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Effects of successive intrastriatal methylmercury administrations on dopaminergic system
Ecotoxicology and Environmental Safety 55 (2003) 173–177
Effects of successive intrastriatal methylmercury administrations on dopaminergic system L.R.F. Faro,a R. Dura´n,b,* J.L.M. Do Nascimento,a D. Perez-Vences,b and M. Alfonsob b
a Departamento de Fisiologia, Centro de Cieˆncias Biolo´gicas, UFPA, Bele´m, PA, Brazil Laboratorio de Fisiologı´a, Departamento de Biologı´a Funcional y Ciencias de la Salud, Facultad de Ciencias, Campus As Lagoas-Marcosende, Universidad de Vigo, Vigo 36200, Spain
Received 31 December 2001; accepted 23 July 2002
Abstract The present study was carried out in order to determine the effects of intrastriatal administration of different doses (40 mM, 400 mM, and 4 mM) of methylmercury (MeHg) on dopaminergic system of rat striatum. Experiments were performed in conscious and freely moving rats using brain microdialysis coupled with liquid chromatography. Intrastriatal administration of MeHg produced significant increases in dopamine (DA) striatal levels (90777%, 18707319%, and 79717534% for the doses of 40, 400 mM, and 4 mM, with respect to basal). The increase in DA levels was associated with significant decreases in extracellular levels of its main metabolites dihydroxyphenylacetic acid (DOPAC) and homovallinic acid (HVA) (65.073.0% and 52.271.3%, respectively) using the dose of 4 mM MeHg, whereas nonsignificant changes in metabolite levels were observed with the doses of 40 and 400 mM MeHg. A second infusion of 4 mM MeHg 24 h after first infusion also produced a rise of DA levels, but this increase was very small as compared with that produced by first infusion (79717534% versus 9857186%). This second infusion of 4 mM MeHg also decreased DOPAC and HVA levels, but this decrease was not significant as compared with that observed after first infusion (65.073.0% and 52.271.3% versus 62.475.2% and 63.477.4%, respectively). We discuss these effects based on a stimulated DA release and/or a decreased DA intraneuronal degradation. r 2003 Elsevier Science (USA). All rights reserved. Keywords: Methylmercury; Dopamine; Striatum; Microdialysis; HPLC
1. Introduction Methylmercury (MeHg) is an ubiquitous environmental pollutant, recognized as a neurotoxic agent that produces many different effects on the living organism, more specifically to the Central Nervous System (CNS), such as paresthesia, ataxia, convulsion, muscular weakness, impairment of vision, hearing, and speech, and partial paralysis (Berlin, 1986). The absorption of methylmercury occurs mainly in the gastrointestinal tract (Glocking et al., 1977). Because of its high lipid solubility, MeHg penetrates the blood–brain barrier and readily diffuses into cell membranes (Felton et al., 1972; Lakowicz and Anderson, 1980). The knowledge of the primary biochemical events associated with MeHg neurotoxicity of CNS is very *Corresponding author. Fax: +34-8681-2556. E-mail address: [email protected] (R. Dura´n).
scarce to date. However, the effects of MeHg could be due to direct actions on the neuron, resulting in disruption of neurotransmission with subsequent sensory and motor deficits (Siriois and Atchison, 1996). Methylmercury is also known to interfere with synaptic transmission at various steps, including synthesis, uptake, release, and degradation of neurotransmitters (Komulainen and Tuomisto, 1981). In vitro, MeHg exposure affects the release of neurotransmitters at the neuromuscular junction, increasing spontaneous transmitter release and decreasing the depolarization-evoked transmitter release (Atchison, 1987). In CNS tissue preparations, MeHg increases spontaneous transmitter release (Bondy et al., 1979; Komulainen and Tuomisto, 1981), and this increase was found to be concentrationdependent in synaptosomes (Minnema et al., 1989). In synaptosomes, MeHg produces a disruption of synaptosomal membrane integrity and an increase in membrane permeability (transient and reversible or only
0147-6513/03/$ - see front matter r 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0147-6513(02)00127-6
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a small fraction of the synaptosomes became leakly) (Minnema et al., 1989). Also consistent with an increase in membrane permeability, and the membrane depolarization that ensues, is the possibility that MeHg alters the conduction of the axonal action potential (Traxinger and Atchison, 1987). Methylmercury is also thought to affect primarily mitochondrial functions and thus energy metabolism of the nervous cell (Komulainen and Bondy, 1987). However, other nonmitochondrial processes may be even more sensitive to MeHg action (Komulainen and Tuomisto, 1981). In addition to MeHg’s effect on mitochondrial enzymes, MeHg interferes with the synthesis, transport, and activity of other enzymatic systems such as glutathione peroxidase, adenylate cyclase, and dehydrogenases (Atchison and Hare, 1994). Moreover, under different in vitro and in vivo experimental conditions, the inhibition of protein synthesis seems to be one of the initial effects of MeHg. Different studies on the distribution of mercury within the CNS of mammals only described the effects of