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Developmental lead exposure: behavioral alterations in the short and long term
Neurotoxicology and Teratology 23 (2001) 489 – 495
Developmental lead exposure: behavioral alterations in the short and long term$ Estefaˆnia G. Moreira, Igor Vassilieff *, Vera Sı´lvia Vassilieff Center for Toxicological Assistance, University Estadual Paulista, Botucatu, Sao Paulo 18618-000, Brazil Received 11 January 2001; received in revised form 15 March 2001; accepted 15 May 2001
Abstract Wistar dams were exposed to 500 ppm of Pb, as Pb acetate, or 660 ppm Na acetate in drinking water during pregnancy and lactation. Male pups at 23 (weaned) or 70 days (adult) of age were submitted to behavioral evaluation and Pb determination. The behaviors evaluated were: locomotor activity (open-field test), motor coordination (rotarod test), exploratory behavior (holeboard test), anxiety (elevated plus maze and social interaction tests), and learning and memory (shuttle box). Pb levels were measured in the blood and cerebral regions (hippocampus and striatum) of dams and pups. The results of the present report demonstrated that exposure to Pb during pregnancy and lactation induces in weaned pups hyperactivity, decreased exploratory behavior, and impairment of learning and memory. These alterations were observed at blood Pb levels in the range that may be attained in children chronically exposed to low levels of Pb (21 ± 3 mg/dl). Regarding adults, the results demonstrated that the regimen of exposure adopted induces anxiety in these animals at nondetectable blood Pb levels. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Lead; Developmental lead exposure; Lead toxicity; Blood lead; Brain lead
1. Introduction Lead (Pb) is a nonessential toxic heavy metal widely distributed in the environment, and chronic exposure to low levels of Pb has been a matter of public health concern in many countries. Pb poisoning exerts its most severe consequences during development due to the intense cellular proliferation, differentiation, and synaptogenesis in the developing brain [6]. Moreover, the developing organism presents a fivefold greater absorption of Pb [27], lacks a functional blood – brain barrier [23], and there is some evidence that formation of Pb – protein complexes, a mechanism for sequestering Pb in mature tissues, is not functioning in fetal brain [23]. In spite of numerous publications addressing Pb-induced behavioral alterations, there are some gaps and inconsistencies in the literature. First, despite the fact that Pb poisoning has been suggested to be related to anxiety [38], this behavior has been neglected in experimental investigation.
Second, the available behavioral reports present conflicting data that render comparison of the studies difficult due to the lack of brain and/or blood Pb determinations in some of them [1,12] and to the presence of undernutrition, or gross morphological changes in others [14,25,35]. Based on the above considerations, this study was carried out in an attempt to provide a comprehensive assessment evaluating the effects of low-level developmental Pb treatment on a wide range of behavioral tasks. Dams were exposed to Pb acetate during pregnancy and lactation and pups were submitted to behavioral evaluation at 23 or 70 days of age. Behavioral assessments included measures of general activity, motor coordination, exploratory behavior, anxiety, learning, and memory. In addition, Pb levels were measured in the blood and cerebral regions (hippocampus and striatum) of dams and pups at both ages.
2. Methods $ This manuscript is part of the PhD proposal that has been developed by E.G. Moreira. * Corresponding author. Tel.: +14-6-821-3048; fax: +14-6-822-1385. E-mail addresses: [email protected] (I. Vassilieff); egmoreira@ bol.com.br (E.G. Moreira).
2.1. Animals and treatment Wistar rats were obtained from the SPF breed at the University of Campinas (UNICAMP) and used as the parent
0892-0362/01/$ – see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 8 9 2 - 0 3 6 2 ( 0 1 ) 0 0 1 5 9 - 3
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generation. The animals were mated at the age of 70 –90 days (two females and one male per cage). On pregnancy day 0 (determined by the presence of sperm in vaginal smears), the dams were divided into Na (13 dams)- or Pb (16 dams)exposed group and were housed singly. The drinking water of dams was adulterated with 500 ppm Pb, as Pb acetate, or 660 ppm Na acetate to equalize acetate exposure for the two groups. To prevent the formation of Pb precipitate, 0.5 ml of glacial acetic acid was added while stirring to prepare 1000 ml of both solutions (Na and Pb). The treatment lasted throughout pregnancy and lactation. At birth, all litters were culled to eight pups. Whenever possible, only male rats were kept within the litter and females were kept just to maintain equal litter sizes. Pups were weaned at 22 days of age on tap water. Maternal body weights, fluid, and food consumption were measured on a weekly basis throughout pregnancy and lactation. Additionally, pup weights at birth and during the first 3 weeks of age were also recorded on a weekly basis. At weaning, the dams were euthanized and blood and brain samples were taken for Pb determination. The pups were submitted to behavioral evaluation only once in their lives: at 23 or 70 days of age. Littermates were not used within a group, but they were used across groups. Immediately after the behavioral evaluation, the pups were euthanized and blood and brain samples were taken for Pb determination. Lights in the animal room were set on a 12:12-h cycle with temperature maintained at 22 ± 2C. The animals were fed with regular lab chow. 2.2. Behavioral evaluation At 23 days, Na- and Pb-exposed pups were divided into two groups. Group 1 was submitted to the elevated plus maze and open-field tests and Group 2 to the holeboard and shuttle-avoidance tasks. At 70 days, the pups were divided into four groups. Group 1 was submitted to the elevated plus maze and open-field tests; Group 2 to the holeboard and shuttle-avoidance tasks; Group 3 to the social interaction test; and Group 4 to the rotarod. The behavioral observations were blind, and carried out under low-intensity light. 2.2.1. General activity General activity was evaluated in the open-field test. Briefly, the apparatus [5] consisted of a circular surface of wood surrounded by a wooden wall 28 cm in height. The diameter of the surface used for the evaluation of weaned rats was 39 cm and, for the adults, 97 cm. The surface was painted white and divided into 19 similar parts. The apparatus was uniformly illuminated with red lights. Each animal was placed individually in the center of the arena and the following variables were recorded during a 3-min session: ambulation (count of floor units entered), rearing (count of times that the animal stood on its hind legs), grooming (time, in seconds, used for the animal to groom), and freezing (time, in seconds,
