ToxSci Advance Access originally published online on March 19, 2007
Toxicological Sciences 2007 97(2):265-278; doi:10.1093/toxsci/kfm061
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Evaluating Transport of Manganese from Olfactory Mucosa to Striatum by Pharmacokinetic Modeling
* CIIT Centers for Health Research, Research Triangle Park, North Carolina 27709
The Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisonsin 53706
1 To whom correspondence should be addressed. Fax: (919) 558-1300. E-mail: tleavens{at}ciit.org.
Received December 28, 2006; accepted March 8, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Increased brain manganese (Mn) following inhalation can result from direct transport via olfactory neurons and blood delivery. Human health risk assessments for Mn should consider the relative importance of these pathways. The objective of this study was to develop a pharmacokinetic model describing the olfactory transport and blood delivery of Mn in rats following acute MnCl2 or MnHPO4 inhalation. Model compartments included the olfactory mucosa (OM), olfactory bulb, olfactory tract and tubercle, and striatum. Intercompartmental transport of Mn was described as ipsilateral, anterograde movement to deeper brain regions. Each compartment contained free and bound Mn and included blood influx and efflux. First-order rate constants were used to describe transport. Model parameters were estimated by comparing the model with published experimental data in rats exposed by inhalation to 54MnCl2 or 54MnHPO4 with both nostrils patent or one nostril occluded. The model-derived elimination rate constant from the OM was higher for the chloride salt (0.022 per hour) compared with the phosphate salt (0.011 per hour), consistent with their relative solubilities. Rate constants for Mn transport among the other compartments were similar for both Mn forms. Our results indicate that direct olfactory transport provided the majority of Mn tracer in the olfactory regions during the 21 days following exposure to 54MnHPO4 and 8 days following exposure to 54MnCl2. Only a small fraction of Mn tracer from the tract and tubercle was predicted to be delivered to the striatum, 3 and 0.1% following 54MnHPO4 or 54MnCl2 exposure, respectively.
Key Words: manganese; olfactory transport; pharmacokinetic model.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Manganese (Mn) is an essential element that is a component of several enzymatic systems in the body including the metalloenzymes arginase, glutamine synthetase, phosphoenolpyruvate decarboxylase, and Mn superoxide dismutase. Mn is ubiquitous in the environment (0.1% of earth's crust), where it is found as a component of a variety of minerals including sulfides, oxides, carbonates, phosphates, silicates, and borates. Emissions of Mn into the environment can occur during mining, crushing, and smelting of ores during steel production, during manufacturing of Mn-containing products, and during use of Mn-containing products such as agricultural and garden applications of certain fertilizers and fungicides. Another source of Mn emission that recently has received increased scrutiny over concerns of potential health effects is the fuel additive methylcyclopentadienyl Mn tricarbonyl (MMT) (Davis, 1998
; Kaiser et al., 2003). Combustion of MMT in automobile engines results in the release of various Mn-containing compounds, mainly Mn sulfate, Mn phosphate, and Mn tetraoxide, into the environment. Because inhalation exposure has been shown to result in greater amounts of Mn reaching the brain than ingestion (Andersen et al., 1999), the use of MMT has become a public health concern with a priority of determining the effects from exposure to ambient Mn.
Chronic inhalation exposure to high concentrations of Mn in occupational settings has been shown to result in adverse neurological, respiratory, and reproductive effects (IPCS, 1999). The majority of cases have been documented in miners (Pal et al., 1999). The neurotoxic effects of Mn result in extrapyramidal symptoms resembling Parkinson disease, such as dystonia and severe gait disturbances. Although Mn is known to accumulate in the basal ganglia in humans, the mechanism of Mn neurotoxicity as well as the potential for Mn to cause neurotoxicity at lower, environmentally relevant concentrations is not known (Davis, 1999). Concern over potential neurotoxic effects from inhalation of Mn from ambient air has lead to a need to quantitate the deposition of Mn in the brain and ascertain pharmacokinetic mechanisms involved in brain delivery.
