p.1 

OAR Box 1131 

Prepped by Ollie Stewart 

Document Number: 

141)11-1-21 

Docket Number: 

A-83-22 

Printed 4/21/2008 2:34:13 PM Sheet 141 of 162 
P.2 

1 ijjjUJJ.P <' ; 

• A : 

Neurohehaxioral Toxicology and Tera'ioli 0'« ufxt I 3 10'"• \\A[ 

hgy\\o\}li%. 499-'S09[tl98S.

 r XrlkhS Internal 

Pr~SS-^S> 

ional Inc. Primed in Ihc U.S.A. 0:75-l?W)/85$J.OO + .00 

JEPAAiRTpbcJCET j 

Neurobehavioral and Neurochemical 

Abnormalities of Pre- and Postnatally 

Lead-Exposed Rats: Zinc, Copper and 

Calcium Status 

MARIO BARALDI,1 PAOLA ZANOLI, TIZIANA ROSSI, PAOLA BORELLA,* 

EVA CASELGRANDI* AND FELICE PETRAGLIAt 

Institute of Pharmacology, Chair of Hygiene,* Department of Obstetrics and Gynecology^ 

University of Modena, 41100 Modena, Italy 

BARALDI, M., P. ZANOLI, T. ROSSI. P. BORELLA, E. CASELGRANDI AND F. PETRAGLIA. Neurobehavioral 

and neurochemical abnormalities of pre- and postnatalh lead-exposed rats: Zinc, copper and calcium status. 

NEUROBEHAV TOXICOL TERATOL 7(5)499-509, 1985.—Although diverging opinions have been expressed, there is a 

body of evidence of a cause-effect relationship between chronic exposure to lead and altered behavior in children. There 

are several problems in the interpretation of the behavioral and biochemical anomalies described in experimental models of 

lead intoxication due mainly to the.different experimental conditions used. One of the major criticisms is linked with the 

claim that in several studies too high levels of exposure were used, hence inducing the suspicion that the effects specifically attributed to lead intoxication could be related to unspecific actions. Here we report that, using an experimental model 

of pre- and postnatal intoxication of rats exposed to a very low amount of lead (I mg/kg/day), in parallel with behavioral 

anomalies, there are slight changes in cerebral dopaminergic and GABAergic receptors. A new finding was the discovery 

that in this condition there is a markedly increased number of opiate receptors in the hypothalamus (and other brain areas) 

in parallel with a decrease of /3-endorphin and an increase of Met-enkephalin. Therefore, low levels of lead intoxication 

which could be pertinent to the human situation, seem to induce neurotoxic effects which may be responsible for the 

behavioral anomalies in rats and, maybe, in children. 

Analgesia Dopamine receptors /3-Endorphin GABA receptors Met-enkephalin Lead nitrate 

Locomotor activity Morphine Opiate receptors Perinatal exposure Sodium nitrate 

EXPOSURE to high levels of lead induces an 

encephalopathic state characterized by brain hemorrhages, 

seizures and coma (for a review see [34]). There are controversial reports on the pathogenetic role of lead in so called 

"minimal brain dysfunction" (MBD), a syndrome which 

mainly affects children and is characterized by hyperactivity, short attention span and easy distractability [22-24]. In < 

past attempts to reproduce the syndrome experimentally 

there was little concern to use doses of lead which could be 

related to the human situation, where hyperactivity becomes 

apparent after asymptomatic lead intoxication. At the same 

time it is very difficult to compare the behavioral and 

biochemical effects of lead intoxication reported in different 

papers because of the great variability in the dose of lead, 

route of administration, duration of exposure", etc. Critical 

points seem to be related to the use of high doses of lead, 

which may lead to nonspecific effects, as well as lack of 

concern for second ion effects, the date at which exposure to 

the metal began, the age at which behavioral and biochemical tests were performed, variability due to sex, monitoring 

of lead levels in blood and brain tissues, interaction with 

other cations and, last but not least, accurate assay oflead in 

biological specimens. 

Let us briefly discuss a few of these points. As far as the 

level of chronic lead exposure is concerned, it must be 

pointed out that in many studies of "low level" lead toxicity 

the doses oflead used are so high (10-200 mg/kg/day) that the 

relevance of the results to subclinical lead poisoning in children is questionable. It has been argued in fact that the observed behavioral and biochemical alterations in some of 

these experiments could in fact result from malnutrition 

[17,53] and inhibition of energy metabolism [16]. 

On the basis of clinical studies in children [42,48] 300 ju.g 

of elemental lead is regarded as the maximum daily permissible intake from all sources and doubling of this dose is 

enough to produce adverse metabolic effects. Blood levels 

ranging between 10 and 20 /xg/dl have been described as 

inducing an alteration of heme synthesis [18,66] in leadexposed children. In rats blood levels of 11 jug/dl have been 

described as resulting in detectable histological lesions in 

'Requests for reprints should be addressed to Prof. Mario Baraldi, Associate Professor of Toxicology, Institute of Pharmacology, University of Modena, Via Campi 287, 41100 Modena, Italy. 

V' 499 
P.3 

500 BARALDI ETAL. 

TABLE 1 

EXPERIMENTAL DESIGN OF CHRONIO L.EAD EXPOSURE OF RAT DAMS DURING GESTATION 

A N D LACTATIO N • •-- - •-, 

Dams 

mated 

12 

12 

12 

Pregnant 

6/12 

5/12 

8/12 

Treatment 

water 

NaNO, 

Pb(N03)s) 

Dose 

mg/kg 

daily 

— 

1 

1 

Volume 

ml/kg 

2 

2 

2 

Dosing 

oral 

oral 

oral 

System 

gastric 

intubation 

gastric 

intubation 

gastric 

intubation 

Duration of 

treatment 

Weeks 

6 

6 

6 

renal proximal tubule cells [28]. In immature rats greater lead 

toxicity than in adults may be caused by higher retention 

[43], reduced elimination rate [39] and preferential localization in cerebral tissues [55]. Moreover, it has been reported 

that the immature CNS is particularly vulnerable to toxic 

effects of lead [34]. From these data one can surmise that 

there is no reason to expose animals to large amounts oflead 

in order to mimic the toxic effects (MBD) induced in children 

by pre- and postnatal exposure to asymptomatic doses. 

In the most widely used animal model of chronic lead 

poisoning, exposure is achieved via the drinking water by 

using lead acetate [12,13]. However the use oflead acetate 

could be questioned since the acetic acid component of lead 

acetate has been shown to be capable of masking the effects 

oflead on both body weight and locomotor activity [9]. 

From this last finding it is clearly imperative to control the 

effects of the second ion of the lead compounds used, especially when high doses oflead salts are administered. Several 

animal studies have been carried out on the effect of lead 

acetate on the activity of rat pups exposed pre- and 

postnatally, but some authors reported no change in level 

activity [44,64], others hypoactivity [25,65] and others 

hyperactivity [30,57] attributable to lead. If methodological 

differences among experiments may contribute to these inconsistencies, it could well be that the acetic component of 

lead acetate played a part in the variability of the results. 

Various animal experimental models have been developed to investigate the neurobehavioral and neurochemical 

effects due to lead exposure during prenatal and postnatal 

life. These animal models of lead intoxication have produced 

a very complicated literature, as recently pointed out by 

Winder and Kitchen [82], who reviewed the biochemical effects which have been described as being associated with 

lead toxicity in the CNS. 

