by Andersen et al.(1984)Go to describe glutathione induction.

Upregulation of thyroidal NIS also affects thyroid ClO4- uptake, and hence the measured thyroid ClO4- concentrations in the drinking water study, as both ClO4- and I- are transported by the same symporter (Wolff, 1964Go, 1998Go). Thus, increased thyroid ClO4- uptake was modeled in the same manner as I-, increasing the value for Vmaxc_TFp with dose and applying the resulting M-M fit to the model.

Sensitivity analysis of chemical-specific parameters.
A sensitivity analysis was run after setting the model parameters as described above to explore the influence of the various parameters on model predictions. The model was run to determine the change in each of the two chosen dosimetrics, average serum ClO4- concentration (AUC: area under the curve) and total thyroid iodide uptake, resulting from a 1% change in the value of each kinetic parameter. In an effort to determine the effect of NIS saturation on relative parameter importance, the sensitivity analysis was performed at two ClO4- doses, presumably representing unsaturated and saturated symporter states. Thus sensitivity analysis for the serum ClO4- AUC predictions were run at drinking water doses of 0.1 and 10.0 mg-kg-day. Because none of the iodide doses used in this model are expected to saturate the NIS, thyroid iodide sensitivity analysis was run only at the dose at which the experiments were run (1.87 ng/kg 125I-). Total thyroid iodine concentration was measured at 8 h post-iodide dosing. The following equation shows the calculation of the sensitivity coefficient for each parameter.


where A is the serum AUC with 1% increased parameter value, B is the serum AUC at the starting parameter value, C is the parameter value after 1% increase, and D is the original parameter value.

Model equations.
The following equations represent the distribution of iodide within the thyroid, in the absence of competitive inhibition. Perchlorate uptake into the thyroid is described similarly, but without the organification (ClProdi) and hormone secretion (ClSecri) terms.







RATSi, RATFi, RATLi, and RABndi are the rates of change in the amount of inorganic iodide in the thyroid stroma, follicle, colloid (lumen) and the rate of change in the amount of organic or incorporated iodine in the total thyroid, respectively. PATFi, PATLi and PTFi, PTLi are the PAs and effective partition coefficients for the stroma:follicle and follicle:colloid membranes, respectively. RupTFi and RupTFi are the active uptake rates of iodide into the follicle and colloid. VmaxTFi, VmaxTLi and KmTFi, KmTLi are the maximum velocities and affinity constants for transport of iodide into the follicle and colloid. QT represents fractional blood flow to the thyroid capillary bed. CAi, CTSi, CTi, and CTLi are the iodide concentrations in arterial plasma, thyroid stroma, follicle, and colloid.

The inhibited thyroid is described in the same manner as shown above, except that the M-M terms for active uptake are modified to account for competitive inhibition. The following equation gives an example of the description of competitive inhibition of iodide uptake by perchlorate. As before, RupTFi represents the rate of active iodide uptake into the thyroid follicle. This rate is modified by the affinity of transport mechanism in the follicle for ClO4- (KmTFp) and the concentration of ClO4- in the stroma (CTSp). Inhibition of iodide uptake in other tissues with NIS is described in the same manner as the thyroid follicle inhibition.


The model description for active uptake of iodide into the GI contents with competitive inhibition by ClO4- is given below. The skin compartment is modeled in the same manner. Equations for perchlorate would be similar to those of iodide without the terms for competitive inhibition. Here RAGBi RAGi and RAGJi represent the rates of change in the gut capillary blood, GI tract, and GI contents, respectively. QG is the regional blood flow to the GI, RMR is the rate of oral dosing, and CGBi CGi and CGJi are the total iodide concentration in the GI blood, tract and contents. Finally, partitioning of iodide between the GI blood and tract and the GI tract and contents is described using the partition coefficients (PGi and PGJi) and permeability area cross products (PAGi and PAGJi).





Model equations for compartments without active uptake (shown for the liver, below) were modeled in the as flow-limited, using only partitioning and blood flow to control tissue iodide and perchlorate concentrations. In the following equations, RALi is the rate of change in the amount of total iodide in the liver, QL is the fractional blood flow to the liver, CLi is the concentrationof iodide in the liver and PLi is the blood:liver partition coefficient. The kidney and fat are modeled similarly.



    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Model Parameterization
Perchlorate data from the drinking water distribution study were used to determine kinetic parameters for ClO4- in the pregnant and fetal rat. Upregulation of NIS transport of ClO4- into the thyroid was accounted for as described in the Materials and Methods section. Figure 2Go shows the resulting model predictions for maternal serum, thyroid, skin, and GI content ClO4- concentrations, as well as the fetal serum and skin ClO4- concentrations, together with measured data from the drinking water study on GD 20 at 0.01, 0.1, 1.0, and 10.0 mg ClO4-/kg-day. In the case of maternal GI contents, fetal serum, and fetal skin, the perchlorate levels in the 0.01 mg ClO4-/kg-day were below analytical detection. In these and subsequent plots, solid lines indicate the model prediction and cross-bars indicate the mean ± SD of measured data.

Maternal mammary gland and GI tract concentrations, as well as the fetal total GI (tract plus contents), were available at the 10 mg/kg-day dose only. Placental ClO4- concentrations were detectable only at the 1.0 and 10 mg/kg-day dose. The ClO4- levels in these tissues from all other dose groups were below analytical detection limits. Therefore, these data were used to verify the applicability of assigned partition coefficients to the model. Figure 3Go demonstrates that the PBPK model adequately simulates the data in these tissues.



View larger version (45K):
[in this window]
[in a new window]
 
FIG. 3. Perchlorate concentration in maternal (a) GI tract and (b) mammary gland at 10.0 mg ClO4-/kg-day, (c) placenta at 10.0 and 1.0 mg ClO4-/kg-day, and (d) fetal total GI (tract plus contents) at 10.0 mg ClO4-/kg-day dose on GD 20. Perchlorate concentrations at the lower doses were below the analytical detection limit.

 
Iodide parameters were determined by fitting the model to data from the iv radiolabeled iodide kinetic study. PA values were adjusted to describe the behavior of iodide data, where increasing PA values toward 1.0 l/h-kg generally increased the rate at which uptake and clearance in a particular tissue occurred, and decreasing PA slowed uptake and clearance. Clearance values for incorporation of inorganic iodide into hormones (ClProdCi) and clearance of the hormonal iodine (ClSecrCi) were determined from the fit of the model simulated incorporated iodine concentrations in the thyroid versus kinetic data after an iv injection of inorganic radioiodide. Figure 4Go shows the simulations for various maternal tissues versus measured 125I- data.




