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Kniironmrntul Health Pemp^eiiies 

Vol. I2,pp. lt'J-123, 1975 

amd Beiia^sora 

s m the Rat 

e to Lead 

durinq Chronic 

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by Lawrence W. Reiter,* George E. Anderson,* John W. 

Laskey,* and Daniel F. Cahill* 

Young male and femule Sprague-Dawley rats were given drinking water containing 5 or 

50 ppm Ph for 10 days prior to mating. Pregnant females were continued on these regimens 

throughout gestation and lactation. After weaning the offspring were similarly exposed 

through adulthood. Keflex development, body weights, and locomotor activity were 

measured in the offspring. Significant delays were noted in the development ofthe righting reflex at 5 and 50 ppm and in eye opening at 50 ppm. No difference was observed in 

development of the startle reflex at either dose. Mean body weights of treatment groups 

during thi.s developmental period were not significantly different from controls. Locomotor activity was measured in adult males utilizing a residential ma/e . Moth levels oflead 

produced a significant reduction in locomotor activity. When groups were treated with (Iamphetamine (1.0 mg/kg subcutaneous), lead treatment caused a dose-related diminution 

in the amphetamine-induced hyperactivity. 

These results indicate that rats exposed to low levels of lead from conception until 

adulthood show a delay in nervous system development. As adults, these animals exhibit 

hypoactivity and decreased responsiveness to amphetamine. 

Introduction 

Load toxicosis in children has been implicated in 

hyperkinesis (/) and mental retardation (2). These 

findings have raised the possibility that chronic, 

low-level exposure to lead may produce "silent" 

bruin damage. Animal experiments, using a variety 

of species, have demonstrated that perinatal exposure to lead produces similar behavioral changes 

including hyperactivity, peripheral ataxia, and aggressiveness (-J-5). These experiments, however, 

have employed dietary levels of lead ranging from 

1200 to 27,000 ppm. 

The present study was undertaken to test for 

possible developmental and behavioral changes 

produced by continuous pre- and postnatal exposure to low levels of lead. The results indicate 

lhat behavioral changes can be produced by levels 

as low as 5 ppm in tbe diet. 

' K\|a- -l iiiirtit.il I tii ilt >|*.> Laboratory. National Kiivironnn-n 

I al Ui'-M-'ircli Center, U.S. Kiivii'oimiriital Protection Agency, 

It. -.-..l.-li Triancle Park. N.<\ L>771 I. 

December 1975 

Methods 

Male and female Sprague-Dawley rats, obtained 

from Blue Spruce Farms (Altamont, N.Y.), were 

randomly assigned to one of three treatment 

groups: 0, 5, or 50 ppm lead administered in the 

drinking water in the acetate form. Animals were 

pretreated for a period of 40 days, which allowed 

lead concentrations to approach steady-state levels 

in soft tissues (6). Following pretreatment, animals 

were mated and pregnant females were continued 

on lead throughout gestation and lactation. At 

weaning, the offspring were similarly exposed 

through 180 days. These Fi generation rats were 

mated at 90 days of age, and the resulting Fj 

generation animals were exposed to the same lead 

regimen. 

Five pups from each of 4-G litters per treatment 

group in both the Fi and Fa generations were tested 

for the ages at development of the startle response, 

tbe righting reflex and the age of eye opening. The 

startle response was evoked by a click (generated 

by a toy clicker) sounded immediately behind the 

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head of an animal held suspended by the nape of 

the neck. A positive response consisted ofa muscle 

jerk in any of the extremities. 

The righting reflex was elicited by holding the 

rat upside down by the nape ofthe neck and the tail 

and dropping it from approximately 30 cm above 

some wood shavings. A response was considered 

positive if the animal righted in mid-air and landed 

on all four feet. 

Age at eye opening was recorded with complete 

opening of both eyes. 

Five pups from each of 9-11 litters (Fi + Fa)per 

treatment group were tested daily between 8:30 

A.M. and 10:30 A.M. The litter means were used to 

calculate the mean day of development of each 

response, for each treatment group. 

Locomotor activity of adult males was recorded 

at approximately 120 days of age in a residential 

ma/e (Fig. 1) described initially by Norton et al. 