Three major systems of neurotransmission have been extensively studied: the dopaminergic, the cholinergic and the 

GABAergic system, while less attention has been devoted to 

the brain opiatergic system. Several studies have provided 

evidence of a catecholaminergic dysfunction due to lead 

poisoning, but there are conflicting results since both inhibition and stimulation of dopaminergic function have been reported. These discrepancies have been attributed to differences in duration of exposure and doses of lead administered, suggesting that high doses of lead inhibit the 

dopaminergic system while relatively low doses activate it 

[81,82]. Though this hypothesis needs further experimental 

demonstration it is noteworthy in this context that the opposite regulation of dopamine levels in different brain areas 

reported by Govoni et al. [31,32] in pre- and postnatally 

lead exposed rats (2.5 g/1 in drinking water) could well explain the variable results reported in the literature on 

dopamine turnover. As far as dopaminergic receptor studies 

are concerned, Lucchi et al. [45] have described an increased density of receptors in the striatum (where dopamine 

synthesis was reduced) and a down-regulation in the nucleus 

accumbens (where dopamine synthesis was increased) when 

3H-sulpiride (a ligand believed to be specific for dopamine D, 

receptors) was used, while no change was found using 'Hspiperone (a ligand which binds both D, and D, receptors but 

with preferential labeling of D2). The chronic decreased and 

enhanced presence of dopamine in the two nuclei would be 

responsible for the increased and decreased number of 

dopamine receptors in the two areas described respectively. 

Several studies (for a review see [71,82]) have been performed to demonstrate the possible implication of functional 

changes in the cholinergic system in lead toxicity. However, 

since the hypofunction of this system reportedly was linked 

only to exposure to very high doses of lead, it has been 

suggested that it is unlikely that lead has a specific neurochemical toxic effect on this system [82]. Studies on brain 

GABA metabolism in animal models using very high doses of 

lead (5 g or 10 g Pb/I in drinking water, equivalent to about 

118-126 mg/kg) have described reduced uptake and release 

of GABA, changes in GABAtransaminase and glutamic acid 

decarboxylase as well as increased GABA synthesis [72]. 

The effects oflead on GABAergic enzymes, however, were 

not confirmed by Govoni et al. [31] using a lower, but still 

high dose oflead (2.5 g/1, equivalent to about 54 mg/kg/day). 

In the same study Govoni et al. [32] reported an increase in 

GABA receptors in the cerebellum and a decrease in the 

striatum. It has been suggested that the neurotoxicity of lead 

could be partially related to the lead-induced increase in 

delta-amino-levulinic acid, which is a weak displacer of 

GABA binding [74]. It is tempting to suggest in this regard 

that an increased chronic presence of this agent could, directly or indirectly by stimulating GABA release and inhibiting GABA uptake [14,15], down regulate GABA receptors in 

certain brain areas. 

Finally, the effect of lead intoxication on the opiatergic 

system has only been studied in the striatum by measuring 

Met-enkephalin levels. While Govoni et al. [32] and Memo et 

al. [51] have reported an increase in this neuropeptide after 

90 days of lead acetate exposure (2.5 g/1, equivalent to 1350 

ppm or about 54 mg/kg/day), Clayton et at. [19] found de-
P.4 

LOW-LEVEL LEAD NEUROTOXICITY 

Mil 

TABLE 2 

EFFECT OF LEAD EXPOSURE ON GESTATION AND PARTURITION 

% of Body weight 

Treatment Days of Number of died or (g) of pups 

(I mg/kg/os/day) gestation pups malformed pups at birth 

water 

NaNO, 

PrXNO,)., 

21.81 ±0.18 

22.60 ± 0.24 

22.13 ± 0.30 

12.67 ± 0.67 

8.20 ± 2.06 

10.38 ± 0.91 

6.14 ± 0.11 

6.55 ± 0.32 

6.23 ± 0.44 

Values are reported as mean ± S.E.M. 

TABLE 3 

LEAD, ZINC AND COOPER CONCENTRATIONS IN URINES OF LACTATING 

MOTHERS POISONED WITH LEAD (I mg/kg/day/os) DURING 

PREGNANCY AND LACTATION 

Concentrations in urines§ (mean ± SEM) 

Treatment 

Zinc 

(Mg) 

Copper 

(Mg) 

Lead 

(ng) 

Urine 

output/rat 

(ml) 

Mean ± SD 

water 

NaNO, 

PlHNO^j 

2.19 ±0.4 

2.64 ± 0.5 

3.18 ±0.2 

1.51 ± 0.3 

1.11 ±0.09 

1.55 ± 0.3 

13.0 ± 3.5 

9.3 ± 0.3 

83.3 ± 14.5* 

5.6 ± 1.2 

4.0 ± 1.8 

5.3 ± 1.3 

SUrine was collected for 6 hr from rats in metabolic cages. 

Student's r-Test: *p<0.001. 

I 

r~ 

creased enkephalin levels after exposure to lower doses of 

lead (300 and 100 ppm) and for a shorter period (from conception to weaning). In this regard it is also interesting to 

mention that altered responses to morphine have been reported in rats by Mitchell et al. [54] after an acute treatment 

with lead. All these important observations taken together 

seem to indicate that an imbalance between different excitatory and inhibitory neurotransmission systems could be 

operative in the neurobehavioral effects oflead intoxication. 

In attempting to explain the multiplicity of effects induced by 

lead on the CNS, little concern has been devoted to the 

possibility that some of the neurotoxic effects of lead might 

be due to interference with homeostasis of other divalent 

cations such as calcium and zinc. It has been suggested that 

the effects of lead on monoaminergic functions could be related at the neuronal level to inhibition of mitochondrial calcium uptake: whether this effect results in an increased or 

decreased calcium-dependent release of dopamine is still 

under debate, since experimental support for both these opposite effects has been provided in different models of lead 

intoxication [73, 80, 81]. Further, exposure to doses oflead 

ranging from 200 to 1350 ppm [46, 47, 52] has been described 

as depressing tissue concentrations of zinc and as increasing 

urinary zinc excretion [79], an effect which could be linked 

with the well proved renal toxicity exerted by these doses of 

lead. 

AIM 

The purposes of our study were: (1) to find a threshold 

dose of lead which could mimic in pre- and postnatally exposed rats the hyperactivity syndrome described in children; 

(2) to perform at the same time neurobehavioral and 

neurochemical assessment in the brain by studying the functional status of neurotransmitter and neuropeptide systems; 

(3) to monitor the levels of lead in urine, blood and brain 

areas together with the levels of other divalent cations such 

as zinc, copper and calcium, which seem to modulate receptor functions in the brain [6, 21, 50, 59, 75, 77]. 

ANIMAL MODELS 

In view of all the above mentioned considerations and 

data from our preliminary studies on lead accumulation in . 

the blood and brain after exposure to varying doses oflead, 

we decided to study the effects of a very low dose of lead on 

rat pups pre- and postnatally exposed through maternal dosing. For this purpose female Wistar rats were treated daily 

by gastric intubation with 1 mg/kg of Pb (N03)2 from mating 

to the end of lactation (Table 1). The offspring, after weaning, underwent the same treatment till 60 days of age. Two 

other groups of rats used as controls were treated with 

NaNO., to test for the second ion effect of the Pb (N03 ) 2 and 

with deaionized water to test for the effect of the solvent 

alone. 

RESULTS AND DISCUSSION 

The level oflead exposure used in this study did not affect 

the body weight of the dams during gestation and lactation, 

the duration of pregnancy or the number or weight of litters 

delivered in comparison with controls (Table 2). 

As reported elsewhere [8], no significant differences were 
502 BARALDI ET AL. 

seen between lead exposed dams and controls at the end of 

the nursing period in urine and plasma concentrations of zinc 

and copper determined by an atomic absorption spectrophotometric method previously described [5] (Tables 3 

and 4), while the excretion oflead was increased about 8 fold 

(Table 3) and level of lead in whole blood was practically 

doubled (Table 4). 

