TOXICOLOGICAL SCIENCES 73, 256 –269 (2003)
DOI: 10.1093/toxsci/kfg080
Copyright © 2003 by the Society of Toxicology
PBPK Predictions of Perchlorate Distribution and Its Effect on Thyroid
Uptake of Radioiodide in the Male Rat
Elaine A. Merrill, *,1 Rebecca A. Clewell,† Jeffery M. Gearhart,‡ Peter J. Robinson,‡ Teresa R. Sterner,* Kyung O. Yu,§
David R. Mattie,§ and Jeffrey W. Fisher§ ,2
*Operational Technologies Corporation, Dayton, Ohio 45432; †GeoCenters, Inc., Wright-Patterson AFB, Ohio 45433; ‡ManTech Environmental
Technology, Inc., Dayton, Ohio 45437; and §AFRL/HEST Operational Toxicology Branch, Wright-Patterson AFB, Ohio 45433
Received November 19, 2002; accepted March 3, 2003
Due to perchlorate’s (ClO 4 –) ability to competitively inhibit
thyroid iodide (I –) uptake through the sodium-iodide symporter
(NIS), potential human health risks exist from chronic exposure
via drinking water. Such risks may include hypothyroidism, goiter, and mental retardation (if exposure occurs during critical
periods in neurodevelopment). To aid in predicting perchlorate’s
effect on normal I – kinetics, we developed a physiologically-based
pharmacokinetic (PBPK) model for the adult male rat. The model
structure describes simultaneous kinetics for both anions together
with their interaction at the NIS, in particular, the inhibition of I –
uptake by ClO 4 –. Subcompartments and Michaelis-Menten (M-M)
kinetics were used to describe active uptake of both anions in the
thyroid, stomach, and skin. Separate compartments for kidney,
liver, plasma, and fat were described by passive diffusion. The
model successfully predicts both 36ClO 4 – and 125I – kinetics after iv
doses of 3.3 mg/kg and 33 mg/kg, respectively, as well as inhibition
of thyroid 125I – uptake by ClO 4 – after iv doses of ClO 4 – (0.01 to 3.0
mg/kg). The model also predicts serum and thyroid ClO 4 – concentrations from 14-day drinking water exposures (0.01 to 30.0 mg
ClO 4 –/kg/day) and compensation of perchlorate-induced inhibition of radioiodide uptake due to upregulation of the thyroid. The
model can be used to extrapolate dose metrics and correlate
observed effects in perchlorate toxicity studies to other species and
life stages, such as rat gestation (Clewell et al., 2003). Because the
model successfully predicts perchlorate’s interaction with iodide, it
provides a sound basis for future incorporation of the complex
hypothalamic-pituitary-thyroid feedback system.
Key Words: perchlorate, radioiodide, thyroid, inhibition, sodium
iodide symporter, PBPK, model.
Ammonium perchlorate is an oxidizing agent used in solid
rocket and missile fuels, pyrotechnics, and air bag inflators.
Past use and disposal practices of the ammonium salt have
resulted in accidental groundwater contamination by its extremely stable dissociation product, perchlorate (ClO 4 –).
Groundwater concentrations have been detected in ranges as
1
To whom all correspondence should be sent at GeoCenters, Inc., 2856 G
Street, Building 79, Wright-Patterson AFB, OH 45433. Fax: (937) 255-1474.
E-mail: [email protected].
2
Present address: Department of Environmental Health Science University
of Georgia, Athens, GA 30602.
256
high as 630,000 to 3,700,000 ppb near Las Vegas, Nevada, but
most groundwater detections throughout other states have been
below 20 ppb (Motzer, 2001). The current detection limit for
perchlorate in water is 1 ppb (EPA Method 314).
Perchlorate’s similarity to iodide (I –) in charge and size
allows the anion to competitively inhibit I – uptake by the
sodium-iodide symporter (NIS) into the thyroid (Wolff, 1998).
NIS actively transports both Na ⫹ and I – from extracellular fluid
into the thyroid epithelial cell and other tissues (Ajjan et al.,
1998). Decreased intrathyroidal iodide eventually results in a
drop in circulating iodide-containing thyroid hormones, triggering a series of compensatory mechanisms by the hypothalamus-pituitary-thyroid axis. These mechanisms include pituitary secretion of thyroid releasing hormone, which signals
increased production of thyroid-stimulating hormone (TSH) by
the hypothalamus. In turn, TSH increases the synthesis of NIS
in the thyroid, thereby restoring intrathyroidal iodide levels.
TSH also directly promotes hormone production by increasing
the synthesis of thyroid peroxidase, an enzyme required for
iodide organification (Spitzweg et al., 1998). Because ClO 4 – is
a potent inhibitor of thyroid iodide uptake, there is concern that
chronic exposure to the low levels of ClO 4 – found in some
drinking water supplies may result in adverse human health
effects, such as hypothyroidism, leading to goiter or even
impaired neurodevelopment from gestational or neonatal exposure (Delange, 2001).
Perchlorate does not appear to be metabolized in the body.
Anbar et al. (1959) administered 36Cl 18O 4 – to rats and reported
that less than 0.1% of the dose appeared in urine as 36Cl – and
36
Cl 18O 3 –. Iodine is readily absorbed from the upper GI tract,
distributed throughout extracellular fluid, and cleared mainly
through the thyroid, where it is incorporated into hormones or
by the kidneys, where it is excreted (Hays and Wegner, 1965).
In rats, Yu et al. (2002) reported 99.5% 36ClO 4 – and 78% 125I –
excreted in urine within 48 and 24 h after administered doses,
respectively. DiStefano and Sapin (1987) reported between 1%
and 7% of activity from administered radiolabeled thyroid
hormones excreted in rat feces. Iodine excretion is not regulated by any iodine-conserving feedback system; therefore,
regular dietary intake is important.
PBPK PREDICTIONS OF PERCHLORATE DISTRIBUTION AND THYROID IODIDE INHIBITION
Literature regarding the toxicity of ClO 4 – from chronic lowlevel exposures and its effect on the hypothalamus-pituitarythyroid axis is limited. High-level drinking water exposures
(0.2–10.0 mg ClO 4 –/kg/day for 14 and 90 days) have shown
increased TSH and decreased thyroid hormone levels in rats
(Siglin et al., 2000). Yu et al. (2002) reported elevated TSH in
rats after 14 days of drinking water exposure, ranging from 0.1
to 10 mg/kg/day, and decrements in T 4 and free thyroxine (fT 4)
from 1.0 to 10 mg/kg/day. In adult humans, short-term studies
have shown little or no significant change in TSH and fT 4
levels after 2 weeks of drinking water exposure from 0.007 to
0.05 mg/kg/day (Greer et al., 2002). A significant drop in fT 4,
intrathyroidal iodine and an increase in thyroglobulin (Tg)
were reported from subchronic high exposures (900 mg/day for
4 weeks) (Brabant et al., 1992). Yet the length of chronic low
level perchlorate exposure required to cause significant hormone deficiencies is definitely not known. A retrospective
epidemiologic study on children in three Chilean cities with
groundwater levels of ⬍4 , 5–7, and 100 –120 mg ClO 4 –/l
revealed significantly higher fT 4 but normal TSH in the two
cities with highest concentrations (Crump et al., 2000).
Currently, the U.S. Environmental Protection Agency (EPA)
is conducting a risk assessment to determine a safe reference
dose (RfD) and drinking water level for ClO 4 –. To assist in
predicting perchlorate’s effect on normal I – kinetics, we have
developed a physiologically based pharmacokinetic (PBPK)
model for the adult male rat. The model describes active uptake
of I – and ClO 4 –, as well as ClO 4 – induced inhibition of I –
uptake into NIS-containing tissues (thyroid, gastric mucosa,
and skin). The model also simulates distribution of both anions
in the kidney, liver, fat, serum, and remaining richly and slowly
perfused tissues, as well as elimination from the kidneys.
