Concurrent exposure to aluminum and stress during pregnancy in rats: Effects on postnatal development and behavior of the offspring

Neurotoxicology and Teratology 27 (2005) 565 – 574 www.elsevier.com/locate/neutera

Concurrent exposure to aluminum and stress during pregnancy in rats: Effects on postnatal development and behavior of the offspring M. Teresa Colomina a,b, Jose L. Roig a, Margarita Torrente a,b, Paloma Vicens a,b, Jose L. Domingo b,* b

a Department of Psychology, Psychobiology Unit, ‘‘Rovira i Virgili’’ University, Sescelades Campus, 43007 Tarragona, Spain Laboratory of Toxicology and Environmental Health, School of Medicine, ‘‘Rovira i Virgili’’ University, San Lorenzo 21, 43201 Reus, Spain

Received 25 January 2005; received in revised form 26 May 2005; accepted 2 June 2005 Available online 18 July 2005

Abstract The present study was conducted to assess the potential combined influence of maternal restraint stress and aluminum (Al) exposure on postnatal development and behavior in the offspring of exposed rats. Female rats were concurrently exposed to 0 (control group), 50 or 100 mg/kg/day of Al administered as Al nitrate nonahydrate in drinking water with citric acid (355 or 710 mg/kg/day) for a period of 15 days prior to mating with untreated males. Aluminum exposure was maintained throughout the gestational, lactational and post-weaning periods. On days 6 – 20 of gestation, one-half of the pregnant animals in each group were restrained for 2 h/day. Food consumption and maternal body weight were decreased in the groups exposed to restraint only or combined with the highest Al dose. All of the animals were allowed to deliver and wean their offspring. The pups were evaluated for physical development and neuromotor maturation. Moreover, open-field activity, passive avoidance, and spatial learning in a water maze were also determined on postnatal days 30, 35 and 60, respectively. Body weight of pups treated with 100 mg/kg/day of Al was decreased relative to controls from postnatal day 12 through 21, sexual maturation was delayed in Al treated females and in males exposed to 100 mg/kg/day. Forelimb grip strength was reduced in males exposed to 100 mg/Al/kg/ day and in females exposed to this Al dose plus prenatal restraint. Learning in a passive avoidance task indicated facilitated performance for Al treated rats at 100 mg/kg/day combined with prenatal restraint as evidenced by longer avoidance latencies, while learning in a water maze task showed a shorter latency to find the platform on acquisition day 2 for Al treated rats. However, no effects of Al on water maze performance were detected during the retention probe trial in which the only effect noted was an increase in the platform quadrant swim time for the prenatal restraint group. In general terms, the results of the present study did not show a notable influence of maternal restraint on the Al-induced postnatal developmental and behavioral effects in the offspring of prenatally Al-exposed rats. D 2005 Elsevier Inc. All rights reserved. Keywords: Aluminum; Maternal restraint; Rats; Postnatal development; Behavior

1. Introduction It is well known that regardless of the host, the route of administration, or the speciation, aluminum (Al) can be a potent neurotoxicant [34,43,47]. Moreover, it is also well established that parenteral exposure to Al during pregnancy can cause a developmental syndrome that includes resorptions and death, skeletal and soft tissue abnormalities, and growth retardation [15,18]. However, because until the last * Corresponding author. Tel.: +34 977 759380; fax: +34 977 759322. E-mail address: [email protected] (J.L. Domingo). 0892-0362/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ntt.2005.06.014

few decades oral Al bioavailability had been considered low, there was little concern about the potential for embryo/fetal consequences following Al ingestion. In recent years, a number of studies have demonstrated that developmental Al exposure at high doses that led to reduced maternal weight gain can also produce growth retardation, delayed ossification, and perhaps malformations in the offspring [7,13,30,37,39]. The severity of these effects is dependent on the form of Al administered [2]. Moreover, it has been shown that gestational and lactational exposure to doses of Al that do not produce maternal toxicity can result in persistent neurobehavioral deficits in the offspring of some mammals.

