Predicting Neonatal Perchlorate Dose and Inhibition of Iodide Uptake in the Rat during Lactation Using Physiologically-Based Pharmacokinetic Modeling

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

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

Received March 5, 2003; accepted May 1, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Perchlorate (ClO4-), a contaminant in drinking water, competitively inhibits active uptake of iodide (I-) into various tissues, including mammary tissue. During postnatal development, inhibition of I- uptake in the mammary gland and neonatal thyroid and the active concentration ClO4- in milk indicate a potentially increased susceptibility of neonates to endocrine disruption. A physiologically based pharmacokinetic (PBPK) model was developed to reproduce measured ClO4- distribution in the lactating and neonatal rat and predict resulting effects on I- kinetics from competitive inhibition at the sodium iodide symporter (NIS). Kinetic I- and ClO4- behavior in tissues with NIS (thyroid, stomach, mammary gland, and skin) was simulated with multiple subcompartments, Michaelis-Menten (M-M) kinetics and competitive inhibition. Physiological and kinetic parameters were obtained from literature and experiment. Systemic clearance and M-M parameters were estimated by fitting simulations to tissue and serum data. The model successfully describes maternal and neonatal thyroid, stomach, skin, and plasma, as well as maternal mammary gland and milk data after ClO4- exposure (from 0.01 to 10 mg/kg-day ClO4-) and acute radioiodide (2.1 to 33,000 ng/kg I-) dosing. The model also predicts I- uptake inhibition in the maternal thyroid, mammary gland, and milk. Model simulations predict a significant transfer of ClO4- through milk after maternal exposure; approximately 50% to 6% of the daily maternal dose at doses ranging from 0.01 to 10.0 mg ClO4-/kg-day, respectively. Comparison of predicted dosimetrics across life-stages in the rat indicates that neonatal thyroid I- uptake inhibition is similar to the adult and approximately tenfold less than the fetus.

Key Words: PBPK model; lactation; perchlorate; iodide; inhibition; milk.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Perchlorate (ClO4-), the soluble anion of ammonium and potassium perchlorate, is a thyroid iodide uptake inhibitor known to be present in several United States drinking water sources, ranging in concentrations from less than 4 ppb to more than 3700 ppm in some Las Vegas water samples (Motzer, 2001Go). Human health concerns arise from the fact that ClO4-, being similar in size and shape to iodide (I-), is able to bind to the sodium-iodide symporter (NIS), thus reducing the amount of iodide taken up into the thyroid. Thyroid hormones are synthesized from I- in the thyroid and are responsible for regulating metabolism. In the adult, lack of iodide causes reduced thyroxine (T4) and triiodothyronine (T3) levels and can eventually lead to hypothyroidism (Wolff, 1998Go). Furthermore, since these hormones are required for normal physical and mental development, exposure to thyroid inhibitors during the period of rapid growth in late gestation and early infancy could result in long-term consequences (Howdeshell, 2002Go; Porterfield, 1994Go). Congenital hypothyroidism and gestational iodide deficiency are known to cause delayed development and, in severe cases, lowered IQ, mental retardation, and even cretinism (Delange, 2000Go; Haddow et al., 1999Go; Howdeshell, 2002Go; Klein et al., 1972Go; Porterfield, 1994Go). In the case of perchlorate contamination of drinking water sources, the question is whether ClO4- is capable of inducing these same developmental effects and at what level of exposure the fetus or infant may be at risk of adverse effects.

In order to help answer these questions, several studies have been conducted in rats involving chronic and short-term perchlorate exposure during gestation, lactation, and in adult males at a variety of doses (Bekkedal et al., 2001Go; Mahle et al., 2002Go, 2003Go; York et al., 1999Go, 2001Go; Yu et al., 2002Go). None of the available studies show the same extent of adverse effects from perchlorate exposure as are known to occur in iodide deficiency. However, consolidating these various data sets into a quantitative measure of risk to the perchlorate-exposed infant is quite difficult due to the variations in study design, as well as the rapid physical and biochemical changes taking place during lactation and infancy. To incorporate these kinetic, physiological, and biochemical data into a predictive tool for perchlorate, iodide, and perchlorate-induced inhibition kinetics, a physiologically based pharmacokinetic (PBPK) model was developed in the lactating and neonatal rat. Together with the concurrent PBPK models developed for the male rat (Merrill et al., 2003Go), pregnant and fetal rat (Clewell et al., 2003Go), and the adult human (Merrill et al., 2001Go), the models can be used to compare internal dose metrics, such as ClO4- concentration in the serum across developmental life stages and species. Thus, the models provide a means for extrapolating predicted kinetics and measures of dose to the potentially more sensitive and often overlooked subpopulation, the human fetus and infant (Clewell and Gearhart, 2002aGo).

Although the hormone feedback system of the postnatal rat is independent of the mother (Howdeshell, 2002Go; Potter et al., 1959Go; Vigouroux and Rostaqui, 1980Go), the effect of maternal perchlorate exposure is intricately tied to neonatal risk. In fact, there are several unique factors that must be accounted for when attempting to quantify risk to the nursing neonate. For example, the lactating mammary gland contains active NIS that concentrates iodide in the milk, ensuring an adequate supply of iodide to the newborn. However, since ClO4- competitively inhibits iodide binding to NIS, maternal exposure during lactation also inhibits iodide transfer in the milk, as has been noted in several species including the rat, goat, rabbit, and cow (Brown-Grant, 1957Go; Cline et al., 1969Go; Grosvenor, 1963Go; Lengemann, 1965Go; Potter et al., 1959Go). It is also possible that this binding of perchlorate to NIS, which inhibits iodide uptake, is responsible for concentration of ClO4- in milk. Intra-laboratory studies have shown milk ClO4- levels to be consistently higher than the maternal plasma, and the neonate was found to have significant blood ClO4- concentrations after nursing from the exposed dams (Yu et al., 2001Go). Thus, the infant would be at risk not only from the diminished iodide intake from the milk, but also from the significant doses of ClO4- received from the milk, and the resulting additional inhibition of iodide uptake at the neonatal thyroid NIS. The PBPK model described here is able to account for the changing physiology of the lactating dam and pup, as well as the resulting impact of these physiological changes on experimentally determined iodide and perchlorate kinetics in order to provide a meaningful, quantitative estimate of two previously uncharacterized determinants of neonatal risk: the relative ClO4- dose to maternal and neonatal rats, and the dose-response relationship between perchlorate exposure and iodide inhibition in the maternal thyroid and milk. At this stage, the model includes only a rudimentary description of endogenous I- kinetics and incorporation into thyroid hormones, sufficient to reproduce the kinetics of radioiodide.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Supporting Experiments
All supporting experiments were performed on timed-pregnant rats and pups of the Sprague-Dawley strain (Crl: CD, Charles River Laboratory, Raleigh, NC). Animals were housed in individual light, heat, and humidity controlled cages and were kept on a 12 h light/dark cycle with access to water and food ad libitum. Euthanization was performed by CO2 asphyxiation on either postnatal day (PND) 5 or 10 and tissues were collected for analysis of ClO4- or radioiodide content using the methods described in Narayanan et al.(2003)Go, respectively. In all experiments, pup serum was pooled by sex within individual litters due to small sample volumes. Pup skin, gastrointestinal (GI) tract, GI contents, and maternal tissues were analyzed individually. 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.

Perchlorate drinking water study.
Pregnant dams (n = 12 per group) were given either deionized water or water containing perchlorate from gestational day (GD) 2 through the day of sacrifice. Daily measurements of body weight and water intake were taken to ensure consistent dosing at levels of 0.0, 0.01, 0.1, 1.0, and 10.0 mg ClO4-/kg-day. Maternal serum, thyroid and milk, and neonatal serum were collected from the PND 10 groups and analyzed for ClO4- content. On PND 5, six of the dams from each dosing regimen were given a tail vein injection of 33 µg/kg 125I- 2 h prior to sacrifice. Maternal and neonatal serum, skin, GI contents and GI tract, as well as maternal thyroid and mammary gland, were collected from these PND 5 rats and analyzed for ClO4- and 125I-. Thyroid hormones (free and total T4 and T3) and TSH were also measured in the serum of PND 5 rats that were not dosed with radioiodide.

