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Chelating effect of novel pyrimidines in a model of aluminum intoxication
JOURNAL OF
Inorganic Biochemistry Journal of Inorganic Biochemistry 99 (2005) 1853–1857 www.elsevier.com/locate/jinorgbio
Chelating effect of novel pyrimidines in a model of aluminum intoxication J.R. Missel, M.R. Schetinger, C.R. Gioda, D.N. Bohrer, I.L. Pacholski, N. Zanatta, M.A. Martins, H. Bonacorso, V.M. Morsch * Departamento de Quı´mica, Centro de Cieˆncias Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil Received 11 April 2005; received in revised form 21 June 2005; accepted 27 June 2005 Available online 10 August 2005
Abstract Long time ago aluminum (Al) was considered as a non-toxic element and its use had no restrictions. However, over the last two decades, scientific publications have indicated that Al is a toxic element. In line with this, aluminum accumulation in the organism is associated with a variety of human pathologies. Efficient therapeutics approach to treat Al intoxication are still not available, but there is a consensus that chelation therapy is the procedure to be used. However, the development of new chelating agents are highly desirable to improve the efficacy of the treatment of Al intoxication. The present study evaluates the chelating effect of two novel pyrimidines: 4-tricloromethyl-1-H-pyrimidin-2-one (THP) and (4-methyl-6-trifluoromethyl-6-pyrimidin-2-il)-hydrazine (MTPH) in a mice model of aluminum intoxication and compares their efficacy with those of desferrioxamine (DFO), a classical agent used for treat Al accumulation. The animals were exposed to aluminum by gavage (0.1 mmol aluminum/kg/day) 5 days/week for 4 weeks. At the end of this period, DFO was injected i.p. and the novel pyrimidines were given by gavage at 0.2 mmol/kg/day for five consecutive days. Aluminum concentration in tissues (brain, liver, kidney and blood) was determined by graphite furnace atomic absorption spectroscopy (GFAAS). The results showed that when administered by gavage, aluminum accumulated in the brain, kidney and liver of mice. MTPH was able to decrease aluminum levels in aluminum plus citrate animal groups, whereas THP was inefficient for this purpose. However, the novel pyrimidines used in this study were unable to surpass the aluminum chelating property of DFO. Thus, new studies must be performed utilizing other chelating agents which can decrease aluminum toxicity. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Aluminum; Chelators; Pyrimidines; Intoxication
1. Introduction Aluminum (Al), the third most abundant element in the EarthÕs crust, is a non-essential and toxic metal in humans [1–4]. Due to its abundance every organism presents small quantities of aluminum [4–6] and it can be found practically in all tissues of mammals as the brain, liver, kidney, heart, blood and bones [1–3,7]. Aluminum may interfere with various metabolic processes, in which
*
Corresponding author. Tel.: +555532208665; fax: +55552208978. E-mail address: [email protected] (V.M. Morsch).
0162-0134/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2005.06.025
Ca2+, Mg2+, Fe3+ (in transferrin and ferritin) and Fe2+ (gastrointestinal absorption) are involved [8]. Aluminum accumulation has been associated with a variety of human pathologies such as anemia, osteodystrophy, encephalopathy, joint diseases, muscular weakness, ParkinsonÕs and AlzheimerÕs diseases [3,8–10]. While the role of aluminum in some of these disorders is controversy, its role in the syndrome of encephalopathy by dialysis, osteomalacy and microcytic anemia is well established [11–16]. The treatment commonly used in aluminum disorders is desferrioxamine (DFO), which is a chelator with great capacity to decrease Al body burden by increase its excre-
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tion in the urine. This compound is usually employed in iron accumulation and since there are chemical and physical similarities among aluminum and iron (charge, ionic radius and protein binding) it has been used in cases of aluminum accumulation [17]. However, DFO therapy is associated with undesirable effects, it is very expensive and it is only efficient intravenously or subcutaneously [9]. In view of this, many researchers have directed their efforts to develop compounds that should be able to decrease aluminum levels in the body, with the same efficiency as DFO, but with oral administration. Recently, a large deal of interest has been shown in 3hydroxy-4-pyridinones (3,4-HPs), bidentate chelating compounds used for removing toxic metal ions (e.g., Al3+ and Fe3+) in the body [10,17]. In a study performed by Chaves et al. [18], it was demonstrated that derivative compounds of N-alkyl-arylamine hydroxypyridinone were able to sequester aluminum and based on this property the authors considered that they could be applied to clinical trials. A substance named Feralex-G (2-deoxy-2-(N-carbamoylmethyl-[N 0 -2 0 -methyl-3 0 -hydroxypyrid-4 0 -one]glucopyranose) also has shown a potential clinical efficacy to chelate aluminum in physiological conditions in subjects with AlzheimerÕs disease [19]. The chelation of aluminum by combining desferrioxamine and 1,2-dimethyl-3-hydroxypyrid-4-one (L1) given to animals after aluminum loading was performed by Blanusa et al. [20]. The objective of this study was to verify the capacity of two novel compounds: 4-tricloromethyl-1-H-pyrimidin-2-one (THP) and (4-methyl-6-trifluoromethyl-pyrimidin-2-il)-hydrazine (MTPH) to reduce aluminum concentration in the brain, liver, kidney and blood of mice by comparing their efficiencies with DFO, the usual aluminum chelator used in aluminum disorder.
