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Effects of low dose methylmercury administration during the postnatal brain growth spurt in rats
Neurotoxicology and Teratology 29 (2007) 282 – 287 www.elsevier.com/locate/neutera
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Effects of low dose methylmercury administration during the postnatal brain growth spurt in rats Addolorata Coluccia a , Pietro Borracci a , Arcangela Giustino a , Mineshi Sakamoto b , Maria Rosaria Carratù a,⁎ a
Department of Pharmacology and Human Physiology, Medical School, University of Bari, Policlinico, Piazza G. Cesare 11, 70124 Bari, Italy b Department of Epidemiology, National Institute for Minamata Disease, 4058-18 Hama, Minamata, Kumamoto 867-0008, Japan Received 14 July 2006; received in revised form 17 October 2006; accepted 17 October 2006 Available online 25 October 2006
Abstract Male Sprague-Dawley rats from eight litters were orally administered 0.75 mg/kg/day methylmercury (MeHg) chloride from postnatal day (PD) 14 to PD 23. One male pup per litter from eight different litters per treatment group was used. Each pup was used only for a single behavioral test and tested once. The MeHg dose level resulted in Hg brain concentrations of 0.82 ± 0.05 μg/g tissue (n = 4). Locomotor behavior was studied in the Opto-Varimex apparatus by testing rats (n = 8) weekly from PD 24 to PD 45. Performance of rats (n = 8) on learning paradigm was analysed on PD 90. MeHg treatment induced a significant reduction in the number of rearings without altering the distance travelled, the resting time and the time spent in the central part of the arena. Results of conditioned avoidance task showed that, unlike control rats, MeHg-treated animals did not show improvement over blocks and never reached a level of performance that would indicate significant learning had taken place. The present results show that low level exposure to MeHg during late brain growth spurt induces subtle and persistent motor and learning deficits, further underlining the serious potential hazard for the exposed children. © 2006 Elsevier Inc. All rights reserved. Keywords: Methylmercury; Neurodevelopment; Locomotor behavior; Associative learning; Rat
1. Introduction Methylmercury, an organic methylated form of mercury, is one of the most hazardous environmental pollutants. The major source of MeHg exposure to the general population is typically through consumption of contaminated fish and other food products [26]. MeHg, absorbed from the gastrointestinal tract, is easily transported across the blood-brain barrier and placenta. Once it is demethylated in the brain, elemental mercury bioaccumulates in the brain tissue [6]. Aschner et al. [1,2] have demonstrated cysteine-facilitated transport of MeHg into the brain, and they also have identified the presence of a neutral aminoacid transport system in astrocytes, which is capable of mediating MeHgcysteine uptake. In rats, the maturation of astrocytes occurs during postnatal developmental stages [36] which correspond to the third trimester of pregnancy in humans [29]. ⁎ Corresponding author. Tel.: +39 080 5478455; fax: +39 080 5478444. E-mail address: [email protected] (M.R. Carratù). 0892-0362/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ntt.2006.10.005
As in humans [8,39], the developing brain in several species ranging from monkeys to mice appears far more susceptible to MeHg than the mature brain [3]. While methylmercury poisoning has been clearly associated with severe neurotoxic effects in both animal studies and human poisoning episodes, the evidence for developmental impairments associated with lower level exposure is less clear [25]. Frank human poisoning, both prenatal and postnatal, has been associated with severe mental retardation. Following exposure of the community of Minamata (Japan) and Iraqi population, the clinical course of surviving victims ranged from cases characterized by low birthweight, microcephaly, profound developmental delay, cerebral palsy, deafness, blindness and seizures [15] to ones with much milder effects including weakness, increased muscle tone, abnormal plantar reflexes, and delays in intellectual and motor development [22]. Severe neurodevelopmental effects were observed in children exposed in utero even when mothers were asymptomatic or had very mild symptoms. Despite the limitations to determine the incidence of developmental disorders
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associated with various exposure levels, the Iraqi population studies identified some dose-related effects of MeHg and raised concern that lower levels of MeHg might be associated with developmental delays [3,24]. The behavioral deficits observed in animal models of MeHg exposure also range from severe to mild depending on the exposure level. Studies reviewed by Burbacher et al. [3] show that, as in humans, also in nonhuman primates and rodents signs of severe toxicity including blindness, seizures, spasticity, quadriplegia may be observed in the most affected animals, whereas more subtle neurobehavioral deficits may be observed in the less affected ones. In this regard, MeHg effects on visual evoked potentials, swimming behavior, open field activity, motor coordination and performance of animals on various learning paradigms have been reported in rodents following preor postnatal exposure to this toxic compound. Therefore, on the basis of clinical and experimental evidence, it is well established that fetuses and neonates are high-risk groups for MeHg exposure, and the MeHg effects may be more or less severe depending on the duration and level of exposure at different developmental stages [11,28]. In this regard, the implications of low level exposure are still controversial [9,21], and the lowest dose of MeHg that might impair neurodevelopment is still unknown. Another intriguing aspect is that, following prenatal or postnatal exposure to MeHg, an infant may develop psychomotor deficits as the nervous system matures. Therefore, great concern has also been raised about the long-term impact of exposure [27], since neurologic effects may continue to manifest themselves throughout life, particularly in aging population. Concerning the critical periods of exposure during development, differences exist between humans and rodents. Rapid brain growth occurs primarily during the third trimester of pregnancy in humans, whereas in rats it occurs after parturition [38]. In particular, cerebellum as well as certain structures important in learning and memory function do not undergo extensive development until late gestation and early postnatal life [28]. While traditional views link dorsolateral prefrontal cortex with complex cognition and cerebellum with motor function, in humans and monkeys there is significant cross-talk between these two brain areas, thus implying a much closer relationship between cognitive and motor development [10]. Therefore, taking into account that the lowest dose of MeHg that might impair neurodevelopment is still unknown, the present study was primarily designed to explore, in the rat, the neurobehavioral effects of a low-level exposure to MeHg during the postnatal brain growth spurt. Taking into account that both motor and cognitive development display protracted developmental timetables and that deficits in both domains are often associated in the same developmental disorders [10], the effects of MeHg on motor and cognitive tasks were evaluated.
