Influence of Dietary Manganese on the Pharmacokinetics of Inhaled Manganese Sulfate in Male CD Rats

David C. Dorman1,, Melanie F. Struve, R. Arden James, Brian E. McManus, Marianne W. Marshall and Brian A. Wong

CIIT Centers for Health Research, 6 Davis Drive, P.O. Box 12137, Research Triangle Park, North Carolina 27709–2137

Received October 12, 2000; accepted December 20, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Concerns exist as to whether individuals with relative manganese deficiency or excess may be at increased risk for manganese toxicity following inhalation exposure. The objective of this study was to determine whether manganese body burden influences the pharmacokinetics of inhaled manganese sulfate (MnSO4). Postnatal day (PND) 10 rats were placed on either a low (2 ppm), sufficient (10 ppm), or high (100 ppm) manganese diet. The feeding of the 2 ppm manganese diet was associated with a number of effects, including reduced body weight gain, decreased liver manganese concentrations, and reduced whole-body manganese clearance rates. Beginning on PND 77 ± 2, male littermates were exposed 6 h/day for 14 consecutive days to 0, 0.092, or 0.92 mg MnSO4/m3. End-of-exposure tissue manganese concentrations and whole-body 54Mn elimination rates were determined. Male rats exposed to 0.092 mg MnSO4/m3 had elevated lung manganese concentrations when compared to air-exposed male rats. Male rats exposed to 0.92 mg MnSO4/m3 developed increased striatal, lung, and bile manganese concentrations when compared to air-exposed male rats. There were no significant interactions between the concentration of inhaled MnSO4 and dietary manganese level on tissue manganese concentrations. Rats exposed to 0.92 mg MnSO4/m3 also had increased 54Mn clearance rates and shorter initial phase elimination half-lives when compared with air-exposed control rats. These results suggest that, marginally manganese-deficient animals exposed to high levels of inhaled manganese compensate by increasing biliary manganese excretion. Therefore, they do not appear to be at increased risk for elevated brain manganese concentrations.

Key Words: manganese; inhalation dietary interactions; biliary elimination; toxicokinetics; rat..


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Manganese is an essential trace mineral that is normally present in all mammalian tissues in concentrations ranging from 0.3 to 2.9 µg Mn/g wet tissue weight (Rehnberg et al., 1982Go). Major dietary sources of manganese include grains, rice, nuts, and legumes (ATSDR, 1992Go). Manganese is required for the normal development and function of the central nervous system. A wide variety of brain enzymes, including mitochondrial manganese-superoxide dismutase and glial glutamine synthetase, either contain manganese or are activated by it (Wedler, 1996Go). With exposure to excess amounts, manganese accumulates in the human basal ganglia and may damage neurons resulting in an extrapyramidal movement disorder known as manganism (Barbeau, 1984Go; Beuter et al., 1994Go; Ingersoll et al., 1995Go). Clinically, manganism resembles Parkinson's disease, yet important differences exist between these two disease syndromes (Calne et al., 1994Go; Olanow et al.; 1996Go; Pal et al., 1999Go). Manganese-induced neurotoxicity primarily arises in humans as a consequence of the chronic inhalation of high concentrations of respirable airborne manganese (> 5 mg/m3) as may occur in manganese mines (Pal et al., 1999Go).

In contrast to the numerous reports describing manganese toxicity following inhalation exposure in humans, there are relatively few reports of manganism arising from water or dietary sources. In part, this trend is due to the relatively low levels of manganese found in these media. For example, most diets in North America result in manganese intakes below the current reference dose of 10 mg/day (Finley and Davis, 1999Go). Water concentrations of manganese typically range from 1 to 100 µg/l with most values below 10 µg/l (Keen and Zidenberg-Cherr, 1994Go). On rare occasions, manganese toxicity results from ingestion. For example, Kawamura and coworkers (1941) and Kondakis et al. (1989) documented outbreaks of manganism in Japan and Greece due to the consumption of water from wells contaminated with extremely high levels of manganese (1.8 to 14 mg Mn/l).

A number of physiological mechanisms also exist to maintain relatively constant tissue manganese concentrations despite wide fluctuations in oral manganese intake (Schroeder et al., 1966Go). When dietary manganese levels are high, adaptive changes often include reduced gastrointestinal absorption of manganese, enhanced manganese liver metabolism, and increased biliary and pancreatic excretion of manganese (Britton and Cotzias, 1966Go; Davis et al., 1993Go; Finley and Davis, 1999Go; Mahoney and Small, 1968Go; Malecki et al., 1996Go; Papavasiliou et al., 1966Go). Thus, the fraction of ingested manganese retained by the body is tightly regulated in order to maintain normal tissue manganese concentrations under different dietary conditions. It has not been clearly established whether inhaled manganese is controlled similarly in individuals with different manganese body burdens. Absent such data, concerns exist that individuals with either deficient or excessive manganese tissue burdens may be at increased risk for manganese toxicity following inhalation exposure (Weiss, 1999Go).

Manganese deficiency in animals results in skeletal abnormalities and an irreversible congenital ataxia syndrome (Freeland-Graves and Llanes, 1994Go). This syndrome has not been reported to occur spontaneously in humans. Suboptimal manganese status, however, may occur in patients with epilepsy, osteoporosis, or exocrine pancreatic insufficiency, in individuals undergoing chronic hemodialysis, and in children with Perthes' disease or phenylketonuria (Carl and Gallagher, 1994Go; Freeland-Graves and Llanes, 1994Go). Relative manganese excess may also occur. People on strict vegetarian diets, individuals consuming manganese-enriched mineral supplements, and patients undergoing total parenteral nutrition often have elevated manganese intake when compared to the general United States population (Finley and Davis, 1999Go; Fitzgerald et al., 1999Go; Nagatomo et al., 1999Go). Manganese concentrations in the nervous system are influenced by hepatic manganese metabolism. For example, Hauser and coworkers (1996) have reported that some individuals with chronic hepatic encephalopathy have elevated blood manganese concentrations and develop brain magnetic resonance imaging changes consistent with elevated basal ganglial manganese levels.

