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Neurotoxic relationship between dopamine and iron in the striatal dopaminergic nerve terminals
Brain Research 858 Ž2000. 26–32 www.elsevier.comrlocaterbres
Research report
Neurotoxic relationship between dopamine and iron in the striatal dopaminergic nerve terminals Marti Santiago, Esperanza R. Matarredona, Luis Granero, Josefina Cano, Alberto Machado
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Departamento de Bioquımica, Bromatologıa ´ ´ y Toxicologıa, ´ Facultad de Farmacia, UniÕersidad de SeÕilla, SeÕille 41012, Spain Accepted 14 December 1999
Abstract The neurotoxic effect of dopamine ŽDA. and ironŽIII. on DAergic terminals in striatum has been studied by intracerebral microdialysis technique. Twenty-four hours after surgery Žday 1., DA andror ironŽIII. with and without DA reuptake inhibitor, nomifensine, were perfused for 1 h. Forty-eight hours after surgery Žday 2., MPPq 1 mM was perfused for 15 min and the output of DA was measured, its amount being directly proportional to the remaining striatal DAergic terminals, supported by tyrosine hydroxylase immunohistochemistry technique. Perfusion of exogenous DA, as well as ironŽIII. 10 and 100 mM, did not produce any neurotoxic effect. However, perfusion of ironŽIII. Ž333 and 1000 mM. produced a concentration-dependent toxic effect. Co-perfusion of ironŽIII. at non-toxic concentration Ž100 mM. with DA Ž15 mM. produced a toxic effect. Elevation of the endogenous extracellular levels of DA by inhibiting its uptake with nomifensine increased the neurotoxic effect of ironŽIII. in a dose-dependent manner. The use of tetrodotoxin after elevation of DA with nomifensine partially prevented the neurotoxic effect of its co-perfusion with ironŽIII. Ž100 mM.. These results suggest that DAergic system could be synergistically damaged by DA and ironŽIII.. Thus, alterations in the clearance of DA from extracellular space along with an increase of iron may have significant consequences for DAergic system toxicity. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Microdialysis; Dopamine release; Iron neurotoxicity; Nomifensine; 1-Methyl-4-phenylpyridinium; Striatum; Rat
1. Introduction The involvement of dopamine ŽDA. in neurotoxicity has been previously described w9,19,20,28x. The DA release induced by methamphetamine Žm-AMPH. appears to be the key element in the development of the DAergic toxicity of this drug w11x. In m-AMPH-treated animals, a direct correlation exists between the amount of released DA and the extent of neuronal loss w30x. This DA toxic effect could be produced through its metabolism and autooxidation. The enzymatic deamination of DA by monoamine oxidase leads to the production of hydrogen peroxide and dihydroxyphenylacetic acid w25x. If not reduced by cellular mechanisms, hydrogen peroxide can react with transition metal, such as iron, to form hydroxyl radical w6x, with the consequent cell damage w17x. On the other hand, autooxidation of DA produces reactive semiquinones or quinones and toxic reduced forms of oxygen w5x in a reaction that occurs spontaneously in the ) C orresponding [email protected]
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presence of iron or enzymatically w15,18x. Indeed, it has been demonstrated that DA and L-DOPA caused neuronal death in tissue culture in the presence of iron w40x. Iron is an essential element involved in numerous biological processes as part of proteins, enzymes and cofactors w7x. High concentrations of free ionic iron can, however, be neurotoxic w16x. Direct injection of iron into the substantia nigra experimentally produces selective damage to nigrostriatal DAergic neurons, resulting in a behavioral and biochemical model of Parkinson’s disease w1,2,24,38x. Increased iron levels have been observed in several neurological disorders, e.g., Parkinson’s and Huntington’s diseases w13,14,22x. Therefore, the balance of iron in the brain is of vital importance. The present study is concerned with the possible mechanisms of the neurotoxic effect of DA and ironŽIII. on the striatal DAergic terminals. Experiments were carried out for 2 consecutive days. On the first day, DA andror iron were perfused for 1 h through the microdialysis probe implanted in the striatum. The neurotoxic effect of the drugs’ perfusion was assessed by perfusion of MPPq 1 mM for 15 min in the second day w29,33x. As demonstrated
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by tyrosine hydroxylase immunohistochemistry, the amount of DA measured after MPPq 1 mM perfusion correlated well with the remaining DAergic terminals.
2. Materials and methods 2.1. Animals and drugs treatment Animals were male albino Wistar rats weighing 270– 320 g at the time of probe implantation. The rats were kept, three or four rats per cage, at constant room temperature Ž22 " 28C. and relative humidity Ž60%. with a 12-h light–dark cycle and unlimited access to food and water. Experiments were carried out in accordance with the Guidelines of the European Union Council Ž86r609rEU., following the Spanish regulations ŽBOE 67r8509-12, 1988. for the use of laboratory animals and approved by the Scientific Committee of the University of Sevilla. The following drugs were used: MPPq iodide and nomifensine maleate ŽResearch Biochemical, Natick, MA, USA., tetrodotoxin and DA hydrochloride ŽSigma, St. Louis, MO, USA. and ironŽIII. chloride anhydrous ŽMerck, Darmstadt, Germany..
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steady baseline of levels in four consecutive samples Žindicated in legends as control values, which are expressed in femtomoles per minute., drugs were administered and sampling was continued for 2.5 h thereafter. All drugs were dissolved in Ringer’s solution. On day 1, DA andror ironŽIII. with or without DA reuptake inhibitor, nomifensine, were perfused for 1 h. On day 2, MPPq 1 mM was perfused for 15 min. IronŽIII. 1 mM solution was adjusted to pH 6.0, adding 5 ml of NaOH 1 Mrml Ringer solution. 2.3. Chemical assays DA level in dialysates was analysed by high-performance liquid chromatography ŽHPLC. with electrochemical detection. A Kontron 420 pump was used in conjunction with a glassy carbon electrode set at y780 mV ŽAntec, the Netherlands.. A Merck Lichrocart cartridge Ž125 = 4 mm2 . column filled with Lichrospher reversephase C 18 5 mM material was used. The mobile phase consisted of a mixture of 0.05 M of sodium acetate, 0.4 mM of 1-octanesulphonic acid, 0.3 mM of Na 2 EDTA and 70 ml methanolrl, adjusted to pH 4.1 with acetic acid. All reagents and water were HPLC-grade. The flow rate was 0.8 mlrmin and the detection limit for DA was 5 fmol per injection.