Lead was measured by an atomic absorption spectrometer (Perkin-Elmer med 5000) equipped with HGA 400 

graphite furnace, following the procedure of Fernandez [27]. 

The reliability of our method was controlled [78] by the 

Health and Safety directorate of the Community European 

Commission (C.E.C.). 

These data are of value in order to ensure that at this level 

of exposure lead does not induce any apparent significant 

change in adult pregnant female rats. No significant differences were noted in growth (Table 5), development of the 

righting reflex or age of eye opening (data not shown) between lead exposed and control pups during the nursing 

period. 

In this context it is noteworthy that only a few days after 

weaning (30 days of age) the pups directly exposed to lead by 

intubation showed a statistically significant decrease in body 

weight when compared with controls. Since this difference 

was no longer apparent at 60 days of age (Table 5), it is likely 

that this phenomenon is to be attributed to a greater reaction 

than controls oflead exposed pups to the weaning-associated 

stress and to manipulations linked with the gastric intubation 

procedure of the treatment. 

An open-field test performed at 15 days of age (Table 6) 

showed a significant increase in both number of crossedsquares and pivots in the lead exposure group in comparison 

with both groups of controls. The open-field test was repeated at 40 days of age and showed practically the same 

results, both male and female lead-treated rats being more 

active than controls. However comparison between groups 

showed a significant difference only in the group of male 

rats. These data seem to be similar to human responses to 

lead, since in the human situation, boys exhibit hyperkinetic 

syndrome more frequently than girls [83]. The hyperactivity 

of lead-treated rats at 40 days of age was confirmed by 

measuring locomotor activity in photocell-equipped boxes 

during 5 minute tests repeated 3 times at intervals of 30 minutes and evaluating only the data of the last test in order to 

avoid adaptation and curiosity interferences with the 

locomotion parameter (data not shown). These findings 

taken together seem to demonstrate that daily exposure to 1 

mg/kg/day of lead nitrate during pre- and postnatal life induces border-line behavioral effects which do not appear to 

be related to nonspecific effects due to general toxicity or 

inhibition of energy metabolism, both in the nursing dams 

and in the offspring. 

As shown in Tables 8 and 9 urinary excretion oflead at 40 

days of age was increased about 7 fold in lead-exposed pups, 

while the whole blood level was twice as high as in controls. 

When compared with the level observed in the dams, these 

data indicate an equivalence between the maternal dosing 

and the direct treatment of the pups with lead. Further, the 

levels oflead in 4 brain areas tested (Fig. 1) were found to be 

about 8 times higher than in controls, a finding which confirms the tendency of lead [55] to accumulate in the brain 

during the early stages of pre- and postnatal life already described. On the other hand no statistically significant 

changes in urinary excretion of copper and zinc were found 

in the lead-exposed pups, a finding which seems to indicate, 

TABLE 4 

CONCENTRATIONS OF ZINC AND COPPER IN PLASMA AND OF 

LEAD IN WHOLE BLOOD OF LEAD-POISONED MOTHER RATS 

DURING GESTATION AND LACTATION (I mg/kg/DAY 

BY ORAL INTUBATION) 

Treatment 

Water 

NaNO, 

Pb(N03), 

Values are 

*p<0.05. 

Plasma 

(Mg/100 ml) 

Zinc Copper 

133.0 ± 1.8 

128.6 ± 4.4 

119.7 ± 5.3 

reported as 

117.6 ± 2.9 

116.0 ± 5.1 

125.7 ± 5.5 

mean ± S.E.M.; 

Whole Blood 

(Mg/100 ml) 

Lead 

2.63 * 0.33 

2.70 ± 0.36 

5.87 ± 0.96* 

Student's r-Test: 

along with the normal urine output, that this level of lead 

exposure does not result in overt renal toxicity. 

While no change was found in plasma copper concentrations, the level of plasma zinc was found to be enhanced in 

the lead-exposed group. This increased presence of circulating zinc was quite surprising but was corroborated by the 

finding that zinc was increased also in the 4 brain areas 

tested (Fig. 1). A modest increase of zinc, which did not 

reach statistical significance, was found also in the cerebellum, hippocampus and cortex of NaN03 treated rats. 

Moreover, an increased presence of calcium, which was 

statistically significant in cerebellum and cortex, was found 

at the same age (40 days) in the brains of lead-exposed rats. 

Since no change in the level of copper was found in the blood 

or in the 4 brain areas tested, it is likely that the increased 

zinc and calcium are not due to a lead-induced general increase in divalent cations entering across the gut and bloodbrain barrier, but rather to specific mechanisms that need 

further elucidation. 

The use of higher doses oflead acetate has been described 

as inducing no change or indeed a decrease in zinc homeostasis in the brain: it could be that, as suggested for other 

lead-induced phenomena [82], a very low level oflead exposure may produce opposite effects to those found after exposure to high levels. In this regard it is noteworthy, however, 

that the content of zinc and calcium, tested in the whole 

brain of 60 day old lead-exposed rats, appeared to be normal 

in comparison with the values of control rats (Table 10). 

Whatever the mechanism by which exposure to low doses of 

lead results in increased presence of zinc and calcium, it is 

clear from the last finding mentioned that this lead-linked 

phenomenon is confined to the early stages of pre- and 

postnatal life. 

The finding that a low level of lead exposure was able to 

induce threshold behavioral effects without implication of 

apparent malnutrition problems prompted us to study the 

functional activity of some of the neurotransmitter systems 

previously suggested as being implicated in the neurotoxicity 

of lead. 

As regards the functional activity of brain GABAA receptors, which were studied according to a method previously 

described using 'H-GABA as a ligand [2], we found (Table 

11) that exposure to the low level of lead we used significantly reduced only the low affinity component of the 

sodium-independent 'H-GABA binding saturation curves 

performed in cortical brain membranes. This finding allowed 

A I 
P.6 

LOW-LEVEL LEAD NEUROTOXICITY 503 

TABLE 5 

BODY WEIGHT OF PUPS NOURISHED BY LEAD-POISONED MOTHERS 

Mother daily 

treatment 

(1 mg/kg/os) 

Water 

NaNO, 

Pb(NOa)2 

4m 

11.7 ± 0.6 

11.4 ± 0.5 

10.8 ± 0.5 

Body 

8m 

17.4 ± 0.5 

18.0 ± 0.3 

16.7 ± 0.3 

weight (g) of pups 

32.4 ± 0.4 

31.8 ± 0.6 

30.0 ± 0.6 

at day: 

24,h 

51.7 ± 1.1 

51.7 ± 1.4 

49.6 ± 1.3 

30,h 

68.2 ± 2.1 

64.9 ± 1.8 

58.7 ± 1.3* 

6 

232.4 ± 7.2 

235.0 ± 8.5 

238.6 ± 8.8 

60,h 

9 

165.3 ± 9.4 

178.3 ± 12.0 

175.7 ± 6.6 

Values are reported as mean ± S.E.M.: Student's '-Test: *p<0.01 vs. controls. 

TABLE 6 

OPEN FIELD BEHAVIOR OF BABY RATS (15 DAYS OF AGE) 

NOURISHED BY LEAD-POISONED DAMS 

Treatment Test ses- Pivots 

(1 mg/kg/os No. of siontime Entries (mean ± 

daily) tested pups (min) (mean ± S.E.M.) S.E.M.) 

water 

NaNO, 

Pb(NO,)2 

18.3 ± 5.5 

22.4 ± 3.5 

30.3 ± 4.1* 

2.2 ± 0.3 

3.4 ± 0.6 

4.0 ± 0.5* 

Student's /-Test: *p<0.01 vs. controls. 