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the Declaration of Helsinki and the “Guide for the Care and Use of Laboratory Animals” as adopted and promulgated by the National Institutes of Health. Primiparous Sprague-Dawley female rats (Harlan, S. Pietro al Natisone, Italy) weighing 250–280 g were used. The animals were allowed free access to food and water, were housed at constant room temperature (20–22 °C) and exposed to a light cycle of 12 h/day (08.00 h–20.00 h). Pairs of females were placed with single male rats in the late afternoon. The day on which sperm were present was designated day 0 of gestation (GD 0). Within 24 h after birth, eight litters were reduced to a standard size of eight pups per litter, and one male pup per litter from different litters was used. Pups were weaned at 24 days of age. Male offsprings were orally administered 0.75 mg/kg/day methylmercury chloride on PD14 for 10 consecutive days. MeHg and L-cysteine (SIGMA, Milan, Italy), in a molecular ratio 1:1, were dissolved in 10% condensed milk and were administered with a microman-pipette (Gilson) for the sucklings, according to the method of Sakamoto et al. [32]. Control animals received cysteine only for 10 days. One male pup per litter from eight different litters per treatment group was used. Each pup was used only for a single behavioral test and tested once. 2.2. Mercury determination procedure On the day after the final treatment, four MeHg-exposed rats (one rat per litter from four different litters) were deeply anesthetized by intraperitoneal injection of pentobarbital. In order to flush out blood from the brain, the rats were thoroughly perfused via the heart with physiological saline for 5 min. Then the brains were removed and kept at −80 °C until use. Total Hg concentrations were determined according to the oxygen combustion–gold amalgamation method [18], using a Mercury Analyzer MV 250R (Sugiyama-gen Environmental Science, Tokyo, Japan). 2.3. Observation of deaths, hind-limb crossing, body and brain weight monitoring Rats were examined, weighed and deaths noted on a daily basis throughout the treatment period. The development of hind-limb crossing was also evaluated. An experimenter, blind to the animal's treatment condition, held a rat by the base of the tail for 2–5 s (perpendicular to the floor) and recorded the position of the hind limbs [20]. Results were coded with a “0” (normal) or a “1” (impaired). Brain weights were monitored on PDs 24 and 90 (one rat per litter from five different litters per tested age and per treatment group).
2. Methods
2.4. Locomotor activity
2.1. Animals and treatment schedule
Motor activity was recorded in an Opto-Varimex apparatus linked to an IBM PC (Columbus Instruments, Columbus, OH) according to the method described by Wedzony et al. [35]. The apparatus consisted of a cage (42 × 42 × 30 cm) equipped with
The experiments have been conducted in accordance with guidelines released by Italian Ministry of Health (D.L. 116/92),
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15 infrared emitters (spaced at 2.65-cm intervals) located on the x and y axes 2–3 cm above the floor of the cage (depending on the size of the animal) and an equivalent number of receivers located on the opposite walls. A further line of emitter/receiver pairs was located ≈ 5 cm (depending on the size of the animal) above the floor of the cage to detect vertical movements (i.e., rearings). Each interruption of a beam generates an electric impulse scored by a digital counter. 2.4.1. Procedure The amount of time spent in ambulatory activity was analyzed by using AUTO-TRACK software (Columbus Instruments, Columbus, OH). Ambulatory activity, defined as a trespass of three consecutive photo-beams, and resting time, calculated as the amount of time during which there were no ambulatory movements, were recorded. Vertical activity was measured by recording the number of horizontal beams that were broken by the rearings of the animal. Furthermore, the amount of time spent in the central part of the arena was analyzed in the first min of test by using the Time In Square (TIS) analysis (AUTO-TRACK software). TIS analysis divides the Opto-Varimex cage area into a four by four grid of sixteen squares. The time the subject is in a square is calculated for each cage interval. The time is calculated to one-tenth of a second accuracy using the raw X–Y coordinate data file. Tests were carried out in a 1 × 1 × 2 m sound-attenuating cabin (Amplifon G-type cabin) illuminated by a 20-W white light source suspended 2 m above the apparatus. Background noise of 42 dB sound pressure level was produced by a fan. Animals were subjected to a 10-min session on the day after the final MeHg treatment and every week thereafter. Individuals were tested on the same day of the week at the same time of the day each time. Each experimental group consisted of eight animals.
