ToxSci Advance Access originally published online on April 19, 2006
Toxicological Sciences 2006 92(1):201-210; doi:10.1093/toxsci/kfj206
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Tissue Manganese Concentrations in Young Male Rhesus Monkeys following Subchronic Manganese Sulfate Inhalation
CIIT Centers for Health Research, 6 Davis Drive, PO Box 12137, Research Triangle Park, North Carolina 27709-2137
1 To whom correspondence should be addressed. Fax: (919) 558-1300. E-mail: dorman{at}ciit.org.
Received November 23, 2005; accepted March 2, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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High-dose human exposure to manganese results in manganese accumulation in the basal ganglia and dopaminergic neuropathology. Occupational manganese neurotoxicity is most frequently linked with manganese oxide inhalation; however, exposure to other forms of manganese may lead to higher body burdens. The objective of this study was to determine tissue manganese concentrations in rhesus monkeys following subchronic (6 h/day, 5 days/week) manganese sulfate (MnSO4) inhalation. A group of monkeys were exposed to either air or MnSO4 (0.06, 0.3, or 1.5 mg Mn/m3) for 65 exposure days before tissue analysis. Additional monkeys were exposed to MnSO4 at 1.5 mg Mn/m3 for 15 or 33 exposure days and evaluated immediately thereafter or for 65 exposure days followed by a 45- or 90-day delay before evaluation. Tissue manganese concentrations depended upon the aerosol concentration, exposure duration, and tissue. Monkeys exposed to MnSO4 at
0.06 mg Mn/m3 for 65 exposure days or to MnSO4 at 1.5 mg Mn/m3 for 15 exposure days developed increased manganese concentrations in the olfactory epithelium, olfactory bulb, olfactory cortex, globus pallidus, putamen, and cerebellum. The olfactory epithelium, olfactory bulb, globus pallidus, caudate, putamen, pituitary gland, and bile developed the greatest relative increase in manganese concentration following MnSO4 exposure. Tissue manganese concentrations returned to levels observed in the air-exposed animals by 90 days after the end of the subchronic MnSO4 exposure. These results provide an improved understanding of MnSO4 exposure conditions that lead to increased concentrations of manganese within the nonhuman primate brain and other tissues. Key Words: Parkinson disease; secondary; manganese poisoning; pharmacokinetics; inhalation exposure; macaca mulatta.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Excessive manganese accumulation within the human striatum, putamen, and globus pallidus occurs following high-dose inhalation or oral exposure (Aschner et al., 2005
). Excessive brain manganese accumulation can lead to neuronal injury and loss of dopaminergic neurons within these sites. Clinical signs associated with human manganese neurotoxicity include gait abnormalities, postural instability, micrographia, dystonia, rigidity, and bradykinesia. Manganese neurotoxicity most often results from the chronic inhalation of extremely high concentrations of airborne manganese (> 1 mg/m3), as may occur in manganese mines, battery producing plants, and certain other workplaces. Individuals receiving total parenteral nutrition and patients with hepatobiliary insufficiency are also at increased risk for manganese neurotoxicity (Aschner et al., 2005). Manganese neurotoxicity has garnered increased interest within the scientific community. In particular, the increasing worldwide use of methylcyclopentadienyl manganese tricarbonyl (MMT) as a gasoline fuel additive has stimulated the debate (Kaiser, 2003). Automobiles that use MMT emit manganese primarily in the phosphate and sulfate forms with smaller amounts of manganese oxides also being discharged (Aschner et al., 2005).
Increased brain manganese concentration is a critical step in the pathogenesis of manganese-induced neurotoxicity (Aschner et al., 2005). Our laboratory recently completed a series of experiments to evaluate the pharmacokinetics of manganese phosphate (as hureaulite) and manganese sulfate (MnSO4) in young adult male rats following subchronic (6 h/day, 5 days/week, for 13 weeks) inhalation exposure (Dorman et al., 2004). Nominal MnSO4 exposure concentrations of 0, 0.01, 0.1, and 0.5 mg Mn/m3 and a single hureaulite exposure concentration of 0.1 mg Mn/m3 were used. Rats were evaluated at the end of the 90-day exposure and at 45 days post-exposure. Elevated manganese concentrations were observed in the olfactory bulb, lung, and pancreas following 90 days of MnSO4 exposures to 0.01 mg Mn/m3. Rats exposed to MnSO4 (0.1 mg Mn/m3) had lower lung, but higher olfactory bulb, striatum, and cerebellum manganese concentrations than did hureaulite-exposed rats. These results confirm that inhalation exposure to soluble forms of manganese results in higher brain manganese concentrations than those seen with a relatively insoluble form of manganese.
