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Intern. J. Neuroscience, 114:1133–1145, 2004Copyright Taylor & Francis Inc.ISSN: 0020-7454 / 1543-5245 onlineDOI: 10.1080/00207450490475959

ANTIOXIDANT EFFECTS OF TAURINE,VITAMIN C, AND VITAMIN E ON

OXIDATIVE DAMAGE IN HIPPOCAMPUSCAUSED BY THE ADMINISTRATION OF

3-NITROPROPIONIC ACID IN RATS

ERIKA RODRÍGUEZ-MARTÍNEZDepartamento de FisiologíaFacultad de Medicina, Universidad Nacional Autónomade México, México

CONCEPCIÓN RUGERIO-VARGASDepartmento de Biología Celular y TisularFacultad de Medicina, Universidad Nacional Autónomade México, México

ALBA I. RODRÍGUEZGossett Neurology LaboratoriesHenry Ford Health SystemDetroit, Michigan, USA

GABINO BORGONIO-PÉREZSELVA RIVAS-ARANCIBIADepartamento de FisiologíaFacultad de Medicina, Universidad Nacional Autónomade México, México

Received 12 December 2003.Address correspondence to Dr. Selva Rivas-Arancibia, Departamento de Fisiología, Facultad

de Medicina, Universidad Nacional Autónoma de México, A. P. 70-250. México, D.F. C.P. 04510.E-mail: srivas@servidor.unam.mx

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The administration of 3-nitropropionic acid increases reactive oxygenspecies (ROS). Antioxidant defense mechanisms buffer these ROS con-verting them into non-damaging compounds. Taurine and vitamins Cand E are antioxidants that play a role in the defense against cellulardamage. This study examines the antioxidant effect of taurine, vitaminC, and vitamin E on acute hippocampal damage caused by 3-NP. Ani-mals treated with 3-NP increased lipid peroxidation levels and astro-cytic damage in the hippocampus. Administration of taurine, vitamin C,and vitamin E partially protected from oxidative damage, indicate thatwhile all substances had antioxidant effects, only taurine showed mor-phological protection in surviving cells.

Keywords antioxidants, hippocampus, 3-NP, oxidative stress, taurine, vi-tamin C, vitamin E

Oxidative stress is a contributing factor to alteration caused in neuro-degenerative processes. The hippocampus is the brain structure mostvulnerable to oxidative stress. Approximately 50% of the axon-spineinterfaces are associated with astrocytes (Aranque et al., 2001). Theyplay an important role in metabolic and structural functions. Thereare many functional interactions between astrocytes and neurons,like the potential role in integrating synaptic signals and providingfeedback responses. In the adult, glial cells maintain neuronal ho-meostasis, reparation, and synaptic plasticity.

The 3-nitropropionic acid (3-NP) administration increases reac-tive oxygen species (ROS). However, the mechanisms of oxidativedamage are not fully understood (Schulz et al., 1996; La Fontaine etal., 2000). 3-NP, a mitochondrial toxin that is an irreversible inhibi-tor of succinate dehydrogenase (SDH), inhibits complex II of therespiratory chain and the tricarboxylic acid cycle, which leads todecreases in ATP synthesis and activation of NMDA receptors. Italso causes excitotoxicity and increases the level of free radicals(Brouillet et al., 1999; Lafon-Cazal et al., 1993), leading to alter-ations in cell membranes (lipoperoxidation), DNA and protein dam-age, and cell death (Ames, 1997; Luft, 1991). Systemic administra-tion of 3-NP causes damage to cells in the caudate putamen, globuspallidus, substantia nigra, and hippocampus (Beal et al., 1993; Hamilton& Gould, 1987). 3-NP induces excitotoxicity-mediated neuronal deathin which intracellular and intramitochondrial calcium influx takepart (Wullner et al., 1994). The astrocytes are more sensitive thanneurons to cellular calcium overload induced by this toxin (Fukuda

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Antioxidants Ameliorate 3-NP Damage to Hippocampus 1135

et al., 1998). This is exacerbated by impairment in glutamate uptakein the glial cell. Nishino and colleagues (1997) convincingly dem-onstrate a greater vulnerability of astrocytes to 3-NP. The astrocytesexpress functional receptors for many different neurotransmittersthat lead to changes in intracellular calcium levels (Verkhrastsky &Kettenmann, 1996; Verkhrastsky et al., 1998).