acute administrations and were performed with tissue samples using radioactive compounds of mercury (Rodier and Kates, 1988). Mercury is accumulated mainly in the following CNS regions: cortex, striatum, hippocampus, brain stem, cerebellum, and spinal cord (Moller-Madsen, 1994). The signs produced by the administration of mercury in different forms could be associated with alterations in the motor function. According to studies indicating that those nuclei associated with the motor systems showed a high concentration of mercury (Moller-Madsen, 1994), the striatum is a major target for the study of mercury effects on the brain. Because MeHg seems to influence the neurotransmission and to be a potent neurotoxin in the CNS, we decided to study its effects on the in vivo release of dopamine (DA), the main striatal neurotransmitter, and its main metabolites, dihydroxyphenylacetic acid (DOPAC) and homovallinic acid (HVA), from rat striatum using a microdialysis technique coupled to high-performance liquid chromatography (HPLC) with electrochemical detection.
2. Methods Female Sprague–Dawley rats (weight range 240– 260 g) were used for the experiments. Animals were housed (four per cage) under controlled conditions of temperature (221721C) and photoperiod (light:dark 14:10 h), with food and water available ad libitum. All experiments were performed in accordance with the Guidelines of the European Union Council (86/609/EU) for the use of laboratory animals.
For microdialysis sampling, animals were anesthetized with chloral hydrate (400 mg/kg via i.p.) and placed in a Narishige SR-6 stereotaxic apparatus for the implantation of a guide-cannula. Microdialysis probe (CMA/12, 3-mm membrane length, Stockholm, Sweden) was inserted through the guide-cannula into the left striatum according to the following coordinates from Bregma: A/P +2.0 mm; L +3.0 mm; V +6.0 mm. Placement of probes within the striatum was confirmed histologically after the experiments, using the stereotaxic atlas of Pellegrino et al. (1979) as reference. The first experiments were initiated 24 h after surgery. Continuous perfusion was performed with a Ringer solution (147 mM NaCl, 4 mM KCl, 3.4 mM CaCl2; pH 7.4) using a CMA/102 infusion pump (CMA/Microdialysis, Stockholm, Sweden) at an infusion rate set at 2 ml/min. The samples obtained from the microdialysis procedure were collected every 15 min (30 mL) by means of a CMA/142 microsampler (CMA/Microdialysis, Stockholm, Sweden). The experiments were performed during 4 h with awake, conscious, and freely moving animals. Different doses of MeHg were dissolved in the perfusion medium and applied locally in the striatum via the dialysis probe after obtaining a stable output for DA and metabolites (DOPAC and HVA). After three basal perfusates (45 min), the striatum was perfused with MeHg for 60 min at concentrations of 40, 400 mM, and 4 mM. After this time, the perfusate was then switched back to the unmodified perfusion medium and the measurements were continued for an additional period of 135 min. In the following experiments, the effects of large concentrations of MeHg on the DA, DOPAC, and HVA extracellular levels were verified using a second infusion of 4 mM MeHg, 24 h after the first infusion. In this case, we used the same experimental conditions as described above. The levels of DA, DOPAC, and HVA were measured by means of HPLC with electrochemical detection. Samples obtained from the microdialysis procedure were injected into a Hewlett-Packard Series 1050 Liquid Chromatograph, using a Rheodyne 7125 injection valve. The isocratic separation of DA, DOPAC, and HVA was made using Spherisorb ODS-1 reversed-phase columns (10 mm particle size). The eluent (pH 4.0) was prepared as follows: 70 mM KH2PO4, 1 mM octanesulfonic acid, 1 mM EDTA, and 5% methanol. The flow rate was 1 mL/min. The detection of the substances was achieved by means of an ESA Coulochem 5100A electrochemical detector (MA, USA) at a potential of +400 mV. The chromatograms obtained allowed the determination of DA, DOPAC, and HVA with a run time of 15 min. The data were corrected for the percentage of recovery for every microdialysis probe, which was
L.R.F. Faro et al. / Ecotoxicology and Environmental Safety 55 (2003) 173–177
similar for the different probes and substances analyzed (15% for DA, 20% for DOPAC, and 25% for HVA). The average of substance concentrations in the three samples before MeHg administration was considered the basal level. Basal levels were considered 100% in order to compare the different responses of DA and metabolites after MeHg administration. The results are shown as the mean7SEM of four to eight experiments, expressed as a percentage of the basal levels. Statistical analysis of the results was performed using a repeated-measures ANOVA and Student–Newman– Keuls multiple range test, considering the following significant differences: Po0:05; Po0:01; and Po0:001:
3. Results 3.1. Basal extracellular levels of DA, DOPAC, and HVA The basal output of dialysate DA and its metabolites from striatum before the first infusion of MeHg (day 1) and 24 h after MeHg administration (day 2) are shown in Table 1. Basal values of DA, DOPAC, and HVA 24 h after infusion of 4 mM MeHg were smaller than those observed on day 1.
