Increase of striatal dopamine release by cadmium in nursing rats and its prevention by dexamethasone-induced metallothionein

Toxicology 131 (1998) 145 – 154

Increase of striatal dopamine release by cadmium in nursing rats and its prevention by dexamethasone-induced metallothionein Elsa Y. Gutie´rrez-Reyes a,1, Arnulfo Albores a, Camilo Rı´os b,* a

Seccio´n de Toxicologı´a Ambiental, Departamento de Farmacologı´a y Toxicologı´a, CINVESTAV-IPN, Mexico City 07300, Mexico b Departamento de Neuroquimica, Instituto Nacional de Neurologı´a Manuel Velasco Sua´rez, A6e. Insurgentes Sur No. 3877, Mexico City, Mexico Received 13 April 1998; accepted 17 September 1998

Abstract Repeated daily intraperitoneal (i.p.) administrations of cadmium (CdCl2, 1 mg/kg per day for 5 days) increased striatal dopamine (DA) release (180% of controls) and turnover (150% of controls) in 13-day-old rats. Cd treatment also increased striatal metallothionein (MT) content (161%), Cd (127%) and lipid peroxidation (LPO, 190%). In addition, Cd treatment decreased striatal tyrosine hydroxylase (TH) activity ( −28%), and such an effect may result from D-2 receptor blockade as a consequence of excessive dopamine release, since sulpiride (a specific D-2 receptor antagonist) administration to Cd-treated rats abolished the effect of Cd on TH. No effect was observed on striatal monoamine oxidase (MAO) activity. Dexamethasone (Dx) treatment increased striatal MT content and caused no effect on either DA release or turnover. However, Dx administration prevented the effects caused by Cd, including the increased DA release and enhanced striatal lipid peroxidation. These results indicate that toxic effects on the brain are to be expected as a result of Cd exposure and that Dx administration can attenuate them. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cadmium; Corpus striatum; Dexamethasone; Dopamine; Developmental toxicity; Metallothionein; Neurotoxicity

1. Introduction * Corresponding author. Tel.: +52-60-64040; Fax: +5252-80095; e-mail: [email protected]. 1 Present address: Departamento de Farmacologia, Facultad de Medicina, Universidad Nacional Francisco de Miranda, Coro, Venezuela.

Cadmium (Cd) is a toxic metal known to damage a number of tissues, including the nervous system (Gabbiani et al., 1967; Singhal et al., 1976;

0300-483X/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 9 8 ) 0 0 1 2 6 - 7

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Rastogi et al., 1979; De Castro et al., 1996). This element has a long biological half-life, therefore, prolonged exposure to environmental Cd constitutes a health hazard and results in long-term toxic effects in animals and humans. Studies with animals have shown that Cd administration produces motor hyperactivity (Wong and Klaassen, 1982), increased aggressive behavior (Arito et al., 1981) impaired social memory processes (Holloway and Thor, 1988), and altered drinking behavior (de Castro et al., 1996). Cd inhibits Na + , K + -ATP-ase activity and dopamine (DA) uptake in brain synaptosomes (Hobson et al., 1986; Rajanna et al., 1990). Cd exposure also causes lipid peroxidation (LPO) in brain and other tissues (Ochi et al., 1987; Rajanna et al., 1990; Kumar et al., 1996), and LPO is thought to be an early intracellular event after Cd exposure (Muller and Ohnesorge, 1982; Muller 1986; Bagchi et al., 1996). However, the mechanism by which Cd causes LPO is not fully understood. In humans, epidemiological studies have shown behavioral changes in children environmentally exposed to Cd (Thatcher et al., 1982; Bonithon-Kopp et al., 1986). However, direct effects of Cd on the brain neurochemistry has not been studied to date. On the other hand, Cd administration induces metallothionein (MT) synthesis in brain and other organs (Bremner and Davies, 1975; Ebadi, 1986). MT is a low-molecular-weight protein which binds essential (i.e. zinc and copper) or toxic (i.e. Cd and Hg) metals. In addition, MT is bound to Cd in several tissues (Webb and Cain, 1982; Kagi and Schaffer, 1988; Misra, et al., 1996). MT isoforms are believed to play an important role in the metabolism of essential metals (Webb and Cain, 1982) and in the detoxification of or tolerance to toxic metals (Leber and Mija, 1976; Min et al., 1987). This protein can be induced by steroids, metals and other factors (Hidalgo et al., 1988; Kagi and Schaffer, 1988; Morselt, 1991), including dexamethasone (Dx). In fact, Dx is an excellent MT inducer in the brain (Palmiter et al., 1992). Because of the importance of DA as a neurotransmitter involved in motor control and some learning and memory processes (Beninger, 1983; Lee, 1984), we decided to study the effect of repeated administrations of Cd on the release and

turnover of striatal DA, and the possible modulation of cadmium neurotoxicity by dexamethasoneinduced metallothionein in nursing rats.