There is a substantial literature base describing species differences in neurological responses following high-dose manganese exposure. Manganese-exposed monkeys develop distribution patterns for this metal within the brain that mimic those seen in heavily exposed people (Eriksson et al., 1992; Newland et al., 1989; Olanow et al., 1996). Like humans, monkeys exposed to high air manganese concentrations also have reduced dopamine and 3,4-dihydroxyphenylacetic acid concentrations in their striatum and pallidus and they develop dopaminergic neuron loss (Olanow et al., 1996). Similar regional distributions of manganese in the brain, neurochemical changes, and neuropathological responses have been inconsistently observed in manganese-exposed rodents (Aschner et al., 2005). In addition, rodents do not develop behavioral syndromes comparable to those seen in manganese-poisoned humans or monkeys (Boyes and Miller, 1998).
When considered together, the evidence suggests that monkeys are a more appropriate animal model to study manganese neurotoxicity (Newland, 1999). This study was therefore conducted to improve our understanding of MnSO4-exposure conditions that lead to increased concentrations of manganese within the central nervous system and other tissues in rhesus monkeys. Our experimental design allowed characterization of temporal- and dose-response relationships between manganese inhalation and manganese concentrations achieved in the brain and other tissues, and led to identification of tissues that accumulate manganese.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental design.
This study was performed in accordance with the U.S. Environmental Protection Agency's Good Laboratory Practice Standards for Inhalation Exposure Health Effects Testing (40 CFR Part 79.60). MnSO4 aerosol concentrations of 0.18, 0.92, and 4.62 mg MnSO4/m3, corresponding to 0.06, 0.3, and 1.5 mg Mn/m3, were generated for this study. Control animals were exposed to filtered air. Exposures were conducted for 6 h/day, 5 days/week. A group of monkeys (cohort 1) were exposed to air (n = 6) or MnSO4 at 0.06 (n = 6), 0.3 (n = 4), or 1.5 mg Mn/m3 (n = 4) for 65 exposure days. Necropsies were performed the day following the last inhalation exposure (i.e., 18–23 h after termination of the final inhalation exposure). Another eight monkeys were exposed to 1.5 mg Mn/m3 for 65 exposure days and held for 45 or 90 days before evaluation (i.e., post-exposure recovery groups). A second group (n = 4 monkeys per time point) of monkeys (cohort 2) was exposed to MnSO4 at 1.5 mg Mn/m3 for either 15 or 33 exposure days and euthanized the day following their last exposure.
Our lowest exposure MnSO4 concentration (0.06 mg Mn/m3) is below the current 8-h threshold limit value for inhaled manganese of 0.2 mg Mn/m3 that has been established by the American Conference of Governmental Industrial Hygienists. The lowest exposure concentration used in this study is also > 2000-fold higher than typical air manganese concentrations observed in the ambient air samples obtained in Canadian cities where MMT was used extensively in gasoline (Aschner et al., 2005). Additional endpoints evaluated in this study, but not presented in this manuscript, include histologic evaluation of the respiratory system (Dorman et al., 2005) and magnetic resonance imaging (MRI) of the brain (Dorman, unpublished observations).
Chemicals.
Manganese (II) sulfate monohydrate (MnSO4•H2O) was obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI). Manganese sulfate is a relatively water-soluble, pale pink, crystalline powder that contains approximately 32% manganese. Unless otherwise noted, all other chemicals were purchased from Sigma-Aldrich (St Louis, MO).
Animals.
This study was conducted under federal guidelines for the care and use of laboratory animals (National Research Council, 1996) and was approved by the CIIT Institutional Animal Care and Use Committee. Thirty-six male rhesus monkeys purchased from Covance Research Products, Inc. (Alice, TX) were used in this study. Monkeys were 17–22 months old at the time of their arrival at CIIT. Animals were screened for the nasal parasite Anatrichosoma spp. and other significant simian pathogens (e.g., herpes B, simian immunodeficiency virus, tuberculosis), and were given a thorough clinical examination including an evaluation of pre-exposure blood samples for routine hematology and clinical chemistry. The results of these evaluations were within normal limits. Animals were between 20 and 24 months of age at the start of the inhalation exposure. Randomization of animals to treatment groups occurred prior to the start of the inhalation exposure and was based upon a weight randomization procedure. Animals were acclimated to the facility for at least 43 days prior to the start of the first inhalation exposure.
Animal husbandry.
