Effect of chronic ingestion of lead on the central cholinergic system in rat brain regions

TOXICOLOGY

Effect

AND

APPLIED

of Chronic

PHARMACOLOGY

Ingestion System

ARVIND T. MODAK,

%,340-347

(1975)

of Lead on the Central in Rat Brain Regions

SUSAN T. WEINTRAUB,

AND WILLIAM

Cholinergic

B. STAVINOHA

Department of Pharmacology, Division of Toxicology, The University of Texas Health Center at San Antonio, San Antonio, Texas 78284 Received May 22,1975; acceptedJuly 8,197s

Effect of Chronic Ingestionof Lead on the Central CholinergicSystem in Rat Brain Regions.MODAK, A. T., WEJBTRAUB, S. T. AND STAVINOHA, W. B. (1975). Toxicol. Appl. Pharmacol. 34,340-347. The effect of chronic lead ingestion on the central cholinergic systemof immature rats was investigated.Rats weregiven 1% lead acetatesolution in the drinking water before and after weaning. Tap water and sodium acetate controls were used. The growth of rats treated with lead was significantly inhibited as comparedto controls. After chronic lead ingestion,a significant decrease in cholinesteraseactivity was seenin the medulla-pons,midbrain, and diencephalon, and a significant increase in choline acetyltransferase activity wasfound in the medulla-pons,hippocampus,and cerebralcortex. The only brain region exhibiting a changein acetylcholine content after chronic lead administration was the diencephalon,wherea significant increasewas observed.Thus, significant changesin the central cholinergic systemhave beenfound after chronic lead ingestion.

There is a remarkable vulnerability of the immature central nervous system to lead poisoning. Chronic ingestion of lead by children greatly increases the body lead burden and can cause chronic hyperkinetic aggressive behavior. This vulnerability is probably due in part to the inability of deveIoping gastrointestinal epithelial celb to reject multivalent cations such as lead, strontium, and iron (Forbes and Reina, 1972). Impairment of function in the cholinergic system has been implicated in several studies on lead. Kostial and Vouk (1957) found decreasedoutput of acetylcholine from the superior cervical ganglia after lead treatment. In addition, Silbergeld et al. (1974) found that lead diminished acetylcholine release at the neuromuscular junction. In order to evaluate the effects of chronic lead treatment on immature animals, Pentschew and Garro (1966) introduced an experimental protocol which was further

developed by Silbergeld and Goldberg (1973) in which suckling rats could be indirectly exposed to lead when the mothers were given a lead solution in place of drinking water. Using this model Silbergeld and Goldberg (1974) did find indications of cholinergic-

aminergic impairment. The present report describes the effects of chronic lead treatment on cholinesterase (ChE) and choline acetyltransferase (CAT) activities and acetylcholine (ACh) content in rat brain regions. Copyright 0 1975 by Academic Press, Inc. AU rights of reproduction in any form reserved. Printed in Great Britain

340

LEAD

AND

THE CHOLINERGIC

SYSTEM

341

METHODS

Fifteen “timed pregnant” Sprague-Dawley rats (ARS Sprague-Dawley, Madison, Wis.) were obtained on day 16-17 of gestation and were caged individually. All rats were allowed free access to food (Wayne Lab Blox) and tap water and were maintained in a 12-hr light-dark cycle with lights being turned on at 0700 hr. Upon parturition, the mothers and pups were divided into three groups. Two groups of mothers were given either 1% lead acetate (Fisher, certified) or 1% sodium acetate (Fisher, certified) in their drinking water. The third group received tap water. The litters were normalized to six pups per mother 48 hr after birth and, upon weaning, the pups were separated according to sex. The administration of lead acetate or sodium acetate in the drinking water of the pups was continued until the termination of the experiment at 60 days post-parturition. Enzyme Activities Cholinesterase (ChE, acylcholine acylhydrolase, EC 3.1.1.8) activity was determined by the radioisotope method of Siakotos et al. (1969) as described by Stavinoha et al. (1973). After decapitation, the brain was removed and was dissected into the pineal gland, cerebellum, medulla-pons, midbrain, diencephalon, hippocampus, striatum, and cerebral cortex. Ten percent (w/v) homogenates in 0.4 M sucrose were made of all brain regions except the pineal gland. All regional brain homogenates were diluted to 1% (w/v) with distilled water with the exception of the striatum which was diluted to 0.25 % (w/v). A 1% homogenate of the pi neal gland was made directly in distilled water. Choline acetyltransferase (CAT; acetyl-Co A: choline-0-acetyltransferase; EC 2.3.1.6) activity was assayed by a modification of the method of Bull and OderfeldNowak (1971) as described by Stavinoha et al. (1973). After decapitation, the brain was removed and dissected into the cerebellum, medulla-pons, diencephalon, striatum, hippocampus, and cerebral cortex. Brain samples were prepared as 10% (w/v) homogenates in cold distilled wat er. Acetylcholine Content For measurement of acetylcholine, 6-set microwave irradiation was used to concurrently kill the rat and inactivate brain enzymes (Stavinoha et al., 1973). The cerebellum, medulla-pons, midbrain, diencephalon, striatum, and hippocampus were homogenized in 1 ml of 15 ‘A (v/v) 1 N formic acid in acetone and the cerebral cortex was homogenized in 2 ml of the same mixture. An appropriate amount of 0.3 mM butyrylcholine iodide was added to each sample as an internal standard. The measurement of acetylcholine was accomplished by pyrolysis-gas chromatography of the periodide-precipitated salts (Stavinoha and W eintraub, 1974). Lead Content The concentration of lead in blood was determined by a modification of the method of Delves (1970). All blood samples were sonicated with a Sonifier cell disruptor Model W185 (Heat Systems-Ultrasonic, Plainview, N.Y.) by using a microtip. Blood from untreated animals was used to prepare standard samples with concentrations of 20-80 pg/lOO ml lead nitrate (Fisher, certified). Twenty-microliter aliquots of blood samples