(7). Groups of three littermates were tested in the 

maze for a period of 5 days. Four mazes were 

housed in a sound-attenuated room maintained on 

a 12 hr light/12 hr dark cycle beginning at 6:00 

A.M. Food and water were available ad libitum. 

The daily experimental procedure was as follows: 

Groups were introduced into the mazes at 10:00 

A.M. and removed at 9:00 A.M. the following day, 

the mazes were cleaned, food and water were 

replenished , an d the animals were then 

reintroduced into the mazes at 10:00 A.M. Activity 

counts were recorded at 1-hr intervals by utilizing a 

Xerox minicomputer. 

Kna>i(i-: I Schematic representation of the i'i-siileiiti.'il maze 

UM-<I lo measure locomotor activity. Tlie maze <on-ists of a 

series ol iiiierconncrlcil alleys 110 cm wide. Ill cm high) converging on ti central upenfield and covered with transparent 

acrylic plastic equipped with a central raised area over the 

open field. The overall dimensions are 7li cm wide x (iO cm 

deep. I.on.mutton is detected by eight phototrarisistor 

( + ('infrared light-emitting diode (ol pairs. 

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Maze activity was also recorded following the 

administration of (/-amphetamine HCl (4'rng/kg) 

administered subcutaneously (SC) on day 5, 20 min 

prior to reintroducingjhejinimals into the maze. 

Group means, for both'reflex development and 

locomotor activity, were compared by tising the ttest. 

Results 

No differences between the Fi and F2 generations were observed in any parameter and results 

for both generations have been combined. 

The effects of lead treatment on development 

are shown in Table 1. Development of the startle 

response, which occurs normally at 12 days of age, 

was not significantly altered by lead treatment. On 

the other hand, both eye opening and development 

of the righting reflex were significantly delayed (p 

< 0.05) by lead administration. Eye opening was 

delayed approximately 1 day by tho 50 ppm treatment, whereas the righting reflex was delayed 1 day 

by 5 ppm and 2 days by 50 ppm. 

Table 1. Effects oflead administration on reflex development and eye opening in the rat. 

Pb dose, 

ppm" 

0 

5 

50 

No. of 

litter; .\* 

11 

!i 

10 

Mean dav 

Startle 

1 l.-S + 0.2 

12.0 ± 0.2 

12.4 ± 0.2 

nf development ± S.K. 

t y e opening 

15.2 + 0.2 

15.1 ± 0.2 

|5.H±0.2'' 

Kighting 

17.7 i 0.2 

18.8 ±0.-1'' 

19.8 + 0.7' 

a

[)ose giv en a; ppm of lead in the drinking water. 

*W ~ number ot litters (5 aniniaU'lirter) 

''Significantly different from control, r-test,/> < 0.05. 

Body weights for male rats during this period of 

developmental testing are shown in Figure 2. No 

consistent differences in body weights of the treatment groups were observed, although the 50 ppm 

lead group was significantly heavier than controls 

on days 15 and 18. Females showed similar growth 

curves. 

Figure 3 shows representative 23-hr time-interval histograms for the various treatment groups 

taken on day 3 in the maze. 

Animals showed an initial high level of exploratory activity during the first hour in the maze. 

Following this initial period of exploration, activity 

showed a normal circadian rhythm with low diurnal (photolight) activity and high nocturnal 

(photodark) activity. Data obtained during these 

various periods, i.e., exploratory, diurnal, and nocturnal as well as total activity were analyzed for 

lead effects 

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Kl(a.'UK 2. Growth curves for control and lead-treated male 

rats during the period of developmental testing:(») control; 

(A) 5 ppm Pb; (n) 50 ppm Pb. Kach point represents the 

mean (± S.E.) for 12 animals. 

The compiled data for maze activity on the first 

four successive days of testing are presented in 

Figure 4. Activity on the first clay in the maze was 

higher than on subsequent days. This difference 

was observed in all treatment groups and during 

all time periods. This elevated activity represents 

the animal's increased reactivity due to the novel 

environment. By the second day, the animals 

became "established" in the maze and subsequent 

activity levels were consistent from day to day. 