Understanding the impact of chronic displacement of I –
during prolonged exposure to ClO 4— contaminated drinking
water is the focus of this and other ongoing research efforts.
Currently, the model is used in extrapolating dose metrics to
reproductive stages (Clewell et al., 2001) and to other species,
including adult humans (Merrill et al., 2001). The model
structure also forms the basis for future model developments
that will include subsequent effects on thyroid hormone homeostasis.
MATERIALS AND METHODS
The kinetic studies by Yu et al. (2002) served as the main data sets used in
the development of this model. A brief summary of their experiments is
provided below. Each study utilized adult male Sprague-Dawley rats (330 ⫾
35 g, N ⫽ 6 rats per group), which were dosed as described and euthanized by
CO 2 asphyxiation at selected time points for tissue collection. All time-course
data described were utilized in estimating model parameter values, with the
exception of I – inhibition data, which were used for model validation. The
36
ClO 4 – and 125I – doses used in the kinetic studies (Yu et al., 2002) were
selected as tracer studies. Due to the low specific activity of 36ClO 4 –, a dose
100 times that used for the 125I – studies was required for reliable measurements.
257
Experiments
Radiolabeled perchlorate ( 36ClO 4 –) time-course kinetics. Naive rats received a tail vein iv dose of 3.3 mg/kg 36ClO 4 – and were euthanized at 0.5, 6,
12, 24, 32, and 48 h post dosing. The thyroid, stomach tissue and contents,
intestinal tissue and contents, muscle, skin, liver, kidney, plasma, and red
blood cells were analyzed for 36ClO 4 – using a liquid scintillation counter
(LSC). Urine was collected from the 24- and 48-h time point animals.
Radiolabeled iodide ( 125I –) time-course kinetics. Rats were administered a
tail vein iv dose of physiological saline (control group) or 33 mg/kg 125I – (with
carrier) in physiological saline. At 5, 15, and 30 min and 1, 2, 6, 9, 24, 32, 48,
and 96 h post dosing, rats were euthanized; thyroids and serum were collected.
Urine voids were collected from the 24-h group. The same study was repeated
to obtain additional tissues (thyroid, serum, skin, and stomach contents) at 30
min and 2 and 6 h post dosing.
Perchlorate-induced inhibition of thyroid 125I – uptake time-course kinetics. Rats received one of five tail vein iv doses of ClO 4 – (0.0, 0.01, 0.1, 1.0,
and 3.0 mg/kg), followed by an iv challenge of 125I – (33 mg/kg with carrier) 2 h
later. Thyroids were collected at 5, 15, and 30 min and 1, 2, 6, 9, and 24 h post
dosing with 125I – and counted for 125I – activity.
Inhibition of thyroid 125I – uptake after drinking water exposure to ClO 4 –.
Rats were exposed via drinking water for 1, 5, and 14 days to ClO 4 – doses of
0.0, 1.0, 3.0, and 10.0 mg/kg/day. In addition to the dose groups described in
Yu et al. (2002), a 30.0 mg/kg/day group was exposed in the same manner
(unpublished data). Daily doses were verified by measuring the amount of
water consumed. At the end of day 14, all dose groups were challenged with
a single iv dose of 33.0 mg/kg 125I – (with carrier) and euthanized 2 h post 125I –
dosing. Serum and thyroid glands were collected for ClO 4 – analyses at the end
of days 1, 5, and 14 for the 3.0, 10.0, and 30.0 mg/kg dose groups (N ⫽ 6 per
group) and at the end of day 14 for the 0.0 and 1.0 mg/kg dose groups.
Thyroids, collected on day 14 from all dose groups, were also analyzed for
125 –
I.
Analytical Methods
Analyses of 36ClO 4 – and 125I – in urine and tissues were performed using a
liquid scintillation counter (LSC) and g-counter, respectively, as explained in
Yu et al. (2002). Cold ClO 4 – concentrations in urine were analyzed using
high-pressure liquid chromatography (HPLC), as described elsewhere (Fisher
et al., 2000). To determine whether any of the ClO 4 – was being metabolized,
Yu et al. (2002) performed both ClO 4 – and chlorate (ClO 3 –) analyses on
thyroid and serum samples, using isocratic and gradient chromatographic
conditions. Chlorate was not detected.
Model Structure
Early model development was based on work by Fisher et al. (2000).
Several compartmental models have been developed for iodide metabolism
(Berman et al., 1968; Degroot et al., 1971; Hays and Wegner, 1965). However,
because these models are not physiologically based, their utility for extrapolating across species and life stages is limited. In addition, this work represents
part of a series of new PBPK models that predict both perchlorate and iodide
kinetics, and the anion interaction at the NIS. Nearly identical model structures
were used to describe the distribution of both anions (Fig. 1), given their
similar size and ionic charge. Multiple subcompartments were used to describe
both active and passive transport of both anions in NIS-containing tissues
(thyroid, stomach, and skin), whereas single compartments were used to
describe passive diffusion through non-NIS tissues (kidneys, liver, fat, and the
remaining lumped rapidly and slowly diffused tissues).
Model structures of the stomach and skin were identical between anions.
The stomach contains the capillary bed, stomach wall, and contents. Skin
contains two subcompartments, representing the capillary bed and the skin
tissue. Other tissues also exhibit NIS, such as the salivary glands and choroid
plexus (Brown-Grant, 1961; Spitzweg et al., 1998). However, the anion pools
in these other tissues are not large enough to affect serum levels significantly
and, therefore, were not included in the model.
258
MERRILL ET AL.
FIG. 1. Schematic of PBPK model for perchlorate and iodide distribution.
Bold arrows indicate sites of active uptake at either NIS or the thyroid apical
iodide channel (with exception of bold arrow for plasma binding of ClO 4 – and
I –). Small arrows indicate passive diffusion.
Differences in model structure between anions exist in the thyroid and blood
compartments. In the thyroid, I – is organified (thyroid hormone production),
whereas ClO 4 – is unreactive and eventually diffuses from the thyroid back into
the systemic circulation (Anbar et al., 1959; Wolff, 1998). Therefore, for
iodide, the thyroid consists of three subcompartments, representing the stroma,
the follicle, the colloid, as well as a separate compartment representing all
bound (organified) iodide in the thyroid. For perchlorate, only the stroma,
follicle, and colloid were included in the thyroid structure. Thyroid perchlorate
time-course data reveal an initial rapid phase in thyroid uptake and equilibrium, presumably between the stroma and follicle, and a slower phase of
equilibrium and clearance between the follicle and lumen (Chow and Woodbury, 1970; Yu et al., 2002). A two-compartmental thyroid failed to fit this
behavior (see Results section). Similarly, several compartmental models for
iodide have also described the necessity for a three-compartmental thyroid
(Lee et al., 1982), which captures the early phase of iodide uptake, consisting
of trapping and organification processes and a slower phase of hormone
accumulation and secretion.
The major difference in the blood compartments is that there is considerable
reversible binding between plasma proteins and ClO 4 – but not I –. Passive
diffusion between plasma and red blood cells (RBCs), however, occurs for
both anions. Equilibrium dialysis studies in rat plasma performed at the
University of Georgia show greater than 99% binding of ClO 4 – in plasma at
concentrations ⱕ100 mg/l and approximately 50% bound at concentrations
ⱖ500 mg/l (see Table 1) (J. W. Fisher, personal communication). MichaelisMenten (M-M) kinetics were used in the model to describe the association of
the free ClO 4 – fraction to unspecific plasma binding sites, and a first-order rate
was used for the dissociation. In the case of iodide, shortly after an injection
of radiolabeled iodide, 88% of plasma iodide radioactivity exists as free
unorganified iodide (Yu et al., 2002). The remaining fraction is incorporated
into either thyroid hormones or nonhormonal iodinated proteins and is analytically indistinguishable from the “free” iodide. Unlike ClO 4 –, however, both
the free and incorporated iodine fractions are taken up into tissues. Therefore,
for simplifying the interpretation of such radiolabeled iodine data, the model
lumps the fractions of free and bound iodide into a single pool for “total
iodide” in the plasma. Thus, it was not necessary to include binding of I – to
serum proteins in order to describe radioiodine kinetics at this point. In the
thyroid itself, however, it is important to distinguish between free and organified (incorporated) iodide, due to the fact that approximately 90% is incorporated (Yu et al., 2002).
