Maternal Milk as Methylmercury Source for Suckling Mice: Neurotoxic Effects Involved with the Cerebellar Glutamatergic System

C. B. Manfroi*, F. D. Schwalm*, V. Cereser*, F. Abreu*, A. Oliveira{dagger}, L. Bizarro{dagger}, J. B. T. Rocha{ddagger}, M. E. S. Frizzo*,§, D. O. Souza* and M. Farina,1

* Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, 90035-003, Porto Alegre, RS, Brazil; {dagger} Laboratório de Psicologia Experimental, Neurociências e Comportamento, Instituto de Psicologia, Universidade Federal do Rio Grande do Sul, 90035-003, Porto Alegre, RS, Brazil; {ddagger} Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil; § Departamento de Ciências Morfológicas, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, 90035-003, Porto Alegre, RS, Brasil; and Departamento de Análises Clínicas e Toxicológicas, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, Campus Universitário, Camobi, 97105-900, Santa Maria, RS, Brazil

Received April 15, 2004; accepted June 10, 2004


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Methylmercury (MeHg) is a highly neurotoxic compound and several studies have reported intoxication signs in children whose mothers were exposed to this environmental toxicant. Although it is well established that the in utero exposure to MeHg causes neurological deficits in animals and humans, there is no evidence of the exclusive contribution of lactational exposure to MeHg as a possible cause of neurotoxicity in the offspring. In this study, we investigated the exclusive contribution of MeHg exposure through maternal milk on biochemical parameters related to the glutamatergic homeostasis (glutamate uptake by slices) and to the oxidative stress (total and nonprotein sulfhydryl groups, nonprotein hydroperoxides, glutathione peroxidase and catalase activities) in the cerebellum of suckling mice (Swiss albino). The same parameters were also evaluated in the cerebellum of mothers. Our results showed, for the first time, that lactational exposure to MeHg caused a high percent of inhibition (50%) on glutamate uptake by cerebellar slices in pups. Contrarily, this effect was not observed in mothers, which were submitted to a direct oral exposure to MeHg (15 mg/l in drinking water). In addition, behavioral/functional changes were observed in the weaning mice exposed to MeHg. It was observed an increase in the levels of nonprotein hydroperoxides in cerebellum, and this increase was negatively correlated to the glutamate uptake by cerebellar slices. This study indicates that (1) the exposure of lactating mice to MeHg causes inhibition of the glutamate uptake by cerebellar slices in the offspring; (2) this inhibitory effect seems to be related to increased levels of hydroperoxide.

Key Words: methylmercury; maternal milk; glutamate; oxidative stress; hydroperoxide; antioxidant enzymes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mercury is present in the environment due to natural events (volcanic eruptions), as well as anthropogenic sources (burning of coal and chemical processing). Within the environment, mercury exists in three different chemical forms: elemental mercury vapor, inorganic mercury salts, and organic mercury (Clarkson, 1997Go). The distribution, metabolism, and toxicity of mercury are largely dependent upon its chemical form. In this regard, organic mercury compounds, such as methylmercury (MeHg), have been extensively studied since they are able to reach high levels in the central nervous system (CNS) leading to neurotoxic effects (Aschner and Clarkson, 1988Go; Clarkson et al., 2003Go).

During the early postnatal period the brain is extremely sensitive to external agents, including MeHg. At this developmental stage, a substantial acceleration of the synthesis of cerebral RNA, DNA, protein, and myelin is observed (Gottlieb et al., 1977Go). In particular, this period is characterized by intense gliogenesis (mainly astrocytes, important to glutamate uptake and MeHg storage). In this regard, it is important to state that the exposure of pregnant women to MeHg can lead to indirect intoxication of their sons (Harada, 1995Go; Weihe et al., 2002Go) and some studies suggest that the fetal exposure to MeHg causes neurological deficits in the offspring (Grandjean et al., 1997Go; Murata et al., 2004Go).