Public attention to manganese neurotoxicity is increasing and has been stimulated in part by the use of the unleaded gasoline additive methylcyclopentadienyl manganese tricarbonyl (MMT). Exposure to the combustion products of MMT is of primary concern since the parent chemical is photosensitive and rapidly degrades in the air. Modern automobiles that are equipped with catalytic converters and use MMT-containing fuel emit manganese primarily in the phosphate and sulfate (MnSO4) forms, although lesser amounts of manganese oxides (e.g., Mn3O4) may also be discharged (Lynam et al., 1999Go; Ressler et al., 1999Go; Zayed et al., 1999aGo).

A critical issue in assessing the risks of chronic, low-level manganese exposure arising from MMT use is the ability to develop a predictive pharmacokinetic model for inhaled manganese (Andersen et al., 1999Go). Such predictive pharmacokinetic models will relate lung, brain, and other tissue manganese concentrations with exposure concentrations of MMT combustion products. Ideally, this model will account for differences in the chemical species of manganese, the route of exposure, and potentially susceptible human subpopulations. Individuals with either relative manganese deficiency or excess could represent susceptible subpopulations at risk for developing elevated tissue manganese concentrations following the inhalation of MnSO4 and other MMT combustion products.

Few investigators have examined whether dietary manganese levels can influence the pharmacokinetics of inhaled manganese. Moore and coworkers (1975) evaluated tissue manganese concentrations in rats fed either a 5- or 42.2-ppm manganese diet and exposed subchronically (8 h/day for 56 consecutive days) to irradiated exhaust from an automobile engine using gasoline containing MMT. These authors reported that brain, kidney, and liver manganese concentrations were lower in air-exposed control animals fed a 5-ppm manganese diet compared to animals fed the 42.2-ppm manganese diet. When compared with air-exposed controls, animals fed either the 5- or 42.2-ppm manganese diet and exposed to irradiated exhaust (containing approximately 117 µg Mn/m3) developed a 45 or 84% increase in total brain manganese concentrations, respectively. However, the results of this study were not analyzed in such a manner as to determine whether diet and inhalation interactive effects occurred. Although not determined directly during this study, the most likely form of manganese in the engine exhaust was the tetroxide, Mn3O4 (Ter Haar et al., 1975Go).

Our laboratory has recently characterized the pharmacokinetics of manganese phosphate, manganese sulfate, and manganese tetroxide in rats following 14 consecutive days of inhalation exposure (Dorman et al., 2001Go; Vitarella et al., 2000Go). These studies showed that inhalation exposure to more soluble forms of manganese (e.g., MnSO4) results in higher brain and liver manganese concentrations than those achieved following exposure to a more insoluble form of manganese (e.g., Mn3O4). Each of these studies used a standard rodent diet (NIH-07) containing approximately 100 ppm manganese. Thus these studies did not address the fundamental question of whether different dietary manganese levels can influence the pharmacokinetics of inhaled MMT combustion products. The present study was therefore undertaken to address this research need. Our hypothesis was that brain and other target tissue manganese concentrations resulting from inhalation exposure would be influenced by dietary manganese concentrations. However, our data do not support this hypothesis.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Experimental design.
A schematic representation of the experiment is presented in Figure 1Go. This study used a 3 x 3 factorial arrangement of treatments in a split-plot design. One factor was diet at 3 levels (2, 10, and 100 ppm manganese), and the other factor was exposure to MnSO4 at 3 inhalation exposure levels (0, 0.092, or 0.92 mg MnSO4/m3). Main plots were litters that were assigned on postnatal day (PND) 10 to one of 3 dietary groups containing either 2, 10, or 100 ppm manganese and 50 ppm iron (n = 8 litters/diet). Subplots were two PND 77 ± 2 male rats from each litter that were randomly assigned to each of the 3 MnSO4 exposure groups.



View larger version (21K):
[in this window]
[in a new window]
 
FIG. 1. Overview of study design and assignment of individual male and female littermates to experimental end points.

 
Female littermates were used to determine the effect of dietary treatment on manganese body burden. Striatal and liver concentrations were determined in the F1 female rats on PND 21, 49 ± 1, 63 ± 2, 77 ± 2, and 91 ± 2 (n = 1 rat/litter/time point). The F1 male littermates were used to determine the effect of dietary manganese treatment on the pharmacokinetics of inhaled MnSO4. Male littermates were exposed by whole-body inhalation for 6 h/day on 14 consecutive days to MnSO4 at concentrations equivalent to 0, 0.03, or 0.3 mg Mn/m3. End-of-exposure tissue manganese concentrations (n = 1 rat/litter/MnSO4 exposure group) and whole-body elimination of a 54Mn tracer (n = 1 rat/litter/MnSO4 exposure group) were then determined in the MnSO4-exposed male rats.