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Body weight, forelimb and hindlimb grip strengths, negative geotaxis and thermal sensitivity have been reported to be affected in post-weaning mouse pups after high dietary Al exposure during gestation and lactation [17,23]. Learning changes have also been observed in prenatally exposed rabbits where a biphasic effect, improvement at low doses and impairment at high doses, was described [46]. In the developing nervous system of guinea pigs, Al was found to accumulate in the spinal cord, brainstem and cerebellum in an age related manner [26]. The question of whether Al exposure directly alters nervous system function is unclear as high dietary Al during development has been shown to impair the ability of nursing mouse pups to retain absorbed Fe and Mn [27]. Both of these consequences (i.e., Al accumulation and/ or imbalance in essential elements) can induce neurobehavioral alterations in mammals [21,27]. Studies in rodents have demonstrated that maternal stress during pregnancy may also be associated with adverse effects on embryo/fetal development [41,45] and impaired learning in male offspring [38]. Interestingly, it has been found that interactions between maternal stress and some metals can enhance the potential developmental toxicity of these elements [16]. In a previous study, we found that maternal restraint in pregnant mice could enhance the Alinduced embryo/fetal toxicity (reduced fetal body weight, increase in the number of litters with morphological defects) at doses that were toxic to the dam [10]. In a subsequent investigation in which all of the pregnant mice were allowed to deliver and wean their offspring, a decrease in the indices of viability and lactation were observed in the pups whose dams were concurrently exposed to Al and restraint stress. Moreover, although no significant effects of maternal Al plus restraint on the behavior of the offspring were noted, a significant influence of restraint on Al-induced developmental effects, decreased body weight, delayed eye opening, and sexual maturation and diminished forelimb grip strength, was found [11]. Because Al is ubiquitous, exposure to this element is unavoidable. Pregnant women may be potentially exposed to Al through the diet (including drinking water), dust and soil ingestion, and some medications, while they may also be concurrently exposed to various types of stress, either at home or in the workplace. Given the fact that both maternal stress and Al can induce behavioral changes in mammals [11,12], the purpose of the present study was to evaluate whether maternal stress might enhance the potential adverse effects of oral Al exposure on the postnatal development and behavior of the offspring of rats.

2. Method 2.1. Subjects and husbandry Sexually mature male and female Sprague – Dawley rats (250 –280 g) were obtained from Criffa (Barcelona, Spain).

The animals were quarantined for 7 days after shipping and housed in plastic cages in an animal room, which was maintained at a temperature of 22 T 2 -C, a relative humidity of 50 T 10%, and a 12-h light/dark automatic light cycle (light: 0800– 2000 h). All animals were allowed free access to food (Panlab rodent chow, Barcelona), containing 41.85 T 7.39 Ag Al/g, and tap water. After the quarantine period, female rats were assigned to four experimental groups by stratified randomization so that body weights were equivalent across all groups. The female rats were exposed to Al in the drinking water for a period of 15 days previous to mating with untreated males. Aluminum exposure was maintained during the gestation, lactation and throughout the life of the rats. Female rats were mated with males (2 : 1) until copulation was detected. Finding of a sperm plug indicated copulation and the day of detection was designated as Day 0 of gestation. Pregnant rats were allowed to deliver and wean their pups until postnatal day 21. The use of animals and the experimental protocol were approved by the Animal Care and Use Committee of the ‘‘Rovira i Virgili’’ University (Tarragona, Spain). 2.2. Treatment Aluminum was administered as aluminum nitrate nonahydrate [(AlNO3)3.9H2O] that was dissolved in the drinking water of the rats at doses of 0, 50, and 100 mg Al/kg/day. In order to enhance the gastrointestinal absorption, doses of 355 and 710 mg/kg/day of citric acid (E. Merck, Darmstadt, Germany) were added to the drinking water of the groups exposed to 50 and 100 mg/kg/day of Al, respectively. Controls received tap water supplemented with 710 mg/kg of citric acid. The selection of the doses of Al and citric acid was based on previous studies performed in our laboratory [31]. Body weight and fluid intake were measured weekly to adjust the doses in order to maintain a constant Al intake. During gestation, fluid intake and body weight were measured on days 0, 6, 15 and 21. The experimental groups were distributed as follows: Aluntreated animals were unrestrained and designated as unrestrained control group. Al-unexposed rats were restrained daily for 2 h (1000 –1200 h) on days 6 –20 of pregnancy (restrained control group). Unrestrained Altreated animals received Al nitrate nonahydrate in the drinking water at doses of 50 and 100 mg/kg/day, respectively. Finally, Al-restrained animals received Al nitrate nonahydrate at doses of 50 and 100 mg/kg/day, respectively, followed by restraint stress for 2 h/day on days 6 – 20 of pregnancy. Restraint consisted of placing the rats in metacrilate cylindrical holders (Letica Scientific Instruments, Barcelona, Spain) and maintaining the rats in a prone position for two hours while periodically monitoring them by observation [9]. Restraint was used as a stressor model because it is one of the most widely used animal models of stress [3,5,14,29,42,44].

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2.5.1. Open field activity An activity test was conducted on postnatal day 30. General motor activity was measured in an open-field apparatus, consisting of a wood 1 1 m square surrounded by a 47 cm-high dark colored wall. During the test, rats were allowed to move freely around the open-field and to explore the environment for 15 min. The path of the animals was recorded by a video camera (Sony CCD-IRIS model) that was placed above the square and was connected to a VHS videocassette recorder (Panasonic AG-5700 model). The video tracking program EthoVision\ (Noldus Information Technologies, Wageningen, The Netherlands) was used to measure the total distance traveled and the number of rearings (a measure of vertical activity). The number of defecations was also recorded as a measure of the rat’s reactivity to being placed in an open-field.

