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TOXICOLOGICAL SCIENCES 83, 25–43 (2005)
doi:10.1093/toxsci/kfi017
Advance Access publication October 27, 2004
PBPK Model for Radioactive Iodide and Perchlorate Kinetics and
Perchlorate-Induced Inhibition of Iodide Uptake in Humans
Elaine A. Merrill,*,1 Rebecca A. Clewell,*,2 Peter J. Robinson,† Annie M. Jarabek,‡ Jeffery M. Gearhart,† Teresa R. Sterner,§
and Jeffrey W. Fisher¶,3
*Geo-Centers, Inc., Wright-Patterson AFB, Ohio 45433; †ManTech Environmental Technology, Inc., Dayton, OH 45437; ‡National Center for Environmental
Assessment (NCEA), U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711; §Operational Technologies Corp., Dayton,
Ohio 45432; and ¶AFRL/HEST, Wright-Patterson AFB, Ohio 45433
Received April 29, 2004; accepted September 17, 2004
Detection of perchlorate (ClO4) in several drinking water
sources across the U.S. has lead to public concern over health effects
from chronic low-level exposures. Perchlorate inhibits thyroid
iodide (I) uptake at the sodium (Na1)-iodide (I) symporter
(NIS), thereby disrupting the initial stage of thyroid hormone synthesis. A physiologically based pharmacokinetic (PBPK) model was
developed to describe the kinetics and distribution of both radioactive I and cold ClO4 in healthy adult humans and simulates the
subsequent inhibition of thyroid uptake of radioactive I by ClO4.
The model successfully predicts the measured levels of serum and
urinary ClO4 from drinking water exposures, ranging from 0.007 to
12 mg ClO4/kg/day, as well as the subsequent inhibition of thyroid
131 I uptake. Thyroid iodine, as well as total, free, and proteinbound radioactive I in serum from various tracer studies, are
also successfully simulated. This model’s parameters, in conjunction with corresponding model parameters established for the male,
gestational, and lactating rat, can be used to estimate parameters in a
pregnant or lactating human, that have not been or cannot be easily
measured to extrapolate dose metrics and correlate observed effects
in perchlorate toxicity studies to other human life stages. For example, by applying the adult male rat:adult human ratios of model
parameters to those parameters established for the gestational
and lactating rat, we can derive a reasonable estimate of corresponding parameters for a gestating or lactating human female. Although
thyroid hormones and their regulatory feedback are not incorporated in the model structure, the model’s successful prediction of free
and bound radioactive I and perchlorate’s interaction with free
radioactive I provide a basis for extending the structure to address
the complex hypothalamic-pituitary-thyroid feedback system.
In this paper, bound radioactive I refers to I incorporated into
The views expressed in this paper are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency.
The U.S. Government has the right to retain a nonexclusive royalty-free copyright covering this article.
1
To whom all correspondence should be addressed at GeoCenters, Inc.,
2729 St., Bldg. 837, Wright-Patterson AFB, OH 45433. Fax: (937) 9049610. E-mail: [email protected].
2
Current address: CIIT Centers for Health Research, Research
Triangle Park, NC 12137.
3
Current address: Environmental Health Science, University of
Georgia, Athens, GA 30602.
Toxicological Sciences vol. 83 no. 1
#
thyroid hormones or iodinated proteins, which may or may not be
bound to plasma proteins.
Key Words: pharmacokinetics; human; perchlorate; radioactive
iodide; inhibition; thyroid.
Advances in detection sensitivity of ion chromatography have
revealed widespread contamination of ground and drinking water
with perchlorate (ClO4) across the United States (Motzer,
2001; Urbansky and Schock, 1999; U.S. Environmental Protection Agency, 2003). The bulk of this contamination is associated
with the use of ammonium perchlorate (NH4ClO4) as an oxidizing agent in missile and rocket fuel. Ammonium perchlorate is
also used in pyrotechnics (fireworks) and air bag inflators. The
salt is readily soluble in water, and its dissociation product, the
perchlorate anion (ClO4), is very stable under environmental
conditions and very mobile in most media (Motzer, 2001).
Perchlorate is not metabolized in the body (Anbar et al., 1959;
Yu et al., 2002). However, because ClO4 has a similar hydrated
ionic radius and carries the same charge as iodide (I), it is able to
affect biological systems by inhibiting I uptake into the thyroid
by the sodium-iodide symporter (NIS) (Anbar et al., 1959;
Brown-Grant and Pethes, 1959). While it is known that
ClO4 competes with I for NIS binding sites, whether ClO4
is actually translocated into thyrocytes is the subject of debate
(Eskandari et al., 1997; Riedel et al., 2001). The weight of
evidence at this time, however, suggests ClO4 is a competitive
inhibitor of thyroid I uptake, replacing I as a substrate of NIS
and crossing the basolateral membrane (Clewell et al., 2004,
Van Sande et al., 2003). Reduced I uptake may lead to a disturbance in the first stage of normal thyroid hormone genesis.
Hence, there is reasonable concern that chronic exposure to
low levels of ClO4 in drinking water could induce thyroid
hormone deficiencies and subsequent thyroid disorders.
NIS resides in the basolateral membrane of thyroid epithelial
cells and simultaneously transports two Na1 and one I ion
from extracellular fluid (plasma) into the thyroid epithelial
cell (Spitzweg et al., 2000). NIS is expressed in the thyroid
and other tissues including the GI tract, skin, mammary tissue,
Society of Toxicology 2005; all rights reserved.
26
MERRILL ET AL.
and placenta. However, only in the thyroid is I organified to
form thyroid hormones and iodinated proteins (Ajjan et al.,
1998; Spitzweg et al., 1998). Thyroid hormone homeostasis
is maintained through a complex feedback mechanism. A drop
in circulating serum thyroid hormone levels signals the pituitary
to produce more thyroid stimulating hormone (TSH), which in
turn stimulates NIS expression.
In rats, a decrease in free thyroxine (fT4) and subsequent
increase in TSH occur quickly (within one day) after acute
ClO4 exposures (Wyngaarden et al., 1952; Yu et al., 2002).
In humans, thyroid hormone conservation is more efficient,
although thyroid hormone status is very dependant on the iodine
status and life stage under consideration. Little or no significant
change in T4, fT4, and TSH was seen in adults after 2 weeks
of controlled exposure to ClO4 via drinking water at 0.007 to
0.05 mg/kg/day (Greer et al., 2002) and 0.14 mg/kg/day
(Lawrence et al., 2000), despite significant levels of thyroid
I uptake inhibition. However, significant drops in fT4,
intrathyroidal iodine, as well as an increase in serum thyroglobulin (Tg) have been reported in humans after high levels of
exposure (900 mg/day) for 4 weeks (Brabant et al., 1992). The
dynamics of thyroid hormone homeostasis is very different for a
late gestation fetus or neonate. Empirical measurement of
intrathyroidal stores of thyroid hormone in human fetuses and
neonates have shown that the amount of thyroid hormone stored
in the colloid is less than that required for a single day (van den
Hove et al., 1999). The extent of chronic low-level ClO4 exposure required to cause significant hormone deficiencies in
humans is not yet known. Thus, the question facing risk assessors
and regulatory agencies is: what concentrations of perchlorate
could be considered problematic? It is known that I deficiency
during the fetal and neonatal period affects physical and mental
development (Laurberg et al., 2000; Porterfield, 1994). In the
adult, effects of I deficiency are less dramatic. Clinical and
subclinical hypothyroidism is often overlooked due to the vague
symptoms associated with the condition. Yet, hypothyroidism
occurs in over 10% of older women and is associated with
cognitive impairment (Volpato et al., 2002). The development
of hyperthyroidism, especially in multinodular goiters with
autonomous nodules, is also associated with long-term mild
to moderate I deficiency in adults. Hyperthyroidism is also
often overlooked in the elderly and, if left untreated, may
lead to cardiac arrhythmias, impaired cardiovascular reserves,
osteoporosis, and other abnormalities (Laurberg et al., 2000).
Therefore, perchlorate-induced I deficiency may represent a
public health concern not only during perinatal development,
but also in the elderly and subpopulations with already
compromised thyroid function.
