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Regional distribution of lead, zinc, iron and copper in suckling and adult rat brains
180
Brain Research, 251 (1982) 180-182
Elsevier Biomedical Press
Regional distribution of lead, zinc, iron and copper in suckling and adult rat brains REIKO KISHI, TOSHIKO IKEDA, HIROTSUGU MIYAKE, EIJI UCHINO, TOSHIHUMI TSUZUKI and KATSUHIRO INOUE Department of Public Health, Sapporo Medical College and Hokkaido Institute of Public Health, Sapporo 060 (Japan)
(Accepted July 13th, 1982) Key words: lead - - zinc - - iron - - copper - - distribution - - brain development
The flamelessatomic absorption method was applied to reveal regional differencesin heavy metals between suckling and adult rat brains. The lead concentrations in the suckling brains were considerably different from those in the adult rats. The levels of lead concentration in the cerebrum and cerebellum of the normal suckling rats were 3-4 times higher than those of adult rats. Iron concentrations in the cerebrum of the suckling brains were approximately half those in the adult brains. Zinc and copper concentrations in medulla from the suckling rats were higher than those in the same region from the adult rats. It is well known that the risk of lead poisoning is especially high in very young children. Until a model using suckling rats was developed, lead encephalopathy had been recognized to occur only in subhuman primates 13. Developmental differences in lead concentration in the brain are suspected to be the cause of lead encephalopathy. In recent years, it has been reported that lead, zinc and copper accumulate in the hippocampus far in excess of the average contents of the whole brain, as assessed by histochemical and analytical techniques 4-6. There have been no data available, however, with respect to the distribution and the levels of lead within specific regions and specific age groups. In order to determine the regional variation of lead in lead-poisoned rats, a serious of studies using atomic absorption spectrophotometry was undertaken. During these studies, considerable differences in lead concentration and distribution in suckling as compared to adult brains were found and are reported in the present paper. The distribution of 3 other metals, possibly influencing the pharmacokinetics of lead, were also determined. Suckling and adult male Wistar rats were raised in our laboratory and were housed in stainless-steel cages. Standard laboratory chow (Oriental Kobo) was used. Metal concentrations in the diet and in the water administered to the rats were analyzed. Lead, 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
zinc, and copper concentrations in the diet were 0.27/zg/g, 51.8 Fg/g, 120 Fg/g and 8.5/~g]g, respectively, while those of water were 0.01 /~g/g, 0.02 #g/g, 0.05/~g/g and 0.01 #g/g respectively. There was no evidence of lead contamination. These animals were sacrificed by heart puncture under pentobarbital anesthesia. Brains were removed and divided carefully into 7 regions using the method of Glowinski and Iversen 7. These 7 regions included: (1) the cerebellum; (2) medulla oblongata; (3) hypothalamus; (4) midbrain; (5) striatum; (6) hippocampus; and (7) cortex. The 'medulla oblongata' in this study corresponded to the medulla oblongata and the ports: the part of the diencephalon; the 'striatum' included the putamen nucleus and caudate nucleus and the globus pallidus nucleus (i.e. the basal ganglia of the telencephalon without amygdala): the cortex corresponded to the telencephalon without the 'striatum' and included the white and gray matter of the cerebral cortex. Analyses were performed by using a variant AA 175 type flameless atomizer equipped with a D2 lamp background corrector. A mixture of nitric acid and perchloric acid was used for the wet digestion of samples. The standard addition method was applied for the determination in order to eliminate matrix interferences. Table I shows the mean concentrations of each
