Bull. Environ. Contam. Toxicol. (1984) 32:157-165 �9 1984 Springer-Verlag New York Inc.

Regional Interrelationships of Zinc, Copper, and Lead in the Brain Following Lead Intoxication

Shafiq-ur-Rehman and Om Chandra

Department of Pharmacology, J. N. Medical College, Aligarh Muslim University, Aligarh--202 001, India

Among heavy metals of industrial and toxicological importance, lead, zinc and copper have probably the widest distribution in the human environment. These metal ions have a non-homogeneous pattern of distribution in the central nervous system (CNS). It has been shown that the normal concentration of these metal ions in the CNS is disturbed following zinc--intoxication (Shafiq-ur-Rehman et al. 1982). Zinc and copper are essential trace metals involved in various metabolic processes of numerous enzymes, neurotransmitters and hormonal functions. The concentration of lead is not regular throughout the CNS. Furthermore, lead is known to influence neuro-transmitter mecha- nism and certain enzyme functions (Govoni et al. 1980; Chandra and Shafiq-ur- Rehman 1982; Nakagawa et al. 1980; Shafiq-ur-Rehman 1983). Recently, we have shown that the lead-induced behavioural complexities are GABAergic mediated (Shafiq-ur-Rehman and Chandra 1983). Many studies are available suggesting that lead poisoning is associated with altered metabolism of essential trace metals (Niklowitz and Yeager 1973; Michaelson and Sauerhoff 1973; Shafiq-ur-Rehman 1983). There are a relatively less number of studies available on the effect of metal ions on their brain tissue concentration when given alone or in combination. In the present study, we have demonstrated the effect of lead alone and in combination with zinc or copper on the regional concentration of zinc, copper and lead ions in the CNS and blood.

MATERIALS AND METHODS

The present study was carried out on albino rabbits (1-1.5 kg), six in each group, of either sex. The animals were provided with standard pellet laboratory food (Hindustan Lever Laboratory Feeds, India) and water ad-libitum and both control and experimental animals were kept under identical diurnal conditions of light (12-hour light-dark cycle), temperature (25-27~ and relative humidity (48-50%). The experimental animals were divided into five groups. First group was administered intraperitoneally with 8 mg elemental lead per kg body weight as lead acetate once daily for 7 consecutive days. Similarly group 2, 3, 4 and 5 were injected with elemental copper (1 mg/kg),

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Tab le 1. Lead levels (~g/g wet tissue or ,g/ml blood, Mean-4-S. E . ) i n

different regions of the CNS and blood following i. p. injections of lead (8 mg/kg) alone and in combination with zinc (1 mg/kg) or copper (Img/kg) daily for 7

consecutive days in rabbits. Figure in parentheses indicates ~o change.

I II III IV Regions Control Lead Ac Lead Ac Lead Ac

Alone + + Cupric Ac Zinc Ac

Cerebral 1.02-4-0.26 1.944-0.21 2.034-0.24 1.804-0.22 cortex ( + 90.20)~ ( + 4.64) b (--7.22) b

Cerebellum 1.044-0.18 2.364-0.12 2.464-0.32 4.724-0.41 (+ 126.92) a (+4.24) b ( + 100.00) b

Brain stem 0.984-0.11 1.544-0.17 2.464-0.22 3.264-0.33 ( + 57.14) a ( + 59.74) b ( + 111.69) b

* * :g~

Hippocampus 4.114-0.38 7.28 4-0.62 6.924-0.60 11.45 4- I. 10 (+77.13) a (--4.95) b (+ 57.28) b

Amygdala 8.664-0.67 12.464-l.30 8,204-0.78 13.064-1.44 (+43.88) a (--34.19) b (+4.82) b

Hypothalamus 22.424-1.86 58.464-4.30 45.244-2.80 42.274-3.78 (+ 160.75) a (--22.61) b (--27.69) b

Spinal cord 4.024-0.36 8.964-0.72 9.244-1.22 ll.66dzi.00 (+ 122.89) a (+ 3.13) b (+ 30.13) b

Blood 0.434-0.05 5.424-0.41 3.66-4-0.26 5.364-0.62 (+ 1160.47) a (--32.47) b (--1.11) b

* ~--- P/_0.05; ** = P /_0.01; *** ~ P / 0.001.

a ~ comparison with group I.

b = comparision with group II.

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zinc (1 mg/kg), lead (8 mg/kg) plus copper (1 mg/kg), and lead (8 mg/kg) plus

zinc (1 mg/kg), as their acetate salt, respectively. The control group received the same volume of physiological saline.