that the animal remained immobile, often in a crouching posture, with eyes wide open, and irregular respiration). 2.2.2. Motor coordination Motor coordination was evaluated on the rotarod. Rats were trained on the rotarod (Ugo Basile, Italy) at 5 rpm for 2 min, three times on the day prior to the experiment. In the test day, animals were placed on the bar at 5 rpm and the time each remained on the rotating rod was recorded. The time limit was fixed at 2 min [15]. 2.2.3. Exploratory behavior Exploratory behavior was evaluated in the holeboard. The apparatus was an age-specific open-field arena with four equally spaced holes 3 cm in diameter in the floor, as described by File and Wardill [19]. Each rat was placed individually in the center of the arena for 5 min, during which were recorded head-dip count and head-dipping duration, in seconds. A head dip was scored if both eyes disappeared into the hole. Head-dipping duration data are expressed as total duration during the 5-min session. 2.2.4. Anxiety Anxiety was evaluated in the elevated plus maze and social interaction tests. The elevated plus maze apparatus consisted of two open arms, 50 10 cm (length width) and two closed arms, 50 10 50 cm (length width height) with an open roof arranged such that the two arms of each type were opposite to each other. Weaned rats were evaluated in a similar apparatus, but the measures were 30 5 cm for the open arms and 30 5 15 cm for the closed arms. The maze was elevated from the floor. For the test, each animal was placed in the center of the maze, facing one of the closed arms and the number of entries into, and the time spent in the open and the closed arms were registered for 5 min [33]. For the social interaction test, adult rats exposed to the same regimen were housed in pairs for 7 days prior to the test. The test consisted of familiarizing each pair (cagemates) of rats with the arena for a period of 8 min on 2 consecutive days. On the third day, each rat was randomly assigned to an unfamiliar partner according to weight. Both members of a pair had been submitted to the same exposure regimen. These animals were placed in the arena to observe social interaction behavior for 5 min. Social interaction time (in seconds) per pair of rats was measured as the time spent sniffing the partner, climbing over and crawling under the partner, mutual grooming, genital investigation, and following and walking around the partner [30]. Aggressive behavior was not considered to be a social interaction behavior. 2.2.5. Learning and memory Learning and memory was evaluated in the active shuttle-avoidance task. Rats were subjected to three sessions in the shuttle box separated by a 24-h interval. In each session, rats were presented with 50 tones at randomly selected intervals of 10 –50 s. Each tone lasted 5 s, and was followed
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Table 1 Lead content in tissues from dams and their 23- and 70-day-old pups Dams
Blood (mg/dl) Hippocampus (ng/g) Striatum (ng/g)
23-day-old pups
70-day-old pups
Na
Pb
Na
Pb
Na
Pb
< 0.1 (15) 11.3 ± 6.7 (12) 22.4 ± 13.1 (12)
41.24 ± 5.98 (17) 370.4 ± 61.5* (10) 412.5 ± 70.7* (8)
< 0.1 (10) 215.0 ± 22.9 (6) 258.2 ± 42.9 (6)
21.24 ± 3.31 (9) 616.3 ± 46.7* (9) 593.7 ± 39.3* (10)
< 0.1 (14) 64.0 ± 34.2 (11) 79.1 ± 32.6 (10)
< 0.1 (12) 218.5 ± 34.2* (11) 356.3 ± 45.4* (11)
Data are expressed as mean ± S.E. The number of samples is given in parentheses. * P < .01 when compared to Na-exposed group by Student’s t test.
by a 10-s, 0.5-mA footshock. If the rat ran to the other compartment of the box during the tone, the shock scheduled for that trial was cancelled. The differences among sessions in the number of shuttle-avoidance responses were interpreted as learning/memory.
Pb concentration was determined by atomic absorption spectrophotometry with a Zeeman-corrected graphite furnace (Model Spectra AA-220Z, Varian, Australia). A standard addition method was applied for the determination to eliminate possible matrix interference.
2.3. Pb analysis
2.4. Statistical analysis
Animals were anesthetized with pentobarbital sodium (60 mg/kg ip) and blood collected from the heart. After perfusion through the ascending aorta with 0.9% saline solution, the brain was removed and dissected according to the method described by Glowinski and Iversen [22] in order to isolate the hippocampus and striatum. The tissues were maintained at 0 –4C throughout the dissection procedure. The wet weight of tissues was taken immediately after the dissection and they were stored in sealed containers at 20C until use. Blood and brain tissues were prepared for Pb analysis by a microwave dissolution procedure utilizing a DGT-100 plus microwave digestion apparatus (Provecto, Brazil). To the blood, 5 ml concentrated nitric acid and 0.5 ml hydrogen peroxide were added to the digestion vessel containing 1 ml of blood. To the brain areas, 0.5 ml concentrated nitric acid was added to 2-ml vessels containing the weighed sample. The tissue digestion procedure produced clearly digested samples (no turbidity or undigested tissue fragments). The recovery of externally added Pb to control samples was found to be consistently greater than 96%.
The litter was considered to be the experimental unit in all statistical analyses performed. Data from body weight, food and solution consumption, and shuttle-avoidance task were evaluated by profile analysis, a type of repeated multivariate ANOVA [32], which evaluates the interaction between treatment and period. The remaining data were evaluated by Student’s t test. To meet the assumptions for parametric analysis, countable data were subjected to square root transformation before analysis, but they are presented as nontransformed data. Differences between groups were considered significant if P < .05.
3. Results 3.1. Blood and brain Pb levels Table 1 shows the mean Pb levels in the blood and brain areas of dams at weaning and pups at 23 and 70 days of age. Data referring to Pb levels in brain areas were compared by
Table 2 Body weight and total fluid and food consumption of dams during pregnancy and lactation Pregnancy
Lactation
Week 1
Week 2
Week 3
Week 4
Week 5
Week 6
Body weight (g) Na (13) Pb (16)
220 ± 5 216 ± 6
244 ± 6 239 ± 6
308 ± 5.5 283 ± 10
242 ± 2 243 ± 4
256 ± 5.5 253 ± 4.5
244 ± 6 244 ± 5.5
Fluid intake (ml) Na (13) Pb (16)
222 ± 9.5 194 ± 12
267 ± 11.5 232 ± 13
347 ± 11 283 ± 19
396 ± 21.5 337 ± 16.5
542 ± 25 478 ± 18
686 ± 28 620 ± 15
Food intake (g) Na (13) Pb (16)
143 ± 2 134 ± 6.5
161 ± 12 148 ± 4
164 ± 6 156 ± 5.5
264 ± 10 232 ± 8
350 ± 8 326 ± 12
423 ± 9.5 415 ± 13
Data are expressed as mean ± S.E. The number of dams is given in parentheses. Compared to Na-exposed group, Pb-exposed group presented significantly decreased fluid consumption during the whole treatment period ( P < .05, profile analysis). Regarding body weight and food consumption, there was no statistical difference between Na- and Pb-exposed group ( P >.05, profile analysis).