View larger version (40K):
[in this window]
[in a new window]
 
FIG. 4. (a) Total radioiodine, incorporated iodine, and inorganic iodide in maternal thyroid and radioiodide concentration in maternal (b) serum, (c) GI contents and skin, (d) mammary gland, and (e) placenta after iv dose of 2.19 ng/kg 125I-.

 
Clearance values for the transfer of iodide between the placenta and fetal blood were determined by visually optimizing the fit of the fetal serum prediction to the data, while maintaining the fit of the maternal blood, placenta, and fetal tissue data simulations. Figure 5Go shows the model simulation versus fetal 125I- data. Fetal thyroid iodide uptake increases rapidly in the final days of gestation (Carpenter, 1959Go; Feldman et al., 1961Go). In order to describe this uptake, the model simulation was fit to fetal thyroid uptake data reported in Feldman et al.(1961)Go 24 h after administration of 131I- to the dam on days 17, 18, and 19 of gestation (Fig. 5dGo). The time-dependent parameters were then incorporated into the model using simple linear interpolation between data points to estimate the value of the parameter at any point in time.



View larger version (21K):
[in this window]
[in a new window]
 
FIG. 5. Radioiodide concentration in fetal (a) serum, (b) skin, and (c) total GI after iv dose of 2.19 ng/kg 125I- to dam. (d) Amount of radioiodide in the fetal thyroid 24 h after maternal iv dose on GD 17, 18, and 19 (Feldman et al., 1961Go). Cross-bars indicate the mean ± SD of data, while squares indicate reported averages.

 
Model Validation
Model simulations were tested against data sets collected under different conditions than the data used for model parameterization to test the predictive capability of the model. Model predictions for iodide kinetics were tested against data collected in other laboratories and in different strains of rats from the literature. Although the limited data do not allow testing of the entire acute kinetic time course, they do allow validation of tissue iodide concentrations at specific time points (e.g., 2 and 24 h postdosing). Finally, the model’s ability to describe both acute perchlorate and iodide kinetics and the interaction between the anions (inhibition of thyroidal iodide uptake) was tested against acute inhibition data collected in our laboratory and in literature after iv dosing with both ClO4- and radioiodide.

Data of Versloot et al. (1997)Go.
A simulation was performed with the model, using the exposure conditions of Versloot et al. (1997Go; an iv injection of 10 µCi or 1.74 ng/kg carrier free 125I- in pregnant Wistar rats on GD 19). This study provided a data set on an additional day of gestation (GD 19 vs. GD 20), as well as an additional time point (24 h) and included measurements of the total fetal burden, which had not previously been determined. The model was able to simulate the data in the maternal thyroid, mammary gland, and placenta, and fetal thyroid (Figs. 6a–6dGo). The model prediction of total body burden (Fig. 6eGo) underpredicted the rest of body iodide content in the fetus after removal of the thyroid. However, the model simulation is within a factor of two from the measured data.




View larger version (31K):
[in this window]
[in a new window]
 
FIG. 6. Radioiodide in maternal (a) thyroid, (b) mammary gland, and (c) placenta, plus fetal (d) thyroid and (e) total body (minus thyroid) after a single iv injection of 125I- to GD 19 Wistar dam. The model simulation is shown versus the mean ± SD at the 4 and 24 h data points in the thyroid and the 24 h data point in the other tissues (Versloot et al., 1997Go).

 
Data of Sztanyik and Turai (1988)Go.
A simulation of data reported in Sztanyik and Turai (1988)Go was performed with the model to test the ability of the model to predict I- distribution from a lower dose than was used in model parameterization, as well as a different species of radiolabeled iodide (131I- vs. 125I-). Sztanyik and Turai administered an ip injection of 0.36 ng/kg carrier free 131I- to pregnant CFY albino rats on GD 20 and measured tissues at 24 h postdosing. The model reproduced the placenta data well at 24 h postdosing without changing any kinetic parameters. The model prediction of the total fetal body burden was slightly high, but was within a factor of two from the measured data. Figure 7Go shows the model simulation versus the measured values of 131I- in the placenta and total fetal body.



View larger version (10K):
[in this window]
[in a new window]
 
FIG. 7. Radioiodide concentration in (a) placenta and (b) total fetus after an iv injection to the GD 20 CFY albino dam (0.36 ng/kg). The model simulation is shown versus the mean ± SD of the data at 24 h after exposure (Sztanyik and Turai, 1988Go).

 
Drinking water inhibition data.
Inhibition of thyroid iodide uptake as well as iodide distribution in several maternal and fetal tissues were measured in the perchlorate drinking water study after maternal exposure to levels of 0.0, 0.01, 0.1, 1.0, and 10.0 mg ClO4-/kg-day for 18 days (see Materials and Methods). Due to upregulation, none of the thyroid iodide concentrations measured after 18 days of perchlorate dosing were significantly different from the control levels. However, iodide data from the control group (0.0 mg/kg-day ClO4-) were useful for testing model predictions at 125I- doses more than four orders of magnitude greater than those used to parameterize the model (33,000 ng/kg vs. 2.19 ng/kg). Table 3Go illustrates the ability of the model to predict iodide uptake in several tissues in the dam and fetus on GD 20.


View this table:
[in this window]
[in a new window]
 
TABLE 3 Model-Predicted Radioiodide Concentration in Maternal and Fetal Tissues versus Measured Data (mean ± SD)
 
Upregulation of thyroid NIS activity was modeled as described in the Materials and Methods section. Using this equation, it was possible to describe the increase in iodide uptake based on the perchlorate dose in chronic exposure scenarios. Neither the measured data nor the model showed any inhibition in iodide concentrations in the maternal thyroid 2 h postdosing with 125I- after 18 days of exposure to 0.01, 0.1, 1.0, and 10.0 mg/kg ClO4- in drinking water. Thus, the model was able to reproduce the upregulation of thyroid NIS activity resulting from subchronic perchlorate exposures in a semiempirical manner. Due to the lack of necessary data, this model does not attempt to describe the time-dependent nature of thyroid upregulation, nor does it account for physiological changes apart from increased NIS, such as increased serum binding or decreased deiodination. Thus, the present model represents a heuristic approximation of thyroid upregulation resulting from prolonged ClO4- exposure. A more complete description would require considerable additional experimental data, including time course data, hormone levels, and endogenous iodine information, in order to properly simulate this time-dependent regulation.