2. Material and methods 2.1. Animals Male mice 6–7 weeks old, weighing 25–30 g, from our own stock and fed on a standard diet with tap water ad libitum were used. Mice were kept at room temperature (22–25 °C) in a dark–light cycle of 12:12 in plastic cages. 2.2. Material The novel chelators tested were 4-tricloromethyl-1-Hpyrimidin-2-one (THP), prepared according to Pacholski et al. [21] and (4-methyl-6-trifluoromethyl-pyrimidin-2il)-hydrazine (MTPH) synthesized according to Zanatta et al. [22] (Fig. 1). DFO and nitric acid were obtained from Sigma (St. Louis). All others reagents were of analytical grade.
Fig. 1. Chemical structure of pyrimidinic compounds.
2.3. Treatment Mice were divided into three groups: one group received 0.1 mmol/kg/day aluminum (in a form of aluminum sulfate, dissolved in ultra-purified water) by gavage during 4 weeks, 5 times a week (n = 20); the second group received aluminum plus sodium citrate in the same dose (n = 20) and the third group received only citrate 10 mg/kg/day (n = 20). After 4 weeks, each group was divided into four groups, one of which received the chelator MTPH (n = 5) and another received TPH (n = 5) 0.2 mmol/kg, (dissolved in ultra-purified water) for five consecutive days by gavage. A third group received DFO (n = 5), administered by intraperitoneal (i.p) route, at a dose of 0.15 mmol/kg/day and a control group received saline (0.9%) (n = 5) 5 days by gavage. The mice were anesthetized with ether then sacrificed by decapitation and the samples were stored in plastic flasks until analysis. 2.4. Contamination control Glass recipients were not used because they could contaminate the samples due to aluminum present in their composition. Flasks were left at least 48 h in a cleaning solution (HNO3/ethanol mixture 1:9, v/v) and washed with ultra-purified water [4,23]. Animal decapitation, sample and reagent preparations were carried out on a Trox clean bench class 100. 2.5. Sample preparation and aluminum determination Tissues were dissolved with diluted sub-boiling distilled nitric acid. Samples were placed in weighed flasks. The flasks were tightly closed and maintained at room temperature for 72 h for complete sample digestion. The samples were diluted with water (1:1). The aluminum concentration was measured using a Varian SpectrAA atomic absorption spectrophotometer with a deuterium background corrector, a Varian GTA-100 graphite furnace and an auto sampler. Pyrolytic graphite-coated tubes with LÕvov platforms were used. The pyrolysis and atomization temperatures were 1000 and 2500 °C, respectively. The hollow cathode lamp used
J.R. Missel et al. / Journal of Inorganic Biochemistry 99 (2005) 1853–1857
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had 10 mA at the 309.3 nm resonance line, the spectral slit width was set at 0.7 nm, and the volume injected was 5 ll. The purging gas was argon with a stop flow at the time of atomization and the absorbance signal was measured in the peak area [3,24]. 2.6. Statistical analysis Statistical analysis was performed by one-way ANOVA, followed by Tukey–Kramer Multiple test when the F-test was significant (P < 0.05) to verify the differences among groups.