Active uptake of ClO 4 – and I – by NIS in the thyroid, skin, and gastric
mucosa and by the apical iodide channel in the thyroid was described using
Michaelis-Menten (M-M) kinetics for nonlinear processes (Fig. 1, bold arrows)
(Chow et al., 1969; Kotani et al., 1998; Wolff, 1998). Permeability area
cross-products and partition coefficients were used to describe the movement
of the anions (ClO 4 – and I –) between the capillary bed, tissue, and inner
compartments (Fig. 1, small arrows), which results from the inherent electrochemical gradient within the tissues. First-order clearance rates were used to
describe the organification of iodide in the thyroid and the subsequent release
of the bound iodide into systemic circulation.
Passive diffusion using partitions and blood flows were used to describe
movement of both anions into the kidney, liver, and fat, because these tissues
do not contain NIS. Urinary clearances were modeled using first-order clearance rates from the kidneys. Fecal excretion of both anions was not significant
(Hays and Wegner, 1965; Yu et al., 2002). Although the current model does
not encompass thyroid hormone regulation, the liver is the major site of
extrathyroidal deiodination and, therefore, was maintained as a separate compartment for future model development. Fat was included as an essentially
exclusionary compartment because large variations in fat content among
humans, as well as dramatic changes during growth and reproduction in
females, could alter ClO 4 –/I – kinetics. This enhanced the model’s capacity for
extrapolations to other species, genders, or reproductive stages.
Physiological parameters. Tissue volumes, V(s), and blood flows, Q(s),
for most compartments were derived from Brown et al. (1997), except for the
cellular dimensions of the thyroid, which were obtained from Malendowicz
and Bednarek (1986) (Table 2). Tissue volumes were scaled linearly with BW,
and Q(s) was multiplied by BW 3/4.
Chemical-specific parameters. The values and sources of chemical-specific parameters used in the model are provided in Table 3. Partition coefficients (P) and blood flows (Q) were used to describe compartments with
flow-limited diffusion. Partition coefficients for both anions were calculated
from tissue/plasma ratios at 24-h time points from Yu et al. (2002), when
available. Tissue/plasma concentrations that were not available from Yu et al.
(2002), such as those for iodide partitioning into kidney, liver, skin, and RBCs,
were obtained from other literature sources (see Table 3). In the case of I –
partitioning into fat, data were not available. Therefore, due to similar polarity,
the fat/blood value reported by Pena et al. (1976) for ClO 4 – was used.
Clearance values (Cl) for first-order rates of urinary excretion, dissociation
rates from serum binding proteins, rates of organification of thyroid iodide, and
secretion of bound iodine from the thyroid into systemic circulation were
determined by fitting model simulations to available time-course data at
various doses.
For compartments with nonlinear uptake of the anions, effective partition
coefficients (P) (reflecting either approximate tissue/serum ratios or electrical
TABLE 1
Percentage ClO 4 – Bound in Plasma
Serum ClO 4 – concentration (ng/ml)
% bound
10.07 ⫾ 0.11
49.57 ⫾ 1.03
100.43 ⫾ 1.42
251.01 ⫾ 0.11
496.30 ⫾ 6.07
100.71 ⫾ 1.11
99.14 ⫾ 2.06
100.43 ⫾ 1.42
50.20 ⫾ 0.02
49.63 ⫾ 0.61
Note. N ⫽ 2 (Fisher, 2002).
PBPK PREDICTIONS OF PERCHLORATE DISTRIBUTION AND THYROID IODIDE INHIBITION
259
TABLE 2
Physiologic Parameters for the Male Rat
Male Rat
Source
Tissue volumes
Body weight BW (kg)
Slowly perfused VSc (% BW)
Richly perfused VRc (% BW)
Fat VFc (% BW)
Kidney VKc (% BW)
Liver VLc (% BW)
Stomach tissue VGc (% BW)
Gastric juice VGJc (% BW)
Stomach blood VGBc (% VG)
Skin tissue VSkc (% BW)
Skin blood VSkBc (% VSk)
Total thyroid VTc (% BW)
Thyroid follicle VTFc (% VT)
Thyroid colloid VTLc (% VT)
Thyroid blood VTBc (% VT)
Plasma VPlasc (% BW)
Red blood cells VRBCc (% BW)
Blood flows
Cardiac output QCc (L/h/kg)
Slowly perfused QSc (% QC)
Richly perfused QRc (% QC)
Fat QFc (% QC)
Kidney QKc (% QC)
Liver QLc (% QC)
Stomach QGc (% QC)
Skin QSkc (% QC)
Thyroid QTc (% QC)
0.3
74.6
11.0
7.4
1.7
5.5
0.54
1.68
4.1
19.0
2.0
0.0077
59.9
24.4
15.7
4.1
3.3
Yu et al., 2002
Brown et al., 1997
Brown et al., 1997
Brown et al., 1997
Brown et al., 1997
Brown et al., 1997
Yu et al., 2002
Yu et al., 2002
Altman and Dittmer, 1971a
Brown et al., 1997
Brown et al., 1997
Malendowicz and Bednarek, 1986
Malendowicz and Bednarek, 1986
Malendowicz and Bednarek, 1986
Malendowicz and Bednarek, 1986
Altman and Dittmer, 1971b; Brown et al., 1997
Altman and Dittmer, 1971b; Brown et al., 1997
14.0
24.0
76.0
6.9
14.0
17.0
1.61
5.8
1.6
Brown et al., 1997
Brown et al., 1997
Brown et al., 1997
Brown et al., 1997
Brown et al., 1997
Brown et al., 1997
Malik et al., 1976
Brown et al., 1997
Brown et al., 1997
potential gradients) and permeability area cross-products, PA(s), were used to
describe passive diffusion. Simulations reveal that alterations in PA values
have a direct impact on the uptake portion of the curve, with lower values
indicating slower uptake. Therefore, PA(s) were visually optimized to the
uptake portion of the time-course data after setting P to the experimentally
determined values in Table 3.
Measured anion concentration gradients between the cellular subcompartments of the thyroid were not available. Therefore, effective partitions of the
anions between the cellular subcompartments of the thyroid (stroma/follicle
and follicle/lumen) were based on electrochemical gradients measured by
Chow and Woodbury (1970) at three doses of ClO 4 –. The difference between
the stroma and follicle can be interpreted as an effective P for charged
moieties, such as ClO 4 – and I –, hindering the entry of negatively charged ions
into the follicle. The approximately equal and opposite potential from the
follicle to the colloid enhances passage of the anions into the colloid, creating
an effective P ⬎ 1. The equivalence between electrical potential differences,
f i–f o, and ionic concentrations, c i and c o, were estimated in the manner of
Kotyk and Janacek (1977), as follows:
␾ i ⫺ ␾ o ⫽ 2.303
RT
co
log
zF
ci
where R is the gas constant, T is the absolute temperature, F is the Faraday
constant, and subscripts i and o refer to “inside” and “outside” the cellular
membranes. At 37°C (2.303 RTF ⫽ 61.6 mV) for a singly charged ion (z ⫽ 1),
this becomes:
␾ i ⫺ ␾ o ⫽ 61.6 log
co
共mV兲
ci
In general, the ratio of species concentrations between two media is given by
the partition coefficient (K p), where K p ⫽ c oc i. Thus, Equation 2 becomes:
␾ i ⫺ ␾ o ⫽ 61.6 log K p
or
K p ⫽ 10 ␾ i⫺ ␾ oⲐ61.6
From Chow and Woodbury (1970), the potential difference for the stroma/
follicle interface ranges from –58 to –51 mV. Therefore, from the above
equation, K p for a monovalent, negatively charged ion is between 0.114 and
0.149. Similarly, for the follicle/lumen interface, f i–f o ranges from ⫹50 to ⫹58
mV, rendering the effective K p between 6.48 and 8.74 (Table 3). These values
were also used to describe the effective partitioning of I –, because both anions
have the same ionic charge.
