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Keywords:

  • antioxidant defense;
  • excitotoxin;
  • neurotoxicity;
  • N-methyl-d-aspartate receptor;
  • quinolinic acid;
  • selenium

Abstract

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  2. Abstract
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Quinolinic acid (QUIN), a well known excitotoxin that produces a pharmacological model of Huntington's disease in rats and primates, has been shown to evoke degenerative events in nerve tissue via NMDA receptor (NMDAr) overactivation and oxidative stress. In this study, the antioxidant selenium (as sodium selenite) was tested against different markers of QUIN-induced neurotoxicity under both in vitro and in vivo conditions. In the in vitro experiments, a concentration-dependent effect of selenium was evaluated on the regional peroxidative action of QUIN as an index of oxidative toxicity in rat brain synaptosomes. In the in vivo experiments, selenium (0.625 mg per kg per day, i.p.) was administered to rats for 5 days, and 2 h later animals received a single unilateral striatal injection of QUIN (240 nmol/µL). Rats were killed 2 h after the induction of lesions with QUIN to measure lipid peroxidation and glutathione peroxidase (GPx) activity in striatal tissue. In other groups, the rotation behavior, GABA content, morphologic alterations, and the corresponding ratio of neuronal damage were all evaluated as additional markers of QUIN-induced striatal toxicity 7 days after the intrastriatal injection of QUIN. Selenium decreased the peroxidative action of QUIN in synaptosomes both from whole rat brain and from the striatum and hippocampus, but not in the cortex. A protective concentration-dependent effect of selenium was observed in QUIN-exposed synaptosomes from whole brain and hippocampus. Selenium pre-treatment decreased the in vivo lipid peroxidation and increased the GPx activity in QUIN-treated rats. Selenium also significantly attenuated the QUIN-induced circling behavior, the striatal GABA depletion, the ratio of neuronal damage, and partially prevented the morphologic alterations in rats. These data suggest that major features of QUIN-induced neurotoxicity are partially mediated by free radical formation and oxidative stress, and that selenium partially protects against QUIN toxicity.

Abbreviations used
DTNB

5,5-dithiobis-2-nitrobenzoic acid

GPx

glutathione peroxidase

GSH

reduced glutathione

LP

lipid peroxidation

NMDAr

N-methyl-d-aspartate receptor

OPA

o-phthaldialdehyde

QUIN

quinolinic acid

ROS

reactive oxygen species

TBA

thiobarbituric acid

TBA-RS

thiobarbituric acid-reactive substances

Excessive activation of glutamate receptors in the mammalian brain represents a cytotoxic mechanism that is potentially involved in neurodegenerative processes (Coyle and Puttfarcken 1993). Quinolinic acid (2,3-pyridine dicarboxylic acid; QUIN) is a well known NMDA receptor receptor (NMDAr) agonist (Schwarcz et al. 1984) that exerts selective neurotoxicity through excitotoxic (Stone 1993) and oxidative (Santamaría et al. 2001b) mechanisms. QUIN has been shown to produce a wide variety of toxic effects in the brain, such as depletion of GABA, excessive increases in cytosolic Ca2+ concentration, ATP exhaustion, neuronal oxidative stress and cell death (Foster et al. 1983; During et al. 1989; Santamaría and Ríos 1993). Recently, attention has been focused on the ability of QUIN to exert part of its neurotoxic actions through an NMDAr-independent mechanism that involves oxidative stress and peroxidative damage (Štípek et al. 1997; Behan et al. 1999; Cabrera et al. 2000; Santamaría et al. 2001b). Some of these mechanisms include the formation of QUIN–iron (II) complexes that mediate generation of ROS (Goda et al. 1996; Stípek et al. 1997; Murakami et al. 1998; Iwahashi et al. 1999), alteration of the profiles of some endogenous antioxidants (Rodríguez et al. 2000) and the direct generation of hydroxyl radical in striatal tissue (Santamaría et al. 2001b). These findings are of particular interest because use of antioxidants may represent a potential therapeutic strategy against QUIN toxicity.