2.3. Physical and functional assessment Pregnant rats were allowed to deliver and wean their offspring. At birth, the following data were recorded: length of gestation, number of live and dead pups, sex, and individual pup weight. After these measures were taken, litter sizes were reduced to eight pups. A sex ratio of 4M : 4F was attempted; otherwise, the selection of pups was random. Body weight was again measured on postnatal days 4, 8, 12 and 21. The day of occurrence for pinna detachment, incisor eruption, eye opening, vaginal opening, and testes descent were also monitored. 2.4. Neuromotor maturation To assess general behavioral effects, pups were subjected to a routine testing battery. A surface righting reflex test was conducted on postnatal days 4, 5 and 6. Pups were placed on their back on a horizontal board and were released. Time to return to the normal dorso-ventral position was measured. A negative geotaxis test was conducted on postnatal days 7, 8, and 9 in which the animals were placed on a 30- inclined screen, and the time spent turning upward was recorded. A forelimb grip strength test was also conducted in one pup of each sex from each litter on postnatal days 10– 13. It was carried out with a grip strength meter from Ugo Basile (Comerio, Italy). The grip strength meter is a force transducer that is connected to a peak amplifier. As the rats are pulled backward across the grasping trapeze they will instinctively grasp anything to attempt to stop this involuntary backward movement. The peak pull force exerted on the trapeze prior to the overcoming of the rats grip strength is recorded on every trial. The maximum force from three consecutive trials was used as the measure of grip strength.

2.5.2. Passive avoidance Learning in a passive avoidance test was conducted on postnatal day 35. The apparatus consisted of a shuttle box separated into two compartments by a wall and a sash door (Ugo Basile, Comerio, Italy). One compartment (40  20 cm) was illuminated (24 v– 10 w) and the other (40  20 cm) was dark. Animals were placed in the illuminated compartment, and after a period of 60 s, the door was pulled up. After the rats spontaneously entered the dark compartment (recorded as T1), the door was shut 1 s after crossing and the animals received an electric shock of 1 mA for 3 s. Twenty-four hours later, the same procedure was repeated with a delay period of 10 s before opening the door. The time elapsed before entering the dark compartment (maximum 5 min) was recorded as T2 [12].

2.5. Post-weaning tests 2.5.3. Water maze On postnatal day 60, learning was evaluated in a water maze with a hidden platform. Because nutritional parameters (food and water intake) were altered during gestation

The following tests were evaluated in one male and one female of each litter, the same cohort of animals were tested in all the behavioral tests.

Table 1 Effects of aluminum, restraint, and combined aluminum and restraint on maternal toxicity in pregnant rats Doses of Al (mg/kg/day)

0

Restraint

0

50

100

+

50

100

+

+

No. of dams

15

13

11

17

9

10

Food consumption (g/dam) on gestation days 0 – 6 Food consumption (g/dam) on gestation days 7 – 15 Food consumption (g/dam) on gestation days 16 – 21 Water consumption (ml) on gestation days 0 – 6 Water consumption (ml) on gestation days 7 – 15 Water consumption (ml) on gestation days 16 – 21 Dam body weight (g) on gestation day 0 Dam body weight (g) on gestation day 6 Dam body weight (g) on gestation day 15 Dam body weight (g) on gestation day 21 Body weight change (g) during gestation period (0 – 21)

137.5 T 21.1 234.2 T 26.2a 171.5 T 25.1 134.5 T 18.1 303.3 T 73.6 225.2 T 25.9 280.1 T 9.8 308.2 T 13.2 356.6 T 15.4a 446.8 T 19.9ac 166.7 T 22.7a

124.3 T 21.5 188.1 T 24.6b 160.4 T 23.6 118.0 T 25.1 242.9 T 76.8 205.0 T 27.0 271.6 T 11.6 297.8 T 8.9 329.5 T 12.2b 403.1 T 20.0bc 131.5 T 17.2bc

132.8 T 9.8 214.6 T 21.6ab 175.4 T 18.5 115.4 T 13.5 284.7 T 71.7 237.0 T 61.2 279.5 T 20.4 304.7 T 22.7 347.4 T 29.0ab 436.5 T 36.4ac 157.0 T 23.0ac

124.4 T 16.4 231.4 T 29.3ac 168.0 T 22.5 142.6 T 43.9 271.0 T 54.0 222.1 T 27.3 272.5 T 17.8 309 T 18.0 349.2 T 22.2ab 427.4 T 25.3c 154.9 T 20.5ac

139.7 T 10.1 206.8 T 33.4ab 172.1 T12.6 133.5 T 9.0 284.8 T 34.6 219.8 T 28.3 273.3 T 12.5 296.8 T 30.7 339.3 T 15.2ab 413.3 T 24.8abc 140.0 T 27.2abc

127.7 T 13.8 190.8 T 20.2bc 144.8 T 16.8 120.8 T 16.2 249.0 T 70.6 197.3 T 39.1 270.0 T 19.0 299.9 T 18.5 327.8 T 19.8b 390.4 T 28.0b 120.4 T 16.1b

Data are given as means T SD. Values in the same row not showing a common superscript (a, b, c) are significantly different at P < .05.