2.2. Surgery and brain dialysis 2.4. Immunohistological eÕaluation Microdialysis in the corpus striatum was performed with an I-shaped cannula w37x. The exposed tip of the dialysis membrane was 4 mm. The dialysis tube Ži.d.: 0.22 mm; o.d.: 0.31 mm. was prepared from polyacrylonitrilersodium methalyl sulphonate copolymer ŽAN 69, Hospal, Bologna, Italy.. The DA and ironŽIII. in vitro recoveries of the membrane were 20.3 " 1.6% Ž N s 5. and 3.5 " 0.5% Ž N s 4., respectively. The probe was stereotaxically implanted into both corpus striata with coordinates from bregma point and dura ŽArP q0.6, LrM 2.8, VrD 6.0 w32x., during general chloral hydrate Ž400 mgrkg, i.p.. and local lidocaine Ž10% wrv in water. anaesthesia. Following surgery, animals were housed individually in plastic cages Ž35 = 35 = 40 cm3 .. The perfusion experiments were carried out 24 Žday 1. and 48 Žday 2. h after implantation of the probe. Microdialysis and subsequent chemical analysis were performed using an automated on-line sample injection system w42x. The corpus striatum was perfused at a flow rate of 3.0 mlrmin, using a microperfusion pump Žmodel 22, Harvard Apparatus, South Natick, MA, USA., with a Ringer’s solution containing Žin mM.: NaCl, 140; KCl, 4.0; CaCl 2 , 1.2; and MgCl 2 , 1.0, pH s 6.0. With the help of an electronic timer, the injection valve was held in the load position for 15 min, during which the sample loop Ž40 ml. was filled with dialysate. The valve then switched automatically to the injection position for 15 s. This procedure was repeated every 15 min, which was the time needed to record a complete chromatogram. After establishing a
Tyrosine hydroxylase ŽTH. immunohistology was carried out 24 h after perfusion of Ringer’s solution for 3 h, alone or co-perfused with MPPq 1 mM for 15 min. Rats were perfused through the heart under deep anaesthesia Žchloral hydrate. with 150–200 ml of 4% paraformaldehyde in phosphate buffer, pH 7.4. The brains were removed, and then cryoprotected serially in sucrose in PBS, pH 7.4; first in 10% sucrose for 24 h and then in 30% sucrose until sunk Ž2–5 days.. The brains were then frozen in isopentane at y158C, and 25-mm sections were cut on a cryostat and mounted onto gelatine-coated slices. All incubations and washes were in Tris buffer saline ŽTBS., pH 7.4, unless otherwise noted. All works were done at room temperature. Sections were washed and then treated with 0.3% hydrogen peroxide in methanol for 30 min, washed again, and incubated in a solution containing TBS and 1% horse serum for 60 min in a humid chamber. Slices were drained and further incubated with mouse anti-bTH ŽBoehringer-Mannheim, 1:200. in TBS containing 1% horse serum and 0.25% Triton X-100 for 24 h. Sections were then incubated for 2 h with biotinylated horse anti-mouse IgG ŽVector Laboratories, 1:200. followed by a second 1-h incubation with ExtrAvidinw –Peroxidase solution ŽSigma, 1:100.. The antibody was diluted in TBS containing 0.25% Triton X-100, and its addition was preceded by three 10-min rinses in TBS. The peroxidase was visualised with a standard diaminobenzidinerhydrogen peroxidase chromogen reaction for 5 min.
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2.5. Statistics Difference on day 1 between DA extracellular output in control values and drugs treatment was compared by Kruskal–Wallis ANOVA by ranks, and, where appropriate Ž H value greater than the 95% confidence level., comparison of the means was carried out using the Mann–Whitney U-test. The same statistics were used for comparison of the sum of DA output collected from five consecutive samples after MPPq perfusion on day 2 in the different treatments.
3. Results On day 1, DA control data before drug treatment were very similar among the different experiments carried out on this study, being as follows Žin fmolrmin.: 14.2 " 1.3 Ž n s 53. for basal value of DA release; 107.5 " 10.2 Ž n s 5. for perfusion of nomifensine 20 mM; and 314.6 " 27.6 Ž n s 19. for perfusion of nomifensine 100 mM. On day 2, DA control data, before MPPq 1 mM for 15 min perfusion, were similar or even higher than data found on
Fig. 2. ŽA. Effect of perfusion Žhorizontal bar. of ironŽIII. 10, 100, 333 and 1000 mM on the striatal extracellular output of DA Žday 1.. ŽB. Twenty-four hours later Žday 2., MPPq 1 mM was perfused. Data are mean"S.E.M. Žvertical bars. of the sum of five consecutive DA output samples after MPPq perfusion, expressed as femtomoles per minute Ž ns 4–8.. Statistical significance ŽKruskal–Wallis followed by the Mann–Whitney U-test.: U P - 0.05; UU P - 0.01, compared with the control value in ŽA. and with control-Ringer data in ŽB..
Fig. 1. ŽA. Effect of perfusion Žhorizontal bars. of Ringer Žopen circles. or MPPq 1 mM Žfilled circles. on the striatal extracellular output of DA Žday 1.. DA 15 and 50 mM were also perfused for 1 h Žcollection times 60, 75, 90 and 105 min, data not shown.. ŽB. Twenty-four hours later Žday 2., MPPq 1 mM was perfused. Data are mean"S.E.M. Žvertical bars. of the sum of five consecutive DA output samples after MPPq perfusion, expressed as femtomoles per minute Ž ns 5–8.. Statistical significance ŽKruskal–Wallis followed by the Mann–Whitney U-test.: UU P - 0.01, compared with control values in ŽA. and with the controlRinger data in ŽB..
day 1: 17.0 " 1.0 fmolrmin Ž n s 62.. The exception was found when MPPq Ž4.8 " 0.7 fmolrmin, n s 7., ironŽIII. 333 mM Ž7.4 " 1.0 fmolrmin, n s 4. and ironŽIII. 1000 mM Žundetectable levels, n s 4. were perfused on day 1. Twenty-four hours after surgery Žday 1., perfusion of MPPq 1 mM alone for 15 min Žcontrol-MPPq. produced a massive increase in the extracellular output of DA ŽFig. 1A.. Fig. 1B shows the sum of five consecutive DA output samples Žcollection times 60, 75, 90, 105 and 120 min. after MPPq perfusion for 15 min on day 2. There was a clear reduction in the extracellular output of DA when MPPq was included in the Ringer solution compared with the perfusion of only Ringer solution Žcontrol-Ringer.. DA 15 Žgiving a value of 30 000 fmolrmin in dialysate. and 50 mM Žgiving a value of 100 000 fmolrmin in dialysate. were perfused for 1 h on day 1 Ždata not shown.. On day 2, MPPq 1 mM was perfused for 15 min. Previous perfusion of DA 15 mM on day 1 produced on day 2, after MPPq perfusion, the highest increase in the extracellular output of DA ŽFig. 1B.. Perfusion of DA 50 mM on day 1 produced an increase of DA on day 2 similar to that of control-Ringer ŽFig. 1B..
M. Santiago et al.r Brain Research 858 (2000) 26–32
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Fig. 3. Photomicrograph of horizontal sections Žcryostat-cut sections. through striatum after TH immunostaining. ŽA. Rat perfused with ironŽIII. 100 mM for 1 h. ŽB. Rat perfused with ironŽIII. 333 mM for 1 h. ŽC. Rat perfused with ironŽIII. 1000 mM for 1 h. Scale bar: 200 mm.