181 metal in the 7 regions obtained from the 2 groups of rats sacrificed at 22 days and 290 days of age, respectively. The lead content of the brain from suckling rats was relatively higher than that from adult rats. Lead concentrations in the cortex, the cerebellum and the medulla oblongata from the suckling rats were significantly different from those in adult rats (P < 0.01, P < 0.005 and P < 0.05, respectively), being about 3 times higher. From Table I it can be seen that the distribution of lead in the brain was characteristically uneven. The regional differences in lead concentration by analysis of variance were statistically significant, with that in the cortex being the lowest in both suckling and adult brains. Lead concentrations in the cerebellum, hippocampus, striatum and hypothalamus from suckling brains were significantly higher than those in the cortex (being P < 0.05, P < 0.05, P < 0.05, P < 0.01, respectively). Similar results were also observed in adult brains. Lead concentrations in the hippocampus, striaturn and hypothalamus were significantly higher than that in the cortex (being P < 0.05, P < 0.05, P < 0.01, respectively). It has been confirmed that only small amounts of lead accumulate in the adult brain 1, probably due to
the slow passage of lead ions through the bloodbrain barrier. A possible explanation for the higher cortex and cerebellar lead concentrations in suckling rats as compared to adult rats therefore may be the existence of a fragile and not yet developed bloodbrain barrier in the young. This explanation agrees well with pathological studies which have shown that developing capillaries are most vulnerable to the adverse effects of leadlL The lead distribution in the brains of the adult rats was quite similar to that reported by Grandjean for 4 adult men without occupational exposure to leads . The levels of lead concentration in the hippocampus from the adult brains were 10 times higher than those in the cerebrum. This result for the hippocampus in our study is in good agreement with the levels reported by Fjerdingstad et al. 5. In addition, however, we also observed, from the results of our experiments, relatively higher concentrations presented in the hippocampus, hypothalamus and medulla oblongata in suckling brains and in the striatum and hypothalamus in adult brains. The mean lead concentrations of the whole brain of rats sacrificed at 22 days of age and 290 days of age were 0.80 + 0.11 #g/g and 0.46 -4- 0.26 #g/g, respectively. The lead levels at 22 days of age were a
TABLE I Regional distribution of lead, iron, zinc and copper in rat brain (#g/g) at 22 and 290 days of age Each value represents the mean q- S.E.M. of 7 rats.
Brain region
Lead 22 days
Cortex 0.394-0.09** Cerebellum 1.034-0.17"** Medulla oblongata 1.144-0.23§* Midbrain 0.784-0.15 Hippocampus 1.534-0.38 Striatum 1.404-0.30 Hypothalamus 1.424-0.21
Iron 290 days
22 days
Zinc 290 days
22 days
0.094-0.02 11.094-0.74"** 20.434-0.90 14.274-0.23 0.354-0.05 18.404-3.14 23.29~0.37 12.724-0.80
Copper 290 days
22 days
290 days
15.304-0.65 3.024-0.25 3.194-0.15 10.794-0.22 2.394-0.08 3.054-0.37
0.364-0.08 15.634-2.64 0.694-0.24 15.21-+-2.79
16.104-0.16 13.414-1.42"* 8.094-0.94 3.284-0.19" 2.624-0.16 19.414-0.77 13.644-1.21 10.394-0.75 2.804-0.22 3.394-0.45
0.914-0.10 27.994-9.42 1.104-0.23 10.934-2.32
26.474-8.14 13.834-1.72 12.274-2.12 14.064-1.08
21.844-0.78 3.684-0.52 3.234-0.50 15.364-3.05 3.224-0.48 3.294-0.60
1.054-0.34 14.214-2.47
17.884-1.57 11.134-0.65
10.464-0.74 2.544-0.18 3.844-0.53
§ n = 6. One extreme observation was tested by the statistical method of Stefansky and Grubbs la, was confirmed as an outlier, and was treated as a missing value. * Significantly higher concentration than in 290 days of age (P < 0.05). ** Significantly higher concentrations than in 290 days of age (P < 0.01). *** Significantly higher concentrations than in 290 days of age (P < 0.005).