Radioiodide and inhibition kinetic studies.
Lactating dams (six control, six inhibition) were dosed via tail vein injection to 125I- (average dose = 2.10 ng/kg) on PND 10 and euthanized at 0.5, 2, 4, 8, and 24 h postdosing. Two h prior to the administration of 125I-, dams from the inhibition group were given a 1.0 mg/kg ClO4- iv and euthanized at 0.5, 1, 2, 4, 8, 12, and 24 h post-125I- dosing. This particular ClO4- dose was chosen to be large enough dose to significantly affect iodide uptake, based on inhibition of thyroid iodide uptake 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). Maternal and neonatal serum, skin, GI contents and GI tract, and maternal thyroid and mammary gland tissue were collected from both control and perchlorate dosed groups and analyzed for 125I- with a gamma counter.

Direct pup radioiodide dosing.
PND 10 Sprague-Dawley pups (n = 6) were given a po gavage of 125I- (0.001mg/kg) in water. Pups and nursing dams were euthanized at 0.5, 1, 2, 4, 8, and 24 h postdosing. Neonatal serum, thyroid, skin, GI contents and GI tract, as well as maternal serum and thyroid were harvested and analyzed for radioiodide content.

Model Structure
All model code was written in ACSL (Advanced Continuous Simulation Language, Aegis Technologies Group, Inc., Huntsville, AL). The model structure is based on those of the male rat and gestation models for iodide and perchlorate kinetics developed concurrently by Merrill et al.(2003)Go and Clewell et al.(2003)Go. The maternal model (Fig. 1AGo) consists of compartments for plasma, thyroid, skin, GI, kidney, liver, fat, mammary gland and milk, plus compartments for the combined slowly and richly perfused tissues. The thyroid, GI and mammary gland are described with three subcompartments representing the stroma, follicle and colloid in the thyroid, the capillary bed, GI tissue and GI contents in the GI, and the capillary blood, tissue, and milk in the mammary gland. For iodide, additional compartments representing the organified (hormone-bound) iodine were included in the thyroid and serum (Fig. 1BGo). Skin is 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, milk, and GI contents, was described with Michaelis-Menten (M-M) type kinetics for saturable processes (bold arrows in Fig. 1Go). Permeability area cross products 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 with partitions and blood flows. Plasma binding of the inorganic anions (I- and ClO4-) was simulated using a saturable term for association of the anions to binding sites in the plasma and a first order clearance rate for dissociation from plasma binding sites. Urinary clearance and transfer of anions between the dam and pup were represented by first order clearance rates.



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FIG. 1. (A) Schematic of perchlorate PBPK model for lactating dam (left) and neonate (right). Model structure for sister iodine model is similar. Differences are contained within the inset area outlined with the dashed line. (B) Thyroid and serum compartments for radioiodide, with additional compartments for the incorporation of iodide into hormones in the thyroid and secretion of the hormones into the serum incorporated iodine compartment as is discussed in the Methods section.

 
Due to the short time frame of the radioiodide experiments, the kinetic behavior of total radioiodine was assumed to behave as free iodide in all compartments other than the thyroid and plasma. Inorganic iodide and perchlorate were then modeled in the same manner, based on the similar size and charge of the ions and their shared affinity for NIS (Wolff, 1998Go). The thyroid, skin, GI and mammary gland contain active NIS and were therefore defined separately in the structure of the model (Kotani et al., 1998Go; Spitzweg et al., 1998Go). The thyroid, mammary gland, milk, skin and GI contents have also been found to maintain higher concentrations of ClO4- and I- than the plasma (Brown-Grant, 1961Go; Brown-Grant and Pethes, 1959Go; Chow et al., 1969Go; Halmi and Stuelke, 1959Go; Wolff, 1998Go; Zeghal et al., 1995Go). 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), the small amounts of anions present in these tissues do not affect plasma concentrations. These tissues were therefore combined in the richly or slowly perfused compartments.

In order to describe free thyroidal iodide, it was necessary to account for the incorporation of iodide into hormones in the thyroid and the secretion of this incorporated iodine into the blood. Our goal was to describe this process with enough detail to predict time course data, while keeping the model as parsimonious as possible. Thus, thyroid hormone production, or the incorporation of iodide into hormones, is described using a first order clearance (ClProdci) of inorganic iodide from the thyroid follicle to the incorporated thyroid iodine compartment. Secretion of the incorporated iodine into the plasma is also described with a first order clearance (ClSecrci) from the incorporated thyroid iodine compartment to the incorporated plasma iodine compartment (Fig. 1BGo). It was not necessary to include a description of hormone incorporation in the perchlorate model, since ClO4- is not organified or metabolized in vivo (Anbar et al., 1959Go).

The incorporated plasma iodine compartment represents combined plasma hormonal iodine, including free T3 and T4 as well as protein bound T3 and T4, but does not attempt to predict individual hormone kinetics. Studies of iodine distribution from dietary intake suggest that the majority (>80%) of serum iodine is, in fact, incorporated into hormones or bound to plasma proteins (Stolc et al., 1973bGo). In contrast, studies in our laboratory found that inorganic iodide accounts for approximately 80% of the total measured plasma radioiodine up to 24 h after an administered 125I- dose (Mahle et al., 2002Go). This apparent discrepancy may be explained by the slow incorporation of administered or ingested iodide into hormones over time. Endogenous serum iodine data primarily reflect hormone-incorporated iodine, since the system is at steady state. This normal iodine turnover is well established in the animal when the radioiodide is introduced. However, the kinetics reflected in the radiolabeled iodide time course is primarily due to binding of the free anion to plasma proteins, uptake of the anion into various tissues, and urinary clearance. Hormone incorporation would have little effect on these radioiodide kinetics, due to the long time-frame required to incorporate the radioiodide into hormones in the thyroid and the slow secretion of these newly produced radiolabeled hormones into the serum. The model is able to reconcile these data by including an incorporated iodine compartment in the plasma, into which the hormone-incorporated iodine enters as it is secreted from the thyroid. A generic first order rate (ClDeiodci) is then used to describe the overall deiodination accomplished in the various tissues, allowing the inorganic iodide to re-enter the free plasma compartment.

Some description of plasma binding was required for both anions in order to adequately reproduce the available data. The inclusion of binding to plasma proteins is especially important in the case of perchlorate. In fact, at low serum concentrations (<=100 µg/l), approximately 99% of the anion is bound to plasma proteins and at higher concentrations (>=500 µg/l), 50% is bound (J. W. Fisher, personal communication). Binding of perchlorate to plasma proteins has also been measured in both human and bovine serum (Carr, 1952Go; Scatchard and Black, 1949Go). Iodine also binds to plasma proteins, but to a lesser extent than ClO4-. Therefore, the model includes a description for the binding of I- and ClO4- to plasma proteins. Competition of the two anions for plasma binding sites was also included in the model.

In addition to the reported presence of NIS in the mammary gland (Spitzweg et al., 1998Go), studies of perchlorate-induced inhibition of iodide uptake in milk and mammary tissue support the conclusion that a transport mechanism similar to that of the thyroid exists in the mammary gland (Brown-Grant, 1957Go; Grosvenor, 1963Go; Potter et al., 1959Go). Furthermore, hormones produced during lactation, such as prolactin, regulate the mammary gland NIS activity (Tazebay et al., 2000Go). Shennan and Peaker (2000)Go also found evidence of a second anion transport mechanism in the secretory cells of the mammary gland, suggesting that this transporter is also able to move iodide and perchlorate against a concentration gradient. This second anion channel is represented in the model as active uptake into the milk compartment.