Perfusion of ironŽIII. 10 mM for 1 h did not modify the release of DA ŽFig. 2A.. Higher doses of ironŽIII. Ž100, 333 and 1000 mM. produced a slight increase in the release of DA. IronŽIII. 1000 mM produced a transient increase followed by a long-lasting decrease in the release of DA ŽFig. 2A.. Increase in the extracellular output of DA produced after perfusion of MPPq 1 mM for 15 min on day 2 was dependent upon the concentration of ironŽIII. perfused on day 1 ŽFig. 2B.. Rats perfused with ironŽIII. 10 and 100 mM on day 1 produced higher increases in the extracellular output of DA after MPPq perfusion than control-Ringer rats on day 2. Rats perfused with ironŽIII. 333 and 1000 mM on day 1 produced lower increases in
Fig. 4. Effect of perfusion Žhorizontal bar. of MPPq 1 mM for 15 min, 24 h Žday 2. after different perfusions carried out on day 1 Žsee X labels, in micromolar., on the striatal extracellular output of DA. Nomifensine was included in the Ringer solution during the whole experiment on day 1. Data are mean"S.E.M. Žvertical bars. of the sum of five consecutive DA output samples after MPPq perfusion, expressed as femtomoles per minute Ž ns 4–8.. Statistical significance ŽKruskal–Wallis followed by the Mann–Whitney U-test.: UU P - 0.01, compared with control-Ringer data.
the extracellular output of DA after MPPq perfusion than control-Ringer rats on day 2. The highest dose of ironŽIII. perfused on day 1 was very toxic, as indicated by the low increase in the extracellular output of DA observed after MPPq perfusion on day 2 ŽFig. 2B.. The effect of 1 h perfusion of several iron concentrations on TH immunoreactivity 24 h later is shown in Fig. 3. IronŽIII. 100 mM did not produce any change in tyrosine hydroxylase immunopositive staining. However, ironŽIII. 333 and 1000 mM produced, respectively, a slight Ž333 mM. and a marked Ž1000 mM. decrease in TH immunoreactivity around the track of the cannula, which is proportional to the HPLC data for DA measured on day 2. Next, we co-perfused on day 1 non-toxic ironŽIII. concentration Ž10 and 100 mM. with a non-toxic DA concentration Ž15 mM.. Iron ŽIII. 10 mM co-perfused with DA 15
Fig. 5. Photomicrograph of horizontal sections Žcryostat-cut sections. through striatum after TH immunostaining. ŽA. Rat perfused with DA 15 mM for 1 h. ŽB. Rat perfused with DA 15 mM plus ironŽIII. 100 mM for 1 h. Scale bar: 200 mM.
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mM did not produce any toxic effect ŽFig. 4.. However, co-perfusion of ironŽIII. 100 mM with DA 15 mM produced a clear reduction of DA extracellular output on day 2 after MPPq perfusion ŽFig. 4.. Perfusion of DA 15 mM for 1 h showed on day 2 TH immunopositive staining in the tissue around the cannula ŽFig. 5.. When we co-perfused iron 100 mM with DA 15 mM on day 1, a lack of TH immunoreactivity around the cannula was observed ŽFig. 5.. As we previously showed, co-perfusion of ironŽIII. 10 mM with DA 15 mM on day 1 did not produce any toxic effect on DAergic terminals. Addition of nomifensine, an inhibitor of DA reuptake, in the Ringer solution on day 1 produced a toxic effect, as observed by the reduction in the extracellular output of DA after MPPq perfusion on day 2 ŽFig. 4.. To check whether an increase in endogenous DA is neurotoxic, we co-perfused nomifensine at different concentrations Ž20 and 100 mM. with a non-toxic Ž100 mM. ironŽIII. concentration. Nomifensine 100 mM was perfused alone for 1 h on day 1. On day 2, perfusion of MPPq 1 mM for 15 min produced an increase in the extracellular output of DA similar to control-Ringer data ŽFig. 6.. The presence of nomifensine 20 mM partially prevented the effect produced on DA release on day 1 by ironŽIII. 100 mM perfusion Ždata not shown.. On day 2, the presence of nomifensine 20 mM during perfusion of ironŽIII. 100 mM on day 1 produced a smaller increase in the extracellular output of DA after MPPq perfusion than in the absence of nomifensine ŽFig. 6.. Co-perfusion of a higher concentration of nomifensine Ž100 mM. with ironŽIII. 100 mM on day 1 produced a greater decrease in the extracellular output of DA on day 2 after MPPq perfusion than
Fig. 6. Effect of perfusion Žhorizontal bar. of MPPq 1 mM for 15 min, 24 h Žday 2. after different perfusions carried out on day 1 Žsee X labels, in micromolar., on the striatal extracellular output of DA. Nomifensine was included in the Ringer solution during the whole experiment on day 1. Data are mean"S.E.M. Žvertical bars. of the sum of five consecutive DA output samples after MPPq perfusion, expressed as femtomoles per minute Ž ns 4–7.. Statistical significance ŽKruskal–Wallis followed by the Mann–Whitney U-test.: UU P - 0.01, compared with ironŽIII. 100 mM data. aaP - 0.01, compared with ironŽIII. 100 mMqnomifensine 100 mM data.
nomifensine 20 mM or in the absence of nomifensine ŽFig. 6.. Therefore, co-perfusion of ironŽIII. 100 mM with different doses of nomifensine showed a dose-dependent neurotoxicity. In the presence of nomifensine 100 mM, TTX 2 mM, a fast sodium channel inhibitor, was perfused for 1 h before its co-perfusion with ironŽIII. 100 mM on day 1. On day 2, MPPq perfusion produced a higher extracellular output of DA in the presence than in the absence of tetrodotoxin 2 mM ŽFig. 6.. Perfusion of TTX 2 mM alone for 1 h on day 1 produced a strong decrease in the extracellular output of DA to nearly undetectable levels Ždata not shown.. On day 2, perfusion of MPPq 1 mM for 15 min produced an increase in the extracellular output of DA similar to control-Ringer data.
4. Discussion The DAergic system is highly susceptible to oxidative damage w21x. DA itself is able to produce hydrogen peroxide and other oxygen radicals either by autooxidation or by enzymatic reactions involved in its metabolism w5,25x. The involvement of DA on several toxic reactions has been extensively described w39x. The massive DA overflow in striatum produced by perfusion of MPPq w33x could be involved in MPTPrMPPq toxicity, since concurrent administration of haloperidol with MPTP significantly enhanced the depression of the activity of tyrosine hydroxylase in the striatum caused by MPTP w27x. Therefore, in an attempt to mimic the effects of a MPPq perfusion in the striatum, we perfused DA throughout the dialysis probe. We calculate that the increase in extracellular output of DA measured after MPPq 1 mM perfusion is similar to a perfusion of DA 15 mM through the microdialysis cannula, given the fact that in vitro recovery of DA is about 20%. In order to evaluate the damage induced by the perfusion of DA carried out on day 1, we perfused MPPq for 15 min 24 h later Žday 2.. We consider the released DA on day 2 after MPPq perfusion as an index of the surviving DAergic terminals of day 1. In contrast to the toxic action of DA ascribed by other groups w3,9x, DA perfusion did not damage the DAergic terminals. The most likely explanation for this discrepancy could be differences in the DA concentration used. These observations indicate that DA may not be uniquely responsible for its described toxic effects. Most of the DA-induced reactions to form free radicals involve the presence of iron, a metal that enhances free radical production by reducing hydrogen peroxide. Toxicity of iron in the nigrostriatal system has been reported w16x. One of the hallmarks in neurodegenerative diseases is a marked increase in iron in the affected brain regions Žfor review, see Ref. w12x.. In fact, iron chelation — removal of free radicals produced by iron with scavengers or spin