All animals were housed in animal rooms or exposure chambers within CIIT's animal facility. This facility is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International. A certified primate chow (# 5048) diet from Purina Mills (St Louis, MO) was fed twice a day (total daily amount fed was approximately 4% of the animal's body weight). Mean (± SEM) manganese concentrations determined in the primate chow were 133 ± 14 ppm. Manganese intake occurring from the ingestion of the base diet was approximately 6.2 mg/kg/day. Dietary supplements including fruits (e.g., oranges, raisins, apples), vegetables (e.g., carrots), and treats (e.g., honey, candies, cereal, fruit juices) were also provided to the monkeys each day. These supplements provided an additional 0–200 µg Mn per serving. Reverse osmosis purified water was available ad libitum. Manganese concentrations in the majority of water samples (36/50 samples) were below assay detection limits (< 0.24 µg Mn/l), while the highest manganese water concentration was 8.9 µg Mn/l. During nonexposure periods, domiciliary stainless steel cages (0.4 m2 x 0.8 m tall) suitable for housing macaque monkeys (Lab Products, Inc., Seaford, DE) were used to individually house monkeys. On each exposure day, animals were transferred to 0.2 m2 x 0.6 m tall stainless steel cages (Lab Products, Inc.) that were designed to fit within the 8-m3 inhalation chambers. Animals were moved back to their domiciliary cages after the end of each 6-h exposure. Additional details concerning these animals and their husbandry have been recently published (Dorman et al., 2005).
Manganese exposures.
Manganese (II) sulfate monohydrate (MnSO4•H2O) was obtained from Aldrich Chemical Company, Inc. Four 8-m3 stainless steel and glass inhalation exposure chambers were used. Methods describing chamber monitoring as well as generation and characterization of the MnSO4 aerosol have been previously described (Dorman et al., 2004, 2005).
Necropsy procedures.
Food was withheld overnight prior to necropsy. Monkeys were anesthetized with ketamine (20 mg/kg, im, Fort Dodge Animal Health, Fort Dodge, IA), and blood was collected from a peripheral vein using plastic syringes with hypodermic needles. A small (
50–80 µl) aliquot was used to determine the packed cell volume. Additional blood samples were collected for complete blood cell counts, routine clinical chemistries, evaluation of basal levels of luteinizing hormone (LH), and determination of red blood cell glutathione (GSH) concentrations. Following blood collection, monkeys were euthanized with pentobarbital (80–150 mg/kg, iv, Henry Schein Inc., Port Washington, NY) followed by exsanguination. Following euthanasia, the lungs and other thoracic organs were removed, weighed, and inspected for gross lesions, and the left lung separated for use in manganese analyses. Brains were removed and divided on the midsagittal plane with anatomical structures identified using a published atlas (Martin and Bowden, 2000). The following brain structures were collected for determination of manganese concentrations: pituitary gland, olfactory bulb, olfactory tract, olfactory cortex, caudate, putamen, globus pallidus, cerebellum, trigeminal nerve, white matter, and frontal cortex. Samples of the following tissues were also collected for manganese analyses: olfactory epithelium, heart, femur, skullcap (parietal bone), liver, pancreas, kidney, skeletal muscle (quadriceps femoris m), testes, gall bladder contents (i.e., bile) and urine. All samples were stored in individual plastic vials or bags, frozen in liquid nitrogen, and stored at approximately –80°C until chemical analyses were performed.
Hematology, clinical chemistry, and serum electrolyte analyses.
Hematology, clinical chemistry, and serum electrolyte analyses were performed by LabCorp (Research Triangle Park, NC) using standard test methods. Heparinized blood (500 µl) used for the determination of total and oxidized red blood cell GSH was diluted with an equivalent volume of phosphate buffered saline. The sample was then centrifuged for 5 min (100 g), in a refrigerated (4°C) microcentrifuge. A 100-µl aliquot of the cell pellet was then added to 900 µl of 5% trichloroacetic acid in water. The sample was then recentrifuged for 5 min (10,000 g) in a refrigerated (4°C) microcentrifuge. The supernatant was then used for the evaluation of total red blood cell GSH, reduced GSH, and oxidized glutathione disulfide (GSSG) using published methods (Anderson, 1985). Total GSH was determined using 5,5'-dithiobis-2-nitrobenzoic acid as the substrate for GSH reductase. Formation of the reaction product, 5-thio-2-nitrobenzoic acid, was measured at 405 nm using a Roche Cobas Fara II chemical analyzer (Roche Diagnostics Systems, Branchburg, NJ). GSSG was assayed after derivatization of GSH to remove it from the reaction. Derivatization was done by neutralizing the pH of the 100-µl sample with 6 µl triethanolamine and incubating the reaction for 60 min with 2 µl 2-vinylpyridine. Remaining oxidized GSH was then determined as previously described, thus providing an estimate of total GSH.