2.6. Hot plate test The technique was described previously by Forman [13]. Prior to the start of each experiment, animals were brought to the procedure room and allowed to acclimate for 60 min. In this test, a rat is placed on a copper plate heated to a temperature of 55 ± 0.5 °C (UGO Basile, Comerio, VA-Italy). The elapsed time between being placed on the hot plate and reacting to the heated surface by either licking its paws or jumping (all four paws) is measured and expressed as the reaction time. Animals were not permitted to remain on the hot plate for more than 30 s. The experiments were conducted in 90-day-old male rats. Each experimental group consisted of eight animals. 2.7. Statistical analysis Body weight gain, locomotor activity and active avoidance conditioning (four 25-trial blocks) were analysed by two-way ANOVA for repeated measures. Furthermore, the results of active avoidance task of 100 trials were fitted with a best-fit line (linear regression analysis). Differences in the slope of the regression lines were evaluated by the unpaired t-test. Data relative to the time spent in the central part of the OptoVarimex cage in the first minute of the test were converted in logarithms to correct for heterogeneity of variance. Student's t-tests were used to compare brain weights as well as brain weights corrected for changes in body weights and hot plate data. Individual comparisons were performed by Bonferroni's, Tukey's or Dunnett's Multiple Comparison tests where appropriate. Fisher's exact-test was used where appropriate. 3. Results
2.5. Active avoidance conditioning
3.1. Mercury exposure assessment
The procedure has been described previously by Salmi et al. [34]. Experiments were performed by using a device consisting of a two-way avoidance box (UGO Basile, Comerio, VA-Italy) housed inside an isolation cubicle (Coulbourn Instruments, Allentown, PA). Each avoidance box was divided into two compartments connected by an opening of 9 × 12 cm and operated by electromechanical programming equipment. The conditioning stimulus (CS) was an 80 dB tone for 10 s. When the CS was on, the animals had to cross to the other side of the shuttle box apparatus (conditioned avoidance response, CAR) to turn the CS off and to avoid the unconditioned stimulus (US). The US, a 2 s positive half-wave constant current of 0.5 mA intensity, was initiated if the animal failed to make an escape response. Additionally, the latency to escape the aversive stimulus was recorded. Male adult rats (90 days of age) were subjected to a 100 trials session (four 25-trial blocks), with a 50 s intertrial interval. Each experimental group consisted of eight animals. Because a confounding variable in the active avoidance task is the animal sensitivity to foot shock, MeHg-treated rats were also subjected to the hot plate test.
Hg concentration (mean ± S.E.M.) in the brain of MeHgexposed animals 24 h after the final treatment was 0.82 ± 0.05 μg/g tissue (n = 4). 3.2. Observation of deaths, hind-limb crossing, body and brain weight monitoring MeHg treatment did not alter the rate of survival and did not cause overt neurological signs, such as hind-limb crossing (data not shown). Moreover, an overall two-way ANOVA for repeated measures of male rat weight gain gave the following results: Ftreatments = 2.44, df = 1/14, n.s.; Fages = 107.60, df = 9/126, p b 0.0001; Ftreatments × ages = 2.27, df = 9/126, p b 0.05. Individual comparisons (Tukey's Multiple Comparison test) did not show any significant change in body weight gain between MeHgtreated and control rats (Fig. 1). Furthermore, no differences in brain weight were observed (PD24: CTRL = 1.46 ± 0.09; MeHg = 1.34 ± 0.10; PD90: CTRL = 1.74 ± 0.04; MeHg = 1.72 ± 0.06). Finally, when examining brain weight corrected for changes in body weight, statistical analysis did not show any significant difference (PD24: CTRL = 0.026 ± 0.001; MeHg =
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Fig. 1. Effects of postnatal MeHg exposure on body weight gain throughout the treatment period. Data represent mean values ± S.E.M.
0.024 ± 0.0006; PD90: CTRL = 0.004 ± 0.0007; MeHg = 0.004 ± 0.0009). 3.3. Locomotor activity The results obtained in the open field test failed to show any general motor deficit in MeHg-exposed rats. Overall two-way ANOVAs did not show any significant difference for distance
Fig. 2. Effects of postnatal MeHg exposure on distance travelled (A), time spent in the central part of the arena (B) and rearings (C) in male rats on the day after the final MeHg treatment and every week thereafter. Data represent mean values ± S.E.M. ⁎p b 0.05; ⁎⁎p b 0.01 vs. control group.
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travelled (Ftreatments = 2.45, df = 1/14, n.s.; Fweeks = 19.57, df = 3/ 42, p b 0.0001; Ftreatments × weeks = 0.67, df = 3/42, n.s.), resting time (Ftreatments = 4.54, df = 1/14, n.s.; Fweeks = 6.23, df = 3/42, p b 0.005; Ftreatments × weeks = 0.45, df = 3/42, n.s.) and time spent in the central part of the arena in the first min of the test (Ftreatments = 1.00, df = 1/14, n.s.; Fweeks = 2.16, df = 3/42, n.s.; Ftreatments × weeks = 1.78, df = 3/42, n.s.) between MeHg-treated and control groups. However, an overall two-way ANOVA for rearings showed the following difference: Ftreatments = 12.97, df = 1/14, p b 0.005; Fweeks = 5.07, df = 3/42, p b 0.005; Ftreatments × weeks = 0.49, df = 3/42, n.s. Individual comparisons (Bonferroni's Multiple Comparison test) showed that MeHg exposure significantly decreased rearings with respect to control rats (p b 0.05 for 1st and 2nd week; p b 0.01 for 3rd week after the final MeHg administration) (Fig. 2). 3.4. Active avoidance conditioning An over-all two-way repeated measures ANOVA for CARs showed the following differences: (i) between treatments (F = 1.43; df = 1/14; n.s.), (ii) between blocks (F = 8.91; df = 3/ 42; p b 0.0001), (iii) between treatments × blocks (F = 3.68; df = 3/42; p b 0.05). Within-group comparisons (Dunnett's Multiple Comparison test) showed that control animals significantly improved in performance over blocks (3rd block = p b 0.05; 4th block = p b 0.01), providing evidence of associative learning. In contrast, MeHg-treated animals never reached a level of performance that would indicate significant
Fig. 3. Effects of postnatal MeHg exposure on active avoidance learning (A) and escape latency (B) in 90-day-old rats. Data represent mean values ± S.E.M. °p b 0.05; °°p b 0.01 vs. first block (Dunnett's multiple comparison test). CARs = Conditioned Avoidance Responses.