TABLE 7 

OPEN HELD BEHAVIOR OF 40 DAYS OLD FEMALE AND MALE RATS NOURISHED BY LEAD-POISONED MOTHERS AND 

TREATED FOR 10 DAYS WITH LEAD BY ORAL INTUBATION 

Treatment 

(1 mg/kg/os 

daily) 

water 

NaNO, 

PtKNO,), 

No. of 

tested pups 

00 00 00 

Test 

session time 

(min) 

3 

3 

3 

Male 

Entries Pivots 

12.7 ± 6.3 2.5 ± 0.2 

14.7 ± 7.1 3.2 ± 3.7 

28.5 ± 7.4* 4.5 ± 1.9* 

Female 

Entries Pivots 

36.2 ± 9.7 

36.2 ± 5.9 

43.5 ± 10.1 

6.5 ± 1.2 

5.4 ± 1.8 

8.2 ± 2.5 

All reported values are mean ± S.E.M. 

Student's /-Test: *p<0.01 vs. controls. 

5* 

= • 

% 

TABLE 8 

LEAD, ZINC AND COPPER CONCENTRATIONS IN URINES OF PUPS 

(AGED 40 DAYS) NOURISHED BY LEAD-POISONED MOTHERS TILL WEANING 

AND EXPOSED FOR 10 DAYS TO LEAD (1 mg/kg/DAY BY ORAL INTUBATION) 

Treatment 

Concentrator! in urines§ (mean ± SEM) 

Zinc 

(Mg) 

Copper 

(Mg) 

Lead 

(ng) 

Urine 

output/rat 

(ml) 

Mean ± SD 

water 

NaNO, 

Pb(NO,)2 

1.21 ±0.41 

1.36 ± 0.37 

1.71 ± 0.22 

0.76 ± 0.04 

0.65 ± 0.09 

0.82 ± 0.14 

8.2 ± 1.5 

8.0 ± 2.5 

58.0 ± 3.0* 

2.2 ± 0.8 

3.3 ± 0.9 

2.5 ± 0.4 

§Urine were collected for 6 hr from rats in metabolic cages. 

Student's r-Test: *p<0.01. 
P.7 

504 BARALDI £7 AL. 

TABLE 9 

CONCENTRATIONS (MEAN * S.E.M.) OF ZINC AND COPPER IN 

PLASMA AND OF LEAD IN WHOLE BLOOD OF PUPS (AGED 40 

DAYS) NOURISHED BY LEAD-POISONED MOTHERS (I mg/kg/day/os) 

TILL WEANING AND EXPOSED FOR 10 DAYS TO LEAD (I mg/kg/DAY 

BY ORAL INTUBATION) 

co*w* CALCIUM 

Treatment 

water 

NaNO, 

P1XNO,), 

Plasma 

(Mg/100 ml) 

Zinc Copper 

120.1 ± 3.0 162.5 ± 10.3 

119.8 ± 1.2 161.5 ± 15.9 

156.6 ± 11.7* 179.0 ± 24.2 

Whole Blood 

(Mg/100 ml) 

Lead 

2.27 + 0.15 

1.67 + 0.32 

5.15+ 1.0* 

Student's /-Test: *p<0.05. 

5 • M.'A'.'.'JIL—* 

inniiiiir^** 

o » 4 • i a • inaano * » « •otooitoxnnoiDoao 

yafr dry waigM 

FIG. 1. Lead, zinc, copper and calcium concentrations in brain 

areas of baby rats (40 days of age) nourished by lead poisoned 

mothers (I mg/kg/day/os) and treated (I mg/kg/day/os) for 10 days 

after weaning. Controls were exposed to water or NaNO:l. The columns represent the mean values (±S.E.M.) of 8 determinations 

done in triplicate. The concentrations are expressed as ftg/g of dry 

weight. Black, white and dashed columns represent the values of 

water, NaNO, and Pb (NO,).., treated rats,.respectively. Student's 

/-test: *p<0.05 at least vs. water and NaNO, treated rats. 

TABLE 10 

METAL CONCENTRATIONS IN WHOLE BRAINS OF 60 DAY OLD RATS PRE AND 

POSTNATALLY EXPOSED TO LEAD (1 mg/kg/day/os) 

Pb Zn Cu Ca 

Water 

NaNO, 

Pb(NO,)2 

0.35 ± 0.5 

0.37 ± 0.8 

1.42 ± 0.4* 

62.2 ±6. 1 

66.8 ± 4.2 

55.6 ± 3.3 

11.9 ± 1.3 

9.8 ± 2.1 

11.5 ± 1.8 

201.8 ± 7.3 

193.5 ± 6.2 

195.9+ 10.1 

Mean ± S.E.M. are expressed as jug/g dry weight. 

Student's/-Test: *p<0.001. 

us to test other brain areas: the GABA binding was found to 

be reduced also in the brainstem and in the cerebellum but not 

in the hippocampus. Although it seems impossible from the 

present data to give a complete explanation of the mechanism whereby the low affinity GABA receptors are affected 

by lead exposure, we can tentatively suggest, in line with the 

hypothesis of Silberged and Lamon [74], that a lead-induced 

increased presence in the brain of delta-aminolevulinic acid 

could be responsible for this down-regulation. In addition, or 

alternatively, we can suggest that the increased presence of 

zinc, which has recently been shown mainly to modulate low 

affinity GABA receptors [6], could, at least in part, be responsible for this change in GABAA receptor characteristics. 

The increased presence of calcium in the brain tissues does 

not seem to play a role in this phenomenon, since calcium 

has been described [21,49] as modulating GABAA high affinity binding sites, an effect which requires, to be optimized, 

an incubation temperature of 37CC, which was not used in the 

present experiments. Though these hypotheses need to be 

borne out by further experiments, we may conclude from the 

present data that there is a lower functional activity of the 

inhibitory GABAergic system in lead-exposed rats in comparison with controls. 

Study of the binding characteristics of dopamine receptors in the striatum of lead-exposed rats at 40 days of age 

using

 :,H-spiperone as a ligand and (-)sulpiride as a binding 

displacer, as previously described [4], showed that there is a 

modestly increased affinity of the dopamine receptors 

labeled by

 :lH-spiperone (mainly D2 type) as expressed by a 

reduced value of the K„ without any change in the maximum 

number (Bmax) of binding sites (Fig. 2) in comparison with 

controls. From this finding we may surmise that the low level 

of lead exposure used here (25 ppm) may induce a modest 

chronic reduction of dopamine in the synaptic cleft, which in 

turn might explain the increased affinity of dopamine receptors. This hypothesis is presently under investigation by 

measuring the neurochemical correlates of the dopamine receptor study in our model. Preliminary data from these 

studies indicate that dopamine synthesis is reduced in the 

striatum of our lead exposed rats. These data seem to be in 

line with the previously reported reduction of dopamine release [80] in the corpus striatum induced by chronic exposure to low concentrations of PbCO., (40 ppm) and of 

dopamine synthesis [51] induced in a dose-related manner by 

lead exposure (216 and 1350 ppm). 

It has been suggested [80] that the inhibitory effect oflead 
P.8 

LOW-LEVEL LEAD NEUROTOXICITY 505 

t | 

TABLE 11 

•H-GABA BINDING KINETIC CHARACTERISTICS IN CORTICAL 

SYNAPTIC MEMBRANES OF PUPS (40 DAYS OF AGE) NOURISHED 

BY LEAD-POISONED MOTHER RATS DURING PRE AND 

POSTNATAL LIFE AND EXPOSED TO LEAD FOR 10 DAYS AFTER 

WEANING (MEAN ± SD) 

Treatment 

(1 mg/kg/ 

day/os) 

water 

NaNO, 

Pi-XNO,).;) 

•H35.7 ± 0.5 

35.2 ± 0.7 

38.8 ± 0.8 

GABA binding characteristics* 

Bnax 1 K|)2 Bmax2 

0.625 ± 0.02 280 + 24 3.5 + 0.4 

0.600 ± 0.04 304 + 12 3.3 ± 0.3 

0.650 ± 0.05 282 + 18 1.7 ± 0. I§ 

*K„=dissociation constant in nM. 

