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Chem. Res. Toxicol. 1997, 10, 360-363
Communications Accumulation of Manganese in the Brain of Mice after Intravenous Injection of Manganese-Based Contrast Agents Bernard Gallez,*,† Christine Baudelet,† Jacques Adline,† Muriel Geurts,‡ and Nathalie Delzenne§ Laboratory of Medicinal Chemistry and Radiopharmacy, Catholic University of Louvain, Avenue Mounier 73.40, B-1200 Brussels, Belgium, Laboratory of Pharmacology, Catholic University of Louvain, Avenue Hippocrate 54, B-1200 Brussels, Belgium, and Laboratory of Toxicologic and Cancerologic Biochemistry, Catholic University of Louvain, Avenue Mounier 73, B-1200 Brussels, Belgium Received December 3, 1996X
Because the manganese-based contrast agents used in magnetic resonance imaging are unstable in vivo, some concern exists about the potential toxicity coming from the Mn2+ released by the complexes. This potential problem arises because the manganese is known to accumulate in the brain of people intoxicated by this metal (manganism): this central accumulation leads to neurological disorders (i.e., parkinsonism-like syndrome). The aim of this study was to assess the amount of Mn found in the brain after administration of MnCl2 or different chelates of Mn in normal mice as well as in mice with impaired biliary elimination. Male NMRI mice received an intravenous injection in a caudal vein of 5 µmol/kg of 54Mn compounds as MnCl2, manganese-diethylenetriaminepentaacetate (Mn-DTPA), or manganese-dipyridoxal diphosphate (Mn-DPDP). The radiolabeled complexes (1:1) were prepared by direct chelation (MnDTPA) or transchelation of preformed complex (Mn-DPDP), and the radiochemical purity was assessed by paper chromatography. The mice were killed at various times post-exposure (0-3 months), and the radioactivity present in the organs was determined by gamma counting. For each compound analyzed in the present study, we observed an accumulation of Mn (0.250.3% of the amount injected/g of tissue) in the mouse brain, reaching a plateau after 24 h, while the Mn content in the liver was decreasing with time. The amount of Mn accumulated in the brain remained unchanged 1 month later, but decreased to 40% of the maximum amount 3 months after the exposure. In mice whose bile ducts had been ligated 24 h before the administration of the manganese compound, we observed, 1 week after the injection, an amount of manganese accumulated in the brain 2 times higher than in normal mice.
Introduction Intensive work has been devoted in designing new magnetic resonance imaging (MRI)1 contrast agents. These can be divided into two categories: particulate agents (superparamagnetic iron oxide) and soluble paramagnetic agents. In the latter class, paramagnetic gadolinium complexes are the most extensively investigated compounds, and four of them are now commercially available. To reduce the toxicity of the free metal ion, gadolinium needs to be chelated to form highly stable metal complexes (1). Manganese(II) possesses five unpaired electrons and is also a potential candidate for enhancing the contrast in MRI. Chelation of Mn(II) is necessary to decrease the * Address correspondence to this author at the Laboratory of Medicinal Chemistry and Radiopharmacy, Avenue Mounier 73.40, B-1200 Brussels, Belgium. Phone: 32 2 7647348. Fax: 32 2 7647363. E-mail:
[email protected]. † Laboratory of Medicinal Chemistry and Radiopharmacy. ‡ Laboratory of Pharmacology. § Laboratory of Toxicologic and Cancerologic Biochemistry. X Abstract published in Advance ACS Abstracts, March 15, 1997. 1Abbreviations: MRI, magnetic resonance imaging; DTPA, diethylenetriaminepentaacetate; DPDP, dipyridoxal diphosphate.