Selenium, an essential dietary element for mammals, is known to be present in the active center of glutathione peroxidase (GPx), an antioxidant enzyme that protects membrane lipids and macromolecules from oxidative damage produced by peroxides (Rotruck et al. 1973; Harman 1993). Selenium is also required for the catalytic activity of mammalian thioredoxin reductase, another important antioxidant enzyme (Marcocci et al. 1997; Lee et al. 2000). In addition, selenium has been shown to protect against methamphetamine-induced neurotoxicity (Imam et al. 1999). Moreover, positive clinical responses obtained during therapy with selenium and other antioxidants in neurodegenerative diseases have provided substantial evidence for the important role of free radicals and oxidative stress in pathologic processes (Halliwell and Gutteridge 1984; Westermarck and Santavouri 1984). Of particular interest are studies performed with ebselen, a selenium derivative with GPx-like activity, which exhibited protective effects against QUIN-induced oxidative damage (Rossato et al. 2002a,b). In view of the promising therapeutic properties of selenium and the well known involvement of oxidative stress events in QUIN-induced neurotoxicity, the present study was carried out to investigate the effect of selenium on different markers of neurotoxicity induced by QUIN in rats using both in vitro and in vivo approaches. For in vitro experiments, the antioxidant effects of selenium were tested in synaptosomes from whole brain and from three different brain regions exposed to QUIN. We used this preparation because synaptic fractions behave as functional entities and have been widely used to characterize QUIN-induced oxidative damage (Santamaría et al. 1997b, 2001a). For in vivo experiments, the effects of selenium on QUIN neurotoxicity were tested using behavioral, biochemical and morphologic markers.

Materials and methods

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Animals

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Adult male Wistar rats (250–300 g) were used in the study. Animals were housed five per cage in acrylic box cages and provided with food and water ad libitum. The housing room at the vivarium (facilities of the Instituto Nacional de Neurología y Neurocirugía, Mexico) was maintained under constant conditions of temperature (25 ± 3°C), humidity and light cycle (12 : 12 light : dark schedule). All experimental manipulations were performed according to the ‘Guidelines for the Use of Animals in Neuroscience Research’ from the Society of Neuroscience.

Chemicals

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Deionized water was used for preparation of all reagents and solutions. QUIN, selenium as Na2SeO3, apomorphine, bovine albumin, HEPES, sucrose, o-phthaldialdehyde (OPA), 3-mercaptopropionic acid, 2-mercaptoethanol, thiobarbituric acid (TBA), and a kit of l-amino acid standards for liquid chromatography were purchased from Sigma Chemical Co. (St Louis, MO, USA). All other reagents were obtained from E. Merck (Mexico City, Mexico) and J. T. Baker (Mexico City, Mexico).

Isolation and incubation of brain synaptosomes

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Synaptosomal fractions were obtained either from whole brain or from specific brain regions (striatum, hippocampus and cortex) by the modified methods of Dodd et al. (1981) and Gary and Whittaker (1962). Animals were killed by decapitation and their brains rapidly removed. Whole brains or dissected regions (Glowinski and Iversen 1966) were homogenized with 20 volumes (w/v) of 0.32 m sucrose and centrifuged at 3000 g for 10 min. The supernatants were centrifuged again at 3000 g for 15 min and pellets were gently resuspended in HEPES buffer and frozen at − 70°C until the experiments. Aliquots of 970 µL were incubated at 37°C for 60 min in the presence of 100 µm QUIN and/or increasing concentrations (5–250 µm) of Na2SeO3, and final volumes were 1000 µL. This concentration of QUIN have been demonstrated to produce significant increases in lipid peroxidation (LP) and damage associated with ROS generation (Santamaría et al. 1997b, 2001a).

Measurement of LP by the TBA assay

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The formation of TBA-reactive substances (TBA-RS) in synaptosomes was measured according to a method modified (Santamaría et al. 2001a) from previously described procedures (Ríos and Santamaría 1991). Synaptosomal fractions (1000-µL samples) previously incubated in the presence of QUIN, selenium, or both, were mixed with 2 mL TBA reagent (containing 0.375 g TBA, 15 g trichloroacetic acid and 2.5 mL HCl in 100 mL H2O), and final solutions were heated in a boiling water bath for 30 min and centrifuged at 3000 g for 15 min. Optical density was then measured in supernatants at 532 nm in a Perkin-Elmer Lambda 20 spectrometer (Perkin-Elmer Co., Norwalk, CT, USA). Protein concentrations were assayed according to a method described previously (Lowry et al. 1951). Results are expressed as nmoles TBA-RS per milligram protein.