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learning had taken place (Fig. 3A). A further evidence was obtained by modeling the slope of the 100 trials with a best-fit line. The linear regression analysis yielded the following results: CTRL: r2 = 0.6045; slope = 0.06130 ± 0.005, df = 98, p b 0.0001; MeHg: r2 = 0.1031, slope = 0.01156 ± 0.003, df = 98, p b 0.001. Statistical comparison (unpaired t-test) showed a significant difference between the slope of the control group and that of the MeHg-treated group (t = 5.567; df = 198; p b 0.0001). Moreover, an over-all two-way repeated measures ANOVA for escape latency showed the following differences: (i) between treatments (F = 0.94; df = 1/14; n.s.), (ii) between blocks (F = 7.04; df = 3/42; p b 0.0001), (iii) between treatments × blocks (F = 3.39; df = 3/42; p b 0.05). Individual comparisons (Tukey's Multiple Comparison test) showed that MeHg-exposed group did not exhibit any significant difference in the time to escape shock with respect to the age-matched control group (Fig. 3B). 3.5. Hot plate Statistical analysis (Student's t-test) showed that MeHg treatment did not influence the reaction time to the heated surface in the hot plate test (data not shown). 4. Discussion The present findings show that low level exposure to methylmercury during the postnatal brain growth spurt induces subtle and long lasting neurobehavioral dysfunction mainly consisting of motor and learning deficits. MeHg administration to suckling rats for ten consecutive days (PD 14–23) did not result in overt neurological signs of intoxication in comparison with the age-matched cysteinetreated (control) rats. Moreover, no change in the rate of survival was observed at the exposure level used in the present study. No significant difference in body weight gain was observed between MeHg-treated and the age-matched control rats. In this regard, previous data [33] have shown that dose levels between 1-3 mg/kg/day from PD 1 to PD 30 do not induce body weight loss which can be observed with a dose as high as 5 mg/kg/day inducing also ataxia. When MeHg-treated rats were subjected to the open field test (weekly from PD 24 until PD 45), only a significant and persistent reduction in the number of rearings was observed with respect to controls. Rearing is a measure dependent on motor ability, exploratory behavior and emotionality [12,19]. Concerning emotionality, an effect of MeHg treatment can be excluded since the time spent by treated rats in the central part of the arena was not altered with respect to controls. Therefore, the reduced number of rearings observed in MeHg-treated rats may be interpreted as due to motor deficits (specifically weakness in the hind limbs) and impairment in exploratory activity. Impairment in vertical activity has been also observed in young rats exposed in utero to a single dose (8 mg/kg) of MeHg [4], thus underlining that, due to the protracted timetable of motor development, both prenatal and postnatal exposure may result in motor deficits. Rearing is also a measure dependent on balance and equilibrium. In this regard, it is
worth noting that deficits of motor coordination in the rotarod test have been observed in rats exposed to higher doses (1– 3 mg/kg) of MeHg from PD 1 to PD 30 [33]. Results of the conditioned avoidance test provide evidence of a learning deficit in MeHg-treated rats. Unlike control animals showing significant improvement in performance over blocks, treated rats never reached a level of performance that would indicate significant learning had taken place. Hughes and Annau [17] have shown that the number of trials to reach criterion on a two-way avoidance task was significantly increased for MeHgexposed mice at estimated brain doses between 2–4 ppm. The MeHg exposure level used in the present study results in brain Hg concentration of 0.82 μg/g tissue (b1 ppm), which corresponds to the “low-dose groups” on the basis of the estimated brain dose for the Collaborative Behavioral Teratology Study and related studies reporting MeHg-induced deficits in neurobehavioral development [3]. Interestingly, the dose-response study by Sakamoto [33] shows deficits in the passive avoidance task at Hg brain levels higher than 3 ppm which induce also neuronal degeneration in brain regions, such as hippocampus and amygdala, associated with memory function [7]. In our study Hg levels were not determined in control brains. Although there is evidence that mercury can be found in rodent feed [37], only trace amount of this compound are expected in control brains since rats were fed a diet containing Hg 0.06 mg/kg. Finally, the active avoidance task involves also an emotionality factor (response to electric shock). Indeed, this test contains elements of conflict because the animal has to initiate a response towards a location where it previously experienced a noxious stimulus. The fact that MeHg-exposed rats subjected to the two-way avoidance test have the same initial escape latency as the controls, suggests that the emotional response to pain remains unimpaired. Also, and more compellingly, there was no difference between the groups in hot-plate latencies, which supports the lack of difference in pain sensitivity. Gender-related susceptibility to MeHg neurotoxicity has not been addressed in the present study. However, epidemiological [16,23] and experimental studies [14,31] report greater developmental effects in males than in females, likely due to gender-related differences in the activity of antioxidant brain defences as well as in MeHg metabolism. A further consideration should be discussed regarding the environmental enrichment (EE). In our study, animals were reared in typical laboratory environments. Based on the observation that, in rats, EE results in improved performance on learning tasks [30], further investigations are needed in order to explore whether an EE program is able to minimize or prevent the adverse effect of MeHg exposure on cognitive development. This facet raises great concern about the influence of environment on MeHg effects in the exposed children since the environment during the first several years of life in humans may also have profound effects on structure and function of brain areas critical for social and emotional behavior [28]. In conclusion, the results show that brain mercury levels less than 1 ppm during the postnatal growth spurt affects motor and learning ability in young adult rats, thus suggesting a much closer relationship between motor and cognitive development. Since
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Short communication
Effects of low dose methylmercury administration during the postnatal brain growth spurt in rats Addolorata Coluccia a , Pietro Borracci a , Arcangela Giustino a , Mineshi Sakamoto b , Maria Rosaria Carratù a,⁎ a