Animals.
The study was conducted under federal guidelines for the care and use of laboratory animals (National Research Council, 1996) and was approved by the Institutional Animal Care and Use Committee of the Chemical Industry Institute of Toxicology. Fifty primiparous pregnant (sperm-plug-positive = gestation day [GD] 0) Crl:CD®(SD)BR rats were purchased on GD 14 from Charles River Laboratories, Inc. (Raleigh, NC) for this experiment. All presumed pregnant rats were initially fed a pelleted, semipurified AIN-93G certified diet from Bio-Serv (Frenchtown, NJ) that nominally contained 10 ppm manganese and 50 ppm iron. Pregnant rats and lactating dams with their pups were individually housed in polycarbonate cages with stainless steel lids (Laboratory Products, Inc., Rochelle Park, NJ) with Alpha-DriTM cellulose fiber chip bedding (Shepherd Specialty Papers, Kalamazoo, MI). Following parturition, 24 litters with at least 6 male and 4 female F1 rats per litter were assigned, on PND 10, to one of 3 AIN-93G certified diets (Bio-Serv, Frenchtown, NJ) containing 2, 10, or 100 ppm manganese and 50 ppm iron. All diets were fed ad libitum except during inhalation exposures. Reverse osmosis purified water containing < 0.002 µg Mn/ml was also available ad libitum. On PND 21, each litter was weaned, and rat pups were individually identified with an eartag, and maintained on their respective diets throughout the duration of the study. All parental (F0) female rats were euthanized after weaning on PND 23 ± 2. Weaned F1 animals were individually housed in suspended stainless steel cages. The animal rooms and exposure chambers were maintained at 22 ± 2°C, relative humidity of 30–70%, and an air flow rate sufficient to provide 10–15 air changes per h. Fluorescent lighting was controlled by automatic controls (lights on 0600–1800 h). The general condition of all animals was checked daily. Clinical examinations and body weights were recorded at least once weekly.

Manganese exposures.
The 1-m3 stainless steel and glass inhalation exposure chambers (Lab Products, Seaford, DE), airflows, chamber temperature and humidity monitoring procedures, animal rotation schedule, and methods used to check for uniformity of distribution of the aerosol have been previously described (Vitarella et al., 2000Go). The MnSO4 atmospheres were generated using a commercially available dry powder (Aldrich Chemical Co., Milwaukee, WI). A Wright Dust Feeder packed to 2000 psi, with a 20-psi air delivery pressure, and a Trost Airjet Mill (Garlock Corp., Newton, PA) were used to generate the initial aerosol. The MnSO4 aerosol was delivered into an approximately 38-l stainless steel prechamber (fitted with a 85Kr discharging unit) that allowed settling of larger (>10 µm) particles. The aerosol then went through a polyvinyl chloride distribution line tuned to allow equal residence time of particles in the pathway to each 1-m3 exposure chamber. The aerosol was diluted to the appropriate concentrations in the inlet air stream to each 1-m3 inhalation chamber. The aerosol in the prechamber and in each 1-m3 inhalation chamber was monitored continuously using an optical particle sensor (Realtime Aerosol Monitor, Model RAM-S, MIE Inc., Bedford, MA) precalibrated for a standard dust (Arizona road dust). The particle size distribution was measured with an aerosol particle spectrometer (APS) (Aerodynamic Particle Sizer, TSI, Inc., Minneapolis, MN), and, whenever possible, a microorifice uniform deposit cascade impactor (MOUDI, MSP Corp., Minneapolis, MN) was used to confirm the APS results. The APS uses a time-of-flight method to determine aerodynamic diameter of individual particles and is capable of sampling the low concentration aerosols used in this study. The APS reports the count median aerodynamic diameter (CMAD) and geometric standard deviation ({sigma}g). The traditionally reported mass median aerodynamic diameter (MMAD) was calculated from the CMAD and {sigma}g using the standard Hatch-Choate equations (Hinds, 1982Go). Nominal MnSO4 exposure concentrations were 0, 0.092, and 0.92 mg MnSO4/m3, corresponding to 0, 0.03, and 0.3 mg Mn/m3. The target particle size distribution was 1.5–2 µm MMAD with a {sigma}g < 2. Control groups were exposed to HEPA-filtered air only. The mean daily inhalation chamber temperature was maintained between 21.7 and 23.8°C, and relative humidity was maintained at 48 to 54%.

Tissue collection and manganese analysis.
Whenever possible, striatal and liver manganese concentrations were determined in CO2-euthanized F1 female rats on PND 21, 49 ± 1, 63 ± 2, and 77 ± 2 (n = 1 rat/litter/time point). Litters without at least 5 live F1 female rats per litter were sampled in a manner to maintain an adequate sample size at the latest sampling time points. On PND 91 ± 2, the remaining F1 female rats (n = 1 rat/litter) were fasted overnight and anesthetized with pentobarbital (50 mg/kg, ip). The common bile duct was then cannulated using polyethylene (PE-10) tubing (Intramedic, Sparks, MD). Bile (~0.5–1 ml) was collected from individual animals for approximately 1 h. A 3- to 5-ml sample of blood was collected by cardiac puncture immediately following the collection of bile. Serum was then obtained by centrifugation of clotted blood at room temperature for 20 min at 3000 g. The F1 female rats were then euthanized by exsanguination, and the striatum, olfactory bulb, cerebellum, lung, liver, proximal portion of the left femur, and ovary samples were collected for tissue manganese determination.

Tissue manganese concentrations were also determined in MnSO4-exposed F1 male rats. The PND 91 F1 male rats (n = 1 rat/litter/MnSO4 exposure group) were fasted overnight prior to their last inhalation exposure. Immediately after their last (i.e., 14th) inhalation exposure, the F1 male rats (n = 1 rat/litter/MnSO4 exposure group) were anesthetized with pentobarbital (50 mg/kg, ip), and a sample (~1-ml) of bile and serum was collected using previously described methods. The F1 male rats were then euthanized by exsanguination, and striatum, olfactory bulb, cerebellum, lung, liver, proximal portion of the left femur, and testes samples were collected for tissue manganese determination.

All tissue, serum, and bile samples had their weight or volume measured and were placed in high purity linear polyethylene vials, frozen in liquid nitrogen, and stored at approximately –70°C until analysis. Total tissue manganese concentrations were determined by neutron activation analysis at the Nuclear Energy Services, North Carolina State University. Samples were irradiated individually for 20 to 60 s at a flux of 1.5 x 1013 n/cm2-s. After a monitored decay, each sample vial was counted for 200 s on Ortec 38% and 42% GeLi detectors coupled to Ortec Omnigam-N computerized gamma detection systems. Method blanks and reference liver samples obtained from the National Institute of Standards and Technology (Gaithersburg, MD) were also irradiated and analyzed with the samples.