Bm,lx=maximum number of binding sites in pmol/mg protein. 

§Student's /-Test: p<0.0l. 

on dopamine release could be due to competition between 

lead and calcium. Lead has been shown to decrease the calcium uptake of brain mitochondria, an effect which leads to 

accumulation of free intracellular calcium [29]. The present 

demonstration that there is indeed an accumulation of calcium in the brain tissues of lead-exposed rats, albeit only 

indicative since the metabolism of the different pools of calcium is very complicated, would seem to give support to the 

above mentioned hypothesis. We have not yet performed a 

challenge with dopaminomimetic drugs on our lead-exposed 

rats, but it is noteworthy that Winder et al. [81] have reported increased responsiveness to stimulation of locomotor 

activity by apomorphine and only marginal changes with 

amphetamine in rats exposed to a relatively low level oflead 

(300 ppm). This finding seems to give credit to the hypothesis 

that the increased affinity of post-junctional dopamine receptors could be due to a lead-dependent partial impairment of 

dopamine turnover at the pre-junctional level. 

The spontaneous locomotor hyperactivity observed could 

be explained by the demonstration given by Shaywitz et al, 

[69] that partial destruction of DA-containing neurons in 

neonatal rats leads to hyperactivity which appears between 

12 and 22 days of age. 

In summary, taken together these biochemical findings 

seem to indicate that a modest imbalance between increased 

functional activity of the striatal postsynaptic dopamine receptors and decreased activity of inhibitory GABAergic system develops during lead intoxication which could explain 

the border-line behavioral effects described in this study. 

Interestingly Memo et al. [51] and Govoni et al. [32], who 

originally reported an increased presence of Met-enkephalin 

in the striatum of lead-exposed rats, have suggested that the 

effect of lead on nigro-striatal neurons could be a consequence of other lead-induced neurotransmitter alterations, 

i.e., the striatal opiatergic system. Enkephalins, which are 

present in the striatum [36], have in fact been postulated as 

inhibitory modulators of dopamine release [26, 35, 63]. 

Though ontogenic studies of the derangement of the developing opiatergic and dopaminergic systems in lead-intoxicated 

rats will solve the problem of priority, at this stage of our 

knowledge we can also hypothesize that lead induced alterations of the dopaminergic system could be responsible for 

the increase in Met-enkephalin by analogy with similar 

phenomena described by Hong et al. [35]. The increase in 

Met-enkephalin in lead-exposed rats together with the pro-

'H-SPIPERONE >p«clllc binding 

(fmoJ/mg prat.) 

FIG. 2. Scatchard plot of 'H-spiperone binding to striatal membranes of pups (40 days of age) nourished by lead-poisoned mothers 

(i mg/kg/day/os) and treated with lead (1 mg/kg/day/os) for 10 days 

after weaning. Controls were exposed to an equivalent treatment 

with NaNO, or water. The values (mean+S.E.M.) of the affinity 

constant (K„) expressed as nM, and of the maximum number of 

binding sites (Bmax) expressed as fmol/mg prot. were: Water (•) 

K„=0.18±0.02, Bmax=148±4; NaNO, (•) K„=0.18±0.03, 

Bma,,= 162+5; Pb (NO,)2 (A) KD=0.08±0.02 (a), Bm„ = 150±4. Student's /-test: (a)p<0.01 vs. water and NaNO,. 

TABLE 12 

/S-ENDORPHIN CONCENTRATIONS IN PITUITARY LOBES OF PUPS 

(40 DAYS OF AGE) NOURISHED BY LEAD-POISONED MOTHERS 

AND TREATED (1 mg/kg/day/os) FOR 10 DAYS AFTER WEANING 

(MEAN + S.D.) 

Treatment 

Water 

NaNO, 

Pb(NO,)2 

^-Endorphin (ng/gland) 

Anterior Neurointermediate 

lobe lobe 

520.3 + 31.5 803.4 ± 63.5 

585.8+ 58.1 1043.6 ±239.1 

1360.9 + 128.2* 2187.8 + 320.2* 

Student's /-Test: */><0.001. 

TABLE 13 

HYPOTHALAMIC 0-ENDORPHIN AND MET-ENKEPHALIN 

CONCENTRATIONS OF PUPS (40 DAYS OF AGE) NOURISHED BY 

LEAD-POISONED MOTHERS AND TREATED (1 mg/kg/day/os) FOR 10 

DAYS AFTER WEANING (MEAN * S.E.M.) 

Treatment 

/3-Endorphin 

(ng/mg prot.) 

Met-Enkephalin 

(ng/mg prot.) 

Water 

NaNO, 

Pb(NO,)2 

36.0 + 5.2 

24.3 + 6.8 

10.5 + 3.5* 

6.43 + 1.0 

5.84 + 1.3 

11.50± 2.4* 

Student's/-Test: *p<0.01. 
P.9 

506 BARALD I ETAL. 

TABL E 14 

INCREASED ANTINOCICEPTIVE RESPONSES TO MORPHINE ADMINISTRATION 

OF PUPS (40 DAYS OF AGE) NOURISHED BY LEAD-POISONED MOTHERS AND 

TREATED (I mg/kg/day/os) FOR 10 DAYS AFTER WEANING 

Chronic 

pretreatment 

Water 

NaNO, 

PtXN03)2 

Reaction time§ in sec 

0 

2.8 + 0.2 

3.4 + 0.3 

4.2 ± 0.5* 

Treatment: 

Morphine 

(5 mg/kg IP) 

Morphine 

(5 mg/kg IP) 

Morphine 

(5 mg/kg IP) 

(mean ± S.E.M.) 

30 

4.0 ± 0.4 

5.2 + 0.5* 

7.2 ±0.lt 

at: 

60 min 

3.2 + 0.09 

4.7 ± 0.3* 

5.9 ± 0.5* 

§Using hot plate test (56 + 0.5°C). 

Student's/-Test: *p<0.05, tp<0.001. 

posal that this peptide may mediate euphoric states [10] and 

facilitate maze performance in rats [40], suggested to us that 

we test the functional status of opiate receptors and assay 

the level of endogenous opiates in the hypothalamus and 

pituitary. As shown in Fig. 2, we found a dramatic increase 

of opiate receptors labeled by

 3H-naloxone in the hypothalamus of lead-exposed rats (at 40 days of age) in comparison 

with the two control groups. The

 3H-naloxone binding study 

was performed in brain membrane preparations as previously described [3]. The number of binding sites, but not the 

affinity constant, of the lead treated group was significantly 

higher than in the case of groups of rats treated with NaN03 

or water. Furthermore, increased

 3H-naloxone binding was 

also found in the brainstem and striatum (data not shown). 