Table 1 Basal values (mean7SEM, n ¼ 8) expressed as nanograms of substance/15 min for DA, DOPAC, and HVA before first infusion of methylmercury (Day 1) and 24 h after first infusion (Day 2)a
DA DOPAC HVA a
Day 1
Day 2
0.3470.08 5.9071.10 6.0070.9
0.2070.03* 4.8570.33 3.2470.28*
Significant differences: Po0:05 with respect to basal on day 1.
175
3.2. Effect of methylmercury infusion The different doses of MeHg produced a concentration-related increase in the striatal output of DA (Fig. 1A). Experiments carried out on day 1 showed that 40, 400 mM, and 4 mM of MeHg produced maximum increases 30 min after the beginning of MeHg perfusion (90777%, 18707319%, and 79717534% of basal levels, respectively). Basal values recovered 135 min after MeHg administration. The highest dose of MeHg assessed (4 mM) caused a significant decrease in extracellular levels of DOPAC and HVA in striatum 45 min after the beginning of MeHg administration (65.0373% and 52.271.3% of decrease related to basal levels, respectively) (Figs. 1B and C). The other remaining concentrations of MeHg (40 and 400 mM) had no significant effects on the acidic metabolites of DA. 3.3. Effect of a second infusion of 4 mM methylmercury on DA, DOPAC, and HVA In these experiments, the rats exposed to 4 mM of MeHg on day 1 were also exposed to 4 mM of MeHg 24 h later. Fig. 2 shows the effects of a second infusion of MeHg, these effects being characterized by increased DA output 90 min after the beginning of 4 mM MeHg perfusion (9857186% with respect to basal levels). However, this increase was very small as compared with that produced by the first MeHg infusion (Fig. 1A). Therefore, the maximal increase (about 8000%) observed after the first infusion dropped dramatically after the second infusion (about 1000%), as shown in Fig. 2A. DOPAC and HVA striatal levels after the second infusion of 4 mM MeHg showed a small decrease: 62.4275.2% and 63.4177.4%, respectively (Fig. 2B and C). This decrease in DOPAC and HVA levels was not significant as compared with the decrease observed
Fig. 1. Maximal increase (mean7SEM) in DA (A), DOPAC (B), and HVA (C) levels in striatal microdialysis samples taken after perfusion of different doses of MeHg (40, 400 mM, and 4 mM). The results (n ¼ 4 8) were expressed as a percentage of the basal levels (100%). Basal levels were considered as the mean of substance concentrations in the three samples before MeHg perfusion. Significant differences: Po0:05; Po0:01; and Po0:001 with respect to the basal. aPo0:01 comparing the doses of 400 mM with 40 mM; bPo0:001 comparing the doses of 4 mM with 400 and 40 mM.
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Fig. 2. Maximal increase (mean7SEM) in DA (A), DOPAC (B), and HVA (C) levels in striatal microdialysis samples obtained on day 1 and day 2 after administration of 4 mM MeHg. The results are expressed as a percentage of the basal levels (100%) (n ¼ 428). Basal levels were considered as the mean of substance concentrations in the three samples before MeHg perfusion. Significant differences: Po0:05; Po0:01; and Po0:001 with respect to the basal; aPo0:01 comparing the effects of day 1 and day 2.
after first infusion (Fig. 2B and C). Moreover, the experiments carried out 24 h later showed that the effects of MeHg were not only less pronounced than those of day 1 but also delayed (data not shown).