54Mn tracer studies.
Immediately after the last MnSO4 inhalation exposure, the remaining F1 male rats from each exposure group (n = 1 rat/litter/MnSO4 exposure group) were given 0.5 µCi of 54MnCl2 (in sterile saline) by tail vein injection (0.4 ml injection volume). Whole-body gamma spectrometry was performed immediately after injection and at 1, 2, 4, 6, 9, and 12 weeks after 54Mn tracer administration. An integrated spectrometry system, consisting of a multichannel analyzer with 2 perpendicular 10.2-cm diameter sodium iodide scintillation detectors, preamplifier, amplifier, and high voltage power supply (Nuclear Data, Inc., Schaumburg, IL), was used to determine total radioactivity of 54Mn in the animal's body. Animals were placed in plastic restraint tubes during whole-body gamma spectrometry (live time < 2 min).

Pharmacokinetic estimates were made after fitting a nonlinear regression curve to the individual whole-body 54Mn concentration-time profiles data by model-dependent methods (WinNonlin, Pharsight Corp., Cary, NC). The use of a two-compartment model provided the best-fit curve to the data (Vitarella et al., 2000Go). Pharmacokinetic parameters were calculated from standard kinetic formulas (Shargel and Yu, 1985Go) and included estimates of total body clearance (Clb), initial and terminal phase elimination half-lives (t1/2{alpha} and t1/2ß), and the area under the curve (AUC0 -> {infty}).

Statistics.
Following an assessment for homogeneity of variance (Levene's test), the data for quantitative, continuous variables (e.g., body weights, tissue manganese concentrations) collected from all female rats and male rats prior to their assignment to the inhalation phase of the study were intercompared for the 3 diet groups by analysis of variance (ANOVA). A Tukey's honestly significant difference test was used to perform pairwise multiple comparisons. All experimental data obtained from MnSO4-exposed F1 male rats (e.g., tissue manganese concentrations, pharmacokinetic parameters) were analyzed by an analysis of variance appropriate for a split-plot experimental design to test for the main effects of diet and exposure and also their interactions. If the diet`s main effect was significant, a Tukey's honestly significant difference test was used to determine which diet levels were different. If the exposure`s main effect was significant, a Dunnett's test was used to compare the two MnSO4 exposure levels to the air-exposed controls. Statistical analyses were performed using SAS Statistical Software (JMP, SAS Institute, Inc., Cary, NC). Unless otherwise noted, data presented are for the mean values ± standard error of the mean (SEM). A probability value of 0.01 was used for Levene's test, while p < 0.05 was used as the critical level of significance for all other statistical tests.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MnSO4 Test Atmospheres
Based upon optical particle sensor results, the actual MnSO4 chamber concentrations (mean ± SD) were 0.101 ± 0.011 and 0.90 ± 0.17 mg/m3 for the target concentrations of 0.092 and 0.92 mg MnSO4/m3, respectively. The grand mean (± SD) for gravimetric filter concentrations were 0.014 ± 0.01, 0.12 ± 0.03, and 0.82 ± 0.16 mg/m3 for the target concentrations of 0, 0.092, and 0.92 mg MnSO4/m3, respectively. The aerosol size distribution measured by the aerosol particle spectrometer in the exposure chambers on a given day was consistent with less than a 3% difference in CMAD between chambers. The average CMAD for the 2 exposure chambers on 2 separate days were 0.98 and 0.89 µm. The calculated MMAD for these days were 1.4 and 1.07 µm, respectively. A {sigma}g of 1.42 and 1.29 were observed on these 2 days. On a different day, a single cascade impactor from the 0.92 mg MnSO4/m3 chamber indicated a size distribution with MMAD and {sigma}g of 2.1 µm and 1.7, respectively.

Body Weight Gain, Clinical Signs, and Necropsy Findings
At 8 weeks after the start of the diet, male rats given the 2-ppm diet from PND 10 to 77 ± 2 had significantly lower PND 77 body weights than those observed in rats given the 100-ppm manganese diet (Fig. 2Go). Short-term (14-day) inhalation exposure to MnSO4 did not affect F1 male rat body weight (data not shown). Adverse treatment-related clinical effects were not observed in any animals.



View larger version (16K):
[in this window]
[in a new window]
 
FIG. 2. Mean (± SEM) body weights of growing male and female rats given manganese-deficient (2-ppm) or sufficient (10- or 100-ppm) diets. *Rats given the 2-ppm manganese diet had significantly lower PND 77 body weights than rats given the 100-ppm manganese diet (p < 0.05).

 
Tissue Manganese Concentrations in F1 Female Rats following Dietary Manipulation
Liver and striatal manganese concentrations in PND 21–91 F1 female rats are presented in Figure 3Go. Peak liver manganese concentrations in neonatal female rats given the 100-ppm manganese diet were observed at weaning on PND 21. Liver manganese concentrations in animals from all dietary treatment groups appeared to stabilize by PND 49. Female rats maintained on the marginally manganese-deficient diet (2 ppm) had lower liver manganese concentrations than those observed in animals fed a manganese-sufficient diet (10 or 100 ppm). When compared with female rats given diets containing the level of manganese (10 ppm) recommended by the National Research Council (NRC, 1995Go), animals fed the marginally manganese-deficient diet did not develop decreased liver manganese concentrations until PND 63. When compared with female rats given the high-normal manganese diet (100 ppm), however, animals fed the 2-ppm manganese diet maintained decreased liver manganese concentrations throughout the PND 21–91 observation period. Striatal manganese concentrations were unaffected by dietary manipulation.