Treatment of animals and striatal lesions

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Na2SeO3 was dissolved in water and the pH was adjusted to 7.0. Animals were divided into four groups with 5–9 rats per group. Groups II and IV were pretreated with selenium (0.625 mg per kg per day, i.p.) for 5 days, whereas the animals in group I (control) received distilled water i.p. Groups III and IV also received a single unilateral injection of QUIN (240 nmol/µL) into the right corpus striatum 2 h after the last selenium injection, whereas controls received the same volume of sterile saline. Stereotaxic coordinates were 0.5 mm anterior to bregma, 2.6 mm lateral to bregma and 4.5 mm ventral to the dura, according to the rat brain atlas of Paxinos and Watson (1998). This procedure was performed subsequently for each experimental analysis. Depending on the purpose of the experiment, animals were killed by decapitation either 120 min (for LP and GPx activity assays) or 7 days (for GABA assay, histologic examination and cell counting) after QUIN injection, brains were quickly removed on ice and the striata were dissected for analysis. Rats employed for behavioral evaluation (6 days after QUIN treatment) were also used 1 day later for histologic examination.

Fluorometric measurement of LP

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In contrast to the in vitro experiments, LP was estimated in vivo in striatal tissue from lesioned rats, by measuring the lipid fluorescent products formation 2 h after QUIN injection, according to a modified method (Santamaría and Ríos 1993) from an original report (Triggs and Willmore 1984). This method has been demonstrated to be more suitable than the TBA-RS assay for in vivo purposes (Gutteridge and Halliwell 1990). The striatal tissues were dissected, weighed and homogenized in 3 mL saline solution (0.9% NaCl). Aliquots of 1 mL were added to 4 mL of a chloroform–methanol mixture (2 : 1 v/v). After stirring, the mixtures were placed on ice for 30 min to allow phase separation. Fluorescence in the chloroform layer was then measured in each sample in a Perkin-Elmer LS50B luminescence spectrometer (Perkin-Elmer Co., Norwalk, CT, USA), using excitation and emission wavelengths of 370 and 430 nm respectively. The spectrometer sensitivity was adjusted to 300 units with a quinine standard solution (0.1 µg/mL). Results are expressed as fluorescent units (relative fluorescence intensity) per gram of wet tissue.

Measurement of GPx activity

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Striatal GPx activity was determined by a method based on the non-enzymatic oxidation of reduced glutathione (GSH) (Hafeman et al. 1974). We measured the activity of this major antioxidant enzyme because of its known dependency on both selenium presence and concentration (Marcocci et al. 1997). Briefly, striatal tissues were obtained from dissected brains and homogenized in phosphate buffer (pH 7.4), as reported previously (Rodríguez-Martínez et al. 2000). Aliquots (30 µL) were incubated at 37°C in a phosphate buffer (pH 7.0) containing 2.0 mm GSH and 0.01 m NaN2 (final volume 2 mL). After 5 min, 1 mL 1.25 mm H2O2 was added to the incubation medium. After 3 min, 1 mL of the mixture was removed and added to 4 mL metaphosphoric acid. The samples were centrifuged at 1500 g for 15 min and supernatants (1 mL) were added to phosphate buffer (pH 7.0) containing 0.5 mL 5,5-dithiobis-2-nitrobenzoic acid (DTNB). Optical density was determined at 512 nm in a Perkin-Elmer Lambda 20 spectrometer 2 min after the addition of DTNB. A standard curve was constructed using increasing concentrations of GSH + EDTA (pH 7.0) + DTNB. One enzyme activity unit was defined as a 50% decrease in GSH in 60 min after the decrease in GSH from the non-enzymatic reaction had been subtracted.

Circling behavior counting

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Rotations were recorded in lesioned animals following previous protocols (Santamaría et al. 1996, 1997a). Six days after QUIN injection, animals from all treatment groups were given apomorphine (1 mg/kg, s.c.) and separated into individual acrylic box cages. Five minutes later, the number of rotations was recorded every 5 min for 60 min (Schwarcz et al. 1979; Norman et al. 1990). Each rotation was defined as a complete 360° turn. At the striatal lesion coordinates employed in this study, an apomorphine challenge to rats produces circling behavior ipsilateral to the lesioned side (Susel et al. 1989; Santamaría and Ríos 1993; Santamaría et al. 1996, 1997a).