The kidney and liver were separately defined within the structure of the model in order to describe the rapid urinary clearance of the anions and to allow for future elaboration of the model that would address hormone metabolism in the liver. A fat compartment was also included to account for the possible effect of changing fat volume on the kinetics due to the hydrophilic nature of both anions. 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 (Chow and Woodbury, 1970Go).

The basic structure of the neonatal model (Fig. 1AGo) is similar to that of the lactating rat, excluding the mammary gland. In order to simplify the model, all pups from a single litter were combined together within the model structure. Neonatal dose is described as a first order transfer rate between the maternal milk and pup GI contents. The anions are then recirculated to the mother through the pup urine based on the work of Samel and Caputa (1965)Go, showing that lactating dams ingest approximately 60% of the neonate’s iodine dose while grooming their pups.

Perchlorate-induced inhibition of iodide uptake was included in the maternal and neonatal thyroid follicle and colloid, GI contents and skin, as well as the maternal mammary gland and milk. Literature sources have reported inhibition of iodide uptake into gastric juice of the male rat (Halmi and Stuelke, 1959Go) and the milk of the lactating rat (Brown-Grant, 1957Go; Grosvenor, 1963Go; Potter et al., 1959Go). Studies in our laboratory have also shown consistent evidence of significant inhibition of iodide uptake in neonatal GI and skin, and slight inhibition in the maternal skin and mammary gland (Mahle et al., 2002Go).

Dosing Procedures
In order to simulate the daily dosing regimen of the perchlorate drinking water experiment, a pulse function in ACSL was used to introduce drinking water to the GI contents of the lactating dam at a constant rate for 12 h per day (1800 to 0600 h). The neonate was dosed continuously throughout the day from the maternal milk. Both the pup milk dose and the oral bolus dose were introduced into the GI contents of the neonate utilizing pulse functions. Tail vein injections were simulated by introducing the anions into the iv serum compartment. Dosing for the dietary iodine studies (Stolc et al., 1973aGo,bGo) was based on the iodine content of the feed and supplemented water, as well as published water, milk, and dietary intake data (Stolc et al., 1966Go) in both the maternal and neonatal rat throughout the postnatal time period, assuming a constant (24 h/day) intake for both dam and neonate.

Model Parameters
Model equations are described in the Appendix. Whenever possible, physiological and kinetic parameters were obtained from literature or experiments. Allometric scaling was generally employed to account for differences in parameters due to variations in body weight of male, female, and neonatal rats. Tissue volumes were scaled linearly by body weight (BW). Blood flows, maximum velocities, permeability area cross products (PA), and clearance values were scaled by BW0.75. Pup values were scaled in a similar manner to the maternal parameters. Physiological and chemical-specific parameters were scaled first by the body weight of an individual pup, as described above, and were then multiplied by the total number of neonates to represent the value for the entire the litter.

Physiological parameters.
The physiological description of maternal and neonatal rats during lactation is based on the work of Fisher et al.(1990)Go, measurements from our laboratory and published physiological data. Due to the nonuniform changes in tissue volume and body weight during lactation, it was necessary to include gender and life stage-specific physiological descriptions whenever possible. Parameters that were not available specifically for the lactating female or neonate were described by adjusting male rat values by body weight. Final values for the physiological parameters and sources from which they were obtained are listed in Table 1Go.


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TABLE 1 Physiological Parameters
 
Lactation-specific changes in tissue volumes and suckling rates were included in the model using ACSL TABLE functions, which employ linear interpolation between available data points. Maternal body weight was assumed to increase by 12% between PND 1 and 10, based on the daily measurements of dams in the perchlorate drinking water study described previously. Since there was no significant difference in the average daily body weights between the five dose groups, the average body weight from all doses were used. The relative volume of the mammary tissue increased from 4.4% on PND 2 to 5.6, 6.3, and 6.6% of the maternal body weight on PND 7, 14, and 21, respectively (Knight et al., 1984Go). Maternal body fat increased from 12.4 to 15.2% of the body weight between parturition and PND 2, with a subsequent decrease to 6.9% of the body weight from PND 2 to 16 (Naismith et al., 1982Go). Values for body fat content on PND 2 and 16 were taken from the measured values of Naismith et al.(1982)Go. Maternal body fat at parturition was calculated from the previously developed PBPK model for the pregnant rat (Clewell et al., 2003Go). Thus, the relative volume of the maternal body fat in the model increases slightly between PND 1 and 2 and then decreases after PND 2. The rate of milk production was assumed to be equal to the suckling rate described below.

As in the maternal tissues, TABLE functions were used to interpolate between reported data points for the changing body weight, suckling rate, and relative tissue volumes in the neonate. Growth of the neonate is directly dependent on the ingestion of milk.Stolc et al.(1966)Go measured both pup body weight and the milk ingestion of suckling rats from birth through weaning. Although the author used a different strain of rats than was used in our studies, the pup body weights were nearly identical between the two studies. Thus, the more comprehensive, published data set of Stolc et al.(1966)Go was used for these physiological parameters. Values for neonatal body fat were based on the data of Naismith et al.(1982)Go, showing a rapid increase from 2.7 to 11% BW between PND 2 and 16 and a subsequent decrease to the adult value of 4.61% (Brown et al., 1997Go).

Increase in neonatal thyroid volume was based on the work of Florsheim et al.(1966)Go, who reported relative thyroid volumes of 0.013, 0.015, 0.012, 0.014, 0.013, 0.013, and 0.013% body weight for neonates on PND 1 through 5, 7, and 11, respectively. The model also describes changing thyroid stroma, follicle, and colloid fractions over time based on the work of Conde et al.(1991)Go, who measured the fractional volumes on postnatal days 0, 5, 10, 15, 20, 25, 30, 60, and 120. Relative tissue volumes of the skin, GI tract, liver, and kidney were modeled based on measured body and tissue weights on PND 1, 5, 10, 20, 30, and 64 (Palou et al., 1983Go). Relative skin volume increased from 19.3 to 20.8% BW from PND 1 to 20 and then decreased to 19% BW by PND 30. Similar trends were also seen in the GI, kidney, and liver, with increasing volume (with respect to body weight) peaking at PND 30, 20, and 30, respectively.

All maternal blood flows that were not directly affected by the changes induced by lactation were scaled allometrically from the adult male rat parameters. TABLE functions were used to describe the changing in cardiac output and fractional blood flow to the mammary tissue throughout lactation, according to the data of Hanwell and Linzell (1973)Go and the neonatal cardiac output, hematocrit, and regional blood flows, based on the data of Rakusan and Marcinek (1973)Go. Among other tissues, Rakusan and Marcinek (1973)Go measured fractional blood flows to the kidney, liver, skin, stomach, and large and small intestines in 1, 30, 60, and 140 day-old rats. Relative blood flow to the neonatal kidney, liver, and total GI are modeled as increasing from 3.6, 4.5, and 4.6 to 15.5, 5.2, and 6.8% cardiac output, respectively, in the first 60 days. Blood flow to the skin remains relatively constant after birth, at approximately 11% of the cardiac output.

Chemical-specific parameters.
Chemical-specific parameters (Table 2Go) for perchlorate and iodide in tissues other than the mammary gland were kept as similar as possible to those used in the PBPK models for the male and pregnant rat (Clewell et al., 2003Go; Merrill et al., 2003Go) in order to facilitate comparison of the models and extrapolation between life-stages. As in the previous models, binding of I- to NIS was given a Km of 4.0 x 106 ng/l based on the work of Gluzman and Niepomniszcze (1983)Go in human thyroid slices. 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 NIS. The second transporter located at the apical membrane in the thyroid was studied by Golstein et al.(1992)Go, who measured a Km of approximately 4.0 x 109 ng/l for iodide (KmTLi) in bovine thyroid. In the model, a somewhat 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. The Km for the second active transport mechanism in the mammary gland (KmMk), for milk uptake, was set by fitting the model simulation to available mammary gland and milk data.