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traps — protects the degeneration induced by iron w23,26,36x. In the present report, we studied the possible neurotoxic interaction between iron and DA. Firstly, we evaluated the toxicity of iron itself by means of the perfusion of different ironŽIII. concentrations Ž10, 100, 333 and 1000 mM in the perfusion fluid or 0.35, 3.5, 12 and 35 mM, respectively, at extracellular level. to study its toxic action. Perfusion of ironŽIII. Ž10 and 100 mM. on day 1 produced a slight increase in DA overflow, but did not affect the DA overflow after MPPq perfusion on day 2, even though DA overflow was higher than in control-Ringer data. This effect could be explained, at least in part, by the activation of the tyrosine hydroxylase enzyme by iron at these nonneurotoxic concentrations. However, higher ironŽIII. concentrations Ž333 and 1000 mM. produced a toxic effect in a concentration-dependent manner. The 333 mM concentration resulted in a reduction in DA extracellular output of 50% after MPPq perfusion as compared with control animals, whereas ironŽIII. 1000 mM totally damaged DAergic terminals, since there was no increase in DA extracellular output after MPPq perfusion. These observations were corroborated by tyrosine hydroxylase immunohistochemistry. An additive or synergistic effect of DA and iron on their toxicity to DA neurons has been described w31,41x. To test this possible effect, we carried out co-perfusion of DA with ironŽIII. at concentrations which, separately, did not produce any damage to DAergic terminals. Under these conditions, co-perfusion produced an evident disruption of DAergic terminals, as shown by MPPq perfusion data on day 2 and tyrosine hydroxylase immunohistochemistry. Since several toxic processes for DAergic neurons that occur inside the neuron have been described, the question is whether the toxic action of iron is mainly produced inside or outside DAergic neurons. DA uptake system is an efficient mechanism, but it seems to have little selectivity, in as much as it produces the uptake of different molecular compounds w4x. Thus, through a mechanism dependent on the DA carrier, iron could bind to DA and come into DAergic neurons, producing a toxic action inside them by increasing free radicals. To test this hypothesis, the effect of the selective DA uptake inhibitor, nomifensine, was examined. Nomifensine 20 mM produced a 10–15-fold increase in the basal extracellular output of DA. We used two ironŽIII. concentrations: one without Ž100 mM. and another with effect Ž333 mM, data not shown. on DAergic terminals. In both cases, co-perfusion with nomifensine induced an increase in iron-induced toxicity. An increase in the nomifensine concentration produced more damage to DAergic terminals. These results suggest that DA reuptake is not necessary for DA–iron-induced toxicity and that an increase in endogenous DA through inhibition of DA uptake is more toxic than DA administered exogenously. To test the latter,
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a decrease in the release of DA, in the conditions mentioned above, was induced with tetrodotoxin, which inhibits the sodium conductance. In this case, a partial protection after MPPq perfusion on day 2 was found. These results demonstrate that the DAergic system could be synergistically damaged by DA and iron and that this damage could be extraneuronally produced. In aged rats, an increase in iron in substantia nigra w35x and in other brain regions w10x has been described. We have previously reported that the oxidative stress in substantia nigra increases during ageing w8x and that this damage increased when aged rats were treated for several months with nomifensine w34x. Therefore, alterations in the clearance of DA from the extracellular space, along with an increase of iron, may have significant consequences for functioning of the DAergic system. Therapy directed to upregulate antioxidative systems should be considered in aging and neurodegenerative diseases such as Parkinson’s disease. Acknowledgements This work was supported by grant 96-1142 from Fondo de Investigaciones Sanitarias, Spain. E.R.M. and L.G. thank Ministerio de Educacion ´ y Ciencia and Consellerıa ´ de Cultura, Educacion ´ y Ciencia, Generalitat Valenciana, Spain, for pre- and post-doctoral fellowships, respectively. References w1x G. Arendash, G. Sengstock, C. Olanow, S. Barone, A. Dunn, Intranigral iron infusion as a model for Parkinson’s disease, in: W. Woodrum, A. Nonneman ŽEds.., Toxin-Induced Models of Neurological Disorders, Plenum, New York, 1994, pp. 175–212. w2x D. Ben-Shachar, M.B.H. Youdim, Intranigral iron injection induces behavioral and biochemical ‘‘parkinsonism’’ in rats, J. Neurochem. 57 Ž1991. 2133–2135. w3x D. Ben-Shachar, R. Zuk, Y. Glinka, Dopamine neurotoxicity inhibition of mitochondrial respiration, J. Neurochem. 64 Ž1995. 718–723. w4x M. Bougria, J. Vitorica, J. Cano, A. Machado, Implication of dopamine transporter system on 1-methyl-4-phenylpyridinium and rotenone effect in striatal synaptosomes, Eur. J. Pharmacol. 291 Ž1996. 407–415. w5x C.C. Chiueh, H. Miyake, M.-T. Peng, Role of dopamine autooxidation, hydroxyl radical generation, and calcium overload in underlying mechanisms involved in MPTP-induced parkinsonism, Adv. Neurol. 60 Ž1993. 251–258. w6x C.C. Chiueh, D.L. Murphy, H. Miyake, K. Lang, P.K. Tulsi, S.J. Huang, Hydroxyl free radical ŽPOH. formation reflected by salicylate hydroxylation and neuromelanin: in vivo markers for oxidative injury of nigral neurons, Ann. N.Y. Acad. Sci. 679 Ž1993. 370–375. w7x R.R. Crichton, Inorganic Biochemistry of Iron Metabolism, Ellis Horwood, 1991. w8x C.P. De la Cruz, E. Revilla, J.L. Venero, A. Ayala, J. Cano, A. Machado, Oxidative inactivation of tyrosine hydroxylase in substantia nigra of aged rats, Free Radical Biol. Med. 20 Ž1996. 53–61. w9x F. Filloux, J.J. Townsend, Pre- and postsynaptic neurotoxic effects of dopamine demonstrated by intrastriatal injection, Exp. Neurol. 119 Ž1993. 79–88. w10x S.J. Focht, B.S. Snyder, J.L. Beard, W. Van Gelder, L.R. Williams, J.R. Connor, Regional distribution of iron, transferrin, ferritin, and
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w28x B.A. McLaughlin, D. Nelson, M. Erecinska, M.F. Chesselet, Toxicity of dopamine to striatal neurons in vitro and potentiation of cell death by mitochondrial inhibitor, J. Neurochem. 70 Ž1998. 2406– 2415. w29x N. Naiman, H. Rollema, E. Johnson, N. Castagnoli Jr., Studies on 4-benzyl-1,2,3,6-tetrahydropyridine, a non-neurotoxic analogue of the parkinsonian-inducing agent, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, Chem. Res. Toxicol. 3 Ž1990. 133–138. w30x S.J. O’Dell, F.B. Weihmuller, J.F. Marshall, Methamphetamine-induced dopamine overflow and injury to striatal dopamine terminals: attenuation by dopamine D1 and D 2 antagonists, J. Neurochem. 60 Ž1993. 1792–1799. w31x D. Offen, I. Ziv, A. Barzilai, S. Gorodin, E. Glater, A. Hochman, E. Melamed, Dopamine–melanin induces apoptosis in PC12 cells; possible implications for the etiology of Parkinson’s disease, Neurochem. Int. 31 Ž1997. 207–216. w32x G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, San Diego, 1986. w33x H. Rollema, G. Damsma, A.S. Horn, J.B. De Vries, B.H.C. Westerink, Brain dialysis in conscious rats reveals an instantaneous massive release of striatal dopamine in response to MPPq, Eur. J. Pharmacol. 126 Ž1986. 345–346. w34x M. Romero-Ramos, J.A. Rodrıguez-Gomez, J.L. Venero, J. Cano, ´ ´ A. Machado, Chronic inhibition of the high-affinity dopamine uptake system increases oxidative damage to proteins in the aged rat substantia nigra, Free Radical Biol. Med. 23 Ž1997. 1–7. w35x A.J.I. Roskams, J.R. Connor, Iron, transferrin, and ferritin in the rat brain during development and aging, J. Neurochem. 63 Ž1994. 709–716. w36x M. Santiago, E.R. Matarredona, L. Granero, J. Cano, A. Machado, Neuroprotective effect of the iron chelator desferrioxamine against MPPq toxicity on striatal dopaminergic terminals, J. Neurochem. 68 Ž1997. 732–738. w37x M. Santiago, B.H.C. Westerink, Characterization of the in vivo release of dopamine as recorded by different types of intracerebral microdialysis probes, Naunyn-Schmiedeberg’s Arch. Pharmacol. 342 Ž1990. 407–414. w38x G.J. Sengstock, N.H. Zawia, C.W. Olanov, A.J. Dunn, G.W. Arendash, Intranigral iron infusion in the rat. Acute elevations in nigral lipid peroxidation and striatal dopaminergic markers with ensuing nigral degeneration, Biol. Trace Elem. Res. 58 Ž1997. 177–195. w39x A.H. Stokes, T.G. Hastings, K.E. Vrana, Cytotoxic and genotoxic potential of dopamine, J. Neurosci. Res. 15 Ž1999. 659–665. w40x M. Tanaka, A. Sotomatsu, H. Kanai, S. Harai, DOPA and dopamine cause cultured neuronal death in the presence of iron, J. Neurol. Sci. 101 Ž1991. 198–203. w41x C. Velez-Pardo, M.J. Del Rio, G. Ebinger, G. Vauquelin, Monoamine ´ and iron-related toxicity: from ‘‘serotonin-binding proteins’’ to lipid peroxidation and apoptosis in PC12 cells, Gen. Pharmacol. 31 Ž1998. 19–24. w42x B.H.C. Westerink, G. Damsma, H. Rollema, J.B. De Vries, A.S. Horn, Scope and limitations of in vivo brain dialysis: a comparison of its application to various neurotransmitter systems, Life Sci. 41 Ž1987. 1763–1776.
