Supplementary Data

Basic model structure common for all the life stages during gestation and lactation

To simulate life stage specific processes, additional compartment(s) were added to the six 

existing compartments of lung, nose, brain, bone, liver, and a lumped body compartment (rest of body) in 

the adult male model (Figure 2). The mammary gland was added for the adult female and lactating female

and was described using tissue binding parameters same as the rest of body compartment. In the pregnant 

female, the placenta was additionally modeled to simulate the gestation. The placenta doesn’t have Mn 

binding capacity. In the fetus, the lung, brain, bone, liver, and rest of body compartment were modeled 

(Figure 2). The neonate and child were modeled using the same tissue compartment structure as the adult 

male (Figure 2). 

Three representative brain regions along with the brain blood were explicitly described to 

simulate Mn kinetics in the brains of the pregnant and lactating mother, fetus, neonate, and child as in the 

adult male. The globus pallidus, cerebellum, and olfactory bulb were included based on the criteria of 

being a target for potential Mn neurotoxicity, a representative of general brain tissue, or a region with 

expected contributions from direct transport from olfactory epithelium, respectively. The pituitary gland 

was not included in the present model due to limited information on tissue Mn kinetics in this brain 

region in early life in humans. 

Mn exposure for the mother and neonate/child was either through diet only or simultaneously 

through diet and inhalation. Only a small fraction of dietary Mn is taken into the liver (FdietUp). The 

dietary intake for the suckling neonate was through breast-milk. The FdietUp was appropriately varied 

from the adult male model to account for the known gender difference in gut absorption. The impacts of 

early age and/or the molecular forms of Mn in infant food sources on the gut absorption of Mn were also 

modeled by changing neonatal FDietUP for each type of milk, either breast-milk or infant formulas,

accordingly. 
In the current model, a certain portion of unabsorbed Mn (Fent) is transiently stored in gut 

epithelial cells. They are not absorbed to the systemic circulation due to the rapid turnover rate of gut 

epithelium in adults, while they do in neonates as a result of slower turnover of the enterocytes in their gut

compared to the adults. This delayed absorption in neonates was dependent on an absorption rate constant 

delGI in the current model. The sloughing rate of gut epithelial cells is reflected by a rate constant of kent,

which describes the slow loss of Mn from the gut epithelium to feces (Figure 2). 

For simulating inhalation exposure, the absorption of deposited Mn in the lung and nasal cavity to 

the systemic circulation was rapid both in the mother and neonate/child as the adult male model. A direct 

transport from olfactory epithelium to the olfactory bulb region in the brain was included for all the life 

stages except the fetus. 

After entering the systemic circulation, Mn is then subsequently cleared into the tissues. This 

clearance was described as an asymmetric bi-directional diffusion for the brain. The free Mn in systemic 

blood rapidly equilibrates with free Mn in the brain tissue blood. The flux between brain tissue-blood and 

brain tissue are diffusion limited, the rate of which are determined by kin and kout, respectively (Figure 2; 

Table 2). These values are region specific and are responsible for the preferential increase in free Mn in 

the mid-brain region seen in excess Mn exposures. The diffusion limitation in the brain was considered 

reflecting the role of blood-brain barrier in controlling Mn flux. The feature of asymmetrical diffusion

was brain region specific, which was rationalized by region-specific expression of transporter proteins for 

essential element in the brain and the underlying mechanisms for the reported preferential influx of Mn 

into the mid-brain in over-exposure conditions. These brain diffusion rate constants were varied during 

perinatal period to reflect maturational changes both in the blood-brain barrier integrity and in the types/ 

levels of Mn transporter expression that are likely in parallel with the needs for this element during the 

active neurodevelopment. For other tissues, a rapid and symmetrical flux of Mn between the systemic 

blood and tissue compartment was used and thus the tissue Mn is determined by the tissue binding 

characteristics.
Once distributed into the tissue, Mn is bound to the cellular components that require Mn for a 

variety of biological functions and thereby it is not readily crossing cell membranes. This incorporation of 