Although it is well established that the in utero exposure to MeHg causes neurological deficits in animals (Sakamoto et al., 2002aGo) and humans (Grandjean et al., 1997Go), there is no evidence of the exclusive contribution of lactational exposure to MeHg as a possible cause of neurotoxicity in the offspring. In fact, studies correlating the exposure of mothers to MeHg with neurotoxic effects in the offspring (Sakamoto et al., 2002aGo) do not separate these two phases of indirect exposure (transplacental and through maternal milk), making difficult to comprehend the real contribution of both phenomena to the development of neurotoxicity.

Oxidative stress (Ou et al., 1999Go), impairment of intracellular calcium homeostasis (Danbolt, 2001Go; Sirois and Atchison, 2000Go), and inhibition of glutamate uptake by astrocytes (Aschner et al., 2000Go; Farina et al., 2003bGo) are mechanisms involved in MeHg-induced neurotoxicity. In this regard, a recent study of our group (Porciúncula et al., 2003Go) demonstrated the inhibitory effect of MeHg in glutamate uptake by synaptic vesicles from rat brain. In addition, we also showed the inhibitory effect of the oral exposure to MeHg on glutamate uptake by cerebral slices from adult mice (Farina et al., 2003aGo). Thus, the hazardous effects of MeHg on glutamate homeostasis both under in vitro and in vivo conditions are well described.

As pointed out above, oxidative stress is one of the main mechanisms related to MeHg-induced neurotoxicity. Yee and Choi (1994)Go showed a decrease in the activity of antioxidant enzymes and an increase in the levels of superoxide anion and hydrogen peroxide in several cellular fractions of brain of mice exposed to MeHg. Moreover, the same authors (Yee and Choi, 1996Go) observed an increase of reactive oxygen species and a decrease in glutathione levels in cultures of cerebral oligodendrocytes, astrocytes, and neurons of rats after in vitro exposure to MeHg.

Although the molecular mechanisms involved in MeHg-induced neurotoxicity are numerous, there is a relationship between some of them. In fact, MeHg-induced glutamatergic excitotoxicity causes hyperactivation of N-methyl-D-aspartate (NMDA) type glutamate receptors, leading to an increase in the Na+ and Ca2+ influxes (Choi, 1992Go). Increased intracellular Ca2+ levels are associated with the generation of reactive oxygen species, which are hazardous to the structural components of cells (Lafon-Cazel et al., 1993Go). Conversely, reactive oxygen species, which are excessively generated in the presence of MeHg, inhibit the transport of excitatory amino acids into the astrocytes (Allen et al., 2001Go), leading to excitotoxicity.

Taking into account the absence of studies showing the real contribution of the lactational MeHg exposure to the development of neurotoxicity in the offspring, and the relationship between the pro-oxidative properties of MeHg and its effects on glutamatergic transmission, this study was aimed to investigate the neurotoxic effects of the exclusive lactational exposure to MeHg in suckling mice. Biochemical parameters related to the oxidative stress were analyzed in cerebellum and correlated to the glutamate uptake by cerebellar slices. These variables were analyzed in offspring, as well as in their respective mothers. Since exposure to MeHg during gestational and suckling periods can result in behavioral alterations, we also examined the presence of negative signs of behavioral changes in offspring exposed to MeHg only during lactation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals. L-[3H]glutamate (48 Ci/mmol) was from Amersham International (UK). Methylmercury (II) chloride was from Aldrich Chemical Co. (Milwaukee, WI). All other chemicals were of analytical grade from Merck (Darmstadt, Germany).

Animals. Adult Swiss Albino mice (male and female), 90 days old, from our own breeding colony were maintained at 25°C, on a 12:12 h light/dark cycle, with free access to food (Nuvital, PR, Brazil) and water. The breeding regimen consisted of grouping three virgin females with one male for five days. Pregnant mice were selected and housed individually in opaque plastic cages.