Twenty four hours after the last injection, the samples of blood were collected directly from the heart by means of heparinized 1-ml syringe before sacrificing the animal by decapitation under Nembutal anaesthesia. The brain and spinal cord were taken out and meninges were removed carefully together with

surface blood vessels. The brain was further dlssected into cerebral cortex, cerebellum, brain stem, hippocampus, amygdala and hypothalamus. The samples of blood, spinal cord and various parts of the brain were subjected to wet-ashing, according to the method of Shafiq-ur-Rehman et al. (1982) with slight modifications, in concentrated nitric acid and perchloric acid (3 ml HNO~ and I ml of 72% HCIO 4 diluted 1:1 with deionized water for 1 g of fresh tissue/or 1 ml of blood). This was placed in an oven for 1 hour at 40~ for 2 hours at 70~ and then over-night at ll0~ The digested samples were diluted with 1N-HNO3 for final estimation of lead, zinc and copper by an atomic absorption spectrophotometer (PYE-UNICAM-SP-2900) as described elsewhere (Shafiq-ur-Rehman 1983).

The chemicals used in this study were of analytical grade and were obtained

from B. D. H. (India). All the results were subjected to statistical analysis performing Student's t-test. Changes were considered significant when P was at least <0.05.

RESULTS AND DISCUSSION

In a dose of 8 mg/kg administered i .p. daily for 7 consecutive days, lead produced a t61% increase of lead levels in the hypothalamus, 127% in the cerebellum, and 123~o in the spinal cord. Other areas where significant changes were detected included the cerebral cortex (+90~ hippocampus (+77~o) and amygdala (+ 44%). Blood showed a significant increment (1160%) of lead

level (Table 1).

When lead (8 mg/kg) was combined with copper (1 mg/kg), significant increment of lead concentration was discernible in the brain stem only. Interestingly, the hypothalamus and amygdala showed a significant decrement (22% and 34% respectively). However, the concentration of lead was significantly reduced (32~/o) in the blood (Table 1 ) .

When higher does of copper (8 mg/kg) were administered along with lead (8 mg/kg) all the animals developed convulsions and died within 60 minutes.

Following combined lead (8mg/kg) and zinc (1 mg/kg) administration,

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Table 2. Copper levels (~g]g wet tissue or , g / e l blood, Mean-bS.E.) in different regions of the CNS and blood following i .p. administration of lead (8 mg/kg) alone and in combination with copper (1 mg/kg) daily for 7 consecutive days. Figure in parentheses indicates % change.

I II III VI Regions Control Lead Ac Cupric Ac Lead Ac

Alone Alone Cupric Ac

Cerebral 2.36::t:0.21 3.324-0.26 5.264-0.42 6.214-0.72 cortex (+40.68) ~ (+ 122.88) a (+ 18.06) 4

Cerebellum 2.014-0.12 3.41-4-0.33 5,86--I-0.40 9.464-0.86 (+ 69,65) a (+ 191,54) a (+61.43) ~'

Brain stem 3.22~0.32 2.12-b0.22 5,624-0.38 8.20+0.79 (--34.16) a (+ 74,53) a (+45.91) b

Hippocampus 6.28:k0.59 12.464-1.23 14.484-1.12 18.924-1.27 (+ 98.41) a (+ 130.57) a (+ 30.66) b

Amygdala 17.82~: 1.27 12.214-0.89 32.404-2.10 37.364-2.86 (--31.48) ~ (+81.82) '~ (+ 15.31) b

Hypothalamus 19.61:]:1.24 13.244-1.02 26.664--92.23 25.424-2.54 (--32.48) a (+ 35.95) a (--4.65) b

Spinal cord 6.46::k0.68 5.204-0.82 8,524-0.94 9.244-1.20 (-- 19,50) a (+ 31.89) a (+ 8.45) 4

Blood 1.86:~0.28 0.42-4-0.03 4,544-0.52 5.02~0.61 (--77.42)~ (+ 144.09) ~ (+ 10.57) a

* = P /0 ,05 ; **----- p / -0 .01 ; *** = P /_0,001.

a ~ comparison with group I.

b = comparison with group III.

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significantly increased concentrations of lead were noticeable in the cerebellum (100%), brain stem (112%), hippocampus (57%) and spinal cord (30%). The hypothalamus, however, showed a reduced (28%) level of lead ions (Table 1).

After the administration of a higher dose of zinc (8 mg/kg) in combination with lead (8 mg/kg) the animals were hyperexcitable, restless, incoordinated and died

within 6 to 12 hours.

