TOXICOLOGICAL SCIENCES 108(1), 22–34 (2009)
doi:10.1093/toxsci/kfn264
Advance Access publication December 19, 2008
Manganese Tissue Dosimetry in Rats and Monkeys: Accounting
for Dietary and Inhaled Mn with Physiologically based
Pharmacokinetic Modeling
Andy Nong,*,1 Michael D. Taylor,† Harvey J. Clewell, III,* David C. Dorman,*,‡ and Melvin E. Andersen*
*The Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina 27709; †Afton Chemical, Richmond, Virginia 23219; and ‡College of
Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606
Received August 15, 2008; accepted December 10, 2008
Manganese (Mn) is an essential nutrient required for normal
tissue growth and function. Following exposures to high concentrations of inhaled Mn, there is preferential accumulation of Mn in
certain brain regions such as the striatum and globus pallidus. The
goal of this research was to complete a physiologically based
pharmacokinetic (PBPK) model for Mn in rats and scale the
model to describe Mn tissue accumulation in nonhuman primates
exposed to Mn by inhalation and diet. The model structure
includes saturable tissue binding with association and dissociation
rate constants, asymmetric tissue permeation flux rate constants
to specific tissues, and inducible biliary excretion. The rat PBPK
model described tissue time-course studies for various dietary Mn
intakes and accounted for inhalation studies of both 14-day and
90-day duration. In monkeys, model parameters were first
calibrated using steady-state tissue Mn concentrations from rhesus
monkeys fed a diet containing 133 ppm Mn. The model was then
applied to simulate 65 exposure days of weekly (6 h/day; 5 days/
week) inhalation exposures to soluble MnSO4 at 0.03 to 1.5 mg
Mn/m3. Sensitivity analysis showed that Mn tissue concentrations
in the models have dose-dependencies in (1) biliary excretion of
free Mn from liver, (2) saturable tissue binding in all tissues, and
(3) differential influx/efflux rates for tissues that preferentially
accumulate Mn. This multispecies PBPK model is consistent with
the available experimental kinetic data, indicating preferential
increases in some brain regions with exposures above 0.2 mg/m3
and fairly rapid return to steady-state levels (within several weeks
rather than months) after cessation of exposure. PBPK models
that account for preferential Mn tissue accumulation from both
oral and inhalation exposures will be essential to support tissue
dosimetry-based human risk assessments for Mn.
Key Words: manganese; nonhuman primates; dose-dependent
regulation; saturable tissue binding; asymmetrical diffusional flux;
biliary induction; inhalation exposures; PBPK.
1
To whom correspondence should be addressed at The Hamner Institutes for
Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709-2137. Fax:
(919) 558-1300. E-mail: [email protected].
Manganese (Mn), a common element in the environment,
naturally occurs in air, soil, and water. Mn is used in the
production of steel and other alloys, which accounts for > 90%
of its global demand (U.S. Geological Survey, 2008). Other
industrial uses of Mn include welding and metal working,
battery production, glass and ceramics manufacturing, and the
octane-enhancing gasoline additive methylcyclopentadienyl
manganese tricarbonyl (MMT).
As an essential nutrient, Mn is required for maintaining the
proper function and regulation of many biological processes.
Mn is a component in various enzymes such as Mn superoxide
dismutase and glutamine synthetase (Cotzias, 1958; Takeda,
2003). Additionally, Mn has a role in immune function,
regulation of blood sugar, production of cellular energy,
reproduction, digestion, bone growth, carbohydrate metabolism, and blood clotting (Aschner, 2000). Mn is present in all
tissues at substantial concentrations due to sufficient daily
exposure (Aschner et al., 2005).
As with other compounds, Mn toxicity may occur with
excessive exposure. Toxicity has been reported from exposure to
Mn-containing dusts in miners (Cotzias, 1958; Pal et al., 1999).
Prolonged exposure to high levels of inhaled Mn can result in the
onset of a neurological syndrome known as manganism. The
neurotoxic response presents with motor symptoms resembling,
but distinguishable from, those of Parkinson’s disease (Lee,
2000; Pal et al., 1999). In cases of Mn toxicity, mid-brain
structures which influence motor control, such as the striatum and
globus pallidus, accumulate Mn and are considered target tissues
for Mn-induced neurotoxicity (Pal et al., 1999).
Generally, humans receive their daily Mn intake from the
diet (ATSDR, 2000). Three to ten percent of ingested Mn is
systemically absorbed from the gut with elimination primarily
by the liver via bile (ATSDR, 2000). Homeostatic mechanisms
regulate substantial variations in dietary Mn without adverse
consequence (U.S. EPA, 1994). Even though the body exerts
control of Mn uptake, increases in blood and brain Mn levels
and Mn-induced neurotoxicity have been reported from various
sources and routes of exposure, including inhalation in
Ó The Author 2008. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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PBPK MODELING OF MN IN ADULT RAT AND MONKEY
occupational settings, ingestion of drinking water high in Mn
(Ljung and Vahter, 2007), in persons with impaired clearance
of Mn because of liver disease (Burkhard et al., 2003; Spahr
et al., 1996), and in individuals receiving prolonged parenteral
nutrition (Iinuma et al., 2003; McKinney et al., 2004).
Uncertainty regarding the homeostatic control of inhaled Mn
has prompted concerns that the long-term inhalation of even
low levels of Mn in ambient air may present a risk to public
health due to the possible accumulation of inhaled Mn in
sensitive target tissues over time (U.S. EPA, 1994). Because
similar neurological responses arise from different intake
routes, the assessment of the risk from long-term exposure to
low ambient levels of inhaled Mn must also include
consideration of all intake routes for this metal and the manner
in which various routes contribute to increases in Mn
concentrations in target regions with in the central nervous
system. A dosimetry-based assessment of Mn characterizing
the increase in brain Mn concentrations from ingestion and
inhalation could properly take into account essentiality as well
as toxicity of Mn (Andersen et al., 1999).
