- Email: [email protected]
26Al incorporation into the tissues of suckling rats through maternal milk
Nuclear Instruments and Methods in Physics Research B 223–224 (2004) 754–758 www.elsevier.com/locate/nimb
26
Al incorporation into the tissues of suckling rats through maternal milk S. Yumoto
a,*
, H. Nagai b, K. Kobayashi c, W. Tada b, T. Horikawa b, H. Matsuzaki c
a
c
Yumoto Institute of Neurology, Kawadacho 6-11, Shinjuku-ku, Tokyo 162-0054, Japan b College of Humanities and Sciences, Nihon University, Tokyo 156-8550, Japan Research Center for Nuclear Science and Technology, University of Tokyo, Tokyo 113-0032, Japan
Abstract Aluminium (Al) is highly neurotoxic and inhibits prenatal and postnatal development of the brain in humans and experimental animals. However, Al incorporation into the brain of sucklings through maternal milk has not yet been well clarified because Al lacks a suitable isotope for radioactive tracer experiments. Using 26 Al as a tracer, we measured 26 Al incorporation into the brain of suckling rats by accelerator mass spectrometry. Lactating rats were subcutaneously injected with 26 AlCl3 from day 1 to day 20 postpartum. Suckling rats were weaned from day 21 postpartum. From day 5 to day 20 postpartum, the 26 Al levels measured in the brain, liver, kidneys and bone of suckling rats increased significantly. After weaning, the amounts of 26 Al in the liver and kidneys decreased remarkably. However, the 26 Al amount in the brain had diminished only slightly up to 140 days after weaning. 2004 Elsevier B.V. All rights reserved. PACS: 87.59.Qx Keywords: Accelerator mass spectrometry (AMS);
26
Al; Aluminium toxicity; Alzheimer’s disease
1. Introduction Aluminium (Al) inhibits prenatal and postnatal development of the brain in humans and experimental animals [1,2]. Al administration to experimental animals during gestation and lactation causes retardation of neurological development and deficits in neurobehavioral performance in
*
Corresponding author. Tel.: +81-3-3341-3406; fax: +81-33358-4867. E-mail address: [email protected] (S. Yumoto).
their offspring [3]. Neurological impairment persists in the weaned offspring at Al doses which do not produce any detectable signs of maternal toxicity [4]. Al is highly neurotoxic and can result in the degeneration of nerve cells in the brain [5]. Excess exposure to Al causes dialysis dementia in patients on dialysis treatment and Al encephalopathy in Al industry workers. Al has been reported to be one of the risk factors for Alzheimer’s disease (senile dementia of Alzheimer type) and amyotrophic lateral sclerosis [6,7]. It is assumed that the neurotoxicity of Al is due to its presence in the brain [8]. However, Al
0168-583X/$ - see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2004.04.140
S. Yumoto et al. / Nucl. Instr. and Meth. in Phys. Res. B 223–224 (2004) 754–758
incorporation into the brain of sucklings has not been well clarified because Al lacks a suitable isotope for radioactive tracer experiments and a sensitive analysis technique. The application of accelerator mass spectrometry (AMS) to the analysis of 26 Al (t1=2 ¼ 7:16 105 years) has enabled the study of Al metabolism under physiological conditions [9,10]. One of the advantages of using AMS is that it measures individual atoms and not radioactivity. Since almost all naturally occurring Al is 27 Al, newly administered 26 Al can be distinguished from endogenous Al (27 Al) by AMS. In addition, as few as 5 · 1018 g (5 attograms) of 26 Al can be detected by this method. Previously, we injected 26 Al intraperitoneally into eight-month-old adult rats as a tracer, and used AMS to demonstrate 26 Al incorporation into the brain through the blood–brain barrier [9,11]. Approximately 17% of the 26 Al measured in the brain was incorporated into the cell nuclei [11]. In this study, we injected 26 AlCl3 subcutaneously into lactating rats to measure 26 Al incorporation into tissues (including the brain) of suckling rats through maternal milk by using AMS. After weaning, the metabolism of the 26 Al taken up into the rat tissues via milk was also studied by AMS.
2. Materials and methods 2.1. Animals Male and female Wistar rats were purchased from Clea Japan Inc., Tokyo, Japan. The rats were fed a standard diet (MF food pellets, Oriental Yeast Co., Tokyo, Japan) and tap water ad libitum, and maintained in light (lights on 0600–1800) and temperature (20–22 C) controlled rooms. Three female rats weighing 240–250 g each at the start of the experiment were put together with two male breeder rats, and vaginal smears were examined the following morning. The day on which sperm was found in the smear was designated as day 1 of gestation. All animal experiments in this study were performed according to the Tokyo University Guidelines for the Care and Use of Laboratory Animals.