3. Results It was observed that the groups overloaded with aluminum plus citrate showed the greatest accumulation of this element in the brain, liver and kidney. Accumulation in the kidney of the aluminum plus citrate group increased 6-fold compared to the aluminum group and similarly there was an increase of 3-fold in the liver and 2-fold in the brain (Figs. 2–4). In the blood, the use of citrate did not cause an increase in aluminum concentration (Fig. 5). The results for the three treatment groups in the brain are shown in Fig. 2. It can be observed that both chelators promoted a decrease in aluminum levels and the group that received aluminum plus citrate presented highest aluminum content. In comparison to the control group, the reduction caused by the two substances was quite similar: 41% and 43% for MTPH and 97 and 89% for DFO in aluminum and aluminum plus citrate overloaded animals, respectively. In the liver, MTPH was able to decrease the aluminum concentration only in the group with the highest
Fig. 2. Aluminum concentration in the brain (lg/g) of mice. *P < 0.05 compared to saline; **P < 0.05 compared to saline and to the aluminum group (Tukey–Kramer multiple comparison test). Each result represents the means ± standard deviation for five animals per group. Al determinations were done in duplicates by GFAAS.
Fig. 3. Aluminum concentration in the liver of mice. *P < 0.05 compared to saline; **P < 0.05 compared to saline and to the aluminum group (Tukey–Kramer multiple comparison test). Each result represents the means ± standard deviation for five animals per group. Al determinations were done in duplicates by GFAAS.
Fig. 4. Aluminum concentration in the kidney of mice. *P < 0.05 compared to saline; **P < 0.05 compared to saline and to the aluminum group (Tukey–Kramer multiple comparison test). Each result represents the means ± standard deviation for five animals per group. Al determinations were done in duplicates by GFAAS.
Fig. 5. Aluminum concentration in the blood of mice. Each result represents the means ± standard deviation for five animals per group. Al determinations were done in duplicates by GFAAS.
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concentration (aluminum plus citrate). Groups that received DFO presented lower aluminum levels (67% reduction compared to the control group) than MTPH groups (reduction of 41%) (Fig. 3). In the kidney (Fig. 4), both compounds caused a decrease in the aluminum concentration in the aluminum plus citrate group, however, DFO was more efficient, decreasing the aluminum concentration from 5.61 to 1.25 lg/g (77.5%), and MTPH to 4.36 lg/g (22.3%). MTPH and DFO presented no changes in the aluminum levels in the blood in all groups tested (Fig. 5). The second novel pyrimidine utilized in this study, THP, was unable to decrease aluminum level in any of the tissues studied (Table 1).
4. Discussion There are many studies that disclose important findings about new substances with chelating action on aluminum [2,9,10,20]. In general, the compounds have a common structure: aromatic rings, in which two nitrogen atoms take the place of two CH groups in the relative meta position. These compounds are generically named pyrimidines and their main compounds produced are pyrimidinones and hydroxypyrimidinones [25–27]. MTPH and THP are included in this class of substances (Fig. 1). The data of this study can confirm the capacity of MTPH in chelating aluminum in the brain, liver and kidney of mice. Some dietary components, like a citrate anion, ascorbate, malate, lactate and tartarate have a fundamental influence on aluminum absorption. Such chemical species may increase aluminum body burden in subjects with chronic renal failure [2,6,7,10,28,29]. For citrate, these properties have been attributed to the chelating action [2,28,30,31]. In line with this, here we showed that oral administration of 0.1 mmol Al3+/kg/day in the absence of citrate did not increase Al burden when compared with the control group. However, when animals received aluminum plus citrate, Al levels rose significantly in relation to the con-