View larger version (18K):
[in this window]
[in a new window]
 
FIG. 3. Mean (± SEM) liver and striatal manganese concentrations in growing female rats. *Indicates that rats given a marginally manganese-deficient diet (2 ppm) had decreased liver manganese concentrations when compared with rats maintained on manganese-sufficient diets (10 or 100 ppm). {dagger}Indicates that rats given the 100-ppm manganese diet had significantly higher liver manganese concentrations when compared with rats maintained on either the 2- or 10-ppm manganese diet. (p <= 0.05).

 
Tissue manganese concentrations in PND 91 ± 2 F1 female rats are presented in Table 1Go. Equivalent liver and bile manganese concentrations were observed in rats given manganese-sufficient diets (10 or 100 ppm). PND 91 female rats maintained on the manganese-deficient diet (2 ppm) had decreased liver manganese concentrations and reduced biliary manganese excretion when compared to rats fed either manganese-sufficient diet (10 or 100 ppm). PND 91 female rats maintained on the manganese-deficient diet (2 ppm) also had lower cerebellar manganese concentrations when compared to rats fed the NRC-recommended level of manganese (10 ppm). Although cerebellar manganese concentrations were also higher in PND 91 female rats given the 100-ppm manganese diet, this increase was not statistically significant when compared with female rats given the 2-ppm manganese diet (p = 0.065). All other PND 91 female rat tissue manganese concentrations were unaffected by dietary manipulation.


View this table:
[in this window]
[in a new window]
 
TABLE 1 Mean Tissue Manganese Concentrations in PND 91 ± 2 F1 Female Rats Given a 2-, 10-, or 100-ppm Manganese Diet Beginning on PND 10
 
Tissue Manganese Concentrations in F1 Male Rats following Dietary Manipulation and MnSO4 Inhalation
End-of-exposure tissue manganese concentrations are presented in Table 2Go. No interactions between dietary manganese level and manganese inhalation exposure concentration were observed. An overall main treatment effect related to inhalation exposure was observed on some tissue manganese concentrations (Table 3Go). Male rats exposed to 0.092 mg MnSO4/m3 had elevated lung manganese concentrations when compared to air-exposed male rats. Male rats exposed to 0.92 mg MnSO4/m3 developed increased striatal, lung, and bile manganese concentrations when compared to air-exposed male rats. An overall main treatment effect related to diet was also observed in some tissues (Table 4Go). Rats given a marginally manganese-deficient diet (2 ppm) had decreased cerebellar manganese concentrations compared with levels observed in rats given the high-normal manganese diet (100 ppm). Male rats given the marginally deficient manganese diet (2 ppm) also developed decreased liver manganese concentrations when compared with animals given the diet containing the current NRC-recommended level of manganese (10 ppm). Surprisingly, similar liver manganese concentrations were observed in rats given either the 2- or 100-ppm manganese diet (p = 0.089).


View this table:
[in this window]
[in a new window]
 
TABLE 2 End-of-Exposure Mean Tissue Manganese Concentrations in MnSO4-Exposed Male Rats
 

View this table:
[in this window]
[in a new window]
 
TABLE 3 End-of-Exposure Overall Mean Tissue Manganese Concentrations Used to Test for an Inhalation-Related Main Effect
 

View this table:
[in this window]
[in a new window]
 
TABLE 4 End-of-Exposure Overall Mean Tissue Manganese Concentrations Used to Test for a Diet-Related Main Effect
 
54Mn Tracer Pharmacokinetics following MnSO4 Inhalation
The whole-body elimination of the 54Mn tracer given immediately following the two-week MnSO4 inhalation exposure could be mathematically described using a two-compartment open model. Mean values for the pharmacokinetic parameters are presented in Table 5Go. A statistically significant diet and inhalation interaction was observed for only the AUC. An overall main treatment effect related to diet was observed for some pharmacokinetic parameters (Table 6Go). Rats given either the 10- or 100-ppm manganese diet had significantly lower AUC and increased manganese clearance rates when compared with rats given the 2-ppm manganese diet (Fig. 4Go). An overall main treatment effect related to inhalation exposure was likewise observed for AUC and whole-body 54Mn clearance rates (Table 6Go). Rats exposed to 0.92 mg MnSO4/m3 had significantly lower AUC and increased manganese clearance when compared with air-exposed control rats (Fig. 4Go). Rats exposed to 0.92 mg MnSO4/m3 also had a shorter initial phase elimination half-life (t1/2{alpha}); however, this effect was marginally significant (p = 0.054). Terminal phase elimination half-lives (t1/2ß) were not affected by either dietary manganese levels or MnSO4 inhalation.


View this table:
[in this window]
[in a new window]
 
TABLE 5 Selected Pharmacokinetic Parameters Describing 54Mn Elimination in Rats Exposed Repeatedly (14 exposures) to MnSO4
 

View this table:
[in this window]
[in a new window]
 
TABLE 6 Pharmacokinetic Parameter Overall Means Used to Test for Dietary and Inhalation Exposure Main Effects
 


View larger version (19K):
[in this window]
[in a new window]
 
FIG. 4. Whole-body elimination of a 54Mn tracer in male CD rats following repeated inhalation exposure to MnSO4. Mean ± SEM (n = 8 rats/exposure concentration). *Indicates increased whole-body clearance rate in MnSO4-exposed rats (0.92 mg MnSO4/m3) when compared to their respective air-exposed controls (p <= 0.05).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The objective of this study was to determine whether manganese body burden influences the pharmacokinetics of inhaled MnSO4. To achieve this goal, we used an experimental design that included 3 dietary levels (2, 10, and 100 ppm manganese) and 3 MnSO4 exposure levels (0, 0.092, and 0.92 mg MnSO4/m3). Our results suggest that tissue manganese concentrations are not influenced by an interaction between the level of manganese found in the diet and short-term MnSO4 inhalation.