Research report
Neurotoxic relationship between dopamine and iron in the striatal dopaminergic nerve terminals Marti Santiago, Esperanza R. Matarredona, Luis Granero, Josefina Cano, Alberto Machado
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Departamento de Bioquımica, Bromatologıa ´ ´ y Toxicologıa, ´ Facultad de Farmacia, UniÕersidad de SeÕilla, SeÕille 41012, Spain Accepted 14 December 1999
Abstract The neurotoxic effect of dopamine ŽDA. and ironŽIII. on DAergic terminals in striatum has been studied by intracerebral microdialysis technique. Twenty-four hours after surgery Žday 1., DA andror ironŽIII. with and without DA reuptake inhibitor, nomifensine, were perfused for 1 h. Forty-eight hours after surgery Žday 2., MPPq 1 mM was perfused for 15 min and the output of DA was measured, its amount being directly proportional to the remaining striatal DAergic terminals, supported by tyrosine hydroxylase immunohistochemistry technique. Perfusion of exogenous DA, as well as ironŽIII. 10 and 100 mM, did not produce any neurotoxic effect. However, perfusion of ironŽIII. Ž333 and 1000 mM. produced a concentration-dependent toxic effect. Co-perfusion of ironŽIII. at non-toxic concentration Ž100 mM. with DA Ž15 mM. produced a toxic effect. Elevation of the endogenous extracellular levels of DA by inhibiting its uptake with nomifensine increased the neurotoxic effect of ironŽIII. in a dose-dependent manner. The use of tetrodotoxin after elevation of DA with nomifensine partially prevented the neurotoxic effect of its co-perfusion with ironŽIII. Ž100 mM.. These results suggest that DAergic system could be synergistically damaged by DA and ironŽIII.. Thus, alterations in the clearance of DA from extracellular space along with an increase of iron may have significant consequences for DAergic system toxicity. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Microdialysis; Dopamine release; Iron neurotoxicity; Nomifensine; 1-Methyl-4-phenylpyridinium; Striatum; Rat
1. Introduction The involvement of dopamine ŽDA. in neurotoxicity has been previously described w9,19,20,28x. The DA release induced by methamphetamine Žm-AMPH. appears to be the key element in the development of the DAergic toxicity of this drug w11x. In m-AMPH-treated animals, a direct correlation exists between the amount of released DA and the extent of neuronal loss w30x. This DA toxic effect could be produced through its metabolism and autooxidation. The enzymatic deamination of DA by monoamine oxidase leads to the production of hydrogen peroxide and dihydroxyphenylacetic acid w25x. If not reduced by cellular mechanisms, hydrogen peroxide can react with transition metal, such as iron, to form hydroxyl radical w6x, with the consequent cell damage w17x. On the other hand, autooxidation of DA produces reactive semiquinones or quinones and toxic reduced forms of oxygen w5x in a reaction that occurs spontaneously in the ) C orresponding [email protected]
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presence of iron or enzymatically w15,18x. Indeed, it has been demonstrated that DA and L-DOPA caused neuronal death in tissue culture in the presence of iron w40x. Iron is an essential element involved in numerous biological processes as part of proteins, enzymes and cofactors w7x. High concentrations of free ionic iron can, however, be neurotoxic w16x. Direct injection of iron into the substantia nigra experimentally produces selective damage to nigrostriatal DAergic neurons, resulting in a behavioral and biochemical model of Parkinson’s disease w1,2,24,38x. Increased iron levels have been observed in several neurological disorders, e.g., Parkinson’s and Huntington’s diseases w13,14,22x. Therefore, the balance of iron in the brain is of vital importance. The present study is concerned with the possible mechanisms of the neurotoxic effect of DA and ironŽIII. on the striatal DAergic terminals. Experiments were carried out for 2 consecutive days. On the first day, DA andror iron were perfused for 1 h through the microdialysis probe implanted in the striatum. The neurotoxic effect of the drugs’ perfusion was assessed by perfusion of MPPq 1 mM for 15 min in the second day w29,33x. As demonstrated
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by tyrosine hydroxylase immunohistochemistry, the amount of DA measured after MPPq 1 mM perfusion correlated well with the remaining DAergic terminals.
2. Materials and methods 2.1. Animals and drugs treatment Animals were male albino Wistar rats weighing 270– 320 g at the time of probe implantation. The rats were kept, three or four rats per cage, at constant room temperature Ž22 " 28C. and relative humidity Ž60%. with a 12-h light–dark cycle and unlimited access to food and water. Experiments were carried out in accordance with the Guidelines of the European Union Council Ž86r609rEU., following the Spanish regulations ŽBOE 67r8509-12, 1988. for the use of laboratory animals and approved by the Scientific Committee of the University of Sevilla. The following drugs were used: MPPq iodide and nomifensine maleate ŽResearch Biochemical, Natick, MA, USA., tetrodotoxin and DA hydrochloride ŽSigma, St. Louis, MO, USA. and ironŽIII. chloride anhydrous ŽMerck, Darmstadt, Germany..