Tissue manganese concentrations.
Tissue manganese concentrations were determined by graphite furnace atomic absorption spectrometry with a Perkin Elmer AAnalyst 800 Atomic Absorption spectrometer equipped with AA WinLab software (version 4.1 SP) using previously published methods (Dorman et al., 2004). Samples (10–30 mg) were digested in 16M nitric acid (1 ml) prior to manganese analysis using a CEM MARS5 Microwave Accelerated Reaction System (CEM, Matthews, NC). Some samples required additional dilution (up to 10-fold) in nitric acid prior to analysis.
Data analysis and statistics.
The data for quantitative, continuous variables were compared for the exposure and control groups by tests for homogeneity of variance (Levene's test), ANOVA, and Dunnett's multiple comparison procedure for significant ANOVA. The ANOVA for the clinical chemistry and hematology data was performed on the parameter value obtained from the difference between an animal's pre-exposure and post-exposure test values. In the event, the Levene's test was significant, then the data were transformed using a natural log (ln) transformation. If the Levene's test remained significant, then the data were analyzed by nonparametric statistics (Wilcoxon/Kruskal-Wallis). Statistical analyses were performed using SAS Statistical Software (Cary, NC). A probability value of < 0.01 was used for Levene's test, while < 0.05 was used as the critical level of significance for all other statistical tests. Unless otherwise noted, data presented are mean values ± SEM.
Elimination half-lives for manganese in several tissues were estimated for animals exposed to 1.5 mg Mn/m3 for 65 exposure days using standard kinetic formulas (Shargel and Yu, 1999). Prior to analysis, mean tissue manganese concentrations were corrected for background manganese concentrations present in the air-exposed monkeys. This analysis was performed on tissues that were significantly increased following the 65th exposure and 90 days thereafter had a mean manganese concentration higher than that present in the air-exposed animals.
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RESULTS |
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Manganese Test Atmospheres
No significant differences in the test aerosol characteristics were observed between the two exposure cohorts. Based upon optical particle sensor results, the overall average concentrations (± SD) for the MnSO4 atmospheres were 0.19 ± 0.01, 0.97 ± 0.06, 4.55 ± 0.33 (cohort 1), and 4.45 ± 0.35 (cohort 2) mg/m3 for the target concentrations of 0.18, 0.92, and 4.62 mg MnSO4/m3. The geometric mean diameters and geometric standard deviations (
g) of the MnSO4 atmospheres were determined to be 1.04 µm (g = 1.51), 1.07 µm (g = 1.54), 1.12 µm (g = 1.58), and 1.04 µm (g = 1.50) for the target concentrations of 0.06, 0.3, 1.5 (cohort 1), and 1.5 (cohort 2) mg Mn/m3, respectively. The calculated mass median aerodynamic diameters (MMAD) were 1.73, 1.89, 2.12, and 1.72 µm for the target concentrations of 0.06, 0.3, 1.5 (cohort 1), and 1.5 (cohort 2) mg Mn/m3, respectively. Particles of unknown composition (arising from animal dander and other background sources) were present in the control chamber at an overall average concentration (± SD) of 0.004 ± 0.002 mg/m3. The calculated MMAD for the particles in the control chamber was 3.88 µm.
Clinical Observations, Body Weights, and Organ Weights
Subchronic inhalation exposure to MnSO4 did not affect body weight gain (data not shown) or terminal body weight (Table 1). Clinical signs observed in the monkeys were of minimal veterinary concern (e.g., alopecia or pulling of hair on the arms and legs, intermittent abnormal stool) and were not related to MnSO4 inhalation (data not shown). Mean (± SEM) absolute organ weights and hematocrits are summarized in Table 1. No statistically significant difference from control in absolute organ weights was observed with any organ in animals exposed to MnSO4 for 65 days and then assessed immediately thereafter. Because the animals continued to grow, evaluation of post-exposure organ weights was confounded by the animal's increase in body weight. There was a statistically significant decrease (approximately 17%) in the relative heart weight (relative to body weight) in monkeys evaluated 90 days after the end of a 13-week exposure to MnSO4 at 1.5 mg Mn/m3. No other statistically significant differences in relative organ weight (relative to either body weight or brain weight) were observed in the MnSO4-exposed animals versus controls (data not shown).