In order to better understand the effect of occupational and
environmental exposure to perchlorate on the hypothalamuspituitary-thyroid (HPT) axis, a few studies have been performed that directly correlate hormone changes to quantitative
perchlorate doses. In two occupational health studies at
U.S. ClO4 production facilities, workers were exposed to
ammonium perchlorate (NH4ClO4) dust in the air. Perchlorate
exposure levels were estimated from monitoring breathing
zone air over full work shifts (Gibbs et al., 1998; Lamm et al.,
1999). Gibbs and coauthors categorized exposure groups based
on job tasks and air monitoring results. Controls, selected
from an associated plant, were not exposure free, but had
exposures estimated to be several orders of magnitude below
any of the ‘exposed’ groups. The researchers found no elevation in pre- and post-shift serum TSH, and no drop in serum
free thyroxine (fT4) among any of the exposed workers. In the
study by Lamm et al. (1999), control or ‘comparison group’
subjects worked at the same facility but at unrelated processes
and were believed to have very low exposure to perchloratecontaminated particulates. Daily perchlorate doses were estimated from breathing zone air monitoring of respirable
particulates over full work shifts and by urinary measurements.
No significant differences in triiodothyronine (T3) and T4 were
reported between the exposure and comparison groups. However, the mean pre- and post-shift urine perchlorate measurements from the comparison group averaged, respectively, 64%
and 22% of those from the lowest ‘exposed’ group.
Ecological epidemiological studies on neonatal screening
data from California, Nevada, and Arizona health departments
have resulted in conflicting data. Studies by Lamm and
Doemland (1999) and Li et al. (2000) showed no increase
in incidence of congenital hypothyroidism or decrease in
neonatal T4 associated with ClO4 in drinking water up to
15 mg/l. In contrast, the studies of Schwartz (2001) and
Brechner et al. (2000) both found effects on newborn thyroid
hormones from exposures at similar environmental levels (1 to
15 mg/l). A retrospective study of school-age children and
newborns in three Chilean cities with drinking water concentrations of 54, 5–7, and 100–120 mg ClO4/l revealed significantly higher fT4 but normal TSH in the two cities with
highest ClO4 concentrations (Crump et al., 2000), a result
opposite to what might be expected. While dietary I levels of
the three Chilean populations were within normal range, their
urinary excretion levels were increased. Hence, the increase in
fT4 may represent an adaptive effect.
These human epidemiological studies, while informative as to
the specific populations in which they were performed, have
been of limited utility in aiding the extrapolation across species,
populations, or exposure scenarios. Furthermore, differences in
route of exposure and lack of adequate adjustment for particle
dosimetry (Gibbs et al., 1998; Lamm et al., 1999), ambiguities in
level of exposure and exposure misclassification (Crump et al.,
2000; Gibbs et al., 1998; Lamm et al., 1999) make these data sets
of questionable use for predictive purposes (U.S. Environmental
Protection Agency, 2003). Laboratory animal data, however,
have indicated effects on developmental neurotoxicity, thyroid
hormones, and thyroid histopathology that have raised concern
for human health at various life stages (U.S. Environmental
Protection Agency, 2003). Unfortunately, measurements of
critical neuropsychological effects, such as IQ or physical
PBPK MODEL FOR INHIBITION OF IODIDE UPTAKE IN HUMANS BY PERCHLORATE
development in children exposed to ClO
4 in drinking water, are
not available. Therefore, to assist in evaluating the potential
effects of ClO4 in humans, a human PBPK model corresponding to those developed for the male, pregnant, and fetal rat
(Clewell et al., 2003a,b; Merrill et al., 2003) is proposed.
When used together, these models allow the entire body of
ClO4 literature (both animal and human) to be used to more
effectively predict perchlorate-induced changes in thyroid I
uptake across species, life-stage, and exposure doses.
The focus of this and ongoing modeling efforts with ClO4 is
to integrate animal and human data and to evaluate quantitatively
the impact of chronic exposure to perchlorate-contaminated
drinking water. This physiological model focuses on the first
step in the process, perchlorate-induced inhibition of I uptake
in the thyroid. The model describes the kinetics and distribution
of both radioactive I and ClO4 in healthy adult humans and
simulates the subsequent inhibition of thyroid uptake of radioactive I by ClO4. Distinct thyroid hormones and their regulatory feedback are not yet incorporated into the model structure,
although free and organified I (representing combined thyroid
hormones and nonhormonal iodoproteins) in plasma and thyroid
secretions are described separately. Hence, in addition to predicting perchlorate’s ability to inhibit thyroid uptake of radioactive I, the model represents significant work toward
establishing a basis for quantifying the effect of ClO4 on the
total amount of circulating thyroid hormones.
METHODS
Supporting studies. As mentioned above, the proposed model was developed concurrently with other PBPK models, which describe several life stages in
the rat, and shares several parameter values for which human data do not exist.
However, whenever available, human data, obtained from Hays and Solomon
(1965), Degroot et al. (1971), and Greer et al. (2002), were used for establishing
I and ClO4 parameters. Hays and Solomon (1965) studied early radioactive
I kinetics in nine healthy males, who received an iv dose of 3.44 3 103 ng
131 I /kg. Frequent measurements of 131I in the thyroid, aspirated gastric secretions, plasma, and cumulative urine samples were taken for 3 h post-dosing. To
examine the effect of gastric uptake on I kinetics, Hays and Solomon repeated
these same measurements on the same subjects, without aspirating gastric juices.
These data were used for establishing radioactive I parameters describing early
time-course behavior in the thyroid, stomach, urine, and plasma. Degroot et al.
(1971) administered a tracer dose of 131I to a health subject and measured
thyroid 131I uptake, total and protein-bound 131I (PBI) in plasma, and urinary
131 I excretion for up to 11 days post-dosing. His data set further served in
establishing I parameters, describing thyroid production and secretion of
incorporated radioactive I into the systemic circulation.
Data supporting the development of the ClO4 portion of the model were
obtained from Greer et al. (2002). In brief, Greer and colleagues administered 0.5,
0.1, or 0.02 mg/kg-day ClO4 in drinking water for 14 days to twenty-four
euthyroid subjects (n 5 8, four males and four females at each level) in a
‘main study.’ Four equal portions of the daily dose were ingested approximately
every 4 h from 8 A.M. to 8 P.M. Individual baseline serum and urine samples were
collected 1 or 2 days prior to beginning the 14 days of ClO4 dosing. During
ClO4 exposure, serum samples were collected at the following approximate
times in the ‘main’ study: day 1 at 12 and 4 P.M., day 2 at 8 A.M., 12 and 5 P.M., day 3
at 9 A.M., day 4 at 8 A.M. and 12 P.M., day 8 between 8 and 9 A.M., day 14 at 8 A.M.,
12 and 5 P.M. and on post-exposure days 1, 2, 3 and 14. Twenty-four-h urine
27
collections were taken on exposure days 1, 2, 14, and post-exposure days 1
through 3. Eight- and 24-h thyroid radioactive I uptake (RAIU) measurements
were taken on the baseline day, exposure days 2 and 14, and post-exposure day 1,
using an oral dose of 100 mCi of 123I.
An additional set of thirteen subjects were later exposed in a more limited
‘uptake’ study. Six women and one man were tested in this ‘uptake’ study at a
lower dose of (0.007 mg/kg-day), and two additional subjects each were tested at
the previous doses. In this ‘uptake study,’ serum samples were taken on days 8
and 14 and urine collected on day 14. RAIU measurements were also made in
these subjects following 123I ingestion on the day prior to perchlorate exposure
(baseline measurements) and at 9 A.M. on exposure day 14 and post-exposure
day 1 (Greer et al., 2002). The age of the subjects in both the ‘main’ and ‘uptake’
studies ranged from 18 to 57 years with a mean of 38 (SD 6 12) years.
The serum and urine samples were shipped to the Air Force Research Lab
(AFRL/HEST) at Wright Patterson Air Force Base for ClO4 analyses. The
analytical method for the analyses was similar to that described in Narayanan
et al. (2003). However, an important distinction between the analytical method
used in this study and that of Narayanan and colleagues is the mobile phase
concentration used. These concentrations were 80 mM NaOH for serum and from
60 to 120 mM NaOH for urine (depending on the sample). The range in mobile
phase concentrations for the urine samples was required to attain proper
sensitivity. Eight- and 24-h thyroid RAIU values were provided directly from
the measurements of Greer et al.
Model validation studies. Iodide model parameters were validated through
predictions of protein-bound iodine (PBI) data from Scott and Reilly (1954). Data
used in validating model predictions of serum ClO4 were obtained from a recent
unpublished drinking water study conducted by Drs. Georg Brabant and Holger
Leitolf of the Medizinische Hochschule, Hanover, Germany. Seven healthy
males ingested 12.0 mg/kg-day ClO4 for 2 weeks. The daily ClO4 dose
was dissolved in 1 l of drinking water and divided into three equal portions,
which were ingested at approximately 7 A.M., 12 P.M., and 7 P.M. each day for 14
days. Serum specimens were collected on exposure days 1, 7, 14, and postexposure days 1 and 2. The serum samples from Brabant and Leitolf were
also analyzed for perchlorate by AFRL/HEST. Data used in validating model
predictions of cumulative urinary ClO4 were obtained from Durand (1938),
Eichler (1929), and Kamm and Drescher (1973). In these studies healthy
males received a single oral dose of potassium perchlorate, ranging from 635
to 1400 mg ClO4.