Inhibition of iodide uptake.
The inhibition of iodide uptake into the thyroid and placenta were simulated against data collected in our laboratory on GD 20, using the same kinetic parameters derived previously. Figure 8Go shows the model prediction of thyroidal (a) and placental (b) iodide uptake with and without perchlorate inhibition. The model is able to predict inhibition in the thyroid gland up to 24 h after dosing with iodide. The ability of the model to predict perchlorate-induced inhibition of thyroid iodide uptake is dependent on accurate simulations of both iodide and perchlorate kinetics. Thus, the appropriateness of the model perchlorate and iodide parameters is supported by the fit of the model simulation to these inhibition data.



View larger version (17K):
[in this window]
[in a new window]
 
FIG. 8. Iodide concentration in (a) maternal thyroid and (b) placenta with and without 1.0 mg/kg ClO4- iv dose 2 h prior to an iv dose of 1.87 ng/kg 125I- to the dam (Clewell et al., 2001Go). The top simulation (solid line) and data (solid box) indicates the control group. The lower simulation (dotted line) and data (open circle) indicate the perchlorate-dosed inhibition group.

 
Inhibition data of Sztanyik and Turai (1988)Go.
Further validation of model-predicted inhibition was validated against additional perchlorate dose levels (3.0 and 6.0 mg/kg KClO4) by testing the model-predicted fetal iodide uptake with and without ClO4- against data from Sztanyik and Turai (1988)Go. In this study, GD 20 pregnant rats were given an ip tracer dose of 131I-. At 20, 60, or 120 min post-131I dosing, an ip dose of KClO4 (either 3.0 or 6.0 mg) was administered to the dams. The uptake of radioiodide in the whole fetus was then measured 24 h after the 131I- dose. The model was able to accurately predict the measured inhibition of fetal uptake in two of the three studied groups on GD 20. The third group showed only slightly less inhibition than the model predicted. Table 4Go shows the model predicted inhibition of iodide uptake in the whole fetus versus the ranges (mean ± 1 SD) of measured values given in Sztanyik and Turai (1988)Go. Mean values are given in parentheses. The ability of the model to simulate these data supports its usefulness in predicting inhibition of placental transfer and fetal thyroid uptake.


View this table:
[in this window]
[in a new window]
 
TABLE 4 Model-Predicted Inhibition of Iodide Uptake in Total Fetus versus Data of Sztanyik and Turai (1988)Go
 
Data of Brown-Grant (1966)Go.
Brown-Grant (1966)Go administered 0.25 and 1% potassium perchlorate solution (approximately 200 and 800 mg/kg respectively) by oral gavage for one to five days (GD 2 to 8) in Wistar rats, and observed 131I thyroid:plasma ratios of between 1.8 and 4.1. Since these large perchlorate doses are sufficient to effectively swamp the NIS, the measured thyroid:plasma ratios primarily reflect the extent to which thyroid iodide uptake is dependent upon partitioning. As described previously, the PBPK model describes partitioning based on the measured electrochemical potentials of Chow and Woodbury (1970)Go. Using these previously calculated partition coefficients and permeability area cross products, the model predicts thyroid: plasma ratios of 3.9 and 3.6, which are within the range of measured values reported by Brown-Grant (1966)Go, thus supporting the validity of the method for calculating partition parameters.

Sensitivity analysis.
Sensitivity analysis performed at 0.1 and 10.0 mg ClO4-/kg-day drinking water revealed a dose-dependent difference in model sensitivity to various parameters. At 0.1 mg/kg-day, the maternal serum is primarily dependent on serum binding and urinary clearance parameters with a greater sensitivity to the serum binding. Sensitivity coefficients for all other parameters were less than 0.1. At the 10.0 mg/kg-day dose, however, only the urinary clearance remained significant, with a sensitivity coefficient of -0.84. Fetal serum is influenced by several model parameters at the 0.1 mg/kg-day dose, including placental transfer, placental active uptake, placental diffusion, fetal serum binding, and maternal urinary clearance parameters. At the higher dose (10.0 mg/kg-day), where active placental uptake and serum binding are likely saturated, only the placental transfer, placental diffusion, and maternal urinary clearance maintain significant sensitivity coefficients. Figure 9Go shows the calculated sensitivity coefficients for maternal and fetal serum ClO4- AUC on GD 20. Results of the sensitivity analysis for thyroid iodide uptake (not shown) were similar, in that all of the parameter sensitivities were less than one in absolute values; however, model predictions of this metric were sensitive to a much larger number of input parameters. This result is not unexpected due to the fact that the uptake prediction is for a specific point in time after administration of the radioiodide, and the rate of distribution into all tissues can affect the time-dependent result (as compared to an average, or AUC, measure, which reflects steady-state behavior). Thus the validation of the model with data on thyroid iodine uptake and inhibition provides a stringent test of the model parameterization.



View larger version (27K):
[in this window]
[in a new window]
 
FIG. 9. Calculated sensitivity coefficients for model parameters with respect to serum perchlorate AUC at drinking water doses of 0.1 and 10.0 mg/kg-day.

 
Internal dosimetrics.
The validated model was used to calculate internal dosimetrics corresponding to perchlorate dosing in the pregnant rat. These internal measures of ClO4- dose include the AUC for ClO4- in the maternal serum and relative fetal dose after repeated drinking water exposure, as well as inhibition of iodide uptake in the thyroid after acute dosing. These internal dosimetrics were then compared to the ClO4- serum AUC and iodide percent inhibition in the male rat (calculated from the model of Merrill et al., 2003Go) to provide insight on relative exposure at different life stages. Tables 5Go and 6Go show the dosimetric comparisons among the adult male, pregnant, and fetal rat for drinking water serum levels, acute iodide inhibition, and fetal ClO4- dose. The models predict a significant transfer of maternal ClO4- to the fetus. They also indicate that the greatest inhibition is predicted to be in the fetal thyroid.


