Physiologically Based Pharmacokinetic 

Modeling of Fetal and Neonatal

Manganese Exposure in Humans

Describing Manganese Homeostasis

during Development

Miyoung Yoon, PhD

Senior Research Investigator

The Hamner Institutes for Health Sciences

For Technical Advisory Panel Meeting

October 31, 2013

At The Hamner Institutes for Health Sciences
Outline

• Recap of critical features of Mn kinetics and 

homeostasis in adults – essentiality vs. toxicity

• Brief summary of Rat PBPK models for Mn during 

gestation and lactation

• Human PBPK models for gestation and lactation 

based on the parallelogram approach 

• Model evaluation

• Summary

• Use of human developmental model to support risk 

assessment
Factors to be Considered in a PBPK 

Model for Mn in Any Life Stage

• Mn is an essential nutrient with multiple adverse effects 

noted when levels in diet are inadequate

− Normal tissue Mn concentrations are substantial

− Required for variety of physiological processes and an important 

cofactor for various enzymes in the brain

− Homeostasis mechanisms to control the body Mn

despite of large fluctuation in intake in diet

• CNS Toxicity in humans occurs with overwhelmed or 

impaired homeostasis due to high-dose exposure or disease 

conditions

– Mn in the specific brain region (globus pallidus) – internal dose in the 

target
Determinants of Mn homeostasis

(Dietary Essentiality)

Sequestration &

Turnover

Trans-membrane 

transport 

Mnf

Mnb B + Mnf

Mn present in the tissue primarily in bound form (associated with 

enzymes and mitochondria; Free Mn is kept low by tissue 

storage/transport processes and at the levels of uptake and 

excretion; free Mn is responsible for toxicity

Gastro-intestinal 

control of Uptake 

(normal fluctuation)

Biliary 

Excretion 

(basal)

Diet 

ka

kd

kin

kout

Utilized in the 

Tissue

Key Model Structure: Saturable Tissue Storage 

and Asymmetric Diffusion

Mnfree

Mnfree + Bf Mnbound

ka

kd

kin kout

Cart Cven

Mntotal = Mnfree + Mnbound

Bmax = Bf + Mnbound

Above tissue capacity -> overwhelmed normal homeostasis -> 

increase in free Mn, which leads to induction of biliary excretion 

Excess Mn

Mnf

High dose Exposure

(e.g., inhalation) 
5

Concerns for Potentially Increased Exposure 

during Development

• Maternal to fetal and neonatal transfer of Mn

• Immature homeostasis suggested from animal studies

– Very low biliary excretion compared to adults observed for 

steady state exposure

– Higher fractional uptake from the gut compared to adults 

for steady state exposure

• Higher demand in Mn for proper development

• Therefore, concerns for potentially increased delivery to the 

brain (target tissue for toxicity) under the high dose exposure 

have raised -- PBPK models can be used to describe early life 

Mn homeostasis to address these questions
6

Summary of Rat modeling

Describing Mn Homeostasis 

during Gestation and Lactation
Extending the Adult Model to 

Gestation Period

Dam

Mnf + B MnB

+

Diet

Biliary 

Excretion

Placenta

Inhalation

Developing Fetus

Mnf + B MnB

A PBPK model structure that 

successfully described Mn

kinetics in the dam and 

fetuses during gestation 

• Mn homeostasis during gestation at steady state

• Mn transfer to the fetus is efficient 

• active transporters for essential metals 

present in the placenta 

• Changes tissue Mn requirement based on 

developmental stage specific needs 

• Higher uptake to the fetal brain - higher 

expression of transporters in fetal brain; Mn

requirement is high in developing brain

• Bone acts as pre-natal storage site for Mn
GD20 Placenta GD20 Fetal Brain

Describing Mn Homeostasis during Fetal 

Development

• Transfer of adequate amount of this ‘essential element’ to the fetus to 

ensure proper development, while protecting fetus from excess Mn

challenge 

• Different fetal transfer characteristics between normal vs. excessexposure in the mother
Uptake and Excretion of Mn in Neonates