Predictions of ClO4-induced inhibition of thyroid I uptake were validated
with the RAIU data of Greer et al. (2002) and ClO4 discharge data by Gray et al.
(1972). Lastly, the model’s ability to predict thyroid I uptake and inhibition in
hyperthyroid individuals was tested against data from Stanbury and Wyngaarden
(1952).
Model structure. The described PBPK model (Fig. 1) conforms to the
structure of the concurrently developed model for the male rat (Merrill et al.,
2003), with the exception of the newly added plasma subcompartment for bound
I, included for completeness. For both I and ClO4, tissues containing NIS
(thyroid, skin, and stomach) were described as compartments with nonlinear
saturable uptake kinetics (Anbar et al., 1959; Chow et al., 1969; Kotani et al.,
1998; Perlman et al., 1941; Slominski et al., 2002; Wolff, 1998). Other NIScontaining tissues, such as the salivary glands, choroid plexus, ovaries, mammary
glands, and placenta (Brown-Grant, 1961; Honour et al., 1952; Spitzweg et al.,
1998) were lumped with the slowly and richly perfused tissues, as either their
anion pools are too small to significantly affect plasma levels (Cserr et al., 1980),
or they are not applicable to the nonpregnant human. In development of the
model, a quantitative sensitivity analysis showed no significant effect of either
the gastric or thyroid uptake parameters on serum levels. Since the organ volumes
are small and relative activity of NIS in the choroids plexus and salivary glands,
etc. are even lower, we would expect these tissues to also have little to no effect on
plasma concentrations.
The stomach includes three subcompartments for capillary blood, stomach
wall, and contents. Skin is described with two subcompartments for capillary
blood and skin tissue. The thyroid is described with three subcompartments to
describe the disposition of ClO4 in the gland representing stroma, follicle, and
28
MERRILL ET AL.
FIG. 1. PBPK model for perchlorate and iodide. Bold arrows indicate active uptake at NIS sites (with exception of plasma binding, also indicated with a bold
arrow). Small arrows indicate passive diffusion.
lumen. The necessity for three subcompartments has been demonstrated
elsewhere (Clewell et al., 2004; Merrill et al., 2003). Unlike ClO4, which
eventually diffuses from the thyroid back into systemic circulation unchanged
(Anbar et al., 1959; Wolff, 1998), most I is quickly incorporated into monoiodothyrosine (MIT) and diiodothyrosine (DIT) in the thyroid follicle, which in
turn combine to form thyroid hormones, triiodothyrosine (T3) and thyroxine (T4),
which are then secreted into circulating blood. Endogenous iodine content in the
normal thyroid is about 10,000 mg, of which greater than 90% is organic and 5–
10% is free (Berman, 1967). Therefore, a fourth subcompartment, representing
all ‘incorporated’ I in the entire thyroid (i.e., total incorporated I in the
stroma, follicle, and lumen) is included. The incorporation of free I into thyroid
hormones and iodinated precursors, and the subsequent secretion of incorporated
I from the thyroid into venous blood were modeled with first-order rates. Passive
diffusion is governed by the electrochemical gradients formed by the variation in
anion concentration across thyroid subcompartments and wasdescribed with
permeability area cross products and partition coefficients, (symbolized as
small arrows in Fig. 1). Active uptake at NIS sites and between the thyroid
follicle and lumen was describe using Michaelis-Menten (M-M) parameters
(Fig. 1, bold arrows).
Fecal elimination of the anions is minimal and therefore not included in the
model. As mentioned earlier, ClO4 is fully eliminated in the urine unchanged.
Yu et al. (2002) reported 97% of ClO4 eliminated in urine within 26 h after
dosing with 3.0 mg/kg in rats. Fecal excretion of I comes from the liver breaking
down thyroid hormones and its secretion of the metabolic products into the bile,
which enters the intestines. Therefore, when Hays (1993) administered 125I-T4
orally to seven healthy male subjects, she found that ‘the fraction of fecal
29
PBPK MODEL FOR INHIBITION OF IODIDE UPTAKE IN HUMANS BY PERCHLORATE
radioactivity attributed to 125I was 0.55 6 0.35’. But, administration of radioactive thyroid hormones is very different from administering radioactive I.
When radioactive I is given orally, nothing is available for the liver to break
down; virtually all of the radioactive I is excreted by the kidneys. As reported in
Elmer (1938), Scheffer (1937) measured 2.8–9.0% of the total radioactive I
excreted in the feces. They noted that this amount was highly variable. For
example, after fasting fecal radioactivity may be undetectable. Braverman
and Utiger (1991) stated ‘fecal excretion of dietary iodine is negligible’ (5 mg/
day). Fecal elimination should be included in future expansion of the model,
which will include thyroid hormone metabolism and homeostasis.
The rapid urinary clearance of ClO4 and radioactive I, seen in both rats (Yu
et al., 2002) and humans (Greer et al., 2002), was described with a kidney
compartment. Urinary clearance could have easily been describe from the
free anion portion in the blood; however, significant levels of deiodination
occur in the kidneys, which would be critical in future model extrapolations
to simulate thyroid hormone metabolism and clearance. Similarly, the liver
compartment was maintained separately because it is the major site of extrathyroidal deiodination and it may be required in future pharmacodynamic elaborations of the model. At this point, the inclusions of these compartments did not
add a great deal of complexity or uncertainty to the model.
Because both anions are highly hydrophilic, fat acts as an exclusionary compartment. Given the large variability in human body fat, it was important to
explore the contribution of this compartment to the overall anion kinetics. In
addition, rapidly changing fat content during reproduction made the compartment important for extrapolation across life stages (Clewell et al., 2001,
2003a,b). The perfusion-limited compartments (e.g., kidney, liver, fat, slowly
perfused, and richly perfused) were each described using partition coefficients
and blood flows.
The structure of the plasma compartments for ClO4 and I are similar. Both
include passive diffusion between plasma and red blood cells (RBCs); however,
reversible binding between plasma proteins and ClO4 and I are slightly
different. In human serum, ClO4 binding to plasma proteins has also been
demonstrated (Hays and Green, 1973; Scatchard and Black, 1949). Approximately 95–98% of endogenous plasma iodine is reported as protein-bound iodide
(PBI) in human serum (Rapport and Curtis, 1950; Underwood, 1977). However,
unlike bound ClO4, the bound I fraction primarily represents nonexchangeable, covalently bound, ‘incorporated I’ secreted from the thyroid (e.g., hormonal iodine and iodinated proteins, including tri- and diiodothyronine) rather
than simply inorganic I bound to carrier proteins. In fact, it has been shown that
approximately 90% of endogenous PBI represents T4 and 5% represents T3
(Berkow et al., 1977). Michaelis-Menten kinetics were used in the model to
describe the association of the free ClO4 and I fractions to unspecific plasma
binding sites, and first-order rates were used for their dissociations. In the case
of I, however, dissociation represents both deiodination of hormones and
disassociation of I from serum proteins. Hormone deiodination occurs at several
sites throughout the body, but in the absence of better data, it was lumped together
as one first-order rate. Although unlabelled I is not included in the model, the
behavior of tracer doses of radioactive I is expected to follow that of endogenous
I. This is because the average levels of plasma inorganic iodine (PII) are
expected to be around 0.40 6 0.23 mg/dl (Elte et al., 1983), well below the
NIS Km value (described below). Therefore, the NIS will not be saturated in
the ‘average’ person.