4. Discussion The present study reported that in vivo exposure to MeHg was able to produce a significant concentrationdependent increase of DA extracellular levels in striatum of rats, as well as significant decreases in both DOPAC and HVA striatal levels. Thus, the question was to know whether the effect of 4 mM MeHg was the same when a first infusion and a second infusion 24 h later were compared. In previous articles (Faro et al., 1997, 1998), we have reported that i.p. chronic and acute administration of MeHg induced significant increases in striatal DA output. The effect of systemic administration of MeHg on the striatal dopaminergic system could be due to action of this toxicant on several organs and functions, which could affect CNS activity. However, the in situ intrastriatal MeHg administration is a more direct way to determine the MeHg effect on the dopaminergic system. Our results indicate that MeHg affects the dopaminergic neurons of striatum, producing an increase in DA striatal levels. There are some possible targets for the increases in DA levels induced by MeHg. Thus, a possible mechanism by which DA levels were increased after intrastriatal administration of MeHg could be due to a direct action of the toxicant on the dopaminergic neurons at different levels: membrane, vesicular release, reuptake system, and so on (Komulainen and Tuomisto, 1981). However, these effects of MeHg on the spontaneous DA release could also occur intraneuronally, since MeHg is lypophillic and it can readily cross the nerve membrane.
In addition to increasing DA release, MeHg could also decrease DA reuptake (Komulainen and Tuomisto, 1981), increasing DA extracellular levels. Since DOPAC is produced intraneuronally from DA, the decrease of DA reuptake could explain the reduced levels of DOPAC observed after MeHg administration. Since the basal levels of DA and its metabolites were lower on day 2 compared with day 1, it could be possible to consider that the first administration of MeHg produced some neuronal damage. However, since on day 2 the second MeHg administration induced a lower increase in DA striatal levels (about 1000% on day 2 versus about 8000% on day 1), with similar decreases in both metabolites (nonsignificant changes on day 2 compared to day 1), we deduced that MeHg could affect those mechanisms involved in DA release. Moreover, the results from day 2 also could indicate that MeHg probably affected DA release rather than DA reuptake and metabolism. In conclusion, the intrastriatal administration of 4 mM MeHg produced significant increases in the release of DA from rat striatal tissue, which were associated with significant decreases in the extracellular levels of DOPAC and HVA. We could explain our results considering a stimulation of DA release and/or a decrease in DA reuptake. Moreover, we have also suggested that MeHg administration could affect the mechanisms involved in DA release.
Acknowledgments This research was supported by grants from Xunta de Galicia and University of Vigo (Spain). Lilian Faro acknowledges CNPq (Brasil) for a research grant. The authors thank Dr. J.L. Soengas for his help in the preparation of the article.
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References Atchison, W.D., 1987. Effects of activation of sodium and calcium entry on spontaneous release of acetylcholine induced by methylmercury. J. Pharmacol. Exp. Ther. 241, 131–139. Atchison, W.D., Hare, M.F., 1994. Mechanism of methylmercuryinduced neurotoxicity. FASEB J. 8, 622–629. Berlin, M., 1986. Mercury. In: Friberg, L., Nordberg, G., Vouk, V. (Eds.), Handbook on the Toxicology of Metals. Elsevier, Amsterdam, pp. 387–444. Bondy, S., Anderson, C.L., Harrington, M.E., Prasad, K.N., 1979. Effects of organic and inorganic lead and mercury on neurotransmitter high-affinity transport and release mechanisms. Environ. Res. 19, 102–111. Faro, L.R.F., Dura´n, R., do Nascimento, J.L.M., Alfonso, M., Picanc-o-Diniz, C.W., 1997. Effects of methylmercury on the in vivo release of dopamine and its acidic metabolites DOPAC and HVA from striatum of rats. Ecotoxicol. Environ. Saf. 38, 95–98. Faro, L.R.F., do Nascimento, J.L.M., Alfonso, M., Dura´n, R., 1998. Acute administration of methylmercury changes the in vivo dopamine release from rat striatum. Bull. Environ. Contam. Toxicol. 60, 632–638. Felton, J.S., Kahn, E., Salick, B., van Natta, F.C., Whitehouse, M.W., 1972. Heavy metal poisoning: mercury and lead. Ann. Intern. Med. 76, 779–792. Glockling, F., Hosmane, N.S., Mahale, V.B., Swindall, J.J., Magos, L., King, T.L., 1977. Mono, bis-, and tris-(trimethylsilyl)methyl derivatives of mercury. J. Chem. Res., 1201–1256.