(Ballatori et al., 1987)

Inducible biliary 

excretion in case of 

excess Mn

administration –

indicating neonatal 

homeostasis at stedy

state is different from 

adults

(Keen et al., 1986)

Indicating much higher 

fractional uptake and 

lower excretion of Mn

compared to adults

Very low excretion rate 

until PND16-18 and a 

rapid increase 

afterwards 

Excretion Retention
Extending the Adult Model to Neonates

Dam

Mnf + B MnB

+

Diet

Biliary 

Excretion

Milk

Inhalation

Mammary Gland

Tested two different 

descriptions on Mn

kinetics in the suckling 

pups 

Growing Pups

Mnf + B MnB No excretion

Biliary 

Excretion

Inhalation

Initially, the model was 

tested with parameters 

based on immature 

homeostasis in 

neonates High absorption

Higher influx

Transfer from 

the mother 

through milk
Simulation of Milk and Neonatal Mn

We were able to simulate milk Mn over a range of exposure 

concentrations, but not the tissue Mn in neonates with the 

initial structure (too high for inhalation) – better interpretation 

of the observation required
To Properly Simulate Neonatal Homeostasis

Age-appropriate physiological processes that 

control Mn homeostasis (at steady-state) were 

incorporated in the model

• Enhanced Mn uptake in gut in neonates

• Different tissue binding characteristics 

compared to the adults due to increased 

Mn needs in brain during fetal and 

postnatal development 

• Low, but inducible biliary excretion in 

neonates
Altered Homeostasis in Neonates only 

Apparent during Lactation at Steady State

• Higher uptake in the gut

• to maintain body Mn, fractional absorption from milk is 

higher than adults due to lower Mn in milk compared to 

solid food

• due to slower turnover of enterocytes that prolongs 

retention of Mn in the gut (enterocytes)

• Although the basal level is very low, when challenged with 

excess Mn, biliary excretion is increased resulting in similar 

homeostatic response to maintain tissue Mn in neonates like 

in adults 
Extending the Adult Model to Neonates

Dam

Mnf + B MnB

+

Diet

Biliary 

Excretion

Milk

Inhalation

Mammary Gland

Incorporating the 

appropriate descriptions 

on neonatal homeostasis 

both at steady state and 

excess Mn

Growing Pups

Mnf + B MnB +

Biliary 

Excretion

Inhalation loss from gut

Depicting and describing 

the features of early life 

Mn homeostasis
Illustration of Neonatal Mn Homeostasis

• Rapid maturation of biliary excretion 

and gut uptake around initiation of 

solid food consumption and/or 

weaning 
All the Information Integrated in the Model...

Simulation of Tissue Mn in Growing Animals

Time Dependent Changes

Inhalation at Mn 0.5 mg/m3
The Key to Describe Perinatal Mn Kinetics

• Maternal to offspring transfer – Placental and 

lactational

• Fetal homeostasis at the level of placental transfer

• Tissue specific Mn demand during fetal and 

postnatal development factored in the model

• Neonatal homeostasis during lactation is 

dependent Mn exposure levels to maintain stable 

body Mn 
Summary of the Rat Modeling

• Diverse information on Mn and other essential 

elements kinetics during gestation and lactation 

were successfully integrated into the PBPK model 

• The model can provide insights into the Mn

tissue levels in developing animals for dietary 

(steady state) and dietary plus inhalation (high 

dose) exposures
Describing Mn Kinetics in 

Humans during Gestation 

and Lactation
Adults

Developmental

Species differences 

in developmental 

biology related 

with Mn

Species specific 

physiology of 

pregnancy and 

lactation

Developing Human Gestation and Lactation Models 

A Parallelogram Approach

Examining 

Critical 

determinants of 

Mn kinetics in the 

body

Features of human model based on successful rat description, human tissue Mn

observations, and the species differences in key processes
Features of Human Developmental Model