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Student’s t test. Statistical analysis could not be carried out for the blood data because of the lack of variation in the Na group (all scores were < 0.1 mg/dl for blood). However, in this case, a statistical test was not necessary to determine differences between groups. In the dams, exposure to Pb resulted in higher Pb blood concentration and significant increases in Pb levels in the hippocampus [t(20) = 6.4, P < .0001] and striatum [t (18) = 6.6, P < .0001]. Pb-exposed 23-day-old pups presented increased Pb levels in the blood, hippocampus [t(13) = 6.6, P < .0001], and striatum [t (14) = 5.5, P < .0001], whereas in 70-day-old pups, Pb was not detected in the blood, but was still significantly increased in the hippocampus [t (20) = 3.2, P < .01] and striatum [t (19) = 4.9, P < .0001]. 3.2. Body weight and fluid and food consumption Profile analysis showed that the Pb regimen employed in the present report did not affect either body weight gain [ F(1,27) = 0.76, P > .05] or food consumption [ F(1,27) = 3.9, P >.05] of dams (Table 2). Profile analysis also showed that there was a period effect for both body weight gain [ F(5,23) = 123.8, P < .05] and food consumption [ F(5,23) = 258.8, P < .05], reflecting the fluctuations that occur in these measures during the pregnancy and lactation periods. Regarding fluid consumption, profile analysis demonstrated that Pb-exposed dams presented decreased fluid intake [ F(1,27) = 9.87, P < .05] compared to Na-exposed dams during the whole treatment period (Table 2). As observed for weight gain and food consumption, there was also a period effect for fluid intake [ F(5,22) = 258.8, P < .05]. Profile analysis showed that the Pb regimen employed did not influence the weight of pups either at birth, or during the first 3 weeks after birth [ F(1,27) = 0.89, P >.05]. Obviously, there was a period effect [ F(3,25) = 401.43, P < .05] reflecting the weight gain of pups with aging (Table 3).
Table 4 Effect of the exposure to Pb during pregnancy and lactation on the behavior of weaned rats (23 days old) in the open-field, holeboard, and elevated plus maze tests Na
Pb
Open-field Ambulation (count) Rearing (count) Grooming (s) Freezing (s)
(n = 7) 81.3 ± 6.1 30.3 ± 2.7 5.1 ± 1.4 0.4 ± 0.1
(n = 7) 100.4 ± 5.2* 30.1 ± 3.5 3.8 ± 1.1 0.4 ± 0.3
Holeboard Head dip (count) Head dipping (s)
(n = 7) 10.7 ± 1.1 19.5 ± 2.6
(n = 7) 6.9 ± 1.4* 10.6 ± 2.7*
Elevated plus maze Percentage of open arm entries Percentage of time spent on open arms
(n = 8) 29.8 ± 2.4 12.5 ± 2.3
(n = 8) 31.0 ± 2.7 15.4 ± 2.9
Data are expressed as mean ± S.E. The number of pups is given in parentheses. All the data represent the total count or duration by session. * P < .05, Student’s t test.
In the shuttle box, the profile analysis showed that pups exposed to Na and Pb presented different profiles of
3.3. Behavioral evaluation of 23-day-old pups Student’s t test demonstrated that exposure to Pb during pregnancy and lactation increased ambulation [t (12) = 2.4, P < .05] in the open-field test; decreased head dip [t(12) = 2.2, P < .05] and head dipping [t (12) = 2.4, P < .05] in the holeboard test, and did not influence the behavior in the elevated plus maze test (Table 4). Table 3 Body weight of pups at birth and during the first 3 weeks after birth
Na (n = 13) Pb (n = 16)
Birth
Week 1
Week 2
Week 3
6.2 ± 0.1 5.8 ± 0.5
14.2 ± 0.5 13.4 ± 0.5
23.9 ± 0.8 23.3 ± 0.7
34.5 ± 1.1 33.2 ± 1.3
Data are expressed as mean ± S.E. The number of samples is given in parentheses. There was no difference between Na- and Pb-exposed pups ( P >.05, profile analysis).
Fig. 1. Effect of the exposure to Pb during pregnancy and lactation on the behavior of (A) weaned (23 days old) and (B) adult rats (70 days old) in the shuttle box. Data are expressed as mean ± S.E. of seven to eight animals in each group. * P < .05 when compared to day 1 of the same treatment and # P < .05 when compared to day 3 of Na-exposed group; profile analysis.
E.G. Moreira et al. / Neurotoxicology and Teratology 23 (2001) 489–495 Table 5 Effect of the exposure to Pb during pregnancy and lactation on the behavior of adult rats (70 days old) in the open-field, holeboard, and elevated plus maze tests Na
Pb
Open-field Ambulation (count) Rearing (count) Grooming (s) Freezing (s)
(n = 9) 82.6 ± 4.6 14.2 ± 1.6 0.34 ± 0.2 0.2 ± 0.2
(n = 7) 89.6 ± 6.7 13.4 ± 1.7 0.21 ± 0.16 0.3 ± 0.3
Holeboard Head dip (count) Head dipping (s)
(n = 10) 8.2 ± 0.5 10.0 ± 1.0
(n = 7) 10.6 ± 1.1* 11.7 ± 1.1
Elevated plus maze Percentage of open arm entries Percentage of time spent on open arms
(n = 8) 34.6 ± 2.3 41.3 ± 5.7
(n = 7) 29.4 ± 2.8 28.4 ± 3.1**
Data are expressed as mean ± S.E. The number of pups is given in parentheses. All the data represent the total count or duration by session. * P < .05, Student’s t test. ** P < .09, Student’s t test.
response [ F(2,12) = 4.78, P < .05]. Na-exposed pups presented a significant increase in the number of shuttleavoidance responses on the third day of the test when compared to the first day [ F(2,6) = 9.5, P < .05]. In the Pb-exposed group, such a difference was not detected [ F(2,5) = 3.1, P >.05] (Fig. 1A). 3.4. Behavioral evaluation of 70-day-old pups Student’s t test demonstrated that exposure to Pb during pregnancy and lactation increased head dip in the holeboard test [t(15) = 2.2, P < .05] and did not influence the behavior of adult pups in the open-field and elevated plus maze tests (Table 5). However, in the elevated plus maze, a statistical tendency ( P < .09) was observed. Compared with the control group, the percentage of time spent in the open arms tended to be decreased in the Pb-exposed group. In the social interaction test, Pb-exposed pups showed decreased
Fig. 2. Effect of the exposure to Pb during pregnancy and lactation on the behavior of adult rats (70 days) in the social interaction test. Data are expressed as mean ± S.E. of five pairs of rats in each group. * P < .05; Student’s t test.