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high acute toxicity of the free metal ions (2). One Mn complex, Mn-DPDP (dipyridoxal diphosphate), is at the present time used in clinical trials (Phase III) (3, 4). However, Mn complexes are relatively unstable in vivo and are dissociated in the biological media (5, 6). Indeed, the intrinsic stability of the manganese complexes is lower than that of the lanthanides (7, 8). Moreover, manganese can be displaced from the complex by other divalent cations such as Ca, Mg, or Zn (5). Finally, unlike gadolinium, manganese is an essential nutrient for mammals, and organisms have developed special carrier systems and selective cellular uptake mechanisms (9), suggesting that there are endogenous chelators that would compete with the ligand for manganese. Because of this manganese release within the body, some concern exists about the potential long-term toxicity associated with the use of Mn-based contrast agents (5, 10). Free manganese was shown to be teratogenic (11, 12) and mutagenic in several models (13, 14). This teratogenic effect was indeed demonstrated after the injection of the complex Mn-DPDP (12). Problems related to a chronic exposure to manganese are well documented. The central nervous system is primarily © 1997 American Chemical Society
Communications
Chem. Res. Toxicol., Vol. 10, No. 4, 1997 361 of 9‰ NaCl containing 2.5 mM MnCl2 (Aldrich, Milwaukee, WI). The radiolabeled complexes (1:1) were prepared by direct chelation (Mn-DTPA) or transchelation of preformed complex (Mn-DPDP). Chemical structures of the chelating agents are shown in Figure 1. 54Mn-DTPA was obtained by mixing 54MnCl with a 10 mM phosphate buffer (pH 7) containing 2.5 2 mM DTPA (Aldrich, Steinheim, Germany) 2.5 mM and MnCl2. 54Mn-DPDP was prepared by transchelation (19): to 50 µL of Mn-DPDP (Byk-Gulden, 145 µmol/mL) was added 10 µL of 54MnCl . The volume was adjusted to 3 mL with water. The 2 radiochemical purity of the compounds was monitored by paper chromatography (Whatman no. 1; eluent: methanol/water 7/3), and the radioactivity on the chromatogram was analyzed with a Bioscan System 200 Imaging Scanner. After 30 min, the radiochemical purity was found constant and greater than 95%. The solutions for in vivo purposes were prepared the day of the experiment. In Vivo Studies. A total of 130 male NMRI mice (weight: 30-40 g) received an iv injection in a caudal vein of 5 µmol/kg (recommended clinical dose) of 54Mn compounds as MnCl2, MnDTPA, or Mn-DPDP. Five mice were used for each experiment (per time and per compound studied). The mice were killed by cervical dislocation at various times post-exposure (0-3 months). The liver and the brain were sampled. After the organs were weighed, the radioactivity (γ-emission) of the samples was measured using a Packard 5230 scintillation spectrometer. The radioactivity measurements were corrected for the half-live of 54Mn (T ) 310 days) because this factor starts to be nonnegligible for mice killed a long time after the injection. The group of mice (30 mice) with bile duct ligation received the following treatment 24 h before the injection of the 54Mn compounds. They were anesthetized with an intramuscular injection of a mixture containing xylazine (10 mg/kg) and ketamine (200 mg/kg). Laparotomy was performed on all these mice with a small incision under the ribs. The total biliary obstruction was obtained by isolating the common bile duct above the pylorus, and ligating the duct with a double suture. After the surgery, the mouse skin was stitched up. The mice were killed at various times post-injection of manganese (0-1 week). The liver and the brain (including cerebellum) were sampled. In order to confirm the impairment in the biliary elimination, a spectrophotometric dosage of the total bilirubin was performed: approximately 300 µL of blood was sampled into Eppendorf tubes containing 30 µL of a 0.5% sodium citrate solution and centrifuged at 4 °C at 2500g for 8 min. The total bilirubin concentration was measured in the plasma using an Elitech Diagnostics kit (Sees, France). This method is based on the enzymatic oxidation of bilirubin into biliverdin by bilirubin oxidase (20).
Figure 1. Structures of the chelating agents. Top: DPDP. Bottom: DTPA.
affected: psychiatric symptoms and disorders of the extrapyramidal system have been reported (15-17). These effects are related to the accumulation of Mn in the brain (18). Although this accumulation is known after exposure to manganese ions, such as MnCl2, or manganese oxides, no study has shown until now an accumulation of Mn in the brain after administration of chelates which are potential MRI contrast media. The purpose of this study is to prepare radiolabeled Mn complexes, and to assess the amount of Mn found in the brain after iv injection of these compounds in mice. Because manganese is almost exclusively eliminated through the biliary system, the amount of Mn is expected to be higher in subjects possessing an impaired biliary elimination. Because that may be of particular importance in patients with impaired hepatic function (10), we also studied the accumulation of Mn in mice whose bile ducts had been ligated before the administration of the Mn compound.