An affinity constant (Km) of 4.0 ⫾ 1.2 ⫻ 10 6 ng/l for I – at the NIS was
derived by Gluzman and Niepomniszcze (1983) from thyroid slices of five
normal humans. They noted little variation in Km(s) between thyroid specimens from other species. For example, average Km(s) for bovine and porcine
specimens were 5.0 and 3.9 ⫻ 10 6 ng/l, respectively (Gluzman and Niepomniszcze, 1983) and Km(s) from rat thyroid specimens varied from 3.17 to
10.2 ⫻ 10 6 ng/l (Wolff, 1964). Wolff and Maurey (1963) found that iodide’s
affinity for NIS varied little across different tissues. This is supported by
Kosugi et al. (1996), who reported a Km for iodide of 4.4 ⫻ 10 6 ng/l in Chinese
hamster ovary cells. Therefore, the Km value for I – measured by Gluzman and
Niepomniszcze (1983) was used in all NIS compartments.
Both anions compete with each other for the NIS receptor. However, ClO 4 –
is known to have a much greater affinity for NIS than I – and, thus, is
preferentially transported (Wolff, 1998; Wolff and Maurey, 1963). Several
studies support this. Halmi and Stuelke (1959), found ClO 4 – to be 10 times as
effective as I – in depressing thyroid and gastric juice to blood I – ratios in the
rat. Similarly, Harden et al. (1968) found human saliva/plasma radioiodide
260
MERRILL ET AL.
TABLE 3
Chemical-Specific Parameters for Male Rat
Partition coefficients (unitless)
Slowly perfused/plasma PS
Richly perfused/plasma PR
Fat/plasma PF
Kidney/plasma PK
Liver/plasma PL
Gastric tissue/gastric blood PG
Gastric juice/gastric tissue PGJ
Skin tissue/skin blood PSk
Thyroid follicle/stroma PT
Thyroid lumen/follicle PDT
Red blood cells/plasma PRBC
Maximum velocity, Vmaxc (ng/h/kg)
Thyroid follicle VmaxcTF
Thyroid lumen VmaxcTL
Skin VmaxcS
Gastric VmaxcG
Plasma binding VmaxcB
Affinity constants, Km (ng/L)
Thyroid lumen KmTL
Thyroid follicle KmTF
Skin KmS
Gastric (stomach) KmG
Plasma binding KmB
Permeability area cross-products, PA (l/h/kg)
Gastric blood to gastric tissue PAGc
Gastric tissue to gastric juice PAGJc
Skin blood to skin tissue PASkc
Plasma to red blood cells PARBCc
Thyroid follicle to stroma PATFc
Thyroid lumen to follicle PATLc
Clearance values, Cl (l/h/kg)
Urinary Excretion CLUc
Plasma Unbinding Clunbc
Hormone Production Clhormc
Hormone Secretion Clsecrc
Perchlorate
Iodide
0.31
0.56
0.05
0.99
0.56
0.70
1.70
1.0
0.15
8.00
0.73
0.21
0.40
0.05
1.00
0.44
1.0
3.50
0.70
0.15
8.00
1.00
1.0 ⫻ 10 3
2.0 ⫻ 10 4
5.0 ⫻ 10 5
2.0 ⫻ 10 4
3.4 ⫻ 10 3
5.4 ⫻ 10 4
4.0 ⫻ 10 6
5.0 ⫻ 10 5
2.0 ⫻ 10 6
1.0 ⫻ 10 2
Fit
Fit
Fit
Fit
Fit
1 ⫻ 10 8
1.8 ⫻ 10 5
1.8 ⫻ 10 5
1.7 ⫻ 10 5
1.1 ⫻ 10 4
1.0 ⫻ 10 9
4.0 ⫻ 10 6
4.0 ⫻ 10 6
4.0 ⫻ 10 6
—
Chow and Woodbury, 1970; Golstein et al.,
Gluzman and Niepomniszcze, 1983; Kosugi
Gluzman and Niepomniszcze, 1983; Kosugi
Gluzman and Niepomniszcze, 1983; Kosugi
Fit
1.00
0.80
0.80
1.00
6.0 ⫻ 10 –5
0.01
1.00
0.10
0.10
1.00
1.0 ⫻ 10 –4
4.0 ⫻ 10 –7
Fit
Fit
Fit
Fit
Fit
Fit
0.07
0.032
—
—
0.05
—
0.10
1.2 ⫻ 10 –6
Fit
Fit
Fit
Fit
concentrations to be seven times lower after equimolar doses of ClO 4 – than
after I –. Lazarus et al. (1974) also demonstrated this effect on saliva/plasma
iodide in mice. Both the salivary glands and stomach are useful for estimating
inhibitory effects on iodide uptake by monovalent anions at NIS, because
organic binding of iodide does not occur in these tissues. Lastly, Kosugi et al.
(1996) actually measured a ClO 4 – Km of 1.5 ⫻ 10 5 ng/l in Chinese hamster
ovary cells. Based on these findings, the measured Km reported by Kosugi et
al. (1996) was initially used. However, visual optimization of thyroid ClO 4 –
data indicated that a slightly higher Km value of 1.8 ⫻ 10 5 ng/l was appropriate
(Table 3). Therefore, due to perchlorate’s considerably lower Km and, thus, its
greater affinity for NIS than that of I –, inhibition of ClO 4 – uptake by I – is
insignificant and not included in the model.
The apical membrane of the thyroid follicle exhibits an additional I – transport channel. A Km of approximately 4.0 ⫻ 10 9 ng/l, describing I – transport
from the bovine thyroid follicle to the colloid (KmTL i in our model notation)
was estimated by Golstein et al. (1992). Using this value, however, the model
underestimates thyroid iodide concentrations at and beyond 8 h post iv-dosing.
A slightly lower Km of 1.0 ⫻ 10 9 ng/l was required to fit thyroid iodide data.
As with NIS, this apical channel also appears to be sensitive to inhibition by
ClO 4 – (Golstein et al., 1992). Model simulations of both thyroid I – inhibition
and thyroidal ClO 4 – levels supported a KmTL p value of 1.0 ⫻ 10 8 ng/l,
approximately 10 times less than that of I – (KmTL i). Radiolabeled perchlorate
Source
Halmi et al., 1956; Yu et al., 2002
Halmi et al., 1956; Yu et al., 2002
Pena et al., 1976
Perlman et al., 1941; Yu et al., 2002;
Perlman et al., 1941; Yu et al., 2002
Yu et al., 2002
Yu et al., 2002
Perlman et al., 1941; Yu et al., 2002
Chow and Woodbury, 1970
Chow and Woodbury, 1970
Rall et al., 1950; Yu et al., 2002
1992
et al., 1996
et al., 1996
et al., 1996
data presented by Chow and Woodbury (1970) also supports this value. After
ip administration of 200 mg 36ClO 4 –/kg in rats, Chow and Woodbury measured
thyroid saturation at approximately 1.8 ⫻ 10 8 ng/l, indicating saturation of the
lumen.