493
social interaction time [t (8) = 2.4, P < .05] (Fig. 2) and, in the rotarod, there was no statistical difference between the two groups, i.e., pups from both groups were able to remain on the rotating bar for 2 min (data not shown). In the shuttle box, the profile analysis showed that pups exposed to both Na and Pb presented the same profile of response [ F(1,13) = 1.35, P >.05]. Na- and Pb-exposed groups presented a significant increase in the number of shuttle-avoidance responses on the third day of the test [ F(2,5) = 17.97, P < .05 and F(2,5) = 15.25, P < .05, respectively] (Fig. 1B).
4. Discussion This study was carried out to provide a comprehensive assessment of the effects of low-level Pb exposure only during pregnancy and lactation on the behavior of weaned and adult pups submitted to a wide range of tasks. The regimen of Pb exposure employed in the present report caused no effect on the weight of pups at birth or on the weight gain of pups and dams, and therefore, undernutrition, a common confounding variable in teratology studies [26,29], could be ruled out as a causative agent for the behavioral changes observed. The mean blood Pb levels in dams, 23- and 70-day-old pups were, respectively, 40 ± 6, 21 ± 3, and lower than 0.1 mg/dl. The 23-day-old pups’ result is within the range of the lowest Pb blood level at which behavioral abnormalities are detectable in rodents (15 – 20 mg/dl) and children (10 – 15 mg/dl), as estimated by Davis et al. [9]. Regarding brain Pb levels, in this work, we analyzed Pb content in hippocampus and striatum, two brain regions that have been widely related to behavioral and cognitive manifestations [11,18,20,28]. Since it is known that the differential sensitivity to Pb neurotoxicity in the different brain regions is not due to a preferential Pb accumulation, but is possibly due to alteration of biochemical or cellular processes that are uniquely associated with, or greatly enhanced in a particular region [39], the importance of our result is to provide quantitative data in order to make possible comparisons among different studies. In weaned rats, Pb treatment induced increased ambulation in the open field, decreased head dips and head dipping in the holeboard, and decreased shuttle-avoidance response in the shuttle box. These results respectively suggest hyperactivity [3], decreased exploratory behavior [19], and impairment in learning and memory. Exploration is a very important behavior by which an animal gains information about its environment and it is an essential, life-preserving component of an animal’s higher nervous functions [37]. The alterations of cognitive functions observed here (i.e., exploration and learning and memory) seem to be in agreement with both animal and children literature, which generally argue for an association between low levels of Pb exposure and cognitive dysfunction. However, an interpre-
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tation that Pb exposure selectively affected cognitive function is only valid after it has been demonstrated that such impairments were not the result of nonspecific stimulation [8]. Based on this consideration, the impairment of exploration and learning and memory observed in the present report may be attributed to the increased locomotor activity presented by these animals, since this activation can impair focusing on specific targets, as the holes to be explored in the holeboard and the stimuli used for conditioning. Regarding the lack of Pb-exposure effect on anxiety evaluated in the elevated plus maze, we would like to point out that this result does not necessarily mean that weaned rats do not have an alteration in their anxiety state. This is explained because since the validation of elevated plus maze test to measure anxiety in rodents is based on the conflict between exploration and aversion to elevated open places [16], the decrease of exploratory behavior by Pb treatment compromised this prerogative. Moreover, it has been accepted that the evaluation of anxiety in the elevated plus maze is also compromised when there is an increase of locomotor activity [10]. In adult rats, no behavioral differences were observed in the open-field, rotarod or shuttle-avoidance tasks, suggesting no Pb-induced alteration in the general activity, motor coordination, and learning and memory. In the holeboard test, Pb exposure induced an increase in head dip and did not alter head dipping, which is considered to be a more accurate indicator of exploratory behavior [19]. Therefore, we believe that the increased head dip may be a casual result that reached statistical significance. In the elevated plus maze test, the percentage of time spent in the open arms tended to be decreased in the Pb-exposed group, indicating a possible anxiogenic effect. In the social interaction test, which has also been validated to measure anxiety in adult rats [17], Pb-exposed pups presented decreased social interaction time demonstrating that such exposure induces, in fact, anxiety in adult rats. We would like to point out that this study is the first to report lasting effects of early Pb exposure on anxiety. Notably, the behavioral effects were observed in animals that did not exhibit signs of overt toxicity during or after the period of Pb exposure and that had blood Pb levels that were not detectable (i.e., lower than 0.1 mg/dl). This finding provides evidence of a causal relationship between early asymptomatic Pb exposure and anxiety later in life. Locomotor activity and learning and memory have been the most investigated behaviors in Pb exposure. The exposure regimen and behavioral protocols are very variable and have led to different results in the literature [2,4,6,13,24,34,41]. However, there seems to be a general agreement that exposure to Pb may lead to hyperactivity and deficits in learning and memory both in experimental animals and humans [2,7,9,28,31,36]. Our results for 23-day-old pups are in accordance with the general literature, while those of 70 days are not. Most of the studies that have evaluated locomotor activity in adult rats detected an increase in this
behavior [28,31,36,40]. However, in these studies, the animals were weaned on the same solution that their dams had received and this exposure lasted until the behavioral evaluation. In this way, it seems likely that the detection of hyperactivity in adult rats depends on the duration of the exposure. The cessation of the exposure at weaning with the consequent decrease of Pb levels in the blood and brain may lead to functional compensation that may be masking possible hyperactivity in adulthood. Observing the differences in the behavioral effects detected in weaned and adult rats, one could infer why they could have happened. Two factors have already been discussed: the inadequacy of the elevated plus maze to evaluate the anxiety in rats with decreased exploratory behavior and increased locomotor activity and also the possibility of functional compensation in the adult brain after a decrease in the Pb level. Besides these factors, it should be taken into consideration that the 23-day-old brain has not yet reached maturity. It is known that the development of most neurotransmitter systems to adult transmitter and receptor levels takes 4 weeks after birth to be completed in the rat [21,42]. In summary, the results of the present report demonstrated that exposure to Pb during pregnancy and lactation induces hyperactivity, decreased exploratory behavior, and impairment of learning and memory in weaned rats, and anxiety in adult rats. These alterations were observed in weaned rats, at blood Pb levels in the range that may be attained in children chronically exposed to low levels of Pb and also in adult rats, at nondetectable blood Pb levels.
Acknowledgments We thank Mr. Eric Jose Cardoso do Nascimento for his technical assistance. This work was supported by FAPESP (98/15373-5 and 99/09410-8).