Frank manganese deficiency has been rarely reported in humans, therefore, the lowest level of dietary manganese used in this study (2 ppm) was intended to provide a marginally deficient level of manganese as may occur in humans with suboptimal manganese status. The 10-ppm manganese diet used meets current rodent dietary guidelines (NRC, 1995Go). The high-normal diet contains supplemental manganese at a level (100 ppm) similar to that found in the NIH-07 rodent diet used in previous manganese inhalation studies conducted by our laboratory (Dorman et al., 2001Go; Vitarella et al., 2000Go).

As expected, the feeding of the 2-ppm manganese diet was associated with a number of effects, including reduced body weight gain, decreased liver manganese concentrations, and reduced whole-body manganese clearance rates. Male rats given either the marginally deficient diet or the diet containing the current NRC-recommended level (10 ppm) of manganese had equivalent growth rates resulting in similar terminal body weights. However, male rats given the 2-ppm diet had pre-MnSO4 exposure (PND 77) body weights that were significantly approximately 5% lower than those observed in rats given the 100-ppm manganese diet. This observation likely reflects both growth-retarding and growth-promoting effects associated with diets that are either low or high in manganese (Keen et al., 1999Go).

Despite developing modest decreases in pup growth, manganese concentrations in most tissues remained unaffected by our dietary manipulations. This finding most likely reflects the use of a low manganese diet (2 ppm) rather than a more severely depleted diet containing < 1 ppm manganese. For example, Malecki and coworkers (1994) reported a 40 to 50% decrease in tissue manganese concentrations in rats fed a 1-ppm manganese diet for 2 months. It is also likely that a more profound alteration in tissue manganese concentration would have occurred if a longer period of dietary restriction were used in our study. Animals will develop frank manganese deficiency under these more extreme dietary conditions. However, as stated earlier, clinical manganese deficiency is rarely reported in humans. Thus, our results are most relevant for people with suboptimal levels of manganese in their diets.

Although manganese concentrations in many tissues were unaffected, our dietary manipulations did induce altered hepatic metabolism of manganese. Liver manganese concentrations observed in the F1 female littermates maintained on the 100-ppm manganese diet were highest at weaning and stabilized at a lower concentration by PND 49. This decline in liver manganese level was probably associated with the maturation of biliary manganese excretion mechanisms that emerge in rats near weaning (Keen et al., 1981Go; Miller et al., 1975Go; Rehnberg et al., 1982Go). Despite this decline, liver manganese concentrations in PND 63–91 female F1 rats given manganese-sufficient diets (10 or 100 ppm) remained 33 to 40% higher than those observed in female rats given the 2-ppm manganese diet. Our results also show that biliary excretion of manganese was influenced by oral manganese intake. When compared to animals maintained on manganese-sufficient diets, female rats kept on the marginally manganese-deficient diet had reduced bile manganese concentrations. Similarly, male rats kept on the marginally manganese-deficient diet had decreased biliary manganese excretion as evidenced by their reduced whole-body elimination of a 54Mn tracer when compared with rats given manganese-sufficient diets.

The highest MnSO4 exposure level used in the present study (0.92 mg MnSO4/m3) is several orders of magnitude higher than those found in the ambient air or in metropolitan areas where the gasoline additive MMT has been widely used for the past 10 years (Clayton et al., 1999Go; Loranger and Zayed, 1997Go; Pellizzari et al., 1999Go; Zayed et al., 1999bGo). Short-term exposure of rats to 0.92 mg MnSO4/m3 is associated with elevated olfactory bulb, lung, and testis manganese concentrations in male rats maintained on an open diet containing approximately 100 ppm manganese (Dorman et al., 2001Go). The manganese exposure concentrations used in our study are orders of magnitude higher than those found in the ambient air or in metropolitan areas where the gasoline additive MMT has been widely used for the past 10 years (Clayton et al., 1999Go; Loranger and Zayed, 1997Go; Pellizzari et al., 1999Go; Zayed et al., 1999bGo). Our previous results suggested that the lowest exposure level (0.092 mg MnSO4/m3) would not alter manganese concentrations in any of the tissues examined in our present study. The results of our current study are qualitatively similar to this previous experiment. In our previous experiment, for example, rats exposed to 0.92 mg MnSO4/m3 had striatal manganese concentrations that were approximately 26% higher than their respective air-exposed controls, although this increase was not found to be statistically significant. In our present experiment, we observed a similar 33% increase in striatal manganese concentrations that was statistically significant. Similar trends were observed with other tissues. Although tissues identified as having increased manganese concentrations differ between these two experiments, these differences likely reflect the small sample sizes used in these experiments rather than a biologically significant difference in manganese metabolism or distribution.

Manganese concentrations in the brain and other target tissues may be influenced by biliary excretion of manganese. For example, liver pathology or surgical or pharmacological manipulations that result in biliary stasis can result in increased brain manganese concentrations (Davis et al., 1998Go, Hauser et al., 1996Go). The use of a 2-ppm manganese diet was intended to induce a state of relative manganese deficiency resulting in reduced biliary excretion of manganese. Our observation of decreased whole-body 54Mn clearance rates and reduced biliary manganese concentrations in rats given the marginally deficient manganese diet indicate that this objective was indeed achieved. Therefore we were surprised that we did not find an interaction between dietary manganese level and inhalation exposure to manganese. Our inability to detect an interaction is most likely the result of an animal's ability to adjust its manganese clearance rates in response to the MnSO4 inhalation exposure. For example, despite being in a relatively manganese-deficient state, rats given the 2-ppm manganese diet and exposed to 0.92 mg MnSO4/m3 increased their rate of manganese excretion by approximately 45% when compared to their respective air-exposed control group. This observation suggests that, at least initially, animals will not retain inhaled manganese to compensate for relative nutritional deficiencies in this essential metal. Likewise, rats given either the 10- or 100-ppm manganese diet and exposed to 0.92 mg MnSO4/m3 also increased their manganese clearance rates when compared to their respective air-exposed control groups. Collectively, these data provide strong evidence that dose-dependent biliary excretion of manganese will serve to regulate the percentage of inhaled manganese retained by manganese-deficient animals, thereby limiting available manganese that could result in increased systemic tissue manganese concentrations. Additional studies evaluating route-to-route differences between the metabolism of inhaled and ingested manganese should be able to determine exposure conditions under which these homeostatic mechanisms begin to fail, resulting in elevated brain manganese concentrations. Our results suggest that even moderately manganese-deficient individuals are not likely to develop elevated brain manganese concentrations following short-term inhalation exposure to moderately high levels of manganese.