GABA detection by HPLC

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Estimation of the striatal contents of GABA was performed in this study because striatal GABA depletion is considered to be one of the main markers of QUIN neurotoxicity (Beal et al. 1986). According to previous reports (Santamaría et al. 1996, 1997a), 7 days after QUIN administration, animals from all treatment groups received a well known glutamate decarboxylase inhibitor, 3-mercaptopropionic acid (1.2 mmol/kg, i.v.), in order to prevent a post-mortem increase in GABA (Van der Heyden and Korf 1978). Two minutes later, rats were killed by decapitation and their brains removed. Right striata were dissected on an ice-cold plate and homogenized in 15 volumes of methanol–water (85% v/v). Samples were then centrifuged (3000 g for 15 min) and aliquots of the corresponding supernatants were stored at − 4°C until chromatographic analysis. GABA levels were assayed by HPLC with fluorometric detection, as described previously (Fleury and Ashley 1983; Smith and Panico 1985). For precolumn derivatization, 100 µL of OPA reagent [containing 5 mg OPA, 626 µL methanol, 5.6 mL borate buffer 0.4 m (pH 9.5) and 25 µL 2-mercaptoethanol] was added to 100 µL of the striatal supernatant. After stirring for 1 min, 20 µL of the mixture was injected into a BAS CC-5 liquid chromatograph with an OPA-HS Alltech reversed-phase column (3 µm particle size) (Alltech Associates, Inc., Deerfield, IL, USA). Linear gradient programming was used to elute OPA-amino acids from 10 to 65% methanol. The gradient mixture comprised 50 mm sodium acetate buffer solution (pH 5.9) containing 1.5% v/v tetrahydrofuran and HPLC-grade methanol. Fluorescence was detected with a BAS FL-45 A fluorescence detector (Bioanalytical Systems, West Lafayette, IN, USA).

Histological evaluation

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On day 7 after QUIN administration, rats from all groups were anesthetized with 0.3 mL chloral hydrate 3.5% i.p. and transcardially perfused with phosphate-buffered saline containing heparin (1/500 v/v), followed by 10% v/v formaldehyde at 4°C. Brains were removed, post-fixed in 10% formalin for 24 h and then immersed in paraffin. Tissues were sectioned on a microtome, and sections (5–7 µm) from the lesion zone exhibiting profound neuronal loss (Roberts and DiFiglia 1989) were obtained every 100 µm, covering a total distance of 2 mm (1 mm anterior and 1 posterior to the needle tract). All sections were stained with cresyl violet in order to identify cell bodies (Luna 1968) at 20 × magnification.

Quantitative assessment of lesions

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Cell counting was performed according to a previous report (Rodríguez et al. 1999). The number of neuronal cells – either preserved, damaged, or total – was obtained as an average of 10 randomly selected fields of three sections per rat brain. The general criteria for scoring damaged neurons included the presence of shrunken and hyperchromatic nuclei (pyknotic nuclei), as well as the occurrence of swollen, edematous neurons with cytoplasmic vacuolation. Other important marker of tissue damage considered were the presence of interstitial edema and enlargement of the lateral ventricle. In order to avoid operator bias in selecting fields, random numbers were obtained from a personal computer to select sections. The observer was always unaware of treatments. The ratio of neuronal damage per field was calculated as the number of damaged cells as a proportion of the total number of cells multiplied by 100.

Statistical analysis

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Except for behavioral and cell counting results, which were analyzed by Kruskal–Wallis anova followed by Mann–Whitney U test (Siegel 1980), all data were analyzed by one-way anova followed by the Tukey's test (Steel and Torrie 1980). Values of p < 0.05 were considered statistically significant.

Effect of selenium on QUIN-induced LP in synaptosomes from whole rat brain

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Figure 1(a) shows the effect of increasing concentrations of selenium on QUIN-induced LP in synaptosomes from whole rat brain. The mean basal levels of TBA-RS formation was 3.14 ± 0.47 nmol per mg protein. Incubation of synaptosomes in the presence of QUIN alone resulted in a significant increase in LP compared with basal levels (247% vs. control). Selenium exhibited a partial concentration-dependent effect on QUIN-induced LP: selenium concentrations of 5, 10 and 25 µm significantly decreased the peroxidative action of QUIN (− 32, − 25 and − 31% respectively vs. QUIN alone), whereas the higher concentrations (50, 100 and 250 µm) blocked the oxidative effect of QUIN (− 46, − 49 and − 45% respectively vs. QUIN alone). The highest selenium concentration (250 µm) tested alone in synaptosomes had no effect on the basal levels of LP (3.36 ± 0.34 nmol TBA-RS per mg protein).

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Figure 1. Concentration–response effect of selenium (Se) on QUIN-induced lipid peroxidation determined by thiobarbituric acid-reactive substance (TBA-RS) formation in rat synaptosomes from whole brain (a), striatum (b), hippocampus (c) and cortex (d). Each bar represents the mean ± SEM from 7–10 experiments. Horizontal lines represent basal TBA-RS formation (control). ap < 0.05, Ap < 0.01 versus control (without QUIN and selenium); bp < 0.05, Bp < 0.01 versus QUIN alone (one-way anova followed by Tukey's test).