TABLE 1
Human Physiological Parameters
Physiological parameters
Tissue volumes
Bodyweight BW (Kg)
Total slowly perfused
VSc (%BW)
Total 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)
Thyroid VTtotc (%BW)
Thyroid follicle VTFc
(%VTtot)
Thyroid lumen VTLc
(%VTtot)
Thyroid blood VTBc
(%VTtot)
Plasma Vplasc (%BW)
Red blood cells VRBCc
(%BW)
Blood flows
Cardiac output QCc
(l/hour-kg)
Fat QFc (%QC)
Kidney QKc (%QC)
Liver QLc (%QC)
Stomach QGc (%QC)
Human
70.0
65.1
12.4
M 21.0
F 32.7
0.44
2.6
1.7
Source
Subject-specific where provided
Brown et al., 1997
Brown et al., 1997
Brown et al., 1997
Brown et al., 1997
Brown et al., 1997
Brown et al., 1997
0.071
Licht and Deen, 1988
4.1
Altman and Dittmer, 1971a
3.7
Brown et al., 1997
8.0
Brown et al., 1997
0.03
57.3
Yokoyama et al., 1986
Brown et al., 1986
15.0
Brown et al., 1986
27.6
Brown et al., 1986
4.4
3.5
16.5
5.2
17.5
22.0
1.0
Thyroid QTc (%QC)
Skin QSkc (%QC)
Remaining slowly
perfused QS (%QC)
1.6
5.8
13.0
Remaining richly
perfused QR (%QC)
33.0
Marieb, 1992; Altman and
Dittmer, 1971b
Marieb, 1992; Altman and
Dittmer, 1971b
Brown et al., 1997; Hanwell and
Linzell, 1973
Brown et al., 1997
Brown et al., 1997
Brown et al., 1997
Leggett and Williams, 1995;
Malik et al., 1976
Brown et al., 1997
Brown et al., 1997
Based on total of 24%
QC (5.8 1 5.2)% (Brown et al.,
1997)
Based on total of 76% QC (17.5 1 22 1 1 1 1.6)%
(Brown et al., 1997)
Model Parameters
Physiological parameters. Tissue volumes and blood flows are presented in
Table 1. Considerable variability was reported for some parameters, such as
blood flow to the stomach (QG), which can increase 10-fold in response to
enhanced functional activity (secretion and digestion) (Granger et al., 1985).
The blood flows used represent estimates of resting values. Human data on the
volume of the stomach capillary bed (VGBc) were not available. Therefore, a
value derived from rat stomach data (Altman and Dittmer, 1971a) was allometrically scaled as described below. Mean values reported in the literature were
used for all other physical parameters.
Total thyroid volume was obtained from ultrasound measurements on 57
healthy adults conducted by Yokoyama et al. (1986). Thyroid volume was
positively correlated with both body weight and age, with weight having the
most pronounced influence. The proportions of the thyroid follicular epithelium,
lumen, and stroma were estimated from histometric measurements of patients at
necropsy reported by Brown et al. (1986). Their findings on the histological
features of thyroids showed overlapping distributions without evidence of significant differences between sexes. Significant sex differences, however, are
30
MERRILL ET AL.
TABLE 2
Chemical Specific Parameters for Human Modela
Partition coefficient, P (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 tissue/thyroid blood PT
Thyroid lumen/thyroid tissue PDT
Red blood cells/plasma PRBC
Max capacity, Vmaxc (ng/h-kg)
Thyroid lumen VmaxcTL
Thyroid follicle VmaxcTF
Skin VmaxcS
Gut VmaxcG
Plasma binding VmaxcB
Affinity constant, Km (ng/l)
Thyroid lumen KmTL
Thyroid KmTF
Iodide
Perchlorate
Source
0.21
0.40
0.05
1.09
0.44
0.50
3.50
0.70
0.15
7.00
1.00
0.31
0.56
0.05
0.99
0.56
1.80
2.30
1.15
0.13
7.00
0.80
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
7.0 3 107
1.5 3 105 6 8.2 3 104
6.0 3 105
9.0 3 105
2.0 3 102
2.5 3 105
5.0 3 104
1.0 3 106
1.0 3 105
5.0 3 102
Fitb
Fitb
Fitb
Fitb
Fit2
1.0 3 109
4.0 3 106
1.0 3 108
1.6 3 105
Skin KmSk
4.0 3 106
2.0 3 105
Gut KmG
4.0 3 106
2.0 3 105
7.8 3 105
1.8 3 104
Golstein et al., 1992
Gluzman and Niepomniszcze, 1983;
Wolff, 1998
Gluzman and Niepomniszcze, 1983;
Wolff, 1998
Gluzman and Niepomniszcze, 1983;
Wolff, 1998
Fitb
0.2
2.0
0.01
1.0
6.0 3 104
1.0 3 104
0.6
0.8
1.0
1.0
1.0 3 104
0.01
Fitb
Fitb
Fitb
Fitb
Fitb
Fitb
0.11
—
9.0 3 104
0.01
1.2 3 106
0.13 6 0.05
0.025
—
—
—
Fitb
Fitb
Fitb
Fitb
Fitb
Plasma binding KmB
Permeability area cross product,
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
Follicle to thyroid blood PATFc
Lumen to thyroid follicle PATLc
Clearance values, Cl (l/h-kg)
Urinary excretion ClUc
Plasma unbinding Clunbc
Deiodination Cldeiodc
Hormone production Clhormc
Hormone ClSecrc
a
b
All parameters listed are notated in the model by either an i (for iodide) or p (for perchlorate) following the parameter name (e.g., PRi, PRp, etc.).
Visual fits were obtained by optimizing the value for each parameter in question against available data with all other parameters held constant.
noted in total body fat (Brown et al., 1997), requiring a gender-specific value for
this parameter.
Chemical-specific parameters. Terminology and values of chemicalspecific parameters are provided in Table 2. These parameters were kept as
consistent as possible with those used in the male and pregnant rat models
(Clewell et al., 2003b; Merrill et al., 2003). Across the models, partition coefficients were nearly identical, although differences exist in parameters describing
maximum capacities for the nonlinear uptake of the anions. The partitions for the
thyroid subcompartments were based on electrical potentials measured within
the thyroid stroma, follicular membrane, and lumen after ClO4 dosing (Chow
and Woodbury, 1970). Electrical potential differences can be interpreted as
effective partition coefficients for charged moieties, such as ClO4 and I,
hindering entrance of negatively charged ions from the stroma into the follicle,
while the equal and opposite potential from the follicle to the lumen enhances
passage of negatively charged species into the lumen and indicates an effective
partition coefficient greater than one. Using Chow and Woodbury’s measured
electrical potentials at the stroma:follicle and follicle:lumen interfaces, effective
partitions were calculated, as described in Merrill et al. (2003). These values were
also used to describe the uptake of I based on the fact that I and ClO4 have
the same ionic charge and similar ionic radii and therefore respond similarly to an
electrochemical gradient.
A M-M affinity constant (KmTFi) for I at the NIS of 4.0 3 106 ng/l was
derived by Gluzman and Niepomniszcze (1983) from human thyroid slices. The
authors noted little variation between normal and pathological thyroid
PBPK MODEL FOR INHIBITION OF IODIDE UPTAKE IN HUMANS BY PERCHLORATE
specimens, or between specimens of different species. Wolff (1998) reported that
I Km(s) were similar across different NIS-containing tissues. This was supported by Kosugi et al. (1996), who reported a similar Km value of 4.4 3 106 ng/l
for I affinity for NIS in Chinese hamster ovary cells. Therefore, the value
reported by Gluzman and Niepomniszcze was also used to describe iodide’s
affinity in the skin and stomach.
Kosugi et al. (1996) also measured perchlorate’s affinity for NIS and
reported a Km of 1.5 3 105 ng/l, approximately ten times lower than the
that for I, indicating a greater affinity for perchlorate. Several other studies
agree that perchlorate’s affinity for NIS is approximately ten times greater
than that of I (Halmi and Stuelke, 1959; Harden et al., 1968; Lazarus et al.,
1974; Wyngaarden et al., 1952). Based on this information, a KmTFp value
was set to 1.6 3 105 ng/l, adjusting the literature value slightly based on the
model fits to ClO4 data in NIS-containing tissues. This values lies between
that measured by Kosugi et al. and that required in the corresponding male rat
model (1.8 3 105 ng/l).
The apical membrane of the thyroid follicle also exhibits a selective I
channel, believed to be pendrin. Pendrin is a transmembrane glycoprotein,
which facilitates both chloride and I efflux across the apical membrane
(Mian et al., 2001; Scott et al., 1999). Golstein et al. (1992) measured a Km
of 4.0 3 109 ng/l, for I transport from the bovine thyroid follicle into the lumen
(KmTLi). However, as in the corresponding rat model, a slightly lower KmTLi of
1.0 3 109 ng/l was required to fit thyroid I data at later time points (48 h).
Golstein et al. (1992) reported that this apical channel also appears sensitive to
ClO4 inhibition, suggesting a lower Km for ClO4 (KmTLp) than for I. A
KmTLp value of 1.0 3 108 ng/l was derived from Chow and Woodbury’s (1970)
data, as described in Merrill et al. (2003).