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TABLE 2 Chemical-Specific Parameters
 
The Km value for ClO4- transport by NIS was given a value of 1.5 x 105 ng/l for all relevant compartments. This value is based on the assumption that perchlorate acts as a competitive inhibitor of iodide uptake and is, in fact, transferred into the tissues via NIS (Clewell and Gearhart, 2002bGo). Therefore, the Km value for perchlorate transport by NIS would be equal to its Ki value. Kosugi et al.(1996)Go measured the Ki for ClO4- at 1.5 x 105 ng/l. This value was adjusted slightly to obtain the best fit of thyroid perchlorate to the drinking water data, resulting in a Km of 2.0 x 105ng/l. 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). Likewise, the Km values for perchlorate transport by the second anion channels in the thyroid and mammary gland were also nearly a factor of 10 less than that of iodide, resulting in values of 1.0 x 108 and 1.0 x 106 ng/l for KmTLp and KmMkp, respectively.

The values for Vmax vary significantly across species and tissues with NIS (Gluzman and Niepomniszcze, 1983Go; Wolff, 1998Go) and were, therefore, determined by fitting the model simulations to available data in the various tissues of PND 5 and 10 rats. In the perchlorate drinking water study, the nonlinearity of tissue ClO4- concentrations across doses in compartments with NIS suggests that the symporter is saturated between the 1.0 and 10.0 mg/kg-day doses. Thus at doses below saturation (<=1.0 mg ClO4-/kg-day), the active transport via NIS would drive tissue concentrations and were therefore used to set Vmax values. For iodide, kinetic data were taken at doses well below the saturation of NIS. Thus, the time course data from the kinetic studies were used to determine values for Vmax in tissues with active uptake.

Partitioning of ClO4- and I- into tissues results from the electrochemical potential present across tissue membranes (Chow and Woodbury, 1970Go). Theoretical effective partition coefficients were calculated from measured electrical potentials presented by Chow and Woodbury (1970)Go using the equations given in Kotyk and Janacek (1977)Go. Calculations are described in detail in the male rat perchlorate model (Merrill et al., 2003Go). Ranges for the partition coefficients corresponding to the stroma:follicle and follicle:lumen membrane diffusion were estimated to be 0.11 to 0.15 and 6.48 to 8.74, respectively. Based on the fit of the model simulation to the data and the calculated values above, values of 0.15 and 7.0 were used for PTFi and PTLi, respectively.

As mentioned previously, the perchlorate drinking water data indicate that NIS transport is saturated between the 1.0 and 10.0 mg ClO4-/kg-day doses. Therefore, at 10.0 mg/kg-day, ClO4- uptake into the various tissues would be predominantly determined by the passive diffusion parameters. Thus, parameters describing partitioning of ClO4- into the tissues were obtained by fitting the model simulation to the highest dose group data from the drinking water study. In the cases where data were not available in the lactating or neonatal rat, such as the muscle (slowly perfused), liver (richly perfused), kidney, and red blood cells, values were obtained from those used in the adult male rat model (Merrill et al., 2003Go). The partition coefficient for perchlorate in fat was measured in the laying hen (Pena et al., 1976Go). Other tissues in the hen, such as the muscle and kidney, were found to have similar partition coefficients to those of the rat.

Iodide partition coefficients and PA values were calculated from the tissue:blood ratios measured during the clearance phase of data for the tissue of interest either from literature or experimental data in the rat. The partitioning parameters for the muscle (slowly perfused), liver (richly perfused), kidney, and red blood cells were given the same values measured in the male rat (Merrill et al., 2003Go) and the value for partitioning of iodide into the fat was given the same value as ClO4-.

Parameters for plasma binding were determined by fitting the model to time course data in the case of iodide and the 0.01 and 0.1 mg/kg-day drinking water data in the case of ClO4-, due to the fact that binding was most prevalent at the lower doses. Urinary clearance of ClO4- was determined from fitting the serum at the 10.0 mg/kg-day dose group, where binding had little effect on serum concentrations. First-order clearances for incorporation of iodide into thyroid hormones and hormone secretion were determined by the fit of the model to the incorporated and free thyroid iodide time course data. Parameters for binding of inorganic iodide to plasma proteins were determined by the fit of the model simulation to initial portion of the serum radioiodine data. Later time points were assumed to be more affected by hormone secretion and deiodination rates, as sufficient time had passed to allow for incorporation of the administered radioiodide into hormones. Urinary iodide clearance was determined from the fit of the model to serum inorganic iodide time course data.

Upregulation of thyroid NIS activity.
At the time of data collection in the drinking water study, rats had been exposed to ClO4- throughout gestation and up to the day of sacrifice. At this point, upregulation of the thyroid activity is evidenced by decreased T4 and elevated TSH levels in the serum at all doses, as well as a lack of noticeable thyroid iodide uptake inhibition (Yu et al., 2001Go). Since increased TSH upregulates thyroid iodide uptake by increasing the number and activity of NIS (Wolff, 1998Go), the value for VmaxcTFi, which corresponds to the maximum capacity of 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 the data were fitted with an M-M equation. This equation then was used in the model to describe the induction of NIS upregulation with dose, in a similar manner to the description used by Andersen et al.(1984)Go to describe enzyme 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 VmaxcTFp with dose and applying the resulting M-M fit to the model.

Dietary iodine model.
The dietary iodine model is identical to that of the radioiodine model. Dosing is accomplished as a constant intake of iodine through water and diet. All chemical-specific parameters are assumed to be the same as those determined from the radioiodide kinetics. The radioiodide, endogenous iodine and perchlorate models operate independently in tissues with passive diffusion, and are linked through competitive uptake in tissues with NIS (e.g., thyroid follicle). Thus, in tissues governed by passive diffusion, tissue:blood ratios are identical for the radiolabeled and dietary iodine models. The interaction of the three models through competitive inhibition at NIS allows us to explore the effect of varying dietary intake on both radiolabeled iodine kinetics and perchlorate-induced inhibition of radioiodide uptake in the thyroid.

Sensitivity analysis of chemical-specific parameters.
A sensitivity analysis was run after finalizing the model parameters as to examine the relative influence of each of the chemical-specific parameters on model predictions. The model was run to determine the change in the average serum ClO4- concentration (AUC: area under the curve) and total thyroid iodide uptake at 8 h postdosing, 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 (0.1 and 10.0 mg-kg-day, respectively). Since the iodide doses used in this model are not expected to saturate the NIS, thyroid iodide sensitivity analysis was run only at the dose used in the kinetic experiments (2.1 ng/kg 125I-). 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
Model Parameterization
The ClO4- data from the drinking water study were used to determine kinetic parameters for ClO4- in the lactating and neonatal rat. Upregulation of NIS transport of ClO4- into the thyroid was accounted for as described in the Methods section. Figure 2Go shows the model simulations for ClO4- concentrations in the maternal and neonatal serum, maternal thyroid, GI contents, mammary gland, and milk, versus measured data from the drinking water study on PND 5 and 10 at 0.01, 0.1, 1.0, and 10.0 mg ClO4-/kg-day. The upregulated values for Vmaxc_TFp were used in the simulation of the thyroid as described in the Methods section. In these and subsequent plots, solid lines indicate the model prediction and cross-bars indicate the mean ± SD of measured data.



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FIG. 2. Perchlorate concentration in maternal (A) serum (Clewell et al., 2001Go), (B) thyroid (upregulated) (Clewell et al., 2001Go), (C) GI contents, (D) mammary gland, (E) milk (Clewell and Gearhart, 2002aGo), and (F) male neonatal serum (Clewell and Gearhart, 2002aGo) at the 0.01, 0.1, 1.0, and 10.0 mg/kg-day doses on PND 5 and 10. Solid lines indicate model prediction. Cross-bars indicate mean ± SD of the measured data.