182 little higher than that reported previously by Mykkanen et al. 11. This discrepancy might have been caused by methodological differences in dissection of the brain. The concentration of iron irt the rat brains was also the highest in the hippocampus. In human studies, iron is found to be in higher concentration in the basal ganglia (particularly the globus pallidus, putamen and caudate nucleus) 9. The values of iron obtained in the rat striatum, which includes the globus pallidus, putamen and caudate nucleus, on the other hand, were relatively low. The concentration of iron in the cortex of suckling rats was significantly lower than that from adult brains. The levels in the cortex from adult brains were approximately twice as great than those in the same region in the sucklings. These findings are in agreement with human data indicating that iron rose approximately 2-fold compared to what it had been perirtatally 15. Zinc concentrations were distributed almost uniformly in each of the regions of the suckling brains measured. There were no regional differences among any of the regions of the suckling brains. Zinc concentrations were considerably different from those in suckling rats. The regional differences among the 7 regions of the adult brains as measured by analysis of variance 1 Barry, S. I. and Mossman, D. B., Lead concentrations in human tissues, Brit. J. industr. Med., 27 (1970) 339-351. 2 Brun, A. and Brunk, U., Heavy metal localization and age related accumulation in the rat nervous system, Histochemie, 34 (1973) 333-342. 3 Crawford, I. L. and Connor, J. D., Zinc in maturing rat brain: hippocampal concentration and localization, J. Neurochem., 19 (1972) 1451-1458. 4 Danscher, G., Fjerdingstad, E.J., Fjerdingstad, E. and Fredens, K., Heavy metal content in subdivisions of the rat hippocampus (zinc, lead and copper), Brain Research, 112 (1976) 442-446. 5 Fjerdingstad, E. J., Danscher, G. and Fjerdingstad, E., Hippoeampus: selective concentration of lead in the normal rat brain, Brain Research, 80 (1974) 350-354. 6 Fjerdingstad, E. J., Danscher, G. and Fjerdingstad, E., Changes in zinc and lead content of rat hippocampus and whole brain following intravital dithizone treatment as determined by flameless atomic absorption spectrophotometry, Brain Research, 130 (1977) 369--373. 7 Glowinski, J. and Iversen, L. L., Regional studies of catecholamines in the rat brain-I. The disposition of [aH]norepinephrine, [aH]dopamine and [aH]dopa in various regions of the brain, J. Neurochem., 13 (1966) 655-669. 8 Grandjean, P., Regional distribution of lead in human
were statistically significant (P < 0.05). The highest zinc level in adult brains was found in the hippocampus and the lowest in the medulla. The distribution of zinc in the brain regions seems to be in accordance with the few records of zinc distribution in adult rat brainsZ,3,10. The zinc concentrations in the medulla from the suckling rats were significantly higher than those in the same region from the adult rats. The copper distribution pattern in the suckling rats was almost the same as that in the adult rats, although the copper concentration in the medulla of the suckling brains was significantly higher than that in the adult brains. When the elemental relationship among the 4 elements was investigated, it was observed that lead concentrations in each of the regions from the suckling rats correlated significantly with those of iron (r : 0.32, P < 0.05). There was no such relationship for adult rats or among other metals. This relationship of lead and iron in the suckling rat might, in part, be explained by the fact that acute lead encephalopathy in the suckling rat has been described as a vasculopathy 1~, and that lead becomes localized in capillary-microvessel fractions even when present at very low levels 14.
brains, ToxicoL Lett., 2 (1978) 65-69. 9 Harrison, W. W., Nestsky, M. G. and Brown, M. D., Trace elements in human brain; copper, zinc, iron and magnesium, Clin. Chim. Acta, 21 (1968) 55-60. 10 Kozma, M. and Ferke, A., Trace element localization and changes in zinc and copper concentrations during postnatal development of the rat CNS, Acta Histochem., 65 (1979) 219-227. 11 Mykkanen, H. M., Dickerson, J. W. T. and Lancaster, M. C., Effect of age on the tissue distribution of lead in the rat, Toxicol. appl. PharmacoL, 51 (1979) 447-454. 12 Pentshew, A. and Garro, F., Lead encephalo-myelopathy of the suckling rat and its modifications on porphyrinopathic nervous disease, Acta Neuropath., 6 (1966) 266-278. 13 Snedecor,W. G. and Cockran, G. W., Statistical Methods, 7th Edn., Iowa State University Press, Ames, IA, 1980, pp. 279-280. 14 Toews, A. D., Kolber, A., Hayward, J., Krigrrmn, M. R. and Morell, P., Experimental lead encephalopathy in the suckling rat: concentration of lead in cellular fractions enriched in brain capillaries, Brain Research, 147 (1978) 131-138. 15 V61ki, A., Berlet, H. and Ule, G., Trace elements (Cu, Fe, Mg, Zn) of the brain during childhood, Neuropiidiatrie, 3 (1974) 236-242.