Department of Pharmacology and Human Physiology, Medical School, University of Bari, Policlinico, Piazza G. Cesare 11, 70124 Bari, Italy b Department of Epidemiology, National Institute for Minamata Disease, 4058-18 Hama, Minamata, Kumamoto 867-0008, Japan Received 14 July 2006; received in revised form 17 October 2006; accepted 17 October 2006 Available online 25 October 2006
Abstract Male Sprague-Dawley rats from eight litters were orally administered 0.75 mg/kg/day methylmercury (MeHg) chloride from postnatal day (PD) 14 to PD 23. One male pup per litter from eight different litters per treatment group was used. Each pup was used only for a single behavioral test and tested once. The MeHg dose level resulted in Hg brain concentrations of 0.82 ± 0.05 μg/g tissue (n = 4). Locomotor behavior was studied in the Opto-Varimex apparatus by testing rats (n = 8) weekly from PD 24 to PD 45. Performance of rats (n = 8) on learning paradigm was analysed on PD 90. MeHg treatment induced a significant reduction in the number of rearings without altering the distance travelled, the resting time and the time spent in the central part of the arena. Results of conditioned avoidance task showed that, unlike control rats, MeHg-treated animals did not show improvement over blocks and never reached a level of performance that would indicate significant learning had taken place. The present results show that low level exposure to MeHg during late brain growth spurt induces subtle and persistent motor and learning deficits, further underlining the serious potential hazard for the exposed children. © 2006 Elsevier Inc. All rights reserved. Keywords: Methylmercury; Neurodevelopment; Locomotor behavior; Associative learning; Rat
1. Introduction Methylmercury, an organic methylated form of mercury, is one of the most hazardous environmental pollutants. The major source of MeHg exposure to the general population is typically through consumption of contaminated fish and other food products [26]. MeHg, absorbed from the gastrointestinal tract, is easily transported across the blood-brain barrier and placenta. Once it is demethylated in the brain, elemental mercury bioaccumulates in the brain tissue [6]. Aschner et al. [1,2] have demonstrated cysteine-facilitated transport of MeHg into the brain, and they also have identified the presence of a neutral aminoacid transport system in astrocytes, which is capable of mediating MeHgcysteine uptake. In rats, the maturation of astrocytes occurs during postnatal developmental stages [36] which correspond to the third trimester of pregnancy in humans [29]. ⁎ Corresponding author. Tel.: +39 080 5478455; fax: +39 080 5478444. E-mail address: [email protected] (M.R. Carratù). 0892-0362/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ntt.2006.10.005
As in humans [8,39], the developing brain in several species ranging from monkeys to mice appears far more susceptible to MeHg than the mature brain [3]. While methylmercury poisoning has been clearly associated with severe neurotoxic effects in both animal studies and human poisoning episodes, the evidence for developmental impairments associated with lower level exposure is less clear [25]. Frank human poisoning, both prenatal and postnatal, has been associated with severe mental retardation. Following exposure of the community of Minamata (Japan) and Iraqi population, the clinical course of surviving victims ranged from cases characterized by low birthweight, microcephaly, profound developmental delay, cerebral palsy, deafness, blindness and seizures [15] to ones with much milder effects including weakness, increased muscle tone, abnormal plantar reflexes, and delays in intellectual and motor development [22]. Severe neurodevelopmental effects were observed in children exposed in utero even when mothers were asymptomatic or had very mild symptoms. Despite the limitations to determine the incidence of developmental disorders
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associated with various exposure levels, the Iraqi population studies identified some dose-related effects of MeHg and raised concern that lower levels of MeHg might be associated with developmental delays [3,24]. The behavioral deficits observed in animal models of MeHg exposure also range from severe to mild depending on the exposure level. Studies reviewed by Burbacher et al. [3] show that, as in humans, also in nonhuman primates and rodents signs of severe toxicity including blindness, seizures, spasticity, quadriplegia may be observed in the most affected animals, whereas more subtle neurobehavioral deficits may be observed in the less affected ones. In this regard, MeHg effects on visual evoked potentials, swimming behavior, open field activity, motor coordination and performance of animals on various learning paradigms have been reported in rodents following preor postnatal exposure to this toxic compound. Therefore, on the basis of clinical and experimental evidence, it is well established that fetuses and neonates are high-risk groups for MeHg exposure, and the MeHg effects may be more or less severe depending on the duration and level of exposure at different developmental stages [11,28]. In this regard, the implications of low level exposure are still controversial [9,21], and the lowest dose of MeHg that might impair neurodevelopment is still unknown. Another intriguing aspect is that, following prenatal or postnatal exposure to MeHg, an infant may develop psychomotor deficits as the nervous system matures. Therefore, great concern has also been raised about the long-term impact of exposure [27], since neurologic effects may continue to manifest themselves throughout life, particularly in aging population. Concerning the critical periods of exposure during development, differences exist between humans and rodents. Rapid brain growth occurs primarily during the third trimester of pregnancy in humans, whereas in rats it occurs after parturition [38]. In particular, cerebellum as well as certain structures important in learning and memory function do not undergo extensive development until late gestation and early postnatal life [28]. While traditional views link dorsolateral prefrontal cortex with complex cognition and cerebellum with motor function, in humans and monkeys there is significant cross-talk between these two brain areas, thus implying a much closer relationship between cognitive and motor development [10]. Therefore, taking into account that the lowest dose of MeHg that might impair neurodevelopment is still unknown, the present study was primarily designed to explore, in the rat, the neurobehavioral effects of a low-level exposure to MeHg during the postnatal brain growth spurt. Taking into account that both motor and cognitive development display protracted developmental timetables and that deficits in both domains are often associated in the same developmental disorders [10], the effects of MeHg on motor and cognitive tasks were evaluated.