Treatment. In the first day after parturition (postnatal day 1), 14 dams were randomly assigned to one of two groups (control and Hg) of seven animals each. Pups (eight per litter) were maintained with their mothers, which were immediately exposed to MeHg through the ingested water. Mothers from Hg group received a solution of MeHg (15 mg/l) diluted in tap water ad libitum as sole source of liquid. MeHg dose was based in previous study (Farina et al., 2003aGo). Mothers from control group received tap water ad libitum. Thus, the exclusive way of offspring exposure to MeHg was through maternal milk. The possibility of direct consumption of MeHg-containing drinking water by pups was excluded because the dispensing ends of the water tubes were not accessible to pups even at postnatal day 21. Liquid and solid ingestions of litter mothers were monitored daily. All experiments were conducted in accordance with the Guiding Principles in the Use of Animals in Toxicology, adopted by the Society of Toxicology in July 1989.

Behavior. At postnatal day 21, two weaning mice from each litter were randomly selected for behavioral experiments—–SHIRPA protocol (Irwin, 1968Go). This standard method provides a behavioral and functional profile by observational assessment of mice. These tests indicate defects in gait or posture, motor control and coordination, changes in excitability and aggression, salivation, lacrimation, piloerection, defecation, muscle tone, and temperature. It also provides a gross measure of analgesia. All parameters are scored to provide a quantitative assessment and all observational assessments were blind.

Tissue preparation. At postnatal day 21, two randomly selected weaning mice, and their respective mothers, were killed by decapitation. Since gender effects are negligible at the weaning period, sex was considered unimportant in selecting the pups. Right cerebellar hemispheres were homogenated (1:7 w/v) in sodium phosphate buffer (50 mM; pH 7.0) and the tissue homogenates were used in the preparation of samples to the determination of the activities of antioxidant enzymes and levels of nonprotein hydroperoxide and sulfhydryl groups from total and nonprotein sources. Left cerebellar hemispheres were used to the preparation of cerebellar slices (0.4 mm) according to Frizzo et al. (2002)Go. It is important to state that the cerebellum was the chosen encephalic structure due to the high MeHg affinity by cerebellar granular cells (Klein et al., 1972Go).

Antioxidant enzymes. Cerebellar homogenates were centrifuged for 10 min at 15,800 x g at 4°C and the supernatant fraction was used.

Glutathione peroxidase (GSHPx) activity was measured by the method of Paglia and Valentine (1967)Go. In short, cerebellar supernatant fraction (200–400 µg protein) was added to the assay mixture and the reaction was started by the addition of H2O2 (final concentration, 0.4 mM). Oxidation of NADPH to NADP + was monitored continuously at 340 nm for 10 min. GSHPx activity was expressed as nmol NADPH oxidized/(min/mg protein), using a molar extinction coefficient of 6.22 x 106 for NADPH.

Catalase activity was measured by the method of Aebi (1974)Go. Cerebellar supernatant fraction (200–500 µg protein) was added to a cuvette and the reaction was started by the addition of 100 µl of freshly prepared 30 mM H2O2 in phosphate buffer (50 mM, pH 7.0; total volume of incubation: 1 ml). The rate of H2O2 decomposition was measured spectrophotometrically at 240 nm during 60 s. The activity of catalase was expressed as k/(s/mg of protein), where k is the rate constant of a first-order reaction.

Determination of tissue nonprotein hydroperoxides and sulfhydryl groups. The levels of nonprotein hydroperoxides were analyzed based on Wolff (1994)Go, with some modifications. In short, an aliquot of the cerebellar homogenate (100 µl) was added to the same volume of 20% trichloroacetic acid. After mixing and centrifugation at 4000 x g during 10 min, at 4°C, the protein pellet was discarded and hydroperoxides levels were determined using the Fe(III) xylenol orange complex formation (Wolff, 1994Go). Total and nonprotein sulfhydryl groups were measured according to Ellman (1959)Go.

Glutamate uptake by cerebellar slices. Experiments on glutamate uptake were based on Frizzo et al. (2002)Go. In short, left cerebellar hemispheres were dissected and coronal slices (0.4 mm) were obtained using a McIlwain tissue chopper. The slices were washed with Hank's balanced salt solution (HBSS) and the sections were finally separated with the help of a magnifying glass for measuring glutamate uptake. Uptake was assessed at 35°C by adding 100 µM glutamate (0.33 µCi L-[3H]glutamate/ml) in HBSS. Uptake was terminated after 7 min by washing twice with ice-cold 1 ml of HBSS and immediately added 0.5 N NaOH, which was kept overnight. Aliquots of lysates were taken for determination of intracellular content of L-[3H]glutamate by scintillation counting and for protein measurement. In order to determine the actual glutamate uptake, parallel experiments were performed under ice and using N-methyl-D-glucamine instead of sodium chloride in the incubation medium, being subtracted from the uptake at 35°C. Glutamate uptake rates were linear with respect to time of incubation for all experimental conditions, according previous standardization (data not shown).