 
Neonatal iodide kinetic parameters were determined by the fit of the model to the data obtained from directly dosing the pup, while maternal parameters were primarily determined from the data obtained by dosing the dam via iv. Transfer from the neonate to the dam was established by fitting the maternal kinetic parameters first and then utilizing maternal data from the pup dosing study to determine the magnitude of iodide transfer to the maternal stomach via pup urine. In the same way, milk transfer was parameterized by first fitting the neonatal parameters from the pup dosing study and then utilizing the pup data from the maternal dosing kinetic study to estimate pup exposure via milk. 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. Model simulations of radioiodide kinetics in maternal and neonatal tissues versus the data obtained from dosing the dams are shown as the control group in the inhibition study (Fig. 3Go). The fit of the model to iodide levels in maternal and neonatal tissues from the direct oral dosing of PND 10 pups are shown in Figure 4Go. The model was able to reproduce data in both maternal and neonatal tissues, whether exposure occurred via maternal iv or oral bolus to the pup. Thus, the model is able to describe both maternal to neonatal and neonatal to maternal iodide transfer, and is also able to reproduce data across exposure routes.



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FIG. 3. Concentration of maternal (A) inorganic radioiodide in serum, (B) plasma bound radioiodine, (C) thyroidal inorganic radioiodide and incorporated radioiodine, (D) mammary gland, and neonatal (E) serum after an iv dose to the dam of 2.10 ng/kg 125I- on PND 10.

 


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FIG. 4. Concentration of neonatal (A) plasma bound radioiodine and plasma inorganic radioiodide, (B) thyroidal incorporated radioiodine and inorganic radioiodide, (C) GI contents (filled circles) and skin (open squares) radioiodide, and (D) maternal total serum and thyroid radioiodine after an oral gavage of 1.0 ng/kg 125I- to the pup on PND 10.

 
Model validation.
Once model parameters were established as described above, the robustness of the model was tested against a variety of data sets taken across different laboratories, rat strains, exposure routes, and time points in lactation. Several published kinetic studies using various isotopes of iodide were used to test the model description of iodide kinetics in the dam and neonate (see below). Accuracy and usefulness of the model perchlorate and iodide descriptions were further validated against studies of perchlorate-induced inhibition of iodide uptake in the maternal thyroid, mammary gland, milk, and neonatal tissues based on the description of competitive binding to NIS.

Radioiodide kinetics on PND 10.
Validation of PND 10 iodide kinetics was performed with the data of Iino and Greer (1961)Go, Samel and Caputa (1965)Go, Vigouroux (1976)Go, and Vigouroux and Rostaqui (1980)Go. In order to simplify comparison of the different data sets, which were originally performed at slightly different dose levels, the data of each study were normalized to a dose of 1.0 ng. The model simulation was then run versus the combined data sets (Fig. 5Go). Figures 5A and 5BGo show the maternal and neonatal thyroid radioiodide levels on PND 10 following an acute 131I- dose to the dam. The model is able to predict the maternal thyroid iodide, but underpredicts pup thyroid iodide levels at the 24 h time point. However, the model prediction is within a factor of two of the measured data. Figures 5C and 5DGo show the model-predicted maternal and neonatal thyroid iodide levels after an ip dose to the pup. The model simulation is able to describe both maternal and neonatal thyroid uptake, again within a factor of two.



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FIG. 5. Amount of radioiodide in (A) maternal and (B) neonatal thyroid after iv dose to dam and in (C) maternal and (D) neonatal serum after iv dose to pup on PND 10 versus the normalized data (1.0 ng 131I-) of (open circles) Iino and Greer (1961)Go, (filled squares) Samel and Caputa (1965)Go, (filled triangles) Vigouroux (1976)Go, and (filled circles) Vigouroux and Rostaqui (1980)Go.

 
The data of Samel and Caputa (1965)Go also allowed validation of the model parameters for urinary output in the pup. Difficulty in separating maternal and pup urine precluded the collection of these data in our own studies. Therefore, urinary clearance values in the model were determined by the fit of the serum to the time course data. Using the previously determined kinetic parameters, the model-predicted urinary iodide, 4 h after an iv dose of 131I- in the PND 10 rat, was 14% of the pup dose, which is close to the range of values (0.61 to 10.9%) given by Samel and Caputa (1965)Go.

Radioiodide kinetics on PND 5.
In order to determine whether the model would provide reasonable predictions of iodide and perchlorate kinetics in younger pups (<PND 10), the model was tested against data obtained in our laboratory after a single iv injection of 125I- on PND 5 of the ClO4- drinking water study. Since the control group did not receive perchlorate during the study, tissue 125I- data from these animals can be used to confirm the model’s ability to predict kinetics in PND 5 rats. Figure 6Go shows that the model-predicted radioiodide concentrations in the maternal serum, thyroid and mammary gland and neonatal serum are in good agreement with the available data on PND 5.



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FIG. 6. Radioiodide concentration in maternal serum, mammary gland, thyroid, and neonatal serum versus the measured data after an iv dose of 33,000 ng/kg 131I– to the dam on PND 5.

 
Upregulation of thyroid NIS activity was modeled against the thyroid iodide levels measured after 23 days of exposure to perchlorate in drinking water at doses of 0.01, 0.1, 1.0, and 10.0 mg ClO4-/kg-day as was described in the Methods section. Using this equation for the upregulation of the follicular Vmaxc (VmaxcTFi), 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 describe the upregulation of thyroid NIS activity resulting from subchronic perchlorate exposures.

Radioiodide kinetics in late lactation.
The model’s ability to simulate iodide kinetics at later time points in lactation (>PND 10) was tested against the normalized data of several literature studies. Figure 7Go shows the model-predicted radioiodide milk:plasma ratio versus the data of Brown-Grant (1957)Go, Grosvenor (1963)Go, and Potter et al. (1959)Go collected on PND 14, 17–20, and 18, respectively. Although the simulation shown in Figure 7Go was run on PND 14, there is no noticeable change in the model-predicted milk:plasma ratio when run at different days in lactation. Despite the changing kinetics (body composition, suckling rate, etc.), the model prediction of relative iodide concentration in the milk remains constant. Although the amount of milk provided to the infant may change during the course of lactation, the concentration of iodide does not. This assumption is supported by the available data, which do not show any increasing or decreasing trend in milk iodide concentrations. Dietary iodine studies by Stolc et al. (1966Go; 1973aGo,bGo) also showed a relatively unchanging milk iodine concentration through the different stages of lactation. The model predictions are in reasonable agreement with the trend suggested by the total composite of the different data sets.



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FIG. 7. Radioiodide milk:plasma concentration after an iv dose to the dam versus the combined literature data. The data points represent individual measurements provided in (filled triangles) Brown-Grant (1957)Go, (filled circles) Grosvenor (1963)Go, and (filled squares) Potter et al.(1959)Go on PND 14, 18, and 17–20, respectively.

 
The maternal urinary iodide was tested against the data of Grosvenor (1960)Go. For the reasons mentioned previously, urine was not collected in our own studies. Thus, model parameters for maternal urinary clearance (ClUci) were determined by fitting the model simulation to serum time course data, while maintaining fits to data in the thyroid, mammary gland, and pup. Using these previously set parameters, the model predicts that 40.8% of the 131I- dose will be excreted within 8 h post-131I- dosing, which is within the range of 36.7 to 42.1% reported in Grosvenor’s 1960 study.

Perchlorate-induced inhibition of iodide uptake.
The ability of the model to predict inhibition of iodide uptake into the thyroid, milk, and other tissues is not only important in calculating risk to the dam and neonate, but is also indicative of the model’s ability to accurately describe both perchlorate and iodide kinetics. Thus, the available data for perchlorate-induced inhibition of iodide kinetics in lactation was used as the final validation of the current model structure. Using the conditions for the inhibition time course study described in the Methods section, the model simulation was run to predict the effect of the administration of 1.0 mg ClO4-/kg on the kinetic behavior of an iv dose of radioiodide (given 2 h post-ClO4- dosing). Figure 8Go shows that the model accurately simulates inhibition of iodide uptake in the maternal thyroid and mammary gland. The simulations of the iodide inhibition kinetics in the mammary gland also suggest that the model is able to predict the affect of perchlorate exposure on the availability of iodide to the neonate. Although NIS inhibition would occur in both maternal GI and skin, neither the data nor the model showed significant difference between the control and ClO4- dosed animals, suggesting that other factors (i.e., diffusion, partitioning) are offsetting the effects of symporter inhibition.