A number of studies have investigated the uptake of Mn in animals from inhalation exposure to a variety of Mn compounds including less soluble forms such as Mn phosphate and Mn oxide and more soluble forms such as Mn chloride and Mn sulfate. In experimental animals following exposure to various forms of Mn, there were increases in Mn brain concentrations, which were form dependent, dose dependent, and region specific (Dorman et al. 2001a,b; Fechter et al., 2002; Normandin et al., 2002, 2004; Salehi et al., 2003; St-Pierre et al., 2001; Tapin et al., 2006; Vitarella et al., 2000). Oftentimes, the brain region with the largest increase in concentration following Mn exposure was the olfactory bulb (OB). Both transport of Mn from the blood following Mn absorption in the lungs as well as direct olfactory transport can contribute to this accumulation (Dorman et al., 2006). Tjälve et al. (1995, 1996) was the first to demonstrate that Mn could be transported via olfactory receptor neurons after intranasal administration of Mn in pike and in rats. Although the exact mechanism of transport along olfactory neurons is not known, other researchers have noted olfactory neuronal transport of Mn into the brain (Brenneman et al., 2000; Cross et al., 2004; Dorman et al., 2002; Elder et al., 2006; Henriksson et al., 1999). The acute inhalation studies by Dorman et al. (2002) with 54MnHPO4, a poorly soluble salt, and by Brenneman et al. (2000) with 54MnCl2, a more soluble salt, have attempted to quantify the relative contribution of blood delivery versus olfactory transport to various brain structures following inhalation exposures to Mn.
Although Dorman et al. (2002) and Brenneman et al. (2000) have demonstrated the relative differences qualitatively in various olfactory regions and the striatum from blood delivery versus olfactory transport of Mn, a quantitative estimate is needed that can be used in risk assessments to address Mn delivery to susceptible brain regions. A pharmacokinetic model describing the flux of Mn via the various pathways could be used to estimate the contributions via blood versus olfactory delivery, and such models eventually could be linked to a physiologically based pharmacokinetic model to describe whole-body distribution of Mn from oral and inhalation exposures. The objective of this article was to develop a pharmacokinetic model in the rat to describe the olfactory transport of Mn while accounting for blood delivery following acute inhalation exposures to Mn. The parameterized model was used to estimate the relative contribution of blood delivery versus olfactory transport in the rat.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The transport model was built to evaluate published data sets for inhalation exposure of rats to Mn (Brenneman et al., 2000
; Dorman et al., 2002). The investigators measured time courses of 54Mn in olfactory compartments on the left and right sides of the olfactory pathway including the olfactory mucosa (OM), OB, and olfactory tract and tubercle (OTT). The concentration of 54Mn in the left and right striatum, which is part of the basal ganglia and includes the caudate nucleus and putamen, was also measured to provide information on Mn accumulation in more distal brain regions following inhalation exposure. The model simulated distribution of Mn via the olfactory neurons and the blood into each of the compartments.
Experimental Data
Brenneman et al. (2000) exposed CD rats by nose-only exposure to 54MnCl2 (0.54 mg Mn/m3; mass median aerodynamic diameter (MMAD) 2.51 µm; specific activity 2.5 nCi/ng) for 90 min, and Dorman et al. (2002) exposed CD rats by nose-only exposure to 54MnHPO4 (0.39 mg Mn/m3; MMAD 1.68 µm; specific activity 0.51 nCi/ng) for 90 min. In each experiment, one group was exposed with both nostrils patent, while a second group was exposed with the right nostril occluded. Thus, the developed model represented right and left sides of the head. Following exposure, the OM, OB, OTT, and striatum along with liver, kidney, and pancreas were collected at 0, 1, 3, 5, and 8 days in both experiments; the 54MnHPO4 study also collected samples on day 21. 54Mn concentrations were measured in tissues by gamma spectrometry. For modeling comparisons, the measured 54Mn concentrations were converted with the specific activity of Mn to total Mn concentrations (ng Mn/g) derived from the 54Mn exposure in the tissues, hereafter referred to as Mn tracer. Statistically significant differences were seen in Mn tracer concentrations in the various olfactory tissues ipsilateral to the right and left OM, while the concentrations in the various regions were similar when both nostrils were patent. For 54MnCl2 experiments, the right and left striatum concentration of Mn tracer increased following the exposure in rats with both nostrils patent; however, the Mn tracer did not increase significantly in either the left or right striatum when the right nostril was plugged. For 54MnHPO4, the right and left striatum concentrations increased following both the unplugged and the plugged exposures.