Taurine is an antioxidant that protects against neuronal damagecaused by oxidative stress (Huxtable, 1992; Rivas-Arancibia et al.,2000). It is synthesized by astrocytes in the brain and participates inthe glial-neuronal interaction. Taurine is released from glial in thehippocampus by osmolarity changes (Decavel & Hatton, 1995) andplays an antioxidant role during excitotoxicity, protecting neuronsfrom glutamate toxicity. This protective mechanism is physiologi-cally important for the brain. After cell damage, taurine synthesis isinduced in high concentrations (Huxtable, 1992; Ogasawara et al.,1994; Rivas-Arancibia et al., 2000). Vitamin E protects cell mem-branes from free radical-induced oxidation, and vitamin C (ascorbicacid) can directly scavenge free radicals in plasma and suppresstheir reactivity (Bendich, 1994, 1996).

Previous studies demonstrated that doses of 3-NP (20 mg/kg)twice each 48 hrs (Rivas-Arancibia et al., 2000), caused cellulardamage and motor alterations, one week after administration. Acuteshort-term effects of 3-NP have not been studied.

Other studies have demonstrated that there is an increase of lipidperoxidation levels after 3-NP administration in acute models (Juárez-Meavepeña et al., 2002). It indicates an oxidative stress state pro-duced by 3-NP. Therefore, the antioxidant administration in initialstages could protect against oxidative damage.

The aim of this work was to study astrocytic oxidative damagecaused by ATP deficit in hippocampus, as well as, evaluated theantioxidant effects of taurine, vitamin C, and vitamin E on hippo-campal damage caused by the administration of 3-NP.

MATERIALS AND METHODS

Sixty male Wistar rats (400 g) were individually housed in acrylicboxes with free access to food (Nutri-cubo; Purina, USA) and water.

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1136 E. Rodríguez-Martínez et al.

They were divided into 5 groups and received one of the followingtreatments: (1) control (0.9% sterile saline i.p.); (2) 20 mg/kg 3-NPi.p. (SIGMA, dissolved in 0.9% sterile saline); (3) 20 mg/kg 3-NP +43 mg/kg taurine i.p. (SIGMA, dissolved in 0.9% sterile saline); (4)20 mg/kg 3-NP + 100 mg/kg vitamin C i.p. (SIGMA, dissolved in0.9% sterile saline), and (5) 20 mg/kg 3-NP + 100 mg/kg vitamin Ei.p. (SIGMA, dissolved in mineral oil). In all treatments pH wasadjusted to 7.4. The antioxidants were administered 10 min after the3-NP injection. Four hours after receiving treatment, animals weresacrificed by decapitation. Following treatment, rats in each groupwere divided into two groups to examine lipid peroxidation or GFAP.

Establishment of 3-NP Experimental Doses and Time

Based on a previous curve dose-response study (5, 10, 15, and 20mg/kg) this study quantified damage at 4 and 8 h using immunohis-tochemistry technique and lipid peroxidation levels. The authorselected a dose of 20 mg/kg and 4 h after administration, quantifiedastrocytic damage. This dose also causes a notable increase inlevels of brain lipid peroxidation at 4 h.