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and lactation in dams exposed to Al 100 mg/kg/day, only rats treated at 0 and 50 mg/kg/day, Al alone or combined with restraint, were tested. The water maze consisted of a blue circular tank (diameter, 1.60 m; height, 0.50 m) divided into four equal-sized quadrants. A blue escape platform was located 1 cm below the water surface and near the center of one of the four quadrants of the maze. The maze was surrounded by curtains with patterns affixed to provide a configuration of spatial cues. The path of the animals was recorded by a video camera (Sony CCD-IRIS model) that was placed above the maze and connected to a VHS videocassette recorder (Panasonic AG-5700 model). The data were analyzed by the video tracking program Etho-Vision\. The rats were subjected to five trials per day for three consecutive days. The maximum duration of each trial was 60 s and each trial was separated by a 60 s intertrial interval. At the beginning of each trial, the rat was placed into the pool with the nose pointing towards the wall from one of five starting positions. These starting positions were chosen at random. If the animal did not locate the

60

2.6. Analysis of Al and other trace elements About 0.3 g of fresh tissue of brain samples (cerebellum, hippocampus, cortex, striatum and brainstem)

A a

Control Al 50 Al50 + Restraint

50

Body Weight (g)

platform within 60 s, the animal was placed on the platform for 30 s. Twenty-four hours after the last training session, retention of the task was assessed by a probe trial, which consisted of a 60 s free swim without the escape platform. The swim-path length and the latency to find the escape platform during the training sessions as well as the total time and the percentage of time spent by the rat swimming in the target quadrant for the probe trial were analyzed as the measures of water maze performance. Results from trials for each day were averaged for every rat. On postnatal day 68, rats were anesthetized and killed by decapitation. Brains were rapidly removed and hand free dissected to obtain samples of cortex, hippocampus, striatum, cerebellum and brainstem (mesencephalon, pons and medulla) to measure Al and trace elements.

a

Restraint Al 100 Al100 + Restraint

a

a b

b

a a

40

ab

Males

b

ab

b

a ac

30

ab

bc ab b

20

10

0

60

Body Weight (g)

50

Day 1

Day 12

Day 16

Day 21

B a

Control

Restraint

Al 50

Al 100

Al 50+ Restraint

Al 100 + Restraint

a

a ac ac

40 Females a

30

a

b

ac a

b b

ab b

b

bc b

b

20

10

0

Day 1

Day 12

Day 16

Day 21

Fig. 1. Body weight of male (A) and female (B) pups during the lactation period. For each day, those values not showing a common letter (a, b, c) are significantly different at P < .05.

M.T. Colomina et al. / Neurotoxicology and Teratology 27 (2005) 565 – 574

from 10 males and 10 females in each group of treatment were predigested with 1 ml of nitric acid (65% Suprapur, E. Merck, Darmstadt, Germany) for 24 h. Samples were then heated at 80 -C for 10 h followed by an additional heating at 130– 150 -C for 30 min. Finally, in presence of 0.5 ml of 70% perchloric acid (E. Merck), solutions were again heated for 4 h, and evaporated almost to dryness. Subsequently, solutions were adjusted to 5 ml volume with deionized water and filtered. Aluminum concentrations were determined by inductively coupled plasma spectrometry (Thermo Jarrell ASH, PolyScan 61 E) according to conditions described previously [32]. The remaining elements (Ca, Zn, Fe) were also analyzed as previously reported [31]. 2.7. Statistical analysis Data were analyzed with the SPSS version 10 software for PC. Gestational data body weight, body weight change, and water and food consumption were analyzed by a oneway analysis of variance (ANOVA). For the developmental study, the litter was the unit of statistical analysis. For the analysis of body weight and developmental landmark data, the mean value of each sex in each litter was used. Body weight was evaluated with a three-way ANOVA for repeated measures, using the age as the repeated measure, and Al treatment, restraint, and sex as factors. Developmental landmarks were evaluated with a two-way analysis of variance (ANOVA), using Al treatment and restraint as factors. Neuromotor development, open-field activity and passive avoidance conditioning data were analyzed with a three-way ANOVA, and with a three-way ANOVA for repeated measures, using the age or the period of time as the

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repeated measure, and Al treatment, restraint, and sex as factors. Significant treatment-related interactions were further analyzed, using the simple effect at each level of interaction, with Tukey corrections for multiple comparisons. A probability value of P < .05 was considered as significant.