2. Materials and methods

2.1. Chemicals CdCl2 was from J.T. Baker de Me´xico, Mexico. Dopamine hydrochloride, dihydroxyphenylalanine free base (L-DOPA), homovanillic acid (HVA), pargyline hydrochloride, imipramine hydrochloride, sulpiride, sodium octyl sulfate, mhidroxybenzylhydrazine (NSD 1015), kynuramine dihydrobromide, 4-hydroxyquinoline (4-HOQ) were all from Sigma Chemical, St. Louis, MO. Sodium metabisulphite was from Aldrich Chemical, Millwakee, WI. Silver standard (Mallinckrodt Specialty Chemicals, Paris, KY), Cd standard (Reference material 3108, National Bureau of Standard, Gaithesburg, MD), methanol, HPLC grade (E. Merck, Mexico) and all others reagents of analytical grade were purchased. Glassware and plastic material were immersed for 24 h in 3% v/v HNO3/H2O, thoroughly rinsed with deionized water and dried in a stove before use.

2.2. Animals Experiments were performed using 13 day-old male and female Wistar rats bred in-house, 0-day old being the day of birth. We choose this age to take advantage of the immaturity of the rat blood-brain barrier. One day after birth, the number of pups per litter was reduced to 8 by random selection. The newborn rats were allowed to remain with their dams throughout the experiment. Rats were kept at 21°C and 40% relative humidity with a 12 h light/dark cycle starting at 0700 h.

2.3. Treatments Each experimental block consisted of four pups from the same litter, injected intraperitoneally (i.p.) at day 13 of age with either: (1) CdCl2 (1 mg of Cd/kg) dissolved in 0.5 ml of saline (0.9% NaCl w/v) (2) Dx (2.5 mg/kg) dissolved in 0.5 ml of

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saline; (3) CdCl2 (1 mg of Cd/kg) and Dx (2.5 mg/kg) each dissolved in saline and (4) saline, 0.5 ml. Injection volume was :0.5 ml for each animal. All treatments were administered daily for 5 consecutive days, however, to investigate Cd acute effects, some animals were dosed once and killed 24 h later. At least five complete blocks were done per experiment. After treatments, all rats were killed by decapitation (between 1000 and 1200 h) and their corpora striata were individually dissected from both brain hemispheres over ice as described by Glowinski and Iversen (1966).

2.4. Blood and tissue cadmium determinations Cadmium in blood was determined as described by Stoeppler and Brandt (1980), with minor modifications (Galicia-Garcia et al., 1997). The striatal Cd content was determined taking the precautions described by Christian (1969) and using the conditions described by Eller and Haartz (1977). Both analyses were performed by graphite furnace atomic absorption spectrophotometry, using a Perkin-Elmer 360 spectrophotometer equipped with an HGA-2200 furnace. Blood Cd results were expressed as mg of Cd/l blood, and striatal Cd data were expressed as ng of Cd/g wet tissue weight.

2.5. Striatal metallothionein measurements Striatal content of metallothionein (MT) was estimated as described by Scheuhammer and Cherian (1986) with minor modifications (Rojas and Rios, 1997). Briefly, tissue samples (about 0.02 g) were homogenized in 300 ml of a phosphate buffer 0.05 M, NaCl 0.375 M mixture (1.5:1 v/v). Then, 250 ml silver nitrate solution (20 ppm) and 400 ml of glycine buffer (0.5 M, pH 8.5) were added. After standing at room temperature by 5 min, 100 ml of rat hemolyzed erythrocytes were added and the mixture boiled for 2 min, then centrifuged at 4000×g for 5 min. The latter step was repeated twice. MT was estimated by measuring the silver content of the supernatant fractions (diluted 1:10 with 3% HNO3 v/v) using an atomic absorption spectrophotometer (Perkin Elmer, model 360) equipped with an HGA-2200 graphite

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furnace. Absorbance signals were recorded on a strip-chart recorder (Perkin Elmer 056). Metallothionein results were expressed as mg of MT/g wet tissue weight.