Lead administration produced a significant (p < 

0.05) decrease in locomotor activity ranging from 

27 to 42% on day 1 and from 11 to 37% on day 4. 

This lead-induced hypoactivity was not dose-related. Both total and nocturnal activity remained 

depressed in both lead-treated groups throughout 

the 4-day period. By day 4, neither lead-treated 

group differed from control during the exploratory 

period and following the first day no group 

differences were found in the diurnal period. 

Twenty minutes prior to testing on day 5, 

animals were given subcutaneous (SC) injections of 

4.0 mg/kg {/-amphetamine HCl. The results presented in Figure 5 represent the change in activity 

(A) following amphetamine administration. The A 

activity was.determined by subtracting tbe activity 

obtained on day 4 (predrug, control levels) from the 

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KICIIKK 3. Representative time-interval histograms from control and lead-treated ani:r.als. Data was obtained on day 3 

in the maze frorr. group- of three adult males. For purpose of 

analysis, the data ha? b^-er. divided into the various activityperiods represented by the shaded areas. 

activity following amphetamine and therefore 

represents the drug-induced changes in activity. 

In control animals, amphetamine produced the 

expected hyperactivity. Since drug was administered during the diurnal period, when activity 

is normally low, the major effect was seen during 

this period. Activity in control animals was elevated to 444^ and 6.52'c of predrug levels, during 

the exploratory and diurnal periods, respectively. 

This was also reflected in an elevation of total activity to 224% of predrug control level. During the 

nocturnal period, however, the control activity fell 

to 80% of the predrug level, demonstrating a partial compensation for the drug-induced hyperactivity. 

Lead-treated animals showed a diminution in 

the amphetamine resjxinse (Fig. 5). When compared to the ^ activity of the control group, both 

treatment groups showed a significant Ip < 0.05) 

decrease in amphetamine-induced activity. This 

decreased responsiveness was also observed when 

activity was expressed as percentage of predrug 

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FICUUK 4. Kffectsof lead administration on locomotor activity in adult male rats: (•) control; (A) 5 ppm Pb; (U) 50 ppm Pb. 

Animals were tested in groups of three, and each point represents the mean (counts/hr +S.K.) for five groups of animals. 

Data are presented for the various activity periods for the first 4 days in the maze. Points marked '"a" are significantlydifferent from control (/-test,/) < 0.05). 

levels. Whereas control. activity was elevated to 

224, 111, and (iS2% during the total, exploratory, 

and diurnal periods, respectively, the 50 ppm group 

was elevated to K3S, 323, and '.V.lS',r during these 

same periods. As with the controls, treatment 

groups showed less activity during the nocturnal 

period and no significant differences between 

groups were present during this period. 

Discussion 

Hals continuously exposed from conception to 

low levels of lead showed a delay in nervous system 

.development as seen by a delay in the righting 

reflex. I'erinatal exposure has previously been 

reported to cause delayed development (N), but at 

dietary levels which produced a fall in food consumption with a concomitant retardation of 

growth. This undernutrition, which is known to 

delay development (.9), was sufficient to produce a 

similar delay in pair-fed controls. In the present 

study, no differences in growth rates were observed 

during the period of reflex testing which suggests 

that the observed delay in development was :he 

result of a direct effect of lead on the nervous 

system of the pups. 

Daily locomotor activity was depressed during 

four consecutive days of testing in adult leadtreated animals. On the first test day, hypnactivity 

was observed during all periods ofthe daily activity 

cycle. However, on subsequent days no differences 

122 Environmenta l Health Perspective s 

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Finally, the observed attenuation in the response 

of lead-treated animals to amphetamine administration has been consistently observed both in 

mice (15) and rats (I'd). Furthermore, this 

diminished responsiveness has been observed when 

baseline activity is elevated (15), normal (13) or 

depressed (present study) by lead treatment. 

Acknowledgements 

We wish to thank Ms. Miriam Ash for her technical assistance during all phases of this work. 

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Klf'.fHK 5. Kffects of d-amphetamine administration (4.0 

mg kg) on locomotor activity of control and lead treated rats. 