Mn into cellular proteins or tissue constituents and subsequent degradation from those ‘binding sites’ are 

governed by tissue specific association and dissociation rate constants ka and kd, respectively. Total Mn 

in any tissue is therefore, the sum of free and bound forms within the tissue, the ratio between the two 

governed by the dissociation equilibrium constant (kd/ka) as well as the maximum binding capacity 

(Bmax). Once the binding sites are saturated, free Mn will rise swiftly, consequently a rapid increase in 

total tissue Mn occurs. The Bmax and binding rate constants are likely determined not only by the degree 

of needs for Mn in a specific tissue, but also by the nature of those needs, i.e., biological functions 

requiring Mn in that tissue. In this reagard, tissue binding constants were subject to vary during per-and 

postnatal growth in the currenet model. Free Mn is the form that circulates in blood and is available for 

excretion into bile and for placental or lactational transfer. The bound form stays in the tissue and slowly 

dissociates from the binding sites with a rate constant kd, which probably corresponds to biological

degradation rates of cell materials such as Mn-containing enzymes. At all times, the fraction bound in a 

specific tissue was kept over 70% to be consistent with our assumption on the nature of Mn tissue binding. 

We assumed that most of Mn in the tissue is associated with cellular constituents. The fraction bound can 

be changed depending on the Mn content in diet. With a sufficient Mn diet, the fraction of Mn bound in a 

tissue would be lower, while with a marginal Mn diet, it should become higher. It should be noted that the 

fraction bound does not represent the actual occupancy of binding sites, though. 

The description for the free Mn dependent influx control in the globus pallidus region used in the 

adult male model was kept similar when simulating all the life stages in the current model. The kinMax,

the maximal fold of induction, was slightly reduced in the fetus and neonate. The kin50 values were 

retained the same as the adult male due to limited data on this additional layer of brain Mn control, 

especially during gestation and lactation (Table 2). 

Simulating placental transfer and the fetus
All the characteristics of the placental transfer of Mn were retained the same as described in the 

rat gestation model (Yoon et al., 2009a). Transfer of Mn from placenta to fetal venous blood was 

mediated by two pathways, one is an active process described by the maximum rate of transport VmaxC

(μg/hr/kg BWF

0.75) and the affinity constant Km(μg/L); and the other is a diffusion process governed by a 

diffusional clearance rate constant ktrans1C (L/hr/kg BWF

0.75). A minor transfer of Mn from fetal arterial 

blood to placenta also occurs, which is driven by diffusion governed by another clearance rate constant

ktrans2C (L/hr/kg BWF

0.75). Both the active and diffusion processes were assumed to be correlated with 

fetal growth and were scaled to fetal BW0.75. The placenta to maternal blood partition coefficient, PPla, 

reflects the high placenta to blood ratio and the experimentally observed Mn trapping in the placenta. The 

fetus acquires Mn solely from the placenta. The excretion of Mn from the fetus is to the placenta only 

(Figure 2).

The movements of Mn between maternal and fetal circulations were modeled assuming majority 

of transfer being maternal to fetal direction through active transport(s) following the same approach 

applied in the rat model (Yoon, et al., 2009a). Parameters for this placental transfer of Mn were then 

estimated based on the observed 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). The 

estimation criteria was to allow the active process to work near at its maximum capacity at normal steady 

state for the pregnant mother on adequate Mn intake as we did for the rat model (Table 2). To achieve this, 

model parameters for maternal to fetal flux, both for active transport, i.e., VmaxC and Km, and for 

diffusion, i.e., ktrans1C were varied until the active transfer becomes more than 95% of the flux to the 

fetal side with 5% or less contribution from simple diffusion. The active process was assumed to work 

almost at its saturation, at about 75% of its maximum, by adjusting the affinity constant Km to an 