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Hematology and Clinical Chemistry
A statistically significant decrease in the difference between pre- and post-exposure total bilirubin concentrations was observed in monkeys exposed to MnSO4 at 1.5 mg Mn/m3 for 65 exposure days when compared to air-exposed controls. However, end-of-exposure total bilirubin concentrations in the air- and MnSO4-exposed monkeys were 0.15 ± 0.02 and 0.15 ± 0.03 mg/dl, respectively. A twofold higher pre-exposure total bilirubin concentration was present in the monkeys assigned to the high-dose MnSO4 exposure group. Alkaline phosphatase activity was approximately 1.6-fold higher in monkeys exposed to MnSO4 at 1.5 mg Mn/m3 for 33 exposure days and monkeys evaluated 90 days after a 13-week exposure to MnSO4 at 1.5 mg Mn/m3, when compared to controls (524 ± 53 IU/l). Mean corpuscular hemoglobin concentration (MCHC %) was decreased in monkeys exposed to MnSO4 at 1.5 mg Mn/m3 for 15 days (post-exposure value = 33.5 ± 0.3%) and monkeys evaluated 45 days after a 13-week exposure to MnSO4 at 1.5 mg Mn/m3 (post-exposure value = 33.6 ± 0.3%) versus controls (post-exposure value = 35.1 ± 0.1%). The pre-exposure and post-exposure total bilirubin, alkaline phosphatase, and MCHCs were in the normal reference range reported for male rhesus monkeys (Wolford et al., 1986
). The most common post-exposure red blood cell morphological finding was slight anisocytosis. Anisocytosis was found in control and MnSO4-exposed monkeys (data not shown). Additional selected post-exposure clinical chemistry parameters are shown in Table 2. Observed differences in clinical chemistry or hematology parameters are unlikely to be toxicologically significant or related to MnSO4 exposure. Subchronic exposure to MnSO4 did not affect any other hematology or clinical chemistry parameters examined in this experiment (data not shown).
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Tissue Manganese Concentrations
Tissue manganese concentrations in the subchronically exposed monkeys are presented in Table 3. Subchronic exposure to MnSO4 at the lowest exposure concentration (
0.06 mg Mn/m3) resulted in increased manganese concentrations in the olfactory epithelium, olfactory bulb, olfactory cortex, globus pallidus, putamen, white matter, cerebellum, and heart. Monkeys exposed to MnSO4 at the mid-dose ( 0.3 mg Mn/m3) for 65 exposure days developed increased manganese concentrations in all the above tissues, as well as in the olfactory tract, caudate, pituitary gland, kidney, pancreas, lung, bile, blood, and urine. Monkeys exposed to MnSO4 at the highest exposure concentration (1.5 mg Mn/m3) for 65 exposure days additionally had increased manganese concentrations in the frontal cortex, trigeminal nerve, liver, skeletal muscle, and parietal bone.
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Tissue manganese concentrations in the monkeys exposed to the highest (1.5 mg Mn/m3) MnSO4 exposure concentration are presented in Table 4. Increased manganese concentrations were observed in the olfactory epithelium, olfactory bulb, olfactory tract, olfactory cortex, globus pallidus, putamen, caudate, frontal cortex, cerebellum, pituitary, femur, kidney, lung, pancreas, parietal bone, and bile of monkeys exposed to MnSO4 at the highest exposure concentration for 3 weeks (15 exposure days). Manganese concentrations were elevated in the olfactory epithelium, olfactory bulb, olfactory tract, olfactory cortex, globus pallidus, putamen, caudate, frontal cortex, cerebellum, pituitary, trigeminal nerve, heart, kidney, lung, pancreas, parietal bone, testes, blood, and bile of monkeys exposed to MnSO4 at the highest exposure concentration for 6 weeks (33 exposure days).
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Tissue manganese concentrations remained elevated (vs. air-exposed controls) in the olfactory cortex, globus pallidus, putamen, pituitary gland, and blood 45 days after the end of the 13-week exposure (65 exposure days) to MnSO4 at 1.5 mg Mn/m3 (Table 4). All tissue manganese concentrations had returned to levels observed in the air-exposed control animals by 90 days after the end of the exposure.