    ACKNOWLEDGMENTS
 
The authors would like to thank Dr. Jack Weaver (North Carolina State University) for his technical assistance, and the staff of the CIIT animal care facility for their contributions. We also thank Drs. Susan Borghoff, Karrie Brenneman, Paul Foster, Fred Miller, and Owen Moss for their critical review of this manuscript and Dr. Barbara Kuyper for editorial review. This publication is based on a study sponsored and funded by the Ethyl 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.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (919) 558–1300. E-mail: dorman{at}ciit.org. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ATSDR (1992). Toxicological Profile for Manganese. Agency for Toxic Substances and Disease Registry, U.S. Department of Health and Human Services, Atlanta, GA.

Andersen, M. E., Gearhart, J. M., and Clewell III, H. J. (1999). Pharmacokinetic data needs to support risk assessments for inhaled and ingested manganese. Neurotoxicology 20, 161–171.[ISI][Medline]

Barbeau, A. (1984). Manganese and extrapyramidal disorders. A critical review and tribute to Dr. George C. Cotzias. Neurotoxicology 5, 13–35.[ISI][Medline]

Beuter, A., Mergler, D., de Geoffroy, A., Carriere, L., Belanger, S., Varghese, L., Sreekumar, J., and Gauthier, S. (1994). Diadochokinesimetry: A study of patients with Parkinson's disease and manganese-exposed workers. Neurotoxicology 15, 655–664.[ISI][Medline]

Britton, A. A., and Cotzias, G. C. (1966). Dependence of manganese turnover on intake. Am. J. Physiol. 211, 203–206.[Medline]

Calne, D. B., Chu, N.-S., Huang, C.-C., Lu, C.-S., and Olanow, W. (1994). Manganism and idiopathic parkinsonism: Similarities and differences. Neurology 44, 1583–1586.[Abstract]

Carl, G. F., and Gallagher, B. B. (1994). Manganese and epilepsy. In Manganese in Health and Disease (D. J. Klimis-Tavantzis, Ed.), pp. 59–86. CRC Press, Boca Raton, FL.

Clayton, C. A., Pellizzari, E. D., Rodes, C. E., Mason, R. E., and Piper, L. (1999). Estimating distributions of long-term particulate matter and manganese exposures for residents of Toronto, Canada. Atmos. Environ. 33, 2515–2526.[ISI]

Davis, C. D., Schafer, D. M., and Finley, J. W. (1998). Effect of biliary ligation on manganese accumulation in rat brain. Biol. Trace Elem. Res. 64, 61–74.[ISI][Medline]

Davis, C. D., Zech, L., and Greger, J. L. (1993). Manganese metabolism in rats: An improved methodology for assessing gut endogenous losses. Proc. Soc. Exp. Biol. Med. 202, 103–108.[Abstract]

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.[ISI][Medline]

Finley, J. W., and Davis, C. D. (1999). Manganese deficiency and toxicity: Are high or low dietary amounts of manganese cause for concern? Biofactors 10, 15–24.[ISI][Medline]

Fitzgerald, K., Mikalunas, V., Rubin, H., McCarthey, R., Vanagunas, A., and Craig, R. M. (1999). Hypermanganesemia in patients receiving total parenteral nutrition. J. Parenter. Enteral. Nutr. 23, 333–336.[Abstract]

Freeland-Graves, J., and Llanes, C (1994). Models to study manganese deficiency. In Manganese in Health and Disease (D. J. Klimis-Tavantzis, Ed.), pp. 115–120. CRC Press, Boca Raton, FL.

Hauser, R. A., Zesiewicz, T. A., Martinez, C., Rosemurgy, A. S, and Olanow, C. W. (1996). Blood manganese correlates with brain magnetic resonance imaging changes in patients with liver disease. Can. J. Neurol. Sci. 23, 95–98.[ISI][Medline]

Hinds, W. C. (1982). Aerosol Technology. John Wiley and Sons, New York.

Ingersoll, R. T., Montgomery, E. B., and Aposhian, H. V. (1995). Central nervous system toxicity of manganese: I. Inhibition of spontaneous motor activity in rats after intrathecal administration of manganese chloride, Fundam. Appl. Toxicol. 27, 106–113.[ISI][Medline]

Kawamura, R., Ikuta, H., Fukuzumi, S., Yamada, R., Tsubaki, S., Kodama, T., and Kurata, S. (1941). Intoxication by manganese in well water. Kitasato Arch. Exp. Med. 18, 145–169.

Keen, C. L., Ensunsa, J. L., Watson, M. H., Baly, D. L., Donovan, S. M., Monaco, M. H., and Clegg, M. S. (1999). Nutritional aspects of manganese from experimental studies. Neurotoxicology 20, 213–223.[ISI][Medline]

Keen, C. L., Lonnerdal, B., Clegg, M. and Hurley, L. S. (1981). Developmental changes in composition of rat milk: Trace elements, minerals, protein, carbohydrate, and fat. J. Nutr. 111, 226–230.[ISI][Medline]

Keen, C. L. and Zidenberg-Cherr, S. (1994). Manganese toxicity in humans and experimental animals. In Manganese in Health and Disease (D.J. Klimis-Tavantzis, Ed.), pp. 193–205. CRC Press, Boca Raton.