755
2.2. 26 Al transfer from lactating rats to suckling rats through maternal milk Three lactating rats with a litter size of 11 pups each were used in this study. Each lactating rat was subcutaneously injected with 0.2 ml of a solution containing 20 dpm (470 pg) of 26 Al (26 AlCl3 ) and 0.009 mg of 27 Al (27 AlCl3 ) in a 0.45 M sodium acetate–hydrochloric acid buffer (pH 4.3) once daily from day 1 to day 20 postpartum. On days 5, 10, 15 and 20 postpartum, suckling rats were euthanized under ether anaesthesia, and the brain (cerebrum), liver, kidneys, parietal bone and blood were removed from the rats, and used for the measurement of 26 Al by AMS. Before euthanizing suckling rats, they were separated from the lactating rats into other cages, starved for 90 min and then put together with the lactating rats to ingest milk for 20 min. After euthanasia of the suckling rats under ether anaesthesia, freshly ingested milk was removed from the stomach of suckling rats for 26 Al assay. Both male and female suckling rats were used in this study. On day 21 postpartum, suckling rats were separated from lactating rats into other cages, and fed with a standard diet. On days 40 (20 days after weaning), 80 (60 days after weaning) and 160 (140 days after weaning) postpartum, weaned rats were euthanized under ether anaesthesia, and the brain, liver, kidneys, parietal bone and blood were removed from the weaned rats, and used for the 26 Al assay by AMS. After weaning, only female rats were used in this study. 2.3. Al measurements by AMS Each sample was weighed, minced and freezedried with a vacuum dryer. The samples from the brain, liver, kidneys, blood and milk were decomposed with 3 ml of concentrated nitric acid at 80 C for 24 h in a Teflon-sealed vessel inserted into a stainless steel bomb [9,12]. The bone (parietal bone) was mixed with 3 ml of concentrated nitric acid at room temperature for 48 h, and heated at 80 C for 24 h in the Teflon-sealed vessel. (Parietal bone is flat and thin in shape, and is easily decomposed with nitric acid.) After cooling to room temperature, the sample was combined with
756
S. Yumoto et al. / Nucl. Instr. and Meth. in Phys. Res. B 223–224 (2004) 754–758
2 mg of 27 Al as a carrier. (In some of the samples, the amount of 27 Al carrier was reduced to 0.5 or 1 mg to improve the detection limit of 26 Al in the samples.) Then, the sample was mixed with 3 ml of concentrated nitric acid, and heated at 140 C for 12 h in the Teflon-sealed vessel. After cooling to room temperature, the sample was blended with 1 ml of perchloric acid (60%), and heated on a hotplate at 90 C for 4 h. This process was repeated three times. Al fractions were purified by cation exchange, treated with pure ammonia water to convert the Al to Al oxide at 800 C and mixed with an equal mass of pure silver powder. The Al oxide was pressed into a sample holder, and examined with a tandem accelerator at Tokyo University. The 26 Al-AMS system at Tokyo University has the following features. The sensitivity threshold of 26 Al/27 Al is about 1 · 1014 and the reproducibility of measurements is better than ±3%. The minimal detectable amount of 26 Al is about 5 · 1018 g (5 attograms).
Fig. 1. Time course of 26 Al levels in the tissues of suckling rats and weaned rats and in the milk. A lactating rat was subcutaneously injected once daily with 470 pg (20 dpm) of 26 Al from day 1 to day 20 postpartum. The variable, ‘‘26 Al injected’’, of the formula on the ordinate represents the total amount of 26 Al injected into each lactating rat up to and including the time of the measurement. After weaning, ‘‘26 Al injected’’ is constant at 20 · 470 pg. 26 Al levels in the brain ( ), liver ( ;), kidneys (), bone (M), blood (N) and milk (j) were measured by AMS. Each point represents the mean ± SD; n ¼ 3 per group.