A series of publications has detailed the development of both
compartmental and physiologically based pharmacokinetic
(PBPK) models for Mn (Nong et al., 2008; Teeguarden
et al., 2007a, b, c). Key processes required in development of
the PBPK model for Mn in the rat were saturable tissue binding
kinetics and asymmetrical flux into brain regions (Nong et al.,
2008). These kinetic mechanisms allowed the tissue compartments to retain fairly constant Mn during period of low daily
intake while accounting for rapid rises in tissue Mn levels
during periods of high inhalation concentrations observed in
animals (Dorman et al., 2006a).
The present study details the development of a more
complete PBPK model for the adult rat and scale-up of the
rat model to the monkey. The adult rat PBPK model has been
extensively modified from earlier work (Nong et al., 2008) that
included kinetics of Mn in the liver and in a single brain region.
These modifications involve applying saturable binding to all
tissues in the rat, preferential accumulation of Mn in several
brain regions, and respiratory and olfactory uptake based on
regional particle deposition within the respiratory tract. These
model refinements required reparameterization of the rat
model. The result of theses efforts is a more consistent
description of the tissue-specific kinetics of Mn that can be
more confidently extrapolated between the rat and the monkey.
METHODS
Rat model development. The development of the rat Mn model included
the description of saturable binding in tissues as presented previously in Nong
et al. (2008). Although the previous model focused on the liver and brain, this
effort extends the saturable kinetics to all tissues in the adult animal (Fig. 1).
Asymmetrical efflux and influx rate constants account for the preferential rise
of Mn concentration in different brain regions or tissues. A new comprehensive
set of model parameters are provided in the supplementary materials.
23
Physiological parameters for the adult rat were obtained from the literature
(Brown et al., 1997). However, actual tissue measurements in rats served as
basis for calculating weights of specific brain regions (Dorman et al., 2004). In
addition to tissue Mn collected for the striatum, Mn concentrations in the
cerebellum and olfactory bulb were also modeled in the rat studies. Rodent
olfactory and respiratory tissue parameters were from the literature (Kimbell
et al., 1997; Conolly et al., 2000; Kelly et al., 2001; Schroeter et al., 2008).
Lung and nose surface area and tissue thickness were estimated from imaging
models of the rat nasal cavity and lung. These dimensions were used to estimate
the fractional deposition of inhaled Mn particles. The deposition model
assumed that soluble MnSO4 particles deposited in the respiratory region
rapidly dissolve in mucus and tissue, and are absorbed rapidly into the systemic
circulation. Inhaled Mn particles are also deposited on the nasal olfactory
epithelium region. After dissolution of the particles, Mn is transported along the
olfactory nerve into the olfactory brain region (olfactory bulb), olfactory tract
and tubercle (Brenneman et al., 2000). Small amounts of Mn move from these
olfactory tract tissues to more distant brain regions. The olfactory tract tissue
Mn simply equilibrates with brain blood (Leavens et al., 2007).
Mn dietary uptake and biliary elimination were calibrated based on steadystate tissue concentration and tracer 54Mn elimination from several studies as
described in Teeguarden et al. (2007a, c) and Nong et al. (2008). Details of this
calibration phase are in the supplementary materials. Tissue capacity and
diffusion parameters were calibrated to simulate tissue concentrations observed
in rats fed a defined diet while being exposed 6 h/day via inhalation for 14 days
to inhaled Mn concentrations from 0 to 3 mg Mn/m3 (Dorman et al., 2001).
Following model calibration for dietary uptake and short-term exposure,
predictions of tissue concentrations were compared with tissue measurements
from rats in two 90-day inhalation rat studies (Dorman et al., 2004; Tapin et al.,
2006). In predicting tissue Mn for these 90-day exposures, parameters
regulating saturable tissue binding and asymmetrical fluxes in brain regions
were not altered compared with the 14-day study. Biliary excretion rate
constants were fit to be consistent with the high dose inhalation studies based
upon the increased bile Mn concentration observed with increasing inhaled
concentration exposure (Dorman et al., 2001).
Monkey model development. The physiological parameters in the monkey
Mn PBPK model were scaled from the rat model to account for the body
weight, tissue volumes, and blood flows of an adult rhesus monkey (see tables
in Supplementary Materials). Monkey physiological parameters were obtained
from Davies and Morris (1993). Regional brain volumes were from Dorman
et al. (2006b). Body weights from the same study were applied to simulate the
observed tissue Mn concentrations. Although an average adult monkey weighs
approximately 5 kg (Davies and Morris, 1993), the average weight of 3 kg of
the young nonhuman primates in Dorman et al. (2006b) was used in the
simulations. Olfactory and respiratory surface areas and tissue thickness were
from monkey imaging studies (Menache et al., 1997) and derived directly from
human literature values (Conolly et al., 2000; Schroeter et al., 2008).
In contrast to the rodent studies, Mn concentrations were measured in
monkey striatum (putamen and caudate) and globus pallidus. The Mn kinetics
in these two primate brain tissues were similar (Dorman et al., 2006b). For the
present study, globus pallidus Mn concentrations in the monkey were modeled
and compared against the striatum rat simulations.