Brain Research, 251 (1982) 180-182
Elsevier Biomedical Press
Regional distribution of lead, zinc, iron and copper in suckling and adult rat brains REIKO KISHI, TOSHIKO IKEDA, HIROTSUGU MIYAKE, EIJI UCHINO, TOSHIHUMI TSUZUKI and KATSUHIRO INOUE Department of Public Health, Sapporo Medical College and Hokkaido Institute of Public Health, Sapporo 060 (Japan)
(Accepted July 13th, 1982) Key words: lead - - zinc - - iron - - copper - - distribution - - brain development
The flamelessatomic absorption method was applied to reveal regional differencesin heavy metals between suckling and adult rat brains. The lead concentrations in the suckling brains were considerably different from those in the adult rats. The levels of lead concentration in the cerebrum and cerebellum of the normal suckling rats were 3-4 times higher than those of adult rats. Iron concentrations in the cerebrum of the suckling brains were approximately half those in the adult brains. Zinc and copper concentrations in medulla from the suckling rats were higher than those in the same region from the adult rats. It is well known that the risk of lead poisoning is especially high in very young children. Until a model using suckling rats was developed, lead encephalopathy had been recognized to occur only in subhuman primates 13. Developmental differences in lead concentration in the brain are suspected to be the cause of lead encephalopathy. In recent years, it has been reported that lead, zinc and copper accumulate in the hippocampus far in excess of the average contents of the whole brain, as assessed by histochemical and analytical techniques 4-6. There have been no data available, however, with respect to the distribution and the levels of lead within specific regions and specific age groups. In order to determine the regional variation of lead in lead-poisoned rats, a serious of studies using atomic absorption spectrophotometry was undertaken. During these studies, considerable differences in lead concentration and distribution in suckling as compared to adult brains were found and are reported in the present paper. The distribution of 3 other metals, possibly influencing the pharmacokinetics of lead, were also determined. Suckling and adult male Wistar rats were raised in our laboratory and were housed in stainless-steel cages. Standard laboratory chow (Oriental Kobo) was used. Metal concentrations in the diet and in the water administered to the rats were analyzed. Lead, 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
zinc, and copper concentrations in the diet were 0.27/zg/g, 51.8 Fg/g, 120 Fg/g and 8.5/~g]g, respectively, while those of water were 0.01 /~g/g, 0.02 #g/g, 0.05/~g/g and 0.01 #g/g respectively. There was no evidence of lead contamination. These animals were sacrificed by heart puncture under pentobarbital anesthesia. Brains were removed and divided carefully into 7 regions using the method of Glowinski and Iversen 7. These 7 regions included: (1) the cerebellum; (2) medulla oblongata; (3) hypothalamus; (4) midbrain; (5) striatum; (6) hippocampus; and (7) cortex. The 'medulla oblongata' in this study corresponded to the medulla oblongata and the ports: the part of the diencephalon; the 'striatum' included the putamen nucleus and caudate nucleus and the globus pallidus nucleus (i.e. the basal ganglia of the telencephalon without amygdala): the cortex corresponded to the telencephalon without the 'striatum' and included the white and gray matter of the cerebral cortex. Analyses were performed by using a variant AA 175 type flameless atomizer equipped with a D2 lamp background corrector. A mixture of nitric acid and perchloric acid was used for the wet digestion of samples. The standard addition method was applied for the determination in order to eliminate matrix interferences. Table I shows the mean concentrations of each