2.6. Endogenous dopamine release Striatal slices of about 250 mm thickness were obtained manually using a glass guide and a knife (McIlwain, 1975). Striatal slices were continuously moistened with warm (37°C) Krebs-Ringer (KR) solution containing NaCl 118 mM, KH2PO4 1.2 mM, KCl 4.7 mM, CaCl2 2.5 mM, MgSO4 1.17 mM, Glucose 5.6 mM and Tris 25 mM, pH 7.4, saturated with 95% O2/5% CO2. The slices (4–5) from each rat were preincubated for 30 min in oxygenated KR solution before experiments started. DA release assays were performed at 37°C in a temperature-controlled water bath, constantly stirred and oxygenated. To induce potassiumevoked DA release, a depolarizing media (40 mM KCl) containing a reduced NaCl concentration (81.7 mM) to correct for osmolarity changes was used. All media contained also pargyline (50× 10 − 4 M) and imipramine (10 − 4 M), in order to prevent DA degradation and uptake, respectively. DA release was estimated as described by Katz et al. (1969) and McIlwain (1975). Briefly, once equilibrated in 3 ml KR solution, the medium containing the striatal slices was aspirated by a vacuum pump and immediately replaced with fresh KR solution (5 ml). After a 5 min period, spontaneous release of the neurotransmitter was monitored 3 times for 3-min periods by taking 150 ml aliquots. Then, the medium was aspirated and replaced with depolarizing medium (5 ml) and the potassium-stimulated DA release was measured by taking medium aliquots (150 ml) every 3 min up to 21 min. Perchloric acid containing 0.4 mM sodium metabisulphite solution (100 ml) was added to each aliquot as an antioxidant solution. Samples were kept frozen (−5°C) until analyzed for DA content. The DA content in tissue slices was measured after the potassium depolarization as follows: the slices were removed from the medium and sonicated in 400–500 ml of ice-cold perchloric acid-sodium metabisulphite an-

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tioxidant solution. Samples were centrifuged at 4000×g for 5 min and the supernatants kept frozen at −5°C until analyzed for DA content in slices. The percentage of striatal DA released was calculated by dividing the DA content in the media by the DA content in the respective slices. All DA analysis were performed by HPLC with electrochemical detection, as described below.

2.7. Tyrosine hydroxylase assay Tyrosine hydroxylase activity was assessed ex vivo by measuring the accumulation of striatal L-DOPA 30 min after the systemic administration of a centrally active inhibitor of DOPA decarboxylase (NSD 1015, 100 mg/kg) (Kehr, et al., 1972). The acute effect of Cd on TH activity was also studied in a group of rats (n= 6) treated once with Cd and killed 24 h later. The possibility of a feedback effect of excessive DA release on TH activity, mediated by D2 receptors, was investigated in control and 5 day Cdtreated rats (n= 8 per group) by injecting them with sulpiride (20 mg/kg, i.p.) and 1 h later with NSD-1015 (100 mg/kg). Rats were killed 30 min after the last injection and L-DOPA determined (Westerink and de Vries, 1989), as described below.

2.8. Dopamine, homo6anillic acid, and L -DOPA analysis and dopamine turno6er DA, homovanillic (HVA) and L-DOPA concentrations were measured using a Perkin-Elmer LC250 liquid chromatograph equipped with a Metrohm 656 electrochemical detector and a Hewlett-Packard HP-3396 series II integrator, as described previously (Garcı´a et al., 1992). An Alltech Associates (Deerfield, IL), adsorbosphere catecholamine analytical column C18 100× 4.8 mm with 3 mm particle diameter was used. The mobile phase consisted of aqueous phosphate buffer (pH 3.1) which contained 0.2 mM sodium octyl sulfate, 0.1 mM EDTA and 15% v/v of methanol. DA turnover was estimated as the HVA/DA ratio, as described by Cheng and Wooten (1982).

2.9. Monoamine oxidase acti6ity Striatal MAO activity was assayed ex vivo as described by Krajl (1965). Striatal brain tissue was homogenized in 3 ml of deionized water at 4°C. One ml of the homogenate was added to 4 ml of phosphate buffer (0.05 M pH 7.4) containing kynuramine dihydrobromide (100 mg) as substrate. Samples were incubated at 37°C for 30 min. After incubation, reaction was stopped by adding 2 ml of 10% w/v trichloroacetic acid. After cooling and centrifugation (3000×g, 15 min.), an aliquot of 1 ml of the supernatant was added to 2 ml of 1 N NaOH. Fluorescence was then measured using a fluorescence spectrophotometer (MPF-44A Perkin Elmer) at excitation and emission wavelengths of 315 and 380 nm, respectively. In each experiment, calibration curves were constructed by measuring the fluorescence intensity of 4-hydroxyquinoline (4-HOQ) standards. 4-HOQ is the product of MAO activity on kynuramine. MAO activity was expressed as mmol of 4-HOQ/g tissue wet weight.