trol group (Table 1). Most of the hypothesis that try to explain aluminum absorption mechanisms, refer to a much stable and more soluble complex formed between the carboxylic anion and aluminum [5,9,29]. The effectiveness of MTPH in reducing aluminum body burden could be explained by the presence of the hydrazine group in its chemical structure. Hydrazine has an electron pair, which could be used to complex aluminum, acting as a Lewis base. According to Yokel [7], an efficient chelator must be lipophilic enough to cross membranes and sufficient hydrophilic to be excreted by the kidney. Thus, two propositions can be formulated to explain the inefficacy of THP as an aluminum chelator: (1) the difficulty to form an aqueous complex with aluminum and (2) low water solubility. Additional assays of this compound with other solvents and other pH values could disclose this question, and perhaps transform THP into a more efficient aluminum chelator. DFO showed great efficacy as an aluminum chelator, corroborating other reports [10,20,32]. DFO forms a highly stable complex with aluminum [33]. According to Blanusa [20] the use of other chelators in combination with DFO minimizes its collateral side effects. Therefore, although MTPH presented lower efficacy than DFO in chelating aluminum, it would be possible to use this pyrimidinic compound together with other chelating agents, especially DFO. When administered by gavage, aluminum accumulated in the brain, kidney and liver of mice. Most of the studies involving aluminum administered via gavage or i.p. disclose lower levels of aluminum in the brain compared to the other organs. However, and in agreement with our results, Hermenegildo et al. [34] performed an experiment utilizing chronic exposure to Al administered in drinking water and also found higher aluminum levels in the brain compared to kidney, liver and spleen. In conclusion, MTPH was able to decrease aluminum levels in aluminum plus citrate animal groups, whereas THP was inefficient for this purpose. However, the novel
Table 1 Aluminum concentration (lg/g) in the brain, liver, kidney and blood using THP as a chelator of aluminum in mice Groups
[Al3+] brain
[Al3+] liver
[Al3+] kidney
[Al3+] blood
Saline THP
1.25 ± 0.62 0.98 ± 0.45
0.21 ± 0.10 0.27 ± 0.12
0.42 ± 0.08 0.59 ± 0.20
1.35 ± 0.21 1.90 ± 0.31
Aluminum + saline Aluminum + THP
4.02 ± 0.73 3.63 ± 0.31
0.19 ± 0.01 0.30 ± 0.12
1.10 ± 0.40 0.84 ± 0.32
1.65 ± 0.33 1.19 ± 0.38
Citrate + saline Citrate + THP
1.29 ± 0.47 1.31 ± 0.22
0.36 ± 0.15 0.24 ± 0.12
1.21 ± 0.33 1.34 ± 0.28
1.22 ± 0.47 1.25 ± 0.12
Aluminum citrate + saline Aluminum + citrate + THP
6.35 ± 1.29 5.91 ± 1.39
0.81 ± 0.23 0.65 ± 0.20
4.82 ± 2.11 4.36 ± 1.63
1.44 ± 0.22 1.10 ± 0.26
The values represent means ± standard deviation, n = 5. There was not a statistically significant difference between groups. (ANOVA one way P > 0.05).
J.R. Missel et al. / Journal of Inorganic Biochemistry 99 (2005) 1853–1857
pyrimidines used in this work were unable to surpass the DFO aluminum chelating property. Thus, new studies must be performed utilizing other chelating agents which can decrease aluminum toxicity. References [1] J.L. Domingo, M. Go´mez, M.T. Llobet, J. Corbella, Lancet 2 (1988) 1362–1363. [2] J.L. Domingo, M. Go´mez, J.M. Llobet, J. Corbella, Kidney International 39 (1991) 598–601. [3] M.R.C. Schetinger, C.D. Bonam, V.M. Morsch, D. Bohrer, L.M. Valentim, S.R. Rodrigues, Brazilian Journal of Medical and Biological Research 32 (1999) 761–766. [4] M.R.C. Schetinger, V.M. Morsch, D. Bohrer, Structure and Bonding 104 (2003) 99–137. [5] R.J.P. Williams, The Journal of Inorganic Biochemistry 76 (1999) 81–88. [6] R.A. Yokel, Coordination Chemistry Reviews 228 (2002) 97–113. [7] R.A. Yokel, K.A. Meurer, T.L. Skinner, A.M. Frefenburg, Drug Metabolism and Disposition 24 (1996) 105–111. [8] P. Zatta, T. Kiss, M. Suwalsky, G. Berthon, Coordination Chemistry Reviews 228 (2002) 271–284. [9] J.C.B. Nicholas, P.T. Dawes, J.D. Davies, A.J. Freemon, Nephrology Dialysis Transplantation 14 (1999) 202–204. [10] M. Go´mez, J.L. Esparza, J.L. Domingo, P.K. Singh, M.M. Jones, Toxicology 130 (1998) 175–181. [11] R.A. Yokel, Neurotoxicology 21 (5) (2000) 813–828. [12] R.J. Ward, Y. Zhang, R.R. Crichton, Journal of Inorganic Biochemistry 87 (2001) 9–14. [13] A. Campbell, D. Hamai, S.C. Bondy, Neurotoxicology 22 (2001) 63–71. [14] S.S.A. El-Rahman, Pharmacology Research 47 (2003) 189–194.