Protein measurement. Protein was measured by the method of Bradford (1976)Go using bovine serum albumin as standard.

Statistical analysis. Differences between groups (treatment and age effects) were evaluated by one-way ANOVA, followed by Duncan's multiple range tests when appropriate. Two-way ANOVA was also performed: two treatments (control and MeHg) x two different ages (mothers and pups). Pearson Correlation was used to evidence a possible correlation between glutamate uptake and hydroperoxide levels. In the behavioral studies, as the data distribution was not normal, a nonparametric analysis (Mann-Whitney test) was performed to detect differences between groups.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Table 1 shows the values of body weight and solid and liquid ingestions of mothers, as well as body weight of weaning mice at the end of treatment. Solid and liquid ingestions and body weight of mothers did not differ between groups. However, suckling mice indirectly exposed to MeHg presented lower body weight when compared to control group. For mothers, a daily MeHg dose of 8.25 mg/kg (about 6.5 mg Hg/kg) was calculated based on their daily liquid ingestion. This represents a total dose of 136.5 mg of Hg to which the dams were exposed in the 21-day period.


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TABLE 1 Body Weight (Mothers and Offspring) at the End of the Treatment, and Liquid and Solid Ingestions (Mothers)

 
In behavioral tests (SHIRPA protocol), three significant differences were observed for the functional profile in weaning mice (Fig. 1). Exposure to MeHg in lactation increased tremors, increased peripheral analgesia in toe pinch test (gentle lateral compression of mid digit of hind foot with fine forceps), and decreased the ability to grasp with hind legs (Wire Maneuver test).



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FIG. 1. Behavioral profile of weaning mice obtained with SHIRPA protocol. Two weaning mice were randomly selected as representative of their respective litters (seven per group). Since SHIRPA protocol yields individual scores and the data distribution was not normal, a nonparametric analysis was performed to detect differences between groups. *Statistically different (p < 0.05) by Mann-Whitney test.

 
Values of glutamate uptake are depicted in Figure 2. Although there were no differences between the values of glutamate uptake for mothers of both groups, the MeHg-exposed offspring showed lower glutamate uptake values when compared to control.



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FIG. 2. Glutamate uptake by cerebellar slices. The values of glutamate uptake are expressed as nmol/mg protein/min and represented as mean ± SEM. Two weaning mice were randomly selected from each litter and the mean of their values of glutamate uptake was used in statistical analysis as representative of the respective litter. The different numbers (1, 2) above bars indicate significant differences (p < 0.05) between groups (effects of MeHg exposure) by one-way ANOVA. Mothers (n = 7 per group). Offspring (n = 7 litters per group; two pups per litter).

 
Figure 3 represents the cerebellar levels of total sulfhydryl groups, nonprotein sulfhydryl groups (NPSH) and nonprotein hydroperoxide. Although MeHg exposure did not change the levels of total and nonprotein sulfhydryl groups both in mothers and weaning mice, it was observed an increase in hydroperoxide levels in MeHg exposed pups when compared to control. In this regard, two-way ANOVA reveled a significant interaction (F3,27 = 5.75; p = 0.025) between treatment (control and MeHg) and age (mothers and offspring). In addition, a negative correlation (Pearson's coefficient = –0.557; p = 0.025) was observed between hydroperoxide levels and glutamate uptake in weaning mice. For mothers, the correlation between glutamate uptake and hydroperoxide levels was not significant (Pearson's coefficient = – 0.283; p = 0.326). The levels of total and nonprotein sulfhydryl groups were lower and the levels of nonprotein hydroperoxides were higher in the offspring when compared to mothers (Fig. 3).