Initial calibration of the monkey model consisted of adjusting dietary
absorption and biliary excretion to fit Mn tissue levels in control monkeys fed
a commercial diet (133 ppm Mn). Model simulations were then evaluated
against Mn tissue concentrations from a study with rhesus monkeys exposed at
MnSO4 for 90 days (Dorman et al., 2006b). Induction of biliary Mn excretion
by blood increasing Mn concentration was incorporated into the model to allow
simulations to fit the increase in biliary Mn concentrations with increasing
inhaled Mn. Detailed accounts of the calibration of the monkey model are in the
result section.
Mn pharmacokinetic studies. The calibration of model parameters was
undertaken with tissue concentrations from rats in a repeated inhalation exposure study. Adult rats were exposed to MnSO4 to 0, 0.03, 0.3, or 3 mg Mn/m3
for 14 consecutive days (6 h/day) (Dorman et al., 2001). The rats were
24
NONG ET AL.
FIG. 1. (A) The PBPK model structure describing tissue Mn kinetics in adult rats. (B) Inhaled Mn is absorbed through deposition of particles on the nasal and
lung epithelium. Mn in the nose is absorbed largely into the systemic blood and a small portion moves directly to the olfactory bulb. Every tissue has a binding
capacity, Bmax, with affinity defined by association and dissociation rate constants (ka, kd). Free Mn moves in the blood throughout the body and is stored in each
tissue as bound Mn. Influx and efflux diffusion rate constants (kin, kout) allow for differential increases in Mn levels for different tissues. Qp, Qc, Qtissue refer to
pulmonary ventilation, cardiac output, and tissue blood flows.
25
PBPK MODELING OF MN IN ADULT RAT AND MONKEY
acclimated to a 125 ppm Mn diet for 14 days prior to exposure and were kept
on that diet throughout the study.
Simulations of tissue Mn concentrations in rats were compared with data
from two long-term exposure studies as a partial validation. In the first study,
Dorman et al. (2004) exposed adult rats via inhalation for 6 h/day and 5 days/
week for 13 weeks at 0, 0.01, 0.1, 0.5 mg Mn/m3 and reported tissue
concentrations during and following the 13 weeks of exposure. The rats were
acclimated to a 10 ppm Mn diet 30 days prior to exposure. Additional tissue
measurements were made at 45 and 90 days following exposure. A second
study by Tapin et al. (2006) reported tissue Mn concentrations for similar
inhalation exposure conditions (6 h/day and 5 days/week for 13 weeks) to
0, 0.03, 0.3, or 3 mg Mn/m3. These rats were acclimated on a 125 ppm Mn diet
for 14 days prior to inhaled exposure.
The PBPK model was next used to examine tissue Mn concentrations in
rhesus monkeys exposed during a 90-day study (Dorman et al., 2006b). This
study included young (17–24 months old) monkeys exposed to 0, 0.06, 0.3,
1.5 mg Mn/m3 of MnSO4 for 6 h/day and 5 days/week during 13 weeks. The
monkeys were acclimated to a diet containing approximately 133 ppm Mn for
43 days prior to exposure. Tissue measurements were also made at 45 and 90
days following cessation of the highest exposure concentration (1.5 mg Mn/m3).
Estimates of induction of biliary elimination of Mn in the model were based
on the increasing bile and blood concentration with increasing inhalation
exposures collected in this monkey study.
PBPK model structure and equations. The Mn PBPK model structure
included liver, bone, lung, nasal cavity, blood, and brain (cerebellum, olfactory
bulb, striatum, and pituitary). Remaining body tissues were combined into
a single compartment (Fig. 1A). The pituitary was not included for the rat
simulations (no data were collected from this tissue in the rats) but was included
for the monkeys. Steady-state levels of tissue Mn were from rats on constant
dietary Mn (Dorman et al., 2004). The control of Mn levels in tissues with
increasing dietary intake was accounted for by a decrease in the fraction of Mn
absorbed via the gastro-intestinal tract and increase of biliary Mn elimination
(Teeguarden et al., 2007b).
In the model structure, tissues contain both ‘‘free’’ and ‘‘bound’’ Mn in
tissues. Free Mn circulates in the blood throughout the body and bound Mn is
confined in tissues, where it is involved in various metabolic functions. The
total amount of Mn in the tissues is the sum of both free and bound Mn.
Because the binding process is capacity dependent, the mass balance equations
describing Mn tissue uptake and release in the model are
dAt;free
¼ Qt Cart Ct;free þ kd 3 At;bnd ka 3 Bt 3 At;free
dt
ð1Þ
dAt;bnd
¼ kd 3 At;bnd þ ka 3 Bt 3 At;free
dt
ð2Þ
Bt ¼ Bt;max At;bnd
ð3Þ
where At,free and At,bnd are the amount of free and bound Mn in the tissue, Bt is
the available binding capacity for free Mn in the tissue, and ka and kd are the
association and dissociation binding rate constants. The other parameters found
in the equations are tissue blood flow (Qt), arterial blood concentration of free
Mn (Cart), and concentration of free Mn in the tissue (Ct,free).
The distribution between bound and free Mn in each tissue is mainly
determined by the dissociation binding ratio (i.e., kd/ka). Tissue levels of bound
Mn are constrained by the tissue’s maximal binding capacity (Bt,max), which is
the sum of bound Mn (At,bnd) and the available binding capacity for free Mn in
the tissue (Bt). Excursions of free Mn above the maximal binding capacity
causes proportionately greater rises in free Mn in tissues compared with basal
conditions where tissue Mn is mostly in the bound form. Every tissue in the
model has a specific saturable binding capacity which maintains a defined total
Mn levels. Detailed descriptions of the equations for these tissues are in the
supplementary materials.