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[15] A.C. Alfrey, Life Chemistry Reports 11 (1994) 197. [16] P. Zatta, R. Lucchini, S.J.V. Rensburg, A. Taylor, Brain Research 62 (2003) 15–28. [17] M.A. Santos, Coordination Chemistry Review 228 (2002) 187– 203. [18] S. Chaves, M. Gil, S. Marques, L. Gano, M.A. Santos, Journal of Inorganic Biochemistry 97 (1) (2003) 161–172. [19] R.W. Shin, T.P.A. Kruck, H. Murayama, T. Kitamoto, Brain Research 961 (2003) 139–146. [20] M. Blanusa, L. Prester, V.M. Varnai, D. Pavlovie´, K. Kostial, M.M. Jones, P.K. Singh, Toxicology 147 (2000) 151–156. [21] I.L. Pacholski, I. Blanco, N. Zanatta, M.A.P. Martins, Journal of the Brazilian Chemical Society 1 (1991) 118. [22] N. Zanatta, D.C. Flores, C.C. Madruga, D. Faoro, A.F.C. Flores, H.G. Bonacorso, M.A.P. Martins, Synthesis (2003) 894–898. [23] D.N. Bohrer, G. Schwedt, Pharmacie 48 (1993) 676–678. [24] V.L.P. Vieira, J.B.T. Rocha, M.R.C. Schetinger, V.M. Morsch, S.R. Rodrigues, S.M. Tuerlinckz, D. Bohrer, P.C. Nascimento, Toxicology Letters 117 (2000) 45–52. [25] P.S. Dobbin, R.C. Hider, A.D. Hall, P.D. Taylor, P. Sarpong, J.B. Porter, G. Xiao, D. Van Der Helm, Journal of Medicinal Chemistry 36 (1993) 2448–2458. [26] D.L. Ladd, Journal of Heterocyclic Chemistry 19 (1983) 917–992. [27] A.L. Papet, A. Marsura, Synthesis (1992) 478–481. [28] C. Exley, Journal of Inorganic Biochemistry 76 (1999) 133–140. [29] G. Berthon, Coordination Chemistry Reviews 149 (1996) 241–280. [30] G.E. Jackson, South African Journal of Chemistry 35 (1986) 89– 92. [31] R.B. Martin, Clinical Chemistry 32 (1986) 1797–1806. [32] H. Nakamura, P.G. Rose, J.L. Blumer, M.D. Reed, Journal of Clinical Pharmacology 40 (2000) 296–300. [33] L.N. Sheppard, G.J. Kontoghiorges, Drug Research 43 (6) (1993) 659–666. [34] C. Hermenegildo, R. Sa´ez, C. Minoia, L. Manzo, V. Felipo, Neurochemistry International 34 (1999) 245–253.
Inorganic Biochemistry Journal of Inorganic Biochemistry 99 (2005) 1853–1857 www.elsevier.com/locate/jinorgbio
Chelating effect of novel pyrimidines in a model of aluminum intoxication J.R. Missel, M.R. Schetinger, C.R. Gioda, D.N. Bohrer, I.L. Pacholski, N. Zanatta, M.A. Martins, H. Bonacorso, V.M. Morsch * Departamento de Quı´mica, Centro de Cieˆncias Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil Received 11 April 2005; received in revised form 21 June 2005; accepted 27 June 2005 Available online 10 August 2005
Abstract Long time ago aluminum (Al) was considered as a non-toxic element and its use had no restrictions. However, over the last two decades, scientific publications have indicated that Al is a toxic element. In line with this, aluminum accumulation in the organism is associated with a variety of human pathologies. Efficient therapeutics approach to treat Al intoxication are still not available, but there is a consensus that chelation therapy is the procedure to be used. However, the development of new chelating agents are highly desirable to improve the efficacy of the treatment of Al intoxication. The present study evaluates the chelating effect of two novel pyrimidines: 4-tricloromethyl-1-H-pyrimidin-2-one (THP) and (4-methyl-6-trifluoromethyl-6-pyrimidin-2-il)-hydrazine (MTPH) in a mice model of aluminum intoxication and compares their efficacy with those of desferrioxamine (DFO), a classical agent used for treat Al accumulation. The animals were exposed to aluminum by gavage (0.1 mmol aluminum/kg/day) 5 days/week for 4 weeks. At the end of this period, DFO was injected i.p. and the novel pyrimidines were given by gavage at 0.2 mmol/kg/day for five consecutive days. Aluminum concentration in tissues (brain, liver, kidney and blood) was determined by graphite furnace atomic absorption spectroscopy (GFAAS). The results showed that when administered by gavage, aluminum accumulated in the brain, kidney and liver of mice. MTPH was able to decrease aluminum levels in aluminum plus citrate animal groups, whereas THP was inefficient for this purpose. However, the novel pyrimidines used in this study were unable to surpass the aluminum chelating property of DFO. Thus, new studies must be performed utilizing other chelating agents which can decrease aluminum toxicity. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Aluminum; Chelators; Pyrimidines; Intoxication
1. Introduction Aluminum (Al), the third most abundant element in the EarthÕs crust, is a non-essential and toxic metal in humans [1–4]. Due to its abundance every organism presents small quantities of aluminum [4–6] and it can be found practically in all tissues of mammals as the brain, liver, kidney, heart, blood and bones [1–3,7]. Aluminum may interfere with various metabolic processes, in which
*
Corresponding author. Tel.: +555532208665; fax: +55552208978. E-mail address: [email protected] (V.M. Morsch).