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steady baseline of levels in four consecutive samples Žindicated in legends as control values, which are expressed in femtomoles per minute., drugs were administered and sampling was continued for 2.5 h thereafter. All drugs were dissolved in Ringer’s solution. On day 1, DA andror ironŽIII. with or without DA reuptake inhibitor, nomifensine, were perfused for 1 h. On day 2, MPPq 1 mM was perfused for 15 min. IronŽIII. 1 mM solution was adjusted to pH 6.0, adding 5 ml of NaOH 1 Mrml Ringer solution. 2.3. Chemical assays DA level in dialysates was analysed by high-performance liquid chromatography ŽHPLC. with electrochemical detection. A Kontron 420 pump was used in conjunction with a glassy carbon electrode set at y780 mV ŽAntec, the Netherlands.. A Merck Lichrocart cartridge Ž125 = 4 mm2 . column filled with Lichrospher reversephase C 18 5 mM material was used. The mobile phase consisted of a mixture of 0.05 M of sodium acetate, 0.4 mM of 1-octanesulphonic acid, 0.3 mM of Na 2 EDTA and 70 ml methanolrl, adjusted to pH 4.1 with acetic acid. All reagents and water were HPLC-grade. The flow rate was 0.8 mlrmin and the detection limit for DA was 5 fmol per injection.
2.2. Surgery and brain dialysis 2.4. Immunohistological eÕaluation Microdialysis in the corpus striatum was performed with an I-shaped cannula w37x. The exposed tip of the dialysis membrane was 4 mm. The dialysis tube Ži.d.: 0.22 mm; o.d.: 0.31 mm. was prepared from polyacrylonitrilersodium methalyl sulphonate copolymer ŽAN 69, Hospal, Bologna, Italy.. The DA and ironŽIII. in vitro recoveries of the membrane were 20.3 " 1.6% Ž N s 5. and 3.5 " 0.5% Ž N s 4., respectively. The probe was stereotaxically implanted into both corpus striata with coordinates from bregma point and dura ŽArP q0.6, LrM 2.8, VrD 6.0 w32x., during general chloral hydrate Ž400 mgrkg, i.p.. and local lidocaine Ž10% wrv in water. anaesthesia. Following surgery, animals were housed individually in plastic cages Ž35 = 35 = 40 cm3 .. The perfusion experiments were carried out 24 Žday 1. and 48 Žday 2. h after implantation of the probe. Microdialysis and subsequent chemical analysis were performed using an automated on-line sample injection system w42x. The corpus striatum was perfused at a flow rate of 3.0 mlrmin, using a microperfusion pump Žmodel 22, Harvard Apparatus, South Natick, MA, USA., with a Ringer’s solution containing Žin mM.: NaCl, 140; KCl, 4.0; CaCl 2 , 1.2; and MgCl 2 , 1.0, pH s 6.0. With the help of an electronic timer, the injection valve was held in the load position for 15 min, during which the sample loop Ž40 ml. was filled with dialysate. The valve then switched automatically to the injection position for 15 s. This procedure was repeated every 15 min, which was the time needed to record a complete chromatogram. After establishing a
Tyrosine hydroxylase ŽTH. immunohistology was carried out 24 h after perfusion of Ringer’s solution for 3 h, alone or co-perfused with MPPq 1 mM for 15 min. Rats were perfused through the heart under deep anaesthesia Žchloral hydrate. with 150–200 ml of 4% paraformaldehyde in phosphate buffer, pH 7.4. The brains were removed, and then cryoprotected serially in sucrose in PBS, pH 7.4; first in 10% sucrose for 24 h and then in 30% sucrose until sunk Ž2–5 days.. The brains were then frozen in isopentane at y158C, and 25-mm sections were cut on a cryostat and mounted onto gelatine-coated slices. All incubations and washes were in Tris buffer saline ŽTBS., pH 7.4, unless otherwise noted. All works were done at room temperature. Sections were washed and then treated with 0.3% hydrogen peroxide in methanol for 30 min, washed again, and incubated in a solution containing TBS and 1% horse serum for 60 min in a humid chamber. Slices were drained and further incubated with mouse anti-bTH ŽBoehringer-Mannheim, 1:200. in TBS containing 1% horse serum and 0.25% Triton X-100 for 24 h. Sections were then incubated for 2 h with biotinylated horse anti-mouse IgG ŽVector Laboratories, 1:200. followed by a second 1-h incubation with ExtrAvidinw –Peroxidase solution ŽSigma, 1:100.. The antibody was diluted in TBS containing 0.25% Triton X-100, and its addition was preceded by three 10-min rinses in TBS. The peroxidase was visualised with a standard diaminobenzidinerhydrogen peroxidase chromogen reaction for 5 min.
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2.5. Statistics Difference on day 1 between DA extracellular output in control values and drugs treatment was compared by Kruskal–Wallis ANOVA by ranks, and, where appropriate Ž H value greater than the 95% confidence level., comparison of the means was carried out using the Mann–Whitney U-test. The same statistics were used for comparison of the sum of DA output collected from five consecutive samples after MPPq perfusion on day 2 in the different treatments.
3. Results On day 1, DA control data before drug treatment were very similar among the different experiments carried out on this study, being as follows Žin fmolrmin.: 14.2 " 1.3 Ž n s 53. for basal value of DA release; 107.5 " 10.2 Ž n s 5. for perfusion of nomifensine 20 mM; and 314.6 " 27.6 Ž n s 19. for perfusion of nomifensine 100 mM. On day 2, DA control data, before MPPq 1 mM for 15 min perfusion, were similar or even higher than data found on
Fig. 2. ŽA. Effect of perfusion Žhorizontal bar. of ironŽIII. 10, 100, 333 and 1000 mM on the striatal extracellular output of DA Žday 1.. ŽB. Twenty-four hours later Žday 2., MPPq 1 mM was perfused. Data are mean"S.E.M. Žvertical bars. of the sum of five consecutive DA output samples after MPPq perfusion, expressed as femtomoles per minute Ž ns 4–8.. Statistical significance ŽKruskal–Wallis followed by the Mann–Whitney U-test.: U P - 0.05; UU P - 0.01, compared with the control value in ŽA. and with control-Ringer data in ŽB..
Fig. 1. ŽA. Effect of perfusion Žhorizontal bars. of Ringer Žopen circles. or MPPq 1 mM Žfilled circles. on the striatal extracellular output of DA Žday 1.. DA 15 and 50 mM were also perfused for 1 h Žcollection times 60, 75, 90 and 105 min, data not shown.. ŽB. Twenty-four hours later Žday 2., MPPq 1 mM was perfused. Data are mean"S.E.M. Žvertical bars. of the sum of five consecutive DA output samples after MPPq perfusion, expressed as femtomoles per minute Ž ns 5–8.. Statistical significance ŽKruskal–Wallis followed by the Mann–Whitney U-test.: UU P - 0.01, compared with control values in ŽA. and with the controlRinger data in ŽB..
day 1: 17.0 " 1.0 fmolrmin Ž n s 62.. The exception was found when MPPq Ž4.8 " 0.7 fmolrmin, n s 7., ironŽIII. 333 mM Ž7.4 " 1.0 fmolrmin, n s 4. and ironŽIII. 1000 mM Žundetectable levels, n s 4. were perfused on day 1. Twenty-four hours after surgery Žday 1., perfusion of MPPq 1 mM alone for 15 min Žcontrol-MPPq. produced a massive increase in the extracellular output of DA ŽFig. 1A.. Fig. 1B shows the sum of five consecutive DA output samples Žcollection times 60, 75, 90, 105 and 120 min. after MPPq perfusion for 15 min on day 2. There was a clear reduction in the extracellular output of DA when MPPq was included in the Ringer solution compared with the perfusion of only Ringer solution Žcontrol-Ringer.. DA 15 Žgiving a value of 30 000 fmolrmin in dialysate. and 50 mM Žgiving a value of 100 000 fmolrmin in dialysate. were perfused for 1 h on day 1 Ždata not shown.. On day 2, MPPq 1 mM was perfused for 15 min. Previous perfusion of DA 15 mM on day 1 produced on day 2, after MPPq perfusion, the highest increase in the extracellular output of DA ŽFig. 1B.. Perfusion of DA 50 mM on day 1 produced an increase of DA on day 2 similar to that of control-Ringer ŽFig. 1B..