Experimental Procedures Caution: 54Mn is a high-energy γ-emitter. It should be handled with care, and an appropriate shield should be used to protect the personnel. Preparation of 54Mn Compounds for iv Injection. A commercial solution of 54MnCl2 (Dupont de Nemours, Boston, MA; 53 mCi/mg, 2 mCi/mL, solution in HCl, 0.5 M) was used to prepare several solutions for iv injection. 54MnCl2 for iv injection was typically prepared by dilution of 2 µL of 54MnCl2 in 800 µL
Results Table 1 summarizes the data obtained about the amount of 54Mn found in the brain (brain including cerebellum) and in the liver after iv injection of Mn
Table 1. Mn Found in the Liver and in the Brain after iv Injection of MnCl2
54Mn
Compoundsa
MnDTPA
MnDPDP
time
brain
liver
brain
liver
brain
liver
0.25 h 1h 4h 6h 8h 1 day 2 days 1 week 2 weeks 1 month 3 months
0.16 ( 0.05 0.16 ( 0.02 0.19 ( 0.03 nd 0.22 ( 0.01 0.28 ( 0.07 0.29 ( 0.03 0.30 ( 0.04 0.32 ( 0.11 0.30 ( 0.03 0.12 ( 0.02
13.1 ( 6.5 12.4 ( 2.9 7.3 ( 1.6 nd 7.2 ( 1.0 3.9 ( 1.0 2.04 ( 0.46 0.99 ( 0.38 0.45 ( 0.18 0.06 ( 0.01 0.07 ( 0.02
0.11 ( 0.02* 0.11 ( 0.05 0.14 ( 0.02** 0.17 ( 0.03 nd 0.20 ( 0.03** 0.19 ( 0.05*** 0.23 ( 0.07* nd 0.25 ( 0.03 0.10 ( 0.03
7.1 ( 4.3 6.2 ( 3.8** 5.3 ( 0.9* 5.4 ( 0.7 nd 2.7 ( 0.4* 2.4 ( 1.2 0.6 ( 0.20 nd 0.06 ( 0.02 0.010 ( 0.003**
0.14 ( 0.02 0.17 ( 0.03 0.19 ( 0.08 nd 0.22 ( 0.04 0.26 ( 0.04 nd 0.24 ( 0.05** 0.26 ( 0.13 nd nd
7.5 ( 1.4 7.3 ( 2.6* 5.0 ( 3.0 nd 4.6 ( 0.9** 2.6 ( 0.4** nd 0.84 ( 0.19 0.42 ( 0.13 nd nd
a The results in the table are expressed as percent injected dose per gram of tissue (mean ( SD). t test of comparison between Mnchelate and MnCl2 at the same time: (*) p < 0.1; (**) p < 0.05; (***) p < 0.01.
362 Chem. Res. Toxicol., Vol. 10, No. 4, 1997
Figure 2. Manganese found in the liver after injection of 54MnCl (top) or 54Mn-DTPA (bottom) for normal mice (9) and 2 mice whose bile ducts had been ligated 24 h before administration (0). The results are expressed as percent of the injected dose per gram of tissue. Note the dramatic increase in the Mn content for mice with impaired biliary elimination (t test of comparison between normal mice and mice with bile duct ligated: one asterisk, p < 0.1; two askerisks, p < 0.05; three asterisks, p < 0.01).
Communications
Figure 3. Manganese found in the brain after injection of 54MnCl (top) or 54Mn-DTPA (bottom) for normal mice (9) and 2 mice whose bile ducts had been ligated 24 h before administration (0). The results are expressed as percent of the injected dose per gram of tissue. Note the increase in the Mn content in the brain for mice with impaired biliary elimination (t test of comparison between normal mice and mice with bile duct ligated: one asterisk, p < 0.1; two asterisks, p < 0.05; three asterisks p < 0.01).