Immunoreactive 0-endorphin (ir-/3-END) and Metenkephaline (ir-ME) concentrations determined at the same 

age in the medio-basal hypothalamus (MBH), following the 

extraction procedure described by Rossier et al. [67] and the 

radioimmunoassay previously described by one of us [61], 

were found to be significantly lower and higher respectively, 

in lead-exposed rats than in both groups of controls (Table 

12). An explanation for such a reduced presence of ir-/3-END 

in the MBH, which contains the cell bodies where this peptide is synthesised, may be given by the finding (Table 13) 

that the levels of ir-i3-END are markedly increased in the 

anterior and neurointermediate lobes of the pituitary of the 

lead-exposed rats. It is likely, in fact, that a blockade of 

/3-endorphin release from the pituitary directly or indirectly 

elicited by lead could be responsible through activation of a 

feed-back process of reduced synthesis of /3-endorphin in the 

MBH. 

A more complicated explanation of the reduced presence 

of ir-/3-END in the MBH could be that a lead induced chronic 

increase of ir-ME (Table 12) will trigger reduced dopamine 

release [37] and in turn a hyperprolactinemic state [33,37], 

which might affect the synthesis of/3-endorphin in the hypothalamus [58]. Whatever the mechanism by which lead induces this effect, the chronically reduced level of 

/3-endorphin in the hypothalamus and probably in other brain 

areas could induce a supersensitivity phenomenon at the 

opiate receptor level. 

Other factors, however, may be considered as responsible 

for these abnormalities in endogenous opiate levels and 

opiate receptors characteristics. The lead-induced increase 

200 300 400 soo aoo 

*H-MALOXONE f-Mdflc MmMng 

ifmol/mfl p«*o*-l 

FIG. 3. Scatchard plot analysis of

 3H-naloxone binding to hypothalamic membranes of pups (40 days of age) nourished by leadpoisoned mothers (1 mg/kg/day/os) and treated (1 mg/kg/day/os) for 10 

days after weaning. Controls were exposed to an equivalent treatment with NaNO, or water. The values (mean±S.E.M.) of the 

maximum number of binding sites (Bma,), expressed as fmol/mg 

protein, and of the affinity constant (K„), expressed as nM, were: 

water (Q) Ko=2.8±0.3, Bma»=272+U.2; NaNO, (A) KD=4.2±0.4 

(a), Bm„=373+12.3 (a); Pb (NO,)2 (O) KD=2.3±0.5, Bm„= 

670+14.6 (b). Student's /-test: (a)p<0.05 vs. water; (b)p<0.001 vs. 

water and vs. NaNO,. 

of brain concentrations of zinc and calcium in the early 

stages of the life described above, could play a role in these 

abnormalities. Both zinc and calcium have been shown to 

exert in vitro [7,75] and in vivo [7,68] an antinarcotic effect 

by interfering with the binding capacity of opiate receptors 

and/or other factors such as fragments of /3-lipotropin (i.e., 

a-melanocyte-stimulating hormone, adrenocorticotropic 

hormone and its fragments), which have affinity for brain 
P.10 

LOW-LEVEL LEAD NEUROTOXICITY 

opiate receptors and inhibit morphine-induced analgesia [11, 

20, 26, 38]. Moreover, not only lead [56] but also zinc [1] and 

calcium [62] ions have been shown to be implicated in the 

regulation of the cerebral activity of adenylate cyclase, an 

enzyme which has been suggested to play a role in the dependence and tolerance to chronic morphine administration 

[70]. The above mentioned hypersensitivity of the opiate receptors induced by lead exposure was proved to be operative 

in "in vivo" experiments by performing a hot plate test. As 

shown in Table 13, the lead-treated rats had a longer reaction 

time and were supersensitive to the administration of a 

threshold dose of morphine. Incidentally, an increased response to morphine administration of lead-exposed rats has 

been reported by others while this paper was in preparation 

[41]. 

j It must be mentioned that, while the chronic treatment 

with NaNO., did not change the binding characteristics of the 

dopaminergic and GABAergic receptors, it was able to induce a weak by statistically significant increase in the number 

of opiate receptors, which paralleled a slight decrease in 

/3-i:ndorphin, increased reaction time in the hot plate test and 

an increased response to a threshold dose of morphine. 

507 

These data seem to indicate that, at least for the opiatergic 

system, NaNO., is not a completely inert compound. 

This data is not completely surprising since an increased 

presence of Na4 has been shown to induce major changes in 

the binding to opiate receptors [60]. Since, however, there 

are no other data available on the effect of a perinatal chronic 

low exposure to NaN03 on the opiatergic system, this 

NaN03-induced effect needs to be further clarified experimentally. It must, however, be stressed that such a slight effect of NaN03 does not markedly interfere in the Pb (N03)2-

induced effect since the number of opiate receptors found in 

lead treated rats is statistically higher in comparison not only 

with the water but also with the NaN03 treated rats. 

To conclude, the first observation reported here of a 

supersensitivity of the opiatergic system which mediates 

pain perception, in parallel with an increased presence of 

Met-enkephalin in the hypothalamic area which might affect 

directly or indirectly motor functions, seems to be the most 

promising data of our present study. These data, in fact, if 

confirmed by further experiments, may provide a new insight in the understanding of the complex mechanism oflead 

neurotoxicity. 

ACKNOWLEDGEMENT 

This research was supported by Italian C.N.R. grant (No. 

8302976-56). 

REFERENCES 

5. 

6. 

9. 

10. 

11. 

Baba, A., T. Kirara, E. Lee and N. Iwata. Activation of rat 

brain adenylate cyclase by copper plus dithiothreitol. Biochem 

Pharmacol 30: 171-174, 1981. 

Baraldi, M. and M. L. Zeneroli. Experimental hepatic 

encephalopathy: changes in the binding of gamma-aminobutyric 

acid. Science 216: 427-429, 1982. 

Baraldi, M., R. Poggioli, M. Santi, A. V. Vergoni and A. Bertolini. Antidepressant and opiates interactions: pharmacological 

and biochemical evidences. Pharmacol Res Commun 15: 843-

857. 1983. 

Baraldi, M., M. L. Zeneroli, P. Ricci, E. Caselgrandi and E. 

Ventura. Down regulation of striatal dopamine receptors in experimental hepatic encephalopathy. Life Sci 32: 1417-1425, 

1983. 

Baraldi, M., E. Caselgrandi, P. Borella and M. L. Zeneroli. 

Decrease of brain zinc in experimental hepatic encephalopathy. 

Brain Res 258: 170-172, 1983. 

Baraldi, M., E. Caselgrandi and M. Santi. Effect of zinc on 

specific binding of GABA to rat brain membranes. In: The 

Neurobiology of Zinc (Part A), edited by C. J. Frederickson, G. 

A. Howell and E. J. Kasarskis. New York: Alan R. Liss, 1984, 

pp. 59-71. 

Baraldi, M., E. Caselgrandi and M. Santi. Reduction of withdrawal symptoms in morphine-dependent rats by zinc: behavioral and biochemical studies. Neurosci Lett 18: S371, 1984. 

Baraldi, M., P. Zanoli, T. Rossi, M. Santi, P. Borella, E. Caselgrandi and M. Bergomi. Zinc, copper and calcium status of baby 

rats nurished by low lead-exposed mothers: neuro-behavioral 

correlates. Clin Neuropharmacol 7: Suppl 1, 36-37, 1984. 

Barret, J. and P. J. Liversey. The acetic acid component oflead 

acetate: Its effect on rat weight and activity. Neurobehav Toxicol Teratol 4: 105-108, 1982. 

Beluzzi. J. and L. Stein. Enkephalin may mediate euphoria and 

drive-reduction reward. Nature 26: 556-558, 1977. 

Bertolini, A.. R. Poggioli and W. Ferrari. ACTH-induced 

hyperalgesia in rats. Experientia 35: 1216—1217, 1979. 

* . * • 

12. Bornschein, R. L., D. A. Fox and I. A. Michaelson. Estimation 

of daily exposure in neonatal rats receiving lead via dams milk. 