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the Declaration of Helsinki and the “Guide for the Care and Use of Laboratory Animals” as adopted and promulgated by the National Institutes of Health. Primiparous Sprague-Dawley female rats (Harlan, S. Pietro al Natisone, Italy) weighing 250–280 g were used. The animals were allowed free access to food and water, were housed at constant room temperature (20–22 °C) and exposed to a light cycle of 12 h/day (08.00 h–20.00 h). Pairs of females were placed with single male rats in the late afternoon. The day on which sperm were present was designated day 0 of gestation (GD 0). Within 24 h after birth, eight litters were reduced to a standard size of eight pups per litter, and one male pup per litter from different litters was used. Pups were weaned at 24 days of age. Male offsprings were orally administered 0.75 mg/kg/day methylmercury chloride on PD14 for 10 consecutive days. MeHg and L-cysteine (SIGMA, Milan, Italy), in a molecular ratio 1:1, were dissolved in 10% condensed milk and were administered with a microman-pipette (Gilson) for the sucklings, according to the method of Sakamoto et al. [32]. Control animals received cysteine only for 10 days. One male pup per litter from eight different litters per treatment group was used. Each pup was used only for a single behavioral test and tested once. 2.2. Mercury determination procedure On the day after the final treatment, four MeHg-exposed rats (one rat per litter from four different litters) were deeply anesthetized by intraperitoneal injection of pentobarbital. In order to flush out blood from the brain, the rats were thoroughly perfused via the heart with physiological saline for 5 min. Then the brains were removed and kept at −80 °C until use. Total Hg concentrations were determined according to the oxygen combustion–gold amalgamation method [18], using a Mercury Analyzer MV 250R (Sugiyama-gen Environmental Science, Tokyo, Japan). 2.3. Observation of deaths, hind-limb crossing, body and brain weight monitoring Rats were examined, weighed and deaths noted on a daily basis throughout the treatment period. The development of hind-limb crossing was also evaluated. An experimenter, blind to the animal's treatment condition, held a rat by the base of the tail for 2–5 s (perpendicular to the floor) and recorded the position of the hind limbs [20]. Results were coded with a “0” (normal) or a “1” (impaired). Brain weights were monitored on PDs 24 and 90 (one rat per litter from five different litters per tested age and per treatment group).
2. Methods
2.4. Locomotor activity
2.1. Animals and treatment schedule
Motor activity was recorded in an Opto-Varimex apparatus linked to an IBM PC (Columbus Instruments, Columbus, OH) according to the method described by Wedzony et al. [35]. The apparatus consisted of a cage (42 × 42 × 30 cm) equipped with
The experiments have been conducted in accordance with guidelines released by Italian Ministry of Health (D.L. 116/92),
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15 infrared emitters (spaced at 2.65-cm intervals) located on the x and y axes 2–3 cm above the floor of the cage (depending on the size of the animal) and an equivalent number of receivers located on the opposite walls. A further line of emitter/receiver pairs was located ≈ 5 cm (depending on the size of the animal) above the floor of the cage to detect vertical movements (i.e., rearings). Each interruption of a beam generates an electric impulse scored by a digital counter. 2.4.1. Procedure The amount of time spent in ambulatory activity was analyzed by using AUTO-TRACK software (Columbus Instruments, Columbus, OH). Ambulatory activity, defined as a trespass of three consecutive photo-beams, and resting time, calculated as the amount of time during which there were no ambulatory movements, were recorded. Vertical activity was measured by recording the number of horizontal beams that were broken by the rearings of the animal. Furthermore, the amount of time spent in the central part of the arena was analyzed in the first min of test by using the Time In Square (TIS) analysis (AUTO-TRACK software). TIS analysis divides the Opto-Varimex cage area into a four by four grid of sixteen squares. The time the subject is in a square is calculated for each cage interval. The time is calculated to one-tenth of a second accuracy using the raw X–Y coordinate data file. Tests were carried out in a 1 × 1 × 2 m sound-attenuating cabin (Amplifon G-type cabin) illuminated by a 20-W white light source suspended 2 m above the apparatus. Background noise of 42 dB sound pressure level was produced by a fan. Animals were subjected to a 10-min session on the day after the final MeHg treatment and every week thereafter. Individuals were tested on the same day of the week at the same time of the day each time. Each experimental group consisted of eight animals.