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Developmental lead exposure: behavioral alterations in the short and long term$ Estefaˆnia G. Moreira, Igor Vassilieff *, Vera Sı´lvia Vassilieff Center for Toxicological Assistance, University Estadual Paulista, Botucatu, Sao Paulo 18618-000, Brazil Received 11 January 2001; received in revised form 15 March 2001; accepted 15 May 2001
Abstract Wistar dams were exposed to 500 ppm of Pb, as Pb acetate, or 660 ppm Na acetate in drinking water during pregnancy and lactation. Male pups at 23 (weaned) or 70 days (adult) of age were submitted to behavioral evaluation and Pb determination. The behaviors evaluated were: locomotor activity (open-field test), motor coordination (rotarod test), exploratory behavior (holeboard test), anxiety (elevated plus maze and social interaction tests), and learning and memory (shuttle box). Pb levels were measured in the blood and cerebral regions (hippocampus and striatum) of dams and pups. The results of the present report demonstrated that exposure to Pb during pregnancy and lactation induces in weaned pups hyperactivity, decreased exploratory behavior, and impairment of learning and memory. These alterations were observed at blood Pb levels in the range that may be attained in children chronically exposed to low levels of Pb (21 ± 3 mg/dl). Regarding adults, the results demonstrated that the regimen of exposure adopted induces anxiety in these animals at nondetectable blood Pb levels. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Lead; Developmental lead exposure; Lead toxicity; Blood lead; Brain lead
1. Introduction Lead (Pb) is a nonessential toxic heavy metal widely distributed in the environment, and chronic exposure to low levels of Pb has been a matter of public health concern in many countries. Pb poisoning exerts its most severe consequences during development due to the intense cellular proliferation, differentiation, and synaptogenesis in the developing brain [6]. Moreover, the developing organism presents a fivefold greater absorption of Pb [27], lacks a functional blood – brain barrier [23], and there is some evidence that formation of Pb – protein complexes, a mechanism for sequestering Pb in mature tissues, is not functioning in fetal brain [23]. In spite of numerous publications addressing Pb-induced behavioral alterations, there are some gaps and inconsistencies in the literature. First, despite the fact that Pb poisoning has been suggested to be related to anxiety [38], this behavior has been neglected in experimental investigation.
Second, the available behavioral reports present conflicting data that render comparison of the studies difficult due to the lack of brain and/or blood Pb determinations in some of them [1,12] and to the presence of undernutrition, or gross morphological changes in others [14,25,35]. Based on the above considerations, this study was carried out in an attempt to provide a comprehensive assessment evaluating the effects of low-level developmental Pb treatment on a wide range of behavioral tasks. Dams were exposed to Pb acetate during pregnancy and lactation and pups were submitted to behavioral evaluation at 23 or 70 days of age. Behavioral assessments included measures of general activity, motor coordination, exploratory behavior, anxiety, learning, and memory. In addition, Pb levels were measured in the blood and cerebral regions (hippocampus and striatum) of dams and pups at both ages.
2. Methods $ This manuscript is part of the PhD proposal that has been developed by E.G. Moreira. * Corresponding author. Tel.: +14-6-821-3048; fax: +14-6-822-1385. E-mail addresses: [email protected] (I. Vassilieff); egmoreira@ bol.com.br (E.G. Moreira).
2.1. Animals and treatment Wistar rats were obtained from the SPF breed at the University of Campinas (UNICAMP) and used as the parent
0892-0362/01/$ – see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 8 9 2 - 0 3 6 2 ( 0 1 ) 0 0 1 5 9 - 3
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generation. The animals were mated at the age of 70 –90 days (two females and one male per cage). On pregnancy day 0 (determined by the presence of sperm in vaginal smears), the dams were divided into Na (13 dams)- or Pb (16 dams)exposed group and were housed singly. The drinking water of dams was adulterated with 500 ppm Pb, as Pb acetate, or 660 ppm Na acetate to equalize acetate exposure for the two groups. To prevent the formation of Pb precipitate, 0.5 ml of glacial acetic acid was added while stirring to prepare 1000 ml of both solutions (Na and Pb). The treatment lasted throughout pregnancy and lactation. At birth, all litters were culled to eight pups. Whenever possible, only male rats were kept within the litter and females were kept just to maintain equal litter sizes. Pups were weaned at 22 days of age on tap water. Maternal body weights, fluid, and food consumption were measured on a weekly basis throughout pregnancy and lactation. Additionally, pup weights at birth and during the first 3 weeks of age were also recorded on a weekly basis. At weaning, the dams were euthanized and blood and brain samples were taken for Pb determination. The pups were submitted to behavioral evaluation only once in their lives: at 23 or 70 days of age. Littermates were not used within a group, but they were used across groups. Immediately after the behavioral evaluation, the pups were euthanized and blood and brain samples were taken for Pb determination. Lights in the animal room were set on a 12:12-h cycle with temperature maintained at 22 ± 2C. The animals were fed with regular lab chow. 2.2. Behavioral evaluation At 23 days, Na- and Pb-exposed pups were divided into two groups. Group 1 was submitted to the elevated plus maze and open-field tests and Group 2 to the holeboard and shuttle-avoidance tasks. At 70 days, the pups were divided into four groups. Group 1 was submitted to the elevated plus maze and open-field tests; Group 2 to the holeboard and shuttle-avoidance tasks; Group 3 to the social interaction test; and Group 4 to the rotarod. The behavioral observations were blind, and carried out under low-intensity light. 2.2.1. General activity General activity was evaluated in the open-field test. Briefly, the apparatus [5] consisted of a circular surface of wood surrounded by a wooden wall 28 cm in height. The diameter of the surface used for the evaluation of weaned rats was 39 cm and, for the adults, 97 cm. The surface was painted white and divided into 19 similar parts. The apparatus was uniformly illuminated with red lights. Each animal was placed individually in the center of the arena and the following variables were recorded during a 3-min session: ambulation (count of floor units entered), rearing (count of times that the animal stood on its hind legs), grooming (time, in seconds, used for the animal to groom), and freezing (time, in seconds,