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FIG. 8. Iodide concentration in (A) maternal thyroid and (B) mammary gland with and without 1.0 mg/kg ClO4- iv dose 2 h prior to an iv dose of 2.10 ng/kg 125I- to the dam on PND 10. The top simulation (solid line) and data (open squares) indicate the control group. The lower simulation (dashed line) and data (filled squares) indicate inhibition.

 
The model-predicted inhibition of both milk and thyroid iodide was validated against the data of Potter et al. (1959)Go. Potter and coauthors measured the amount of radioiodide (131I-) taken up into the thyroid gland and the combined mammary gland and expressed milk 24 h after dosing with iodide in PND 18 rats. The total effect on iodide uptake was measured after two ClO4- doses given 1 h prior to and 30 min after the radioiodide dose. At these large doses of ClO4-, thyroid iodide uptake is essentially blocked (approximately 99% inhibition). Therefore, it is likely that the NIS transport of iodide into the mammary gland is also completely inhibited. However, unlike the thyroid, the milk still showed significant amounts of iodide (approximately 10% of the control value at 24 h). It is possible that the second mammary transport mechanism is responsible for this difference in tissue response to ClO4- exposure, because it depletes iodine content in the mammary gland to the extent that passive diffusion between the gland and the blood becomes significant. In contrast, passive diffusion of iodine into the thyroid is restricted by the slow clearance via secretion of hormones into the blood, as well as the lower permeability and partitioning of the anions suggested by the model. The ability of the model to predict the combined mammary gland and milk control and ClO4- dosed data of Potter et al. (1959Go; Fig. 9Go) supports the accuracy of the description of iodide partitioning in the mammary gland and the second active transporter. The model predicts the data remarkably well despite significant differences between this study and those used for model development, such as different time points in lactation (PND 18 vs. 10) and a different dosing regimen.



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FIG. 9. Radioiodide concentration in milk with and without ClO4- (Clewell et al., 2001Go). Perchlorate doses of 25 mg and 12.5 mg were given to the dam 30 min before and 4 h after administration of 131I, respectively. The top simulation and data (filled triangles) indicate the control group. The lower simulation and data (filled circles) indicate the inhibition group. Data points represent milk samples from individual dams fromPotter et al. (1959)Go.

 
Dietary iodine.
In three separate experiments, Stolc et al. (1966Go, 1973aGo,bGo) studied the distribution of endogenous iodine in various tissues of the maternal and neonatal rat resulting from a controlled intake of iodine through the diet and water. Model-predictions of the maternal and neonatal tissue iodine concentrations are shown in Figure 10Go versus data collected at water and feed concentrations of 60 ng I/g and 500 ng I/ml, respectively (Stolc et al., 1973aGo), which corresponds to daily doses of 4.8 mg I/kg-day. Of the three dosing levels presented by Stolc and coauthors, this highest dose was chosen to validate the model prediction of normal iodine distribution, due to the fact that it is within the range of normal dietary intake for rats and it is closest to dietary intake expected for our experimental studies (800 ng I/mg food). The two lower dose groups would be considered moderately deficient and deficient iodine diets, resulting in changes in the H-P-T axis in order to affect upregulation as is evidenced by the measured tissue iodine concentrations. Despite a fivefold difference in water iodide concentrations, the serum and thyroid concentrations remain unchanged. Other tissues, including the skin and GI, do show a dose-dependent change in tissue concentration. Thus, since this model does not yet include the pharmacodynamic response of the thyroid axis to dietary insufficiency, it is premature to attempt to predict such data. Using kinetic parameters obtained from acute data, the model is generally able to predict endogenous data within a factor of two of the measured data for tissue concentrations ranging over more than four orders of magnitude (Fig. 10Go).



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FIG. 10. Model predicted tissue iodine vs. measured data ofStolc et al.(1973a)Go with a normal dietary intake (approximately 560 ng/day). Standard deviations were given with the measured maternal data only.

 
While admittedly only a preliminary description, this endogenous iodine model could be exercised to assess the affect of changing dietary intake on predicted acute radioiodide kinetics, as well as predicted perchlorate-induced inhibition of radioiodide kinetics in the perinatal and adult rats. Since the radioiodide, endogenous iodine and perchlorate models operate independently in tissues with passive diffusion, and are linked through competitive uptake in tissues with NIS (e.g., thyroid follicle), only tissues with active uptake are expected to show any change in tissue concentrations with varying dietary intake. From this modeling exercise, it was determined that the effects of changes in dietary iodine intake on acute radioiodide kinetics are likely to be negligible. In fact, significant differences in predicted thyroid concentrations were not observed at feed concentrations as high as 8.0 ppm (more than an order of magnitude higher than standard laboratory diet). Indeed, the first apparent change in predicted inhibition of thyroidal radioiodide uptake is seen at feed concentrations 100 times greater than standard laboratory rat chow.

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 several of the chemical-specific parameters (Fig. 11Go). At 0.1 mg/kg-day, the maternal serum is primarily dependent on serum binding, showing less sensitivity to urinary clearance. All other parameters had calculated sensitivity coefficients less than 0.1. At the 10.0 mg/kg-day dose, binding parameters are no longer important determinants of predicted serum levels. Only the urinary clearance remains significant, with a sensitivity coefficient of –0.87. Neonatal serum ClO4- levels are influenced by several model parameters at the 0.1 mg/kg-day dose, including the parameters defining passive diffusion and active transfer in the mammary gland, milk, and neonatal GI. However, similar to dam, neonatal serum AUC shows the greatest sensitivity to serum binding parameters at this lower dose. At the higher dose (10.0 mg/kg-day), where active uptake into the mammary gland and serum binding are likely saturated, partitioning into mammary gland and milk and urinary clearance (both maternal and neonatal) show the greatest influence on pup serum ClO4- levels. Results of the sensitivity analysis for thyroid iodide uptake (not shown) were similar, in that the magnitude of all of the parameter sensitivities were less than one, although model predictions for 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 effect the time-dependent result [as compared to an average, or AUC (area under the curve), measure, which reflects steady-state behavior]. Thus, the validation of the model with kinetic thyroid iodine uptake and inhibition data provides a reasonable test of the model parameterization.



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FIG. 11. 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.

 
Calculation of Internal Dose Metrics
The model was used to calculate internal dose metrics corresponding to both acute and subchronic perchlorate exposure in the lactating and neonatal rat. These internal measures of dose include: AUC for ClO4- in the maternal and neonatal serum, relative neonatal dose (% maternal dose) and inhibition of thyroid iodide uptake after acute dosing. Table 3Go shows the predicted neonatal dose on PND 10 as % maternal dose and as an amount adjusted for milk intake and pup body weight. The model predicts a significant transfer of maternal ClO4- to the neonate on PND 10 at low maternal doses. In fact, per kg body weight, the PND 10 pup receives a greater dose than the dam (0.07 vs. 0.01 mg/kg BW). Tables 4 and 5GoGo show the dose metric comparisons between the adult male, pregnant, fetal, lactating and neonatal rats for serum ClO4- AUCs from drinking water exposure, as well as thyroid iodide inhibition after acute iv exposures in the adult male, GD 20 pregnant and fetal rats, and the PND 10 lactating and neonatal rats. The internal dose metrics in the lactating and neonatal rat were compared to those of the male (Merrill et al., 2003Go), pregnant, and fetal rat (Clewell et al., 2003Go) in order to provide insight on relative exposure at different life stages. From Tables 4 and 5GoGo, it is apparent that while the lactating rat shows the highest serum ClO4- concentrations, the fetal rat actually shows the greatest inhibition of thyroid iodide uptake.


