Manganese Tissue Dosimetry in Rats and Monkeys - Oxford Journals

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

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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.

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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

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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.

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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

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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


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

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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.


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

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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.

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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


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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,

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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. REFERENCES Andersen, M. E., Gearhart, J. M., and Clewell, H. J., 3rd. (1999). Pharmacokinetic data needs to support risk assessments for inhaled and ingested manganese. Neurotoxicology 20, 161–171. Anjilvel, S., and Asgharian, B. (1995). A multiple-path model of particle deposition in the rat lung. Fundam. Appl. Toxicol. 28, 41–50. Aschner, M. (2000). Manganese in health and disease: From transport to neurotoxicity. In Handbook of Neurotoxicology (E. Massaro, Ed.), pp. 195–209. Humana Press, Totowa, NJ. Aschner, M., and Aschner, J. L. (1991). Manganese neurotoxicity: Cellular effects and blood-brain barrier transport. Neurosci. Biobehav. Rev. 15, 333–340. Aschner, M., Erikson, K. M., and Dorman, D. C. (2005). Manganese dosimetry: Species differences and implications for neurotoxicity. Crit. Rev. Toxicol. 35, 1–32.


33

Aschner, M., and Gannon, M. (1994). Manganese (Mn) transport across the rat blood-brain barrier: Saturable and transferrin-dependent transport mechanisms. Brain Res. Bull. 33, 345–349.

Mahoney, J. P., and Small, W. J. (1968). Studies on manganese. 3. The biological half-life of radiomanganese in man and factors which affect this half-life. J. Clin. Invest. 47, 643–653.

Aschner, M., Gannon, M., and Kimelberg, H. K. (1992). Manganese uptake and efflux in cultured rat astrocytes. J. Neurochem. 58, 730–735.

Malecki, E. A., Radzanowski, G. M., Radzanowski, T. J., Gallaher, D. D., and Greger, J. L. (1996). Biliary manganese excretion in conscious rats is affected by acute and chronic manganese intake but not by dietary fat. J. Nutr. 126, 489–498.

Agency for Toxic Substances Disease Registry (ATSDR). (2000). Toxicological Profile for Manganese. U.S. Department of Health and Human Services, Public Health Service, Atlanta, GA. Brenneman, K. A., Wong, B. A., Buccellato, M. A., Costa, E. R., Gross, E. A., and Dorman, D. C. (2000). Direct olfactory transport of inhaled manganese ((54)MnCl(2)) to the rat brain: Toxicokinetic investigations in a unilateral nasal occlusion model. Toxicol. Appl. Pharmacol. 169, 238–248. Brown, R. P., Delp, M. D., Lindstedt, S. L., Rhomberg, L. R., and Beliles, R. P. (1997). Physiological parameter values for physiologically based pharmacokinetic models. Toxicol. Ind. Health 13, 407–484. Burkhard, P. R., Delavelle, J., Du Pasquier, R., and Spahr, L. (2003). Chronic parkinsonism associated with cirrhosis: A distinct subset of acquired hepatocerebral degeneration. Arch. Neurol. 60, 521–528. Conolly, R. B., Lilly, P. D., and Kimbell, J. S. (2000). Simulation modeling of the tissue disposition of formaldehyde to predict nasal DNA-protein crosslinks in Fischer 344 rats, rhesus monkeys, and humans. Environ. Health Perspect. 108(Suppl. 5), 919–924. Cotzias, G. C. (1958). Manganese in health and disease. Physiol. Rev. 38, 503–532. Davies, B., and Morris, T. (1993). Physiological parameters in laboratory animals and humans. Pharm. Res. 10, 1093–1095. Dorman, D. C., McManus, B. E., Marshall, M. W., James, R. A., and Struve, M. F. (2004). Old age and gender influence the pharmacokinetics of inhaled manganese sulfate and manganese phosphate in rats. Toxicol. Appl. Pharmacol. 197, 113–124. Dorman, D. C., Struve, M. F., Clewell, H. J., 3rd, and Andersen, M. E. (2006a). Application of pharmacokinetic data to the risk assessment of inhaled manganese. Neurotoxicology 27, 752–764. Dorman, D. C., Struve, M. F., James, R. A., Marshall, M. W., Parkinson, C. U., and Wong, B. A. (2001). Influence of particle solubility on the delivery of inhaled manganese to the rat brain: Manganese sulfate and manganese tetroxide pharmacokinetics following repeated (14-day) exposure. Toxicol. Appl. Pharmacol. 170, 79–87. Dorman, D. C., Struve, M. F., Marshall, M. W., Parkinson, C. U., James, R. A., and Wong, B. A. (2006b). Tissue manganese concentrations in young male rhesus monkeys following subchronic manganese sulfate inhalation. Toxicol. Sci. 92, 201–210. Gavin, C. E., Gunter, K. K., and Gunter, T. E. (1999). Manganese and calcium transport in mitochondria: Implications for manganese toxicity. Neurotoxicology 20, 445–453. Iinuma, Y., Kubota, M., Uchiyama, M., Yagi, M., Kanada, S., Yamazaki, S., Murata, H., Okamoto, K., Suzuki, M., and Nitta, K. (2003). Whole-blood manganese levels and brain manganese accumulation in children receiving long-term home parenteral nutrition. Pediatr. Surg. Int. 19, 268–272. Kelly, J. T., Kimbell, J. S., and Asgharian, B. (2001). Deposition of fine and coarse aerosols in a rat nasal mold. Inhal. Toxicol. 13, 577–588. Kimbell, J. S., Godo, M. N., Gross, E. A., Joyner, D. R., Richardson, R. B., and Morgan, K. T. (1997). Computer simulation of inspiratory airflow in all regions of the F344 rat nasal passages. Toxicol. Appl. Pharmacol. 145, 388–398. Leavens, T. L., Rao, D., Andersen, M. E., and Dorman, D. C. (2007). Evaluating transport of manganese from olfactory mucosa to striatum by pharmacokinetic modeling. Toxicol. Sci. 97, 265–278. Lee, J. W. (2000). Manganese intoxication. Arch. Neurol. 57, 597–599. Ljung, K., and Vahter, M. (2007). Time to re-evaluate the guideline value for manganese in drinking water? Environ. Health Perspect. 115, 1533–1538.