that the animal remained immobile, often in a crouching posture, with eyes wide open, and irregular respiration). 2.2.2. Motor coordination Motor coordination was evaluated on the rotarod. Rats were trained on the rotarod (Ugo Basile, Italy) at 5 rpm for 2 min, three times on the day prior to the experiment. In the test day, animals were placed on the bar at 5 rpm and the time each remained on the rotating rod was recorded. The time limit was fixed at 2 min [15]. 2.2.3. Exploratory behavior Exploratory behavior was evaluated in the holeboard. The apparatus was an age-specific open-field arena with four equally spaced holes 3 cm in diameter in the floor, as described by File and Wardill [19]. Each rat was placed individually in the center of the arena for 5 min, during which were recorded head-dip count and head-dipping duration, in seconds. A head dip was scored if both eyes disappeared into the hole. Head-dipping duration data are expressed as total duration during the 5-min session. 2.2.4. Anxiety Anxiety was evaluated in the elevated plus maze and social interaction tests. The elevated plus maze apparatus consisted of two open arms, 50 10 cm (length width) and two closed arms, 50 10 50 cm (length width height) with an open roof arranged such that the two arms of each type were opposite to each other. Weaned rats were evaluated in a similar apparatus, but the measures were 30 5 cm for the open arms and 30 5 15 cm for the closed arms. The maze was elevated from the floor. For the test, each animal was placed in the center of the maze, facing one of the closed arms and the number of entries into, and the time spent in the open and the closed arms were registered for 5 min [33]. For the social interaction test, adult rats exposed to the same regimen were housed in pairs for 7 days prior to the test. The test consisted of familiarizing each pair (cagemates) of rats with the arena for a period of 8 min on 2 consecutive days. On the third day, each rat was randomly assigned to an unfamiliar partner according to weight. Both members of a pair had been submitted to the same exposure regimen. These animals were placed in the arena to observe social interaction behavior for 5 min. Social interaction time (in seconds) per pair of rats was measured as the time spent sniffing the partner, climbing over and crawling under the partner, mutual grooming, genital investigation, and following and walking around the partner [30]. Aggressive behavior was not considered to be a social interaction behavior. 2.2.5. Learning and memory Learning and memory was evaluated in the active shuttle-avoidance task. Rats were subjected to three sessions in the shuttle box separated by a 24-h interval. In each session, rats were presented with 50 tones at randomly selected intervals of 10 –50 s. Each tone lasted 5 s, and was followed
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Table 1 Lead content in tissues from dams and their 23- and 70-day-old pups Dams
Blood (mg/dl) Hippocampus (ng/g) Striatum (ng/g)
23-day-old pups
70-day-old pups
Na
Pb
Na
Pb
Na
Pb
< 0.1 (15) 11.3 ± 6.7 (12) 22.4 ± 13.1 (12)
41.24 ± 5.98 (17) 370.4 ± 61.5* (10) 412.5 ± 70.7* (8)
< 0.1 (10) 215.0 ± 22.9 (6) 258.2 ± 42.9 (6)
21.24 ± 3.31 (9) 616.3 ± 46.7* (9) 593.7 ± 39.3* (10)
< 0.1 (14) 64.0 ± 34.2 (11) 79.1 ± 32.6 (10)
< 0.1 (12) 218.5 ± 34.2* (11) 356.3 ± 45.4* (11)
Data are expressed as mean ± S.E. The number of samples is given in parentheses. * P < .01 when compared to Na-exposed group by Student’s t test.
by a 10-s, 0.5-mA footshock. If the rat ran to the other compartment of the box during the tone, the shock scheduled for that trial was cancelled. The differences among sessions in the number of shuttle-avoidance responses were interpreted as learning/memory.
Pb concentration was determined by atomic absorption spectrophotometry with a Zeeman-corrected graphite furnace (Model Spectra AA-220Z, Varian, Australia). A standard addition method was applied for the determination to eliminate possible matrix interference.
2.3. Pb analysis
2.4. Statistical analysis
Animals were anesthetized with pentobarbital sodium (60 mg/kg ip) and blood collected from the heart. After perfusion through the ascending aorta with 0.9% saline solution, the brain was removed and dissected according to the method described by Glowinski and Iversen [22] in order to isolate the hippocampus and striatum. The tissues were maintained at 0 –4C throughout the dissection procedure. The wet weight of tissues was taken immediately after the dissection and they were stored in sealed containers at 20C until use. Blood and brain tissues were prepared for Pb analysis by a microwave dissolution procedure utilizing a DGT-100 plus microwave digestion apparatus (Provecto, Brazil). To the blood, 5 ml concentrated nitric acid and 0.5 ml hydrogen peroxide were added to the digestion vessel containing 1 ml of blood. To the brain areas, 0.5 ml concentrated nitric acid was added to 2-ml vessels containing the weighed sample. The tissue digestion procedure produced clearly digested samples (no turbidity or undigested tissue fragments). The recovery of externally added Pb to control samples was found to be consistently greater than 96%.
The litter was considered to be the experimental unit in all statistical analyses performed. Data from body weight, food and solution consumption, and shuttle-avoidance task were evaluated by profile analysis, a type of repeated multivariate ANOVA [32], which evaluates the interaction between treatment and period. The remaining data were evaluated by Student’s t test. To meet the assumptions for parametric analysis, countable data were subjected to square root transformation before analysis, but they are presented as nontransformed data. Differences between groups were considered significant if P < .05.
3. Results 3.1. Blood and brain Pb levels Table 1 shows the mean Pb levels in the blood and brain areas of dams at weaning and pups at 23 and 70 days of age. Data referring to Pb levels in brain areas were compared by
Table 2 Body weight and total fluid and food consumption of dams during pregnancy and lactation Pregnancy
Lactation
Week 1
Week 2
Week 3
Week 4
Week 5
Week 6
Body weight (g) Na (13) Pb (16)
220 ± 5 216 ± 6
244 ± 6 239 ± 6
308 ± 5.5 283 ± 10
242 ± 2 243 ± 4
256 ± 5.5 253 ± 4.5
244 ± 6 244 ± 5.5
Fluid intake (ml) Na (13) Pb (16)
222 ± 9.5 194 ± 12
267 ± 11.5 232 ± 13
347 ± 11 283 ± 19
396 ± 21.5 337 ± 16.5
542 ± 25 478 ± 18
686 ± 28 620 ± 15
Food intake (g) Na (13) Pb (16)
143 ± 2 134 ± 6.5
161 ± 12 148 ± 4
164 ± 6 156 ± 5.5
264 ± 10 232 ± 8
350 ± 8 326 ± 12
423 ± 9.5 415 ± 13
Data are expressed as mean ± S.E. The number of dams is given in parentheses. Compared to Na-exposed group, Pb-exposed group presented significantly decreased fluid consumption during the whole treatment period ( P < .05, profile analysis). Regarding body weight and food consumption, there was no statistical difference between Na- and Pb-exposed group ( P >.05, profile analysis).