Four brain regions displaying differential changes of total Mn concentration
with increasing exposure concentrations were simulated in the model: olfactory
bulb, globus pallidus (monkey) or striatum (rat), cerebellum, and pituitary in
the monkeys (Fig. 1A). Differential increases in free Mn in various tissues were
described with asymmetrical diffusional permeability rate constants. The
preferential increase represents a greater Mn influx compared with efflux
exchange between brain blood and brain tissues in various regions. In
combination with saturable binding capacity, the asymmetrical diffusion of Mn
accounts for preferential increases of Mn in specific brain regions or in specific
tissues. The general equations describing the combined kinetic processes for
a specific brain region (XX) are
dAbrb
¼ Qbrain ðCart Cvbrb Þ þ kout 3 AXX;free kin 3 Abrb
dt
ð4Þ
dAXX;free
¼ kout 3 AXX;free þ kin 3 Abrb þ kd 3 AXX;bnd ka 3 BXX 3 AXX;free
dt
ð5Þ
where Abrb and Cvbrb are the amount and concentration of free Mn in the brain
blood, and kin and kout are the permeability diffusional influx and efflux rate
constants. Because the blood volume of the brain accounts for roughly 3% of
the total volume, the total amount of Mn in a specific brain region was
calculated as the sum of 3% of the amount of Mn in brain blood and 97% of the
amount of Mn in brain tissue. A complete description of the equations for each
brain region in the model are in the supplementary materials.
Approximately 8% of the inhaled Mn is deposited on the olfactory
epithelium of the nasal cavity in the rat, whereas 0.5% is deposited in the
monkey olfactory epithelium. Subsequently, free Mn enters the olfactory bulb
via the olfactory neural pathway using a first order rate constant (kNPOB). The
nasal uptake represents the process of Mn transport through the olfactory nerve
and into the olfactory brain regions (Brenneman et al., 2000).
Absorption of Mn particles in the respiratory tract is determined by the
deposition of the particles on the surface epithelium of the nasal cavity and lung
for both rats and monkeys (Fig. 1B). The MnSO4 particles are considered
highly soluble in mucus and tissue based on previous laboratory work (Vitarella
et al., 2000). Deposited material is described as being rapidly absorbed into the
systemic blood, lung tissue or directly into the olfactory bulb via the nasal
epithelium in the model. Absorption is assumed to occur as a soluble Mn,
probably manganous ion. In addition to Mn exposure from dietary uptake and
inhalation, tracer kinetics of an intravenous dose of 54Mn were also examined
to reproduce the terminal half-lives found experimentally in Dorman et al.
(2001). The model calibration with the dietary study is described in the
supplemental materials.
Model simulations and analyses. Model simulations and analyses were
performed in asclXtreme version 2.4 (AEgis Technology Group Inc.,
Hunstville, AL). The estimates of the model parameters with the adult rat
tissue Mn concentrations were optimized using a Nelder-Mead algorithm. The
parameters were chosen based on the best fit of the model output with the data
as measured from the log-likelihood and precision of estimates. Model code is
available from the corresponding author (Dr Andy Nong).
A sensitivity analysis of Mn tissue concentrations in relation to changes in
model parameters was calculated at different inhaled exposure concentrations
(0.0, 0.3, and 3 mg Mn/m3). The sensitivity analysis determined the importance
of specific parameters in controlling tissue Mn at different inhaled concentrations. Normalized sensitivity coefficients were calculated with the central
difference method. Changes in Mn tissue concentrations were calculated for 1%
changes of each parameter.
RESULTS
Rat Model Calibration for Inhaled Exposure
The PBPK model parameters were first fit to be consistent
with the steady-state tissue concentrations in rats fed specific
daily diets (Dorman et al., 2001). Fractional gastro-intestinal
absorption (fdietup) and Mn biliary excretion (kbile) rate were
26
NONG ET AL.
adjusted based on the dietary levels (10 ppm for long-term
exposure or 125 ppm for short-term exposure) because the
processes controlling uptake and elimination of Mn are dosedependent (Teeguarden et al., 2007b). The adjustments to the
model parameters regulating basal total Mn tissue levels
provided estimates of tissue Mn concentrations that were
consistent with steady-state tissue concentrations and with
tracer elimination half-life of approximately 40 days. Detailed
depictions of the model fit with dietary data are found in the
supplementary materials.
Mn deposition and uptake in the respiratory tract were
estimated based on the chemical characteristics of Mn particulates used in the experiments. Although Mn2O4, hureaulite (a
form of MnPO4), and MnSO4 have been used in the various Mn
inhalation studies, the current model structure only simulates
exposure to MnSO4, the most highly soluble form. Animal
studies in conjunction with in vitro work showed that MnSO4
particles were highly soluble in mucus, tissues, and blood
(Dorman et al., 2001; Vitarella et al., 2000). Thus, deposited Mn
was modeled to be rapidly absorbed systemically from lung or
nasal tissues to blood or transported from the nasal olfactory
epithelium to the olfactory tract and into the bulb. The use of the
most soluble form of Mn represents a simplifying approach to
model development, as this form has the highest bioavailability
compared with the other less soluble particulate forms. Adjustments based on apparent solubility of deposited Mn could be
made in order for this model to consider other forms of Mn salts.