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TABLE 3 Model-Predicted Perchlorate Dose to the Pup from Maternal Drinking Water Exposure on PND 10
 

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TABLE 4 Model Predicted Internal Dosimetrics: Serum ClO4- AUC (mg/l) in Male, Pregnant, Fetal, Lactating, and Neonatal Rat from Drinking Water Perchlorate Exposure
 

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TABLE 5 Model Predicted Internal Dosimetrics: %Inhibition of Thyroid Iodide Uptake in Male, Pregnant, Fetal, Lactating, and Neonatal Rat from Acute Perchlorate Exposure
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
The PBPK model is able to describe iodine kinetics across doses, exposure routes, and time points during lactation for both acute dosing and dietary iodine studies. The iodide model was simplified by assuming that radiolabeled iodide could be described as free iodide in all compartments other than the thyroid and plasma. Simulations were performed against total radioiodide concentrations in extrathyroidal tissues and against inorganic and incorporated iodide in thyroid and serum. Despite this simplification, the model is able to describe radiodide data in maternal and neonatal tissues from PND 5 through 18 over doses spanning more than four orders of magnitude, as well as distribution data from dietary iodine intake. Thus, the model description of extrathyroidal tissue iodide uptake based on the transfer of inorganic iodide via NIS predicts the data reasonably well without the added uncertainty or complexity of significant contribution from the uptake of incorporated radioiodine by extrathyroidal tissues.

Although preliminary extrapolation of the acute iodide kinetic model to long-term exposure scenarios compares favorably with the data, this model does not yet address the feedback mechanisms involved in maintaining normal iodine homeostasis. However, the model predicted interaction between dietary and administered radioiodide does indicate that the small variations in laboratory rat chow expected between studies should not affect model predictions of radioiodide distribution or perchlorate-induced inhibition of thyroid radioiodide uptake. At the dietary intake required to affect competition for NIS, serum levels would be high enough (>250 µg/l) to trigger a response from the thyroid known as the Wolff-Chaikoff effect, wherein the thyroid appears to expel inorganic iodide from the cells (Wolff and Chaikoff, 1948Go). In order to address cases of dietary iodine excess or deficiency, future elaborations of the model would have to include a more detailed description of hormone distribution, homeostasis, and regulation. However, the present model should be useful for predicting tissue dosimetry and inhibition of thyroid iodide uptake in the rat and for extrapolation to the average human with a normal iodide diet.

The PBPK model described here also successfully reproduces measured perchlorate and radioiodide distribution kinetics in the lactating rat and neonate. The model simulates ClO4- distribution and transfer via breast milk with reasonable accuracy in drinking water exposures ranging over three orders of magnitude (0.01–10.0 mg/kg-day) and at two time points in lactation, PND 5 and 10. In the absence of acute perchlorate kinetic data in rat lactation, we rely on 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 lactation, most of the remaining chemical-specific parameters remain essentially unchanged from those of the adult male rat. Therefore, we can assume that, like the male rat model, the acute perchlorate kinetics can be adequately described with model structure based on iodide 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. Additionally, 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. The accuracy of the model ClO4- description allows us to answer two vital questions in determining risk across life stages: (1) the dose to the neonate and (2) the relative sensitivity, with respect to tissue dose and thyroid iodide inhibition, compared to the nonpregnant and pregnant adult, as well as to the fetus (Tables 3 through 5GoGoGo).

Experiments in our laboratory have confirmed that perchlorate was indeed transferred to the pup through suckling by the detection of ClO4- in milk, as well as in the neonate serum, GI contents, and skin (Fig. 2Go). However, the small number of data points and the difficulty in determining ingestion of milk, loss through urinary clearance, and other competing processes made the determination of neonatal dose quite difficult by classical methods. Because the model accounts for physiological and kinetic differences, it is able to provide a reasonable estimate of a previously uncharacterized measure of risk, ClO4- dose to the neonate (Table 3Go). In fact, the model predicts that the pup receives a sevenfold greater perchlorate dose than the dam on PND 10 when adjusted for body weight at the lowest experimental dose (0.01 mg ClO4-/kg-day). This difference between the maternal and neonatal dose disappears, however, at higher doses (10 mg ClO4-/kg-day), where toxicity would be expected.

Additional calculations were performed with the model to determine the AUC for perchlorate in serum across doses and life stages. Serum, rather than thyroid, perchlorate concentration was designated as a dose metric for life-stage and species comparison. Perchlorate’s action on the thyroid is the inhibition of iodide uptake, leading to diminished intrathyroidal I- levels and potentially decreased hormone production. Since thyroid iodide uptake inhibition is dependent on serum ClO4- levels, this variable was determined to be a more appropriate dose metric by which to judge relative sensitivity to later effects. By comparing these measures of average serum concentrations across life stages, valuable insight can be gained regarding relative sensitivity to perchlorate exposure. Thus it is evident that, despite the increased dose to the neonate (0.07 vs. 0.01 mg/kg-day in the adult), the PND 10 pup serum average ClO4- concentrations are consistently lower than those of the adult. In fact, a comparison across life stages reveals that the serum ClO4- concentrations of the lactating dam were slightly higher than the male, pregnant, fetal, or neonatal rat, suggesting that lactation may be the time period with the greatest internal perchlorate exposure. This increased serum concentration of ClO4- in the lactating rat, suggested by both model simulations and measured data, is somewhat surprising, considering the additional clearance route provided through the milk, and is likely due to increased serum binding (Iino and Greer, 1961Go).

In developing the perchlorate thyroid model, some assumptions were made concerning the mode of action, as well as in the designation of values for some of the parameters. The model structure is highly dependent on the chosen definition of the mode of action, which in the case of perchlorate involves the competitive binding of the perchlorate ion to NIS, resulting in diminished I- thyroidal uptake and the active concentration of ClO4- in the thyroid cells. Although it has been suggested that ClO4- may not be transferred into the thyrocytes based on electrogenicity studies in oocytes (Soldin, 2002Go), the larger body of evidence suggests otherwise. Specifically, published studies with both radiolobeled and cold perchlorate consistently report thyroid: serum ratios that are greater than 1 and as much as 30 (Chow and Woodbury, 1970Go; Clewell et al., 2002b, 2003Go; Yu et al., 2002Go). Furthermore, ClO4- has been shown in studies in our laboratory to be concentrated in all of the measured extra-thyroidal tissues known to contain NIS, including the GI contents, skin, and milk (see Fig. 2Go and Clewell et al., 2002b; Yu et al., 2002Go). A more detailed justification for the use of competitive inhibition is available elsewhere (Clewell and Gearhart, 2002bGo).

Thus, this model structure is based upon competitive inhibition of iodide at the symporter. However, despite the obvious influence this interpretation has on predicted internal thyroid ClO4- levels, it actually has very little influence on the either thyroid iodide inhibition or serum ClO4- levels. Since the relative thyroid volume is very small, the total amount of chemical is quite low in spite of the high concentrations. Therefore, large changes in predicted thyroid concentrations do not significantly affect blood levels. Additionally, the ability of ClO4- to inhibit iodide uptake is based on the relative affinities and the amount of free ClO4- in the blood, rather than the amount of perchlorate in the thyroid itself. Thus, the active uptake of perchlorate into the thyroid cells, though included for pharmacokinetic accuracy, does not affect the usefulness of the model for comparing life-stage and species differences in the precursors to hormone disruption.

Linking the perchlorate and iodide models via competitive inhibition at the symporter also enables the model to predict ClO4-–induced inhibition of iodide uptake in the maternal and neonatal tissues after acute ClO4- exposure in the lactating rat. Because ClO4- has a greater affinity for NIS than I-, it effectively inhibits uptake not only into the thyroid, but also into the milk, stomach, and skin. The model accurately predicts data on this inhibition of iodide uptake in the maternal thyroid mammary gland and milk from our studies and those of Potter et al. (1959)Go. Although data are not available directly in the neonate, confidence in the model predictions is increased by the ability of the maternal model, as well as the previously described sister models in the male, pregnant, and fetal rat (Clewell et al., 2003Go; Merrill et al., 2003Go), to describe this inhibition using the same mechanistic construct and validated chemical-specific parameters. Thus, the model provides a means for estimating neonatal inhibition of thyroid iodide in the absence of such data.