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Student’s t test. Statistical analysis could not be carried out for the blood data because of the lack of variation in the Na group (all scores were < 0.1 mg/dl for blood). However, in this case, a statistical test was not necessary to determine differences between groups. In the dams, exposure to Pb resulted in higher Pb blood concentration and significant increases in Pb levels in the hippocampus [t(20) = 6.4, P < .0001] and striatum [t (18) = 6.6, P < .0001]. Pb-exposed 23-day-old pups presented increased Pb levels in the blood, hippocampus [t(13) = 6.6, P < .0001], and striatum [t (14) = 5.5, P < .0001], whereas in 70-day-old pups, Pb was not detected in the blood, but was still significantly increased in the hippocampus [t (20) = 3.2, P < .01] and striatum [t (19) = 4.9, P < .0001]. 3.2. Body weight and fluid and food consumption Profile analysis showed that the Pb regimen employed in the present report did not affect either body weight gain [ F(1,27) = 0.76, P > .05] or food consumption [ F(1,27) = 3.9, P >.05] of dams (Table 2). Profile analysis also showed that there was a period effect for both body weight gain [ F(5,23) = 123.8, P < .05] and food consumption [ F(5,23) = 258.8, P < .05], reflecting the fluctuations that occur in these measures during the pregnancy and lactation periods. Regarding fluid consumption, profile analysis demonstrated that Pb-exposed dams presented decreased fluid intake [ F(1,27) = 9.87, P < .05] compared to Na-exposed dams during the whole treatment period (Table 2). As observed for weight gain and food consumption, there was also a period effect for fluid intake [ F(5,22) = 258.8, P < .05]. Profile analysis showed that the Pb regimen employed did not influence the weight of pups either at birth, or during the first 3 weeks after birth [ F(1,27) = 0.89, P >.05]. Obviously, there was a period effect [ F(3,25) = 401.43, P < .05] reflecting the weight gain of pups with aging (Table 3).
Table 4 Effect of the exposure to Pb during pregnancy and lactation on the behavior of weaned rats (23 days old) in the open-field, holeboard, and elevated plus maze tests Na
Pb
Open-field Ambulation (count) Rearing (count) Grooming (s) Freezing (s)
(n = 7) 81.3 ± 6.1 30.3 ± 2.7 5.1 ± 1.4 0.4 ± 0.1
(n = 7) 100.4 ± 5.2* 30.1 ± 3.5 3.8 ± 1.1 0.4 ± 0.3
Holeboard Head dip (count) Head dipping (s)
(n = 7) 10.7 ± 1.1 19.5 ± 2.6
(n = 7) 6.9 ± 1.4* 10.6 ± 2.7*
Elevated plus maze Percentage of open arm entries Percentage of time spent on open arms
(n = 8) 29.8 ± 2.4 12.5 ± 2.3
(n = 8) 31.0 ± 2.7 15.4 ± 2.9
Data are expressed as mean ± S.E. The number of pups is given in parentheses. All the data represent the total count or duration by session. * P < .05, Student’s t test.
In the shuttle box, the profile analysis showed that pups exposed to Na and Pb presented different profiles of
3.3. Behavioral evaluation of 23-day-old pups Student’s t test demonstrated that exposure to Pb during pregnancy and lactation increased ambulation [t (12) = 2.4, P < .05] in the open-field test; decreased head dip [t(12) = 2.2, P < .05] and head dipping [t (12) = 2.4, P < .05] in the holeboard test, and did not influence the behavior in the elevated plus maze test (Table 4). Table 3 Body weight of pups at birth and during the first 3 weeks after birth
Na (n = 13) Pb (n = 16)
Birth
Week 1
Week 2
Week 3
6.2 ± 0.1 5.8 ± 0.5
14.2 ± 0.5 13.4 ± 0.5
23.9 ± 0.8 23.3 ± 0.7
34.5 ± 1.1 33.2 ± 1.3
Data are expressed as mean ± S.E. The number of samples is given in parentheses. There was no difference between Na- and Pb-exposed pups ( P >.05, profile analysis).
Fig. 1. Effect of the exposure to Pb during pregnancy and lactation on the behavior of (A) weaned (23 days old) and (B) adult rats (70 days old) in the shuttle box. Data are expressed as mean ± S.E. of seven to eight animals in each group. * P < .05 when compared to day 1 of the same treatment and # P < .05 when compared to day 3 of Na-exposed group; profile analysis.
E.G. Moreira et al. / Neurotoxicology and Teratology 23 (2001) 489–495 Table 5 Effect of the exposure to Pb during pregnancy and lactation on the behavior of adult rats (70 days old) in the open-field, holeboard, and elevated plus maze tests Na
Pb
Open-field Ambulation (count) Rearing (count) Grooming (s) Freezing (s)
(n = 9) 82.6 ± 4.6 14.2 ± 1.6 0.34 ± 0.2 0.2 ± 0.2
(n = 7) 89.6 ± 6.7 13.4 ± 1.7 0.21 ± 0.16 0.3 ± 0.3
Holeboard Head dip (count) Head dipping (s)
(n = 10) 8.2 ± 0.5 10.0 ± 1.0
(n = 7) 10.6 ± 1.1* 11.7 ± 1.1
Elevated plus maze Percentage of open arm entries Percentage of time spent on open arms
(n = 8) 34.6 ± 2.3 41.3 ± 5.7
(n = 7) 29.4 ± 2.8 28.4 ± 3.1**
Data are expressed as mean ± S.E. The number of pups is given in parentheses. All the data represent the total count or duration by session. * P < .05, Student’s t test. ** P < .09, Student’s t test.
response [ F(2,12) = 4.78, P < .05]. Na-exposed pups presented a significant increase in the number of shuttleavoidance responses on the third day of the test when compared to the first day [ F(2,6) = 9.5, P < .05]. In the Pb-exposed group, such a difference was not detected [ F(2,5) = 3.1, P >.05] (Fig. 1A). 3.4. Behavioral evaluation of 70-day-old pups Student’s t test demonstrated that exposure to Pb during pregnancy and lactation increased head dip in the holeboard test [t(15) = 2.2, P < .05] and did not influence the behavior of adult pups in the open-field and elevated plus maze tests (Table 5). However, in the elevated plus maze, a statistical tendency ( P < .09) was observed. Compared with the control group, the percentage of time spent in the open arms tended to be decreased in the Pb-exposed group. In the social interaction test, Pb-exposed pups showed decreased
Fig. 2. Effect of the exposure to Pb during pregnancy and lactation on the behavior of adult rats (70 days) in the social interaction test. Data are expressed as mean ± S.E. of five pairs of rats in each group. * P < .05; Student’s t test.