View this table:
[in this window]
[in a new window]
 
TABLE 5 Model-Predicted Internal Dosimetrics: Fetal Dose and Serum AUC in Male, Pregnant, and Fetal Rat from Drinking Water Perchlorate Exposure
 

View this table:
[in this window]
[in a new window]
 
TABLE 6 Model-Predicted Internal Dosimetrics: % Inhibition of Thyroid Iodide Uptake in Male, Pregnant, and Fetal Rat from Acute Perchlorate Exposure
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The PBPK model described here successfully simulates perchlorate and radioiodide distribution kinetics in the pregnant rat and fetus by accounting mathematically for physiological changes in gestation, such as nonlinear growth. The model is able to reproduce ClO4- distribution and placental transfer resulting from drinking water exposure at doses ranging over three orders of magnitude (0.01–10.0 mg/kg-day). Although acute kinetic data for perchlorate is not available in the pregnant or fetal rats, in terms of exposure route, drinking water dosing is actually more relevant to the risk assessment. Thus, the model is able to describe distribution to the target tissues and in the serum resulting from exposure via the route that is most applicable to that of humans.

Despite the lack of data regarding the serum binding of ClO4-, the model is able to reproduce both total serum concentrations and tissue levels resulting from only free ClO4- transfer. The model-predicted bound perchlorate levels compare reasonably to the measured in vitro binding data of Fisher (2002, as cited in Merrill et al., 2003Go) in male rat serum. The model predicts 30 and 80% of the ClO4- to be bound at total serum levels of 500 and 50 ng/ml ClO4- as compared to the measured values of 50 and 100%. As expected, the sensitivity analysis suggests that at the 10.0 mg/kg-day dose, the model serum prediction is most sensitive to the value for urinary clearance, while the lower doses (0.1 mg/kg-day) are primarily determined by the binding parameters, indicating that the binding is saturated between the 1.0 and 10.0 mg ClO4-/kg-day dose groups.

In the absence of acute perchlorate kinetic data in rat gestation, we make use of the consistency of this model structure and its parameters with those of the male rat (Merrill et al., 2003Go), which successfully describes such data. Having accounted for differences in physiology due to gestation, most chemical-specific parameters remain essentially unchanged from those of the adult male rat. Therefore, we can assume that, like the male rat model, this model is also able to adequately predict acute perchlorate kinetics. This is further supported by the ability of the model to predict acute iodide inhibition kinetics, as inhibition of thyroid uptake is dependent on free serum perchlorate levels.

The PBPK model was able to predict the ClO4- induced inhibition of iodide uptake in the maternal thyroid and the total fetus after acute ClO4- exposures in the pregnant rat by linking the perchlorate and iodide models via competitive inhibition at the symporter. Although some authors have suggested that ClO4- may not be translocated into the thyroid cell via NIS (Riedel et al., 2001aGo,bGo), the weight of evidence suggests that the anion is a true competitive inhibitor of iodide uptake (Clewell and Gearhart, 2002Go). Based on electrogenicity studies showing that the addition of 500 µM ClO4- to a bathing medium containing I- abolished the existing inward current in an oocyte with NIS (Eskandari et al., 1997Go), Riedel and coauthors (2001aGo,bGo) suggested two potential mechanisms for ClO4- action on the thyroid: (1) that ClO4- blocks I- transport, but is not transferred into the cell, and (2) ClO4- competes with I- and is transferred into the cell by NIS at a 1:1 ratio with Na+. In our model, we utilize the latter proposed mechanism, in which ClO4- competes for binding sites on the NIS and is transferred into the follicle in place of iodide. This assumption is based on measured radiolabeled and cold perchlorate thyroid concentrations with tissue to blood ratios greater than one (Chow and Woodbury, 1970Go; Yu et al., 2002bGo), the consistency of ClO4- accumulation in tissues with NIS, and studies showing that ClO4- affects the internal thyroid iodide as well as the external uptake of iodide by NIS (Hildebrandt and Halmi, 1981Go). The ability of the model to reproduce experimental perchlorate, iodide, and inhibition data from different exposure scenarios (acute vs. drinking water) and over a wide range of doses supports the use of this mode of action as the foundation for the pharmacokinetic models.

The iodide model was simplified by assuming that radiolabeled iodide could be described as free iodide in all compartments other than the maternal thyroid. Simulations were performed against total radioiodide concentrations in extrathyroidal tissues and against inorganic and incorporated iodide in thyroid. Despite this simplification of the model structure, the kinetic behavior of radioiodide in the naïve rat (no ClO4- exposure) was accurately simulated in all measured maternal and fetal tissues over a range of doses spanning nearly five orders of magnitude (0.36 to 33,000 ng/kg) 24 h after a single administration of radiolabeled iodide. Intralaboratory studies found that approximately 80% of serum iodide is in the inorganic form in the pregnant rat as much as 12 h after dosing with radioiodide (Yu et al., 2002aGo). Thus, the model description of extrathyroidal tissue iodide uptake based on the transfer of inorganic iodide via NIS predicts the data reasonably well without significant contribution from the uptake of incorporated iodine.

This PBPK model describes the dosimetry, distribution, kinetic behavior, and interaction between administered radioiodide and perchlorate. As such, a description of dietary iodide and interactions of endogenous and dosed iodide have not been included in the present version of the model. For the purpose of extrapolation of this animal model to human exposure scenarios, the influence of variations in dietary iodide intake will need to be included, in order to make the human predictions more applicable across populations. However, in the rat, iodide intake is easily controlled through the animal’s diet, and given that iodide is an essential nutrient, the mineral is tightly controlled by regulatory mechanisms. Therefore, slight variations in laboratory rat chow are not expected to affect model predictions of distribution or even perchlorate-induced inhibition. Although the mechanism for iodide transfer (NIS) is saturable, the high value for Km dictates that very large doses of iodide would be required to inhibit its own uptake, as opposed to more effective inhibition by the higher affinity ClO4-. In fact, in studies by Wolff and Chaikoff (1948)Go, it was found that the uptake of iodide into the thyroid is linear up to serum iodide levels of 250 µg/l, which is more than six times higher than serum levels of the highest dose groups used in our studies and more than 60 times greater than would be expected from daily iodide intake from a normal rat chow. Thus, this model is useful for predicting tissue dosimetry and inhibition in the rat and for extrapolation to the average human with normal iodide diet. In order to address intraspecies variability and susceptibility to hormone effects due to variations in diet, future elaborations of the model should include endogenous iodide, as well as a more detailed description of hormone distribution, homeostasis, and regulation.