Model Structure
The anatomical regions of the olfactory system and brain (OM, OB, OTT, and striatum) that were experimentally evaluated and included in the model are shown in Figure 1. The model (Figure 2) presumed that Mn would undergo axonal transport from the OM to the OB. Efferent projections from the OM are found within the superficial olfactory nerve layer of the OB. Multiple projections within the rat OB can contribute to Mn movement to the deeper granule cell layers involved in signal transduction to other sites (Shipley et al., 1995). Serial transport of Mn from the OB to the OTT and then to the striatum (a structure containing basal ganglial targets for Mn neurotoxicity) was presumed to occur within our model.
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In the model, each of the regions was represented by a homogeneous, well-mixed compartment. The olfactory transport of Mn tracer was ipsilateral from the OM to the OTT, assuming anterograde movement via primary or secondary olfactory neurons to the OTT. The Mn transported out of the OTT distributed partially to the ipsilateral striatum with the remainder distributed to the rest of brain. Previous studies have shown that anterograde movement of injected Mn occurs in the rat basal ganglia (Sloot and Gramsbergen, 1994
). Neuroimaging studies that followed the movement of Mn further confirm the anterograde movement of this metal (Murayama et al., 2006; Saleem et al., 2002). In addition, neuroimaging studies performed in rats following intranasal administration of Mn are consistent with this model structure (Cross et al., 2004).
Each side of the olfactory pathway and striatum were modeled the same except for the volume of the compartments in the model (Tables 1 and 2) and the initial concentration in the OM depending on whether the right nostril was plugged or unplugged during exposure (Figs. 4 and 5). The model had blood influx and efflux for each of the tissue compartments. Within each compartment, there existed both a free and bound pool of Mn tracer. Only the free pool of Mn tracer was available for either efflux to the blood or olfactory transport. This free pool does not necessarily represent divalent Mn but may also include other forms of Mn such as trivalent Mn, Mn citrate, Mn transferrin, or other forms of Mn, which have been shown to diffuse into the brain and other tissues (Aschner and Gannon, 1994; Crossgrove et al., 2003). The bound Mn represented the sequestration of Mn which occurs in cells (Tiffany-Castiglioni and Qian, 2001). The concentrations of Mn measured immediately following exposure to MnCl2 (Brenneman et al., 2000) or MnHPO4 (Dorman et al., 2002) were the initial compartment concentrations in the model.
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Henriksson et al. (1999)
demonstrated saturable olfactory transport or influx across blood brain barrier, but the Mn concentrations used in those instillation studies were much higher than the inhalation studies used in this work. Henriksson et al. (1999) showed linear concentration-dependent transport at the concentrations observed by Brenneman et al. (2000) and Dorman et al. (2002). Therefore, the rates of transport of Mn between tissue compartments and between bound and free pools were simply first order. The rates of influx and efflux from blood were modeled as first order to represent the active transport into the cells and the slow diffusion from the cells based on the results of Yokel et al. (2003). Because the volume of the blood for each of the olfactory compartments is not known, the influx of Mn from the blood was estimated as a clearance rate, which is a product of the first-order rate constant and blood volume. Tables 1 and 2 contain the symbols used in the model and their description.
The mass balance in the OM was described by the equation:
 | (1) |
) was a product of the first-order influx rate constant (k
) and the blood volume in the mucosa compartment (V):
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The mass balance for the OB was described by the following equation:
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The clearance from the blood to the OB (Cl
OB) was a product of the first-order influx rate constant (k
) and the blood volume in the OB (V):
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The mass balance for the OTT was described by the following equation:
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The clearance from the blood to the OTT (Cl
) was a product of the first-order influx rate constant (k) and the blood volume in the OTT (V):
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The mass balance in the striatum was described by the following equation in which only a fraction (fS) of the Mn leaving the OTT was transported to the striatum:
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The clearance from the blood to the striatum (Cl
) was a product of the first-order influx rate constant (k) and the blood volume in the striatum (V):
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The free concentration of Mn in all compartments (i = OM, OB, OTT, or S) was described as
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Model Parameters
Blood concentrations.
Blood concentrations of 54Mn were not measured in either the Brenneman et al. (2000) or Dorman et al. (2002) studies; however, 54Mn concentrations in the kidney, liver, and pancreas following exposure were reported. The mean concentration in these three organs (Fig. 3) was used to represent the blood concentration. The concentration profile of Mn tracer versus time was used to estimate pharmacokinetic parameters for first-order absorption and elimination into a single compartment.