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Komulainen, H., Bondy, S., 1987. Increased free intrasynaptosomal Ca2+ by neurotoxic organometals: distinctive mechanisms. Toxicol. Appl. Pharmacol. 88, 77–86. Komulainen, H., Tuomisto, J., 1981. Interference of methylmercury with monoamine uptake and release in rat brain synaptosomes. Acta Pharmacol. Toxicol. 48, 214–222. Lakowicz, J., Anderson, C., 1980. Permeability of lipids bilayers to methylmercury chloride: quantification by fluorescence quenching of a carbazole-labeled phospholipid. Chem. Biol. Interact. 30, 309–323. Minnema, D.J., Cooper, G.P., Greenland, R.D., 1989. Effects of methylmercury on neurotransmitters release from rat brain synaptosomes. Toxicol. Appl. Pharmacol. 99, 510–521. Moller-Madsen, B., 1994. Localization of mercury in CNS of the rat. Pharmacol. Toxicol. 75, 1–41. Pellegrino, L.J., Pellegrino, A.S., Cushman, A.J., 1979. A Stereotaxic Atlas of the Rat Brain. Plenum Press, New York. Rodier, P.M., Kates, B., 1988. Histological localization of methylmercury in mouse brain and kidney by emulsion autoradiography of Hg. Toxicol. Appl. Pharmacol. 92, 224–234. Siriois, J.E., Atchison, W.D., 1996. Effects of mercurials on ligandand voltage-gated ion channels: a review. Neurotoxicology 17, 63–84. Traxinger, D.L., Atchison, W.D., 1987. Reversal of methylmercury-induced block of nerve-evoked release of acetylcholine at the neuromuscular junction. Toxicol. Appl. Pharmacol. 90, 23–33.
Effects of successive intrastriatal methylmercury administrations on dopaminergic system L.R.F. Faro,a R. Dura´n,b,* J.L.M. Do Nascimento,a D. Perez-Vences,b and M. Alfonsob b
a Departamento de Fisiologia, Centro de Cieˆncias Biolo´gicas, UFPA, Bele´m, PA, Brazil Laboratorio de Fisiologı´a, Departamento de Biologı´a Funcional y Ciencias de la Salud, Facultad de Ciencias, Campus As Lagoas-Marcosende, Universidad de Vigo, Vigo 36200, Spain
Received 31 December 2001; accepted 23 July 2002
Abstract The present study was carried out in order to determine the effects of intrastriatal administration of different doses (40 mM, 400 mM, and 4 mM) of methylmercury (MeHg) on dopaminergic system of rat striatum. Experiments were performed in conscious and freely moving rats using brain microdialysis coupled with liquid chromatography. Intrastriatal administration of MeHg produced significant increases in dopamine (DA) striatal levels (90777%, 18707319%, and 79717534% for the doses of 40, 400 mM, and 4 mM, with respect to basal). The increase in DA levels was associated with significant decreases in extracellular levels of its main metabolites dihydroxyphenylacetic acid (DOPAC) and homovallinic acid (HVA) (65.073.0% and 52.271.3%, respectively) using the dose of 4 mM MeHg, whereas nonsignificant changes in metabolite levels were observed with the doses of 40 and 400 mM MeHg. A second infusion of 4 mM MeHg 24 h after first infusion also produced a rise of DA levels, but this increase was very small as compared with that produced by first infusion (79717534% versus 9857186%). This second infusion of 4 mM MeHg also decreased DOPAC and HVA levels, but this decrease was not significant as compared with that observed after first infusion (65.073.0% and 52.271.3% versus 62.475.2% and 63.477.4%, respectively). We discuss these effects based on a stimulated DA release and/or a decreased DA intraneuronal degradation. r 2003 Elsevier Science (USA). All rights reserved. Keywords: Methylmercury; Dopamine; Striatum; Microdialysis; HPLC
1. Introduction Methylmercury (MeHg) is an ubiquitous environmental pollutant, recognized as a neurotoxic agent that produces many different effects on the living organism, more specifically to the Central Nervous System (CNS), such as paresthesia, ataxia, convulsion, muscular weakness, impairment of vision, hearing, and speech, and partial paralysis (Berlin, 1986). The absorption of methylmercury occurs mainly in the gastrointestinal tract (Glocking et al., 1977). Because of its high lipid solubility, MeHg penetrates the blood–brain barrier and readily diffuses into cell membranes (Felton et al., 1972; Lakowicz and Anderson, 1980). The knowledge of the primary biochemical events associated with MeHg neurotoxicity of CNS is very *Corresponding author. Fax: +34-8681-2556. E-mail address: [email protected] (R. Dura´n).