This model currently includes only a highly simplified description of the hormone feedback system that controls iodide uptake and upregulation of the inhibited thyroid in order to predict the dose-dependent change in thyroid iodide uptake after long-term exposure to ClO4- in the rat. However, it is important to note that the rat thyroid has a much greater sensitivity to perturbations in the pituitary-thyroid axis than the human. In fact, evidence of upregulation is seen as early as 12 h after administration of ClO4- to a male rat (Merrill et al., 2003Go), whereas human studies have found that after two weeks of ClO4- exposure, TSH levels were not yet elevated and thyroidal iodide uptake showed inhibition similar to that of the control subjects (Brabant et al., 1992Go; Greer et al., 2002Go). Although the human shows similar inhibition to the rat (Greer et al., 2002Go) at equal perchlorate doses, the human system does not respond by upregulating thyroid activity. This species difference is most likely due to the fact that the human has a greater thyroid hormone storage capacity than the rat, resulting from increased thyroid colloid volume and serum binding (thyroxine binding globulin; Brown et al., 1986Go; Dohler et al., 1979Go). Therefore, small perturbations in iodide uptake do not appear to affect hormone balance to the same extent in humans as in rats. Since stable serum ClO4- levels cannot be established in rats for more than a few hours without inducing upregulation of NIS, it is not possible to use rats directly as an animal model for human drinking water ClO4- exposure. However, by using the rat PBPK models to extrapolate to humans, we can mathematically bridge this gap by using the existing rat data and incorporating a longer time course for upregulation in the human model.

Together with a concurrent model developed by Merrill et al.(2003)Go, the present PBPK model is able to define and quantify kinetic differences in the male, pregnant, and fetal rat. The models account for kinetic differences through the description of physiological differences. Most of the kinetic parameters are nearly identical between the male rat and pregnant rat, with the exception of those for thyroid and skin uptake and urinary clearance of iodide. Where different kinetic parameters were necessary in the two models, literature studies were able to provide a justification for these differences. For example, Brown-Grant and Pethes (1959)Go found that the adult male and neonatal rat possess a greater iodide reserve in the skin than the pregnant dam. This difference in life stage kinetics is reflected in the parameters for skin iodide uptake in the pregnant and male rat models (e.g., VmaxcSi = 6.0 x 104 vs. 5.0 x 105, respectively; Merrill et al., 2003Go). This information provided by the models, concerning the variation in perchlorate and iodide kinetics across gender and life stages in the rat, should improve the accuracy of predictions for human perchlorate exposures during gestation (Clewell et al., 2001Go).

A comparison of model-predicted internal dosimetrics provides important information on ClO4- distribution and inhibition kinetics across life stages. It is apparent that the fetus receives a significant portion of the maternal ClO4- dose, while the serum AUCs are similar between the male and pregnant rats. Comparison of thyroid iodide inhibition suggests that the thyroid of the pregnant rat may be somewhat more sensitive to inhibition than that of the male rat. This may be due to the increased loss of iodine during pregnancy, resulting from loss to the fetus and increased urinary output (Versloot et al., 1997Go). The predicted fetal thyroid inhibition was even greater than that of the male and pregnant female, possibly due to the combined effect of inhibition at both the placenta and fetal thyroid. Inhibition in the fetal thyroid is partially offset by the increased fetal serum iodide levels resulting from inhibition in the fetal skin and GI contents. However, the actual risk to the fetus may be better characterized by looking at the effect of perchlorate on iodide levels in the total fetus, due to the fact that fetal iodide stores in extra-thyroidal tissues (e.g., skin) are important in maintaining the needed iodide supply during the transition from intra- to extra-uterine life.

A potentially informative use of this PBPK model is in the correlation of predicted internal dosimetrics to periods in gestation where perchlorate exposure and/or iodide deficiency has been associated with developmental effects. The model can be used to predict tissue dosimetry in effects studies and to pinpoint specific times in gestation when fetal iodide uptake is most critical. A lactation exposure model is currently in development for use with the gestation model to provide a complete kinetic description of the developmental period. When used together, these developmental models could be used to help answer questions concerning susceptible time points and primary routes of exposure during development.


    ACKNOWLEDGMENTS
 
The authors would like to thank Tammie Covington, Harvey Clewell, and Dr. Melvin Andersen for their modeling advice; Latha Narayanan and Gerry Buttler for sample analyses; Charles Goodyear for performing statistical analyses of the data; and Dick Godfrey, Peggy Parish, Susan Young, TSgt Todd Ligman, MSgt Jim McCafferty, Tim Bausman, SSgt Paula Todd, and MSgt Rick Black for technical support. The authors would like to acknowledge Annie Jarabek, LtCol Dan Rogers, Dr. Richard Stotts and the U.S. Air Force for their support of this project and the U.S. Navy for financial support.


    NOTES
 
1 To whom correspondence should be addressed at CIIT Centers for Health Research, 6 Davis Drive, Research Triangle Park, NC 27709-2137. Fax: (919) 558-1300. E-mail: rclewell{at}ciit.org. Back

2 Present address: The University of Georgia, Athens, GA 30602. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Altman, P. L., and Dittmer, D. S. (1971). Volume of blood in tissue: Vertebrates. In Respiration and Circulation, pp. 383–387. Federation of American Societies for Experimental Biology, Bethesda, MD.

Andersen, M. E., Gargas, M. L., and Ramsey, J. C. (1984). Inhalation pharmacokinetics: Evaluating systemic extraction, total in vivo metabolism, and the time course of enzyme induction for inhaled styrene in rats based on arterial blood:inhaled air concentration ratios. Toxicol. Appl. Pharmacol. 73(1), 176–187.[CrossRef][ISI][Medline]

Bakke, J. L., Lawrence, N. L., Robinson, S., and Bennett, J. (1976). Lifelong alterations in endocrine function resulting from brief perinatal hypothyroidism in the rat. J. Lab. Clin. Med. 88, 3–13.[ISI][Medline]

Beaton, G. H., Beare, H., Ryu, M. H., and McHenry, E. W. (1954). Protein metabolism in the pregnant rat. J. Nutr. 54, 291–304.[ISI][Medline]

Bengele, H. H., and Solomon, S. (1974). Devlopment of renal response to blood volume expansion in the rat. Am. J. Physiol. 227(2), 364–368.[Free Full Text]

Berman, M., Hoff, E., Barandes, M., Becker, D. B., Sonenberg, M., Benua, R., and Koutras, D. A. (1968). Iodine kinetics in man—a model. J. Clin. Endocrinol. Metab. 28(1), 1–14.[ISI][Medline]

Brabant, G., Bergmann, P., Kirsch, C. M., Kohrle, J., Hesch, R. D., and von zur Muhlen, A. (1992). Early adaptation of thyrotropin and thyroglobulin secretion to experimentally decreased iodine supply in man. Metabolism 41, 1093–1096.[ISI][Medline]