0162-0134/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2005.06.025
Ca2+, Mg2+, Fe3+ (in transferrin and ferritin) and Fe2+ (gastrointestinal absorption) are involved [8]. Aluminum accumulation has been associated with a variety of human pathologies such as anemia, osteodystrophy, encephalopathy, joint diseases, muscular weakness, ParkinsonÕs and AlzheimerÕs diseases [3,8–10]. While the role of aluminum in some of these disorders is controversy, its role in the syndrome of encephalopathy by dialysis, osteomalacy and microcytic anemia is well established [11–16]. The treatment commonly used in aluminum disorders is desferrioxamine (DFO), which is a chelator with great capacity to decrease Al body burden by increase its excre-
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tion in the urine. This compound is usually employed in iron accumulation and since there are chemical and physical similarities among aluminum and iron (charge, ionic radius and protein binding) it has been used in cases of aluminum accumulation [17]. However, DFO therapy is associated with undesirable effects, it is very expensive and it is only efficient intravenously or subcutaneously [9]. In view of this, many researchers have directed their efforts to develop compounds that should be able to decrease aluminum levels in the body, with the same efficiency as DFO, but with oral administration. Recently, a large deal of interest has been shown in 3hydroxy-4-pyridinones (3,4-HPs), bidentate chelating compounds used for removing toxic metal ions (e.g., Al3+ and Fe3+) in the body [10,17]. In a study performed by Chaves et al. [18], it was demonstrated that derivative compounds of N-alkyl-arylamine hydroxypyridinone were able to sequester aluminum and based on this property the authors considered that they could be applied to clinical trials. A substance named Feralex-G (2-deoxy-2-(N-carbamoylmethyl-[N 0 -2 0 -methyl-3 0 -hydroxypyrid-4 0 -one]glucopyranose) also has shown a potential clinical efficacy to chelate aluminum in physiological conditions in subjects with AlzheimerÕs disease [19]. The chelation of aluminum by combining desferrioxamine and 1,2-dimethyl-3-hydroxypyrid-4-one (L1) given to animals after aluminum loading was performed by Blanusa et al. [20]. The objective of this study was to verify the capacity of two novel compounds: 4-tricloromethyl-1-H-pyrimidin-2-one (THP) and (4-methyl-6-trifluoromethyl-pyrimidin-2-il)-hydrazine (MTPH) to reduce aluminum concentration in the brain, liver, kidney and blood of mice by comparing their efficiencies with DFO, the usual aluminum chelator used in aluminum disorder.
2. Material and methods 2.1. Animals Male mice 6–7 weeks old, weighing 25–30 g, from our own stock and fed on a standard diet with tap water ad libitum were used. Mice were kept at room temperature (22–25 °C) in a dark–light cycle of 12:12 in plastic cages. 2.2. Material The novel chelators tested were 4-tricloromethyl-1-Hpyrimidin-2-one (THP), prepared according to Pacholski et al. [21] and (4-methyl-6-trifluoromethyl-pyrimidin-2il)-hydrazine (MTPH) synthesized according to Zanatta et al. [22] (Fig. 1). DFO and nitric acid were obtained from Sigma (St. Louis). All others reagents were of analytical grade.
Fig. 1. Chemical structure of pyrimidinic compounds.