3. Results 26
The time course of Al levels in the samples from the suckling rats and in rat milk is shown in Fig. 1. The 26 Al levels in the milk were very high, specifically on day 5 postpartum. The 26 Al concentrations in the milk were stationary from day 10 to day 20 postpartum. Approximately 0.2–0.4% of the 26 Al injected into a lactating rat was contained in 1 g of the milk. The 26 Al levels in the bone were the highest in the samples from the suckling rats, and showed almost constant values from day 5 to day 20 postpartum. The 26 Al levels of the liver and kidneys increased significantly from day 5 to day 20 postpartum. In contrast, the 26 Al levels in the blood decreased remarkably from day 5 to day 20 postpartum. This decrease in the blood 26 Al levels was probably due to the development of renal function, which removes Al from the blood, during this stage. The 26 Al levels in the brain (cerebrum) of suckling rats were almost stationary from day 5 to day 20 postpartum. After weaning, the 26 Al levels in the liver, kidneys and bone decreased considerably (Fig. 1). Especially the 26 Al levels measured in the liver and
kidneys had diminished sharply 20 days after weaning. The blood 26 Al levels continued to decrease until 140 days after weaning (day 160 postpartum). On the other hand, the 26 Al levels in the brain decreased only slightly up to 140 days after weaning. As shown in Fig. 2, the liver and
Fig. 2. Growth of tissues in suckling rats and weaned rats. Tissue weights of the brain ( ), liver ( ) and kidneys () are shown. Each point represents the mean ± SD; n ¼ 3 per group.
S. Yumoto et al. / Nucl. Instr. and Meth. in Phys. Res. B 223–224 (2004) 754–758
Fig. 3. Time course of amounts of 26 Al in the tissues of suckling rats and weaned rats. Total amounts of 26 Al in the brain ( ), liver( ) and kidneys () were assayed by AMS. Each point represents the mean ± SD; n ¼ 3 per group.
kidneys gained weight considerably during development from day 5 to day 160 postpartum (the weight of the liver increased 66 times on average; the weight of the kidneys increased 18 times on average). On the other hand, the brain weight showed a modest increase from day 5 to day 160 postpartum (a 4.5-times increase on average). Fig. 3 shows the time course of the amount of 26 Al measured in the brain, liver and kidneys. From day 5 to day 20 postpartum, the amounts of 26 Al in the brain, liver and kidneys of suckling rats increased considerably. After weaning, the amounts of 26 Al in the liver and kidneys decreased considerably. However, the 26 Al amount in the brain diminished only slightly up to 140 days after weaning (day 160 postpartum). 4. Discussion The present study demonstrated that 26 Al subcutaneously injected into lactating rats was transferred to the brain of suckling rats through maternal milk. The amount of 26 Al incorporated into the brain via milk decreased only slightly up to 140 days after weaning. These results show quite a gradual clearance of the Al which had entered the brain of suckling rats through maternal milk. We have reported that as much as 27% of the 26 Al in the brain of suckling rats (on day 20 postpartum) was detected in brain cell nuclei [10].
757
Approximately 89% of the 26 Al incorporated into the brain cell nuclei of adult rats was bound to chromatin [11]. Al has been reported to condense brain chromatin configurations [13]. Al interacts with DNA, and modulates DNA (CCG repeats) topology [14]. Al decreases transcription of mRNA, and inhibits protein synthesis in the brain [15]. Since Al markedly reduces DNA synthesis [16] as well as induces cell death (apoptosis) [17], Al may inhibit not only the generation of nerve cells from their progenitor cells, but also the cell division of glial cells and endothelial cells, which act to maintain nerve cell functions. Many authors have reported that Al intake in infants considerably impairs postnatal neurological development of the brain [1,18]. The human brain continues to grow until adolescence [19]. The areas of the brain that take longest to mature during childhood and adolescence (e.g. the hippocampus, the intracortical association areas, the reticular formation) are the same areas of the brain that show the earliest signs of Alzheimer’s disease [20]. It has been hypothesized that exposure of the brain to neurotoxic substances during early life may