Kondakis, X. G., Makris, N., Leotsinidis, M., Prinou, M., and Papapetropoulos, T. (1989). Possible health effects of high manganese concentrations in drinking water. Arch. Environ. Health 44, 175–178.[ISI][Medline]

Loranger, S., and Zayed, J. (1997). Environmental contamination and human exposure to airborne total and respirable manganese in Montreal. J. Air Waste Manag. Assoc. 47, 983–989.[ISI][Medline]

Lynam, D. R., Roos, J. W., Pfeifer, G. D., Fort, B. F., and Pullin, T. G. (1999). Environmental effects and exposures to manganese from use of methylcyclopentadienyl manganese tricarbonyl (MMT) in gasoline. Neurotoxicology 20, 145–150.[ISI][Medline]

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

Malecki, E. A., Huttner, D. L., and Greger, J. L. (1994). Manganese status, gut endogenous losses of manganese, and antioxidant enzyme activity in rats fed varying levels of manganese and fat. Biol. Trace Elem. Res. 42, 17–29.[ISI][Medline]

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.[ISI][Medline]

Miller, S. T., Cotzias, G. C., and Evert, H. A. (1975). Control of tissue manganese: Initial absence and sudden emergence of excretion in the neonatal mouse, Am. J. Physiol. 229, 1080–1084.[Medline]

Moore, W., Hysell, D., Miller, R., Malanchuk, M., Hinners, R., Yang, Y., and Stara, J. F. (1975). Exposure of laboratory animals to atmospheric manganese from automotive emissions. Environ Res. 9, 274–284.[ISI][Medline]

Nagatomo, S., Umehara, F., Hanada, K., Nobuhara, Y., Takenaga, S., Arimura, K., and Osame, M. (1999). Manganese intoxication during total parenteral nutrition: Report of two cases and review of the literature. J. Neurol. Sci. 162, 102–105.[ISI][Medline]

NRC (1995). Nutrient Requirements for Laboratory Animals, 4th Ed. National Research Council. National Academic Press, Washington, DC.

NRC (1996). Guide for the Care and Use of Laboratory Animals. National Research Council. National Academic Press, Washington, DC.

Olanow, C. W., Good, P. F., Shinotoh, H., Hewitt, K. A., Vingerhoets, F., Snow, B. J., Beal, M. F., Calne, D. B., and Perl, D. P. (1996). Manganese intoxication in the rhesus monkey: A clinical, imaging, pathologic, and biochemical study. Neurology 46, 492–498.[Abstract]

Pal, P. K., Samii, A., and Calne, D. B. (1999). Manganese neurotoxicity: A review of clinical features, imaging, and pathology. Neurotoxicology 20, 227–238.[ISI][Medline]

Papavasiliou, P. S., Miller, S. T., and Cotzias, G. C. (1966). Role of liver in regulating distribution and excretion of manganese. Am. J. Physiol. 211, 211–216.[Medline]

Pellizzari, E. D., Clayton, C. A., Rodes, C. Mason, R. E., Piper, L., Fort, B. F., Pfeifer, G. D., and Lynam, D. R. (1999). Particulate matter and manganese exposures in Toronto, Canada. Atmos. Environ. 33, 721–734.[ISI]

Rehnberg, G. L., Hein, J. F., Carter, S. D., Linko, R. S., and Laskey, J. W. (1982). Chronic manganese oxide administration to preweanling rats: Manganese accumulation and distribution, J. Toxicol. Environ. Health 6, 217–226.

Ressler, T., Wong, J., and Roos, J. (1999). Manganese speciation in exhaust particulates of automobiles using MMT containing gasoline. J. Synchrotron Radiation 6, 656–658.[ISI][Medline]

Schroeder, H. A., Balassa, J. J., and Tipton, I. H. (1966). Essential trace metals in man: Manganese, a study in homeostasis. J. Chronic. Dis. 19, 545–571.[ISI][Medline]

Shargel, L. and Yu, A. B. (1985). Multicompartment models. In Applied Biopharmaceutics and Pharmacokinetics (L. Shargel and A.B. Yu, Eds.), pp. 51–66. Appleton-Century-Crofts, Norwalk, CT,

Ter Haar, G. L., Griffing, M. E., Brandt, M., Oberding, D. G., and Kapron, M. (1975). Methylcyclopentadienyl manganese tricarbonyl as an antiknock: composition and fate of manganese exhaust products. J. Air Pollut. Control Assoc. 25, 858–860.[ISI]

Vitarella D., Wong, B. A., Moss, O. R., and Dorman, D. C. (2000). Pharmacokinetics of inhaled manganese phosphate in male Sprague-Dawley rats following subacute (14-day) exposure. Toxicol. Appl. Pharmacol. 163, 279–285.[ISI][Medline]

Wedler, F. C. (1996). Manganese. In Role of Glia in Neurotoxicology (M. Aschner and H. K. Kimmelberg, Eds.), pp. 155–173. CRC Press, New York.

Weiss, B. (1999). Manganese in the context of an integrated risk and decision process. Neurotoxicology 20, 519–525.[ISI][Medline]

Zayed, J., Hong, B., and L'Esperance, G. (1999a). Characterization of manganese-containing particles collected from the exhaust emissions of automobiles running with MMT additive. Env. Sci. Technol. 33, 3341–3346.[ISI]

Zayed, J., Thibault, C., Gareau, L., and Kennedy, G. (1999b). Airborne manganese particulates and methylcyclopentadienyl manganese tricarbonyl (MMT) at selected outdoor sites in Montreal. Neurotoxicology 20, 151–157.[ISI][Medline]