appropriate value (Table 2). The diffusion clearance parameter from the fetal arterial blood to the 

maternal placenta, ktrans2C, was set the same as ktrans1C. With these feto-placental parameters, we 

were able to successfully recapitulate the reported Mn concentrations in the placenta and in several fetal 

tissues at late or at the end of gestation without inhalation exposure in the mother in humans (Figure 3).
The placenta to blood partition coefficient for Mn, PPla, was estimated to reproduce the 

concentration ratio between the placenta and maternal blood seen at the end of pregnancy and the 

resulting value was 10 (Takser, et al., 2004)(Table 2). A kinetic assessment of Mn using the dually 

perfused human placenta in vitro supported this high PPla. A rapid accumulation of substantial portion of 

Mn was observed within the placental tissue after Mn was added in the perfusate (Miller et al., 1987). 

Simulating lactational transfer and the suckling neonate

The free Mn in mammary gland is secreted into milk with a clearance rate constant CLmilkC

(L/hr/kg BWM

0.75; Table 2) as described in the rat lactation model (Yoon et al., 2009b). The clearance of 

Mn into milk is assumed to be proportional to maternal BW0.75. The rate of milk production (klac, L/hr) 

was assumed to be the same as the milk consumption rate by infants (Casey et al., 1985). The 

concentration of milk Mn (Cmilk, μg/L) is defined as: 

lac

milk f

milk k

CL C _ Mam C



(2)

where CLmilk indicates the lactational clearance of Mn (μg/hr) and Cf_Mam is the free Mn 

concentration in the mammary gland tissue The impacts of early age and/or the molecular forms of Mn in 

infant food sources on gut absorption of Mn were modeled by adjusting neonatal FDietUP for each type 

of milk, either breast-milk or infant formulas, accordingly. Enhanced absorption for breast milk Mn was 

incorporated using a constant FDP so that the actual fraction absorbed in neonate was FDP times FdietUp

(Table 2). 

In addition to the enhanced absorption, the description for Mn movement in gastrointestinal (GI) 

tract in suckling neonate was modeled differently from adults. In the neonate, some of the Mn transiently 

trapped in enterocytes ( Fent) is absorbed to the systemic circulation due to slower turnover of the 

enterocytes (kent) compared to the adults. This delayed absorption was described with an absorption rate 

constant delGI in the current model. Maturation of GI epithelium was assumed to occur at around 
weaning and the sloughing rate reaches the adult level. Otherwise, the suckling neonate was modeled 

following the same Mn kinetic description as adults.

REFERENCES:

Casey, C. E. and Robinson, M. F. (1978). Copper, manganese, zinc, nickel, cadmium and lead in human 

foetal tissues. British Journal of Nutrition 39, 639-646.

Osman, K., AÌŠkesson, A., Berglund, M., Bremme, K., SchuÌˆtz, A., Ask, K. and Vahter, M. (2000). 

Toxic and essential elements in placentas of swedish women. Clinical Biochemistry 33, 131-138.

Takser, L., Lafond, J., Bouchard, M., St-Amour, G. and Mergler, D. (2004). Manganese levels during 

pregnancy and at birth: Relation to environmental factors and smoking in a southwest quebec population. 

Environmental Research 95, 119-125.

Yoon, M., Nong, A., Clewell, H. J., Taylor, M. D., Dorman, D. C. and Andersen, M. E. (2009a). 

Evaluating placental transfer and tissue concentrations of manganese in the pregnant rat and fetuses after 

inhalation exposures with a pbpk model. Toxicological Sciences 112, 44-58.

Yoon, M., Nong, A., Clewell, H. J., Taylor, M. D., Dorman, D. C. and Andersen, M. E. (2009b). 

Lactational transfer of manganese in rats: Predicting manganese tissue concentration in the dam and pups 

from inhalation exposure with a pharmacokinetic model. Toxicological Sciences 112, 23-43.