342

MODAK,

WEINTRAUB

AND

STAVINOHA

were added to preflamed Delves cups (Trace Metals Instruments, Inc., New York, N.Y.). Each cup was placed at the flame edge of a Perk&Elmer Model 403 atomic absorption spectrometer (Perkin-Elmer, Norwalk, Conn.) and the sample was ashed at a temperature where lead does not volatilize. The cup was then moved into the center of the flame. The lead concentration was determined by comparison with the standard curve. Blood samples from treated animals were diluted sufficiently to give lead concentrations which fit into the standard curve. All samples and standards were performed in triplicate. RESULTS

Chronicadministration of 1% lead acetate solutions significantly inhibited the growth of newborn rats. At 60 days of age the mean weight &SD of six rats given lead acetate solution was 165 it 17 g, while rats given tap water weighed 220 2 10 g and rats given sodium acetate solution weighed 225 f 14 g. The blood concentrations of lead for these treated rats was 245 f 43 pg/lOO ml as compared to negligible concentrations in both control groups. During the 60 days of treatment no tremors or convulsions were observed. The effect of chronic lead treatment on cholinesterase activity is shown in Table 1. ChE activity was significantly lower in the medulla-pons, midbrain, and diencephalon of lead-treated rats as compared to the two control groups. The results in Table 2 show that after chronic lead treatment choline acetyltransferase activity was increased in the medulla-pans, hippocampus, and cerebral cortex. The acetylcholine content of the diencephalon was significantly increased following chronic administration of lead (Table 3). DISCUSSION

A study of the effects of lead on the central nervous system is important for evaluation of the consequences of chronic ingestion by children. The choice of a proper animal model for this study is essential because absorption of lead differs in the mature and immature digestive systems. Normally, 5-10 % of ingested lead is absorbed in the adult (Kehoe, 1964). However, the absorption of lead from the intestine of neonates is very high, and 60% of the lead present in the mothers’ milk is taken up (Kostial et al., 1971; Waldron and Stofen, 1974). Immature epithelial cells do not have the ability to reject unwanted multivalent cations and, therefore, ions are unselectively absorbed by the immature gut (Forbes and Reina, 1972). Silbergeld and Goldberg (1973) reported that mice of 40-60 days of age which were given doses of 2-10 mg/ml of lead in the drinking water before and after weaning exhibited significant increases in motor activity. These increases in motor activity were not dose related. The growth and development of these animals were significantly inhibited. In the present study, rats were given 1 ‘A lead acetate in the drinking water before and after weaning. Growth was significantly inhibited by chronic lead treatment, but no overt changes in motor activity were observed. Changes in the central cholinergic system after chronic lead treatment may be reflected in changes in either substrate concentration or enzyme activity. Lead might affect enzyme activity in a number of ways. Lead could produce tissue damage and

Treatment*

ACTIVITY

IN RAT BRAIN

FOLLOWING

LEAD

ACETATE

TABLE

788 *47

(6) 718’ f67

(8)

(6)

295 a114

‘39

(6)

(6) 305 +36

803 +36

311 _+61

(8)

810c +81

(6)

915 +50

(6)

948 253

IN DRINKING

WATER

(8)

65Zc +49

(6)

740 j-49

(6)

727 *54

(8)

486 251

(6)

525 *52

(6)

504 f61

(8)

3655 t-354

(6)

3616 +-266 (5) 3395 f116

(8)

639 *66

(6)

617 +121

(6)

621 +43

Cortex

BEFORE AND AVER

Diencephalon Hippocampus Striatum

Mean Activity *SD“

ADMINISTRATION

1

Cerebellum Medulla-pons Midbrain

REGIONS

(;;

(3) 104

B

$

8;d 8

2 s

+14

2 (4) 103

z

+15

112

Pineal

WEANING

a Values are expressed in prnol ACh hydrolyzed/g tissue/hr for brain tissue of the 60-day animals. Numbers in parentheses indicate the number of rats in each group. b Solution used for drinking water from birth to 60 days of age. c Significantly different from tap water and sodium acetate controls (p < 0.05); Student’s t test.