2.3. Treatment Mice were divided into three groups: one group received 0.1 mmol/kg/day aluminum (in a form of aluminum sulfate, dissolved in ultra-purified water) by gavage during 4 weeks, 5 times a week (n = 20); the second group received aluminum plus sodium citrate in the same dose (n = 20) and the third group received only citrate 10 mg/kg/day (n = 20). After 4 weeks, each group was divided into four groups, one of which received the chelator MTPH (n = 5) and another received TPH (n = 5) 0.2 mmol/kg, (dissolved in ultra-purified water) for five consecutive days by gavage. A third group received DFO (n = 5), administered by intraperitoneal (i.p) route, at a dose of 0.15 mmol/kg/day and a control group received saline (0.9%) (n = 5) 5 days by gavage. The mice were anesthetized with ether then sacrificed by decapitation and the samples were stored in plastic flasks until analysis. 2.4. Contamination control Glass recipients were not used because they could contaminate the samples due to aluminum present in their composition. Flasks were left at least 48 h in a cleaning solution (HNO3/ethanol mixture 1:9, v/v) and washed with ultra-purified water [4,23]. Animal decapitation, sample and reagent preparations were carried out on a Trox clean bench class 100. 2.5. Sample preparation and aluminum determination Tissues were dissolved with diluted sub-boiling distilled nitric acid. Samples were placed in weighed flasks. The flasks were tightly closed and maintained at room temperature for 72 h for complete sample digestion. The samples were diluted with water (1:1). The aluminum concentration was measured using a Varian SpectrAA atomic absorption spectrophotometer with a deuterium background corrector, a Varian GTA-100 graphite furnace and an auto sampler. Pyrolytic graphite-coated tubes with LÕvov platforms were used. The pyrolysis and atomization temperatures were 1000 and 2500 °C, respectively. The hollow cathode lamp used
J.R. Missel et al. / Journal of Inorganic Biochemistry 99 (2005) 1853–1857
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had 10 mA at the 309.3 nm resonance line, the spectral slit width was set at 0.7 nm, and the volume injected was 5 ll. The purging gas was argon with a stop flow at the time of atomization and the absorbance signal was measured in the peak area [3,24]. 2.6. Statistical analysis Statistical analysis was performed by one-way ANOVA, followed by Tukey–Kramer Multiple test when the F-test was significant (P < 0.05) to verify the differences among groups.
3. Results It was observed that the groups overloaded with aluminum plus citrate showed the greatest accumulation of this element in the brain, liver and kidney. Accumulation in the kidney of the aluminum plus citrate group increased 6-fold compared to the aluminum group and similarly there was an increase of 3-fold in the liver and 2-fold in the brain (Figs. 2–4). In the blood, the use of citrate did not cause an increase in aluminum concentration (Fig. 5). The results for the three treatment groups in the brain are shown in Fig. 2. It can be observed that both chelators promoted a decrease in aluminum levels and the group that received aluminum plus citrate presented highest aluminum content. In comparison to the control group, the reduction caused by the two substances was quite similar: 41% and 43% for MTPH and 97 and 89% for DFO in aluminum and aluminum plus citrate overloaded animals, respectively. In the liver, MTPH was able to decrease the aluminum concentration only in the group with the highest
Fig. 2. Aluminum concentration in the brain (lg/g) of mice. *P < 0.05 compared to saline; **P < 0.05 compared to saline and to the aluminum group (Tukey–Kramer multiple comparison test). Each result represents the means ± standard deviation for five animals per group. Al determinations were done in duplicates by GFAAS.
Fig. 3. Aluminum concentration in the liver of mice. *P < 0.05 compared to saline; **P < 0.05 compared to saline and to the aluminum group (Tukey–Kramer multiple comparison test). Each result represents the means ± standard deviation for five animals per group. Al determinations were done in duplicates by GFAAS.
Fig. 4. Aluminum concentration in the kidney of mice. *P < 0.05 compared to saline; **P < 0.05 compared to saline and to the aluminum group (Tukey–Kramer multiple comparison test). Each result represents the means ± standard deviation for five animals per group. Al determinations were done in duplicates by GFAAS.
Fig. 5. Aluminum concentration in the blood of mice. Each result represents the means ± standard deviation for five animals per group. Al determinations were done in duplicates by GFAAS.
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concentration (aluminum plus citrate). Groups that received DFO presented lower aluminum levels (67% reduction compared to the control group) than MTPH groups (reduction of 41%) (Fig. 3). In the kidney (Fig. 4), both compounds caused a decrease in the aluminum concentration in the aluminum plus citrate group, however, DFO was more efficient, decreasing the aluminum concentration from 5.61 to 1.25 lg/g (77.5%), and MTPH to 4.36 lg/g (22.3%). MTPH and DFO presented no changes in the aluminum levels in the blood in all groups tested (Fig. 5). The second novel pyrimidine utilized in this study, THP, was unable to decrease aluminum level in any of the tissues studied (Table 1).