Maximum velocities, Vmax(s), for anion uptake vary between tissues and
species (Bagchi and Fawcett, 1973; Wolff, 1998). Humans tend to have lower
Vmax values than other species (Gluzman and Niepomniszcze, 1983; Wolff
and Maurey, 1961) when expressed per gram of tissue. The Vmax(s)
(ng/h/kg) for I uptake in the thyroid and plasma were estimated by visually
optimizing the clearance portion of the curves to respective time-course data
of Degroot et al. (1971). This was accomplished by keeping all other parameters fixed, while the Vmax value was adjusted so that the model prediction
adequately approximated the observed mean. It may be noted that Vmax
values for the thyroid follicle and lumen differ by up to an order of magnitude from preliminary values, reported in Clewell et al. (2001). This was
attributed to the availability of new data sets, which allowed improved
parameterization.
For tissues lacking time-course data, the Vmax(s) were estimated to yield
kinetics similar to those described by the male rat model (Merrill et al., 2003). For
example, for I kinetics in the stomach and skin, VmaxGi and VmaxSki respectively, were visually optimized to resemble tissue:serum concentration ratios
seen in the rat, while maintaining the fits to human serum data. Because ClO4
data was only available in serum and urine, ClO4 Vmax(s) for NIS-containing
tissues were scaled from the I Vmax(s), using the ratios between corresponding
I and ClO4 Vmax(s) established in the male rat model.
Diffusion, concurrent with active uptake in the stomach, thyroid, and skin,
was described using permeability area cross products (PA) (l/h-kg) and effective
partition coefficients (P). In general, PA values were visually fit to the uptake
portion of the curves, prior to setting Vmax(s), with partition coefficients, and all
other parameters were set to the values in Table 2 and held fixed. The early timecourse data of I in gastric aspirations from Hays and Solomon (1965) were used
to estimate PAGJci, representing 131I transfer from the gastric juice into the
gastrointestinal plasma (l/h-kg). To simulate the removal of gastric aspirations,
the amount of 131I reabsorbed by the stomach wall had to be mathematically
eliminated or set to zero. Once parameters were established using the aspiration
session data, stomach reabsorption was reintroduced, and the permeability cross
product for 131I transfer between gastric blood and tissue (PAGci) was fit to the
corresponding increase in 131I in plasma, thyroid, and urine seen in the control
session (where gastric juices were not aspirated). The permeability area cross
product between the thyroid stroma and follicle, PATFci, was visually optimized
to the uptake portion of the thyroid I data.
31
The first-order clearance rates describing the organification of I shortly
after it enters the thyroid follicle (Clhormci) and the secretion of organic I
into systemic circulation (Clsecrci) were visually optimized to the clearance
portion of thyroid 131I data, as well as the later time points of the plasma PBI
data from Degroot et al. (1971). The first-order rate, describing the body’s
overall deiodination rate (Cldeiodci) was also estimated through visual optimization of the later PBI timepoints, while maintaining the model fit to total
plasma iodine and keeping all other parameters fixed. Later plasma time
points of PBI reflect the contribution of hormone secretion and deiodination
rates due to sufficient lapse of time for radioactive I incorporation into
thyroid hormones and precursors. Therefore, earlier PBI time points were
visually fit to establish parameters for binding of inorganic I to plasma
proteins (e.g., KmBi, VmaxBi). Similarly, reversible plasma binding of perchlorate was described using a first-order rate constant (Clunbp), which was
visually optimized to available serum ClO4 data. Urinary excretion rates for
both anions (ClUi and ClUp) were visually fit to available cumulative urine
data (Degroot, 1971; Greer et al., 2002; Hays and Solomon, 1965). Because
cumulative urinary perchlorate data was available in the Greer et al. study,
ClUp was visually fit to each individual’s data, and the average value was then
used for the model parameter.
Allometric scaling and rate equations. Differential equations used to
simulate radioactive I and ClO4 transport were written and solved in
ACSLTM (Advanced Continuous Simulation Language) (AEgis Technologies,
Austin, TX). To account for body-weight-dependent variables and species extrapolations, allometric scaling was applied to Vmax(s), PA(s), Cl(s), tissue volumes
(V), and blood flows (Q). The variety of dosing regiments and routes were
simulated using various pulse function codes in ACSL.
Rate equations describing I transport in ng/h in the thyroid stroma, follicle
and lumen (colloid) (RATSi, RATFi, and RATLi, respectively), as well as the rate
of change in bound thyroid iodine (RAbndi) are provided below. These equations
demonstrate diffusion-limited uptake, using P(s) and PA(s), and saturable uptake
and competitive inhibition using M-M parameters. Equations used in the other
compartments are expressed similarly.
RATSi ¼ QT3ðCAi CVTSi Þ þ PATFi 3ðCTFi =PTFi CVTSi Þ RupTFi
RATFi ¼ RupTFi þ PATFi 3ðCVTSi CTFi =PTFi Þ
RupTLi þ PATLi 3ðCTLi =PTLi CTFi Þ ðClhormi 3CTFi Þ
RATLi ¼ RupTLi þ PATLi 3ðCTFi CTLi =PTLi Þ
RupTFi ¼
VmaxTFi 3CVTSi
KmTFi 3ð1 þ CVTSp =KmTFp Þ þ CVTFi
RupTLi ¼
VmaxTLi 3CTFi
KmTLi 3ð1 þ CTFp =KmTLp Þ þ CTFi
RAbndi ¼ ðClhormi 3CTFi Þ ðClsecri 3CTbndi Þ
Subscripts i and p identify the anion as either I or ClO4, QT is thyroid blood
flow (l/h), CAi is the arterial blood concentration (ng/l), CVTSi,p is the thyroid
stroma concentration (ng/l), CTFi,p is the follicular concentration (ng/l) of I or
ClO4, and CTbndi is the concentration of incorporated I in the entire thyroid.
PTFi, PTLi, PATFi, and PATLi are the partition coefficients and permeability
cross products describing passive diffusion of I across the basal (follicle:
stroma) and apical (lumen:follicle) membranes. Michaelis-Menten equations
are used to describe the rates of active uptake of I into the follicle by NIS
and into the lumen by the apical I channel (RupTFi and RupTLi, respectively),
including inhibition by ClO4. VmaxTFi, VmaxTLi, KmTFi,p and KmTLi,p are the
maximum velocities (ng/h/kg) and affinity constants (ng/l) for transport of I or
ClO4 into the follicle and lumen. Clhormi and Clsecri are first-order clearance
values (h1) for the organification of I into thyroid hormones and the secretion
of organified I into systemic circulation. Transport of ClO4 through the thyroid
32
MERRILL ET AL.
is calculated similarly, except there are no terms for organification of ClO4
(Clhormi and Clsecri). In addition, inhibition of ClO4 uptake by I is not
included. As described earlier, due to the lower affinity of I (10-fold higher
Km) than that of ClO4, I does not significantly inhibit ClO4 sequestration in
NIS-containing tissues. Example equations of other compartments are shown
elsewhere (Merrill et al., 2003).
Sensitivity analysis of parameters. To assess the relative impact of each
parameter on model predictions, a sensitivity analysis was performed. After
finalizing all model parameters, the model was run at a drinking water dose
below NIS saturation (0.1 mg/kg/day) for 240 h (to ensure equilibrium was
reached) to determine the average serum ClO4 concentration [area under the
curve (AUC)]. The model was then repeatedly rerun, using a 1% increase in each
parameter to determine the resulting change in predicted serum ClO4 concentration AUC, and sensitivity coefficients for each parameter were then calculated
using the equation below.
Sensitivity Coefficient ¼
ðA BÞ=B
ðC DÞ=D
Where A equals serum ClO4 AUC with 1% increased parameter value, B
equals serum ClO4 AUC using original parameter value, C equals parameter
value increased 1% from original value, and D equals the original parameter
value.
RESULTS
Model Parameterization
Iodide kinetics. Effective partition coefficients (P) and affinity constants for the active transport mechanisms (Km), listed in
Table 2, were kept consistent with those used in the male rat
model. Parameterization of other I parameters was obtained
using available human time-course data as described in the
Methods section. Figures 2A through 2D show the model simulations of the early distribution of 131I in plasma, thyroid, gastric juice, and urine during both the control and gastric aspiration
session by Hays and Solomon (1965). Degroot’s plasma, urine,
and thyroid time-course measurements extended nearly 11 days
post-dosing, allowing greater calibration of parameters affecting
both uptake and clearances in the thyroid and plasma. Model
simulations of plasma inorganic I, PBI, and total plasma iodine
are presented in Figures 3A–3C. The model underpredicted
urinary I measured by Degroot et al. (1971) (Fig. 3D). However, considerable variations in I excreted by humans exist, as it
varies with thyroid function and dietary I intake (NRC, 1996).