2.6. Hot plate test The technique was described previously by Forman [13]. Prior to the start of each experiment, animals were brought to the procedure room and allowed to acclimate for 60 min. In this test, a rat is placed on a copper plate heated to a temperature of 55 ± 0.5 °C (UGO Basile, Comerio, VA-Italy). The elapsed time between being placed on the hot plate and reacting to the heated surface by either licking its paws or jumping (all four paws) is measured and expressed as the reaction time. Animals were not permitted to remain on the hot plate for more than 30 s. The experiments were conducted in 90-day-old male rats. Each experimental group consisted of eight animals. 2.7. Statistical analysis Body weight gain, locomotor activity and active avoidance conditioning (four 25-trial blocks) were analysed by two-way ANOVA for repeated measures. Furthermore, the results of active avoidance task of 100 trials were fitted with a best-fit line (linear regression analysis). Differences in the slope of the regression lines were evaluated by the unpaired t-test. Data relative to the time spent in the central part of the OptoVarimex cage in the first minute of the test were converted in logarithms to correct for heterogeneity of variance. Student's t-tests were used to compare brain weights as well as brain weights corrected for changes in body weights and hot plate data. Individual comparisons were performed by Bonferroni's, Tukey's or Dunnett's Multiple Comparison tests where appropriate. Fisher's exact-test was used where appropriate. 3. Results
2.5. Active avoidance conditioning
3.1. Mercury exposure assessment
The procedure has been described previously by Salmi et al. [34]. Experiments were performed by using a device consisting of a two-way avoidance box (UGO Basile, Comerio, VA-Italy) housed inside an isolation cubicle (Coulbourn Instruments, Allentown, PA). Each avoidance box was divided into two compartments connected by an opening of 9 × 12 cm and operated by electromechanical programming equipment. The conditioning stimulus (CS) was an 80 dB tone for 10 s. When the CS was on, the animals had to cross to the other side of the shuttle box apparatus (conditioned avoidance response, CAR) to turn the CS off and to avoid the unconditioned stimulus (US). The US, a 2 s positive half-wave constant current of 0.5 mA intensity, was initiated if the animal failed to make an escape response. Additionally, the latency to escape the aversive stimulus was recorded. Male adult rats (90 days of age) were subjected to a 100 trials session (four 25-trial blocks), with a 50 s intertrial interval. Each experimental group consisted of eight animals. Because a confounding variable in the active avoidance task is the animal sensitivity to foot shock, MeHg-treated rats were also subjected to the hot plate test.
Hg concentration (mean ± S.E.M.) in the brain of MeHgexposed animals 24 h after the final treatment was 0.82 ± 0.05 μg/g tissue (n = 4). 3.2. Observation of deaths, hind-limb crossing, body and brain weight monitoring MeHg treatment did not alter the rate of survival and did not cause overt neurological signs, such as hind-limb crossing (data not shown). Moreover, an overall two-way ANOVA for repeated measures of male rat weight gain gave the following results: Ftreatments = 2.44, df = 1/14, n.s.; Fages = 107.60, df = 9/126, p b 0.0001; Ftreatments × ages = 2.27, df = 9/126, p b 0.05. Individual comparisons (Tukey's Multiple Comparison test) did not show any significant change in body weight gain between MeHgtreated and control rats (Fig. 1). Furthermore, no differences in brain weight were observed (PD24: CTRL = 1.46 ± 0.09; MeHg = 1.34 ± 0.10; PD90: CTRL = 1.74 ± 0.04; MeHg = 1.72 ± 0.06). Finally, when examining brain weight corrected for changes in body weight, statistical analysis did not show any significant difference (PD24: CTRL = 0.026 ± 0.001; MeHg =
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Fig. 1. Effects of postnatal MeHg exposure on body weight gain throughout the treatment period. Data represent mean values ± S.E.M.
0.024 ± 0.0006; PD90: CTRL = 0.004 ± 0.0007; MeHg = 0.004 ± 0.0009). 3.3. Locomotor activity The results obtained in the open field test failed to show any general motor deficit in MeHg-exposed rats. Overall two-way ANOVAs did not show any significant difference for distance
Fig. 2. Effects of postnatal MeHg exposure on distance travelled (A), time spent in the central part of the arena (B) and rearings (C) in male rats on the day after the final MeHg treatment and every week thereafter. Data represent mean values ± S.E.M. ⁎p b 0.05; ⁎⁎p b 0.01 vs. control group.
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travelled (Ftreatments = 2.45, df = 1/14, n.s.; Fweeks = 19.57, df = 3/ 42, p b 0.0001; Ftreatments × weeks = 0.67, df = 3/42, n.s.), resting time (Ftreatments = 4.54, df = 1/14, n.s.; Fweeks = 6.23, df = 3/42, p b 0.005; Ftreatments × weeks = 0.45, df = 3/42, n.s.) and time spent in the central part of the arena in the first min of the test (Ftreatments = 1.00, df = 1/14, n.s.; Fweeks = 2.16, df = 3/42, n.s.; Ftreatments × weeks = 1.78, df = 3/42, n.s.) between MeHg-treated and control groups. However, an overall two-way ANOVA for rearings showed the following difference: Ftreatments = 12.97, df = 1/14, p b 0.005; Fweeks = 5.07, df = 3/42, p b 0.005; Ftreatments × weeks = 0.49, df = 3/42, n.s. Individual comparisons (Bonferroni's Multiple Comparison test) showed that MeHg exposure significantly decreased rearings with respect to control rats (p b 0.05 for 1st and 2nd week; p b 0.01 for 3rd week after the final MeHg administration) (Fig. 2). 3.4. Active avoidance conditioning An over-all two-way repeated measures ANOVA for CARs showed the following differences: (i) between treatments (F = 1.43; df = 1/14; n.s.), (ii) between blocks (F = 8.91; df = 3/ 42; p b 0.0001), (iii) between treatments × blocks (F = 3.68; df = 3/42; p b 0.05). Within-group comparisons (Dunnett's Multiple Comparison test) showed that control animals significantly improved in performance over blocks (3rd block = p b 0.05; 4th block = p b 0.01), providing evidence of associative learning. In contrast, MeHg-treated animals never reached a level of performance that would indicate significant
Fig. 3. Effects of postnatal MeHg exposure on active avoidance learning (A) and escape latency (B) in 90-day-old rats. Data represent mean values ± S.E.M. °p b 0.05; °°p b 0.01 vs. first block (Dunnett's multiple comparison test). CARs = Conditioned Avoidance Responses.