Brown, R. A., Al-Moussa, M., and Beck, J. (1986). Histometry of normal thyroid in man. J. Clin. Pathol. 39, 475–482.[Abstract]

Brown, R. P., Delp, M. D., Lindstedt, S. L., Rhomberg, L. R., and Beliles, R. P. (1997). Physiological parameter values for physiologically based pharmacokinetic models. Toxicol. Ind. Health 13, 407–484.[ISI][Medline]

Brown-Grant, K. (1961). Extrathyroidal iodide concentrating mechanisms. Physiol. Rev. 41, 189–213.[Free Full Text]

Brown-Grant, K. (1966). Failure of orally administered perchlorate to affect deciduoma formation or pregnancy in the rat. J. Reprod. Fertil. 12(2), 353–357.[CrossRef][Medline]

Brown-Grant, K., and Pethes, G. (1959). Concentration of radioiodine in the skin of the rat. J. Physiol. 148, 683–693.[ISI][Medline]

Buelke-Sam, J., Holson, J. F., and Nelson, C. J. (1982). Blood flow during pregnancy in the rat: II. Dynamics of and litter variability in uterine flow. Teratology 26, 279–288.[ISI][Medline]

Carpenter, E. (1959). Development of the fetal rat thyroid with special reference to the uptake of radioactive iodine. J. Exp. Zool. 142, 247–257.[ISI][Medline]

Carr, C. W. (1952). Studies on the binding of small ions in protein solutions with the use of membrane electrodes. I. The binding of the chloride ion and other inorganic anions in solutions of serum albumin. Arch. Biochem. Biophys. 40, 286–294.[CrossRef][ISI][Medline]

Chow, S. Y., Chang, L. R., and Yen, M. S. (1969). A comparison between the uptakes of radioactive perchlorate and iodide by rat and guinea pig thyroid glands. J. Endocrinol. 45, 1–8.[ISI][Medline]

Chow, S. Y., and Woodbury, D. M. (1970). Kinetics of distribution of radioactive perchlorate in rat and guinea-pig thyroid glands. J. Endocrinol. 47, 207–218.[ISI][Medline]

Clewell, R. A., and Gearhart, J. M. (2002). Proposed PBPK model to predict infant exposure: Clewell and Gearhart’s Response. Environ. Health Perspect. 110(11), A15–A16.[ISI][Medline]

Clewell, R. A., Merrill, E. A., and Robinson, P. J. (2001). The use of physiologically based models to integrate diverse data sets and reduce uncertainty in the prediction of perchlorate kinetics across life stages and species. Toxicol. Ind. Health 17, 210–222.[CrossRef][Medline]

Clos, J., Crepel, F., Legrand, C., Legrand, J., Rabie, A., and Vigouroux, E. (1974). Thyroid physiology during the postnatal period in the rat: A study of the development of thyroid function and of the morphogenetic effects of thyroxine with special reference to cerebellar maturation. Gen. Comp. Endocrinol. 23, 178–192.[ISI][Medline]

Conde, E., Martin-Lacave, I., Godalez-Campora, R., and Galera-Davidson, H. (1991). Histometry of normal thyroid glands in neonatal and adult rats. Am. J. Anat. 191, 384–390.[ISI][Medline]

Delange, F. (2000). The role of iodine in brain development. Proc. Nutr. Soc. 59, 75–79.[ISI][Medline]

DiStefano, J. J., III, Malone, T. K., and Jang, M. (1982). Comprehensive kinetics of thyroxine distribution and metabolism in blood and tissue pools of the rat from only six blood samples: Dominance of large, slowly exchanging tissue pools. Endocrinology 111(1), 108–117.[Abstract]

Dohler, K. D., Wong, C. C., and von zur Muhlen, A. (1979). The rat model for the study of drug effects on thyroid function: Consideration of methodological problems. Pharmacol. Ther. 5, 305–318.[CrossRef][ISI]

Eguchi, Y., Fukiishi, Y., and Hasegawa, Y. (1980). Ontogeny of the pituitary-thyroid system in fetal rats: Observations on the fetal thyroid after maternal treatment with goitrogen. Anat. Rec. 198, 637–642.[ISI][Medline]

Eskandari, S., Loo, D. D., Dai, G., Levy, O., Wright, E. M., and Carrasco, N. (1997). Thyroid Na+/I- symporter: Mechanism, stoichiometry, and specificity. J. Biol. Chem. 272, 2730–2738.

Feldman, J. D., Vazquez, J. J., and Kurtz, S. M. (1961). Maturation of the fetal thyroid. J. Biophys. Biochem. Cytol. 11, 365–383.[Abstract/Free Full Text]

Fisher, J., Todd, P., Mattie, D., Godfrey, D., Narayanan, L., and Yu, K. (2000). Preliminary development of a physiological model for perchlorate in the adult male rat: A framework for further studies. Drug Chem. Toxicol. 23, 243–258.[CrossRef][ISI][Medline]

Fisher, J. W., Whittaker, T. A., Taylor, D. H., Clewell, H. J., and Andersen, M. E. (1989). Physiologically based pharmacokinetic modeling of the pregnant rat: A multiroute exposure model for trichloroethylene and its metabolite, trichloroacetic acid: Toxicol. Appl. Pharmacol. 99, 395–414.[ISI][Medline]

Geloso, J. P. (1961). Date de l’entrée en fonction do la thyroide chez lel foetus de rat. C. R. Soc. Biol. (Paris) 155, 1239–1244.[ISI]

Gentry, P. R., Covington, T. R., Andersen, M. E., and Clewell, H. J. (2002). Application of a physiologically-based pharmacokinetic model for isopropanol in the derivation of an RfD/RfC. Reg. Toxicol. Pharmacol. 36, 51–68.[CrossRef][ISI][Medline]

Gluzman, B. E., and Niepomniszcze, H. (1983). Kinetics of the iodide trapping mechanism in normal and pathological human thyroid slices. Acta Endocrinol. 103, 34–39.[ISI][Medline]

Goedbloed, J. F. (1972). The embryonic and postnatal growth of rat and mouse. I. The embryonic and early postnatal growth of the whole embryo. A model with exponential growth and sudden changes in growth rate. Acta Anat. 82, 305–306.[ISI][Medline]

Golstein, P., Abramow, M., Dumont, J. E., and Beauwens, R. (1992). The iodide channel of the thyroid: A membrane vesicle study. Am. J. Physiol. 263, C590–C597.[ISI][Medline]