4. Discussion There are many studies that disclose important findings about new substances with chelating action on aluminum [2,9,10,20]. In general, the compounds have a common structure: aromatic rings, in which two nitrogen atoms take the place of two CH groups in the relative meta position. These compounds are generically named pyrimidines and their main compounds produced are pyrimidinones and hydroxypyrimidinones [25–27]. MTPH and THP are included in this class of substances (Fig. 1). The data of this study can confirm the capacity of MTPH in chelating aluminum in the brain, liver and kidney of mice. Some dietary components, like a citrate anion, ascorbate, malate, lactate and tartarate have a fundamental influence on aluminum absorption. Such chemical species may increase aluminum body burden in subjects with chronic renal failure [2,6,7,10,28,29]. For citrate, these properties have been attributed to the chelating action [2,28,30,31]. In line with this, here we showed that oral administration of 0.1 mmol Al3+/kg/day in the absence of citrate did not increase Al burden when compared with the control group. However, when animals received aluminum plus citrate, Al levels rose significantly in relation to the con-
trol group (Table 1). Most of the hypothesis that try to explain aluminum absorption mechanisms, refer to a much stable and more soluble complex formed between the carboxylic anion and aluminum [5,9,29]. The effectiveness of MTPH in reducing aluminum body burden could be explained by the presence of the hydrazine group in its chemical structure. Hydrazine has an electron pair, which could be used to complex aluminum, acting as a Lewis base. According to Yokel [7], an efficient chelator must be lipophilic enough to cross membranes and sufficient hydrophilic to be excreted by the kidney. Thus, two propositions can be formulated to explain the inefficacy of THP as an aluminum chelator: (1) the difficulty to form an aqueous complex with aluminum and (2) low water solubility. Additional assays of this compound with other solvents and other pH values could disclose this question, and perhaps transform THP into a more efficient aluminum chelator. DFO showed great efficacy as an aluminum chelator, corroborating other reports [10,20,32]. DFO forms a highly stable complex with aluminum [33]. According to Blanusa [20] the use of other chelators in combination with DFO minimizes its collateral side effects. Therefore, although MTPH presented lower efficacy than DFO in chelating aluminum, it would be possible to use this pyrimidinic compound together with other chelating agents, especially DFO. When administered by gavage, aluminum accumulated in the brain, kidney and liver of mice. Most of the studies involving aluminum administered via gavage or i.p. disclose lower levels of aluminum in the brain compared to the other organs. However, and in agreement with our results, Hermenegildo et al. [34] performed an experiment utilizing chronic exposure to Al administered in drinking water and also found higher aluminum levels in the brain compared to kidney, liver and spleen. In conclusion, MTPH was able to decrease aluminum levels in aluminum plus citrate animal groups, whereas THP was inefficient for this purpose. However, the novel
Table 1 Aluminum concentration (lg/g) in the brain, liver, kidney and blood using THP as a chelator of aluminum in mice Groups
[Al3+] brain
[Al3+] liver
[Al3+] kidney
[Al3+] blood
Saline THP
1.25 ± 0.62 0.98 ± 0.45
0.21 ± 0.10 0.27 ± 0.12
0.42 ± 0.08 0.59 ± 0.20
1.35 ± 0.21 1.90 ± 0.31
Aluminum + saline Aluminum + THP
4.02 ± 0.73 3.63 ± 0.31
0.19 ± 0.01 0.30 ± 0.12
1.10 ± 0.40 0.84 ± 0.32
1.65 ± 0.33 1.19 ± 0.38
Citrate + saline Citrate + THP
1.29 ± 0.47 1.31 ± 0.22
0.36 ± 0.15 0.24 ± 0.12
1.21 ± 0.33 1.34 ± 0.28
1.22 ± 0.47 1.25 ± 0.12
Aluminum citrate + saline Aluminum + citrate + THP
6.35 ± 1.29 5.91 ± 1.39
0.81 ± 0.23 0.65 ± 0.20
4.82 ± 2.11 4.36 ± 1.63
1.44 ± 0.22 1.10 ± 0.26
The values represent means ± standard deviation, n = 5. There was not a statistically significant difference between groups. (ANOVA one way P > 0.05).
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