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learning had taken place (Fig. 3A). A further evidence was obtained by modeling the slope of the 100 trials with a best-fit line. The linear regression analysis yielded the following results: CTRL: r2 = 0.6045; slope = 0.06130 ± 0.005, df = 98, p b 0.0001; MeHg: r2 = 0.1031, slope = 0.01156 ± 0.003, df = 98, p b 0.001. Statistical comparison (unpaired t-test) showed a significant difference between the slope of the control group and that of the MeHg-treated group (t = 5.567; df = 198; p b 0.0001). Moreover, an over-all two-way repeated measures ANOVA for escape latency showed the following differences: (i) between treatments (F = 0.94; df = 1/14; n.s.), (ii) between blocks (F = 7.04; df = 3/42; p b 0.0001), (iii) between treatments × blocks (F = 3.39; df = 3/42; p b 0.05). Individual comparisons (Tukey's Multiple Comparison test) showed that MeHg-exposed group did not exhibit any significant difference in the time to escape shock with respect to the age-matched control group (Fig. 3B). 3.5. Hot plate Statistical analysis (Student's t-test) showed that MeHg treatment did not influence the reaction time to the heated surface in the hot plate test (data not shown). 4. Discussion The present findings show that low level exposure to methylmercury during the postnatal brain growth spurt induces subtle and long lasting neurobehavioral dysfunction mainly consisting of motor and learning deficits. MeHg administration to suckling rats for ten consecutive days (PD 14–23) did not result in overt neurological signs of intoxication in comparison with the age-matched cysteinetreated (control) rats. Moreover, no change in the rate of survival was observed at the exposure level used in the present study. No significant difference in body weight gain was observed between MeHg-treated and the age-matched control rats. In this regard, previous data [33] have shown that dose levels between 1-3 mg/kg/day from PD 1 to PD 30 do not induce body weight loss which can be observed with a dose as high as 5 mg/kg/day inducing also ataxia. When MeHg-treated rats were subjected to the open field test (weekly from PD 24 until PD 45), only a significant and persistent reduction in the number of rearings was observed with respect to controls. Rearing is a measure dependent on motor ability, exploratory behavior and emotionality [12,19]. Concerning emotionality, an effect of MeHg treatment can be excluded since the time spent by treated rats in the central part of the arena was not altered with respect to controls. Therefore, the reduced number of rearings observed in MeHg-treated rats may be interpreted as due to motor deficits (specifically weakness in the hind limbs) and impairment in exploratory activity. Impairment in vertical activity has been also observed in young rats exposed in utero to a single dose (8 mg/kg) of MeHg [4], thus underlining that, due to the protracted timetable of motor development, both prenatal and postnatal exposure may result in motor deficits. Rearing is also a measure dependent on balance and equilibrium. In this regard, it is
worth noting that deficits of motor coordination in the rotarod test have been observed in rats exposed to higher doses (1– 3 mg/kg) of MeHg from PD 1 to PD 30 [33]. Results of the conditioned avoidance test provide evidence of a learning deficit in MeHg-treated rats. Unlike control animals showing significant improvement in performance over blocks, treated rats never reached a level of performance that would indicate significant learning had taken place. Hughes and Annau [17] have shown that the number of trials to reach criterion on a two-way avoidance task was significantly increased for MeHgexposed mice at estimated brain doses between 2–4 ppm. The MeHg exposure level used in the present study results in brain Hg concentration of 0.82 μg/g tissue (b1 ppm), which corresponds to the “low-dose groups” on the basis of the estimated brain dose for the Collaborative Behavioral Teratology Study and related studies reporting MeHg-induced deficits in neurobehavioral development [3]. Interestingly, the dose-response study by Sakamoto [33] shows deficits in the passive avoidance task at Hg brain levels higher than 3 ppm which induce also neuronal degeneration in brain regions, such as hippocampus and amygdala, associated with memory function [7]. In our study Hg levels were not determined in control brains. Although there is evidence that mercury can be found in rodent feed [37], only trace amount of this compound are expected in control brains since rats were fed a diet containing Hg 0.06 mg/kg. Finally, the active avoidance task involves also an emotionality factor (response to electric shock). Indeed, this test contains elements of conflict because the animal has to initiate a response towards a location where it previously experienced a noxious stimulus. The fact that MeHg-exposed rats subjected to the two-way avoidance test have the same initial escape latency as the controls, suggests that the emotional response to pain remains unimpaired. Also, and more compellingly, there was no difference between the groups in hot-plate latencies, which supports the lack of difference in pain sensitivity. Gender-related susceptibility to MeHg neurotoxicity has not been addressed in the present study. However, epidemiological [16,23] and experimental studies [14,31] report greater developmental effects in males than in females, likely due to gender-related differences in the activity of antioxidant brain defences as well as in MeHg metabolism. A further consideration should be discussed regarding the environmental enrichment (EE). In our study, animals were reared in typical laboratory environments. Based on the observation that, in rats, EE results in improved performance on learning tasks [30], further investigations are needed in order to explore whether an EE program is able to minimize or prevent the adverse effect of MeHg exposure on cognitive development. This facet raises great concern about the influence of environment on MeHg effects in the exposed children since the environment during the first several years of life in humans may also have profound effects on structure and function of brain areas critical for social and emotional behavior [28]. In conclusion, the results show that brain mercury levels less than 1 ppm during the postnatal growth spurt affects motor and learning ability in young adult rats, thus suggesting a much closer relationship between motor and cognitive development. Since
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