Whereas the Km is similar across NIS-containing tissues and species,
maximum velocity (Vmax) varies significantly (Gluzman and Niepomniszcze,
1983; Wolff, 1998; Wolff and Maurey, 1961). Simulations revealed Vmax(s)
having a greater effect on tissue clearance than on uptake. Tissue clearance is
also affected by substrate concentration and blood flow; however, measured
values are used for these parameters. Therefore, due to the lack of reported
values, ClO 4 – Vmaxc(s) for the thyroid, stomach, and skin were estimated by
visually optimizing the clearance portion of the 36ClO 4 – time-course data and
from thyroid measurements obtained from the drinking water data by Yu et al.
(2002), at dose levels exhibiting saturation (Figs. 2A, 2D, 2E; also Fig. 6B in
Results section). ClO 4 – demonstrated saturability between 1.0 and 3.0 mg/kg/
day. Iodide Vmaxc(s) for the same compartments were also visually fit to the
clearance portion of iodide time-course kinetics (Figs. 3 and 4).
TSH-induced upregulation has been shown to increase the Vmax at thyroidal
NIS by increasing both its activity and half-life (Riedel et al., 2001). Upregulation of thyroid NIS was occurring during the ClO 4 – drinking water studies,
as revealed by elevated TSH levels across doses and compensation of thyroid
iodide inhibition (Yu et al., 2002). To fit the upregulated thyroid uptake,
PBPK PREDICTIONS OF PERCHLORATE DISTRIBUTION AND THYROID IODIDE INHIBITION
261
thyroid ClO 4 – and I – levels were first visually optimized by increasing the
Vmax(s) at the NIS of the thyroid follicular membrane (VmaxTF p and VmaxcTF i) to fit measured values at each dose. The “upregulated” VmaxTF(s) were
plotted against the predicted corresponding free ClO 4 – levels in serum. The
equations from the resulting M-M type curves were used to calculate “upregulated” VmaxTF p and VmaxcTF i in the model.
Sensitivity analysis. An analysis of model parameter sensitivity on the
predicted area under the curve (AUC) of serum ClO 4 – concentrations was
performed. Using a 1% increase in each chemical-specific parameter value, the
model was run at two drinking water ClO 4 – doses, above and below NIS
saturation (0.1 and 10 mg/kg/day). The sensitivity coefficient for each parameter was then calculated, using Equation 4.
Sensitivity Coefficient ⫽
共A ⫺ B兲ⲐB
共C ⫺ D兲ⲐD
Where A equals serum ClO 4 – AUC with 1% increased parameter value, B
equals serum ClO 4 – AUC using original parameter value, C equals parameter
value increased 1% from original value, and D equals the original parameter
value.
Allometric scaling and equations. For ongoing extrapolations of the
model to humans, allometric scaling was applied by multiplying all Vmaxc(s),
PA(s), and Cl(s) by BW 3/4. Simultaneous differential equations in the model
code were written and computed using ACSL (Advanced Continuous Simulation Language) software (AEgis Technologies, Huntsville, AL). Example
equations demonstrating both diffusion-limited uptake, using P(s) and PA(s),
first-order clearance values, Cl(s), and saturable uptake using M-M parameters
are presented in the Appendix.
RESULTS
Parameterization
FIG. 2. Model calibrations (lines) versus actual 36ClO 4 – levels (mean ⫾
SD) in the thyroid (A), serum (B), red blood cells (C), skin (D), gastric
contents (E), gastric tissue (F), kidneys (G), liver (H), slowly perfused tissue
(I), and urine (J) after an iv dose of 3.3 mg/kg 36ClO 4 – (Yu et al., 2002).
Radiolabeled perchlorate 36ClO 4 – time-course kinetics.
Model simulations of various tissues and serum concentrations
were derived using the physiologic and chemical-specific parameters provided in Tables 1 and 2. Figure 2 shows simulations of 36ClO 4 – in various tissues after an iv dose (3.3 mg/kg).
Using partitions (P) and blood flows (Q) derived from the
literature, excellent reproductions of the 36ClO 4 – kinetics in all
flow-limited tissues were achieved, without any fitting required. The stomach, skin, and thyroid concentrations were
simulated by using literature-derived Km and effective P values and by visually optimizing the PA(s) and Vmaxc(s), as
described above. The model reproduced each data set with only
slightly overpredicting data in the clearance portion of the
thyroid time-course data (Fig. 2A).
Radiolabeled iodide ( 125I –) time-course kinetics. As previously stated, the amount of I – in the simulations represents total
iodine in all tissues except the thyroid, where both free I – and
bound iodine are shown (where data are available). Modelpredicted I – kinetics (from 0.033 mg/kg 125I – iv) in the thyroid,
serum, stomach contents, skin, and urine are shown versus
measured data in Figures 3 and 4. Although stomach and skin
data displayed wide variation, the simulated amount of 125I – in
stomach contents and skin indicated rapid uptake and gradual
reabsorption, suggesting that these tissues play an important
role in iodide turnover. The model accurately simulates the
amount of 125I – in the thyroid over 96 h and in serum over 24 h
after dosing (Figs. 3A and 4B).
262
MERRILL ET AL.
FIG. 3. Model calibrations (lines)
versus measured 125I – (mean ⫾ SD) in
thyroid (A), serum (B), stomach contents
(C), and skin (D) of male rats after an iv
dose of 0.033 mg/kg 125I – (Yu et al.,
2002).
Perchlorate drinking water kinetics. To simulate drinking
water exposure, the model was programmed to provide a
continuous oral dose for 12 h/day, assuming rats drink throughout their waking hours. Without taking plasma binding into
account, the model underpredicts serum concentrations at 0.1
mg/kg/day and lower. Therefore, binding parameters (KmB p,
VmaxcB p, and Clunb p) were estimated by fitting serum simulations to data from 0.1 and 1.0 mg/kg/day, in accordance with
the unpublished binding data by Fisher (personal communication). Using these binding parameters with the other ClO 4 –
parameters describing acute 36ClO 4 – kinetics, the model successfully predicted other serum concentrations from subchronic exposure, ranging from 0.01 to 10.0 mg/kg/day (Fig.
5). Upregulation of the thyroid was evident from elevated TSH
levels in the drinking water studies across doses (Yu et al.,
2002). However, this upregulation of the thyroid does not
significantly affect serum concentrations, due to the small size
of the gland. Additionally, TSH does not regulate NIS activity
in any tissues other than the thyroid (Brown-Grant, 1961;
Cavalieri, 1997; Spitzweg et al., 2000). Although not of consequence in this model, upregulation of mammary gland NIS
expression has been noted in lactating rats with normal TSH
levels and appears to be regulated by prolactin and/or oxytocin
(Spitzweg et al., 2000), inferring that other agents may stimulate NIS expression and function. Thus, parameters for the
extrathyroidal tissues should not change from those determined
from the kinetic data. This is consistent with our model; had
VmaxGJ and VmaxSk changed between acute and subchronic
exposures, significant reductions in serum levels, which are not
observed, would be noted.
As stated above, TSH-induced upregulation of thyroid NIS
was apparent in the drinking water studies. As expected, using
the acute thyroid parameters, the model underpredicted thyroid
ClO 4 – concentrations at 3.0 mg/kg/day and higher (Fig. 6A).
By using the “upregulated” VmaxTF p, described in the Materials and Methods section, the model successfully predicted
thyroid ClO 4 – concentrations across doses at day 14 (336 h)
(Fig. 6B). However, the dose-dependent equation does not
account for the time lag of induction, demonstrated by the
gradual increase in ClO 4 – uptake between days 1 and 5 at 3.0
and 10.0 mg/kg/day, suggesting that NIS induction by ClO 4 – is
both time- and dose-dependent.
