Division of Environmental Health Sciences, School of Public Health, and Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York 10032
Received September 13, 1999; accepted November 18, 1999
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ABSTRACT |
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Key Words: manganese; methylcyclopentadienyl manganese tricarbonyl (MMT); toxicokinetics; bioavailability; half-life; clearance; volume of distribution; Sprague-Dawley rats.
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INTRODUCTION |
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Manganese-induced neurologic lesions are located in the globus pallidus and striatum of the basal ganglia. The pallidus and striatum display a marked decrease in myelinated nerve fibers, accompanied by depletion of striatal dopamine (Bonilla, 1980; Eriksson et al., 1987
; Mena et al., 1967
, Olanow et al., 1996
). These neuropathologic alterations have been observed among manganese-intoxicated experimental primates, which display the illustrative neurologic symptoms (Olanow et al., 1996
; Pentschew et al., 1963
). In rodents, the similar neurobiochemical alterations have also been demonstrated. For example, chronic exposure to manganese in rodents has been shown to alter brain dopaminergic neurotransmitters (Bonilla, 1980
; Eriksson et al., 1987
; Gianutsos and Murray, 1982
; Parenti et al., 1986
; Rodriguez et al., 1998
) and inhibits critical enzymes involved in energy production (Seth et al, 1977
; Zheng et al., 1998
, 1999
). Manganese in rat brain may interact with other essential metal ions and cause oxidative stress in targeted brain areas (Ali et al., 1995
; Sloot et al., 1996
). Some researchers have characterized behavioral and pathologic changes in rodents due to chronic manganese exposure (Chandra et al, 1979
; Singh et al., 1974
). Local injection of manganese into rat brain also interferes with motor activity (Brouillet et al., 1993
; Ingersoll et al., 1995
). Moreover, following exposure to MMT, rodents show significant neuroexcitatory toxicity and altered normetanephrine levels (Fishman et al., 1987
; Komura and Sakamoto, 1994
).
In light of the broad application of rodents as acceptable models for the mechanistic studies of manganese neurotoxicity, it is surprising that no toxicokinetic study of either inorganic or organic manganese has been conducted in rodents. Particularly sparse is the knowledge on toxicokinetic parameters such as half-lives, volume of distribution, and clearance in some routinely used animal species. The lack of these fundamental measures has rendered it difficult to establish a physiologically relevant dosing regimen for chronic investigation of manganese toxicity in rodents. This study was therefore undertaken to evaluate the plasma kinetics of manganese in Sprague-Dawley rats following intravenous or oral administration of MnCl2 or following oral administration of MMT.
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MATERIALS AND METHODS |
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Animals.
Male Sprague-Dawley rats were purchased from Harlan Inc. (Indianapolis, IN) and were 210230 g (2 months old) at the time of the experiments. The animals were housed in a temperature-controlled, 12:12 h light/dark room, and were allowed free access to tap water and food (Teklad 4% Mouse-Rat Diet, Teklad, Madison, WI). For the oral dosing study, animals were fasted for 12 h prior to administration of MnCl2 or MMT.
Administration of manganese compounds.
MnCl2 was dissolved in sterile saline for iv or oral administration. For the iv dosing study, MnCl2 was injected via the tail vein at a dose of 6.0 mg Mn/kg (1.0 ml/kg) over approximately 5 sec. For the oral dosing study, MnCl2 was administered by a single gavage at the same dose level. This dose regimen was chosen because it was known to be associated with a significant reduction of succinic dehydrogenase and aconitase in rat brain (Seth et al., 1977; Singh et al., 1974
; Zheng et al., 1998
).
MMT was dissolved in corn oil and administered orally by gavage to rats at a dose of 20 mg/kg (3.3 ml/kg in volume). This dose was equivalent to 5.6 mg Mn/kg.
Collection of blood samples.