The ability of ClO4- to reduce iodide levels in the milk, as well as uptake in thyroid, presents a potentially increased health risk to the neonate. In order to quantitatively determine the effect of maternal ClO4- exposure on the transfer of iodide in breast milk and subsequent neonatal thyroid levels, the model predicted percent inhibition in maternal and neonatal thyroids were compared to those generated for the male, pregnant, and fetal rat with the models of Merrill et al. (2003)Go and Clewell et al. (2003)Go. In spite of the multiple inhibition sites (mammary gland, milk, and thyroid), inhibition in the neonatal thyroid was similar to that of the dam. This may be due to the fact that neonatal serum perchlorate levels are less than those of the dam. From the model estimates given in Table 5Go, the neonate shows less perchlorate-induced inhibition of thyroid iodide uptake compared to the other life stages in the rat. Model estimates suggest that the fetal rat thyroid is most vulnerable to inhibition, with a tenfold greater inhibition than the neonate at the lowest measured dose (0.01 mg/kg ClO4-).

Thus, it is possible to utilize these pharmacokinetic models to develop reasonable estimates of internal dose metrics based on quantitative biological concepts and a variety of data collected in different conditions, species, and life stages. The chosen dose metrics are measures of internal dose, and should be better indicators of relative risk than external dose (i.e., perchlorate intake). However, these internal dose metrics are merely measures of the precursor kinetics and do not give a complete picture of perchlorate’s affect on hormone homeostasis. Indeed, determining which life stage is at greatest risk actually depends on the chosen precursor dose metric. Of those presented in this article, utilizing serum ClO4- levels indicates that the lactating dam is at highest risk, while thyroid iodide inhibition suggests the fetus is the most sensitive life stage.

In reality, many factors must be taken into consideration when assessing the risk associated with perchlorate exposure. For example, although the predicted thyroid inhibitions across life stages indicate that the fetal thyroid is most vulnerable, overall risk to the fetus may actually be less than that of the neonate. This is due to the fact that inhibition of thyroid iodide uptake is only a precursor to hormone disruption. In gestation, maternal thyroid hormones are available to the fetus, as opposed to lactation, where the neonate is responsible for its hormone synthesis. Therefore, the maternal hormones may compensate for the increased inhibition seen in the fetal thyroid resulting in less chance of adverse developmental effects. In fact, in the developmental studies of York et al. (1999Go, 2001)Go, the pregnant dam showed the greatest change in serum TSH and T4 levels. Thus, it is possible that the additional pharmacodynamic interactions (hormone synthesis, metabolism, etc.) could result in a relative risk profile not at all suggested by the preliminary measurements of tissue dose or pharmacokinetic perturbations. For this reason, further elaboration of these models to include hormone homeostasis and the pharamocodynamic interactions is critical to improve the risk assessment for perchlorate.

This model allows the integration of a wide variety of physiological, biochemical, and dosimetry information, to produce parameter estimates consistent with measured perchlorate and iodide kinetic data during important periods of development. In order to further assess model performance, other analytical tools can be applied to the model, including statistical evaluation of the goodness of model fit to present data sets, more comprehensive sensitivity analyses for multiple dosimetrics and assessment of the effects of parameter variability on dose measures. Sensitivity analysis (Fig. 11Go) provides insight into the relative importance of model parameters with respect to specific measures of dose. The variability analysis, performed with known distributions for model parameters, allows the prediction of likely ranges of the dosimetrics within a human population. For the application of risk assessment, incorporation of a more comprehensive evaluation of variability of the more sensitive model parameters will be more important than a formal estimation with the present data sets.

An 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 development fetal/neonatal iodide uptake is most critical.

Further elaboration of these models to extrapolate dosimetry to humans has been explored elsewhere (Clewell et al., 2001Go; Clewell and Gearhart, 2002aGo). Together with the models of Merrill et al.(2001Go, 2003)Go and Clewell et al. (2003)Go, this model can be used to approximate species and life stage kinetic differences at specific doses. Furthermore, since these PBPK models to relate complex pharmacokinetic variables back to the basic physiological and biochemical parameters that are often measurable, we can use the comparative information provided by the PBPK models about the chemical kinetics to develop quantitative estimates of species and life stage differences. Thus, it is possible to extrapolate these models to the population of interest (human gestation and lactation), in order to run simulations for sensitive human populations at a variety of exposure scenarios to estimate internal dose (Clewell et al., 2001Go).


    APPENDIX
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 APPENDIX
 REFERENCES
 
The following equations represent the distribution of iodide within the thyroid, in the absence of competitive inhibition (Equations 1–6GoGoGoGoGoGo). Perchlorate uptake into the thyroid is described similarly, but without the organification (ClProdi) and hormone secretion (ClSecri) terms.


(1)



(2)


(3)


(4)


(5)


(6)

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, CTFi, CTLi, and CTbndi are the iodide concentrations in arterial plasma, thyroid stroma, follicle, colloid, and the incorporated (organified) iodine compartment, respectively.

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.


(7)

The model description of uptake iodide uptake in the milk with competitive inhibition at both transporters is shown in Equations 8 through 12GoGoGoGoGo. Equations for perchlorate would be similar, but without the terms for competitive uptake. RAMBi, RAMi, and RAMki are the rate of change in the amount of iodide in the mammary gland capillary blood, the mammary gland tissue, and the milk, respectively. RupMi and RupMki represent the rate of active uptake of iodide into the mammary gland and the milk. VmaxMi, VmaxMki and KmMi, KmMki are the maximum velocities and affinity constants for the active transport of iodide into the mammary gland and milk. QM represents fractional blood flow to the mammary gland capillary bed. PAMi, PAMki, PMi, and PMki are the permeability area cross products and partition coefficients used to describe the passive diffusion of iodide between the capillary blood and mammary gland, and the mammary gland and milk, respectively. CAi, CMBi, CMi, and CMki are the iodide concentrations in arterial plasma, mammary capillary blood, mammary gland and milk. Ktrans represents the rate of milk production, which is assumed to be equal to the suckling rate.


(8)



(9)


(10)


(11)


(12)

The model description for active uptake of iodide into the GI contents with competitive inhibition by ClO4- is given in Equations 13 through 16GoGoGoGo. 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 GI 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).


(13)


(14)


(15)


(16)

Model equations for compartments without active uptake (shown for the liver, Equation 17Go) 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 concentration of iodide in the liver, and PLi is the blood:liver partition coefficient. The kidney and fat are modeled similarly.


(17)

Model equations describing iodine in the arterial blood are given below (Equations 18–19GoGo). Where RaBndi and RaIncorpi are the rates of change in amount of iodide bound to plasma proteins and hormone incorporated iodine, respectively. VmaxBi and Kmi are the Michaelis-Menten terms for saturable binding of inorganic iodide to plasma proteins. Kmp is the affinity constant for the binding of ClO4- to plasma proteins, which is used to adjust the affinity of iodide in order to mathematically describe the competitive inhibition of the anions for binding sites. Clunbi, Clsecri and Cldeiodi are the first order rate constants for the dissociation of I from plasma proteins, the secretion of thyroid hormones (hormone incorporated iodine) form the thyroid and the whole-body deiodination of thyroid hormones, respectively. CAi and CAp are the concentrations of free inorganic iodide and free ClO4- in the plasma. The binding of perchlorate to plasma proteins would be described in the same manner as iodide (Equation 18Go).


(18)


(19)


    ACKNOWLEDGMENTS
 
The authors would like to thank Tammie Covington, Harvey Clewell, Dr. Melvin Andersen, and Dr. Peter Robinson 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. David Mattie, 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 present address: CIIT Centers for Health Research, Six 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. Back


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