Model Validation
Using the parameters estimated from the various time-course
data presented above, the model’s ability to predict thyroid
PBPK PREDICTIONS OF PERCHLORATE DISTRIBUTION AND THYROID IODIDE INHIBITION
263
FIG. 4. Model calibrations (lines)
versus measured (A) free 125I – and (B)
bound 125I – concentrations (mean ⫾ SD)
in thyroid up to 96 h, (D) total 125I – in
serum, and (C) cumulative 125I – in urine
for up to 24 h following iv administration
of 0.033 mg/kg 125I – (Yu et al., 2002).
inhibition data from Yu et al. (2002) was tested. In addition,
model predictions were compared with available 36ClO 4 – and
radioiodide data from the literature.
Inhibition of thyroid I – uptake after ClO 4 – iv dosing. The
model successfully simulated inhibition of thyroid 125I – uptake
following iv doses of 0, 0.01, 0.1, 1.0, and 3.0 mg/kg ClO 4 –
(Fig. 7) using parameters established from the acute 36ClO 4 –
and subchronic drinking water data. The measured means ⫾
SD for 125I – inhibition in the thyroid were 13.0 ⫾ 18.7, 23.6 ⫾
12.6, 69.7 ⫾ 9.5, and 87.5 ⫾ 2.0% at 2 h post 125I – dosing (4
h post ClO 4 –) (Yu et al., 2002) versus model-predicted reductions of 1.7%, 18%, 76%, and 91% from the predicted radioiodide uptake in controls (without ClO 4 –) at the same time
point for the 0.01, 0.1, 1.0, and 3.0 mg/kg dose groups, respectively. The model simulations lie within the range of measured
data, with exception of slight overprediction of inhibition at 3.0
mg/kg at 4 h post ClO 4 –.
Inhibition of thyroid 125I – uptake after drinking water exposure to ClO 4 –. TSH-induced upregulation of NIS compensated for competitive inhibition of thyroid I – uptake by ClO 4 –
across doses in the drinking water study. Only slight inhibition
was seen at 10 and 30 mg ClO 4 –/kg/day on day 14 (Yu et al.,
2002). The increased NIS activity was modeled using the
upregulated VmaxTF i and VmaxTF p, calculated using M-M
FIG. 5. Model calibrations (lines) versus measured mean ⫾ SD (bars)
serum ClO 4 – concentrations after a) 0.01, b) 0.1, c) 1.0, d) 3.0, e) 10.0, and f)
30.0 mg/kg/day perchlorate via drinking water for 14 days (336 h) (Yu et al.,
2002). The 24- and 120-h time points correspond to 3 (d), 10 (e), and 30 (f)
mg/kg/day.
264
MERRILL ET AL.
FIG. 6. Model validations (lines)
versus measured thyroid ClO 4 – concentrations (means ⫾ SD) during ingestion
of 0.01, 0.1, 1.0, 3.0, 10.0, and 30.0 mg/
kg/day ClO 4 – in drinking water for 14
days (Yu et al., 2002). (A) Simulations
using thyroid parameters established
from acute dosing underpredict measurements at 3.0, 10.0, and 30.0 mg/kg/day.
(B) Simulations across doses using a
dose-dependent upregulated VmaxcTF p.
equations dependent on serum-free ClO 4 – concentrations, as
described in the Materials and Methods section. Using the
upregulated VmaxTF i and VmaxTF p values, the model restores
thyroid uptake of radioiodide across doses to control levels.
Other ClO 4 – and radioiodide studies used for model validation. Using the parameters developed with the data primarily
from Yu et al. (2002), the model predicted various tissue
concentrations of ClO 4 – and radioiodide from other studies.
Urinary ClO 4 – levels measured by Eichler (1929) were successfully predicted at doses of 1.6, 8.0, and 49 mg/kg (Fig. 8).
Chow and Woodbury (1970) gave 0.5, 10.0, and 200 mg
36
ClO 4 –/kg by ip administration to rats and measured radioac-
FIG. 7. Model validations of iodide uptake inhibition in male rats intravenously administered ClO 4 –, followed by 33 mg/kg 125I – with carrier 2 h later.
Model predictions (lines) versus measured thyroid 125I – concentrations
(mean ⫾ SD) after 0, 0.01, 0.1, 1.0, and 3.0 mg/kg ClO 4 – are presented. (Note:
inhibition from 0.0 and 0.01 mg/kg ClO 4 – nearly overlap) (Yu et al., 2002).
tivity in their thyroids and serum. The rats in this study were
functionally nephrectomized by ligation of the renal pedicle of
both kidneys. Therefore, to simulate this condition, the model’s
urinary ClO 4 – excretion (ClU p) was set to zero. The model
adequately predicted thyroid concentrations across doses (Fig.
9A). Predicted serum concentrations at 0.5 and 10.0 mg/kg
were within a factor of 2 from the data (Fig. 9B).
Finally, the model also reproduced iodide data from Perlman
et al. (1941) and Wolff (1951). Perlman et al. studied iodide
distribution in rats after administering 2.63 mg/kg labeled
iodine via stomach tubes. The model predicted their timecourse data for liver accurately and their kidney data within a
factor of less than 1.3 (Fig. 10). Wolff (1951) measured thyroid
iodide clearance in male rats up to 210 h after an ip injection
FIG. 8. Model validations (lines) versus mean cumulative ClO 4 – in urine
(⫹) from male rats (N ⫽ 8) after subcutaneous doses of 1.6, 8.0, and 49 mg/kg
(Eichler, 1929).
PBPK PREDICTIONS OF PERCHLORATE DISTRIBUTION AND THYROID IODIDE INHIBITION
265
FIG. 9. Model validations (lines)
versus measured mean ClO 4 – concentrations (⫹) in thyroid (A) and serum (B) in
male rats (N ⫽ 4 – 6) after ip administration of 0.5, 10.0, and 200 mg/kg 36ClO 4 –
(Chow and Woodbury, 1970).
of 33.0 mCi 131I – (approximately 0.88 ng/kg). Again, the model
successfully predicted the kinetic behavior of I – in the thyroid
(Fig. 11).
– 0.12. Sensitivity coefficients of all other parameters were at
or below an absolute value of 0.01.
DISCUSSION
Sensitivity Analysis
–
Sensitivity analysis on serum ClO 4 AUC levels at 0.1
mg/kg/day indicated plasma binding parameters for maximum
capacity (VmaxB p) and the first-order dissociation rate (Clunb p)
had the greatest influence, with sensitivity coefficients of 0.5
and – 0.32, respectively. Skin parameters exhibited the next
greatest effect on serum ClO 4 – AUC, with sensitivity coefficients of – 0.29, – 0.22, and 0.21 for the skin/plasma partition
coefficient (PSk p), VmaxSk p, and PASk p, respectively. Urinary
clearance (ClU p) followed, with a sensitivity coefficient of
FIG. 10. Model validations (lines) versus measured mean iodide concentrations in kidneys (open circles) and livers (filled circles) of male rats (N ⫽
20) after receiving KI (2.5 mg/kg I –) (Perlman et al., 1941).
Model predictions of internal doses of ClO 4 – show excellent
correspondence with measured tissue concentrations. Thus, we
have confidence in the chosen model description of ClO 4 – and
I – kinetics. The model also stipulates a specific mechanism for
the interaction of perchlorate with iodide at the thyroid and
other tissues, particularly its competition for transport by the
NIS. In this case, too, the model has been found to agree with
the available experimental data related to iodide uptake inhibition after acute ClO 4 – dosing, ranging from 0.01 to 3.0
FIG. 11. Model validation (line) versus measured mean 131I – in the thyroid
(filled circles) of male rats after an ip injection of 0.88 ng 131I – (N ⫽ 6 to 17
per time point) (Wolff, 1951).