Since increasing urinary clearance (ClUi) would result in underprediction of plasma iodine, the value for ClUi was not changed
to fit this data set.
The subjects from the Hays and Solomon (1965) and Degroot
(1971) studies had fasted at least 12 h prior to 131I administration. The VmaxTFi values, visually optimized from these data,
averaged 2.5 3 105 ng/h-kg. Using this value, however, individual 8 and 24 h radioactive I thyroid uptake (RAIU’s) from
Greer and coworkers’ (2002) study were over-predicted. Since
no dietary restrictions were given to the subjects in the Greer
study, differences in dietary I among subjects in the three
studies may account for the differences in uptake. Individually
fit VmaxTFi values using the Greer (2002) data resulted in a
lower average, 1.5 3 105 ng/h-kg, reflecting the high iodine
intake (see Table 3 and Figure 4).
Kinetic data for I in human skin were not available. However, as mentioned earlier, NIS has been identified in human skin
(Slominski et al., 2002). Assumptions that anion uptake and
clearance in human skin were comparable to that in rat skin
[e.g., slow uptake into and diffusion out of the tissue (Merrill
et al., 2003)] lead to improved fits of serum 131I concentrations.
Perchlorate kinetics. Because I parameterization, which
was based on actual human time-course data, resulted in
many values similar to those developed for the male rat, it is
reasonable to expect that the proportional difference between the
rat’s I and ClO4 parameters would also apply to the human’s
ClO4 parameters. Using these proportionally scaled ClO4
parameters for the thyroid, stomach, and skin, cumulative
ClO4 in urine was visually fit for each individual in the 0.5,
0.1, and 0.02 mg/kg/day dose groups from Greer et al., (2002),
resulting in an average urinary clearance constant for ClO4
(ClUcp) of 0.13 6 0.05 l/h-kg (Table 3) (Fig. 5).
As mentioned earlier, reversible binding of ClO4 to nonspecific human plasma proteins has been qualitatively demonstrated
in other studies (Carr, 1952). Additionally, the model indicated
the existence of plasma binding, as without it the model underestimated serum ClO4 at 0.1 mg/kg/day, while simulations at
0.5 mg/kg/day produced adequate fits. Hence, at the lower level,
plasma binding represents a larger proportion of the overall
amount of serum ClO4. Serum levels from the 0.02 mg/kg/
day dose group were below the detection limit and thus could
not be compared to model predictions. The plasma protein affinity for ClO4 (KmBp) was assumed to be similar to that used in
the male rat model, given the same proteins (albumin and prealbumin) appear to be responsible (Carr, 1952; Merrill et al.,
2003) (Fig. 6).
Model Validation
The ability of the model to predict human data from other
experiments, analyzed at different labs, was tested using available data from other independent studies. Using the parameters
in Table 2, the model slightly overpredicted plasma-bound 131I
fractions from several euthyroid patients; however, the prediction was remarkably close (within a factor of 0.75 of the mean
values reported) (Fig. 7). Predictions of cumulative ClO4 in
urine after oral doses of approximately 9.07 mg/kg (Durand,
1938), 9.56 mg/kg (Kamm and Drescher, 1973), and 20 mg/kg
(Eichler, 1929) were excellent (Figs. 8–10).
The model was also able to predict serum ClO4 concentrations at 12 mg/kg-day from the unpublished study performed by
Dr. Georg Brabant at the Medizinische Hochschule, Hanover,
Germany, described previously in the Methods section (Fig. 11).
With the exception of the significantly higher dose, this study
was very similar to Greer et al. (2002). Subjects received
12 mg/kg-day ClO4 in drinking water at or near meal times.
PBPK MODEL FOR INHIBITION OF IODIDE UPTAKE IN HUMANS BY PERCHLORATE
(a)
(b)
(c)
(d)
33
FIG. 2. 131I in serum (A), thyroid (B), gastric juice (C), and urine (D) of nine healthy males after an iv dose of 10 mCi 131I (3.44 ng/kg). Model
simulations (lines) and actual values (stars) are presented for both the control and aspiration sessions (Hays and Solomon, 1965).
The wide range in the observed serum measurements was
believed to reflect variability in the dosing regime, as the experimental protocol was less rigid than that used in Greer et al.
(2002). Table 4 lists the predicted and measured average
serum ClO4 and percent inhibitions on exposure day 14 from
each dose group in Greer et al. (2002).
The competition of I and ClO4 for NIS binding sites was
also successfully predicted by the model. Predictions of thyroid
radioactive I uptake inhibition after 14 days of exposure to
ClO4 in drinking water at 0.5, 0.1, 0.02, and 0.007 mg/kgday, also measured by Greer et al. (2002) in the same subjects,
were well within the mean and standard deviations of the
observed data (Figs. 12A–12D). Using this same set of I and
ClO4 parameters, again the model accurately predicted ClO4
discharge tests performed by Gray et al. (1972) on euthyroid
subjects (Fig. 13).
Lastly, the model’s utility for predicting I and ClO4 kinetics
under altered thyroid function was evaluated. Hyperthyroidism,
34
MERRILL ET AL.
(a)
(b)
(c)
(d)
FIG. 3. Model simulations (lines) and measured
dose of 131I (Degroot et al., 1971).
131 I in plasma (A), protein-bound iodine (B), thyroid (C), and urine (D) of euthyroid adult after a tracer
as manifest in Grave’s disease, is marked by increased thyroid I
uptake, as well as elevated T4, T3, PBI, and TSH (Fenzi et al.,
1985). Gluzman and Niepomniszcze (1983) reported elevated
Vmax(s) at the NIS in thyroid specimens from subjects with
Grave’s disease. As expected, by increasing VmaxcTFi to 5.0 3
106 ng/l-kg, the model successfully simulates the thyroidal 131I
uptake in a male with Grave’s disease (upper line in Fig. 14)
(Stanbury and Wyngaarden, 1952). This value exceeds those
estimated for normal subjects with varying levels of dietary
I by more than a factor of 10. Using this set of thyroid
parameters, including the elevated VmaxcTFi, the model
underpredicts the degree of inhibition after 100 mg KClO4,
but is within a factor of 2 from the data (bottom line Fig. 14).
Sensitivity Analysis
Sensitivity analysis on serum ClO4 AUC levels at 0.1 mg/kg/
day indicated that the urinary clearance (ClUp) had the greatest
influence. The sensitivity coefficient for ClUp was 0.84.
35
PBPK MODEL FOR INHIBITION OF IODIDE UPTAKE IN HUMANS BY PERCHLORATE
TABLE 3
Individually Fit ClUcp(s) and VmaxcTFi(s)
Estimated
ClUcp (l/h-kg)
Estimated
VmaxcTFi (ng/h/kg)
Measured urinary
iodine (mg/day)
Measured serum
iodine (mg/dl)
F
M
M
F
M
M
F
F
0.10
0.09
0.10
0.09
0.09
0.09
0.10
0.10
0.10
0.01
1.34 3 105
1.20 3 105
2.00 3 105
1.80 3 105
2.50 3 105
8.00 3 105
5.00 3 105
1.00 3 105
1.96 3 105
1.35 3 105
303.1
363.0
140.8
282.1
391.8
567.7
305.5
238.8
324.1
124.7
6.5
5.0
5.0
5.5
3.5
6.2
6.8
4.8
5.13
0.95
9
10
11
12
13
14
15
16
F
M
M
F
M
F
F
M
0.10
0.20
0.20
0.12
0.24
0.10
0.13
0.17
0.16
0.05
1.10 3 105
2.20 3 105
6.80 3 104
1.50 3 105
1.20 3 105
1.60 3 105
5.00 3 104
1.20 3 105
1.25 3 105
5.35 3 104
286.4
343.9
413.0
172.9
519.2
104.1
527.4
152.3
314.9
164.2
6.0
5.8
7.0
4.5
5.8
4.2
7.5
3.8
5.58
1.32
17
18
19
20
21
22
23
24
F
F
M
M
M
F
F
M
NAa
0.10
0.15
NA
NA
0.20
0.11
0.06
0.12
0.05
1.50 3 105
1.40 3 105
8.00 3 104
1.50 3 105
1.40 3 105
1.50 3 105
9.00 3 104
8.00 3 104
1.23 3 105
3.28 3 104
212.6
242.2
190.4
277.4
240.8
202.6
323.4
632.7
290.3
144.9
6.5
5.5
7.5
6.0
4.0
7.0
10.2
5.8
6.56
1.81
25
26
27
28
29
30
31
F
M
F
F
F
F
F
Group mean
Group std. dev.