Gotshall R. W., Breay-Pilcher J. C., and Boelcskevy B. D. (1987). Cardiac output in adult and neonatal rats utilizing impedance cardiography. Am. J. Physiol. 253(5 Pt. 2), H1298–H1304.[ISI][Medline]

Greer, M. A., Goodman, G., Pleus, R. C., and Greer, S. E. (2002). Health effects assessment for environmental perchlorate contamination: The dose response for inhibition of thyroidal radioiodide uptake in humans. Environ. Health Perspect. 110, 927–937.[ISI][Medline]

Haddow, J. E., Palomaki, G. E., Allan, W. C., Williams, J. R., Knight, G. J., Gagnon, J., O’Heir, C. E., Mitchell, M. L., Hermos, R. J., Waisbren, S. E., et al. (1999). Maternal thyroid deficiency during pregnancy and subsequent neuropsychological development of the child. N. Engl. J. Med. 341, 549–555.[Abstract/Free Full Text]

Halmi, N. S., and Stuelke, R. G. (1959). Comparison of thyroidal and gastric iodide pumps in rats. Endocrinology 64, 103–109.[ISI][Medline]

Hanwell, A., and Linzell, J. L. (1973). The time course of cardiovascular changes in the rat. J. Physiol. 233, 99–109.

Harden, R. G., Alexander, W. D., Shimmins, J., and Robertson, J. W. (1968). A comparison between the inhibitory effect of perchlorate on iodide and pertechnetate concentrations in saliva in man. Q. J. Exp. Physiol. Cogn. Med. Sci. 3, 227–238.

Hays, M. T., and Wegner, L. H. (1965). A mathematical and physical model for early distribution of radioiodide in man. J. Appl. Physiol. 20, 1319–1328.[ISI]

Hildebrandt, J. D., and Halmi, N. S. (1981). Intrathyroidally generated iodide: The role of transport in its utilization. Endocrinology 108, 842–849.[Abstract]

Honour, A. J., Myant, N. B., and Rowlands, E. N. (1952). Secretion of radioiodine in digestive juices and milk in man. Clin. Sci. 11, 447–463.[ISI]

Howdeshell, K. L. (2002). A model of the development of the brain as a construct of the thyroid system. Environ. Health Perspect. 110(3), 337–348.

Klein, A. H., Meltzer, S., and Kenny, F. M. (1972). Improved prognosis in congenital hypothyroidism treated before age three months. J. Pediatr. 81, 912–915.[ISI][Medline]

Knight, C. H., Docherty, A. H., and Peaker, M. (1984). Milk yield in the rat in relation to activity and size of the mammary secretory cell population. J. Dairy Res. 51, 29–35.[ISI][Medline]

Knight, C. H., and Peaker, M. (1982). Mammary cell proliferation in mice during pregnancy and lactation in relation to milk yield. Q. J. Exp. Phys. 67, 165–177.[ISI]

Kosugi, S., Sasaki, N., Hai, N., Sugawa, H., Aoki, N., Shigemass, C., Mori, T., and Yoshida, A. (1996). Establishment and characterization of a Chinese hamster ovary cell line, CHO-4J, stably expressing a number of Na+/I- symporters. Biochem. Biophys. Res. Commun. 227(1), 94–101.[CrossRef][ISI][Medline]

Kotani, T., Ogata, Y., Yamamoto, I., Aratake, Y., Kawano, J. I., Suganuma, T., and Ohtaki, S. (1998). Characterization of gastric Na+/I- symporter of the rat. Clin. Immunol. Immunopathol. 89, 271–278.[CrossRef][ISI][Medline]

Kotyk, A., and Janacek, K. (1977). Transport of ions. In Membrane Transport: An Interdisciplinary Approach, pp. 243–247. Plenum Press, New York.

Lazarus, J. H., Harden, R. M., and Robertson, J. W. K. (1974). Quantitative studies of the inhibitory effect of perchlorate on the concentration of 36ClO4-, 125I-, and 99mTcO4- in salivary glands of male and female mice. Arch. Oral. Biol. 19, 493–498.[CrossRef][ISI][Medline]

Mahle, D. A., Godfrey, R. J., McCafferty, J. D., Bausman, T. A., Narayanan, L., Parish, M. A., Todd, P. N., Ligman, T. A., Mattie, D. R., and Yu, K. O. (2002). Kinetics of perchlorate and iodide in lactating S-D rats and pups at postnatal day 10. Toxicol. Sci, 66(1-S), 139 (Abstract).[Abstract/Free Full Text]

Malendowicz, L. K., and Bednarek, J. (1986). Sex dimorphism in the thyroid gland. Acta Anat. 127, 115–118.[ISI][Medline]

Merrill, E. A., Clewell, R. A., Gearhart, J. M., Robinson, P. J., Sterner, T. R., Yu, K. O., and Fisher, J. W. (2003). PBPK model for perchlorate-induced radioiodide inhibition in the male rat. Toxicol. Sci. 73, 256–269.

Morreale de Escobar, G., Pastor, R., Obregon, M. J., and Escobar del Rey, F. (1985). Effect of maternal hypothyroidism on the weight and thyroid hormone content of rat embryonic tissues, before and after onset of fetal thyroid function. Endocrinology 117(5), 1890–1900.[Abstract]

Motzer, W. E. (2001). Perchlorate: Problems, detection, and solutions. Environ. Foren. 2, 301–311.[CrossRef][ISI]

Myant, N. B. (1971). The role of thyroid hormone in the fetal and postnatal development of mammals. In Hormones in Development (M. Hamburgh and E. J. W. Barrington, Eds.), pp. 465–471. Appleton-Century-Crofts, New York.

Naismith, D. J., Richardson, D. P., and Pritchard, A. E. (1982). The utilization of protein and energy during lactation in the rat, with particular regard to the use of fat accumulated during pregnancy. Br. J. Nutr. 48, 433–441.[ISI][Medline]

Nataf, B., and Sfez, M. (1961). Debut du fonctionement de la thyroide foetale du rat. C. R. Soc. Biol. 55, 1235–1238.