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Regional effect of selenium on QUIN-induced LP in rat brain synaptosomes

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The effects of selenium on QUIN-induced LP in three rat brain regions are also shown in Fig. 1. Basal levels of TBA-RS formation were 1.94 ± 0.23 nmol per mg protein for the striatum (Fig. 1b), 1.84 ± 0.39 for hippocampus (Fig. 1c), and 0.84 ± 0.09 for cortex (Fig. 1d). Treatment of synaptosomes with QUIN alone produced significant peroxidative effects in the three regions tested, compared with basal LP (42% in striatum, 101% in hippocampus and 34% in cortex vs. respective control). Selenium produced a decrease in the QUIN-induced peroxidative action in the striatum (Fig. 1b) at the lowest concentration tested (5 µm; − 15% vs. QUIN alone), and up to the highest concentration (250 µm; − 20% vs. QUIN). A clear concentration-dependent effect of selenium on QUIN-induced LP was observed only in hippocampus (Fig. 1c); a decrease in QUIN oxidative toxicity was found at selenium concentrations of 5 and 10 µm (both − 19% vs. QUIN alone), and prevention at concentrations of 25, 50 and 100 µm (− 32, − 33 and − 32% respectively vs. QUIN), whereas 250 µm selenium produced a protective effect even below the control value (− 23% vs. control and − 62% vs. QUIN). In cortex (Fig. 1d), except for the 5 µm concentration (− 18% vs. QUIN), selenium had no evident effect on QUIN-induced peroxidation at any concentration tested. Again, the incubation of synaptosomes in the presence of selenium alone (250 µm) had no significant effect on the basal levels of LP (1.86 ± 0.35, 2.01 ± 0.12 and 0.90 ± 0.14 nmol TBA-RS per mg in striatum, hippocampus and cortex respectively).

Bodyweight

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In the in vivo experiments, bodyweight was recorded every day during treatment in animals from all groups. We found no significant changes in body weight in selenium-treated animals (285 ± 15 g at the beginning of the treatment vs. 296 ± 10 g on the last day of selenium administration) compared with control animals (278 ± 19 and 290 ± 19 g respectively). An average of two animals per group died, regardless of the experimental condition. From these observations, we assumed that selenium treatment had no artificial side effects resulting from systemic toxicity of the antioxidant on the markers further evaluated in this study.

Effect of selenium on the QUIN-induced LP in rat striatum

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Figure 2 shows the effect of selenium on QUIN-induced LP in the rat striatum. Mean basal levels of LP were 237.17 ± 19.22 U per g tissue. QUIN alone increased striatal LP by 52% compared with control values, whereas selenium significantly decreased the oxidative toxicity of QUIN (− 23% vs. QUIN alone). Selenium treatment alone had no significant effect on the basal levels of LP (− 8% vs. control).

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Figure 2. Effects of selenium (Se) on QUIN-induced LP (white bars) and striatal GPx activity (black bars) in rats. Animals received selenium (0.625 mg per kg per day, i.p.) for 5 days before a single unilateral intrastriatal injection of QUIN (240 nmol/µL) and LP was measured 120 min after QUIN administration. Each bar represents the mean ± SEM from 5–8 animals. Ap < 0.01 versus control (without QUIN and selenium); bp < 0.05 versus QUIN alone (one-way anova followed by Tukey's test).

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Effect of selenium on GPx activity

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The effect of selenium on striatal GPx activity is also shown in Fig. 2. In control animals, the mean GPx activity was 38.71 ± 4.83 U per mg protein. QUIN had no effect on GPx compared with control values (6% vs. control), whereas both selenium alone and selenium plus QUIN treatments significantly increased the striatal GPx activity compared with either control or QUIN treatments (93 and 77% respectively vs. QUIN alone).

Effect of selenium on QUIN-induced circling behavior

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The effect of selenium on QUIN-induced circling behavior in rats is shown in Fig. 3(a). Control animals (intrastriatally administered with phosphate-buffered saline) showed irritability and behavioral excitement after apomorphine injection, as reported by Norman et al. (1990) and Santamaría et al. (1996), but no circling behavior. QUIN-lesioned rats exhibited a marked number of rotations (208 ± 36 ipsilateral turns per 60 min) after apomorphine challenge, whereas selenium treatment significantly attenuated the rotation behavior in QUIN-lesioned animals (− 40% vs. QUIN). Selenium alone did not produce rotations in control rats after apomorphine administration.