• Basic model structure is the same as the rat developmental 

model 

– Rat models were developed based on the studies with inhaled Mn

exposure for a defined diet

• Extrapolation processes for adults to fetuses/neonates 

were based on

• Rat developmental modeling

• Comparative physiology of developmental processes 

between the rats and humans

• Information on Mn concentration in humans including milk, 

placenta, blood and tissues from nutritional studies and 

cadaver data

21
Human Model Structure: Gestation
Human Model Structure: Lactation
Model Structure and Parameterization I

• Describing dynamics of gestation and lactation physiology 

– Modified/updated from Clewell et al., 1999; Corley et al., 2003; Gentry 

et al., 2002 and 2003; ICRP, 2003; Kiserud et al., 2006 

– Especially for brain growth during development

• Female models

– Higher gut absorption and enhanced biliary excretion in childbearing age women, while no significant gender difference 

observed in tissue Mn

– In association with their reduced iron status compared to the 

same age men based on potential co-absorption with iron 

through DMT-1, but subsequent independent control of body Mn

(based on Finley et al., 1994; Aschner and Aschner, 2005; Finley, 

1999)
Model Structure and Parameterization II

• Modeling inhalation exposure during pregnancy and lactation

– Changes in lung structure and ventilation characteristics 

incorporated to predict particle deposition patterns in 

different ages and life stages from inhalation exposure to 

Mn

– Mn particle deposition during gestation and lactation 

simulated with the MPPD model using female specific 

respiratory parameters and pregnancy-related changes 

(ICRP, 2003) 

– Ventilation changes during pregnancy and lactation in the 

mother and infants/children (Child’s Specific Exposure 

Factors Handbook, 2002)
Early age (age in year) Adult

0.25 yr 1.92 yr 3 yr Male Female Pregnant Male

Alveolar Ventilation

QP (L/hr) 125 189 231 342 285 490

QPC (L/hr/kg BW0.75) 34 27 30 14 13 20

Respiratory parameters

Breathing Frequency

(/min) 39 27 24 12 14 14

Tidal volume (ml) 30 87 121 625 460 584

Deposition Fractions (predicted from MPPD model)

Total deposition 0.61 0.58 0.59 0.63 0.58 0.65

Lung 0.38 0.32 0.32 0.27 0.23 0.26

Nasal

Cavity

Respiratory 0.22 0.25 0.26 0.34 0.33 0.37

Olfactory 0.012 0.013 0.014 0.018 0.018 0.020

Age-dependent Changes in Respiratory Parameters and 

Fractional Deposition of Mn Particles
Model Structure and Parameterization III

Placental Transfer of Mn

• The same structure as in the rat 

• Active transport was inferred by the existence of 

several essential metal transporters in the placenta 

(Leazer and Klaassen, 2003; McArdle et al., 2008)

• Parameters were estimated based on the human data

• Placental and fetal tissue Mn concentrations in late 

gestation and/or at delivery in humans (Casey and 

Robinson, 1978; Osman et al., 2000; Takser et al., 

2004)

• Featuring the placental sequestration of Mn

• The placenta-to-blood ratio of Mn was high in vivo 

and Mn trapping was observed in the placenta exvivo(Takser et al., 2004; Miller et al., 1987)

• Role of placenta as a barrier for excess Mn transfer to 

fetus (saturable nature of active transport)

 

Placenta

 

Developing Fetus

Vmax

Km ktrans2

ktrans1
Model Structure and Parameterization IV

Efficient Transfer of Mn for Fetal Development

Supporting evidence for 

• the efficient transfer of Mn to the fetus in the model - Fetal 

tissue Mn similar to adult levels (Casey and Robinson, 1978; 

Casey et al., 1982; Lehmann et al., 1971; Reiman and 

Minot, 1920; Bonilla et al., 1982; Tingey, 1937)