At the appropriate times, the rats were subjected to light anesthesia with ether. Blood samples (0.30.5 ml) were collected from the orbital sinus through heparin-pretreated glass capillary tube and transferred to an Eppendorf tube. For the MnCl2 study, the blood was collected prior to (as 0 h) and at 0.05, 0.17, 0.33, 0.5, 1, 2, 4, 8, and 12 h following either iv or oral administration. The blood was centrifuged at 5000 x g for 5 min, and the plasma was separated and stored at 20°C until analyzed. The plasma samples were usually analyzed for manganese content within 1 week.
For the MMT study, the procedure for sample collection was identical to that described above, except for the times of blood collection, which were prior to (as 0 h) and at 0.17, 0.5, 1, 2, 4, 8, 12, 24, 48, 120, 168, 288, 384, and 456 h following oral gavage.
Atomic absorption spectrophotometry (AAS) analysis.
Manganese concentrations in plasma were determined by a flameless graphite furnace AAS. Aliquots (50 µl) of plasma samples were diluted (1050 fold) with an appropriate volume of 8% Triton X-100 and 5% EDTA in distilled, deionized water prior to AAS. A Perkin-Elmer Model 3030 Zeeman AAS, equipped with an HGA-600 graphite furnace, was used for quantification. The standard curves were established using freshly made manganese standards on the day of analysis. The detection limit for this method was 0.2 ng Mn/ml of assay solution (Zheng et al., 1998, 1999
).
Toxicokinetic analysis.
Under normal conditions, the rats used in this experiment had baseline plasma manganese concentrations of 0.029 ± 0.011 µg/ml (mean ± SD, n = 13). To eliminate background interference, all plasma data were corrected for this baseline value prior to kinetics calculations.
For the iv dosing study, plasma concentration-time data were analyzed by a two-compartment model represented by the following equation:
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For the oral dosing study, the terminal elimination phases of most tested animals usually comprised 46 data points, which were inadequate for compartment model analysis. Thus, the plasma concentration-time data were analyzed by a noncompartmental method, rather than the compartment-based curve fitting approach. Values of Cmax and Tmax were obtained directly from plasma concentration-time profiles. The apparent first-order disposition rate constant (Ke) was estimated by linear least-squares regression of the data in the terminal phase. From these values, the half-lives were calculated (). The AUC and the total area under the first moment curve (AUMC) were calculated using the linear trapezoidal rule and extrapolating to time infinity. The mean residence time (MRT) and mean absorption or input time (MAT) were calculated as follows:
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Statistics.
All data are presented as mean ± SD. Statistical analysis for comparison of two means was performed using one-way ANOVA. In all cases, a probability level of p < 0.05 was considered as the criterion of significance.
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RESULTS |
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DISCUSSION |
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Given that hepatic clearance represents the majority of manganese systemic clearance, it is conceivable that the total body clearance of manganese would be close to the blood flow of the liver. In fact, by adjusting the body weight (i.e., 250 g), the total body clearance of manganese (1.8 ml/min) in the current study was about 9-fold less than (or 12% that of) the hepatic blood flow (15 ml/min) in rats (Hollinger, 1995). Assuming that the liver clears nearly all manganese ions present in hepatic blood flow, the hepatic extraction ratio (the ratio of hepatic clearance/hepatic blood flow) of manganese would seem unlikely to exceed 12% of manganese passing through the liver. Such a low hepatic extraction ratio is unexpected and might suggest a membrane-limited passive diffusion or transport of manganese ions by the liver. Further studies are needed to address this issue.