266
MERRILL ET AL.
mg/kg, and after subchronic exposure to ClO 4 – via drinking
water.
The closeness of parameter values describing passive diffusion indicates that the kinetics of I – and ClO 4 – are generally
similar. In addition, urinary clearances were very close and
agreed with Eichler and Hackenthal (1962), in that urinary
clearance of ClO 4 – was found to be slightly higher than that of
I –. Values for saturable uptake, however, are somewhat different between anions. Perchlorate exhibits a greater affinity for
NIS than does I –, with a Km approximately 10 times lower than
that of I –. Although both anions can compete for NIS, perchlorate’s lower Km renders inhibition of ClO 4 – uptake by
iodide insignificant, as described by the M-M equations and as
supported by the inhibition data. Vmax(s) and PA(s) for the
skin and stomach, however, were similar between anions, with
the exception of VmaxG(s), for anion transport into stomach
contents, which was an order of magnitude higher for iodide
than for ClO 4 –. However, this value was based on limited and
highly variable data. Thus, it is possible that additional data
would suggest that the VmaxG(s) (for gastric uptake of I – and
ClO 4 –) are actually closer.
Thyroid parameters showed the greatest difference between
the two anions, where VmaxcTF (for transport into the thyroid
follicle) and VmaxcTL (for transport into the colloid or lumen)
were approximately one and three orders of magnitude greater
for I – than for ClO 4 –, respectively. In addition, a lower permeability area cross-product (PATL) was required to describe
diffusion between the follicle and lumen for I – than for ClO 4 –
(0.0001 vs. 0.01 l/h/kg). This slower uptake and clearance of
iodide is expected, due to I – organification in the follicle and
hormone storage in the colloid. The storage of organified I –
also results in much higher thyroid/serum concentrations than
those of ClO 4 –. The rate of organification of I – (Clhorm i) is
relatively fast (0.1/h), whereas the rate of secretion of organified I – into systemic circulation (Clsecr i) is very slow (9.5 ⫻
10 –7/h), thus resulting in a buildup of organic iodine that would
not occur with ClO 4 –.
Serum kinetics were quite different between the anions, due
to the large amount of nonspecific plasma binding of ClO 4 – at
low doses. The literature suggests that albumin and prealbumin
are the major serum binding proteins for ClO 4 –. Shishiba et al.
(1970) found that ClO 4 – interferes with binding of free thyroxine (fT 4) to prealbumin and albumin but not to thyroid binding
globulin (TBG) in human blood. As stated earlier, T 4 was also
displaced in rat serum after ClO 4 – administration (Yamada,
1967; Yamada and Jones, 1968). The ClO 4 – data suggest that
more than one binding site in serum is present. Model-predicted serum ClO 4 – values at 0.1 mg/kg/day were at the low
end of the data, and it is possible that multiple plasma binding
sites are responsible.
To fit the TSH-upregulated thyroid ClO 4 – concentrations
resulting from high drinking water exposures, it was necessary
to use a dose-dependent increase in VmaxcTF p. However, it is
evident from the increase in thyroid ClO 4 – between days 1 and
5 at 3.0 mg/kg/day and higher that the dose-dependent Vmax-
TF p should also be time-dependent in order to capture changing NIS activity. The fact that full compensation of thyroidal
125 –
I inhibition was not seen on day 14 in the 10.0 and 30.0
mg/kg/day of ClO 4 – also suggests that a similar time-dependent
saturable function is also required for VmaxTF i. Other subtle
changes known to result from TSH stimulation include increased follicle size (Conde et al., 1991; Ginda et al., 2000),
total protein, and RNA and DNA content (Pisarev and Kleiman
de Pisarev, 1980). Bagchi and Fawcett (1973) suggest that
variations in Vmax are also affected by intrathyroidal I – and the
magnitude of I – efflux.
The slight underprediction of Chow and Woodbury’s (1970)
serum ClO 4 – levels, from the nephrectomized rats (Fig. 9A),
may suggest increased plasma binding under those conditions.
Measured serum concentrations at each dose level exceed the
plasma binding KmB p of 1.1 ⫻ 10 4 ng/l. However, it does not
necessarily suggest that increased plasma binding is required
under normal physiologic conditions because the model adequately simulates 36ClO 4 – concentrations in various tissues and
serum at lower doses. Analytical differences in measurement
between Chow and Woodbury and Yu et al. (2002) may be
responsible. Another possibility is that blocking kidney discharge, as done by Chow and Woodbury, creates physiologic
effects, such as increased extracellular Na ⫹ (Tietz et al., 1990),
which may possibly affect binding to an extent that the model
cannot account for by simply eliminating urinary clearance.
Chow and Woodbury’s thyroid uptake data displayed an
interesting decrease in thyroid saturation with dose (Fig. 9B).
A 20-fold increase in dose, from 0.5 to 10.0 mg/kg, yielded a
small increase (factor of 2) in thyroid 36ClO 4 – concentration,
whereas an additional 20-fold increase in dose (200 mg/kg)
resulted in a 10-fold increase in thyroid concentration. The
model can physiologically describe this phenomenon, due to
the three-compartmental thyroid, which allows sequestration of
the anions tens of times greater than serum concentrations. At
200 mg/kg, the thyroid reaches an approximate 36ClO 4 – concentration of 1.8 ⫻ 10 8 ng/l. Although this concentration is
three orders of magnitude higher than the Km of NIS (KmTF p),
1.8 ⫻ 10 5 ng/l, it approximately equals the Km of the apical
channel (KmTL p), 1 ⫻ 10 8 ng/l. Therefore, the jump in thyroid
concentration measured by Chow and Woodbury reflects saturation of the lumen compartment and supports our estimated
KmTL p. The model, however, slightly underpredicts the rapid
uptake during the first hour post dosing at 0.5 and 10.0 mg/kg.
It is possible that nephrectomization may also have had an
effect on the rate of 36ClO 4 – uptake in the thyroid.
Accounting for free and bound iodide in the thyroid and
inhibition of NIS iodide transport by perchlorate are initial
steps toward modeling a complex regulatory process for thyroid hormone production. Free and bound thyroidal radioiodide
were accurately predicted from an iv dose of 33 ␮g/kg. In
addition, the model accurately predicted total thyroidal iodide
levels from an ip dose as low as 0.88 ng/kg. The current model,
based on the measured biologic and physiologic characteristics
of the organism, is readily extrapolated across species and
267
PBPK PREDICTIONS OF PERCHLORATE DISTRIBUTION AND THYROID IODIDE INHIBITION
gender (Clewell et al., 2001). Most importantly, the model
accurately predicts serum and thyroid perchlorate levels from
subchronic drinking water exposures (the most common route
of human exposure) from 0.01 to 30 mg/kg/day (90 to 270,000
ppb). Perchlorate-induced inhibition in humans after 2 weeks
of exposure via drinking water, from 0.007 to 0.5 mg/kg/day
(250 to 17,500 ppb, assuming water consumption of 2.0 l/day)
(Greer et al., 2002), has been shown to be more similar to the
acute inhibition seen in the rat (post iv-dosing with ClO 4 –, from
0.01 to 3.0 mg/kg), which this model predicts satisfactorily.
However, after 2 weeks of ClO 4 – exposure, upregulation and
hormone perturbations were not yet seen in the humans.
Clearly, the human’s iodide economy is more efficient than the
rat’s, likely due to greater plasma binding of thyroid hormones
(Dohler et al., 1979), but the extent of low-level perchlorate
exposure required for upregulation and hormone perturbations
in humans is unknown. Therefore, model-based predictions of
serum perchlorate from subchronic exposure and inhibition
from acute exposures may be highly valuable in risk assessment, especially in identifying dose levels that could potentially cause adverse effects, given prolonged exposure.