3. Results 3.1. Maternal effects A one-way ANOVA revealed significant effects of treatment group on food consumption during gestational days 7 –15 [ F(5,49) = 4.67, P = .002], on dam body weight on days 15 –21 [ F(5,60) = 3.74, P = .005; F(5,60) = 6.87, P < .001], and on body weight change during the whole gestational period [ F(5,60) = 7.01, P < .001]. Food consumption and body weight were decreased in the groups exposed to restraint only or combined with Al at 100 mg/kg/day (Table 1). No differences on water intake were observed during the gestational period. However, significant differences between groups were noted in maternal water and food intake during the lactation period [ F(5,66) = 20.11, P < .01; F(5,66) = 19.51, P < .01]. Dams exposed to Al at 100 mg/kg/ day showed a significant decrease in both water and food ingestion during the lactation period when compared to those in the remaining groups (data not shown). 3.2. Fetal outcome and physical maturation There were no differences among groups in the length of gestation, viability (live pups on postnatal day 4/live pups at

Table 2 Effects of aluminum, restraint, and combined aluminum and restraint on physical maturation of the offspring of prenatally exposed rats Doses of Al (mg/kg/day)

0

0

Restraint Length of gestation (days) No. of litters No. of fetuses per litter Viability index (%) Lactation index (%) No. of days at pinna detachment Males Females No. of days at incisor eruption Males Females No. of days at eye opening Males Females No. of days at testes descent No. of days at vagina opening

50

100

+

50

100

+

+

22 15 13.93 T 2.15 100 100

22 13 12.54 T 3.04 98.77 100

22 11 13.82 T 1.83 98.68 100

22 17 13.82 T 2.79 97.02 100

22 9 15.00 T 1.87 97.04 100

22 10 12.40 T 3.20 98.39 100

2.85 T 0.69 2.80 T 0.56

2.60 T 0.52 2.50 T 0.52

2.82 T 0.40 2.82 T 0.40

2.53 T 0.74 2.65 T 0.93

2.89 T 0.33 3.00 T 0.50

2.87 T 0.35 2.80 T 0.42

5.5 T 0.5a 5.5 T 0.5ab

5.3 T 0.5a 5.5 T 0.7ab

6.1 T 0.3b 6.1 T 0.3b

5.3 T 0.7a 5.3 T 0.7a

5.5 T 0.5ab 5.5 T 0.5ab

5.2 T 0.4a 5.2 T 0.4a

14.8 T 0.9 14.8 T 0.9 24.9 T 1.4ab 32.5 T 3.7ac

14.7 T 0.5 14.7 T 0.6 22.8 T 0.4a 40.9 T 3.8bc

15.0 T 1.0 14.8 T 0.6 27.1 T 3.8b 45.9 T 11.2b

14.09 T 0.6 14.7 T 0.5 23.2 T 0.7a 40.6 T 4.4bc

15.5 T 0.7 15.1 T 0.6 27.7 T 2.9b 44.9 T 9.5b

14.7 T 0.6 14.7 T 0.6 23.9 T 1.9a 31.1 T 4.5a

Data are given as means T SD. Values in the same row not showing a common superscript (a, b, c) are significantly different at P < .05.

M.T. Colomina et al. / Neurotoxicology and Teratology 27 (2005) 565 – 574 800

*

700 Male Forelimb grip strength (g)

birth) and lactation index (live pups on postnatal day 21/live pups on postnatal day 4). A three-way (Al  restraint  sex) ANOVA for repeated measures using age as the repeated measure, indicated an interaction between Al and age [ F(10,236) = 12.09, P < .001], as well as an overall effect of Al exposure [ F(2,121) = 24.97, P < .001] on body weight. A more detailed analysis revealed differences between groups in both sexes beginning on postnatal day 12 (Fig. 1). On postnatal days 12, 16 and 21, male and female rats exposed to 100 mg/kg/day Al, alone or combined with prenatal restraint, showed diminished body weight when compared to both the unrestrained and restrained control groups. A diminished body weight was also observed in females exposed to Al at 50 mg/kg/day and prenatal restraint when compared to unrestrained control groups on postnatal days 12 and 16. Developmental landmarks were evaluated for both males and females by a two-way (Al treatment  restraint) ANOVA. No significant differences on pinna detachment or on the number of days at eye opening were observed. For the number of days at incisor eruption an overall effect of Al was observed in males and females [ F(2,74) = 7.17, P = .001; F(2,74) = 5.37, P = .007], while an overall effect of restraint stress was observed only in males [ F(1,74) = 4.53, P = .037]. A more detailed analysis revealed differences between groups on the number of days at incisor eruption for both males and females (Table 2). An overall effect of Al was observed in both males and females in sexual maturation [ F(2,70) = 19.41, P < .001; F(2,74) = 24.29, P < .001], with significant differences between groups on sexual maturation (Table 2).