cause Alzheimer’s disease in old age [21]. In our experiments, we found that the 26 Al levels in the milk of lactating rats were very high. It is worth noting that lactoferrin is markedly rich in milk [22]. Lactoferrin comprises 20–35% of the proteins in breast milk. It shares many structural and functional features with serum transferrin, including an ability to bind iron very tightly, but reversibly. Most of the Al in the blood binds with iron-binding sites of transferrin instead of iron [23]. Since lactoferrin has essentially identical ironbinding sites to those of transferrin [22], it is probable that Al in the milk mostly binds with the iron-binding sites of lactoferrin. Large amounts of Al are used in daily life [24]. Food additives, toothpastes, deodorants and antacids contain high concentrations of Al. From 0.1% to 1% of orally administered 26 Al (26 Al ions or dissolved 26 Al) is absorbed into the human body [25]. In addition, breastfeeding infants are more vulnerable to Al toxicity than adults because of their underdeveloped gastrointestinal barrier to Al absorption and immature renal function to excrete Al from the blood [1,18]. Therefore, women should avoid Al
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S. Yumoto et al. / Nucl. Instr. and Meth. in Phys. Res. B 223–224 (2004) 754–758
intake as much as possible for the health of their infants. We conclude that the 26 Al administered to lactating rats was incorporated into the brain of suckling rats through maternal milk. The 26 Al taken up into the brain of suckling rats decreased only slightly up to 140 days after weaning. Al incorporated into the brain of infants via milk may impair neurological development of the brain, and may provide a clue about the possible involvement of Al in Alzheimer’s disease. Acknowledgements This research was supported by Japan Science and Technology Corporation funds. References [1] N.J. Bishop, R. Morley, B. Chir, J.P. Day, A. Lucas, N. Engl. J. Med. 336 (1997) 1557. [2] G. Muller, M.-F. Hutin, D. Burnel, P.R. Lehr, Biol. Trace Elem. Res. 34 (1992) 79. [3] R.A. Yokel, Toxicol. Appl. Pharmacol. 79 (1985) 121. [4] J.M. Donald, M.S. Golub, M.E. Gershwin, C.L. Keen, Toxicol. Appl. Pharmacol. 11 (1989) 345. [5] A.C. Alfrey, G.R. LeGendre, W.D. Kaehny, N. Engl. J. Med. 294 (1976) 184. [6] D.R. Crapper, S.S. Krishnan, S. Quittkat, Brain (1976) 67. [7] D.R. McLachlan, P.E. Fraser, E. Jaikaran, W.J. Lukiw, in: L.W. Chang (Ed.), Toxicology of Metals, CRC, New York, 1996, p. 387. [8] S. Yumoto, Y. Horino, Y. Mokuno, S. Kakimi, K. Fujii, Nucl. Instr. and Meth. B 109 & 110 (1996) 362.
[9] K. Kobayashi, S. Yumoto, H. Nagai, H. Hosoyama, M. Imamura, Sh. Masuzawa, Y. Koizumi, H. Yamashita, Proc. Jpn. Acad. 66B (1990) 189. [10] S. Yumoto, H. Nagai, H. Matsuzaki, H. Matsumura, W. Tada, E. Nagatsuma, K. Kobayashi, Brain Res. Bull. 55 (2001) 229. [11] S. Yumoto, H. Nagai, M. Imamura, H. Matsuzaki, K. Hayashi,, A. Masuda, H. Kumazawa, H. Ohashi, K. Kobayashi, Nucl. Instr. and Meth. B 123 (1997) 279. [12] S. Yumoto, H. Nagai, H. Matsuzaki, T. Kobayashi, W. Tada, Y. Ohki, S. Kakimi, K. Kobayashi, Nucl. Instr. and Meth. B 172 (2000) 925. [13] P.R. Walker, J. LeBlanc, M. Sikorska, Biochemistry 28 (1989) 3911. [14] K. Latha, S. Anitha, K. Rao, M. Viswamitra, Biochim. Biophys. Acta 1588 (2002) 56. [15] D.M. Nicholls, G.M. Spears, S. Asina, A.C.M. Miller, Int. J. Biochem. Cell Biol. 27 (1995) 365. [16] G.M. Berlyne, A.J. Ben, E. Knoff, R. Yagil, G. Weinberger, D.M. Donovitch, Lancet i (1970) 494. [17] R. Tsubouchi, H.H. Htay, K. Murakami, M. Haneda, M. Yoshino, Biometals 14 (2001) 181. [18] A.B. Gruskin, Adv. Pediatr. 35 (1988) 281. [19] J.L. Conel, in: The Postnatal Development of the Human Cerebral Cortex, Vol. 1–8, Harvard University Press, Cambridge, MA, 1939–1967. [20] V.M. Moceri, W.A. Kukull, I. Emanuel, G. van Belle, E.B. Larson, Neurology 54 (2000) 415. [21] M. Prince, Epidemiology 9 (1998) 618. [22] E.N. Baker, H.M. Baker, R.D. Kidd, Biochem. Cell Biol. 80 (2002) 27. [23] J. Barker, J.P. Day, N.D. Priest, D. Newton, P.V. Drumm, J.S. Lilley, G.W.A. Newton, Nucl. Instr. and Meth. B 68 (1992) 319. [24] S. Yumoto, in: K. Watanuki, K. Yoshioka (Eds.), Global Environment and Human Activity, Maruzen Planet, Tokyo, 1996, p. 41. [25] N.D. Priest, R.J. Talbot, J.G. Austin, J.P. Day, S.J. King, K. Fifield, R.G. Creswell, Biometrics 9 (1996) 221.