2.10. Lipid peroxidation As an index of LPO, lipid fluorescence products (LFP) were measured as described by Santamarı´a and Rı´os (1993). Striatal tissue was carefully weighed and homogenized in 2.5 ml of saline solution. One ml aliquots of the homogenate were placed in glass tubes and 3 ml of chloroformmethanol (2:1 mixture) was added. The tubes were capped, gently mixed and placed on ice for 20 min to allow phase separation. The aqueous phase was discarded and 1 ml of the chloroformic layer was transferred into a quartz cuvette, and 0.1 ml of methanol was added. Fluorescence was measured in a Perkin-Elmer MPF-44A fluorescence spectrophotometer at 370 nm excitation and 430 nm emission. Sensitivity was adjusted to 140 fluorescence units with 0.1 mg/l quinine standard prepared in 0.05 M aqueous sulfuric acid solution prior to the measurement of the samples. Results are expressed as fluorescence units per gram of tissue per ml extracted. A positive control for LFP formation was carried out by injecting FeSO4 intraventricularly to a group of 6 rats, killed 60 min later (Triggs and Willmore, 1984).

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Table 1 Blood Cadmium (Cd) and striatal concentrations of Cd and metallothionein (MT) of rats treated with 5 repeated daily i.p. doses of Cd (1 mg/kg) or Dexamethasone (Dx, 2.5 mg/kg)a Treatment

Blood Cd (mg/l)

Striatal Cd (ng/g of tissue)

Striatal MT (mg/g of tissue)

Control Cd Dx Cd+Dx

0.29 9 0.04 10.029 1.13b 0.27 90.05 10.9 9 1.33b

36.79 0.9 46.590.6b 38.5 9 1.2 53.991c

74.1 9 2.3 119.3 94.4b 158.8 97.0c 158.2 95.4c

a

Values are the mean9 S.E.M. of duplicate analysis for each animal, n =8 animals per group. Different from control group. c Different from Cd-treated group (PB0.05, block-design ANOVA followed by Tukey’s test). b

2.11. Statistical analysis The experiment was conducted as a block design in order to control variability from one set of conditions (four rats from the same litter were used in parallel) to another. Therefore, results were analyzed using a block design analysis of variance followed by the Tukey’s test for multiple comparisons, after checking for homogeneity of variances (Steel and Torrie, 1969).

thereafter. The increase remained about 2-fold above control values for the rest of the experiment up to 21 min (Fig. 1). Dx caused no statistically significant change in striatal DA release as compared to controls. When rats were treated with both Cd+ Dx for 5 days, DA release was similar to that of the group treated with Dx alone (Fig. 1).

3. Results

3.1. Cadmium and metallothionein concentrations Cadmium treatment of both control and Dxtreated rats resulted in 1.3- and 1.5-fold increases in the striatal content of the element, respectively. Blood Cd concentration increased about 36-fold in both Cd-treated groups (Table 1). MT content also increased 1.6- and 2.1-fold in the Cd and Cd +Dx treated rats, respectively (see again Table 1).

3.2. Effects of cadmium on dopamine release and tyrosine hydroxylase acti6ity. There were no differences in the spontaneous release of DA among the treatment groups, showing that there was no increased leakiness of cell membranes as a result of treatments. Cd treatment of growing rats caused a significant increase in K + -evoked DA release from striatal slices, which was statistically significant at 9 min and

Fig. 1. Endogenous dopamine release from striatal slices. Thirteen-days-old rats were treated with either: (1) CdCl2 (Cd, 1 mg of Cd/kg) i.p. daily for 5 consecutive days (2) Dexamethasone (Dx, 2.5 mg/kg) i.p. daily for 5 consecutive days (3) CdCl2 (1 mg of Cd/kg) and Dx (2.5 mg/kg, Cd + Dx), both i.p. daily for 5 consecutive days; or saline solution (control). Results are expressed as mean 91 S.E.M. of n =5 rats per group. Different from all other treatment group values * PB 0.05, ** PB0.01, block design analysis of variance followed by Tukey’s test.