600

*

500

*

400 300 200

*

100

Day 10

Day 11

The effect of treatment on neuromotor development were evaluated by a two-way (Al  restraint) ANOVA for repeated measures (at different ages). No significant effects of Al or restraint stress were observed on surface righting and negative geotaxis (data not shown). Treatment effects were only observed on the forelimb grip strength, where an overall effect of Al was noted in both males and females [ F(2,61) = 3.80, P = .028; F(2,61) = 5.35, P = .007]. Further analyses revealed significant differences existed between groups at 11 and 13 days of age (Fig. 2). Male rats treated with Al at 100 mg/kg/day showed decreased forelimb grip strength at day 11 when compared to control and at day 13 when compared to rats exposed to prenatal stress and treated with Al at 50 mg/kg/day. Female rats exposed to prenatal restraint stress and Al at 100 mg/kg/day, showed decreased forelimb grip strength on days 11 and 13 when compared to prenatally restrained rats. 3.4. Postweaning tests With respect to the activity level measured in an openfield for both male and females, a three-way (Al 

Day 12

Day 13

800

*

700 600 500

*

400

*

300 200

*

100 0

3.3. Neuromotor maturation

Control Restraint Al 50mg Al 100mg Al 50mg + Restraint Al 100mg + Restraint

0

Female Forelimb grip strength (g)

570

Day 10

Day 11

Control Restraint Al 50mg Al 100mg Al 50mg + Restraint Al 100mg + Restraint Day 12

Day 13

Fig. 2. Forelimb grip strength in male (top) and female (bottom) pups during the lactation period. For each day, data accompanied by an asterisk (*) represent a statistically significant difference ( P < .05) from the restraint control group.

restraint  sex) ANOVA for repeated measures, using 5 min interval blocks as a repeated measure did not show significant effects of Al or restraint. A significant interaction of sex and time and an overall effect of sex could be observed [ F(2,135) = 3.92, P = .022; F(1,135) = 11.82, P = .001]. The sex effect consisted of greater overall activity in the females. No effects on total activity or rearings during the entire period (15 min) were noted in either the males or the females (data not shown). To evaluate learning, a passive avoidance test was conducted on day 35. The latency to enter the dark compartment was measured. Differences between latency on acquisition day (T1) and latency on retention day 24 h later (T2) were evaluated by a three-way (Al  restraint  sex) ANOVA for repeated measures, using the day of the test as a repeated measure and the body weight as a covariant. A significant effect of day [ F(1,134) =86.43, P < .001] was observed which

M.T. Colomina et al. / Neurotoxicology and Teratology 27 (2005) 565 – 574

571

trial. A one-way ANOVA revealed effects of Al treatment in both distance traveled and time spent to find the platform [ F (3,75) = 3.10, P = .032; F (3,75) = 4.08, P = .010]. Significant differences between groups were only observed in the time spent to find the platform on acquisition day 2, in which rats exposed to 50 mg Al/kg/ day showed shorter swim latencies than those in the control groups. A one-way ANOVA revealed an overall effect of treatment group on the time spent on the platform quadrant during the retention task [ F(2,75) = 3.09, P = 0.032]. A more detailed analysis revealed significant differences between the unrestrained control group and the prenatally restrained group, which spent more time in the target quadrant, indicating a better retention of the task (Fig. 4).

indicates that the subjects learned the tasks. An interaction between test day and restraint [ F(1,134) = 4.21, P = .042], and an interaction between test day, restraint and aluminum exposure [ F(2,134) = 4.13, P = .018] were observed. An overall effect of aluminum was also noted [ F(2,134) = 3.58, P = .03], while no effects of sex were observed. For this reason, males and females were evaluated together to assess for differences between the groups. Because the data analyzed were not homogeneous, differences were assessed using the Kruskal –Wallis test, while differences between groups were evaluated by the Mann – Whitney U-test. Significant differences were observed in the retention of the task (T2) ( P = .047), and in the number of defecations on day 2 ( P = .035). A more detailed analysis showed significant differences mainly for prenatally restrained rats exposed to 100 mg/kg/day Al. Animals in this group showed increased latencies when compared to the remaining groups (Fig. 3). Spatial learning in rats exposed to Al at 0 or 50 mg/ kg/day alone or combined with prenatal restraint stress was evaluated in a water maze task on postnatal day 60. A three-way (Al treatment  restraint  sex) ANOVA for repeated measures across the three days of acquisition revealed an interaction between the day of acquisition and Al treatment [ F(2,67) = 8.19, P < .001] on the distance swam to find the platform, and an interaction of Al treatment and acquisition day on time spent to find the platform [ F(2,67) = 7.41, P < .001]. Interactions for sex or restraint stress, or overall effects of sex were not observed. For this reason, a more detailed analysis by combining males and females was conducted in order to analyze each day of acquisition and the retention probe

3.5. Trace elements analyses To assess mineral concentrations in exposed rats, Al, Zn, Ca and Fe were measured in cortex, cerebellum, hippocampus, striatum and cerebral trunk (Table 3). A three-way (Al treatment  restraint stress  sex) ANOVA was performed for all the structures and elements evaluated. An overall effect of Al treatment [ F(2,56) = 3.072, P < .001] was observed. Because no differences between sexes were noted, males and females were analyzed together, and a one-way ANOVA for each element in each structure was performed. No evidence of Al accumulation in the exposed rats was found in any of the analyzed brain structures. However, a significant increase in Ca and Zn concentrations were observed for

350

300

T1 T2 b a a

250

a

a

Latency (s)

a 200

150

100

50

0

Control

Restraint

Al 50

Al 100

Al 50 + Restraint

Al 100 + Restraint

Fig. 3. Passive avoidance acquisition (T1) and retention 24 h later (T2) in rats prenatally exposed to Al and restraint stress. Different letters (a, b) indicate statistically significant differences at P < .05.