Elimination of manganese from the monkey brain varied from region to region with the shortest halftime of elimination occurring in the olfactory bulb (4.9 days), intermediate in the globus pallidus, putamen, and caudate (15.7–16.7 days), with slower elimination occurring in the olfactory cortex (19.4 days), pituitary (23.6 days), and cerebellum (32.3 days). The apparent halftime of elimination in the olfactory epithelium, kidney, and heart were 12.9, 18.3, and 27.3 days, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There is overwhelming evidence that the human striatum, globus pallidus, and substantia nigra are the primary target sites for manganese neurotoxicity. In the present study, we found that the globus pallidus, putamen, and cerebellum were among the brain regions that were most likely to accumulate manganese following exposure to the lowest MnSO4 exposure concentration (
0.06 mg Mn/m3) used in this study. The relative magnitude of the increase in end-of-exposure manganese concentration can provide a clue as to whether selective accumulation occurs within a particular brain region. A greater than 3- to 5-fold increase (vs. air-exposed controls) in mean tissue manganese concentration was observed in the globus pallidus, putamen, and caudate of monkeys exposed subchronically (65 exposure days) to MnSO4 at the highest exposure concentration (1.5 mg Mn/m3). In contrast, manganese concentrations in the same animal's frontal cortex and cerebellum, two brain regions not generally associated with manganese neurotoxicity, had less than a threefold increase in manganese concentration (vs. air-exposed controls).
Accumulation of manganese within the basal ganglia of monkeys has been consistently demonstrated in previous inhalation studies. Bird et al. (1984) exposed female rhesus monkeys for 6 h/day, 5 days/week for 12 months to either air or manganese dioxide (MnO2) at 30 mg Mn/m3. Increased putamen and globus pallidus manganese concentrations were observed in the MnO2-exposed monkeys. Coulston and Griffin (1977) exposed rhesus monkeys for approximately 23 h/day for 7 days/week to manganese tetroxide (Mn3O4) at 0.1 mg Mn/m3. An unexposed group of animals were used as controls. Elevated pallidum, basal ganglia, cerebellum, and pons manganese concentrations were observed following the 1-year inhalation exposure. These findings are qualitatively similar to those observed from our present study.
Our study demonstrates that, like rats (Dorman et al., 2004), monkeys exposed subchronically to MnSO4 also develop increased olfactory bulb manganese concentrations. Increased olfactory epithelium, olfactory bulb, and olfactory cortex manganese concentrations occurred in the MnSO4-exposed monkeys. Monkeys subchronically exposed to the highest MnSO4 exposure concentration developed a 5- to >10-fold increase (vs. air-exposed controls) in manganese concentration within the olfactory epithelium and olfactory bulb. As in the basal ganglia, increased (vs. air-exposed controls) manganese concentrations in these tissues were also observed in monkeys exposed to MnSO4 at the highest exposure concentration for 15 exposure days. These results suggest that, like the basal ganglia, the olfactory system of monkeys preferentially accumulates inhaled manganese. Increases in olfactory bulb manganese concentration may not reflect systemic (blood borne) delivery of manganese. Previous studies conducted in our laboratory have shown that delivery of inhaled manganese to the rat olfactory bulb can occur via direct axonal transport along the olfactory nerve (Brenneman et al., 2000). Our results from the present study suggest that direct olfactory nerve transport of manganese from the olfactory epithelium to the olfactory bulb also occurs in nonhuman primates. In addition, MRI images of the monkeys that were exposed to MnSO4 for 65 days also demonstrate manganese accumulation in the olfactory bulb (Dorman, unpublished observations). Although these results suggest that direct olfactory uptake of manganese occurs in nonhuman primates, as in rats (Brenneman et al., 2000; Cross et al., 2004; Dorman et al., 2002), direct delivery of manganese from the monkey olfactory bulb to the basal ganglia was not evident in our MRI studies.
Lewis et al. (2005) reported that the rat trigeminal nerve may also absorb manganese from the nasal cavity. In our study, increased trigeminal nerve manganese concentrations were seen only in the monkeys exposed to MnSO4 at 1.5 mg Mn/m3. Manganese concentrations in the trigeminal (semilunar) ganglion, were, however, appreciably lower than those seen in the olfactory epithelium or olfactory bulb. These findings suggest that the trigeminal nerve likely plays a minor role in the movement of manganese from the primate nasal cavity to other parts of the central nervous system.
Increased pituitary gland manganese concentrations occurred in monkeys exposed to MnSO4 at either 0.3 mg Mn/m3 for 65 exposure days or 1.5 mg Mn/m3 for 15 exposure days. An approximately sevenfold increase (vs. air-exposed controls) in pituitary gland manganese concentration was observed in monkeys exposed to MnSO4 at the highest exposure concentration (1.5 mg Mn/m3) for 65 exposure days. These results suggest that the pituitary gland also preferentially accumulates manganese. Brain imaging studies conducted by Newland et al. (1989) in a cynomolgus monkey exposed to a manganese chloride aerosol also demonstrated manganese accumulation in the pituitary gland. High-dose manganese exposure in people has been associated with decreased libido (Aschner et al., 2005). Because of the known association between testosterone production and male libido (Schill, 2001), we examined serum levels of LH, a protein hormone secreted by the anterior pituitary gland that stimulates production of testosterone by the Leydig cells of the testes. We did not observe an effect of manganese inhalation on LH levels. Laskey et al. (1985) did not observe any change in unstimulated or stimulated LH serum concentrations in neonatal Long-Evans rat pups that were given oral Mn3O4 (0, 71, or 214 µg Mn/g body weight/day) from birth to weaning on day 21. Additional studies may be warranted to determine whether high-dose manganese exposure has any effect on normal pituitary function.