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Figure 3. Effect of selenium (Se) on QUIN-induced circling behavior (a) and striatal GABA depletion (b) in rats. Both markers were assessed in the same animals. The rats received selenium (0.625 mg per kg per day, i.p.) for 5 days before a unilateral intrastriatal QUIN injection (240 nmol/µL). (a) The number of ipsilateral 360° turns was recorded visually for 60 min 6 days after QUIN administration. Each bar represents mean ± SEM from 7–9 animals. Bp < 0.01 versus QUIN alone (Kruskal–Wallis anova followed by Mann–Whitney U test). (b) The striatal GABA content was measured 7 days after the QUIN administration. Each bar represents the mean ± SEM from 7–9 animals. ap < 0.05, Ap < 0.01 versus control (without QUIN and selenium); bp < 0.05 versus QUIN alone (one-way anova followed by Tukey's test).

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Effect of selenium on the striatal levels of GABA

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Figure 3(b) shows the effects of selenium on QUIN-induced changes in the striatal GABA content. Mean baseline GABA content in control animals was 212.79 ± 11.76 µg per g tissue. QUIN produced a significant decrease in GABA levels (− 44% vs. control values), and selenium attenuated this effect by increasing GABA levels (by 35% vs. QUIN alone). Nevertheless GABA levels in the selenium + QUIN group were still significantly lower than those in controls (− 21%), indicating that protection was only partial. Selenium treatment alone had no significant effects on GABA levels (− 5% vs. control values).

Effect of selenium on QUIN-induced striatal morphologic alterations

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The morphologic alterations produced by QUIN in the rat striatum, as well as the effect of selenium on QUIN-lesioned tissues, are shown in Fig. 4. QUIN injection resulted in a remarkable cell loss and atrophy, defined as presence of abundant pyknotic nuclei and interstitial edema in the striatal tissue (Fig. 4b), compared with the striata from control rats (Fig. 4a). Collapse of the neuropil induced by the lesion was associated with a corresponding enlargement of the lateral ventricle on the lesion side, compared with control animals. In contrast, the striatal tissue from rats treated with selenium + QUIN exhibited only a few pyknotic nuclei and a limited number of swollen neurons, and there was no interstitial edema (Fig. 4d). In sections prepared from rats treated with selenium alone (Fig. 4c), we found preserved neuronal nuclei and no evidence of interstitial edema or cell loss.

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Figure 4. Photomicrographs showing striatal tissue from sham (a), QUIN-treated (b), selenium-treated (c), and selenium +QUIN-treated (d) rats. Animals received selenium (0.625 mg per kg per day, i.p.) for 5 days before an intrastriatal QUIN injection (240 nmol/µL). Striatal tissues were collected 7 days after QUIN administration and the sections (5–7 µm) were stained with cresyl violet. Ventricular enlargement (not shown) and necrotic core were seen typically in the lesion group, close to the lesion zone. Normal appearance of cell bodies and neuropil are shown in (a), (c) and (d). Hyperchromatic nuclei (arrows) are seen in (b). Interstitial edema (asterisk) is also shown in (b). Scale bar 10 µm, magnification 20 ×.

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The effect of selenium on the ratio of neuronal damage per field in the striatum of QUIN-lesioned rats is shown in Table 1. QUIN treatment significantly increased the ratio of neuronal damage by 1438% compared with control values whereas selenium treatment significantly decreased this marker (by 70% vs. QUIN alone). Again, administration of selenium alone had no effect on the ratio of neuronal damage compared with basal values.

Table 1.  Effect of selenium on the ratio of striatal neuronal damage in QUIN-lesioned rats
TreatmentNeuronal damage/field
  1. Values are mean ± SEM values of 10 fields per section (three sections per rat) around the lesion site (× 100). Rats were treated with selenium (0.625 mg per kg per day, i.p.) for 5 days before intrastriatal lesioning with 240 nmol/µL QUIN. Tissues were collected 7 days after QUIN injection. *p < 0.01 versus control (without QUIN and selenium);p < 0.05, p < 0.01 versus QUIN alone (Kruskal–Wallis anova followed by Mann–Whitney U test).

Control3.38 ± 3.29
selenium5.41 ± 4.99
QUIN52.02 ± 5.09*
selenium + QUIN15.93 ± 3.01

Discussion

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This study presents strong evidence that QUIN-induced neurotoxicity can be attenuated by the antioxidant selenium both under in vitro and in vivo conditions. The data suggest that a fraction of the neurotoxic pattern elicited by QUIN is mediated by free radical formation and oxidative stress. The in vitro peroxidative action of QUIN was prevented in synaptosomes from whole rat brain, striatum and hippocampus. The in vivo striatal toxicity evoked by QUIN was remarkably reduced as evidenced by effects on LP and more physiologically integrative markers, such as rotation behavior, striatal GABA content and the ratio of neuronal damage.