scarce to date. However, the effects of MeHg could be due to direct actions on the neuron, resulting in disruption of neurotransmission with subsequent sensory and motor deficits (Siriois and Atchison, 1996). Methylmercury is also known to interfere with synaptic transmission at various steps, including synthesis, uptake, release, and degradation of neurotransmitters (Komulainen and Tuomisto, 1981). In vitro, MeHg exposure affects the release of neurotransmitters at the neuromuscular junction, increasing spontaneous transmitter release and decreasing the depolarization-evoked transmitter release (Atchison, 1987). In CNS tissue preparations, MeHg increases spontaneous transmitter release (Bondy et al., 1979; Komulainen and Tuomisto, 1981), and this increase was found to be concentrationdependent in synaptosomes (Minnema et al., 1989). In synaptosomes, MeHg produces a disruption of synaptosomal membrane integrity and an increase in membrane permeability (transient and reversible or only
0147-6513/03/$ - see front matter r 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0147-6513(02)00127-6
174
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a small fraction of the synaptosomes became leakly) (Minnema et al., 1989). Also consistent with an increase in membrane permeability, and the membrane depolarization that ensues, is the possibility that MeHg alters the conduction of the axonal action potential (Traxinger and Atchison, 1987). Methylmercury is also thought to affect primarily mitochondrial functions and thus energy metabolism of the nervous cell (Komulainen and Bondy, 1987). However, other nonmitochondrial processes may be even more sensitive to MeHg action (Komulainen and Tuomisto, 1981). In addition to MeHg’s effect on mitochondrial enzymes, MeHg interferes with the synthesis, transport, and activity of other enzymatic systems such as glutathione peroxidase, adenylate cyclase, and dehydrogenases (Atchison and Hare, 1994). Moreover, under different in vitro and in vivo experimental conditions, the inhibition of protein synthesis seems to be one of the initial effects of MeHg. Different studies on the distribution of mercury within the CNS of mammals only described the effects of acute administrations and were performed with tissue samples using radioactive compounds of mercury (Rodier and Kates, 1988). Mercury is accumulated mainly in the following CNS regions: cortex, striatum, hippocampus, brain stem, cerebellum, and spinal cord (Moller-Madsen, 1994). The signs produced by the administration of mercury in different forms could be associated with alterations in the motor function. According to studies indicating that those nuclei associated with the motor systems showed a high concentration of mercury (Moller-Madsen, 1994), the striatum is a major target for the study of mercury effects on the brain. Because MeHg seems to influence the neurotransmission and to be a potent neurotoxin in the CNS, we decided to study its effects on the in vivo release of dopamine (DA), the main striatal neurotransmitter, and its main metabolites, dihydroxyphenylacetic acid (DOPAC) and homovallinic acid (HVA), from rat striatum using a microdialysis technique coupled to high-performance liquid chromatography (HPLC) with electrochemical detection.
2. Methods Female Sprague–Dawley rats (weight range 240– 260 g) were used for the experiments. Animals were housed (four per cage) under controlled conditions of temperature (221721C) and photoperiod (light:dark 14:10 h), with food and water available ad libitum. All experiments were performed in accordance with the Guidelines of the European Union Council (86/609/EU) for the use of laboratory animals.