Exposure of laboratory animals to high levels of manganese has also been linked with histopathologic and biochemical changes in the seminiferous tubule (Imam and Chandra, 1975; Murthy et al., 1980). In exposed monkeys, however, testicular histopathological changes occurred only after manganese-induced neurotoxicity was observed (Murthy et al., 1980). We did not observe increased testes manganese concentrations following subchronic MnSO4 exposure, although higher manganese levels were seen in the testes following high-dose exposure to MnSO4 for 33 exposure days.
Another objective of our study was to evaluate manganese clearance from the brain and other tissues following cessation of the MnSO4 inhalation. Manganese concentrations in the olfactory cortex, globus pallidus, putamen, pituitary gland, and blood remained elevated 45 days after the end of the subchronic exposure. Manganese concentrations in other brain regions as well as in all other examined tissues and body fluids had returned to levels observed in the air-exposed control animals by this time. The initial elimination half-life for manganese in the juvenile nonhuman primate brain varied from region to region but was consistently 33 days or less and was around 15–16 days in known target sites (e.g., globus pallidus, putamen). This observation is consistent with data presented by Takeda et al. (1995) who reported a biological half-life of manganese in the adult rat brain to be 51–74 days. A very similar elimination halftime of 53 days has been reported in a macaque monkey given MnCl2 via an implanted subcutaneous osmotic minipump (Newland et al., 1987). It should be noted that Newland et al. (1987) also exposed two cynomolgus monkeys to a tracer dose (0.01–0.02 µg Mn) of 54MnCl2 given as an aerosol via an endotracheal tube and then evaluated head and chest 54Mn levels by gamma spectrometry. They observed that clearance of 54Mn from the head was slow with an estimated half-life of elimination greater than 220 days. This estimate may however be confounded by the relatively slow elimination of 54Mn from the skull and may not reflect the rate at which this metal is eliminated from the brain parenchyma.
In addition to an evaluation of manganese concentrations in known target tissues, we also evaluated tissues that are involved in the storage or elimination of manganese. Bone can effectively sequester manganese, and prolonged retention (half-lives > 50 days) of manganese in bone has been reported (Furchner et al., 1966). Monkeys exposed to MnSO4 at the highest exposure concentration (1.5 mg Mn/m3) for 90 days developed increased manganese concentrations in the parietal bone. Manganese concentrations in the femur of animals from this exposure group were also elevated (approximately 140% of control values), although this increase was not statistically significant (p = 0.09). This finding was surprising since our earlier study demonstrated that young male rats subchronically exposed to MnSO4 (at 0.5 mg Mn/m3) developed increased femur manganese concentrations (Dorman et al., 2004).
Manganese intake is known to influence the amount of manganese eliminated in the bile. In the present study, we observed that monkeys exposed to MnSO4 at 0.3 mg Mn/m3 for 65 exposure days developed increased bile manganese concentrations. Increased biliary excretion of manganese developed within 3 weeks of the high-dose MnSO4 exposure and occurred in the absence of significant elevations in liver manganese concentration. Monkeys exposed to MnSO4 at 0.3 mg Mn/m3 for 65 exposure days also developed increased manganese concentrations in the pancreas. This response was also observed in monkeys exposed to MnSO4 at 1.5 mg Mn/m3 for 15 days. Dose-dependent biliary and pancreatic excretion of divalent manganese serves to regulate the percentage of ingested manganese retained by the body and to limit increases in liver and other systemic tissue manganese concentrations (Aschner et al., 2005). Assuming a normal bile flow of 3.5 ml/h in adult rhesus monkeys (Beaudoin et al., 1975) and a specific gravity of bile of 0.998–1.062, we estimate that daily biliary excretion of manganese approached 140–840 µg Mn/day for monkeys exposed subchronically to MnSO4 at 0.06 or 1.5 mg Mn/m3, respectively. The animals' intake of manganese can also be estimated. Assuming a minute volume of 2.2 l/min and a pulmonary absorption fraction of 60% (Andersen et al., 1999), we estimate that daily inhalation intake of manganese was approximately 30–710 µg Mn for monkeys exposed 6 h to MnSO4 at 0.06 or 1.5 mg Mn/m3, respectively. Assuming an average animal weight of 2.5 kg and a net gastrointestinal absorption fraction of 3% (Andersen et al., 1999), we estimate that the diet accounted for a daily intake of approximately 460 µg Mn.