26
Al incorporation into the tissues of suckling rats through maternal milk S. Yumoto
a,*
, H. Nagai b, K. Kobayashi c, W. Tada b, T. Horikawa b, H. Matsuzaki c
a
c
Yumoto Institute of Neurology, Kawadacho 6-11, Shinjuku-ku, Tokyo 162-0054, Japan b College of Humanities and Sciences, Nihon University, Tokyo 156-8550, Japan Research Center for Nuclear Science and Technology, University of Tokyo, Tokyo 113-0032, Japan
Abstract Aluminium (Al) is highly neurotoxic and inhibits prenatal and postnatal development of the brain in humans and experimental animals. However, Al incorporation into the brain of sucklings through maternal milk has not yet been well clarified because Al lacks a suitable isotope for radioactive tracer experiments. Using 26 Al as a tracer, we measured 26 Al incorporation into the brain of suckling rats by accelerator mass spectrometry. Lactating rats were subcutaneously injected with 26 AlCl3 from day 1 to day 20 postpartum. Suckling rats were weaned from day 21 postpartum. From day 5 to day 20 postpartum, the 26 Al levels measured in the brain, liver, kidneys and bone of suckling rats increased significantly. After weaning, the amounts of 26 Al in the liver and kidneys decreased remarkably. However, the 26 Al amount in the brain had diminished only slightly up to 140 days after weaning. 2004 Elsevier B.V. All rights reserved. PACS: 87.59.Qx Keywords: Accelerator mass spectrometry (AMS);
26
Al; Aluminium toxicity; Alzheimer’s disease
1. Introduction Aluminium (Al) inhibits prenatal and postnatal development of the brain in humans and experimental animals [1,2]. Al administration to experimental animals during gestation and lactation causes retardation of neurological development and deficits in neurobehavioral performance in
*
Corresponding author. Tel.: +81-3-3341-3406; fax: +81-33358-4867. E-mail address: [email protected] (S. Yumoto).
their offspring [3]. Neurological impairment persists in the weaned offspring at Al doses which do not produce any detectable signs of maternal toxicity [4]. Al is highly neurotoxic and can result in the degeneration of nerve cells in the brain [5]. Excess exposure to Al causes dialysis dementia in patients on dialysis treatment and Al encephalopathy in Al industry workers. Al has been reported to be one of the risk factors for Alzheimer’s disease (senile dementia of Alzheimer type) and amyotrophic lateral sclerosis [6,7]. It is assumed that the neurotoxicity of Al is due to its presence in the brain [8]. However, Al
0168-583X/$ - see front matter 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2004.04.140
S. Yumoto et al. / Nucl. Instr. and Meth. in Phys. Res. B 223–224 (2004) 754–758
incorporation into the brain of sucklings has not been well clarified because Al lacks a suitable isotope for radioactive tracer experiments and a sensitive analysis technique. The application of accelerator mass spectrometry (AMS) to the analysis of 26 Al (t1=2 ¼ 7:16 105 years) has enabled the study of Al metabolism under physiological conditions [9,10]. One of the advantages of using AMS is that it measures individual atoms and not radioactivity. Since almost all naturally occurring Al is 27 Al, newly administered 26 Al can be distinguished from endogenous Al (27 Al) by AMS. In addition, as few as 5 · 1018 g (5 attograms) of 26 Al can be detected by this method. Previously, we injected 26 Al intraperitoneally into eight-month-old adult rats as a tracer, and used AMS to demonstrate 26 Al incorporation into the brain through the blood–brain barrier [9,11]. Approximately 17% of the 26 Al measured in the brain was incorporated into the cell nuclei [11]. In this study, we injected 26 AlCl3 subcutaneously into lactating rats to measure 26 Al incorporation into tissues (including the brain) of suckling rats through maternal milk by using AMS. After weaning, the metabolism of the 26 Al taken up into the rat tissues via milk was also studied by AMS.
2. Materials and methods 2.1. Animals Male and female Wistar rats were purchased from Clea Japan Inc., Tokyo, Japan. The rats were fed a standard diet (MF food pellets, Oriental Yeast Co., Tokyo, Japan) and tap water ad libitum, and maintained in light (lights on 0600–1800) and temperature (20–22 C) controlled rooms. Three female rats weighing 240–250 g each at the start of the experiment were put together with two male breeder rats, and vaginal smears were examined the following morning. The day on which sperm was found in the smear was designated as day 1 of gestation. All animal experiments in this study were performed according to the Tokyo University Guidelines for the Care and Use of Laboratory Animals.