O’Flaherty, E. J., Scott, W., Schreiner, C., and Beliles, R. P. (1992). A physiologically based kinetic model of rat and mouse gestation: Disposition of a weak acid. Toxicol. Appl. Pharmacol. 112, 245–256.[ISI][Medline]

Palou, A., Remesar, X., Arola, L., and Alemany, M. (1983). Body and organ size and composition during late fetal and postnatal development of rat. Comp. Biochem. Physiol. A 75, 597–601.[CrossRef][ISI][Medline]

Pena, H. G., Kessler, W. V., Christian, J. E., Cline, T. R., and Plumlee, M. P. (1976). A comparative study of iodine and potassium perchlorate metabolism in the laying hen. 2. Uptake, distribution, and excretion of potassium perchlorate. Poult. Sci. 55, 188–201.[ISI][Medline]

Porterfield, S. P. (1994). Vulnerability of the developing brain to thyroid abnormalities: Environmental insults to the thyroid system. Environ. Health Perspect. 102(Suppl. 2), 125–130.

Rakusan, K., and Marcinek, H. (1973). Postnatal development of the cardiac output distribution in rat. Biol. Neonate. 22, 58–63.[ISI][Medline]

Riedel, C., Dohan, O., De la Vieja, A., Ginter, C. S., and Carrasco, N. (2001a). Journey of the iodide transporter NIS: From its molecular identification to its clinical role in cancer. Trends Biochem. Sci. 26, 490–496.[CrossRef][ISI][Medline]

Riedel, C., Levy, O., and Carrasco, N. (2001b). Post-transcriptional regulation of the sodium/iodide symporter by thyrotropin. J. Biol. Chem. 276, 21458–21463.[Abstract/Free Full Text]

Roti, E., Gnudi, A., and Braverman, L. E. (1983). The placental transport, synthesis and metabolism of hormones and drugs which affect thyroid function. Endocr. Rev. 4, 131–149.[ISI][Medline]

Scatchard, G., and Black, E. S. (1949). The effects of salts on the isoionic and isoelectric points of proteins. J. Phys. Colloid Chem. 53, 88–99.[ISI]

Schneidereit, M. (1985). Study of fetal organ growth in Wistar rats from day 17 to 21. Lab. Anim. Lond. 19, 240–244.

Shishiba, Y., Shimizu, T., Yoshimura, S., and Shimizu, K. (1970). [Effect of thiocyanate and perchlorate on free thyroxine fraction]. Nippon. Naibunpi. Gakkai. Zasshi. 46, 16–19.[Medline]

Sikov, M. R., and Thomas, J. M. (1970). Prenatal growth of the rat. Growth 34, 1–14.[ISI][Medline]

Spitzweg, C., Joba, W., Eisenmenger, W., and Heufelder, A. E. (1998). Analysis of human sodium iodide symporter gene expression in extrathyroidal tissues and cloning of its complementary deoxyribonucleic acids from salivary gland, mammary gland, and gastric mucosa. J. Clin. Endocrinol. Metab. 83, 1746–1751.[Abstract/Free Full Text]

Sztanyik, L. B., and Turai, I. (1988). Modification of radioiodine incorporation in the fetus and newborn rats by the thyroid blocking agents. Acta Phys. Hungaria 72, 343–354.

Tazebay, U. H., Wapnir, I. L., Levy, O., Dohan, O., Zuckier, L. S., Zhao, Q. H., Deng, H. F., Amenta, P. S., Fineberg, S., Pestell., R. G., et al. (2000). The mammary gland iodide transporter is expressed during lactation and in breast cancer. Nat. Med. 6, 871–878.[CrossRef][ISI][Medline]

Urbansky, E. T. (1998). Perchlorate chemistry: Implications for analysis and remediation. Bioremediation Journal 2, 81–95.

Urbansky, E. T., and Schock, M. R. (1999). Issue in managing risks associated with perchlorate in drinking water. J. Environ. Manag. 56, 79–95.[CrossRef][ISI]

USEPA. (2002). Report on the Peer Review of the U. S. Environmental Protection Agency’s Draft External Review Document. USEPA EPA/635/R-02/003. 01 Jun 2002. U.S. Environmental Protection Agency, Washington, DC, 412.

Versloot, P. M., Schroder-van der Elst, J. P., van der Heide, D., and Boogerd, L. (1997). Effects of marginal iodine deficiency during pregnancy: Iodide uptake by the maternal and fetal thyroid. Am. J. Physiol. 273, E1121–E1126.[ISI][Medline]

Wolff, J. (1964). Transport of iodide and other anions in the thyroid gland. Physiol. Rev. 44, 45–90.[Free Full Text]

Wolff, J. (1998). Perchlorate and the thyroid gland. Pharmacolog. Rev. 50, 89–105.[Abstract/Free Full Text]

Wolff, J., and Chaikoff, I. L. (1948). Plasma inorganic iodide as a homeostatic regulator of thyroid function. J. Biol. Chem. 174, 555–564.[Free Full Text]

Wolff, J., and Maurey, J. R. (1963). Thyroidal iodide transport: IV. The role of ion size. Biochim. Biophys. Acta 69, 48–58.[CrossRef][ISI][Medline]

Yamada, T. (1967). Effects of perchlorate and other anions on thyroxin metabolism in the rat. Endocrinology 81, 1285–1290.[ISI][Medline]

Yu, K., Mahle, D., Narayanan, L., Godfrey, D., Todd, P., Parish, M., McCafferty, J., Ligman, T., Sterner, T., and Buttler, G. (2001). Tissue distribution and inhibition of iodide uptake by perchlorate in pregnant and lactating rats in drinking water studies. Toxicol. Sci. 60(1-S), 291 (Abstract).[CrossRef]

Yu, K. O., Mahle, D. A., Narayanan, L., Buttler, G. W., Todd, P. N., Parish, P. N., McCafferty, J. D., Ligman, T. A., Sterner, T. R., and Bausman, T. A. (2002a). Kinetics of perchlorate-induced inhibition of iodide uptake in tissues of the pregnant rat and fetus. Toxicol. Sci. 66(1-S), 139 (Abstract).[Abstract/Free Full Text]

Yu, K. O., Narayanan, L., Mattie, D. R., Godfrey, R. J., Todd, P. N., Sterner, T. R., Mahle, D. A., Lumpkin, M. H., and Fisher, J. W. (2002b). The pharmacokinetics of perchlorate and its effect on the hypothalamus/pituitary-thyroid axis in the male rat. Toxicol. Appl. Pharmacol. 182(2), 148–159.[CrossRef][ISI][Medline]

Zeghal, N., Redjem, M., Gondran, F., and Vigouroux, E. (1995). Analysis of iodine compounds in young rat skin in the period of suckling and in the adult. Effect of perchlorate. Arch. Physiol. Biochem. 103, 502–511.[ISI][Medline]