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FIG. 3. Nonprotein sulfhydryl groups (NPSH), total sulfhydryl groups (total –SH) and hydroperoxides from mice cerebellum. The values of NPSH (A), total –SH (B), and hydroperoxides (C) are expressed as nmol/mg protein and represented as mean ± SEM. Two weaning mice were randomly selected from each litter and the means of their values were used in statistical analysis as representative of the respective litter. The different numbers (1, 2) above bars indicate significant differences (p < 0.05) between groups (effects of MeHg exposure) by one-way ANOVA. The different letters (a, b) above bars indicate significant differences (p < 0.05) between control dams and control offsprings by one-way ANOVA. Mothers (n = 7 per group). Offspring (n = 7 litters per group; two pups per litter).

 
The activities of cerebellar catalase and glutathione peroxidase are depicted in Figure 4. MeHg did not affect catalase activity both in mothers and offspring (Fig. 4A). However, a higher activity was observed in offspring when compared to mothers. It is important to state that there was a positive correlation (Pearson's coefficient = 0.472; p = 0.031) between the levels of nonprotein hydroperoxide and catalase activity when mothers and offspring were analyzed together. MeHg exposure inhibited the activity of cerebellar glutathione peroxidase (Fig. 4B) only in mothers. Moreover, glutathione peroxidase activity was higher in mothers when compared to offspring.



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FIG. 4. Cerebellar catalase and glutathione peroxidase activities. Catalase activity (A) is expressed as mmol of H2O2 degraded/min/mg protein. Glutathione peroxidase activity (B) is expressed as nmol NADPH oxidized/min/mg protein. Data are represented as mean ± SEM. Two weaning mice were randomly selected from each litter and the means of their values of enzymatic activity were used in statistical analysis as representative of the respective litter. The different numbers (1, 2) above bars indicate significant differences (p < 0.05) between groups (effects of MeHg exposure) by one-way ANOVA. The different letters (a, b) above bars indicate significant differences (p < 0.05) between control dams and control offsprings by one-way ANOVA. Mothers (n = 7 per group). Offspring (n = 7 litters per group; two pups per litter).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There are several studies showing that the pre- and postnatal exposures to MeHg cause behavioral changes in animals (Goulet et al., 2003Go; Kim et al., 2000Go; Sakamoto et al., 2002aGo). In addition, evidence show the existence of neurological deficits in children whose mothers were exposed to MeHg during the gestational period (Aschner et al., 2000Go; Harada, 1995Go; Grandjean et al., 1997Go; Weihe et al., 2002Go). However, studies on the lactational MeHg exposure are scarce, pointing to transplacental exposure as the main cause of neurotoxicity in MeHg-exposed offsprings. In this regard, a comparative study from the Iraq outbreak showed that prenatal MeHg exposure is more toxic than exposure during lactation (Amin-Zaki et al., 1976Go). In addition, Grandjean et al. (1995)Go reported that MeHg exposure from human milk had no adverse effects on developmental scores in infants and Sakamoto et al. (2002b)Go showed a declining risk of methylmercury exposure to infants during lactation when compared to gestational period. These same researchers (Sakamoto et al., 2002aGo) showed a gradual reduction of blood mercury levels in newborn rats after birth, even when the direct exposure of mothers to MeHg was maintained. The results of our study pointed to neurotoxic effects (biochemical and behavioral changes) as consequence of the exclusive lactational MeHg exposure in suckling mice. From a toxicological point of view, these results represent an important finding because they show, for the first time, that the exposure of mothers to MeHg during the lactational period can, per se, cause neurotoxic effects in the offspring, without the need of previous in utero intoxication. Nevertheless, it is important to state that the dose used was relatively high and the equivalent dose in humans has yet to be established.