M. Santiago et al.r Brain Research 858 (2000) 26–32
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Fig. 3. Photomicrograph of horizontal sections Žcryostat-cut sections. through striatum after TH immunostaining. ŽA. Rat perfused with ironŽIII. 100 mM for 1 h. ŽB. Rat perfused with ironŽIII. 333 mM for 1 h. ŽC. Rat perfused with ironŽIII. 1000 mM for 1 h. Scale bar: 200 mm.
Perfusion of ironŽIII. 10 mM for 1 h did not modify the release of DA ŽFig. 2A.. Higher doses of ironŽIII. Ž100, 333 and 1000 mM. produced a slight increase in the release of DA. IronŽIII. 1000 mM produced a transient increase followed by a long-lasting decrease in the release of DA ŽFig. 2A.. Increase in the extracellular output of DA produced after perfusion of MPPq 1 mM for 15 min on day 2 was dependent upon the concentration of ironŽIII. perfused on day 1 ŽFig. 2B.. Rats perfused with ironŽIII. 10 and 100 mM on day 1 produced higher increases in the extracellular output of DA after MPPq perfusion than control-Ringer rats on day 2. Rats perfused with ironŽIII. 333 and 1000 mM on day 1 produced lower increases in
Fig. 4. Effect of perfusion Žhorizontal bar. of MPPq 1 mM for 15 min, 24 h Žday 2. after different perfusions carried out on day 1 Žsee X labels, in micromolar., on the striatal extracellular output of DA. Nomifensine was included in the Ringer solution during the whole experiment on day 1. Data are mean"S.E.M. Žvertical bars. of the sum of five consecutive DA output samples after MPPq perfusion, expressed as femtomoles per minute Ž ns 4–8.. Statistical significance ŽKruskal–Wallis followed by the Mann–Whitney U-test.: UU P - 0.01, compared with control-Ringer data.
the extracellular output of DA after MPPq perfusion than control-Ringer rats on day 2. The highest dose of ironŽIII. perfused on day 1 was very toxic, as indicated by the low increase in the extracellular output of DA observed after MPPq perfusion on day 2 ŽFig. 2B.. The effect of 1 h perfusion of several iron concentrations on TH immunoreactivity 24 h later is shown in Fig. 3. IronŽIII. 100 mM did not produce any change in tyrosine hydroxylase immunopositive staining. However, ironŽIII. 333 and 1000 mM produced, respectively, a slight Ž333 mM. and a marked Ž1000 mM. decrease in TH immunoreactivity around the track of the cannula, which is proportional to the HPLC data for DA measured on day 2. Next, we co-perfused on day 1 non-toxic ironŽIII. concentration Ž10 and 100 mM. with a non-toxic DA concentration Ž15 mM.. Iron ŽIII. 10 mM co-perfused with DA 15
Fig. 5. Photomicrograph of horizontal sections Žcryostat-cut sections. through striatum after TH immunostaining. ŽA. Rat perfused with DA 15 mM for 1 h. ŽB. Rat perfused with DA 15 mM plus ironŽIII. 100 mM for 1 h. Scale bar: 200 mM.
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mM did not produce any toxic effect ŽFig. 4.. However, co-perfusion of ironŽIII. 100 mM with DA 15 mM produced a clear reduction of DA extracellular output on day 2 after MPPq perfusion ŽFig. 4.. Perfusion of DA 15 mM for 1 h showed on day 2 TH immunopositive staining in the tissue around the cannula ŽFig. 5.. When we co-perfused iron 100 mM with DA 15 mM on day 1, a lack of TH immunoreactivity around the cannula was observed ŽFig. 5.. As we previously showed, co-perfusion of ironŽIII. 10 mM with DA 15 mM on day 1 did not produce any toxic effect on DAergic terminals. Addition of nomifensine, an inhibitor of DA reuptake, in the Ringer solution on day 1 produced a toxic effect, as observed by the reduction in the extracellular output of DA after MPPq perfusion on day 2 ŽFig. 4.. To check whether an increase in endogenous DA is neurotoxic, we co-perfused nomifensine at different concentrations Ž20 and 100 mM. with a non-toxic Ž100 mM. ironŽIII. concentration. Nomifensine 100 mM was perfused alone for 1 h on day 1. On day 2, perfusion of MPPq 1 mM for 15 min produced an increase in the extracellular output of DA similar to control-Ringer data ŽFig. 6.. The presence of nomifensine 20 mM partially prevented the effect produced on DA release on day 1 by ironŽIII. 100 mM perfusion Ždata not shown.. On day 2, the presence of nomifensine 20 mM during perfusion of ironŽIII. 100 mM on day 1 produced a smaller increase in the extracellular output of DA after MPPq perfusion than in the absence of nomifensine ŽFig. 6.. Co-perfusion of a higher concentration of nomifensine Ž100 mM. with ironŽIII. 100 mM on day 1 produced a greater decrease in the extracellular output of DA on day 2 after MPPq perfusion than
Fig. 6. Effect of perfusion Žhorizontal bar. of MPPq 1 mM for 15 min, 24 h Žday 2. after different perfusions carried out on day 1 Žsee X labels, in micromolar., on the striatal extracellular output of DA. Nomifensine was included in the Ringer solution during the whole experiment on day 1. Data are mean"S.E.M. Žvertical bars. of the sum of five consecutive DA output samples after MPPq perfusion, expressed as femtomoles per minute Ž ns 4–7.. Statistical significance ŽKruskal–Wallis followed by the Mann–Whitney U-test.: UU P - 0.01, compared with ironŽIII. 100 mM data. aaP - 0.01, compared with ironŽIII. 100 mMqnomifensine 100 mM data.
nomifensine 20 mM or in the absence of nomifensine ŽFig. 6.. Therefore, co-perfusion of ironŽIII. 100 mM with different doses of nomifensine showed a dose-dependent neurotoxicity. In the presence of nomifensine 100 mM, TTX 2 mM, a fast sodium channel inhibitor, was perfused for 1 h before its co-perfusion with ironŽIII. 100 mM on day 1. On day 2, MPPq perfusion produced a higher extracellular output of DA in the presence than in the absence of tetrodotoxin 2 mM ŽFig. 6.. Perfusion of TTX 2 mM alone for 1 h on day 1 produced a strong decrease in the extracellular output of DA to nearly undetectable levels Ždata not shown.. On day 2, perfusion of MPPq 1 mM for 15 min produced an increase in the extracellular output of DA similar to control-Ringer data.