Lipid Peroxidation Analysis

Lipid peroxidation levels were quantified using a K-ASSAY LPOkit (Kamiya Biomedical Co). Four hours after receiving treatment,animals were sacrificed by decapitation; hippocampus was dissectedon an ice-cold plate and immediately weighed. Each tissue samplewas homogenized in PBS 1:20 and stored at –70°C until day of theassay to measure lipid peroxidation. Homogenates of each samplewere centrifuged. Supernatant was removed, and the enzyme re-agent (ascorbic oxidase and lipoprotein lipase) was added. The mix-ture was incubated for 5 min at a temperature of 30°C and thechromogen reagent (10-N- methylcarbamoyl-3,7-dimethylamino-10-H-phenothiazine (MCDP) was added. The resulting mixture wasincubated for 10 min at a temperature of 30°C. Finally, absorbancewas measured at 675 nm in a spectrophotometer. A two-point cali-bration curve was made using the saline blank (0 nmol/ml) and the50 nmol/ml cumene hydroperoxides standard provided with the kit.The results were then calculated using the following equation, whose

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Antioxidants Ameliorate 3-NP Damage to Hippocampus 1137

linear range for this assay is between 2.0 and 300 nmol/ml: LPO[nmol/ml] = [(Es – Eb) × 50.0/Estd – Eb)], where Es is sampleabsorbance, Estd is absorbance of 50 nmol/ml standard, and Eb isblank absorbance.

Immunohistochemical Analysis

Four hours after receiving treatment, animals were anesthetized withsodium pentobarbital (50 mg/kg i.p.) and perfused transcardiallywith 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4).The brains were post-fixed with 10% formaldehyde for 24 h. Coro-nal sections of the hippocampus were cut at 18 µm on a cryostat,thaw-mounted onto superfrost slides, and processed for GFAPimmunohistochemistry as follows: Sections from each brain werepermeabilized for 1 h at room temperature with 0.03% Triton X-100 (SIGMA), 0.03% Lysine (SIGMA), and 10% normal horseserum for GFAP. After washes with phosphate buffered saline (PBS),sections were incubated overnight with primary antibody at 4ºC in ahumidified chamber. Mouse monoclonal anti-glial fibrillary acidicprotein (GFAP (SIGMA)) (1:200) was used as primary antibody.Sections were then washed and incubated for 2 h at room tempera-ture using biotinylated horse anti-mouse secondary antibody (1:200,Vector). The ABC system (Vectastain ABC Elite Kit, Vector Labo-ratories) with 3,3'-diaminobenzidine (DAB, Pierce) as the chromogenwas used to visualize bound antibody. After that, the tissues werecontrasted with hematoxilin-buffer solution. Representative brainsections from each group were processed in parallel. Following coverslipping with Permount; the sections were examined with a WPI,Inc. H605T Microscope and photographed. The number of astro-cytes cells were quantified, using an average of ten randomly se-lected fields of three sections from each rat.

RESULTS

Lipid Peroxidation Analysis.

A Kruskal-Wallis test showed significant differences in lipid peroxi-dation levels in hippocampus between groups (p < .001). Dunett

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1138 E. Rodríguez-Martínez et al.

tests showed that animals receiving 3-NP had increased lipid peroxidationlevels compared to controls and that animals receiving taurine, vita-min C, or vitamin E after 3-NP injection had significantly lowerlipid peroxidation levels than those receiving 3-NP alone (p < .05)(Figure 1).

Immunohistochemical Analysis

In the control group (Figure 2A and B), GFAP-reactive astrocyteswith well-defined processes were distributed throughout the hippoc-ampus. In contrast, the hippocampus of 3-NP treated rats exhibitedincreased GFAP immunoreactivity per cell (qualitatively), astrocytic

FIGURE 1. Effects of 3-NP administration on lipid peroxidation levels (*p < .05 vs.control; +p < .05 vs. 3-NP). Mean ± SE. The treatment is depicted on the abscissa; lipidperoxidation levels in nmol/ml are depicted on the ordinate (n = 6).