For microdialysis sampling, animals were anesthetized with chloral hydrate (400 mg/kg via i.p.) and placed in a Narishige SR-6 stereotaxic apparatus for the implantation of a guide-cannula. Microdialysis probe (CMA/12, 3-mm membrane length, Stockholm, Sweden) was inserted through the guide-cannula into the left striatum according to the following coordinates from Bregma: A/P +2.0 mm; L +3.0 mm; V +6.0 mm. Placement of probes within the striatum was confirmed histologically after the experiments, using the stereotaxic atlas of Pellegrino et al. (1979) as reference. The first experiments were initiated 24 h after surgery. Continuous perfusion was performed with a Ringer solution (147 mM NaCl, 4 mM KCl, 3.4 mM CaCl2; pH 7.4) using a CMA/102 infusion pump (CMA/Microdialysis, Stockholm, Sweden) at an infusion rate set at 2 ml/min. The samples obtained from the microdialysis procedure were collected every 15 min (30 mL) by means of a CMA/142 microsampler (CMA/Microdialysis, Stockholm, Sweden). The experiments were performed during 4 h with awake, conscious, and freely moving animals. Different doses of MeHg were dissolved in the perfusion medium and applied locally in the striatum via the dialysis probe after obtaining a stable output for DA and metabolites (DOPAC and HVA). After three basal perfusates (45 min), the striatum was perfused with MeHg for 60 min at concentrations of 40, 400 mM, and 4 mM. After this time, the perfusate was then switched back to the unmodified perfusion medium and the measurements were continued for an additional period of 135 min. In the following experiments, the effects of large concentrations of MeHg on the DA, DOPAC, and HVA extracellular levels were verified using a second infusion of 4 mM MeHg, 24 h after the first infusion. In this case, we used the same experimental conditions as described above. The levels of DA, DOPAC, and HVA were measured by means of HPLC with electrochemical detection. Samples obtained from the microdialysis procedure were injected into a Hewlett-Packard Series 1050 Liquid Chromatograph, using a Rheodyne 7125 injection valve. The isocratic separation of DA, DOPAC, and HVA was made using Spherisorb ODS-1 reversed-phase columns (10 mm particle size). The eluent (pH 4.0) was prepared as follows: 70 mM KH2PO4, 1 mM octanesulfonic acid, 1 mM EDTA, and 5% methanol. The flow rate was 1 mL/min. The detection of the substances was achieved by means of an ESA Coulochem 5100A electrochemical detector (MA, USA) at a potential of +400 mV. The chromatograms obtained allowed the determination of DA, DOPAC, and HVA with a run time of 15 min. The data were corrected for the percentage of recovery for every microdialysis probe, which was
L.R.F. Faro et al. / Ecotoxicology and Environmental Safety 55 (2003) 173–177
similar for the different probes and substances analyzed (15% for DA, 20% for DOPAC, and 25% for HVA). The average of substance concentrations in the three samples before MeHg administration was considered the basal level. Basal levels were considered 100% in order to compare the different responses of DA and metabolites after MeHg administration. The results are shown as the mean7SEM of four to eight experiments, expressed as a percentage of the basal levels. Statistical analysis of the results was performed using a repeated-measures ANOVA and Student–Newman– Keuls multiple range test, considering the following significant differences: Po0:05; Po0:01; and Po0:001:
3. Results 3.1. Basal extracellular levels of DA, DOPAC, and HVA The basal output of dialysate DA and its metabolites from striatum before the first infusion of MeHg (day 1) and 24 h after MeHg administration (day 2) are shown in Table 1. Basal values of DA, DOPAC, and HVA 24 h after infusion of 4 mM MeHg were smaller than those observed on day 1.
Table 1 Basal values (mean7SEM, n ¼ 8) expressed as nanograms of substance/15 min for DA, DOPAC, and HVA before first infusion of methylmercury (Day 1) and 24 h after first infusion (Day 2)a
DA DOPAC HVA a
Day 1
Day 2
0.3470.08 5.9071.10 6.0070.9
0.2070.03* 4.8570.33 3.2470.28*
Significant differences: Po0:05 with respect to basal on day 1.
175
3.2. Effect of methylmercury infusion The different doses of MeHg produced a concentration-related increase in the striatal output of DA (Fig. 1A). Experiments carried out on day 1 showed that 40, 400 mM, and 4 mM of MeHg produced maximum increases 30 min after the beginning of MeHg perfusion (90777%, 18707319%, and 79717534% of basal levels, respectively). Basal values recovered 135 min after MeHg administration. The highest dose of MeHg assessed (4 mM) caused a significant decrease in extracellular levels of DOPAC and HVA in striatum 45 min after the beginning of MeHg administration (65.0373% and 52.271.3% of decrease related to basal levels, respectively) (Figs. 1B and C). The other remaining concentrations of MeHg (40 and 400 mM) had no significant effects on the acidic metabolites of DA. 3.3. Effect of a second infusion of 4 mM methylmercury on DA, DOPAC, and HVA In these experiments, the rats exposed to 4 mM of MeHg on day 1 were also exposed to 4 mM of MeHg 24 h later. Fig. 2 shows the effects of a second infusion of MeHg, these effects being characterized by increased DA output 90 min after the beginning of 4 mM MeHg perfusion (9857186% with respect to basal levels). However, this increase was very small as compared with that produced by the first MeHg infusion (Fig. 1A). Therefore, the maximal increase (about 8000%) observed after the first infusion dropped dramatically after the second infusion (about 1000%), as shown in Fig. 2A. DOPAC and HVA striatal levels after the second infusion of 4 mM MeHg showed a small decrease: 62.4275.2% and 63.4177.4%, respectively (Fig. 2B and C). This decrease in DOPAC and HVA levels was not significant as compared with the decrease observed
Fig. 1. Maximal increase (mean7SEM) in DA (A), DOPAC (B), and HVA (C) levels in striatal microdialysis samples taken after perfusion of different doses of MeHg (40, 400 mM, and 4 mM). The results (n ¼ 4 8) were expressed as a percentage of the basal levels (100%). Basal levels were considered as the mean of substance concentrations in the three samples before MeHg perfusion. Significant differences: Po0:05; Po0:01; and Po0:001 with respect to the basal. aPo0:01 comparing the doses of 400 mM with 40 mM; bPo0:001 comparing the doses of 4 mM with 400 and 40 mM.