181 metal in the 7 regions obtained from the 2 groups of rats sacrificed at 22 days and 290 days of age, respectively. The lead content of the brain from suckling rats was relatively higher than that from adult rats. Lead concentrations in the cortex, the cerebellum and the medulla oblongata from the suckling rats were significantly different from those in adult rats (P < 0.01, P < 0.005 and P < 0.05, respectively), being about 3 times higher. From Table I it can be seen that the distribution of lead in the brain was characteristically uneven. The regional differences in lead concentration by analysis of variance were statistically significant, with that in the cortex being the lowest in both suckling and adult brains. Lead concentrations in the cerebellum, hippocampus, striatum and hypothalamus from suckling brains were significantly higher than those in the cortex (being P < 0.05, P < 0.05, P < 0.05, P < 0.01, respectively). Similar results were also observed in adult brains. Lead concentrations in the hippocampus, striaturn and hypothalamus were significantly higher than that in the cortex (being P < 0.05, P < 0.05, P < 0.01, respectively). It has been confirmed that only small amounts of lead accumulate in the adult brain 1, probably due to
the slow passage of lead ions through the bloodbrain barrier. A possible explanation for the higher cortex and cerebellar lead concentrations in suckling rats as compared to adult rats therefore may be the existence of a fragile and not yet developed bloodbrain barrier in the young. This explanation agrees well with pathological studies which have shown that developing capillaries are most vulnerable to the adverse effects of leadlL The lead distribution in the brains of the adult rats was quite similar to that reported by Grandjean for 4 adult men without occupational exposure to leads . The levels of lead concentration in the hippocampus from the adult brains were 10 times higher than those in the cerebrum. This result for the hippocampus in our study is in good agreement with the levels reported by Fjerdingstad et al. 5. In addition, however, we also observed, from the results of our experiments, relatively higher concentrations presented in the hippocampus, hypothalamus and medulla oblongata in suckling brains and in the striatum and hypothalamus in adult brains. The mean lead concentrations of the whole brain of rats sacrificed at 22 days of age and 290 days of age were 0.80 + 0.11 #g/g and 0.46 -4- 0.26 #g/g, respectively. The lead levels at 22 days of age were a
TABLE I Regional distribution of lead, iron, zinc and copper in rat brain (#g/g) at 22 and 290 days of age Each value represents the mean q- S.E.M. of 7 rats.
Brain region
Lead 22 days
Cortex 0.394-0.09** Cerebellum 1.034-0.17"** Medulla oblongata 1.144-0.23§* Midbrain 0.784-0.15 Hippocampus 1.534-0.38 Striatum 1.404-0.30 Hypothalamus 1.424-0.21
Iron 290 days
22 days
Zinc 290 days
22 days
0.094-0.02 11.094-0.74"** 20.434-0.90 14.274-0.23 0.354-0.05 18.404-3.14 23.29~0.37 12.724-0.80
Copper 290 days
22 days
290 days
15.304-0.65 3.024-0.25 3.194-0.15 10.794-0.22 2.394-0.08 3.054-0.37
0.364-0.08 15.634-2.64 0.694-0.24 15.21-+-2.79
16.104-0.16 13.414-1.42"* 8.094-0.94 3.284-0.19" 2.624-0.16 19.414-0.77 13.644-1.21 10.394-0.75 2.804-0.22 3.394-0.45
0.914-0.10 27.994-9.42 1.104-0.23 10.934-2.32
26.474-8.14 13.834-1.72 12.274-2.12 14.064-1.08
21.844-0.78 3.684-0.52 3.234-0.50 15.364-3.05 3.224-0.48 3.294-0.60
1.054-0.34 14.214-2.47
17.884-1.57 11.134-0.65
10.464-0.74 2.544-0.18 3.844-0.53
§ n = 6. One extreme observation was tested by the statistical method of Stefansky and Grubbs la, was confirmed as an outlier, and was treated as a missing value. * Significantly higher concentration than in 290 days of age (P < 0.05). ** Significantly higher concentrations than in 290 days of age (P < 0.01). *** Significantly higher concentrations than in 290 days of age (P < 0.005).