NA
NA
NA
NA
NA
NA
NA
NA
NA
1.35 3 105
1.35 3 105
7.80 3 104
8.00 3 104
1.40 3 105
2.80 3 105
9.00 3 104
1.34 3 105
6.99 3 104
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Total mean
Total std. dev.
0.13
0.05
1.45 3 105
8.17 3 104
309.8
139.9
5.9
1.5
Dose group
0.5 mg/kg/day
Subject
Sex
1
2
3
4
5
6
7
8
Group mean
Group std. dev.
0.1 mg/kg/day
Group mean
Group std. dev.
0.02 mg/kg/day
Group mean
Group std. dev.
0.007 mg/kg/day
Note. From Greer et al., 2002.
a
NA 5 not available.
Serum ClO4 levels at this dose level are also sensitive to plasma
binding parameters for maximum capacity (VmaxBp), the firstorder dissociation rate (Clunbp), and plasma binding affinity
constant (KmBp), with sensitivity coefficients of 0.26, 0.25,
and 0.17, respectively. A comparison of model sensitivity to
these parameters between the concurrently developed male rat
model and this human model are shown in Figure 15. Sensitivity
coefficients of all other parameters were below an absolute
value of 0.1.
DISCUSSION
The validity of the model structure and its parameters are
demonstrated by its ability to predict ClO4 and I in serum
and urine data, as well thyroid I inhibition data from various
studies, which involve various dose levels and routes, while
using a single set of parameters. The model adequately simulates
serum and cumulative urine levels after drinking water exposure
to ClO4 spanning almost four orders of magnitude (0.02 to
36
MERRILL ET AL.
FIG. 4. Model simulations (lines) and observed mean 6 SD (bars) 8 and
24 h RAIU measurements from 31 healthy subjects (Greer et al., 2002).
FIG. 5. Model simulations (lines) and mean and standard deviations of the
observed cumulative ClO4 in urine (cross bars) in male subjects dosed 0.02,
0.1, and 0.5 mg/kg-day (Greer et al., 2002).
12.0 mg/kg-day). Although serum ClO4 levels were not
available at 0.02 mg/kg-day, the model was able to simulate the
cumulative urine from that dose group (Fig. 9). Comparison with
parameters of the rat model indicates that humans have a considerably lower plasma binding capacity (VmaxcBp) for ClO4
FIG. 6. Model simulations (lines) and mean 6 SD (bars) of serum ClO4
in volunteers who received 0.02, 0.1, and 0.5 mg/kg-day via drinking water,
four times per day for 14 days. n 5 8/dose group (Greer et al., 2002). Note:
serum perchlorate levels at 0.02 mg/kg/day were below detection limits.
FIG. 7. Model simulations (lines) and mean 6 SD (bars) of plasma
bound 131I fractions of 25 euthyroid patients after an oral dose of 100 mCi
131 I (Scott and Reilly, 1954).
(approximately 20 times lower). Although binding of ClO4 to
plasma proteins has been directly measured in human serum, it is
not surprising that it would occur to a lesser extent than in the rat.
Carr (1952) tested the ability of ClO4 to bind to various proteins
PBPK MODEL FOR INHIBITION OF IODIDE UPTAKE IN HUMANS BY PERCHLORATE
37
FIG. 8. Model simulation (line) and observed (asterisks) cumulative
ClO4 in urine from a healthy male after an oral dose of 9.56 mg ClO4
(Kamm and Drescher, 1973).
FIG. 10. Model simulation (line) and observed (asterisks) cumulative
amount of ClO4 in urine from a healthy male after an oral dose of
approximately 9.07 mg/kg of ClO4 (Durand, 1938).
FIG. 9. Model simulation (line) and observed (asterisks) cumulative
ClO4 in urine from a healthy male after an oral dose of approximately 20 mg
ClO4/kg (Eichler, 1929).
FIG. 11. Model simulations (lines) and mean 6 SD (bars) of serum
ClO4 concentrations in 5 males during exposure to 12 mg/kg-day of ClO4 in
drinking water. Subjects ingested the drinking water solution three times/day
for 14 days (unpublished data, Brabant and Leitolf).
in human blood, including albumin, pre-albumin and thyroxine
binding globulin (TGB). They found that ClO4 was able to bind
to albumin and prealbumin, but not TBG. Thus, it would be
expected that the ClO4 binding capacity would be greater in
the rat, whose primary binding protein is albumin, than the
human, whose primary binding protein is TBG.
Data available for calibrating and validating serum I were
limited to tracer dose levels. However, the kinetic behavior of I
38
MERRILL ET AL.
TABLE 4
Average Serum Perchlorate and Percent Inhibition Across Dose Groups
Dose
(mg/kg-day)
0.5
0.1
0.02
0.007
BW (Kg)
Serum
ClO4(mg/l)
Predicted serum
ClO4 (mg/l)
Average % inhibition
of 24-h iodide uptake
Predicted % inhibition
of 24-h iodide uptakec
76.1 6 14.3
78.7 6 13.2
78.4 6 18.2
73.2 6 15.4
0.65 6 0.25
0.13 6 .053
5DLa
NAb
0.53
0.11
0.026
0.010
67.4 6 12.1
43.4 6 12.3
18.2 6 12.8
6.0 6 22.03
60.0
38.0
11.3
3.0
Note. Average serum perchlorate and percent inhibition across dose groups on exposure day 14 of drinking water study by Greer et al., 2002.
a
Samples at or below detection limit.
b
At 0.007 mg/kg/day serum samples were not collected. NA 5 not available, RAIU measurements not taken.
c
Measured inhibition was not significantly different from baseline measurements.
is expected to be linear over a wide range of doses. Although the
mechanism of transfer into the tissues with NIS is saturable, the
value of Km (4.0 3 106 ng/l) indicates that very high doses would
be required to saturate this mechanism. Additionally, similar
parameter values and identical model structures in the corresponding rat models of various life stages have yielded validated
serum predictions at dose levels ranging three or more orders of
magnitude for both anions.
Aspects of the model, which were supported in the literature or
laboratory studies but could not be directly observed in humans,
were incorporated if necessary to improve the fit of the model to
available data. For example, active uptake of I and ClO4 into
human skin and the relatively slow diffusion of both anions from
skin back into systemic circulation were incorporated in spite of
a lack of human time-course data. The literature supports this
behavior, as NIS has been identified in human skin (Slominski
et al., 2002), and slow diffusion has been noted with similar
anions, such as pertechnetate. Hays and Green (1973) performed
dialysis studies on intact human tissues with pertechnetate. They
found skin had a relatively slow uptake of pertechnetate peaking
at 18 h and, in fact, more retention after leaching dialysis than
seen in brain, muscle, and serum.
It is possible that the reason elevated I in human skin has not
been reported in clinical radioactive I scans is the difficulty in
differentiating skin radioactivity from background radioactivity. The large volume of the skin allows radioactive I to be
diffused over a large surface. However, this same property
allows the skin to be an important pool for the storage and
slow turnover of I. Simulations with this model demonstrated
that inclusion of active uptake in human skin was required to
simulate serum data. Sensitivity analysis on the corresponding
male rat model indicated that serum ClO4 levels were highly
sensitive to parameters of the skin and plasma binding and
urinary excretion (Merrill et al., 2003).
Kinetic data for establishing parameters for the gastric compartment were limited to the early I data (3 h post-dosing) by
Hays and Solomon (1965). Their gastric juice 131I data indicated rapid transport of I into the gastric mucosa (Fig. 2C).
It is expected that ClO4 uptake in the stomach would behave
likewise, due to the similarity of the anions in size and charge.
The time-course behavior of radioactive iodine in stomach contents of any species is complicated by the fact that it reflects more
than sequestration of radioactive I by NIS. Its appearance also
reflects the accumulation of salivary radioactive I that is swallowed involuntarily throughout the study. Several studies that
examined sequestration of these anions in digestive juices have
all shown high variability in the concentrations measured over
time (Hays and Solomon, 1965; Honour et al., 1952). There is a
tendency for the gastric juice:plasma ratio to be low when the
rate of secretion of gastric juice and saliva is high (Honour et al.,
1952). This is because the increase in secretions does not correspond with upregulation of NIS; therefore, the gastric juice
concentration becomes diluted. Fluctuations in the secretion
rate are probably the most important factor in determining
the pattern of the concentration ratios in individuals. Therefore,
variability in stomach or GI tract parameters between models is
expected. However, the early rise in the gastric juice:plasma
ratio mentioned earlier is a constant feature across these data
sets, whether or not an attempt was made to eliminate contamination of gastric juices by dietary contents or saliva. Animal
data that show both the anion uptake and clearance in the
stomach (Yu et al., 2002), unlike the data in Figure 2C which
only show uptake, indicate that the clearance portion is less
rapid. This model, and the series of different life-stage rat models
(Clewell et al., 2003a,b; Merrill et al., 2003;) consistently
predict this same trend.