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M.T. Colomina et al. / Neurotoxicology and Teratology 27 (2005) 565 – 574 40

A

35

Control Restraint a

Latency to platform (s)

30

Al 50mg

a

Al 50mg + Restraint

25 ab

20

b

15 10 5 0 Day 1

Day 2

Day 3

Control

Time in the platform quadrant (s)

45

B

Restraint Al 50mg

40

Al 50mg + Restraint b

35

ab

30

ab

a

25 20 15 10 5 0

Fig. 4. Latency (mean values T SD) to find a hidden platform in a water maze task during a five days acquisition period by male rats exposed to oral Al (0 and 50 mg/kg body weight) given alone or combined with prenatal stress (A). Time (mean values T SD) in the target quadrant during the probe trial (B). Different letters (a, b) indicate significant differences at P < .05.

almost all the structures analyzed in rats exposed to 50 mg Al/kg/day (Table 3).

4. Discussion Studies in rodents have shown that, during pregnancy, maternal stress from restraint, noise, light, and heat may be associated with adverse maternal effects and adverse embryo/fetal and postnatal development [18,35,38]. Moreover, it is also well known that exposure to certain metal levels during gestation can also cause maternal and developmental toxicity [16]. Prenatal Al exposure at 100 mg/kg/day, either alone or combined with restraint, induced a decrease in body weight from postnatal day 12 and delays in incisor eruption, sexual maturation, vagina opening and testis descent. In recent decades, a number of studies

conducted in rats and mice have shown that maternal oral Al exposure altered performance on a neurobehavioral test battery, specifically impairing negative geotaxis and reducing forelimb and hindlimb grip strength [4,20,22,23]. In the current study, only differences in forelimb grip strength were observed in the groups exposed to the highest Al dose, either alone or plus restraint which would suggest long lasting effects of Al (alone or combined with restraint). Similar results have also been reported in mice exposed to dietary aluminum during development [17,24]. These results in physical maturation and neurobehavioral development could be related to changes in maternal body weight, food consumption during gestation, as well as to changes in water and food consumption during lactation. With respect to this, Golub and Germann [19] showed that Al effects could be exacerbated by a suboptimal maternal diet. In the present investigation, maternal toxicity effects due to Al treatment and/or restraint exposure were observed during the gestational period for restraint only or for restraint combined with the highest Al dose. During the lactation period, a decrease in water and food consumption was also observed in the females exposed to the highest Al dose given alone or combined with restraint stress. Both of these effects (Al exposure or dietary insufficiency) during the gestational and lactational periods can influence the developmental outcome of the offspring. In the current study, prenatal Al exposure, alone or plus restraint, did not cause significant changes in the activity of the offspring measured in an open-field. However, Misawa and Shigeta [36] reported diminished horizontal activity in female rats prenatally exposed to Al chloride at doses of 1800 mg/kg on gestational day 15. The different experimental procedure between the studies and/or the chemical form of Al administered could be the reason for the differences in outcome between the current study and Misawa and Shigeta [36]. With regard to learning in a passive avoidance task, the present results indicate that rats exposed to 100 mg/kg/day Al throughout their life following prenatal restraint stress showed improved performance as evidenced by increased latencies in the passive avoidance retention task. In the water maze task, a similar trend toward facilitated performance was observed in the rats exposed to 50 mg/kg/day of Al alone as evidenced by shorter latencies to find the platform on acquisition day 2. The water maze findings are similar to those reported by Golub et al. [28] in mice exposed to a high Al dose. Other studies have not found learning deficits in delayed spatial alteration [18,25 – 27,24], or avoidance learning ability based on alimentary motivation [6], in adult mice and rats exposed to Al during development. However, Gonda and Lehotzky [33] found that rats prenatally exposed to an intraperitoneal (ip) injection of Al lactate (9.8 mg/kg on gestation days 7 – 15) were impaired in a passive avoidance task. Impaired performance in maze tasks [1] and in a radial arm maze after a high ip Al injection (200 mg/kg) [40] were also reported in adult

M.T. Colomina et al. / Neurotoxicology and Teratology 27 (2005) 565 – 574

573

Table 3 Concentrations (Ag/g) of aluminum, calcium, zinc, and iron in brain regions of rats prenatally exposed to aluminum at 0, 50 and 100 mg/kg/day and restraint stress on gestation days 6 – 20 Doses of Al (mg/kg/day)

0

Restraint Aluminum concentration (lg/g) Cerebellum Striatum Hippocampus Cortex Brainstem