Discussion compounds. For each compound analyzed in the present study, we observed a slow accumulation of Mn in the mouse brain, while the Mn content in the liver is decreasing with time. The total amount found in the brain is approximately 0.3% of the injected dose per gram of tissue for MnCl2, and 0.25% for Mn-DTPA and MnDPDP. This accumulation reaches a plateau 1 day after the injection. The amount found 1 month later is unchanged, showing that the clearance from the central nervous system is very slow for all Mn species. Three months after the injection, the amount of Mn found in the brain is approximately 40% of the maximal Mn content observed. Figure 2 and Figure 3 show the amount of 54Mn found in the liver and in the brain of mice whose bile ducts had been ligated before the administration of the Mn compound compared with control mice. The amount of 54Mn found in the liver is dramatically increased in mice whose bile ducts had been ligated. These data are consistent with a previous MRI observation of Marchal et al. (21). Interestingly enough, the amount found in the brain is statistically not different (p > 0.05, t-test) 1 day and 2 days after the injection, and is approximately 2-fold increased 1 week after exposure (p < 0.01). The impairment in the bile excretion was confirmed by the yellowness of the teguments, a distended bladder, and a high plasma level of total bilirubin (6.8 ( 1.2 mg/dL, mean ( SD).
Manganese is an essential nutrient: this ion plays a role in several metabolism processes and is a cofactor in a variety of enzymes, including pyruvate carboxylase, superoxide dismutase, glutamine synthetase, alkaline phosphatase, and ferment arginase. The brain normally contains a small amount of manganese [4.5-6.2 µmol/ kg in humans (23, 24), 10.2-11.9 µmol/kg in mice (2527)], but an excessive amount can be neurotoxic. Consequently, some concern exists about the potential longterm toxicity associated with the use of Mn-based contrast agents. Our data clearly show that Mn, when released from the complexes Mn-DTPA or Mn-DPDP, is accumulated inside the brain to an extent almost similar (80-85%) to the one observed in MnCl2-injected animals. That means that the chelation by a ligand to form a complex possessing a relatively high stability constant (g1015) is unable to prevent the central accumulation of the metal. Moreover, this accumulation of manganese is dramatically increased 1 week after exposure in mice whose bile ducts had been ligated before the administration of the Mn compound. The potential long-term toxicity associated with the release of free manganese is an important concern (5, 10), but toxicological consequences of such an accumulation are difficult to extrapolate. We will mention for the readers some elements of comparison with known data about acute Mn exposure, on the one hand, and try a rough extrapolation of Mn overload in the brain for a patient, on the other hand.
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Chem. Res. Toxicol., Vol. 10, No. 4, 1997 363
The central toxicity of manganese is generally reported after chronic exposure to relatively high amounts of free manganese, and only few studies deal with acute exposure. To our knowledge, the smallest amount of MnCl2, administered as a single dose and able to produce neurological disorders in monkeys, was 34 µmol/kg (18). No data in humans are available for similar exposures to small amounts of free manganese. This is higher than the dose used in clinical trials: the usual dose is 5-10 µmol/kg (14-16), and, to our knowledge, only one study reported experiments using 25 µmol/kg (22). It is also possible to roughly estimate the overload of manganese in the brain for a patient who receives an iv injection of a Mn complex. For this purpose, we need to assume that the behavior of the Mn complexes, particularly the extent of Mn accumulation in the brain, will be approximately the same in mice and in humans (that is only an assumption). Taking into account the weight of the mouse brain ((500 mg), the amount found in the brain is (0.13% of the injected dose after the injection of a Mn complex in normal mice and 0.2% in mice with bile duct ligated. Considering a human being of 70 kg, and a dose of 5 µmol/kg, he will receive 350 µmol of Mn. Assuming the same extent in the human brain accumulation as in mice, the probable amount which will accumulate in the brain is around 0.45 µmol for a normal patient and 0.7 µmol for a patient with a total biliary obstruction. The manganese concentrations in the brain reported for human beings are in the range of 0.25-0.34 mg/kg (4.5-6.2 µmol/kg) (23, 24), i.e., 5.4-7.4 µmol of Mn for a brain of 1.2 kg. That means an increase of the Mn amount in the brain of 8.5% for a normal patient and 13% for a patient with biliary obstruction. This overload could be higher if patients received a dose larger than 5 µmol/kg or several MRI examinations using contrast media. However, it should be noted that the increase in manganese content inside the brain is within the physiological range of concentrations observed in a normal population (23, 24). Considering our data and the critical problem for extrapolating the potential toxicity in humans, we would like to suggest a systematic follow-up of patients after Mn complex injection in order to evaluate if the toxicological problem is relevant or not. This follow-up should deal with behavior and neurologic signs, instead of the vital signs and usual laboratory parameters which were checked in the first clinical studies (22).
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