182 little higher than that reported previously by Mykkanen et al. 11. This discrepancy might have been caused by methodological differences in dissection of the brain. The concentration of iron irt the rat brains was also the highest in the hippocampus. In human studies, iron is found to be in higher concentration in the basal ganglia (particularly the globus pallidus, putamen and caudate nucleus) 9. The values of iron obtained in the rat striatum, which includes the globus pallidus, putamen and caudate nucleus, on the other hand, were relatively low. The concentration of iron in the cortex of suckling rats was significantly lower than that from adult brains. The levels in the cortex from adult brains were approximately twice as great than those in the same region in the sucklings. These findings are in agreement with human data indicating that iron rose approximately 2-fold compared to what it had been perirtatally 15. Zinc concentrations were distributed almost uniformly in each of the regions of the suckling brains measured. There were no regional differences among any of the regions of the suckling brains. Zinc concentrations were considerably different from those in suckling rats. The regional differences among the 7 regions of the adult brains as measured by analysis of variance 1 Barry, S. I. and Mossman, D. B., Lead concentrations in human tissues, Brit. J. industr. Med., 27 (1970) 339-351. 2 Brun, A. and Brunk, U., Heavy metal localization and age related accumulation in the rat nervous system, Histochemie, 34 (1973) 333-342. 3 Crawford, I. L. and Connor, J. D., Zinc in maturing rat brain: hippocampal concentration and localization, J. Neurochem., 19 (1972) 1451-1458. 4 Danscher, G., Fjerdingstad, E.J., Fjerdingstad, E. and Fredens, K., Heavy metal content in subdivisions of the rat hippocampus (zinc, lead and copper), Brain Research, 112 (1976) 442-446. 5 Fjerdingstad, E. J., Danscher, G. and Fjerdingstad, E., Hippoeampus: selective concentration of lead in the normal rat brain, Brain Research, 80 (1974) 350-354. 6 Fjerdingstad, E. J., Danscher, G. and Fjerdingstad, E., Changes in zinc and lead content of rat hippocampus and whole brain following intravital dithizone treatment as determined by flameless atomic absorption spectrophotometry, Brain Research, 130 (1977) 369--373. 7 Glowinski, J. and Iversen, L. L., Regional studies of catecholamines in the rat brain-I. The disposition of [aH]norepinephrine, [aH]dopamine and [aH]dopa in various regions of the brain, J. Neurochem., 13 (1966) 655-669. 8 Grandjean, P., Regional distribution of lead in human
were statistically significant (P < 0.05). The highest zinc level in adult brains was found in the hippocampus and the lowest in the medulla. The distribution of zinc in the brain regions seems to be in accordance with the few records of zinc distribution in adult rat brainsZ,3,10. The zinc concentrations in the medulla from the suckling rats were significantly higher than those in the same region from the adult rats. The copper distribution pattern in the suckling rats was almost the same as that in the adult rats, although the copper concentration in the medulla of the suckling brains was significantly higher than that in the adult brains. When the elemental relationship among the 4 elements was investigated, it was observed that lead concentrations in each of the regions from the suckling rats correlated significantly with those of iron (r : 0.32, P < 0.05). There was no such relationship for adult rats or among other metals. This relationship of lead and iron in the suckling rat might, in part, be explained by the fact that acute lead encephalopathy in the suckling rat has been described as a vasculopathy 1~, and that lead becomes localized in capillary-microvessel fractions even when present at very low levels 14.
brains, ToxicoL Lett., 2 (1978) 65-69. 9 Harrison, W. W., Nestsky, M. G. and Brown, M. D., Trace elements in human brain; copper, zinc, iron and magnesium, Clin. Chim. Acta, 21 (1968) 55-60. 10 Kozma, M. and Ferke, A., Trace element localization and changes in zinc and copper concentrations during postnatal development of the rat CNS, Acta Histochem., 65 (1979) 219-227. 11 Mykkanen, H. M., Dickerson, J. W. T. and Lancaster, M. C., Effect of age on the tissue distribution of lead in the rat, Toxicol. appl. PharmacoL, 51 (1979) 447-454. 12 Pentshew, A. and Garro, F., Lead encephalo-myelopathy of the suckling rat and its modifications on porphyrinopathic nervous disease, Acta Neuropath., 6 (1966) 266-278. 13 Snedecor,W. G. and Cockran, G. W., Statistical Methods, 7th Edn., Iowa State University Press, Ames, IA, 1980, pp. 279-280. 14 Toews, A. D., Kolber, A., Hayward, J., Krigrrmn, M. R. and Morell, P., Experimental lead encephalopathy in the suckling rat: concentration of lead in cellular fractions enriched in brain capillaries, Brain Research, 147 (1978) 131-138. 15 V61ki, A., Berlet, H. and Ule, G., Trace elements (Cu, Fe, Mg, Zn) of the brain during childhood, Neuropiidiatrie, 3 (1974) 236-242.