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 | (11) |
is the product of the fraction (F) of the administered dose (X) that is absorbed divided by the blood volume (Vblood).
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, fitted to the data to estimate the blood concentration over time for the model. The values for the parameters are given in Prism (Sigma Graphpad, Inc., San Diego, CA) was used to estimate the pharmacokinetic parameters for the blood. Prism uses the Levenberg-Marquardt algorithm to minimize the sum-of-squares in the nonlinear regression. The resulting empirical equation was used to simulate the blood concentrations (Cblood) of Mn tracer following inhalation exposure.
Compartment volumes.
Organ weights of the various olfactory tissue compartments were obtained from unpublished data from the Brenneman et al. (2000) and Dorman et al. (2002) studies. The weights measured on each day for the right and left sides were averaged together for both the plugged and unplugged exposures since they were not significantly different. The mean weights for each side of the olfactory system for the 54MnCl2 and 54MnHPO4 exposures were converted to volumes in the model assuming unit density (
= 1 g/ml) and are shown in Tables 1 and 2.
Rates of elimination, blood influx and efflux, and binding.
The first-order rate constant for the bound to free (kb:f) could not be estimated from the data collected and was set to a low value (1 x 10–6/h) that was similar to the efflux rate to represent slow removal from the bound fraction. The remainder of the parameters for the model were estimated with acslXtreme software (Xcellon, AEgis Technology Group, Huntsville, AL). The parameters estimated for either 54MnCl2 or 54MnHPO4 are listed in Tables 1 and 2. The objective criterion was maximization of the log likelihood function with the Nelder-Mead optimization algorithm. The data were weighted assuming that the error in the data was proportional to the mean during the optimization of the parameters to better simulate concentrations at all time points. The parameters were estimated separately for the 54MnCl2 and 54MnHPO4 exposures. For both forms of Mn, the data from both the unplugged and plugged exposures were used simultaneously in the optimization of the parameters. AcslXtreme uses the variance-covariance matrix generated from the optimization of the parameters to calculate correlation coefficients among the parameters. The correlation coefficients generated from optimization of parameters for 54MnCl2 or 54MnHPO4 were used to assess the interrelationship among the model parameters.
The optimized parameters were used to estimate the percentage of total Mn tracer in the compartments that was attributable to either olfactory transport or blood transport from the blood. The percentage was calculated from the mass of Mn tracer entering the compartment either via direct olfactory transport or from blood divided by the total mass from both the olfactory transport and blood that entered the compartment after exposure. For example, the percentage contribution of Mn tracer in the striatum from olfactory transport was given by:
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The percentage contribution of Mn tracer in the striatum from transport from the blood was given by:
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In both equations, tf is the time following exposure. The percentage contributions were calculated at the latest sampling time point for 54MnCl2 and 54MnHPO4.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The blood concentrations of Mn tracer estimated from the mean of the liver, kidney, and pancreas concentrations differed between the 54MnCl2 and 54MnHPO4 exposures and between the plugged and unplugged exposures (Fig. 3). These differences are reflected in the pharmacokinetic parameters used to describe the systemic blood concentration in the olfactory transport model (Table 3). The unplugged exposure resulted in higher amounts available for absorption into the blood and was likely due to increased minute ventilation rates (Dorman, unpublished data). C
, the initial deposited concentration, was at least twofold higher for the unplugged exposure versus the plugged exposure for both 54MnCl2 and 54MnHPO4. In addition, ka, the absorption constant, was approximately twofold greater for 54MnCl2 than 54MnHPO4. This difference is consistent with the greater solubility of MnCl2 compared with MnHPO4. However, the elimination of Mn tracer from the systemic blood was greater for 54MnHPO4 than 54MnCl2. The simulated blood concentration compared with the experimental data from Brenneman et al. (2000) and Dorman et al. (2002) are shown in Figure 3. As noted earlier, the tissue concentrations simulated with the pharmacokinetic constants given in Table 3 were used as the surrogate for blood concentrations in the olfactory transport model.
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Figures 4–7
show the comparison of the simulations with the optimized parameter values from Tables 1 and 2 for 54MnCl2 and 54MnHPO4 exposure, respectively. For both chemical forms, the striatum was the least accurate of the fits. For 54MnCl2, the set of optimized parameters provided a better simulation of the plugged exposure than the unplugged exposure; however, for 54MnHPO4, both exposure scenarios were described comparably well.