Evidence shows that MeHg-induced neurotoxicity is related to the hyperactivation of the glutamatergic system (Allen et al., 2002Go). In fact, MeHg seems to affect glutamatergic homeostasis both under in vitro (Aschner et al., 2000Go) and in vivo (Farina et al., 2003aGo,bGo) conditions, leading to an increase in the extracellular glutamate levels that, in turn, causes excitotoxicity. The results of the present study point to the cerebellum as a structural target for the inhibitory effects of MeHg on glutamate uptake. Although some studies on rodents have reported delayed or abnormal development of the cerebellum, especially the granular layers, after developmental treatment with MeHg (Chang et al., 1977Go; Choi et al., 1981Go), our finds are the first to show decreased glutamate uptake in this structure associated with increase in pro-oxidative parameters. Taking into account the relationship between cerebellar function and motor activity, evidence shows motor impairment in animals (Inouye et al., 1985Go) and humans (Castoldi et al., 2003Go) after MeHg exposure. In accordance with this, we also observed changes in gross motor behavior, i.e., an accentuated increase in tremors and a decreased ability to grasp with hind legs, which can be linked to changes in glutamate transport in cerebellum.

Another important data from our study is that the inhibitory effect of MeHg on glutamate uptake by cerebellar slices was observed only in weaning mice and not in their respective mothers. Why the fetus displays different neuropathological effects and a higher sensitivity to methylmercury relative to the adult is still unknown (Castoldi et al., 2003Go). However, it is reasonable to suppose that, due to the immaturity of central nervous system in the offspring, higher MeHg levels pass through blood brain barrier to brain and cerebellum. In line with this idea, Watanabe and collaborators (1999)Go showed higher mercury concentrations in the offsprings' brains when compared to the respective mothers, which were directly exposed to MeHg. It is also important to imply that the levels of sulfhydryl-containing biomolecules, such as glutathione (main intracellular antioxidant) and proteins (e.g., metallothioneins), are lower in the cerebellum of weaning mice when compared to their mothers. This phenomenon can be related, at least in part, to the offsprings higher sensitivity to the neurotoxic effects of MeHg.

From a mechanistic point of view, we propose that the inhibitory effect of MeHg on glutamate uptake is related to its pro-oxidative properties. In this regard, MeHg exposure caused an increase in the levels of nonprotein hydroperoxide in the cerebellum of weaning mice. Taking into account that in vitro studies point to hydrogen peroxide as a major inhibitor of the glutamate transport through astrocytes (Allen et al., 2001Go) and that a negative correlation between cerebellar hydroperoxide levels and the glutamate uptake by cerebellar slices was observed in weaning mice, it is reasonable to suppose a relationship between both phenomena. Although the oxidative effects of MeHg toward sulfhydryl groups is well established (Farina et al., 2004Go; Yee and Choi, 1996Go), the lack of effects of MeHg in the total and nonprotein thiol levels in cerebellum can be related, at least in part, to the high levels of sulfhydryl-containing molecules in mouse cerebellum (around 4 and 15 nmol –SH/mg protein for nonprotein and total thiols, respectively). In addition, it is important to state that MeHg is able to interact with nucleophilic sites distinct from thiols groups, including carboxyl groups.

Since cerebellar catalase and GSHPx activities were not changed in MeHg-exposed weaning mice, one could assume that the observed increase in cerebellar hydroperoxide levels in these animals was not related to an impaired peroxide detoxification. This idea corroborates with previous data (Ariza et al., 1998Go) showing that mercury can induce the formation of peroxide without changes on the activity of enzymes that detoxify peroxides. The observed inhibitory effect of MeHg on cerebellar GSHPx from mothers seems to have no direct relationship with glutamate uptake, however, reinforce the hypothesis that this enzyme can act as a molecular target to the toxic effects of MeHg (Farina et al., 2004Go; Watanabe et al., 1999Go).

In conclusion, the results of the present study indicate that the exposure of lactating mice to MeHg causes inhibition of the glutamate uptake by cerebellar slices in the offspring and that this inhibitory effect seems to be related to increased levels of hydroperoxide.


    ACKNOWLEDGMENTS
 
This study was supported by grants from Brazilian National Research Council CNPq -No. 300910/03-7 and FAPERGS -No. 0301963.


    NOTES
 

1 To whom correspondence should be addressed. Fax: + 55-55-220-8753. E-mail: marcelofarina{at}zipmail.com.br.


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 DISCUSSION
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