0

50

100

+ 3.8 T 4.4 13.9 T 17.3 8.5 T 10.6 8.4 T 8.3 6.7 T 9.8

4.0 T 4.4 12.4 T 14.0 13.6 T 14.0 7.2 T 7.2 8.6 T 9.9

3.9 T 4.9 12.9 T 28.8 9.1 T 7.7 3.5 T 2.9 3.7 T 3.3

116.4 T 97.1ab 48.6 T 49.5a 92.7 T 81.3a 84.5 T 52.2a 92.0 T 44.5a

83.9 T 48.1b 44.7 T 47.0a 66.6 T 40.3a 75.5 T 37.3a 124.3 T 72.1a

153.8 T 66.6a 106.2 T 77.5ab 258.6 T 93.6b 192.0 T 70.1b 205.1 T 81.6b

Zinc concentration (lg/g) Cerebellum Striatum Hippocampus Cortex Brainstem

8.83 T 2.7a 4.27 T 2.7a 6.21 T 3.1a 8.97 T 2.8a 4.94 T 2.5a

9.4 T 2.7a 5.3 T 3.5ac 7.7 T 3.7a 11.7 T 2.5b 7.1 T 3.3a

21.7 T 7.3b 31.2 T 29.3b 35.6 T 15.8b 25.4 T 7.1c 21.6 T 7.3b

Iron concentration (lg/g) Cerebellum Striatum Hippocampus Cortex Brainstem

20.9 T 6.3ab 16.5 T 9.1ab 14.1 T 6.3ac 16.7 T 4.4ab 16.5 T 4.7a

19.6 T 5.1ab 14.6 T 1.9a 16.0 T 3.1ab 18.3 T 2.8a 18.7 T 2.6ab

21.1 T 3.1a 16.18 T 9.2ab 19.53 T 5.8bc 18.84 T 3.3a 20.21 T 3.9ab

Calcium concentration (lg/g) Cerebellum Striatum Hippocampus Cortex Brainstem

2.88 T 3.0 15.26 T 27.4 5.44 T 6.5 5.07 T 6.8 2.24 T 3.1

109.6 T 90.3ab 78.3 T 34.2a 127.7 T 82.2a 82.8 T 35.9ac 139.8 T 117.0ab

50

100

+

+

3.66 T 4.6 8.18 T 8.2 2.94 T 4.4 3.18 T 3.8 5.18 T 5.6

6.3 T 8.8 10.2 T 22.7 4.4 T 6.8 3.3 T 5.7 4.1 T 5.5

200.6 T 167.9ab 141.9 T 45.8b 234.4 T 92.4b 138.2 T 36.2bc 261.1 T149.7b

96.8 T 60.3ab 79.4 T 40.2a 86.6 T 64.4a 108.5 T 64.6ac 113.6 T 68.3a

9.01 T 2.7a 8.40 T 5.1c 9.00 T 4.6a 10.60 T 3.8ad 6.21 T 2.9a

19.3 T 3.4b 33.5 T 19.3b 36.6 T 17.1b 22.9 T 5.6c 24.0 T 5.2b

10.6 T 3.1a 8.6 T 5.7ac 11.2 T 7.4a 12.4 T 2.2bd 8.4 T 4.7a

16.8 T 3.0b 11.02 T 3.4b 11.08 T 3.9a 13.79 T 3.9b 16.47 T 7.6ab

21.0 T 1.6a 17.5 T 4.5a 20.22 T 12.1b 18.53 T 2.4a 21.07 T 2.7b

22.0 T 4.7a 16.8 T 3.6a 16.4 T 3.9ab 18.5 T 3.8a 20.1 T 3.2ab

Data are given as means T SD. Values in the same row not showing a common superscript (a, b, c, d) are significantly different at P < .05.

mice prenatally exposed to Al. Taken together, these conflicting findings on learning found in the literature may be a consequence of different doses and routes of exposure. It seems that oral exposure to moderate doses of Al may improve some aspects of learning, while higher doses and/or parenteral exposure may be related to impaired learning. In spite of the potential interest about the influence of maternal stress on the developmental and neurobehavioral toxicity of metals, studies about it are scarce [16]. In the present investigation no remarkable effects of maternal Al exposure plus restraint on the behavior of the offspring could be noted. However, in a previous study we found a significant influence of maternal stress on Al-induced developmental effects in mice, a decrease in fetal body weight and an increase in skeletal defects when Al was administered intraperitoneally at doses that were maternotoxic by themselves [10] In the current study, the chosen Al doses did not result in maternotoxicity when the route of administration was via oral consumption. In general, effects of oral Al are less remarkable than those observed for parenteral administration. However, the oral route is probably a more realistic way of modeling Al exposure in humans. In this sense, and taking into account that both Al and stress could be risk factors for neuropathological processes, especially during aging, it would be interesting

to assess the concurrent effects of Al and stress in aged rats. The results of the present study together with those of our previous investigations with other metals [8,9] seem to indicate that the influence of maternal stress on the effects of prenatal exposure to metals on embryo/fetal toxicity and postnatal development and behavior depends on each specific metal and dose, more than on the general influence of the concurrent exposure.

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