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Consistent with the greater solubility of the chloride form, the rate of transport of Mn tracer from the OM to the OB (k
) was twofold higher for 54MnCl2 compared with 54MnHPO4 (Tables 1 and 2). Except for fraction of OTT efflux to the striatum (fS), the rates of transport of Mn tracer between the remaining compartments were similar for both 54MnCl2 and 54MnHPO4 (Tables 1 and 2). The fraction to the striatum (fS) for 54MnHPO4 was greater than for 54MnCl2, 0.03 versus 0.001, respectively.
For the transport of Mn tracer from the blood, the clearance was greater into the olfactory compartments but not the striatum following 54MnHPO4 exposure compared with 54MnCl2 (Tables 1 and 2). For the striatum, blood clearance was greater for 54MnCl2. This was due to the model predicting less olfactory transport for the 54MnCl2 exposure. As seen in Tables 4 and 5 for the correlation coefficients among the model's parameters, fSand Cl are highly correlated (– 0.99 to – 1.0). Therefore, either pathway (olfactory or blood) could be used to predict the observed increase in Mn tracer concentration in the striatum. Because striatal Mn tracer concentrations did not increase in either side in the plugged 54MnCl2-exposed rats, but did in the unplugged animals, the optimization program attributed this to the difference in the blood concentrations and therefore efflux from the blood to the striatum. However, for plugged animals exposed to 54MnHPO4, the left and right sides differed in the concentration profile with the left side increasing almost to the same concentration as in the unplugged exposures. Therefore, the increase in striatal Mn tracer concentrations was attributed to olfactory transport of 54Mn as the blood concentrations between the plugged and unplugged exposure did not differ as much following this exposure. There was little efflux from the olfactory compartments or striatum into the blood for either 54MnHPO4 or 54MnCl2.
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The percentages of Mn tracer that were transported into each compartment by olfactory transport or blood delivery following plugged or unplugged exposure to 54MnCl2 or 54MnHPO4 were calculated from the model with the optimized rate constants (Table 4). The percentage contributions differed between 54MnHPO4 and 54MnCl2 and among compartments, and for 54MnCl2, the percentage contribution differed between plugged and unplugged exposures.
For 54MnCl2, the majority of Mn tracer in the OB was contributed by direct olfactory transport from the OM for both sides of the olfactory system for both plugged and unplugged exposure. The percentage contributed by olfactory transport decreased in the OTT to approximately 70% for both sides of the olfactory system for the unplugged exposure. However, for the plugged exposure, the OTT on the occluded right side had a smaller contribution (40%) from olfactory transport from the OB. In the plugged exposure, Mn tracer in the right OB ipsilateral to the occluded nostril most likely represents Mn tracer transported from the blood to the OB, which would subsequently be transported via neurons to the OTT. Finally, in the striatum, the majority of the Mn tracer was transported from the blood for both the unplugged and the plugged exposures.
The percentage contribution following 54MnHPO4 exposure differed significantly from 54MnCl2. For the OB, olfactory transport from the OM provided approximately the same percentage Mn tracer as blood delivery for the unplugged exposure. For the plugged exposures, on the left patent side, olfactory transport provided slightly more Mn tracer, whereas on the right occluded side, blood delivery provided slightly more Mn tracer. Unlike 54MnCl2, the OTT for both the unplugged and plugged exposures received the largest percentage of Mn tracer due to direct olfactory transport on both the left and right sides. This is due to the comparable clearances from the OB to the OTT (1.4 x 10–3 ml/h) and from the blood into the OTT (1.6 x 10–3 ml/h) for the 54MnHPO4 exposures but higher concentrations of Mn tracer in the OB compared with the blood (peak concentrations of 40 ng/g compared with 7–9 ng/g, respectively). In addition, olfactory transport provided a larger percentage of Mn tracer to the striatum following 54MnHPO4, again because the predicted clearance from the OTT and the OTT concentration were greater than the blood concentration and clearance to the striatum following 54MnHPO4 exposure.