• developmental period specific requirement – higher 

demand in the brain and bone. Bone as a prenatal storage 

site for Mn due to higher demand for Mn for proper 

skeletal growth in utero (Hurley, 1981); the released Mn

from the prenatal pool re-distributes to other tissues 

during early neonatal period
Model Structure and Parameterization V

Parameterization for Developing Brain

• Prenatal maturation of human brain based on the observed fetal 

and neonatal brain Mn being comparable to the adult and 

comparative neurodevelopmental periods between the rat and 

human (Casey and Robinson, 1978; Casey et al., 1982; Bonilla et 

al., 1982; Tingey, 1937; Clancy et al., 2007; U.S. EPA, 2001)

• Enhanced transport of required materials during brain 

development such as Mn and other essential elements based on 

the higher expression of metal transporters in the brain (Erikson 

et al., 2007;Siddappa et al., 2002)

• Brain diffusion parameters (kin/kout) were adjusted to ensure 

sufficient Mn influx into developing brain along with increased 

incorporation (ka) into the cellular component without negating 

the presence of an immature BBB
Fetal Mn (μg/g)a Adult Mn (μg/g)c

Liver 0.94 ± 0.35 1.20 ± 0.35

Kidney 0.45 ± 0.10 0.56 ± 0.22

Brain 0.16 ± 0.02 (whole brain)

0.36 ± 0.11 (cerebellum)

0.19 – 0.53 (different regions) b

0.32 (whole brain) d

Heart 0.27 ± 0.07 0.21 ± 0.08

Lung 0.22 ± 0.08 0.22 ± 0.09

Muscle 0.13 ± 0.03 0.09 ± 0.05

Bone 0.88 ± 0.21 0.07 ± 0.06

a Casey et al., 1978: 40 fetuses of 22-43 weeks of gestation, New Zealand

b Tingey, 1937: newborn to 2 years old, in adult brain, the highest Mn in striatum

c Sumino et al, 1975: Japanease cadavers

d Reiman & Minot, 1920

Tissue Mn Concentrations in Humans 
Homeostatic Control in Response to Low versus 

Excess Mn in Early Ages

• Both age- and dose-dependent biliary excretion (low 

basal, but inducible at high-dose exposure)

0 ( )

5 0

max

n

art

n

b

n

b art

neonate adult app k C

k C

kbile kbile RE f

neonate





   

• Neonatal tissue Mn comparable to adults while human milk Mn level is relatively 

low compared to other species 

• Higher uptake of milk Mn in the gut - Mn bioavailability determined by types of 

transporters in the gut that recognize different Mn forms in infant milk/diet 

(adjusting fractional uptake in the gut) – important only for describing higher oral 

exposure such as the fortified formula due to very low contribution from milk Mn to 

body Mn levels

maturation pattern of 

biliary function

apparent biliary excretion as a fraction 

of kbile0

fappneonate = 0.001, while fappadult = 1

Mn dose (body 

burden)-dependent 

biliary induction

basal biliary excretion rate with 

normal Mn diet in adults
32

Inhalation Exposure Scenarios

• Continuous inhalation of ambient air Mn (as MnSO4) at three 

different concentrations

• 0 mg/m3 – normal background exposure from diet or 

milk

• 0.001 mg/m3- high ambient air concentration (20-fold 

higher than the current EPA RfC for Mn)

• 0.01 mg/m3 – close to the lower end of the 

occupational exposure range

• Environmental exposure was simulated as a continuous 

inhalation to Mn throughout preconception, pregnancy, and 

lactation to the mother

• For the offspring, the exposure by inhalation on their own 

started at birth and continued during lactation and postnatal 

growth after weaning
Simulation of Placental and Fetal Tissue Mn

Note: Whole brain mn reported in Casey et al., 1978, while 

model simulation indicates fetal globus pallidus concentration.

Dietary Exposure Only (Basal Steady-state)

Model simulation for the end of gestation (270days)
Inhalation of Mn during whole gestation and model simulation for the end of gestation 

Note: Whole brain Mn reported in Casey et al., 1978, while 

model simulation indicates fetal globus pallidus concentration.

Inhalation + Dietary Exposure

Transfer from the Mother to the Fetus

Steady state vs. Environmental Exposure
Simulated Milk Mn Concentrations

• Exclusive breast-feeding for 6 months was assumed.