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Table 2 Striatal tyrosine hydroxylase (TH) activity of rats treated with five repeated daily i.p. doses of Cd (1 mg/kg) or Dexamethasone (Dx, 2.5 mg/kg)a

treatment was an increased HVA/DA content (Table 3). This change was fully antagonized by Dx co-administration.

Treatment

TH activity

TH activity after sulpiride (20 mg/kg)

3.4. Effect of cadmium on monoamine oxidase acti6ity

Control Cd Dx Cd+Dx

0.65 90.02 0.47 9 0.02b 0.60 90.02 0.53 90.03

3.149 0.10 2.989 0.18 2.14 90.13b 2.679 0.15

There were no significant changes in MAO activity among the treatment groups as compared to control group values (results not shown).

a Values are the mean 9S.E.M. of duplicate analysis for each animal, n = 8 animals per group. b Different from control group (PB0.05, block-design ANOVA followed by Tukey’s test).

In addition, Cd administration inhibited the striatal TH activity by 28%, estimated as L-DOPA accumulation (Table 2). This inhibition, however, was not longer observed in the group of animals treated with a presynaptic D-2 receptor blocker (sulpiride, Table 2). In all treatment groups, animals administered Cd only once and killed after 24 h showed no change in either DA release or TH activity (data not shown).

3.3. Effect of cadmium on dopamine and homo6anillic acid content and dopamine turno6er Dexamethasone administration significantly increased striatal DA content by 152% of control animals (Table 3), and this increase remained in animals treated with the combination of Cd plus Dx (170.9% of control values), indicating an idiosyncratic effect of the drug on striatal DA content. The only change attributable to Cd

3.5. Effect of cadmium on lipid peroxidation Striatal lipid peroxidation (LPO) data showed a significant increase (190% of control values) in LFP as a result of Cd treatment (Fig. 2). Animals injected with 250 nmol FeSO4 showed an increase (160%) in LFP formation as compared to control values (Fig. 2). Again, Dx co-administration completely prevented this effect (see Fig. 2).

4. Discussion This study shows that Cd treatment of nursing rats for 5 days was able to increase significantly striatal DA release, but this effect was not observed after 1 day of Cd administration. This result suggests that for Cd to affect dopaminergic function, repeated exposure to the metal is required. The effect of Cd on DA release seems to be pathophysiologically important in vivo, given the observed correlation to reduced TH activity. TH activity is physiologically inhibited via the activity of dopaminergic D-2 presynaptic receptors in the

Table 3 Striatal Dopamine (DA), homovanillic acid (HVA) concentrations and HVA/DA ratio of rats treated with five repeated daily i.p. doses of Cd (1 mg/kg) or Dexamethasone (Dx, 2.5 mg/kg)a Treatment

DA (mg/g of tissue)

HVA (mg/g of tissue)

HVA/DA ratio

Control Cd Dx Cd+Dx

2.5490.33 2.789 0.16 3.869 0.14b 4.3490.39b

0.38 90.04 0.49 9 0.03 0.39 9 0.002 0.48 9 0.03

0.1 90.01 0.14 90.005b 0.09 9 0.03 0.1 90.003

a b

Values are the mean9 S.E.M. of duplicate analysis for each animal, n =8 animals per group. Different from control group (PB0.05, block-design ANOVA followed by Tukey’s test).

E.Y. Gutie´rrez-Reyes et al. / Toxicology 131 (1998) 145–154

Fig. 2. Striatal lipid peroxidation measured as lipid fluorescence product formation. Thirteen-days-old rats were treated with either: (1) CdCl2 (Cd, 1 mg of Cd/kg) i.p. daily for 5 consecutive days (2) Dexamethasone (Dx, 2.5 mg/kg) i.p. daily for 5 consecutive days (3) CdCl2 (1 mg of Cd/kg) and Dx (2.5 mg/kg, Cd +Dx), both i.p. daily for 5 consecutive days (4) FeSO4 (100 nmol/1 ml) single intrastriatal microinjection, or saline solution i.p. (control). Results are expressed as mean 91 S.E.M. of n = 6 rats per group. Different from all other treatment group values * PB 0.05, ** P B0.01, block design analysis of variance followed by Tukey’s test.