Significant elevation of copper level was obtained in the hippocampus, cerebral

cortex and cerebellum, however, the amygdala and hypothalamus and brain

stem exhibited depressed values following lead (8 mg/kg) administration. Copper in a dose of 1 mg/kg significantly elevated its own level in all the brain regions, spinal cord and blood. When the aforementioned doses of lead and eopper were mixtured a further increase in the copper levels were

discernible in the cerebellum, brain stem and hippocampus (Table 2).

Lead exposure (8 mg/kg) produced a significant increase in zinc ions in the hypotalamus and amygdala, and a remarkable depletion of zinc levels in the cerebral cortex, cerebellum and hippocampus. Zinc in a dose of 1 mg/kg caused a significant elevation in the hippocampus, however, an increment in the blood level was also detected. Combined zinc and lead exposure markedly increased the concentration of zinc in the hypothalamus and amygdala while the cerebellum showed depressed contents The concentration of zinc was seen decreased in the blood (Table 3).

Although lead is probably the most studied occupation toxic agent, several questions regarding its toxicity still remain (W. H. O. Technical Report Series 647, 1980, p. p. 72). Better knowledge is required of the factors that increase

or decrease the toxicity of lead with regard to CNS dysfunction. The uneven distribution of biologically important substances could be considered an indica- tion of special physiological role in that area where the concentration is highest. Although highest concentration of lead was detected in the hypo- thalamus when it was administered in a dose of 8 mg/kg i. p. daily for 7 days, combined lead and copper administration caused remarkable depletion of lead level in this region. Thus an interesting antagonism of lead and copper was apparent in this brain area which controls the internal environment of the

body. It is likely that the increase in copper concentration in excess of the

physiological requirement may cause displacement or redistribution of lead ions in the hypothalamus and amygdala. But higher doses of cupric acetate (8 mg/kg) when combined with lead proved fatal within an hour. Gooddy et al. (1975) suggested that at the time of overloading, element interchange system is disturbed and may be responsible for organic consequences. Interestingly, combined lead and zinc administration caused significant elevation of lead ions in the cerebellum, brain stem, hippocampus and spinal cord. It has already been proved that zinc interferes with copper and lead tending to form an

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Table 3. Zinc levels (;g/g wet tissue or ~g/ml blood, Mean 4-S . E . ) i n

different regions of the CNS and blood following i .p . administration of lead (8 mg/kg) alone and in combination with zinc (1 mg/kg) daily for 7 consecutive

days. Figure in parentheses indicates % change.

I II III IV Regions Control Lead Ac Zinc Ac Lead Ac

Alone Alone + Zinc Ac

~g

Cerebral 12.36+1.30 8.424-0.89 13.42-t-1.42 12.264-1.32 cortex ( - - 31.88) ~ ( + 8.58) ~ ( - - 8.64) b

Cerebellum 15.244-1.28 10.07 4-0.81 18.364-1.62 12.344- I. 11 (--33.92) a ( + 20.47) a (--32.79) b

Brain stem 10.284-1.27 9.214-1.22 12.444-1.32 13424-1.42 (--10.41) a (+ 21.01) a ( + 7.88) b

Hippocampus 18.264-1.62 13.254-1.42 24.624-2.14 24.404-2.11 (--27.44) a (+ 34.83) a (--0.89) b

Amygdala 22.54+2.23 43.924-3.19 22.404-2.04 32.144-2.97 (+ 54.92) a (--0.62) a ( + 43.48) b

Hypothalamus 28.924-2.30 58.314-3.69 32064-3.16 66.40+5.21 ( + 101.63) a ( + 10.86) a (+ 107.11) b

Spinal cord 17.44-1-1.39 19.26-t-2.12 18.60-4-2.13 19.034-2.16 ( + 10.44) a (+6.65) a (+2.31) u

BloGd 6.62-t-0.70 5.17-f-0.61 11.45+1.11 16.66-4-1.30 (--21.90) a (+72.96) a (+45.50) u

* = P / '0 .05; ** = P / '0 .01; *** = P / 0.001.

a = comparison with group I.

b ~--- comparison with group III.

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antagonism and agonism association in various brain areas respectively (Shafiq-ur -Rehman et al. 1982).