• Inhalation during lactation both to the mother and the infant

Diet only Diet + Inhalation
Neonatal Tissue Mn with or without 

Inhalation

• Inhalation was simulated starting before pregnancy and continued through gestation, 

lactation, and postnatal period. 

• After 6 months of breast-feeding, dietary Mn intake in children was assumed to at 2 mg 

Mn/day based on published tolerable upper limit of recommended daily Mn dietary intakes 

for children between 1 and 3 years of age (The National Academies, 2001). 

Liver Mn Brain Mn
Predicting Perinatal Brain Mn with Inhalation 

Exposure to Mn

Role of placenta to ensure 

adequate Mn supply for normal 

development, while preventing 

over -exposure

Fetus Infants and children

Homeostatic control to retain 

adequate Mn in response to low 

Mn in human milk

(whole brain)
Model Evaluation with Human Blood Data

At Birth without Inhalation

Umbilical cord or newborn 

blood (μg Mn/g blood)

Model predicted 0.038

Takser et al., 2004 0.034 ± 0.0134

Zota et al., 2009 0.042 ± 0.016

Su et al., 2007 0.044 ± 0.0106

Spencer, 1999 0.041 ± 0.012
Model Evaluation with Human Blood Data

During Postnatal Growth without Inhalation
Model Evaluation with Human Blood Data

Breast Milk vs. Infant Formula 

0.0014 mg/day 0.05 mg/day 0.0023 mg/day 1.15 mg/day

Estimated Daily Mn intake

35 day old infants 3 month old infants

Bioavailability for gut uptake is different between 

breast-milk and Mn fortified formula
Estimation of Mn Daily Doses from Various 

Sources in Different Life Stages

• Daily systemically available dose to the adult, infant (6months), and child (3 years) were 

compared among milk, dietary, and inhaled doses on the selected day.

• Inhalation at 0.01 mg/m3 of Mn was simulated.
Comparison of Internal Dose Metrics in 

Brain in Different Life Stages

• For pregnant mother and fetus, average daily AUCs were calculated during whole gestation.

• For lactation mother and nursing infant, values were from averaging AUCs during breast-feeding 

period (6 months).

• For the adults and a child (3 years), the AUCs were averaged during the 3 years period after weaning.
Mn Dose-Response at the Target 

Comparison across Life Stages
Mn PBPK Model for Human Fetus and Neonate 

Successfully Developed 

• Based upon the rat developmental model in conjunction 

with human adult model (parallelogram)

• By integrating the knowledge on fetal and neonatal Mn

kinetics

- Mn kinetics in the developing organism is determined 

by the requirements for this element during normal 

growth

- Homeostatic controls of Mn kinetics at normal steady 

state are different from those in adult due to different 

sources of Mn and requirements across ages

- Infants and children are able to respond overexposure 

to Mn in a similar way as the adult
Summary

• The current model provides estimates for fetal 

and neonatal Mn target tissue dose metrics 

under various inhalation exposure conditions 

together with dietary exposure (including breastmilk exposure) 

• In general, similar or lower internal exposures to 

Mn in fetal and neonatal brain are predicted 

compared with the adult for the various 

inhalation exposure scenarios examined, which 

represent environmentally relevant exposure 

conditions
Sensitivity/Uncertainty Analysis
Sensitivity/Uncertainty Analysis
Human Developmental PBPK Models for Mn to 

Support Risk Assessment

• Whether children should be regarded as a 

susceptible population for effects of Mn

• Early Life PBPK models for Mn can be used to:

• Characterize Mn transfer through placenta 

and milk

• Evaluate life-stage differences in Mn

pharmacokinetics

• Compare exposures from inhalation, breast 

milk, and formula
Acknowledgements

Andy Nong

Jeffery Schroeter

Mel Andersen

Harvey Clewell

Dave Dorman

Mike Taylor

Jeff Fisher

Michelle Medinsky

Dan Krewski

Funding 

Afton Chemical Company