McKinney, A. M., Filice, R. W., Teksam, M., Casey, S., Truwit, C., Clark, H. B., Woon, C., and Liu, H. Y. (2004). Diffusion abnormalities of the globi pallidi in manganese neurotoxicity. Neuroradiology 46, 291–295. Menache, M. G., Hanna, L. M., Gross, E. A., Lou, S. R., Zinreich, S. J., Leopold, D. A., Jarabek, A. M., and Miller, F. J. (1997). Upper respiratory tract surface areas and volumes of laboratory animals and humans: Considerations for dosimetry models. J. Toxicol. Environ. Health 50, 475–506. Miller, L. V., Krebs, N. F., and Hambidge, K. M. (2000). Development of a compartmental model of human zinc metabolism: Identifiability and multiple studies analyses. Am. J. Physiol. Regul. Integr. Comp. Physiol. 279, 1671–1684. Murphy, V. A., Wadhwani, K. C., Smith, Q. R., and Rapoport, S. I. (1991). Saturable transport of manganese(II) across the rat blood-brain barrier. J. Neurochem. 57, 948–954. National Institute for Public Health and the Environment (RIVM). (2002). Multiple Path Particle Dosimetry Model (MPPD v. 1.0): A model for human and rat airway particle dosimetry. Bilthoven, Netherlands. RIVA Report 650010030. Nies, D. H. (2007). Biochemistry. How cells control zinc homeostasis. Science 317, 1695–1696. Nong, A., Teeguarden, J. G., Clewell, H. J., 3rd., Dorman, D. C., and Andersen, M. E. (2008). Pharmacokinetic modeling of manganese in the rat IV: Assessing factors that contribute to brain accumulation during inhalation exposure. J. Toxicol. Environ. Health A 71, 413–426. Pal, P. K., Samii, A., and Calne, D. B. (1999). Manganese neurotoxicity: A review of clinical features, imaging and pathology. Neurotoxicology 20, 227–238. Roth, J. A., and Garrick, M. D. (2003). Iron interactions and other biological reactions mediating the physiological and toxic actions of manganese. Biochem. Pharmacol. 66, 1–13. Santamaria, A. B., Cushing, C. A., Antonini, J. M., Finley, B. L., and Mowat, F. S. (2007). State-of-the-science review: Does manganese exposure during welding pose a neurological risk? J. Toxicol. Environ. Health B 10, 417–465. Schroeter, J. D., Kimbell, J. S., Gross, E. A., Willson, G. A., Dorman, D. C., Tan, Y. M., and Clewell, H. J., 3rd. (2008). Application of physiological computational fluid dynamics models to predict interspecies nasal dosimetry of inhaled acrolein. Inhal. Toxicol. 20, 227–243. Spahr, L., Butterworth, R. F., Fontaine, S., Bui, L., Therrien, G., Milette, P. C., Lebrun, L. H., Zayed, J., Leblanc, A., and Pomier-Layrargues, G. (1996). Increased blood manganese in cirrhotic patients: Relationship to pallidal magnetic resonance signal hyperintensity and neurological symptoms. Hepatology 24, 1116–1120. Takeda, A. (2003). Manganese action in brain function. Brain Res. Brain Res. Rev. 41, 79–87. Tapin, D., Kennedy, G., Lambert, J., and Zayed, J. (2006). Bioaccumulation and locomotor effects of manganese sulfate in Sprague-Dawley rats following subchronic (90 days) inhalation exposure. Toxicol. Appl. Pharmacol. 211, 166–174. Teeguarden, J. G., Dorman, D. C., Covington, T. R., Clewell, H. J., 3rd., and Andersen, M. E. (2007a). Pharmacokinetic modeling of manganese. I. Dose dependencies of uptake and elimination. J. Toxicol. Environ. Health A 70, 1493–1504.

34

NONG ET AL.

Teeguarden, J. G., Dorman, D. C., Nong, A., Covington, T. R., Clewell, H. J., 3rd, and Andersen, M. E. (2007b). Pharmacokinetic modeling of manganese. II. Hepatic processing after ingestion and inhalation. J. Toxicol. Environ. Health A 70, 1505–1514. Teeguarden, J. G., Gearhart, J., Clewell, H. J., 3rd, Covington, T. R., Nong, A., and Andersen, M. E. (2007c). Pharmacokinetic modeling of manganese. III. Physiological approaches accounting for background and tracer kinetics. J. Toxicol. Environ. Health A 70, 1515–1526. U.S. EPA. (1994). Integrated risk information system, manganese (CASRN 7439-96-5). Available from: www.epa.gov/iris. Accessed March, 2008.

U.S. EPA. (2005). Guidelines for carcinogen risk assessment. National Center for Environmental Assessment, Washington, DC; EPA/630/P-03/001F. Available from: http://epa.gov/ncea. Accessed June, 2008. U.S. Geological Survey. (2008). Mineral commodity summaries. Available from: http://minerals.usgs.gov/minerals/pubs/commodity/manganese/. Accessed March, 2008. Vitarella, D., Moss, O., and Dorman, D. C. (2000). Pulmonary clearance of manganese phosphate, manganese sulfate, and manganese tetraoxide by CD rats following intratracheal instillation. Inhal. Toxicol. 12, 941–957.