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Fig. 2. Maximal increase (mean7SEM) in DA (A), DOPAC (B), and HVA (C) levels in striatal microdialysis samples obtained on day 1 and day 2 after administration of 4 mM MeHg. The results are expressed as a percentage of the basal levels (100%) (n ¼ 428). Basal levels were considered as the mean of substance concentrations in the three samples before MeHg perfusion. Significant differences: Po0:05; Po0:01; and Po0:001 with respect to the basal; aPo0:01 comparing the effects of day 1 and day 2.
after first infusion (Fig. 2B and C). Moreover, the experiments carried out 24 h later showed that the effects of MeHg were not only less pronounced than those of day 1 but also delayed (data not shown).
4. Discussion The present study reported that in vivo exposure to MeHg was able to produce a significant concentrationdependent increase of DA extracellular levels in striatum of rats, as well as significant decreases in both DOPAC and HVA striatal levels. Thus, the question was to know whether the effect of 4 mM MeHg was the same when a first infusion and a second infusion 24 h later were compared. In previous articles (Faro et al., 1997, 1998), we have reported that i.p. chronic and acute administration of MeHg induced significant increases in striatal DA output. The effect of systemic administration of MeHg on the striatal dopaminergic system could be due to action of this toxicant on several organs and functions, which could affect CNS activity. However, the in situ intrastriatal MeHg administration is a more direct way to determine the MeHg effect on the dopaminergic system. Our results indicate that MeHg affects the dopaminergic neurons of striatum, producing an increase in DA striatal levels. There are some possible targets for the increases in DA levels induced by MeHg. Thus, a possible mechanism by which DA levels were increased after intrastriatal administration of MeHg could be due to a direct action of the toxicant on the dopaminergic neurons at different levels: membrane, vesicular release, reuptake system, and so on (Komulainen and Tuomisto, 1981). However, these effects of MeHg on the spontaneous DA release could also occur intraneuronally, since MeHg is lypophillic and it can readily cross the nerve membrane.
In addition to increasing DA release, MeHg could also decrease DA reuptake (Komulainen and Tuomisto, 1981), increasing DA extracellular levels. Since DOPAC is produced intraneuronally from DA, the decrease of DA reuptake could explain the reduced levels of DOPAC observed after MeHg administration. Since the basal levels of DA and its metabolites were lower on day 2 compared with day 1, it could be possible to consider that the first administration of MeHg produced some neuronal damage. However, since on day 2 the second MeHg administration induced a lower increase in DA striatal levels (about 1000% on day 2 versus about 8000% on day 1), with similar decreases in both metabolites (nonsignificant changes on day 2 compared to day 1), we deduced that MeHg could affect those mechanisms involved in DA release. Moreover, the results from day 2 also could indicate that MeHg probably affected DA release rather than DA reuptake and metabolism. In conclusion, the intrastriatal administration of 4 mM MeHg produced significant increases in the release of DA from rat striatal tissue, which were associated with significant decreases in the extracellular levels of DOPAC and HVA. We could explain our results considering a stimulation of DA release and/or a decrease in DA reuptake. Moreover, we have also suggested that MeHg administration could affect the mechanisms involved in DA release.
Acknowledgments This research was supported by grants from Xunta de Galicia and University of Vigo (Spain). Lilian Faro acknowledges CNPq (Brasil) for a research grant. The authors thank Dr. J.L. Soengas for his help in the preparation of the article.
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