Values are presented as A activity (counts/hr ± S.K.) following amphetamine; A activity - (activity on drug day) - (activity on the predrug day). Kach bar. therefore, represents 

the drug-indus-ed locomotor activity for five groups of 

animals. Bars marked "a " are significantly different from 

control (f-test.p < 0.05). 

were observed in diurnal activity and by the fourth 

day. exploratory activity (defined as the first hour 

in the maze) was also at control levels. This transient difference in activity suggests that lead treatment alters the animal's reactivity to a novel situation and that once stable activity is established, 

• •nly nocturnal activity is affected by lead treatment. Similar variations in the responsiveness of 

animals to administration of other chemical agents 

fts a function of time have been described (10). 

Tiiis finding of hypoactivity has added more fuel 

:•< the fire of confusion which consumes the lead 

literature. All possible changes in locomotor activity have been reported following lead treatment: 

hyperactivity ('l.ll), hypoactivity (12), and finally, 

r.o change in activity (l'.l). 

In the present study, animals were tested as 

adults. The possibility exists that the lead effect on 

•activity in the rat is age-dependent with initial hyperactivity in young animals (//) followed by hyj'.activity in the adult. This time course would be 

• "iisistent with the relationship of lead toxicosis 

-villi minimal brain dysfunction in children since 

•;it- hyperactivity in children generally disappears 

•'. i'h age, the so-called "maturational lag" (/'/). 

Trie serial time course of activity changes in lead 

1. David, O., et al. Lead and hyperactivity, Lancet 2: 900 

(1972). 

2. Beattie, A. D., et al. Role of chronic low-level lead exposure in the aetiology of metal retardation. Lancet 1: 589 

(1975). 

3. Pentschew, A., and Garro, F. Lead cncephalnmvc-lopathy 

of the suckling rat and its implications on the 

porphyrinopathic nervous diseases. Acta Neuropath. 

(Berlin) 6: 266 <196S>. 

4. Silbergeld, K. K., and Goldberg, A. M. A lead-induced 

behavioral disorder. Life Sci. 13: 1275 (1973). 

5. Allen, J. R, McWey. P. J., and Suomi, S. J. Pathobinlogical 

and behavioral effects of lead intoxication in the infant 

Rhesus monkey. Knviron. Health Perspect. 7: 2'19 (19741. 

6. Castellino, N., and Aloj. S. Kinetics of distribution and excretion oflead in the rat. Brit. J. Ind. Med. 21: 30S 11964 i 

7. Norton, S., Culver. B.. and Mullenix, P. Measurements of 

the effects of drugs on activity of permanent groups of rats. 

Psychopharmacol. Comm. 1:131 (1975). 

8. Michaelson, I. A., and Sauerhoff, M. VV. An improved 

model of lead-induced brain dysfunction in the suckling 

rat. Toxicol. Appl. Pharmacol. 28:88 (1974). 

9. Smart, J. L., and IXibbing, J. Vulnerability of developing 

brain. II. Kffects of early nutritional deprivation cn reflex 

ontogeny and development of behaviour in the rat. Brain 

Res. 28:"85 (1971). 

10. Reinberg, A., and Halberg, F. Circadian chronopharmacology. Ann. Rev. Pharmacol. 11: 455 (1971). 

11. Sauerhoff, M. VV., and Michaelson, I. A. Hyperactivity and 

brain catecholamines in lead-exposed developing rats. 

Science 182: 1022 (1973). 

12. McLellan, J. S., et al. Developmental toxicology of lead in 

the mouse. Fed. Proc. 33: Abstr. 479 (197-1). 

13. Sobotka, T. J., and Cook, M. P. Postnatal lead acetate exposure in rats: Possible relationship to minimal brain dysfunction. Amer. .1. Ment. U.-fic. 79: 5 (1971). 

I-I. Wender, P. H. Minimal Itrain Dysfunction in Children. 

Wiley-lntcrscii'iuc, New York, 1971 p. 75. 

15. Silbergeld, K. K., and Coldberg, A. M. I*ad-induced 

behavioral dysfunction: an animal model of hyperactivity. 

Kxp. Neurol. 12: llli (1971). 

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