The correlations among estimated parameters for the olfactory transport model are listed in Tables 5 and 6 for 54MnCl2 and 54MnHPO4, respectively. Correlation coefficients with absolute values greater than 0.5 are highlighted. As mentioned previously for the predictions of the Mn tracer concentrations in the striatum, fSand Cl were highly correlated since either pathway could account for the accumulation of Mn tracer in the striatum. The fraction from the OTT following 54MnHPO4 could best be viewed as an upper estimate of the proportion of Mn tracer deposited in the OM, which is transported directly to the striatum via the olfactory pathway. In addition for both 54MnCl2 and 54MnHPO4, the rate constants for olfactory transport and for the rate of free to bound Mn tracer were correlated in the OM, OB, and OTT; correlation values ranged from 0.59 to 0.83. For both 54MnCl2 and 54MnHPO4, the olfactory transport from the OTT (k
) was correlated with blood clearance to the OTT (Cl). In addition, k was correlated with the clearance of blood Mn tracer to the OB (Cl) following 54MnHPO4 exposure versus following 54MnCl2 exposure where kwas positively correlated with olfactory elimination from the OM (k) and negatively correlated with the first-order rate constant for binding in the OM (k
). These differences reflect the difference in the blood versus olfactory contributions for 54MnHPO4 and 54MnCl2. There were negative correlations between the clearance of Mn tracer from blood to the OM (Cl) and OB (Cl) and between the clearance of Mn tracer from blood to the OB (Cl) and OTT (Cl). This observation is due to the blood contribution of Mn tracer available for further transport via olfactory neurons since free Mn tracer in the compartments is available for olfactory transport regardless of original source.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Investigators have noted varying Mn concentrations in regions of the rat brain from oral versus pulmonary exposure to Mn (Roels et al., 1997
). Both direct olfactory transport and blood delivery may contribute to Mn concentrations in the various olfactory regions and possibly other regions of the central nervous system following inhalation exposure (Brenneman et al., 2000; Cross et al., 2004; Dorman et al., 2002; Elder et al., 2006; Henriksson et al., 1999; Tjälve et al., 1996). Tjälve et al. (1996) first raised concerns about Mn gaining access to more remote regions of the central nervous system via direct olfactory transport. They noted that tertiary olfactory connections originating in the anterior olfactory nucleus in the anterior portion of the olfactory peduncle may result in ipsilateral transport to the motor olfactory striatum (stria terminalis and nucleus accumbens septi) and the central amygdaloid nucleus.
Mn is known to cross the blood brain barrier from the systemic circulation and accumulate in various regions of the brain. MnCl2 has been administered iv in rodents and primates to provide magnetic resonance imaging (MRI) images of the central nervous system (Aoki et al., 2004; Pautler and Koretsky, 2002). With iv administration, the OB was the region with the greatest signal enhancement, while other regions of the olfactory system and the basal forebrain showed increased enhancement but to a lesser extent than the bulb (Aoki et al., 2004).
Previous articles have demonstrated qualitatively that Mn delivery via the olfactory pathway to more distant regions in the brain such as the globus pallidus in nonhuman primates or the striatum in rats appears limited (Brenneman et al., 2000; Cross et al., 2004; Dorman et al., 2002; Elder et al., 2006; Henriksson et al., 1999; Tjälve et al., 1996). However, neither the rates of transport via the olfactory system nor the relative contributions of blood delivery versus olfactory transport have been quantified. The purpose of this research was to develop a computational model describing the olfactory transport of Mn in the rat to provide estimates of the relative contribution of olfactory delivery versus blood delivery to brain regions of interest in Mn toxicity.
Consistent with the relative solubility of MnCl2 and MnHPO4, the estimated rate of elimination of Mn tracer from the OM was twofold greater following 54MnCl2 exposure than 54MnHPO4 exposure. The rate constant in the olfactory epithelium is not only a reflection of the rate of movement of Mn along the olfactory neurons out of the epithelium but also represents the rates of dissolution of the particles into the mucosa layer, uptake of Mn into the olfactory neurons, and the movement of Mn along the primary neurons out of the epithelium into the OB. Therefore, the rate-limiting step will be the process that drives the value of the first-order rate constant for the elimination of Mn from the mucosa. At this time, the rate-limiting step is unknown but appears from our analysis to be dependent in part on Mn particle solubility.
The rate constants for transport of Mn tracer from the OB to the OTT and exiting the OTT were similar for both forms of Mn. This observation suggests that either the speciation of Mn is similar in the olfactory system following MnCl2 and MnHPO4 exposures or the rates at which various forms of Mn are transported along the olfactory neurons are similar. Both studies used a divalent form of Mn (Mn2+). Neither the speciation of Mn in the various olfactory and brain regions nor the mechanism by which Mn is transported in the olfactory system is currently known. However, our model simulations revealed some differences in binding of Mn within the compartments and in its blood transport, suggesting that the speciation of Mn may differ following exposure to the chloride or phosphate form.