Fractional depositions of Mn particles in the nasal cavity and
lung (fdepNP, fdepLu) were estimated (Anjilvel and Asgharian,
1995; RIVM, 2002) based on particle size (geometric mean
aerodynamic diameter of 1.2 lm and geometric standard
deviation of 1.5) and density (2.98 g/cm3). The fractions
deposited were calculated in the Multiple-Path Model of Particle
Deposition software (MPPD version 2.0; CIIT, Raleigh, NC;
available at www.thehamner.org). The deposition values indicate significant deposition of Mn in both the nasal cavity and
lung (50–60% of the total inhaled Mn is expected to be retained
during inhalation for these particles).
The PBPK model parameters for tissue Mn binding were
next adjusted to account for increases in total tissue Mn
concentrations in rats inhaling Mn for 6 h/day during 14
consecutive days (Dorman et al., 2001). These model
parameters were determined in relation with the Mn dietary
intake and biliary Mn excretion determined earlier. Optimal
values for the model parameters were consistent with the total
Mn tissue concentration measured in rats exposed at 0.03, 0.3,
or 3 mg Mn/m3 (Fig. 2). Tissue: blood partition coefficients
(Ptissue) and tissue binding rate constants (ka, kd) were adjusted to
fit the Mn concentrations in liver, lung, bone and rest of the body.
Dose-dependencies in kinetics were accounted due to saturation
of the tissue binding with increasing body burdens of Mn.
Influx and efflux permeability diffusional rate constants (kin,
kout) for the regions of the brain were adjusted along with ka
and kd to account for the differential increases in free Mn
FIG. 2. Comparison of tissue concentrations during 14-day inhalation
studies in rats for striatum, cerebellum, and olfactory bulb (Dorman et al.,
2001). The curve represents model simulations and symbols are mean and
standard errors from eight rats/exposure concentration. Each plot contains Mn
tissue concentrations in rats exposed at 0.03, 0.3, or 3 mg Mn/m3.
concentrations (Fig. 2). The difference between the kin and kout
leads to differential relative partitioning between tissue and
blood. Consequently, the model describes larger rises of Mn in
the striatum compared with the cerebellum (Fig. 2). The larger
ratio kin/kout for the striatum (1.7 /h/kg)/1 /h/kg ¼ 1.7) give
PBPK MODELING OF MN IN ADULT RAT AND MONKEY
27
rises to larger tissue-blood gradient of Mn. In contrast, the low
ratio of kin/kout for the cerebellum (1 /h/kg) 8 /h/kg ¼ 0.125)
results in smaller tissue-blood gradients for Mn in cerebellum.
The olfactory bulb had additional input of Mn from the nasal
absorption pathway. The ratio of kin/kout for the olfactory bulb
(0.087 /h/kg) 1.2 /h/kg ¼ 0.67) was intermediate between
striatum and cerebellum. The olfactory nasal deposition was
8% of the inhaled dosed, and the elimination half-life
associated with transfer of Mn from the olfactory epithelial to
the olfactory bulb (kNPOB) was calculated as approximately
2.5 days. The rate of nasal uptake in the model is consistent
with the inhalation exposure Mn tissue concentrations described earlier (Brenneman et al., 2000; Dorman et al., 2001;
Leavens et al., 2007).
Evaluation of Rat Model Predictions
The parameter suite developed to simulate tissue total Mn
concentrations in the 14-day inhalation studies were used to
predict expected tissue Mn concentration following 90-day
exposure in rats. The simulations were compared against two
different inhalation studies (Dorman et al., 2004; Tapin et al.,
2006). Tissue Bmax parameters were altered in proportion to the
basal Mn levels found in the experimental studies. The basal
tissue Mn concentrations for the initial model adjustments were
obtained from rats fed on a specific diet (10 and 125 ppm)
without inhalation exposure. Predictions for the 90-day
inhalation exposure were then obtained without further changes
in tissue binding capacity or permeability diffusional rate
constants.
Model predictions for the highest exposure concentrations
tended to overestimate brain tissue Mn in rats exposed over
a 90-day period (Fig. 3). A better fit to the 90-day studies was
obtained when a small (approximately twofold) increase in the
rate constant for biliary excretion was included for the
simulations. Enhanced biliary induction in the 90-day versus
the 14-day study would be consistent with an adaptive response
that required a longer-term increase in blood Mn. Although the
90-day rat studies did not include direct measures of biliary
Mn, the monkey studies provided more direct support for the
inclusions of adaptive biliary Mn elimination.
Monkey Pharmacokinetics Extrapolation
Physiological parameters were scaled to adult monkey
values prior to any calibration. Blood flows and tissue volumes
were adjusted to represent measurements observed in adult
rhesus monkeys (Davies and Morris, 1993). Cardiac and
pulmonary blood flows, and nasal surface area were then
allometrically scaled (BW75) to the average body weight in the
monkey study (Dorman et al., 2006b). Background levels of
total tissue Mn were set to the control tissue measurements in
the experiments by adjusting biliary elimination and dietary
absorption from a 133 ppm diet fed to the monkeys. Nasal and
pulmonary deposition and absorption rates were scaled in
FIG. 3. Comparison of Mn concentration in the cerebellum, striatum, and
liver at various inhaled Mn concentrations for the adult rat (Tapin et al., 2006).
The curves are model simulations and symbols are means and standard errors
from 30 rats/exposure concentration. The dotted lines represent the addition of
dose-dependent biliary excretion (up to 2.5-fold increase).
proportion to the olfactory and respiratory tissues surface area
and ventilation rates in monkeys.
Induction of biliary elimination of Mn with increasing blood
Mn concentrations was included. A dose-dependent biliary
excretion rate constant was used to describe the increasing bile
28
NONG ET AL.