493
social interaction time [t (8) = 2.4, P < .05] (Fig. 2) and, in the rotarod, there was no statistical difference between the two groups, i.e., pups from both groups were able to remain on the rotating bar for 2 min (data not shown). In the shuttle box, the profile analysis showed that pups exposed to both Na and Pb presented the same profile of response [ F(1,13) = 1.35, P >.05]. Na- and Pb-exposed groups presented a significant increase in the number of shuttle-avoidance responses on the third day of the test [ F(2,5) = 17.97, P < .05 and F(2,5) = 15.25, P < .05, respectively] (Fig. 1B).
4. Discussion This study was carried out to provide a comprehensive assessment of the effects of low-level Pb exposure only during pregnancy and lactation on the behavior of weaned and adult pups submitted to a wide range of tasks. The regimen of Pb exposure employed in the present report caused no effect on the weight of pups at birth or on the weight gain of pups and dams, and therefore, undernutrition, a common confounding variable in teratology studies [26,29], could be ruled out as a causative agent for the behavioral changes observed. The mean blood Pb levels in dams, 23- and 70-day-old pups were, respectively, 40 ± 6, 21 ± 3, and lower than 0.1 mg/dl. The 23-day-old pups’ result is within the range of the lowest Pb blood level at which behavioral abnormalities are detectable in rodents (15 – 20 mg/dl) and children (10 – 15 mg/dl), as estimated by Davis et al. [9]. Regarding brain Pb levels, in this work, we analyzed Pb content in hippocampus and striatum, two brain regions that have been widely related to behavioral and cognitive manifestations [11,18,20,28]. Since it is known that the differential sensitivity to Pb neurotoxicity in the different brain regions is not due to a preferential Pb accumulation, but is possibly due to alteration of biochemical or cellular processes that are uniquely associated with, or greatly enhanced in a particular region [39], the importance of our result is to provide quantitative data in order to make possible comparisons among different studies. In weaned rats, Pb treatment induced increased ambulation in the open field, decreased head dips and head dipping in the holeboard, and decreased shuttle-avoidance response in the shuttle box. These results respectively suggest hyperactivity [3], decreased exploratory behavior [19], and impairment in learning and memory. Exploration is a very important behavior by which an animal gains information about its environment and it is an essential, life-preserving component of an animal’s higher nervous functions [37]. The alterations of cognitive functions observed here (i.e., exploration and learning and memory) seem to be in agreement with both animal and children literature, which generally argue for an association between low levels of Pb exposure and cognitive dysfunction. However, an interpre-
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tation that Pb exposure selectively affected cognitive function is only valid after it has been demonstrated that such impairments were not the result of nonspecific stimulation [8]. Based on this consideration, the impairment of exploration and learning and memory observed in the present report may be attributed to the increased locomotor activity presented by these animals, since this activation can impair focusing on specific targets, as the holes to be explored in the holeboard and the stimuli used for conditioning. Regarding the lack of Pb-exposure effect on anxiety evaluated in the elevated plus maze, we would like to point out that this result does not necessarily mean that weaned rats do not have an alteration in their anxiety state. This is explained because since the validation of elevated plus maze test to measure anxiety in rodents is based on the conflict between exploration and aversion to elevated open places [16], the decrease of exploratory behavior by Pb treatment compromised this prerogative. Moreover, it has been accepted that the evaluation of anxiety in the elevated plus maze is also compromised when there is an increase of locomotor activity [10]. In adult rats, no behavioral differences were observed in the open-field, rotarod or shuttle-avoidance tasks, suggesting no Pb-induced alteration in the general activity, motor coordination, and learning and memory. In the holeboard test, Pb exposure induced an increase in head dip and did not alter head dipping, which is considered to be a more accurate indicator of exploratory behavior [19]. Therefore, we believe that the increased head dip may be a casual result that reached statistical significance. In the elevated plus maze test, the percentage of time spent in the open arms tended to be decreased in the Pb-exposed group, indicating a possible anxiogenic effect. In the social interaction test, which has also been validated to measure anxiety in adult rats [17], Pb-exposed pups presented decreased social interaction time demonstrating that such exposure induces, in fact, anxiety in adult rats. We would like to point out that this study is the first to report lasting effects of early Pb exposure on anxiety. Notably, the behavioral effects were observed in animals that did not exhibit signs of overt toxicity during or after the period of Pb exposure and that had blood Pb levels that were not detectable (i.e., lower than 0.1 mg/dl). This finding provides evidence of a causal relationship between early asymptomatic Pb exposure and anxiety later in life. Locomotor activity and learning and memory have been the most investigated behaviors in Pb exposure. The exposure regimen and behavioral protocols are very variable and have led to different results in the literature [2,4,6,13,24,34,41]. However, there seems to be a general agreement that exposure to Pb may lead to hyperactivity and deficits in learning and memory both in experimental animals and humans [2,7,9,28,31,36]. Our results for 23-day-old pups are in accordance with the general literature, while those of 70 days are not. Most of the studies that have evaluated locomotor activity in adult rats detected an increase in this
behavior [28,31,36,40]. However, in these studies, the animals were weaned on the same solution that their dams had received and this exposure lasted until the behavioral evaluation. In this way, it seems likely that the detection of hyperactivity in adult rats depends on the duration of the exposure. The cessation of the exposure at weaning with the consequent decrease of Pb levels in the blood and brain may lead to functional compensation that may be masking possible hyperactivity in adulthood. Observing the differences in the behavioral effects detected in weaned and adult rats, one could infer why they could have happened. Two factors have already been discussed: the inadequacy of the elevated plus maze to evaluate the anxiety in rats with decreased exploratory behavior and increased locomotor activity and also the possibility of functional compensation in the adult brain after a decrease in the Pb level. Besides these factors, it should be taken into consideration that the 23-day-old brain has not yet reached maturity. It is known that the development of most neurotransmitter systems to adult transmitter and receptor levels takes 4 weeks after birth to be completed in the rat [21,42]. In summary, the results of the present report demonstrated that exposure to Pb during pregnancy and lactation induces hyperactivity, decreased exploratory behavior, and impairment of learning and memory in weaned rats, and anxiety in adult rats. These alterations were observed in weaned rats, at blood Pb levels in the range that may be attained in children chronically exposed to low levels of Pb and also in adult rats, at nondetectable blood Pb levels.
Acknowledgments We thank Mr. Eric Jose Cardoso do Nascimento for his technical assistance. This work was supported by FAPESP (98/15373-5 and 99/09410-8).
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