Inclusion of a bound fraction in each tissue compartment was necessary to slow Mn transport during the postexposure sampling period and is consistent with known tissue kinetics of Mn. Mn is incorporated into various cellular enzymes as well as sequestered in the mitochondria and other subcellular compartments in part to protect the cell from free concentrations of metals (Keen et al., 1999). For the relatively short-time domain modeled in this study, the binding of Mn was in essence irreversible as the tissue concentrations were not sensitive to k
, the rate constant for bound to free Mn. However, as the model is expanded to simulate multiple dosing and longer time domains, more accurate estimates of k likely will be needed to predict Mn distribution in the olfactory compartments and brain.
Different binding constants (k
) for the 54MnCl2 and 54MnHPO4 exposures were necessary to predict the amount of Mn tracer lost from the tissue compartments. The experimental data used in this study (Brenneman et al., 2000; Dorman et al., 2002) showed that the total combined amount of Mn tracer in the OM, OB, OTT, and striatum at 8 days after exposure was only 19% lower than the combined amount present immediately after exposure for 54MnHPO4, whereas following 54MnCl2 it was 40% lower at 8 days after exposure. However, in the OM alone, the amounts lost following exposure to both forms were more similar at 8 days after exposure, 58 versus 68% for 54MnHPO4 and 54MnCl2, respectively. Therefore, the difference in the combined amount lost from the OM, OB, OTT, and striatum was not solely due to lower solubility of the phosphate form in the mucosa.
Another model-predicted difference in Mn tracer transport following 54MnCl2 versus 54MnHPO4 exposure was influx of Mn tracer from the blood to the olfactory compartments and the striatum. For 54MnCl2, the clearance from the blood (i.e., brain influx) was low and varied among the brain regions similar to Crossgrove et al. (2003) who measured variable influx of Mn tracer in different brain regions. For 54MnHPO4, brain influx from the blood was higher in all the regions than that seen following 54MnCl2 exposure and was higher in the olfactory regions than the striatum. This observation is consistent with imaging studies performed following iv administration of Mn in which the OB (in addition to the choroid plexus and ventricles) have the earliest and highest signal intensities (Aoki et al., 2004). One explanation for the difference in blood clearances between the two Mn forms could be different blood speciation of Mn. Crossgrove et al. (2003) showed dependence of brain region influx on the form of Mn in the blood. The speciation of Mn in the blood following inhalation has not been previously studied. Different rates of lung absorption of Mn into the circulation following MnHPO4 versus MnCl2 exposure could possibly result in different blood concentrations of the various forms of Mn and thus result in different distribution properties into and within the tissues.
The model predicted low transport efficiency via the olfactory pathway to the striatum following Mn exposure. Although this has been speculated from the previous work of Brenneman et al. (2000) and Dorman et al. (2002), the model provided a quantitative estimate of 0.1% (54MnCl2) or 3% (54MnHPO4) of Mn tracer eliminated from the OTT being transported directly to the striatum. Based on the concentrations of 54Mn in the striatum, this mass flux represented only a minor contribution to the total Mn tracer (less than 10%) following 54MnCl2 exposure but likely represented approximately 80% of total Mn tracer following 54MnHPO4 exposure. The remaining Mn tracer eliminated from the OTT would be transported to other regions of the brain or cerebral spinal fluid and eventually eliminated to the blood.
The model-predicted higher blood clearance to the striatum (Cl) following 54MnCl2 exposure, which when combined with higher blood concentrations following 54MnCl2 exposure, results in a greater mass flux of Mn tracer into the striatum via blood delivery. Striatum concentrations of Mn tracer measured at 8 days following 54MnHPO4 exposure (Dorman et al., 2002) were similar to concentrations seen 8 days following 54MnCl2 exposure (Brenneman et al., 2000) even though the estimated blood concentrations were lower following the phosphate exposure. In addition, Mn tracer concentrations in the left striatum ipsilateral to the patent nostril during the plugged exposures to 54MnHPO4 increased significantly during the 21 days after exposure. This difference was not seen in the right striatum ipsilateral to the occluded nostril following plugged exposure to 54MnHPO4 or in either side of the striatum following plugged exposure to 54MnCl2. Therefore, the model attributed the Mn tracer in the striatum following 54MnHPO4 exposure to olfactory transport.