Mn elimination observed directly in the higher exposure
concentrations in the monkeys (see figure in Supplementary
Materials). The mathematical relation is described as
kbile ¼ kbile0 þ
kbmax 3 Cartn
kb50 n þ Cartn
ð6Þ
where kbile0 is the basal biliary excretion rate constant for
biliary Mn, kbmax is the maximal excretion rate, kb50 is the
arterial concentration at half the induced level of Mn in the
arterial blood (Cart), and n is the factor defining the slope, with
a larger n-value leading to a steeper slope. This structure for the
biliary induction relationship was motivated by the Hill
equation. In this equation, the Cart is used as a surrogate of
the free Mn concentration in liver. Using the Mn blood levels
has advantages over free liver Mn because Mn blood levels are
directly measurable. Predicted tissue concentrations in monkeys with this scaled model were then compared with Mn
concentrations observed in the monkeys exposed for a 90-day
period (Dorman et al., 2006b).
These predictions were consistent with the rise of tissue Mn
levels observed in monkeys exposed over a 90-day period at
different inhaled concentrations (Fig. 4; Dorman et al., 2006b).
Some additional refinements to brain kinetics in the monkey
were required (see figures in Supplementary Materials). Scaling
the parameters from rat to the monkey body weight did not
produce a proportional rise of Mn tissue levels. Changes to
tissue binding rate constants (ka, kd), binding capacities (Bmax)
and brain regional tissues influx and efflux permeability rate
constants (kin, kout) were required to fit the total tissue Mn
concentration in the monkey dataset. Tissue Mn influxes for the
pituitary and globus pallidus were modified to predict
FIG. 4. Simulated end-of-exposure tissue Mn levels in the arterial blood, liver, pituitary, and globus pallidus of monkeys following 90-day exposures at
various inhaled Mn concentrations compared with data from Dorman et al. (2006b). The curves represent model simulations and symbols are mean and standard
error from four to six monkeys per exposure concentration. Dose-dependent biliary elimination was included with a maximum increase of up to threefold.
PBPK MODELING OF MN IN ADULT RAT AND MONKEY
background Mn levels at steady-state and accumulation at high
inhaled exposure concentrations. Because changing the ratio
between kin and kout rate constants was not sufficient to
reproduce the experimental data, a brain amount-dependent
influx rate constant was introduced as shown in the next
equation.
kin ¼ kin0 þ
kinMax 3 Afree;t
kin50 þ Afree;t
ð7Þ
The monkey transient influx permeability rate constant (kin)
was determined by the basal influx rate constant (kin0),
maximal influx rate constant (kinMax), affinity rate constant
(kin,50), and the amount of free Mn in the brain region (pituitary
or globus pallidus, Afree,t). In this fashion, the tissue influx rate
constant increases as free tissue Mn increases in the brain
regions. A comparison of the fixed and dose-dependent
diffusional influxes with the monkey brain regions is presented
in the supplementary materials.
The consistency of the model predictions with the monkey
data was also noted in examining the rates of loss of Mn from
29
tissue after cessation of the 1.5 mg/m3 exposure (Fig. 5;
Dorman et al., 2006b). In these simulations, the rate constant
for biliary elimination was induced by approximately threefold
at highest exposure concentration (1.5 mg Mn/m3). In our
PBPK representation of Mn kinetics, the accumulation of Mn
in tissues depended on two key processes—transport and
binding. The concentration gradient maintained in tissues was
dependent on the ratio of transport rate constants, kin/kout; the
overall concentration at which the free Mn begins to dominate
in a tissue depends on another ratio of rate constants, kd/ka, an
effective dissociation constant for Mn in the tissues.
Sensitivity Analysis at Various Inhaled Concentrations
Sensitivity of model parameters for the liver, striatum, and
cerebellum was examined in the rat model. Normalized
sensitivity coefficients of changes in predicted concentration
from a 1% change in parameter value were determined for rats
exposed at 0, 0.3, or 3 mg Mn/m3 (see figure in Supplementary
Materials). The sensitivity analysis for liver tissue Mn
concentration revealed a decrease in sensitivity toward tissue
binding constants (Bmax, ka, kd) as inhaled Mn increased, due to
FIG. 5. Simulated tissue Mn levels in the lung, liver, pituitary, and globus pallidus of monkeys exposed at 1.5 mg Mn/m3 are compared with monkey data
from Dorman et al. (2006b). The curves represent simulations and symbols are means and standard errors from four to six monkeys per exposure concentration.
Dose-dependent biliary elimination was included with a maximum increase of up to threefold.
30
NONG ET AL.
saturation of tissue binding between 0.3 and 3 mg/m3. After
binding becomes saturated, the partition of free Mn into tissue
becomes more important than the presence of bound forms of
Mn. Similarly, as the binding capacity in the striatum saturates,
the sensitivity coefficients for Bmax, ka, kd decrease. However,
because of the asymmetry in influx and efflux rate constants,
striatal Mn remains sensitive to kin and kout at all for all three
exposure conditions.
DISCUSSION
Mn PBPK Model Dosimetry
The main purpose for creating these PBPK models for Mn in
rats and monkeys was to develop tools to predict the
relationship between increasing inhaled Mn exposure concentrations and increases of tissue Mn concentration for use in
a dosimetry-based risk assessment of inhaled Mn. Dietary
Mn was included into the simulations as the determinant of
steady-state Mn tissue levels. Defined levels of dietary Mn
were identified in the animals experiments (10–133 ppm) and
intake was set at a 0.05 kg/day/kg body weight daily intake as
suggested in the U.S. EPA guideline (2005). As the major
source of Mn, daily intake for the general human population
ranges from 0.7 to 10.9 mg/day in food and 3–5 mg/day in
water, and cases of deficiencies are rarely observed (Santamaria
et al., 2007). Because of the ubiquitous nature of Mn and the
role of dietary Mn in establishing steady-state tissue concentrations, risk assessments of inhaled Mn must consider the essentiality of Mn from diet to establish the tissue concentrations
that will be altered with increasing levels on inhaled Mn.