Average urinary clearance values were found to be 0.11 l/h-kg
for I and 0.13 l/h-kg for ClO4. However, these values are not
expected to be successfully applied to every euthyroid individual
studied, though the use of these average values should provide a
reasonable prediction of the euthyroid population. Individual
differences in urinary I are expected with variation in thyroid
function and protein-bound I in plasma. Iodide is ultimately
removed or eliminated by two competing mechanisms, thyroidal
uptake and urinary excretion. Thus, a higher amount of excreted
I in urine is indicative of reduced thyroid uptake. Historically,
this relationship has been used to estimate thyroid function.
A cumulative 24-h urinary clearance of less than 30% of a tracer
PBPK MODEL FOR INHIBITION OF IODIDE UPTAKE IN HUMANS BY PERCHLORATE
(a)
(b)
(c)
(d)
39
FIG. 12. Model simulations and observed (mean 6 SD) of 8 and 24 h RAIU measurements before exposure (upper lines and solid squares) and on day 14 of
perchlorate exposure at (A) 0.5 mg/kg-day, (B) 0.1 mg/kg-day, (C) 0.02 mg/kg-day, and (D) 0.007 mg/kg-day (lower lines and open circles) (Greer et al., 2002).
dose is indicative of hyperthyroidism, whereas clearances
exceeding 40% are often associated with normal or decreased
thyroid function. However, a high degree of variability exists
between human subjects. Such a significant degree of overlap
exists in thyroid test results for normal, hyperthyroid, and
hypothyroid patients, that it is often necessary to run several
different additional screens in order to identify subclinical
conditions (NRC, 1996).
In addition to the expected variability in thyroid uptake parameters (VmaxcTFi values ranging from 5.0 3 104 to 5.0 3
105 ng/h-kg) between individuals, variability across data sets
was also noted. However, the difference seen in the average
VmaxcTFi obtained from the Greer et al. (2002) subjects
(1.5 3 105 ng/h-kg) and those from Hays and Solomon (1965)
(2.5 3 105 ng/h-kg) is easily explained by the difference in
experimental conditions between the two studies. Hays and
40
MERRILL ET AL.
FIG. 15. Calculated sensitivity coefficients for model parameters with
greatest impact on serum ClO4 AUC at a drinking water dose of 0.1 mg/kg/
day. Comparison shown for the male rat and human models.
FIG. 13. Model simulation (line) and observed perchlorate discharge tests
performed on euthyroid subjects (Gray et al., 1972).
FIG. 14. Model simulation (line) and observed (asterisks) amount of
I uptake in the thyroid of a male with Graves’ disease after iv dose of
10 mCi 131I before and after 100 mg KClO4 (Stanbury and Wyngaarden,
1952).
131 Solomon’s subjects fasted 12 h prior to the administration of the
131 I , whereas Greer and coauthors’ subjects had no dietary
restrictions prior to 125I administration. As a result, intrathyroidal I levels would have been lower in the fasted individuals,
and as anticipated, the average VmaxcTFi from Hays and
Solomon (1965) would be increased.
Dietary iodine and endogenous inorganic I levels are clearly
important in modeling I and ClO4 kinetics, because excessive
I levels cause the ion to inhibit its own uptake (Wolff and
Chaikoff, 1948). The ability of the model to describe the
bound and free I fractions in the thyroid and serum provides
the basis for subsequent modeling of hormone synthesis and
regulation in humans. Measurements of tracer radioactive I
can be fitted to predict transfer rates. However, the use of
these acute parameters is limited when attempting to describe
long-term thyroid kinetics, unless the existing endogenous I
and the relationships between the regulating hormones are taken
into consideration. Ultimately, such factors as preexisting thyroid conditions and regional dietary iodine might be addressed in
a more comprehensive hormone feedback model. In its present
state, our model is useful in predicting perchlorate’s effect on
thyroid I uptake in what is considered the normal population:
euthyroid individuals with adequate dietary I.
That the model is capable of predicting I uptake in hyperthyroid subjects by increasing the VmaxTFi supports the usefulness
of the current model structure. TSH increases the total amount of
NIS in a membrane, thereby increasing VmaxTFi. Gluzman and
Niepomniszcze (1983) reported elevated Vmax(s) in thyroid
specimens from subjects with Graves’ disease, toxic adenoma,
and dishormonogenetic goiter. In future versions of the model,
the increase of TSH and subsequent effect on this parameter can
be described mathematically in order to predict the dose- and
time-dependent response of the thyroid activity to various
disease states. In specimens from nontoxic nodular goiter,
Hashimoto’s thyroid, or extranodular tissue from toxic adenoma, Vmax(s) were decreased. However, in all subjects
there was little variation in the KmTi. Therefore, one would
not expect the underprediction of thyroid inhibition in the subject
with Graves’ disease to be due to disease-induced lowering of
PBPK MODEL FOR INHIBITION OF IODIDE UPTAKE IN HUMANS BY PERCHLORATE
Km, but rather the increased inhibition is mostly likely due to
simple interindividual differences. Sensitivity analyses performed on the model for the rat indicates that model-predicted
values of inhibition are highly sensitive to even small changes in
Km for ClO4. Thus, it is quite possible that changing Km within
the range of normal values would account for this apparent
discrepancy in the model fit.
The PBPK models developed for perchlorate-induced inhibition have been useful to the ongoing risk assessment of ClO4,
and helped integrate the data from diverse data sets to evaluate
the dose response of adverse effects from low levels of ClO4
exposure (U.S. Environmental Protection Agency, 2003). The
resulting parameters may be used in conjunction with those
established for the male (Merrill et al., 2003) and perinatal
rat models (Clewell et al., 2003a,b) to extrapolate to human
gestational and lactational models (Clewell et al., 2001). In
order to further assess model performance and to facilitate the
use of these models in risk assessment, a more comprehensive
statistical evaluation of model parameters may prove additionally useful. Sensitivity analysis provided insight into the relative
importance of model parameters with respect to specific measures of dose. Comparing the highest sensitivity coefficients
between the male rat and human models indicated that, at low
doses, human serum ClO4 levels are most sensitive to urinary
clearance, whereas the rat’s serum levels are more sensitive to
plasma binding parameters (Fig. 15) (Merrill et al., 2003). The
fact that data was available across multiple doses for establishing
parameter values for urinary clearance and plasma binding adds
confidence to the model’s predictive ability. More useful to the
application of the models, for human dosimetry predictions, is
variability analysis that is performed with known distributions
for model parameters. This tool can be applied to the model to
allow the prediction of likely ranges of the dosimetrics within a
human population.
Modeled effects on hormone regulation are yet to be developed. Perturbations in hormones levels after ClO4 exposure
demonstrate complex differences in the hormone regulatory
mechanisms between rats and humans, which are difficult to
describe (Clewell et al., 2001; Merrill et al., 2001). However,
the current model structures may provide a basis for evaluating
thyroid effects from other environmental contaminants. For
example, excessive exposure to other similarly behaving anions,
such as sodium chlorate, thiocyanate, or nitrate, all found to also
contaminate ground and surface waters, may contribute to environmental anti-thyroid effects in humans (Hooth et al., 2001;
Wolff and Maurey, 1963). Further, the possibility of additive
anti-thyroid effects to those of perchlorate from these cocontaminants may need to be considered (Kahn et al., 2004).
41
studies for perchlorate analyses and Dr. Mel Andersen and Harvey Clewell for
constructive comments. Also acknowledged are Lt. Col. Dan Rogers, Dr. Richard
Stotts, and Dr. Dave Mattie for assistance in obtaining funding for this research
from the U.S. Air Force, U.S. Navy, and NASA. Drs. Andrew Geller and Allan
Marcus are thanked for their critical technical reviews of the draft manuscript.
Lastly, this work would not have been possible without analytical support from
Lt. Eric Eldridge, Latha Narayanan, Gerry Buttler, and SSgt. Paula Todd. Funding for this research was provided by the U.S. Air Force, U.S. Navy, and NASA.
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