When copper levels were determined following lead exposure, its significant elevation was noticeable in the cerebral cortex, cerebellum and hippocampus

and depression in the amygdala, and hypotbalamus. However, combined lead and copper administration caused increment in the hippocampus and brain stem only, the amygdala was unaffected. On the other hand depletion of zinc levels occurred in the cerebellum, cerebral cortex and hippocampus after the administration of lead. In this case combined zinc and lead exposure caused

a concordant increase in zinc levels of hypothalamus and amygdala, the similar

effect was also seen in these regions when lead was administered alone. Thus a heterogeneous distribution of metal ions in various regions of the brain after the administration of lead, zinc or copper, singly and in combination, was discernible. It may be argued that different cell types may accumulate different amounts of metal ions which are regarded to be co-factors related to the particular metabolic processes in the CNS. When the tissues were heavily overloaded with lead, the impaired cellular transport and reabsorption functions occurred resulting redistribution of other ions. Because the most prominent changes of lead toxicosis are cerebral edema associated with an increase in

eerebrospinal fluid pressure, proliferation and swelling of endothelial cells accompanied by dilatation of capillaries and arterioles, proliferation of gliol cells and focal necrosis and neuronal degeneration (Goyer 1981). In this case all these damaging situations reflect the ion-exchange-process where different cell types, cell densities, cell volume and capillarization may have a major part. In agreement with Gooddy and Co-workers (1975) that the metals are

in dynamic state and can interchange in health, toxicity and disease. Moreover, such competitive influence of metal ion interaction can reform an important

mechanism during and after destroying the normal-equillibrium-system of other metal ions. The possibility remains open that a functional coupling between metal ions and certain ligands, eg. hormones, exists and that the

former could alter molecular configuration towards specific high affinity regional

receptor sites in the CNS.

In lead poisoning the concentration of lead was increased in all the brain regions causing significant alterations in the levels of zinc and copper in various brain

areas, spinal cord and blood. Recently, it has been shown that the essential trace metals are primarily influenced in lead poisoning (Shafiq-ur-Rehman 1983). In other experiment, Seth et al. (1976) have demonstrated with repeated injections of lead (8 mg/kg i.p.) on third and fifth day and mice were sacrificed on eleventh day, the levels of lead zinc and copper were not changed significantly in the brain. In the present demonstration, the exposure of lead (8 mg/kg i. p.) was daily for 7 consequtive days and regional study of the brain was performed. Consequently, we obtained depleted concentrations of

zinc ion in the cerebral cortex, cerebellum and hippocampus, these regions did

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not show any significant changes of copper levels except the hippocampus which had an elevated concentration. In both the amygdala and hypothalamus, pb++ exhibited an agonism with zinc and antagonism with copper following lead intoxication. In the blood, an inverse-lead-copper-relationship was noticeable in lead poisoning. From the data obtained in the present study and on the basis of our earlier results (Shafiq-ur-Rehman et al. 1982) it seems reasonable to suggest that the hypothalamus, hippocampus, amygdala and cerebellum are the brain regions most commonly affected by interactions among the metal ions. Consequently redistribution takes place to achieve a novel state of altered equillibrium. The mechanism and kinetics of the redistri- bution of ions is not known.

ACKNOWLEDGEMENTS

Authors are thankful to the Bureau Police Research and Development, Ministry of Home Affairs, Government of India, New Delhi, for their financial assistance in the form of an individual research fellowship grants (No. 69/3/80mTrg/F).

REFERENCES

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Govoni S, Memo M, Lucchi L, Spano P F, Trabucchi M (1980) Brain neuro- transmitter systems and chronic lead intoxication. Pharmacol Res Commun 12 : 447-460

Goyer R A (1981) Lead. In : Bronner F, Coburn J W (eds) Disorders of mineral metabohsm. Academic Press, New York, P. 159.

Michaelson I A, Sauerhoff M W (1973) The effect of chronically ingested inorganic lead on brain levels of Fe, Zn, Cu and Mn of 25 day old rat. Life Sci 13 : 417-428

Nakagawa K, Asami M, Kuriyama K (1980) Inhibition of release of lysosomal enzymes in young rat brain by lead acetate. Toxieol Appl Pharmacol 56 : 86-92

Niklowitz W J, Yeager D W (1973) Interference of Pb with essential brain tissue Cu, Fe, and Zn as main determinant in experimental tetraethyllead eneepha- lopathy. Life Sci 13 : 897-905

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Shafiq-ur-Rehman, Kabir-ud-Din, Hasan M, Chandra O (1982) Zinc, Copper and lead levels in blood, spinal cord and different Parts of thebrain in rabbit : Effect of zinc-intoxication. Neurotoxicology 3 : 195-204

Shafiiq-ur Rehman, Chandra O (1983) Effect of lead on motor activity and regional brain GABA. Psychopharmacology (Accepted)

Shafiq-ur-Rehman(1983) Essential trace metals are primary targets of lead poisoning. Life Sci (Communicated)

Seth T D, Agarwal L N, Satija N K, Hasan M Z (1976) The effect of lead and cadmium on liver, kidney and brain levels of cadmium, copper, lead, manganese and zinc, and on erythrocyte ALA---D activity in mice. Bull Environ Cont Toxi 16:190-196

Received July 21, 1983 Accepted August 23, 1983

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