755
2.2. 26 Al transfer from lactating rats to suckling rats through maternal milk Three lactating rats with a litter size of 11 pups each were used in this study. Each lactating rat was subcutaneously injected with 0.2 ml of a solution containing 20 dpm (470 pg) of 26 Al (26 AlCl3 ) and 0.009 mg of 27 Al (27 AlCl3 ) in a 0.45 M sodium acetate–hydrochloric acid buffer (pH 4.3) once daily from day 1 to day 20 postpartum. On days 5, 10, 15 and 20 postpartum, suckling rats were euthanized under ether anaesthesia, and the brain (cerebrum), liver, kidneys, parietal bone and blood were removed from the rats, and used for the measurement of 26 Al by AMS. Before euthanizing suckling rats, they were separated from the lactating rats into other cages, starved for 90 min and then put together with the lactating rats to ingest milk for 20 min. After euthanasia of the suckling rats under ether anaesthesia, freshly ingested milk was removed from the stomach of suckling rats for 26 Al assay. Both male and female suckling rats were used in this study. On day 21 postpartum, suckling rats were separated from lactating rats into other cages, and fed with a standard diet. On days 40 (20 days after weaning), 80 (60 days after weaning) and 160 (140 days after weaning) postpartum, weaned rats were euthanized under ether anaesthesia, and the brain, liver, kidneys, parietal bone and blood were removed from the weaned rats, and used for the 26 Al assay by AMS. After weaning, only female rats were used in this study. 2.3. Al measurements by AMS Each sample was weighed, minced and freezedried with a vacuum dryer. The samples from the brain, liver, kidneys, blood and milk were decomposed with 3 ml of concentrated nitric acid at 80 C for 24 h in a Teflon-sealed vessel inserted into a stainless steel bomb [9,12]. The bone (parietal bone) was mixed with 3 ml of concentrated nitric acid at room temperature for 48 h, and heated at 80 C for 24 h in the Teflon-sealed vessel. (Parietal bone is flat and thin in shape, and is easily decomposed with nitric acid.) After cooling to room temperature, the sample was combined with
756
S. Yumoto et al. / Nucl. Instr. and Meth. in Phys. Res. B 223–224 (2004) 754–758
2 mg of 27 Al as a carrier. (In some of the samples, the amount of 27 Al carrier was reduced to 0.5 or 1 mg to improve the detection limit of 26 Al in the samples.) Then, the sample was mixed with 3 ml of concentrated nitric acid, and heated at 140 C for 12 h in the Teflon-sealed vessel. After cooling to room temperature, the sample was blended with 1 ml of perchloric acid (60%), and heated on a hotplate at 90 C for 4 h. This process was repeated three times. Al fractions were purified by cation exchange, treated with pure ammonia water to convert the Al to Al oxide at 800 C and mixed with an equal mass of pure silver powder. The Al oxide was pressed into a sample holder, and examined with a tandem accelerator at Tokyo University. The 26 Al-AMS system at Tokyo University has the following features. The sensitivity threshold of 26 Al/27 Al is about 1 · 1014 and the reproducibility of measurements is better than ±3%. The minimal detectable amount of 26 Al is about 5 · 1018 g (5 attograms).
Fig. 1. Time course of 26 Al levels in the tissues of suckling rats and weaned rats and in the milk. A lactating rat was subcutaneously injected once daily with 470 pg (20 dpm) of 26 Al from day 1 to day 20 postpartum. The variable, ‘‘26 Al injected’’, of the formula on the ordinate represents the total amount of 26 Al injected into each lactating rat up to and including the time of the measurement. After weaning, ‘‘26 Al injected’’ is constant at 20 · 470 pg. 26 Al levels in the brain ( ), liver ( ;), kidneys (), bone (M), blood (N) and milk (j) were measured by AMS. Each point represents the mean ± SD; n ¼ 3 per group.