4. Discussion The DAergic system is highly susceptible to oxidative damage w21x. DA itself is able to produce hydrogen peroxide and other oxygen radicals either by autooxidation or by enzymatic reactions involved in its metabolism w5,25x. The involvement of DA on several toxic reactions has been extensively described w39x. The massive DA overflow in striatum produced by perfusion of MPPq w33x could be involved in MPTPrMPPq toxicity, since concurrent administration of haloperidol with MPTP significantly enhanced the depression of the activity of tyrosine hydroxylase in the striatum caused by MPTP w27x. Therefore, in an attempt to mimic the effects of a MPPq perfusion in the striatum, we perfused DA throughout the dialysis probe. We calculate that the increase in extracellular output of DA measured after MPPq 1 mM perfusion is similar to a perfusion of DA 15 mM through the microdialysis cannula, given the fact that in vitro recovery of DA is about 20%. In order to evaluate the damage induced by the perfusion of DA carried out on day 1, we perfused MPPq for 15 min 24 h later Žday 2.. We consider the released DA on day 2 after MPPq perfusion as an index of the surviving DAergic terminals of day 1. In contrast to the toxic action of DA ascribed by other groups w3,9x, DA perfusion did not damage the DAergic terminals. The most likely explanation for this discrepancy could be differences in the DA concentration used. These observations indicate that DA may not be uniquely responsible for its described toxic effects. Most of the DA-induced reactions to form free radicals involve the presence of iron, a metal that enhances free radical production by reducing hydrogen peroxide. Toxicity of iron in the nigrostriatal system has been reported w16x. One of the hallmarks in neurodegenerative diseases is a marked increase in iron in the affected brain regions Žfor review, see Ref. w12x.. In fact, iron chelation — removal of free radicals produced by iron with scavengers or spin
M. Santiago et al.r Brain Research 858 (2000) 26–32
traps — protects the degeneration induced by iron w23,26,36x. In the present report, we studied the possible neurotoxic interaction between iron and DA. Firstly, we evaluated the toxicity of iron itself by means of the perfusion of different ironŽIII. concentrations Ž10, 100, 333 and 1000 mM in the perfusion fluid or 0.35, 3.5, 12 and 35 mM, respectively, at extracellular level. to study its toxic action. Perfusion of ironŽIII. Ž10 and 100 mM. on day 1 produced a slight increase in DA overflow, but did not affect the DA overflow after MPPq perfusion on day 2, even though DA overflow was higher than in control-Ringer data. This effect could be explained, at least in part, by the activation of the tyrosine hydroxylase enzyme by iron at these nonneurotoxic concentrations. However, higher ironŽIII. concentrations Ž333 and 1000 mM. produced a toxic effect in a concentration-dependent manner. The 333 mM concentration resulted in a reduction in DA extracellular output of 50% after MPPq perfusion as compared with control animals, whereas ironŽIII. 1000 mM totally damaged DAergic terminals, since there was no increase in DA extracellular output after MPPq perfusion. These observations were corroborated by tyrosine hydroxylase immunohistochemistry. An additive or synergistic effect of DA and iron on their toxicity to DA neurons has been described w31,41x. To test this possible effect, we carried out co-perfusion of DA with ironŽIII. at concentrations which, separately, did not produce any damage to DAergic terminals. Under these conditions, co-perfusion produced an evident disruption of DAergic terminals, as shown by MPPq perfusion data on day 2 and tyrosine hydroxylase immunohistochemistry. Since several toxic processes for DAergic neurons that occur inside the neuron have been described, the question is whether the toxic action of iron is mainly produced inside or outside DAergic neurons. DA uptake system is an efficient mechanism, but it seems to have little selectivity, in as much as it produces the uptake of different molecular compounds w4x. Thus, through a mechanism dependent on the DA carrier, iron could bind to DA and come into DAergic neurons, producing a toxic action inside them by increasing free radicals. To test this hypothesis, the effect of the selective DA uptake inhibitor, nomifensine, was examined. Nomifensine 20 mM produced a 10–15-fold increase in the basal extracellular output of DA. We used two ironŽIII. concentrations: one without Ž100 mM. and another with effect Ž333 mM, data not shown. on DAergic terminals. In both cases, co-perfusion with nomifensine induced an increase in iron-induced toxicity. An increase in the nomifensine concentration produced more damage to DAergic terminals. These results suggest that DA reuptake is not necessary for DA–iron-induced toxicity and that an increase in endogenous DA through inhibition of DA uptake is more toxic than DA administered exogenously. To test the latter,
31
a decrease in the release of DA, in the conditions mentioned above, was induced with tetrodotoxin, which inhibits the sodium conductance. In this case, a partial protection after MPPq perfusion on day 2 was found. These results demonstrate that the DAergic system could be synergistically damaged by DA and iron and that this damage could be extraneuronally produced. In aged rats, an increase in iron in substantia nigra w35x and in other brain regions w10x has been described. We have previously reported that the oxidative stress in substantia nigra increases during ageing w8x and that this damage increased when aged rats were treated for several months with nomifensine w34x. Therefore, alterations in the clearance of DA from the extracellular space, along with an increase of iron, may have significant consequences for functioning of the DAergic system. Therapy directed to upregulate antioxidative systems should be considered in aging and neurodegenerative diseases such as Parkinson’s disease. Acknowledgements This work was supported by grant 96-1142 from Fondo de Investigaciones Sanitarias, Spain. E.R.M. and L.G. thank Ministerio de Educacion ´ y Ciencia and Consellerıa ´ de Cultura, Educacion ´ y Ciencia, Generalitat Valenciana, Spain, for pre- and post-doctoral fellowships, respectively. References w1x G. Arendash, G. Sengstock, C. Olanow, S. Barone, A. Dunn, Intranigral iron infusion as a model for Parkinson’s disease, in: W. Woodrum, A. Nonneman ŽEds.., Toxin-Induced Models of Neurological Disorders, Plenum, New York, 1994, pp. 175–212. w2x D. Ben-Shachar, M.B.H. Youdim, Intranigral iron injection induces behavioral and biochemical ‘‘parkinsonism’’ in rats, J. Neurochem. 57 Ž1991. 2133–2135. w3x D. Ben-Shachar, R. Zuk, Y. Glinka, Dopamine neurotoxicity inhibition of mitochondrial respiration, J. Neurochem. 64 Ž1995. 718–723. w4x M. Bougria, J. Vitorica, J. Cano, A. Machado, Implication of dopamine transporter system on 1-methyl-4-phenylpyridinium and rotenone effect in striatal synaptosomes, Eur. J. Pharmacol. 291 Ž1996. 407–415. w5x C.C. Chiueh, H. Miyake, M.-T. Peng, Role of dopamine autooxidation, hydroxyl radical generation, and calcium overload in underlying mechanisms involved in MPTP-induced parkinsonism, Adv. Neurol. 60 Ž1993. 251–258. w6x C.C. Chiueh, D.L. Murphy, H. Miyake, K. Lang, P.K. Tulsi, S.J. Huang, Hydroxyl free radical ŽPOH. formation reflected by salicylate hydroxylation and neuromelanin: in vivo markers for oxidative injury of nigral neurons, Ann. N.Y. Acad. Sci. 679 Ž1993. 370–375. w7x R.R. Crichton, Inorganic Biochemistry of Iron Metabolism, Ellis Horwood, 1991. w8x C.P. De la Cruz, E. Revilla, J.L. Venero, A. Ayala, J. Cano, A. Machado, Oxidative inactivation of tyrosine hydroxylase in substantia nigra of aged rats, Free Radical Biol. Med. 20 Ž1996. 53–61. w9x F. Filloux, J.J. Townsend, Pre- and postsynaptic neurotoxic effects of dopamine demonstrated by intrastriatal injection, Exp. Neurol. 119 Ž1993. 79–88. w10x S.J. Focht, B.S. Snyder, J.L. Beard, W. Van Gelder, L.R. Williams, J.R. Connor, Regional distribution of iron, transferrin, ferritin, and
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