One of the perplexing questions in clinical monitoring of manganese toxicity is that blood levels of manganese usually poorly reflect the body burden of manganese and the ensuing disease status. There is a discrepancy between blood manganese and intracellularly distributed tissue manganese. This was apparently the case when our data were compared with those of others (Klaassen, 1974; Takeda et al., 1995
). In the current study, the terminal phase elimination t
following iv injection was about 1.8 h, and by 12 h, the plasma manganese returned to normal levels in all tested animals. Theoretically, such a short t
would not likely result in the accumulation of manganese in the body, assuming that manganese transport among tissues follows the mass balance. However, Takeda et al. (1995), by using autoradiography technique, reported that manganese persistently presented in various brain regions following radiotracer injection. The biologic t
of manganese in the basal ganglia, brain stem, and cerebral cortex was estimated to be between 5174 days. Klaassen also showed that manganese levels in liver after an iv injection dropped by 90% during a 5-day period. However, brain manganese levels were actually slightly increased (Klaassen, 1974
). In primates, the brain t
of manganese from inorganic manganese exposure was found to be as long as 53 days (Newland et al., 1987
). Comparison of our results with these earlier studies indicated that the brain manganese did not seem to decline in response to the blood manganese status. One of the possible explanations is that manganese may readily enter the brain by one-way transport mechanism at the blood-brain barrier and blood-CSF barrier in the choroid plexus (Aschner et al., 1999
). It is also possible that the intracellular binding and sequestration of manganese in the brain may prevent the metal from emigration to the extracellular space. The slow effluent brain manganese thus merges into the background level of plasma manganese. Under this condition, the observed plasma t
would not reflect the true brain burden of manganese. By the same token, this may explain the lack of correlation between blood manganese levels and total body burden of manganese, which is presumably associated with manganese toxicity seen in clinic.
Manganese was rapidly absorbed in the gastrointestinal tract after oral dosing. The absolute bioavailability of manganese in rats was about 34 times higher than that in humans (35%). As an essential element, human minimum daily dietary requirement for manganese ranges from about 2.5 to 5.0 mg/day. In an adult human, the normal blood concentration of manganese lies between 8.616 ng/ml (Aschner et al., 1999). In comparison, the rats, prior to manganese administration in this study, had average blood manganese concentration of about 28.6 ng/ml. Increased bioavailability in rats may explain the higher blood level of manganese in the rats than in humans.
The temporal pattern of plasma manganese following MMT oral administration could be characterized by the substantially delayed Tmax, large AUC, and prolonged t. Concerning the absolute manganese dosage in the dose formula, the MMT dose in this study (20 mg/kg) contained 5.6 mg/kg of manganese, which was comparable to the dose used in MnCl2 study (6 mg/kg). However, AUC in MMT-treated rats was 37-fold higher than that in MnCl2-treated rats. Furthermore, MMT-derived manganese appeared in blood in a much slower rate than did MnCl2-derived manganese (Fig. 2
vs. Fig. 3
). The Tmax in MMT-treated rats was about 30-fold longer than that in rats receiving oral dose of inorganic manganese (Fig. 4
). These results suggest a relatively complete but much slower absorption process following MMT dose administration.
This exceptionally slow absorption of MMT by the gastrointestinal tract was unexpected for MMT's high lipophilicity. As MMT was dissolved in corn oil, the slow release of MMT from the oily vehicle may contribute, at least in part, to the prolonged absorption duration. However, other biologic processes may also be taken into account. For example, unlike the inorganic manganese as the dose formula, MMT-derived manganese mainly accumulates in the lung and produces acute pulmonary hemorrhage edema, which is considered as the primary cause of MMT-induced death in animals (Hanzlik et al., 1980a, McGinley et al., 1987
). Whereas the lung serves as the target organ, the hepatic cytochrome P-450 metabolizing enzymes may play a critical role in toxicokinetics of MMT. The studies by Hanzlik et al., (1980a,b) demonstrated that rats pretreated with phenobarbital yielded a remarkable protection against lung injury and associated lethality caused by orally administered MMT. The same treatment also doubled the rate of excretion of biliary metabolites of MMT. These results suggest that MMT molecules, upon being absorbed from the gastrointestinal tract, undergo extensive first-pass hepatic biotransformation. The question as to what extent MMT or the manganese released from it enters the blood stream remains unanswered. Yet, the hepatic manipulation of MMT appears likely to underlie the distinct Tmax between MMT and MnCl2 in this study. It is also possible that the delayed absorption of MMT may be due to a saturated absorption mechanism in the gastrointestinal wall.