However, the conclusions regarding the transport of Mn tracer to the striatum should be interpreted cautiously since the model overpredicted concentrations of Mn tracer in the tissue at the later time points for the plugged exposures to 54MnCl2 or 54MnHPO4 and the unplugged exposure to 54MnHPO4 (Figs. 4–7). The current model description of first-order transport of Mn from the blood to the striatum may not reflect the actual mechanism of Mn transport in vivo. MRI studies in rats following iv administration of MnCl2 showed rapid enhancement in the choroid plexus and ventricles (Aoki et al., 2004). One day thereafter the images showed enhancement of other brain regions. A more complete model could include this or other pathways by which Mn could reach the brain systemically. In addition, the description of the transport between free and bound Mn may be more complex than simple first-order transport.
Our model structure also represents a simplification of the neural circuits found in the rat olfactory and basal ganglial systems (Heimer et al., 1995; Shipley et al., 1995). For example, the axons from the OB project in the olfactory tract to the anterior olfactory nucleus, the olfactory tubercle, the piriform cortex (primary olfactory cortex), the cortical nucleus of the amygdala, and the entorhinal area (Lledo et al., 2005). Although greatly simplified, our model is consistent with Mn movement within the rat brain. Cross et al. (2002) mapped functional connections in the rat olfactory system with MRI of animals after unilateral intranasal delivery of this metal. They showed significant ipsilateral transport of Mn to the OB, lateral OTT, the piriform cortex in the lateral olfactory cortex, ventral pallidum, amygdala, and the anterior commissure. Cross' findings are also consistent with the tracer studies upon which our model was built (Brenneman et al., 2000; Dorman et al., 2002).
Other compounds including drugs, solvents, metals, and particulates have been shown to be transported directly via the olfactory neurons and trigeminal nerve to the brain (Bagger and Bechgaard, 2004; Brenneman et al., 2000; Chow et al., 2001; Ghantous et al., 1990; Illum, 2000; Larsson and Tjälve, 2000; Mathison et al., 1998; Oberdörster et al., 2004; Tjälve and Henrikkson, 1999; Wang et al., 1998). Limited mathematical modeling has been pursued to quantitate the rates of transport via this route. A model for [14C]-antipyrine, [14C]-diazepam, [3H]-sucrose, or [3H]-verapamil was developed by Graff et al. (2005) to describe the substrates' uptake by p-glycoprotein and distribution through the olfactory epithelium to the CNS of CF1 mice following intranasal instillation. The model was used to describe the time profile of radioactivity in serial sections taken from the brain. Graff et al. (2005) used a series of 20 homogeneous compartments with first-order absorption and diffusion to describe the rostral to caudal transport of the compounds. Blood delivery of the compounds was examined but not included in the final model for nasal administration. Our model was structured similarly with homogeneous compartments and first-order transport; however, our compartments represented discrete regions of the CNS instead of slices. In addition, blood delivery was necessary to describe Mn tracer absorbed from the lungs into the systemic blood and then distributed to the brain.
The model developed in this article for olfactory transport can be combined with a physiologically based pharmacokinetic model to estimate the whole-body distribution of Mn following inhalation or dietary exposure to various forms of Mn. This work is currently underway in our laboratory (Nong et al., 2007). In addition, the model can be expanded to include predictions of the deposition of Mn particles during exposure, which will be useful for predicting chronic exposure scenarios and for extrapolation of the model to humans. Differences between the rodents and humans would include differences in the compartment volumes of the olfactory regions as they are significantly smaller in humans. Mn could be assumed to be transported via the olfactory neurons at the same rate in rats as in humans. The model could then help answer questions of potential contribution of olfactory transport for target tissue doses of Mn from chronic exposure to ambient air concentrations of various forms of Mn.
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ACKNOWLEDGMENTS |
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The authors would like to thank Afton Chemical Company for funding for this work. We acknowledge Melanie Struve and Michael Pulliam at CIIT Centers for Health Research for providing the illustration of the rat nasal cavity and brain. We also appreciate the time and thoughtful reviews by Harvey Clewell, Andy Nong, and members of our Mn Technical Advisory Panel, Drs Michele Medinsky and Jeffrey Fisher. Conflicts of Interest: None reported.
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REFERENCES |
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