3. Results 26
The time course of Al levels in the samples from the suckling rats and in rat milk is shown in Fig. 1. The 26 Al levels in the milk were very high, specifically on day 5 postpartum. The 26 Al concentrations in the milk were stationary from day 10 to day 20 postpartum. Approximately 0.2–0.4% of the 26 Al injected into a lactating rat was contained in 1 g of the milk. The 26 Al levels in the bone were the highest in the samples from the suckling rats, and showed almost constant values from day 5 to day 20 postpartum. The 26 Al levels of the liver and kidneys increased significantly from day 5 to day 20 postpartum. In contrast, the 26 Al levels in the blood decreased remarkably from day 5 to day 20 postpartum. This decrease in the blood 26 Al levels was probably due to the development of renal function, which removes Al from the blood, during this stage. The 26 Al levels in the brain (cerebrum) of suckling rats were almost stationary from day 5 to day 20 postpartum. After weaning, the 26 Al levels in the liver, kidneys and bone decreased considerably (Fig. 1). Especially the 26 Al levels measured in the liver and
kidneys had diminished sharply 20 days after weaning. The blood 26 Al levels continued to decrease until 140 days after weaning (day 160 postpartum). On the other hand, the 26 Al levels in the brain decreased only slightly up to 140 days after weaning. As shown in Fig. 2, the liver and
Fig. 2. Growth of tissues in suckling rats and weaned rats. Tissue weights of the brain ( ), liver ( ) and kidneys () are shown. Each point represents the mean ± SD; n ¼ 3 per group.
S. Yumoto et al. / Nucl. Instr. and Meth. in Phys. Res. B 223–224 (2004) 754–758
Fig. 3. Time course of amounts of 26 Al in the tissues of suckling rats and weaned rats. Total amounts of 26 Al in the brain ( ), liver( ) and kidneys () were assayed by AMS. Each point represents the mean ± SD; n ¼ 3 per group.
kidneys gained weight considerably during development from day 5 to day 160 postpartum (the weight of the liver increased 66 times on average; the weight of the kidneys increased 18 times on average). On the other hand, the brain weight showed a modest increase from day 5 to day 160 postpartum (a 4.5-times increase on average). Fig. 3 shows the time course of the amount of 26 Al measured in the brain, liver and kidneys. From day 5 to day 20 postpartum, the amounts of 26 Al in the brain, liver and kidneys of suckling rats increased considerably. After weaning, the amounts of 26 Al in the liver and kidneys decreased considerably. However, the 26 Al amount in the brain diminished only slightly up to 140 days after weaning (day 160 postpartum). 4. Discussion The present study demonstrated that 26 Al subcutaneously injected into lactating rats was transferred to the brain of suckling rats through maternal milk. The amount of 26 Al incorporated into the brain via milk decreased only slightly up to 140 days after weaning. These results show quite a gradual clearance of the Al which had entered the brain of suckling rats through maternal milk. We have reported that as much as 27% of the 26 Al in the brain of suckling rats (on day 20 postpartum) was detected in brain cell nuclei [10].
757
Approximately 89% of the 26 Al incorporated into the brain cell nuclei of adult rats was bound to chromatin [11]. Al has been reported to condense brain chromatin configurations [13]. Al interacts with DNA, and modulates DNA (CCG repeats) topology [14]. Al decreases transcription of mRNA, and inhibits protein synthesis in the brain [15]. Since Al markedly reduces DNA synthesis [16] as well as induces cell death (apoptosis) [17], Al may inhibit not only the generation of nerve cells from their progenitor cells, but also the cell division of glial cells and endothelial cells, which act to maintain nerve cell functions. Many authors have reported that Al intake in infants considerably impairs postnatal neurological development of the brain [1,18]. The human brain continues to grow until adolescence [19]. The areas of the brain that take longest to mature during childhood and adolescence (e.g. the hippocampus, the intracortical association areas, the reticular formation) are the same areas of the brain that show the earliest signs of Alzheimer’s disease [20]. It has been hypothesized that exposure of the brain to neurotoxic