* Geo-Centers, Inc., Wright-Patterson AFB, Ohio 45433;
AFRL/HEST, Wright-Patterson AFB, Ohio 45433;
Mantech Environmental Technology, Inc., Dayton, Ohio 45437; and
Operational Technologies Corp., Dayton, Ohio 45432
Received March 5, 2003; accepted May 1, 2003
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ABSTRACT |
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Key Words: PBPK model; lactation; perchlorate; iodide; inhibition; milk.
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INTRODUCTION |
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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., 2001; Mahle et al., 2002
, 2003
; York et al., 1999
, 2001
; Yu et al., 2002
). 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., 2003
), pregnant and fetal rat (Clewell et al., 2003
), and the adult human (Merrill et al., 2001
), 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, 2002a
).
Although the hormone feedback system of the postnatal rat is independent of the mother (Howdeshell, 2002; Potter et al., 1959
; Vigouroux and Rostaqui, 1980
), 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, 1957
; Cline et al., 1969
; Grosvenor, 1963
; Lengemann, 1965
; Potter et al., 1959
). 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., 2001
). 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.
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MATERIALS AND METHODS |
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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., 2003), 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) and Clewell et al.(2003)
. The maternal model (Fig. 1A
) 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. 1B
). 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. 1
). 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. 1
), which results from the inherent electrochemical gradients within these tissues (Chow et al., 1969
). 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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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. 1B). 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., 1959
).
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., 1973b). 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., 2002
). 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, 1952
; Scatchard and Black, 1949
). 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., 1998), 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, 1957
; Grosvenor, 1963
; Potter et al., 1959
). Furthermore, hormones produced during lactation, such as prolactin, regulate the mammary gland NIS activity (Tazebay et al., 2000
). Shennan and Peaker (2000)
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, 1970).
The basic structure of the neonatal model (Fig. 1A) 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)
, showing that lactating dams ingest approximately 60% of the neonates 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, 1959) and the milk of the lactating rat (Brown-Grant, 1957
; Grosvenor, 1963
; Potter et al., 1959
). 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., 2002
).
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., 1973a,b
) 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., 1966
) 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), 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 1
.
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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) 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)
was used for these physiological parameters. Values for neonatal body fat were based on the data of Naismith et al.(1982)
, 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., 1997
).
Increase in neonatal thyroid volume was based on the work of Florsheim et al.(1966), 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)
, 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., 1983
). 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) and the neonatal cardiac output, hematocrit, and regional blood flows, based on the data of Rakusan and Marcinek (1973)
. Among other tissues, Rakusan and Marcinek (1973)
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 2) 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., 2003
; Merrill et al., 2003
) 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)
in human thyroid slices. This value for the follicular Km (KmTFi) remained constant across species (Gluzman and Niepomniszcze, 1983
) and tissues (Wolff, 1998
) 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)
, 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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The values for Vmax vary significantly across species and tissues with NIS (Gluzman and Niepomniszcze, 1983; Wolff, 1998
) 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, 1970). Theoretical effective partition coefficients were calculated from measured electrical potentials presented by Chow and Woodbury (1970)
using the equations given in Kotyk and Janacek (1977)
. Calculations are described in detail in the male rat perchlorate model (Merrill et al., 2003
). 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., 2003). The partition coefficient for perchlorate in fat was measured in the laying hen (Pena et al., 1976
). 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., 2003) 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., 2001). Since increased TSH upregulates thyroid iodide uptake by increasing the number and activity of NIS (Wolff, 1998
), 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)
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, 1964, 1998
). 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.
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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.
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RESULTS |
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Radioiodide kinetics on PND 10.
Validation of PND 10 iodide kinetics was performed with the data of Iino and Greer (1961), Samel and Caputa (1965)
, Vigouroux (1976)
, and Vigouroux and Rostaqui (1980)
. 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. 5
). Figures 5A and 5B
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 5D
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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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 models ability to predict kinetics in PND 5 rats. Figure 6 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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Radioiodide kinetics in late lactation.
The models ability to simulate iodide kinetics at later time points in lactation (>PND 10) was tested against the normalized data of several literature studies. Figure 7 shows the model-predicted radioiodide milk:plasma ratio versus the data of Brown-Grant (1957)
, Grosvenor (1963)
, and Potter et al. (1959)
collected on PND 14, 1720, and 18, respectively. Although the simulation shown in Figure 7
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. (1966
; 1973a
,b
) 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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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 models 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 8 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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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. 11). 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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DISCUSSION |
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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, 1948). 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 (0.0110.0 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., 2003), 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 5
).
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. 2). 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 3
). 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. Perchlorates 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, 1961).
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, 2002), 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, 1970
; Clewell et al., 2002b, 2003
; Yu et al., 2002
). 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. 2
and Clewell et al., 2002b; Yu et al., 2002
). A more detailed justification for the use of competitive inhibition is available elsewhere (Clewell and Gearhart, 2002b
).
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). 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., 2003
; Merrill et al., 2003
), 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) and Clewell et al. (2003)
. 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 5
, 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 perchlorates 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. (1999, 2001)
, 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. 11) 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., 2001; Clewell and Gearhart, 2002a
). Together with the models of Merrill et al.(2001
, 2003)
and Clewell et al. (2003)
, 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., 2001
).
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APPENDIX |
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![]() | (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 12. 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 16. 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 17) 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 1819). 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 18
).
![]() | (18) |
![]() | (19) |
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ACKNOWLEDGMENTS |
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NOTES |
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2 Present address: The University of Georgia, Athens, GA.
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