We also observed a significant increase in the urine manganese concentration of monkeys exposed to MnSO4 at 0.3 mg Mn/m3. Although urinary elimination of manganese increased in these MnSO4-exposed monkeys, the contribution that this route of elimination makes toward total body clearance remains low. For example, assuming a urine flow of 0.1 ml/min/kg (Peterson et al., 1988), daily urinary excretion of manganese approached 2 µg Mn/day in our most heavily exposed animals. Similarly, Ishihara and Matsushiro (1986) estimated that in humans, basal urinary manganese elimination was 10- to 20-fold lower than basal biliary excretion rates. Our results are consistent with occupational studies that suggest a weak association between manganese inhalation and urinary levels of this metal (Lucchini et al., 1995; Mergler et al., 1994).
Whole blood, serum, and plasma manganese concentrations have also been evaluated in individuals exposed to airborne manganese, most commonly in an occupational setting. In studies of occupational exposure, blood manganese of exposed workers has been shown to be elevated above that of control subjects repeatedly (Lucchini et al., 1997; Mergler et al., 1994), and in some studies a significant dose-response between blood manganese and exposure indices has been reported (Lu et al., 2005; Lucchini et al., 1995, 1999; Myers et al., 2003). The strength of the observed correlation between blood manganese concentrations and manganese exposure concentrations often depends upon the magnitude and the duration of the exposure. We observed an approximately 2.5-fold increase in the amount of manganese in the blood of monkeys exposed to MnSO4 at 0.3 mg Mn/m3. Increased blood manganese concentrations were observed in monkeys exposed to MnSO4 at 1.5 mg Mn/m3 following 33 and 65 exposure days as well as 45 days after the cessation of the subchronic manganese inhalation. Ulrich et al. (1979) reported elevated whole blood manganese concentrations in squirrel monkeys (Saimiri scuireus) exposed to manganese dioxide (MnO2) at 1152 µg Mn/m3 for approximately 21–22 h/day, 7 days/week for 9 months.
The heart also developed increased manganese concentrations following subchronic exposure to MnSO4 at the lowest exposure concentration. A nearly twofold increase (vs. air-exposed controls) in mean tissue manganese concentration was observed in the heart from animals subchronically exposed to our lowest MnSO4 exposure concentration (0.06 mg Mn/m3). Coulston and Griffin (1977) reported an approximately twofold increase in heart muscle manganese concentrations in monkeys that were exposed semicontinuously to Mn3O4 (at 0.1 mg Mn/m3) for 1 year. This propensity of manganese to accumulate within the heart tissue likely reflects mitochondrial retention of divalent manganese by this tissue (Gavin et al., 1992). In our present study, the heart was also the only organ to demonstrate a change in relative organ weights (vs. body weight) following subchronic exposure to MnSO4. There are limited data to suggest that manganese adversely affects cardiovascular function. Studies conducted to date have largely focused on intravenous manganese chloride and mangafodipir trisodium. Mangafodipir trisodium is an intravenous MRI contrast agent for the detection of lesions in the liver. These studies have shown that high concentrations of divalent manganese antagonizes calcium and can induce negative inotropy, reflex tachycardia, and hypotension (Jynge et al., 1997).
This study provides new information about manganese concentrations in the brain and other tissues following high-dose subchronic manganese inhalation by nonhuman primates. These studies also contribute to our understanding of the inhalation exposure conditions that lead to increased concentrations of the metal within the central nervous system and other target organs and manganese clearance from these sites. Work underway in our laboratory is leading to the development of physiologically based pharmacokinetic models that will assist in extrapolation of our data to environmentally and occupationally relevant exposure conditions. These quantitative models may be useful in assessing expected relationships between tissue concentration and the relatively small alterations in blood manganese concentrations that were observed in the monkeys in the present study.
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ACKNOWLEDGMENTS |
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The authors would like to thank Paul Ross, Carol Bobbitt, John Murphy, and other members of the CIIT Centers for Health Research staff for their contributions. We also thank Drs Mel Andersen, Susan Borghoff, Teresa Leavens, and Owen Moss for their critical review of this manuscript. This publication is based on a study sponsored and funded by the 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 Subsections 79.50 et seq.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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