substances during early life may cause Alzheimer’s disease in old age [21]. In our experiments, we found that the 26 Al levels in the milk of lactating rats were very high. It is worth noting that lactoferrin is markedly rich in milk [22]. Lactoferrin comprises 20–35% of the proteins in breast milk. It shares many structural and functional features with serum transferrin, including an ability to bind iron very tightly, but reversibly. Most of the Al in the blood binds with iron-binding sites of transferrin instead of iron [23]. Since lactoferrin has essentially identical ironbinding sites to those of transferrin [22], it is probable that Al in the milk mostly binds with the iron-binding sites of lactoferrin. Large amounts of Al are used in daily life [24]. Food additives, toothpastes, deodorants and antacids contain high concentrations of Al. From 0.1% to 1% of orally administered 26 Al (26 Al ions or dissolved 26 Al) is absorbed into the human body [25]. In addition, breastfeeding infants are more vulnerable to Al toxicity than adults because of their underdeveloped gastrointestinal barrier to Al absorption and immature renal function to excrete Al from the blood [1,18]. Therefore, women should avoid Al
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intake as much as possible for the health of their infants. We conclude that the 26 Al administered to lactating rats was incorporated into the brain of suckling rats through maternal milk. The 26 Al taken up into the brain of suckling rats decreased only slightly up to 140 days after weaning. Al incorporated into the brain of infants via milk may impair neurological development of the brain, and may provide a clue about the possible involvement of Al in Alzheimer’s disease. Acknowledgements This research was supported by Japan Science and Technology Corporation funds. References [1] N.J. Bishop, R. Morley, B. Chir, J.P. Day, A. Lucas, N. Engl. J. Med. 336 (1997) 1557. [2] G. Muller, M.-F. Hutin, D. Burnel, P.R. Lehr, Biol. Trace Elem. Res. 34 (1992) 79. [3] R.A. Yokel, Toxicol. Appl. Pharmacol. 79 (1985) 121. [4] J.M. Donald, M.S. Golub, M.E. Gershwin, C.L. Keen, Toxicol. Appl. Pharmacol. 11 (1989) 345. [5] A.C. Alfrey, G.R. LeGendre, W.D. Kaehny, N. Engl. J. Med. 294 (1976) 184. [6] D.R. Crapper, S.S. Krishnan, S. Quittkat, Brain (1976) 67. [7] D.R. McLachlan, P.E. Fraser, E. Jaikaran, W.J. Lukiw, in: L.W. Chang (Ed.), Toxicology of Metals, CRC, New York, 1996, p. 387. [8] S. Yumoto, Y. Horino, Y. Mokuno, S. Kakimi, K. Fujii, Nucl. Instr. and Meth. B 109 & 110 (1996) 362.
[9] K. Kobayashi, S. Yumoto, H. Nagai, H. Hosoyama, M. Imamura, Sh. Masuzawa, Y. Koizumi, H. Yamashita, Proc. Jpn. Acad. 66B (1990) 189. [10] S. Yumoto, H. Nagai, H. Matsuzaki, H. Matsumura, W. Tada, E. Nagatsuma, K. Kobayashi, Brain Res. Bull. 55 (2001) 229. [11] S. Yumoto, H. Nagai, M. Imamura, H. Matsuzaki, K. Hayashi,, A. Masuda, H. Kumazawa, H. Ohashi, K. Kobayashi, Nucl. Instr. and Meth. B 123 (1997) 279. [12] S. Yumoto, H. Nagai, H. Matsuzaki, T. Kobayashi, W. Tada, Y. Ohki, S. Kakimi, K. Kobayashi, Nucl. Instr. and Meth. B 172 (2000) 925. [13] P.R. Walker, J. LeBlanc, M. Sikorska, Biochemistry 28 (1989) 3911. [14] K. Latha, S. Anitha, K. Rao, M. Viswamitra, Biochim. Biophys. Acta 1588 (2002) 56. [15] D.M. Nicholls, G.M. Spears, S. Asina, A.C.M. Miller, Int. J. Biochem. Cell Biol. 27 (1995) 365. [16] G.M. Berlyne, A.J. Ben, E. Knoff, R. Yagil, G. Weinberger, D.M. Donovitch, Lancet i (1970) 494. [17] R. Tsubouchi, H.H. Htay, K. Murakami, M. Haneda, M. Yoshino, Biometals 14 (2001) 181. [18] A.B. Gruskin, Adv. Pediatr. 35 (1988) 281. [19] J.L. Conel, in: The Postnatal Development of the Human Cerebral Cortex, Vol. 1–8, Harvard University Press, Cambridge, MA, 1939–1967. [20] V.M. Moceri, W.A. Kukull, I. Emanuel, G. van Belle, E.B. Larson, Neurology 54 (2000) 415. [21] M. Prince, Epidemiology 9 (1998) 618. [22] E.N. Baker, H.M. Baker, R.D. Kidd, Biochem. Cell Biol. 80 (2002) 27. [23] J. Barker, J.P. Day, N.D. Priest, D. Newton, P.V. Drumm, J.S. Lilley, G.W.A. Newton, Nucl. Instr. and Meth. B 68 (1992) 319. [24] S. Yumoto, in: K. Watanuki, K. Yoshioka (Eds.), Global Environment and Human Activity, Maruzen Planet, Tokyo, 1996, p. 41. [25] N.D. Priest, R.J. Talbot, J.G. Austin, J.P. Day, S.J. King, K. Fifield, R.G. Creswell, Biometrics 9 (1996) 221.