Similar models, incorporating physiologic changes in rats
during pregnancy and neonatal growth, have been developed
concurrently by Clewell et al. (in press). These models were
validated with both acute radioiodide doses, ranging from
0.005 to 33 mg/kg, and ClO 4 – drinking water data within the
same dose range as those used to validate this model. Overall,
model parameters were consistent with those of this model,
instilling further confidence in the parameters established here.
Differences found between sex and/or reproductive stages were
all physiologically logical. For example, during pregnancy,
estrogen levels rise sharply (Iino and Greer, 1961), resulting in
increased serum proteins and greater plasma binding of ClO 4 –
in the pregnant rat. The models also account for subtle, yet
important sex differences in thyroid morphology. In the male
rat, the follicle and colloid comprise approximately 60% and
24% of the thyroid volume, whereas in the pregnant dam, the
follicle and colloid both comprise approximately 45% of the
thyroid volume (Conde et al., 1991; Malendowicz and
Bednarek, 1986), reflecting the capacity for increased hormone
production during gestation. Together, these models enable
life-stage differences to be quantified and predictions of internal ClO 4 – dose (and its effect on iodide incorporation into the
thyroid) to be made (Clewell et al., 2001).
This model provides the foundation for development of a
more complex physiologically based dynamic model that
would account for hormone control systems and provide a
basis for the design of further experimental investigations.
Finally, by extrapolating to specific human exposure situations
(including particularly susceptible individuals, such as pregnant women or infants), such extended models will provide the
basis for more accurate and reliable risk assessments of the
effects of perchlorate on thyroid hormone production and homeostasis.
APPENDIX
EXAMPLE OF DIFFERENTIAL EQUATIONS USED IN
THE PBPK MODEL TO DESCRIBE CLO 4 – AND I –
DISTRIBUTION
1. Rate of change (ng/h) in the amount (RAX i) of I – or ClO 4 – in non-NIS
tissues (X):
冉
RAX i ⫽ QX ⫻
CA i ⫺
CX i
PX i
冊
where Q is the blood flow to X th tissue, and CA i is the concentration of either
I – or ClO 4 – (identified by subscripts i or p, respectively) in the arterial blood.
CX i is concentration of iodide (i) or perchlorate (p) in X th tissue.
2. Rates of change (ng/h) in amount of I – in the thyroid stroma, follicle, and
colloid (lumen) (RATS i, RATF i and RATL i, respectively) plus the amount of
bound thyroid I –, RAbnd i:
RATS i ⫽ QT ⫻ 共CA i ⫺ CVTS i 兲 ⫹ PATF i ⫻
RATF i ⫽ RupTF i ⫹ PATF i ⫻
冉
CTF i
PTF i ⫺ CVTS i
CVTS i ⫺ CTF i
PTF i
⫻
冉
冊
冉
冊
⫺ RupTF i
⫺ RupTL i ⫹ PATL i
CTL i
PTL i ⫺ CTF i
RATL i ⫽ RupTL i ⫹ PATL i ⫻
RupTF i ⫽
冉
冊
⫺ 共Clhorm i ⫻ CTF i 兲
CTF i ⫺ CTL i
PTL i
冊
VmaxTF i ⫻ CVTS i
CVTS p
KmTF i ⫻ 1 ⫹
⫹ CVTF i
KmTF p
RupTL i ⫽
冉
冊
VmaxTL i ⫻ CTF i
CTF p
KmTL i ⫻ 1 ⫹
⫹ CTF i
KmTL p
冉
冊
RAbnd i ⫽ 共Clhorm i ⫻ CTF i 兲 ⫺ 共Clsecr i ⫻ CTF i 兲
where subscripts i and p identify the anion I – or ClO 4 –, QT is thyroid blood flow
(l/h), CA i is the arterial blood concentration (ng/l), CVTS i,p is the thyroid
stroma concentration (ng/L) and CTF i,p is the follicular concentration (ng/l) of
iodide or perchlorate. PTF i, PTL i, PATF i, and PATL i are the partition coefficients and permeability cross-products describing passive diffusion of I – across
the basal (follicle/stroma) and apical (lumen/follicle) membranes. M-M equations used to describe the rates of active uptake of I – in the follicle and colloid
(RupTF i and RupTL i, respectively), include inhibition by ClO 4 – at NIS and the
apical iodide channel. VmaxTF i, VmaxTL i, KmTF i,p, and KmTL i,p are the
maximum velocities (ng/h/kg) and affinity constants (ng/l) for transport of I – or
ClO 4 – into the follicle and lumen. Clhorm i and Clsecr i are first-order clearance
values (h –1) for the organification of iodide into thyroid hormones and the
secretion of organified iodide into systemic circulation. Thyroid transport of
ClO 4 – is calculated similarly, with the exception that organification and inhibition of ClO 4 – uptake by iodide are not included. As described earlier, due to
iodide’s lesser affinity (10-fold higher Km) than ClO 4 –, it does not significantly
inhibit ClO 4 – sequestration in NIS-containing tissues.
3. Rates of change (ng/h) in the amount of I – or ClO 4 – in the gastric capillary
bed, tissue, and secretions (juice) (RAGB i, RAG i,and RAGJ i, respectively):
RAGB i ⫽ QG ⫻ 共CA i ⫺ CVGB i 兲 ⫹ PAG i ⫻
冉
CG i
⫺ CVGB i
PG i
冊
268
MERRILL ET AL.
RAG i ⫽ PAG i ⫻
冉
CVGB i ⫺ CG i
PG i
冊
⫹ PAGJ i ⫻
RAGJ i ⫽ RupGJ i ⫹ PAGJ i ⫻
冉
冉
CGJ i
PGJ i ⫺ CG i
CG i ⫺ CGJ i
PGJ i
冊
冊
Brown, R. P., Delp, M. D., Lindstedt, S. L., Rhomberg, L. R., and Beliles, R. P.
(1997). Physiological parameter values for physiologically based pharmacokinetic models. Toxicol. Ind. Health 13, 407– 484.
⫺ RupGJ i
⫹ RMR i
VmaxGJ i ⫻ CG i
RupGJ i ⫽
CG p
KmG i ⫻ 1 ⫹
⫹ CG i
KmG p
冉
冊
where subscripts i and p identify the anion I – or ClO 4 –, QG is stomach blood
flow (l/h), CVGB i,p is the thyroid stroma concentration (ng/l), and CG i,p is the
concentration (ng/l) of iodide or perchlorate in the gastric tissue. PG i, PGJ i,
PAG i, and PAGJ i (l/h/kg) are the partition coefficients and permeability
cross-products describing passive diffusion of I – between the stomach blood
and wall, and between the stomach wall and contents. The rate of active
secretion (ng/h) of I – into gastric juice (or contents)(RAGJ i), including inhibition by ClO 4 – at NIS, was described using an M-M equation with VmaxGJ i
and KmG i,p as the maximum velocity (ng/h/kg) and affinity constant (ng/l),
respectively, for transport of I – or ClO 4 – into gastric juice. Again, transport of
ClO 4 – through the stomach is calculated similarly, with the exception of
inhibition by iodide.
ACKNOWLEDGMENTS
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. We thank Dr. Richard Stotts and Lt. Col. Dan Rogers for assistance
in obtaining research funding; the U.S. Air Force and NASA HQ/EM for
financial support; Annie Jarabek, Mel Andersen, and Harvey Clewell for
modeling advice; and the University of Georgia for plasma binding studies.
Last, this work would not have been possible without laboratory support from
Eric Eldridge, Richard Godfrey, Paula Todd, Tim Bausman, Susan Young,
Latha Narayanan, Gerry Buttler, and Deirdre Mahle.
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