The terminal elimination t and the AUC of plasma manganese in MMT rats were about 12-fold and 37-fold, respectively, higher than those in rats orally administered with MnCl2 (Fig. 4
). Our data are consistent with the observation by Gianutsos et al. (1985), who reported that in adult mice, administration of MMT produced a long-lasting elevation of manganese concentrations in both blood and brain. In a rat model, MMT administration also caused MMT or its metabolites to be accumulated and retained in lung, liver, kidney, and brain (McGinley et al., 1987
; Komura and Sakamoto, 1994
). The prolonged t
and elevated AUC in this study, thus, suggest an accumulation of manganese in the body after exposure to MMT.
Comparison of clearance data between MnCl2 and MMT groups showed that MMT-derived manganese was cleared at a rate of about 4.8-fold slower than that in the MnCl2-dosed group (Table 2). As MMT is highly lipophilic, the main route of elimination of MMT-derived manganese in rats could be different from that of MnCl2-treated rats. Earlier studies have shown that after MMT oral ingestion 73% of the dose as manganese is eliminated in the first 24 h, of which 36% of MMT-derived manganese was present in the urine (Moore et al., 1974
). This ratio was much higher than that in MnCl2-exposed animals, whose urinary elimination was less than 1% of the dose. In phenobarbital-pretreated rats, during the first 48 h after administration of MMT, 7489% of the dose was eliminated in the urine as MMT or its metabolites, and fecal elimination amounted to only 24% of the dose (Hanzlik et al., 1980b
). It was subsequently postulated that the large fraction of administered MMT apparently remains intact as an organometallic complex while undergoing biotransformation and excretion. In contrast to the extensive biliary excretion following inorganic manganese administration (Klaassen, 1974
), a 6-h bile collection period found only about 1011% of the injected manganese dose in the bile (Hanzlik et al., 1980a
). In addition, Komura and Sakamoto (1994) have suggested that MMT has a higher absorption rate from the digestive tract than that of inorganic manganese, although the absolute bioavailability of MMT is still unknown. Thus, a combination of complete absorption, special biotransformation pathway, shifted elimination route, and large tissue retention of MMT may explain the long t
and large AUC observed in MMT-treated rats.
A distinct and significant gender-dependent difference in the toxicokinetic profiles of MMT-derived manganese was observed in the current study. Female rats had higher plasma concentrations of manganese and AUC than did the male rats (Fig. 3). Although the apparent volume of distribution (Vß/F) in males was similar to those in females following oral dosing of MMT (Table 2
), the apparent body clearance (CL/F) of manganese in the males was about twice as much as in the females. The elimination rate constant (Ke) was greater in male than in female rats. Hence, a slower elimination of MMT in female rats may account for the higher plasma manganese concentrations in this sex. As the gender difference in drug metabolism has been demonstrated in many other cases, we postulate that the gender-dependent metabolism of MMT may underlie this observation. Whether this gender difference is also present in humans would be interesting to explore.
In summary, manganese toxicokinetics after iv dosing of MnCl2 appeared to follow a two-compartment disposition model. Manganese in rats displayed a moderate volume of distribution and a relatively short t. The discrepancy between the plasma t
and tissue accumulation of manganese was discussed. Following oral dose administration, manganese was rapidly but poorly absorbed from the gastrointestinal tract. The percentage of the dose absorbed was about 13%. After oral dosing of MMT, the MMT-derived manganese entered the blood circulation at a slower rate, but to a much greater extent as compared to the oral dosing of inorganic manganese. The relatively complete absorption and slow elimination of MMT-derived manganese in MMT-treated rats contribute to its higher plasma concentration-time profiles. The elimination of MMT-derived manganese appears gender dependent.
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ACKNOWLEDGMENTS |
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NOTES |
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