Tissue accumulation of inhaled Mn appears to be associated
with an overwhelmed homeostatic control that operates to
maintain normal Mn within a narrow range of healthful
concentrations. With the inclusion of the dose-dependent
processes, such as saturable binding tissue capacities and
asymmetrical influx/efflux transport, the models captured the
regulation of Mn as observed in the animal dietary and
inhalation studies at multiple exposure concentrations and
exposure durations. Importantly, the extent of increase in tissue
Mn, the rate of clearance from tissues, and the control at near
steady-state levels for low inhaled concentrations of Mn were
all nicely recapitulated with these PBPK models.
The rat and monkey models identify ranges of inhaled
concentrations where Mn levels in target tissues do not increase
appreciably (Fig. 6). Based on 90-day simulations of inhalation
exposures, inhaled concentrations of 0.2 mg Mn/m3 or higher
are required to cause a doubling of rat striatal and monkey
pallidal Mn levels. Two factors are at work here to limit
increases in brain Mn: (1) induction of biliary excretion rate
constant with increasing exposure concentration, and (2) the
relationship between intake from diet versus intake from
inhalation. Dietary intake is closely regulated at the gut and bile
to maintain essential levels of Mn in the body. Although
FIG. 6. Simulated end-of-exposure tissue Mn levels in the rat striatum and
the monkey globus pallidus following 90-day exposures (5 days/week, 6 h/day)
at various inhaled Mn concentrations. The curves represent simulations and
symbols are means and standard errors of animal data. The simulations are
compared with monkey data (dark triangle) from Dorman et al. (2006b) and
with rat data (gray circle) from Dorman et al. (2004) and Tapin et al. (2006).
bypassing controls at the gut, inhaled particles of Mn are
influenced by tissue storage of Mn and biliary excretion. An
important point achieving increases in tissue Mn is the
relationship between net uptake from the diet and incremental
increases in total absorption due to the inhaled burden.
As the inhaled concentration increases above this 0.2 mg/m3,
the intrinsic control mechanisms associated with biliary
excretion cannot keep up with increasing pulmonary uptake.
Tissue binding and biliary clearance processes become
saturated. At these higher inhaled concentrations, free Mn
preferentially accumulates in the tissues (Fig. 7). Direct
measurements of tissue Mn in the animal studies includes
both free and bound Mn. The model structure required binding
and differentiation of Mn (as either free or bound Mn) to
account for tissue Mn from dietary and inhalation exposures.
The toxicologically relevant measures of striatal tissue dose is
most likely free Mn, a metric that is determined by tissue
binding parameters, tissue diffusional parameters, and inducible biliary excretion.
Rat to Monkey Model Extrapolation
The extent of Mn accumulation in rat and monkey tissues
differ because of key physiological and biochemical differences in these species. One significant difference is the 20-fold
difference in nasal olfactory surface area between the rat and
monkey. The difference in surface area of the nasal epithelium
influences the amount of Mn delivered to olfactory bulb
through the olfactory pathway. Although the nose and lung
structure in the model represent a simplification of a previous
PBPK MODELING OF MN IN ADULT RAT AND MONKEY
31
FIG. 7. Simulated end-of-exposure Mn tissue concentrations of total (solid), free (dotted), and bound (gray) in the rat striatum and the monkey globus pallidus
as produced in Fig. 6. The curves represent simulations following 90-day exposure at various inhaled concentrations.
developed model (Leavens et al., 2007), key olfactory and
respiratory absorption processes for airborne Mn were still
included.
Another physiological determinant was the differences in
tissue volumes and blood flows between the rat and monkey.
These physiological differences require sets of species-specific
parameter values for describing endogenous levels of Mn. The
interspecies scaling was based on typical allometric expectations for flows (BW0.75) and volumes (BW). The permeability
rate constants (kkin, kout) were scaled as BW0.25 because they
represent rate constants comprised of a clearance term divided
by a tissue volume. Because tissue binding capacity (Bmax)
terms account for observed tissues Mn concentrations in each
species, they were simply scaled to the tissue volumes. The
tissue binding rate constants (ka and kd) terms represent
presumed association and dissociation processes which are
likely related to incorporation and degradation of specific
macromolecular Mn stores. These parameters were essentially
constant from the rat to the monkey. After accounting for these
physiological differences between rat and monkey, a similar
model structure provided good predictions for both the rat and
monkey.
Additional adjustments were then required besides these
generic scaling approaches. These changes included dose
dependencies of brain uptake rate constants and for biliary
elimination of free Mn. Although inducible biliary excretion of
Mn was suggested by the results in long-term exposure in rats,
the data could be fit adequately without adding the enhanced
uptake with increasing blood Mn. Incorporation of dosedependent uptake mechanisms in the monkey brain are likely
indicative of a more complex control on brain Mn in monkeys
than in rats. Recognition of the differences in factors which
control Mn uptake into target tissues between rat and monkey
is important because the monkey with selective increases in
mid-brain regions and similar toxic responses to Mn appears to
be the better model animal for predicting expected behaviors in
humans.