Because selenium administration increased the GPx activity in control and QUIN-treated rats, it is likely that selenium may be acting by scavenging free radicals through an increased GPx activity, thus providing partial protection against the QUIN-induced striatal toxicity. Although QUIN may affect both the GSH to oxidized glutathione ratio (Rodríguez-Martínez et al. 2000) and glutathione metabolism (Cruz-Aguado et al. 2000), the toxin itself does not directly affect GPx activity (Cruz-Aguado et al. 2000; Rodríguez-Martínez et al. 2000), as has been confirmed by the present findings. In contrast, we have demonstrated that QUIN has the ability to produce hydroxyl radicals in vivo in the rat corpus striatum during the first stages of acute toxicity (Santamaría et al. 2001b) and, therefore, it is likely that enhanced basal GPx activity stimulated by selenium might be responsible in turn for protection against QUIN-induced H2O2 decomposition and further hydroxyl radical-mediated oxidative damage. Experimental evidence to support this suggestion has recently appeared in the literature (Rossato et al. 2002a,b), as ebselen, a seleno-organic compound with GPx-like activity, has been shown to protect rat brain homogenates, striatal homogenates and lesioned striatal tissue from QUIN-induced TBA-RS formation, but not from behavioral alterations. Rossato and co-workers have also described an effect of ebselen and other organochalcogenide compounds on QUIN-induced TBA-RS formation in rat brain homogenates that resembles our findings at comparable selenium concentrations. The main difference between our study and theirs is that repeated selenium administration over several days before QUIN lesion might have stimulated GPx activity directly, enabling more efficient hydrogen peroxide removal, whereas administration of a single dose of ebselen just 15 min before the toxic insult might have not been enough to prevent neurotoxicity probably due to further free radical formation. In addition, the observed lack of effect of ebselen on the behavioral alterations might be explained by the higher intrastriatal dose of QUIN used by Rossato and co-workers (360 nmol), as we have previously demonstrated that a dose of 240 nmol is sufficient to produce significant LP (Santamaría and Ríos 1993; Rodríguez-Martínez et al. 2000) and circling behavior in rats (Santamaría et al. 1996). Moreover, the behavioral assessments of Rossato's group were observed in the absence of a pharmacological challenge, such as the one we employed in this study. Nevertheless, the findings of Rossato's group support the hypothesis that GPx or GPx-like activity may be protective in QUIN-induced toxicity.

Given that selenium is an essential dietary element, its deficiency has been reported to enhance brain susceptibility to oxidative damage, affecting monoamine turnover in the substantia nigra and striatum (Castano et al. 1993). Through its interaction with the glutathione reductase–GPx system, it represents a major defense system against oxidative stress in the brain (Rotruck et al. 1973). Indeed, decreased GPx activity has been reported in degenerative pathologies with oxidative components, such as Parkinson's disease (Hirsch 1992). The therapeutic properties of selenium and other antioxidants have been tested successfully in some neurodegenerative diseases associated with oxidative damage (Halliwell and Gutteridge 1984; Westermarck and Santavouri 1984), as well as cancer and other pathologic processes (Harman 1993). The incidence of different forms of cancer is low in areas where the selenium intake is high. It has been proposed that this interesting inverse correlation between cancer incidence and serum selenium levels is mediated through a reduction in free radical reactions (Ganther 1999). In addition, selenium has also been shown to protect against the methamphetamine-induced neurotoxicity in mice by a free radical-mediated mechanism (Imam et al. 1999).