Toxicol Appl Pharmacol 40: 577-587, 1977. 

13. Bornschein, R. L., I. A. Michaelson, D. A. Fox and R. Loch. 

Evaluation of animal models used to study effects of lead on 

neurochemistry and behavior. In: Biochemical Effects of Environmental Pollutants, edited by S. D. Lee. Ann Arbor MI: 

Science Publications, 1977, pp. 441-460. 

14. Brennan, M. J. and R. C. Can trill. The effect of deltaaminolaevulinic acid on the uptake and efflux of 'H-GABA in 

rat brain synaptosomes. J Neurochem 32: 1781-1786, 1979. 

15. Brennan, M. Y. W. and R. C. Cantrill. d-Aminolaevulinic acid is 

a potent agonist for GABA autoreceptors. Nature 280: 514-515, 

1979. 

16. Bull, R. J. In: Lead Toxicity, edited by L. Singhal and J. A. 

Thomas. Baltimore: Urban and Schwartzenberg, 1980, pp. 

119-168. 

17. Carmichael, N. G., C. Winder and P. D. Lewis. Dose response 

relationship during perinatal lead administration in the rat: a 

model study of lead effects on brain development. Toxicology 

21: 117-128, 1981. 

18. Cavalleri, A., A. Baruffini, C. Minoia and L. Bianco. Biological 

response of children to low levels of inorganic lead. Environ Res 

25: 415-423, 1981. 

19. Clayton, L. B., I. Kitchen and C. Winder. Ontogenic differences in striatal enkephalin levels in rats exposed to low levels 

of lead. Br J Pharmacol 79: 199 P. 1983. 

20. Contreras, P. C. and A. E. Takemori. Antagonism of morphineinduced analgesia, tolerance and dependence by 

a-melanocyte-stimulating hormone. J Pharmacol Exp Ther 229: 

21-26, 1984. 

21. Corda, M. G. and A. Guidotti. Modulation of GABA receptor 

binding by Ca2+. J Neurochem 41: 277-280, 1983. 

22. David, O., J. Clark and K. Voeller. Lead and hyperactivity. 

Lancet 2: 900-903, 1972. 

23. David, O., B. McGann, S. Hoffman, J. Sverd and I. Clark. Low 

lead levels and mental retardation. Lancet 2: 1376-1379, 1979. 
P.11 

508 BARALDI ETAL. 

24. De La Burde, B. and M. S. Choate. Early asymptomatic lead 

exposure and development at school age. J Pediatr 87: 638-642, 

1975. 

25. Driscoll, J. W. and E. Stegner. Behavioral effects of chronic 

lead ingestion on laboratory rats. Pharmacol Biochem Behav 4: 

411-417, 1976. 

26. Ferland, L., K. Fuxe. P. Eneroth and J. A. Gustafsson. Effects 

of methionine-Enkephalin on prolactin release and catecholamine levels and turnover in the median eminence. Eur J Pharmacol 43: 80-90, 1977. 

27. Fernandez, F. J. Micromethod for lead determination in whole 

blood by atomic absorption with use of graphic furnace. Clin 

Chem 21: 558-661, 1975. 

28. Fowler, B. A., C. A. Kimmel, J. S. Woods. E. E. Mc Connell 

and L. D. Grant. Chronic low-level lead toxicity in the rat. 

Toxicol Appl Pharmacol 56: 59-77, 1980. 

29. Goldstein, G. Lead encephalopathy: The significance of lead 

inhibition of calcium uptake by brain mitochondria. Brain Res 

106: 185-188, 1977. 

30. Goiter, M. and I. A. Michaelson. Growth, behavior and brain 

catecholamines in lead-exposed neonatal rats: a reappraisal. 

Science 187: 359-361, 1975. 

31. Govoni, S., M. Memo, P. F. Spano and M. Trabucchi. Chronic 

lead treatment differentially affects dopamine synthesis in various brain areas. Toxicology 12: 343-349, 1979. 

32. Govoni, S., M. Memo, L. Lucchi, P. F. Spano and M. Trabucchi. Brain neurotransmitter system and chronic lead intoxication. Pharmacol Res Commun 12: 447-450, 1980. 

33. Govoni, S., L. Lucchi, F. Battaini, P. F. Spano and M. Trabucchi. Chronic lead treatment affects dopaminergic control of 

prolactin secretion in rat pituitary. Toxicol Lett 20: 237-241, 

1984. 

34. Goyer, R. A. and B. C. Rhyne. Pathological effects on lead. In: 

International Reviews of Experimental Pathology, edited by G. 

W. Richter and M. A. Epstein. New York: Academic Press, 

1973, pp. 2-77. 

35. Hong, J. S., H. Y. T. Yang, W. Fratta and E. Costa. Rat striatal 

methionine-Enkephalin content after chronic treatment with 

cataleptogenic and non-cataleptogenic antischizophrenic drugs. 

J Pharmacol Exp Ther 205: 141-147, 1977. 

36. Hong, J. S., H. Y. T. Yang and E. Costa. On the location of 

Methionine-Enkephalin neurons in rat striatum. Neuropharmacology 16: 451-453, 1977. 

37. Hokfelt, T., S. R. Vincent, C. J. Dalsgaard, M. HerreraMarschitz, V. Ungersted, M. Shultzbergn, I. Christensson and 

L. Terenius. Some aspects on distribution and role of opioid 

peptides in the central and peripheral nervous system. In: Central and Peripheral Endorphins: Basic and Clinical Aspects, 

edited by E. E. Muller and A. R. Gennazzani. New York: 

Raven Press, 1984, pp. 1-16. 

38. Jacquet, Y. T. Opiate effects after adrenocorticotropin or 

^-Endorphin injection in the periaqueductal gray matter of rats. 

Science 201: 1032-1034, 1978. 

39. Jugo, S. Metabolism of toxic heavy metals in growing organism. 

A Review. Environ Res 13: 35-46, 1977. 

40. Kastin, A., E. Scollan, M. G. King, A. Schally and D. H. Coy. 

Enkephalin and a potent analog facilitate maze performance 

after intraperitoneal administration in rats. Pharmacol Biochem 

Behav 5: 691-695, 1976. 

41. Kitchen, I., J. McDowell and C. Winder. Low level lead exposure alters morphine antinociception in neonatal rats. Toxicol 

Lett 22: 119-123, 1984. 

42. King, B. G. Maximum daily intake of lead without excessive 

body lead burden in children. Am J Dis Child 122: 337-340, 

1971. 

43. Kostial, K., D. Kello and G. H. Harrison. Comparative metabolism of lead and calcium in young and adults. Int Arch Arbeitsmed 31: 150-161, 1973. 

44. Krehbiel, D., G. A. Davis, L. M. LeRoy and R. E. Bowman. 

Absence of hyperactivity in lead-exposed developing rats. 

Environ Health Perspect 18: 147-157, 1976. 

45. Lucchi, L., M. Memo, M. L. Airaghi. P. F. Spano and M. 

Trabucchi. Chronic lead treatment induces in rat a specific and 

differential effect on dopamine receptors in different brain 

areas. Brain Res 213: 397-404. 1981. 

46. Mahaffey, K. R., R. Goyer and J. K. Haseman. Dose-response 

to lead ingestion in rats fed low dietary calcium. J Lab Clin Med 

82: 92-100. 1973. 

47. Mahaffey, K. R., S. G. Capar. B. C. Gladen and B. H. Fowler. 

Concurrent exposure to lead, cadmium and arsenic. J Lab Clin 

Med 98: 463-481, 1981. 

48. Mahaffey, K. R. Quantities of lead producing health effects in 

humans: Sources and bioavailability. Environ Health Perspect 

19: 285-295. 1977. 