Kinetics of Mn with Dose-Dependent Mechanisms
With this PBPK representation of Mn kinetics, the
differential increase of tissue Mn concentrations in specific
regions of the monkey brain upon higher concentrations of
exposure is primarily attributed to the differential influx and
efflux of Mn in the brain. The background level of Mn in the
brain regions are defined by the specific saturable binding
capacities. Increases of free Mn in these brain regions are
determined by the regulation of Mn transport via limiting
influx/efflux exchange of Mn between blood and each brain
region. Large proportionate increases of Mn levels in the rat
striatum and monkey globus pallidus were generated with
asymmetrical diffusional rate constants. The extent of asymmetry defined the proportionate increase in one tissue
compared with another after tissue binding became saturated.
As asymmetry becomes larger, the increase in tissue concentration is greater.
Dose-dependent Mn influxes into brain tissues have been
observed previously in cultured rat cells (Aschner and Gannon,
1994; Aschner et al., 1992; Murphy et al., 1991). These in vitro
experiments identified a form of saturable transport uptake of
Mn into cells which is similar to the processes defined in the
present model. A possible explanation for the preferential
influx of Mn in these brain regions may be a requirement for
these brain tissues to maintain Mn at low intake rates and
preferentially maintain Mn during deficiency conditions. In this
fashion, similar mechanisms working to preserve brain Mn
concentrations in critical regions may be responsible for large
influxes and accumulation of Mn in these same regions during
high inhalation concentration exposures.
In contrast to the globus pallidus and striatum, rat and
monkey cerebellum displays small changes of Mn concentrations at any inhaled exposure concentration suggesting
a more limited Mn influx pathway. In other tissues,
accumulation may be related to different processes. For
example, increases of Mn in the olfactory bulb is largely
attributable to nasal olfactory uptake. Without nasal transport,
32
NONG ET AL.
the olfactory bulb would have very similar dose dependency as
the cerebellum (Leavens et al., 2007).
Dose-dependent mechanisms of Mn regulation occurring
over a wide range of Mn exposure concentrations are also
included in the PBPK model. Various processes allow for
different levels of regulation of Mn based on the contribution
of inhaled and ingested Mn. At low inhaled concentration and
constant dietary intake, tissue Mn concentrations are controlled
by high affinity tissue binding while tissue loss is dependent on
the slow dissociation rate constant and biliary elimination. At
exposures above 0.2 mg/m3 for both rat and monkey where
the binding capacity in tissues becomes saturated, increases
in tissue concentration are preferentially due to rise of free
Mn. Free Mn is eliminated from tissue primarily by the kout term
and whole body elimination occurs by inducible biliary excretion.
The current model associates increased biliary excretion with free
Mn in blood. Dose-dependent biliary excretion of Mn has been
consistently observed in experimental rodent studies (Dorman
et al., 2001; Malecki et al., 1996) and in preloading Mn
experiments with humans (Mahoney and Small, 1968). The
combination of these control mechanisms provide a biologically
consistent description of the pharmacokinetics of Mn at different
inhaled and dietary exposures in two different species.
This regulation of tissue Mn likely represents contributions
from several biological processes. The present model suggests
saturable binding would occur from the presence of cellular
components that store or release Mn at concentration levels
which tissues exceed homeostatic control. Asymmetric transport could be explained either by metal transporters (e.g.,
DMT1 or transferrin) compatible with other essential elements
(Roth and Garrick, 2003), co-mediated transport with calcium
channels (Gavin et al., 1999), or even zinc-like membrane
transporters (Nies, 2007). Other compartmental PK models for
essential elements, such as zinc (Miller et al., 2000), have
focused on kinetic behaviors seen at low intake levels with
concerns for deficiency states. The challenge with essential
element models appears to be in capturing the transitions
between adequacy, with active processes to retain the metal, and
excess, where the body appears to activate or engage dosedependent processes to protect against excursions of the free
metal that might lead to toxic sequelae. Although further
experiments are necessary to determine the specific transporters
involved in uptake and efflux from tissues and the nature of the
binding sites within tissues, these PBPK models have captured
the main dose-dependent characteristics of Mn disposition in rats
and monkeys as well as provided a structure to organize and
parameterize an equivalent description in humans.
Conclusion
These elaborated PBPK models for Mn in rats and monkeys
not only describe the slow clearance of Mn associated with
essential dietary ingestion but more importantly reproduce the
rapid tissue intake of free Mn in the blood from high
concentration of inhaled particulates and the subsequent rapid
clearance of Mn to return at basal levels (a feature that has not
previously been included in kinetic models of essentiality).
These key features of our models provide confidence that the
dynamics of the processes regulating Mn disposition in
laboratory animals are sufficiently well-characterized to
warrant development of similar models for human exposures,
and consideration of such models as cornerstones of future risk
assessments. In a risk assessment framework for essential
elements, use of validated PBPK models will allow characterization of the contribution of diet and inhalation to changes in
free Mn concentration in target tissues in human populations.
Dosimetry-based approaches for risk assessments of essential
elements should provide a better consideration of both
essentiality and excess, thus accounting for the biology of
essential elements better than risk assessment approaches that
simply apply uncertainty factors to a point-of-departure
obtained from toxicological or epidemiological studies.
SUPPLEMENTARY DATA
Supplementary data are available online at http://toxsci.
oxfordjournals.org/.
FUNDING
This work was supported by Afton Chemical Corporation in
satisfaction of registration requirements arising under Section
211 (a) and (b) of the Clean Air Act and corresponding
regulations at 40 CFR Substance 79.50 et seq.
ACKNOWLEDGMENTS
The authors would like to thank Drs Miyoun Yoon, Jerry L.
Campbell, Jr, Michelle A. Medinsky, Daniel Krewski, and
Jeffrey W. Fisher for many helpful comments during the
preparation of this manuscript.
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