1% Lead acetate

1% Sodiumacetate

Tap water

CHOLINESTERASE

TABLE

2

0.98 f0.18 (5) 0.75 k0.13 (8) 0.90 kO.14 O-9

Cerebellum 10.67 f0.62 (6) 10.95 k1.08 (9) 12.45= kO.70 (10)

Medulla-pons 5.89 kO.71 (6) 5.53 +0.57 (9) 5.63 -10.73 (10)

Diencephalon

5.09 +0.61 (5) 5.02 +0.57 (9) 5.93” +0.92 (10)

18.9 k2.31 (6) 17.09 k1.73 (9) 17.26 k2.09 (10)

5.77 kO.86 (5) 5.87 kO.94 (9) 6.83’ kO.58 (9)

Cortex

WATER BEFORE

Striatum

IN DRINKING

Hippocampus

Mean activity +SD”

IN RAT BRAIN REGIONS FOLLOWINCI LEAD ACETATE ADMINISTRATION AND AFTER WEANING

c Significantly different from tap water and sodium acetate controls (p < 0.05); Student’s t test.

bSolutionusedfor drinkingwaterfrom birth to 60daysof age.

n Values are expressed in pm01 ACh synthesized/g tissue/hr for brain tissue of the 60-day animals. Numbers in parentheses indicated the number of rats in each group.

1% Lead acetate

1% Sodiumacetate

Tap water

Treatmentb

CHOLINE ACETYLTRANSFERASE ACTNITY

% 8 P

i

F

ic

2

5 pFf

5.8 +0.7 (4) 5.9 +0.9 (5) 4.9 +1.2 (5)

Cerebellum -___ 31.4 23.3 (4) 34.9 k4.8 (5) 34.1 +1.0 (5)

Medulla-pons 34.4 51.7 (4) 35.4 +4.0 (5) 34.3 +2.4 (5)

Midbrain 28.3 22.4 (4) 29.7 +3.3 (5) 35.3c _+3.9 (5)

Diencephalon

+SD’

25.8 k2.7 (4) 24.4 k2.7 (4) 28.6 *5.3 (4)

Hippocampus 66.0 59.7 (4) 68.9 210.3 (5) 74.6 +9.2 (5)

Striatum

19.0 +1.7 (4) 21.1 +3.1 (4) 19.1 k3.2 (4)

Cortex

n Values are expressed in nmol ACh/g tissue for brain tissue of the 60-day animals. Numbers in parentheses indicate the number of rats in each group. b Solutions used for drinking water from birth to 60 days of age. c Significantly different from tap water and sodium acetate controls (p i 0.05); Student’s t test.

1% Lead acetate

1% Sodium acetate

Tap water

Treatment*

Mean concentration

TABLE 3 ACETYLCHOLINE CONTENT IN RAT BRAIN REGIONS FOLLOWING LEAD ACETATE ADMINISTRATION IN DRINKING WATER BEFORE AND AFTER WEANING

9 3 Ei

E g

Q P

ki

%

G %

346

MODAK,

WEINTRAUB

AND

STAVINOHA

release membrane-bound enzymes or lead might act directly on the enzyme itself. Increases in serum transaminases, aldolase, and catalase, and decreases in serum alkaline phosphatase and carbonic anhydrase have been reported after lead administration. Thus, lead can either stimulate or inhibit enzyme activity (de Bruin, 1971; Waldron and Stofen, 1974). We have found that lead changed the activity of the synthetic enzyme of the central cholinergic system in an opposite direction from the change in activity of the degradative enzyme. Cholinesterase, the enzyme which is responsible for physiological destruction of acetylcholine, had significantly lower activity in the medulla-pons, midbrain, and diencephalon in rats chronically treated with lead as compared to control. This agrees with previous findings that serum ChE was suppressed in human beings exposed to high concentrations of lead in the environment, in patients with lead poisoning and in experimental animals treated with lead (de Bruin, 1971). In contrast to the effects of lead on ChE, choline acetyltransferase activity was significantly increased in the medulla-pons, hippocampus, and cerebral cortex after chronic ingestion of lead. Chronic administration of lead acetate solution before and after weaning produced a significantly elevated acetylcholine concentration in the diencephalon. This increase correlates with the significant decrease in cholinesterase activity in the diencephalon. It is interesting to note that although ChE activity was significantly decreased in the medulla-pons and CAT activity was increased, the steady state concentration of acetylcholine was unchanged in that brain region. Thus, chronic administration of lead produced changes in the three parameters of the cholinergic system studied. The enzyme changes were consistent in that cholinesterase activity was decreased and choline acetyltransferase activity was increased in the brain regions which exhibited changes. The only region showing a change in acetylcholine content after chronic lead treatment was the diencephalon where the increase in acetylcholine concentration coincided with a decrease in cholinesterase activity. Further work on the dynamics of the interaction of lead and the cholinergic system seen here is necessary to understand the consequences of chronic lead ingestion. ACKNOWLEDGMENTS The technical assistance of Ernest Loredo is gratefully acknowledged. This work was supported in part by NIH Grant No. 2ROl NSO-9356-04. REFERENCES DE

BRUIN,

A. (1971). Certain biological effects of lead upon the animal organism. Arch.

Environ. Hlth. X%,249-264.

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THE CHOLINERGIC

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