49. Majewska. M. D. and D. M. Chuang. Modulation by calcium of 

•y-aminobutyric acid (GABA) binding to GABA A and GABA„ 

recognition sites in rat brain. Mol Pharmacol 25: 352-359, 1984. 

50. Marzullo, G. and A. J. Friedhoff. An inhibitor of opiate receptor 

binding from human erythrocytes identified as a glutathionecopper complex. Life Sci 21: 1559-1568. 1977. 

51. Memo. M., L. Lucchi, P. F. Spano and M. Trabucchi. Dosedependent and reversible effects of lead on rat dopaminergic 

system. Life Sci 28: 795-799. 1981. 

52. Michaelson, I. A. and M. W. Sauerhoff. The effect of chronically ingested inorganic lead on brain levels of Fe, Zn, Cu and 

Mn of 25 day old rats. Life Sci 13: 417-428, 1973. 

53. Michaelson, I. A. In: Lead Toxicity, edited by L. Singhal and J. 

A. Thomas. Baltimore: Urban and Schwartzemberg, 1980, pp. 

301-365. 

54. Mitchell, W. G, S. L. Cookson and J. D. Mann. Effects oflead 

on behavioural responses to morphine in juvenile rats. Clin Res 

29: 892 A. 1982. 

55. Momcilovic, B. and K. Kostial. Kinetics of lead retention and 

distribution in suckling and adult rats. Environ Res 8: 214-220, 

1974. 

56. Nathauson, B. R. and F. E. Bloom. Lead induced inhibition of 

brain adenyl cyclase. Nature 255: 419-420, 1978. 

57. Overmann, S. R. Behavioral effects of asymptomatic lead exposure during neonatal development in rats. Toxicol Appl Pharmacol 41: 459-471, 1977. 

58. Panerai. A. E., J. Sawynok, F. S. LaBella and H. Friassen. 

Prolonged hyperprolactinemia influences /3-endorphin and 

met-enkephalin in the brain. Endocrinology 106: 1804-1808, 

1980. 

59. Pasternak. G. W., A. M. Snowmna and S. H. Snyder. Selective 

enhancement of "H-opiate agonist binding by divalent cations. 

Mol Pharmacol 11: 725-744, 1975. 

60. Pert, C. B., G. Pasternak and S. H. Snyder. Opiate agonists and 

antagonists discriminated by receptor binding in brain. Science 

162: 1359-1361, 1973. 

61. Petraglia. F.. A. Penalva. V. Locatelli, D. Cocchi. A. E. 

Panerai. A. R. Genazzani and E. E. Muller. Effects ofgonadectomy and gonadal steroids replacement on pituitary and plasma 

endorphin levels in rats. Endocrinology 111: 1224-1229. 1982. 

62. Piascik, M. T., M. F. Piascik. R. I. Hitzemann and J. D. Potter. 

Ca*+-dependent regulation of rat caudatum nucleus adenylate 

cyclase and effects on the response to dopamine. Mol Pharmacol 20: 3 \9-tt5, 1981. 

63. Poliand, H.. S. C. Llorens-Cortes and J. C. Schwartz. 

- Enkephalin receptors on dopaminergic neurones in rat striatum. 

Nature 268: 745-747, 1977. 

64. Rafaels, L. S.. R. L. Bornscheim, I. A. Michaelson. R. K. Loch 

and G. F. Barker. Drug induced activity in lead-exposed mice. 

Pharmacol Biochem Behav 10: 95-104, 1979. 

65. Reiter, L. W., G. E. Anderson, J. W. Laskey and D. F. Cahill. 

Developmental and behavioral changes in the rat during chronic 

exposure to lead. Environ Health Perspect 12: 119-123. 1975. 

66. Roels, H., J. P. Buchet. R. Lauwerys, G. Hubermont, P. 

Bruaux. F. Claeys-Thoreau. A. Lafohtaine and J. VanOvershelde. Impact of air pollution by lead on the heme biosynthetic 

pathway in school-age children. Arch Environ Health 31: 310-

316, 1976. 
' -TM,^TM^^IJ....-.^-^1^ -

P.12 

LOW-LEVEL LEAD NEUROTOXICITY 509 

67. Rossier. J., A. Bayon, T. M. Vaigo, N. Ling, R. Guillemin and 

F. Bloom. Radioimmunoassay of brain peptides: evaluation of a 

methodology. Life Sci 21: 847-852, 1977. 

68. Sanghvi, I. S. and S. Gershon. Morphine dependent rats: blockade of precipitated abstinence by calcium. Life Sci 18: 649-654, 

1976. 

69. Shaywitz, B. A., R. D. Yager and J. H. Klopper. Selective brain 

dopamine depletion in developing rats: an experimental model 

of minimal brain dysfunction. Science 191: 305-307, 1976. 

70. Sharma, S. K., W. A. Klee and M. Niremberg. Dual regulation 

of adenylate cyclase accounts for narcotic dependence and 

tolerance. Proc Natl Acad Sci USA 72: 3092-3096, 1975. 

71. Shih, T. and I. Hanin. Chronic lead exposure in immature 

animals: neurochemical correlates. Life Sci 23: 877-888, 1978. 

72. Silbergeld, E. K., L. T. Miller. S. Kennedy and N. Eng. Lead 

GABA and seizures. Effects of subencephalopathic lead exposure on seizure sensitivity and GABAergic function. Environ 

Res 19: 371-382, 1979. 

73. Silbergeld. E. K. and R. E. Hruska. In: Low Level Lead Exposure: The Clinical Implications of Current Research, edited by 

H. L. Needleman. New York: Raven Press, 1980. pp. 135-158. 

74. Silbergeld, E. K. and J. M. Lamon. The role of altered haem 

synthesis in the neurotoxicity of lead. J Occup Med 25:680-684, 

1980. 

75. Stengaard-Pederson, K. Inhibition of Enkephalin binding to 

opiate receptors by zinc ions: possible physiological importance 

in the brain. Acta Pharmacol Toxicol 50: 213-220, 1982. 

76. Terenius, L., W. H. Gispen and D. De Wied. ACTH-like peptides and opiate receptors in the rat brain: structure-activity 

studies. Eur J Pharmacol 33: 395-399, 1975. 

77. Usdin, I. B.. I. Creese and S. H. Snyder. Regulation by cations 

of •1H-spiperidol binding associated with dopamine receptors of 

rat brain. J Neurochem 34: 669-676, 1980. 

78. Vahter, M. Assessment of human exposure to lead and cadmium through biological monitoring. WHO-UNEP-National 

Institute of Environmental Medicine and Department of Environmental Hygiene-Karolinska Institute, Stockholm, 1982. 

79. Victery, W.. D. Thomas. P. Schoeps and A. J. Vander. Lead 

increases urinary zinc excretion in rats. Biol Trace Element Res 

4:211-219, 1982. 

80. Wince, L. C, C. H. Donodan and A. J. Azzaro. Alterations in 

the biochemical properties of central dopamine synapses following chronic postnatal PbCO, exposure. J Pharmacol Exp Ther 

214: 642-650, 1980. 

81. Winder, C. The interaction between lead and catecholaminergic 

function. Biochem Pharmacol 31: 3717-3721, 1982. 

82. Winder, C. and I. Kitchen. Lead neurotoxicity: a review of the 

biochemical, neurochemical and drug induced behavioral evidence. Prog Neurobiol 22: 59-87, 1984. 

83. Zimmering, R. T., R. G. Burringht and P. Donovick. Effects of 

pre-natal and continued lead exposure on activity levels in the 

mouse. Neurobehav Toxicol Teratol 4: 9-14, 1982.