synaptic cleft (Kehr et al., 1972), thus, an excessive release of dopamine induced by Cd could activate those receptors to inhibit enzyme activity. This explanation is supported by the observed blockage of the Cd effect on TH by sulpiride pretreatment, as this drug is an specific D-2 receptor antagonist. Increased DA release from striatal slices was by no means an unexpected finding, despite the fact that Cd ion is reported as a Ca2 + antagonist, and therefore a reduced DA release could be expected if Cd is acting as a calcium channel blocker, as occurs, for example, with acetylcholine release in vitro (Cooper and Manalis, 1984). However, in adrenal medulla preparations, Cd is able to evoke DA release both in the presence and in the absence of calcium (Kanthasamy et al., 1995). A possible explanation of the Cd-induced enhancement of striatal DA release is that some mecha-

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nisms are acting in vivo that result in an increased efficacy of DA release. An enhanced activation of calmodulin by Cd (Behra and Gall, 1991), which in turn can increase catecholamine exocytosis (Lin et al., 1990), has been described and it is also a possible mechanism to explain our results. Cd also mobilizes intracellular calcium (Yamagami et al., 1994) and this effect can contribute to the higher DA release observed. A potentiation of ATP-evoked dopamine release has also been reported in phaeochromocytoma cells exposed to Cd (Ikeda et al., 1996), thus, the participation of purinoceptors in cadmium-induced enhancement of dopamine release should not be discarded. In agreement with results presented here, earlier reports of Rastogi et al. (1979) suggested that Cd treatment increased the physiological utilization of catecholamines in the rat brain. In addition, several reports suggest that Cd exposure produces a significant increase of rodent locomotor activity (Wong and Klaassen, 1982). Motor behavioral effects could be explained by the excessive dopamine release observed in this study, since DA is involved in motor control (Lee, 1984) and hyperactivity (Costall et al., 1977). The observed increase in DA turnover rate after Cd treatment could result from increased DA release from the presynapsis exerted by the metal, that removes the neurotransmitter from this structure and accelerates the replacement of it by newly synthesized DA. Released DA could be then metabolized by glial or neuronal elements. Results of previous studies have shown significantly increased lipid peroxidation and oxidative stress in rat brain after Cd administration (Rajanna, et al., 1990; Manca et al., 1991; Shukla et al., 1996). This effect was particularly evident in the striatum (Pal et al., 1993). The mechanism of Cd-induced LPO is not fully understood, however, a contributing factor could be the enhancement of both DA release and turnover rate, as DA catabolism produces hydrogen peroxide (Kehrer, 1993), a well-known oxygen active molecule. Some other conditions involving lipid peroxidation, as for example vitamin E deficiency, can also increase DA release in the corpus striatum (Castan˜o et al., 1993a,b).

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In the blood of Cd-exposed rats, Cd concentrations were about 10 mg/l, similar to those reported in the human population environmentally exposed to high amounts of the metal (ATSDR, 1993). Thus, neurotoxic effects induced by Cd might be found in people exposed to the metal under these conditions. The Cd content in striatum was also significantly increased by Cd treatment, specially in those rats treated with Cd+ Dx. This result suggests that Cd is being accumulated by induced MT, a protein with a high affinity for this metal. It is important to note that the main neurotoxic effects of Cd exposure observed in the present study (dopaminergic hyperfunction and increased LPO) were completely abolished by Dx, a wellknown brain MT inducer (Palmiter et al., 1992), indicating that both effects could be mediated by cadmium not bound to metallothionein, in agreement with reports in the literature (Pal et al., 1993). The molar ratio of striatal Cd/MT concentrations, calculated from Table 1, was different in Cd and Cd+ Dx groups (0.016 and 0.011, respectively), suggesting that cadmium bound to MT is slightly higher in the Cd+ Dx group than in the Cd alone group. Thus, Dx neuroprotective effects observed in the present study might be attributed to a higher capacity of striatal MT to chelate Cd and to inactivate it. Results presented in this work show that Cd exposure at a relatively low dose is able to increase central dopaminergic function of the growing rat. Further studies are needed to clarify whether or not these changes are related to the behavioral changes observed in animals exposed to the metal. The exact mechanisms producing the dopaminergic hyperfunction observed here also deserve further study. However, the participation of MT as a protective mechanism against Cadmium-induced neurotoxicity is suggested by our results.

Acknowledgements The authors thank to ‘Fundacio´n Gran Mariscal de Ayacucho’ and Universidad Nacional ‘Francisco de Miranda’, Venezuela, for financial support to EYG-R.

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