On the other hand, both QUIN and the kynurenine pathway have been largely proposed to be involved in the pathogenesis of neurodegenerative (Stone and Connick 1985; Moroni et al. 1986; Schwarcz et al. 1988; Stone 2001), infectious, inflammatory and non-inflammatory diseases (Heyes et al. 1990, Heyes et al. 1992; Heyes and Lackner 1990; Ogawa et al. 1992). QUIN has also been used as a model to reproduce the neurochemical and histopathologic features of Huntington's disease (Schwarcz et al. 1983; Beal et al. 1986; Bruyn and Stoof 1990; Hantraye et al. 1990; Ferrante et al. 1993). Several published reports have considered the toxic actions of QUIN predominantly related with NMDAr overactivation (Stone 1993; Susel et al. 1989), through a typical process of excitotoxicity. However, more recently, several other studies have shown that QUIN is also capable of inducing oxidative injury itself, potentially involving a pattern of toxicity that might be partially independent of NMDAr activation (Behan et al. 1999; Cabrera et al. 2000; Rodríguez et al. 2000; Santamaría et al. 2001b). We have recently demonstrated the ability of QUIN to modify the profiles of some endogenous antioxidants such as the content of GSH and oxidized glutathione, and the copper–zinc-dependent superoxide dismutase activity in striatal tissue (Rodríguez et al. 2000), as well as the ability of QUIN to generate hydroxyl radical in the rat corpus striatum during the early stages of acute neurotoxicity (Santamaría et al. 2001b). Remarkably, the QUIN-induced formation of hydroxyl radical, although sensitive to the NMDAr antagonist MK-801, was not completely blocked, emphasizing that at least a small fraction of the QUIN-induced oxidative toxicity occurs independently of NMDAr activation. Further evidence for a role of oxidative stress in QUIN-induced neurotoxicity is based on the ability of QUIN to form a QUIN–iron (II) complex that mediates the hydroxyl radical formation and is responsible for in vitro DNA chain breakage and LP (Goda et al. 1996). It has also been shown that QUIN and some related compounds enhance the Fenton reaction in phosphate buffer, leading to hydroxyl radical formation (Iwahashi et al. 1999). This is reinforced by the observation that QUIN and other pyridine compounds are able to inhibit the auto-oxidation of the ferrous ion, accounting for a potential oxidative factor (Murakami et al. 1998). In addition, it is known that the in vitro QUIN-induced LP in nerve tissue is dependent on the formation of such QUIN–iron (II) complexes (Stípek et al. 1997). Furthermore, ferrous iron modulates QUIN-mediated [3H]MK-801 binding to rat brain synaptic membranes (Št'astnýet al. 1999). Consequently, it is likely that the intrinsic ability of QUIN to act as a chelator may help to explain its oxidant nature. Interestingly, it has also been reported that the use of nitric oxide synthase inhibitors, such as Nω-nitro-l-arginine methyl ester (Pérez-Severiano et al. 1998) and Nω-nitro-l-arginine (Santamaría et al. 1997b, 1999), protect against QUIN-induced neurotoxicity in vitro and in vivo, which implicates the role of nitric oxide radicals in the pattern of toxicity evoked by QUIN.

Free radical scavengers and antioxidant enzyme activity inducers, such as melatonin (Southgate et al. 1998; Behan et al. 1999; Southgate and Daya 1999; Cabrera et al. 2000) and deprenyl (Behan et al. 1999), have been shown to protect nerve tissue against QUIN oxidative toxicity under in vitro and in vivo conditions. The free radical scavenger OPC-11117 has also been shown to attenuate QUIN-induced nuclear factor kappa B activation and apoptosis in the rat striatum (Nakai et al. 1999). This is of particular interest as there is evidence that selenium can inhibit the transcription factor nuclear factor kappa B (Makropoulos et al. 1996), by controlling the half-life of I kappa B alpha (Kretz-Remy and Arrigo 2001), which may constitute an additional mechanism of cell protection against QUIN-induced apoptosis. Moreover, selenium, as sodium selenite, has also been demonstrated to suppress hydrogen peroxide-induced cell apoptosis through inhibition of Apoptosis signal-regulating kinase 1/c-Jun N-terminal kinase (ASK1/JNK) and activation of phosphatidylinositol 3′-kinase/protein kinase Akt (PI3-K/Akt) pathways (Yoon et al. 2002). These findings are consistent with an active role for selenium in cell signaling via the inhibition of ROS formation, thus preventing cell damage.

In summary, all data gathered in the present study and by other investigators (Noack et al. 1998; Heron and Daya 2000; Stone et al. 2000; Platenik et al. 2001; Santamaría et al. 2001c) support an active role of free radicals and oxidative stress in QUIN-induced neurotoxicity. The present study also demonstrates that selenium provides partial protection against QUIN-induced neurotoxicity, suggesting that some physiopathological consequences of QUIN toxicity in the brain may be attenuated by preventing the actions of free radicals. The results of this work therefore support the use of antioxidants as therapeutic agents against neurodegenerative processes evoked by excitotoxic and oxidative events.

Acknowledgements

  1. Top of page
  2. Abstract
  3. References

The authors wish to express sincere gratitude to Dr Marisela Méndez-Armenta and Dr Jorge Guevara for their excellent assistance. This work was supported by CONACyT Grant 40689-M (A.S., Mexico).

References

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