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Antioxidants Ameliorate 3-NP Damage to Hippocampus 1139

swelling and shortening of processes (Figure 2C). Although therewere fewer astrocytes present, the 3-NP + Taurine hippocampusconserved morphology, showing more similarity to controls (Figure2D). The hippocampus of animals treated with 3-NP + vitamin Cshowed a decrease of process size and increased GFAP immunore-activity per cell (qualitatively) (Figure 2E) and that of animals treated

FIGURE 2. Light photomicrographs of astrocytes immunolabeled for GFAP. (A) Con-trol 4×, the rectangle indicate hippocampus area that is shown in 40×. (B) Control 40×,(C) 3-NP 40×, (D) 3-NP + Taurine, (E) 3-NP + Vitamin C, (F) 3-NP + Vitamine E.(→) Astrocytes. Bars represent 50 Πm.

E F

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1140 E. Rodríguez-Martínez et al.

with 3-NP + vitamin E exhibited astrocytic swelling, shortening ofprocesses, and increased GFAP immunoreactivity in each cell (Fig-ure 2F), although these effects were not as severe as in the 3-NPgroup.

Astrocyte Number

A Kruskal-Wallis test showed significant differences in number ofastrocytes in hippocampus between groups (p < .01). Dunett testsshowed that animals receiving 3-NP had decreased number of astro-cytes compared to controls and that animals receiving taurine, vita-min C, or vitamin E after 3-NP injection had a significantly lowernumber of astrocytes than those receiving 3-NP alone (p < .05)(Figure 3).

FIGURE 3. Effects of 3-NP administration on number of astrocytes in hippocampus(*p < .05 vs. control; +p < .05 vs. 3-NP). Mean ± SE. The treatment is depicted on theabscissa; number of astrocytes/field are depicted on the ordinate (n = 6).

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Antioxidants Ameliorate 3-NP Damage to Hippocampus 1141

DISCUSSION

Recent studies implicate oxidative stress in 3-NP-induced neurotox-icity (La Fontaine et al., 2000; Binienda & Kim, 1997) leading toincreased lipid peroxidation levels (Nishino et al., 1997). In thepresent study, 3-NP administration caused energetic deficit, whichproduces oxidative stress in hippocampus. It is shown by dramaticincreases in lipid peroxidation levels in this structure (Figure 1).Hippocampus is vulnerable to oxidative stress and 3-NP toxicity.The damage in other brain areas is found also with systemic admin-istration, that is, striatum, frontal cortex, cerebellum, and so on.Nevertheless, intrastriatal administration is used frequently as a modelof Huntington disease.

Results showed damage on astrocytic cell caused by acute oxida-tive stress. Actually, an astrocyte can make contact with a neuronand a capillary. For this reason, they are principal targets of sys-temic 3-NP administration (Alexi et al., 1998; Rivas-Arancibia etal., 2000). Due to the physiological role that astrocytes play, theyare essential for neuronal survival and very important in neuro-degenerative processes.

In the present study, increased GFAP immunoreactivity and as-trocytic damage in the hippocampus was evident 4 h after adminis-tration of the toxin (Figure 3). As astrocytes lose the capacity tomaintain their internal homeostasis and normal morphology, theyenter a process that begins with hypertrophy and edema, and leadsto cellular death (Kreutzberg et al., 1989). Results showed that anti-oxidants may be able partially to prevent this damage. These ex-periments show that the number of astrocytes decrease in all treat-ments. This could be interpreted as tissue damage increasing (Figure2). However, the morphological integrity of hippocampal astrocytesthat survive was maintained when taurine was administered to 3-NPtreated animals. It could mean that there is a maintenance of survi-vor cellular function (Figure 3D). The authors have also shown thisneuroprotective effect in striatum (Rivas-Arancibia et al., 2001).

Treatment with taurine, vitamin C, and vitamin E prevented in-crease in lipid peroxidation levels. The fact that antioxidants ame-liorated these alterations indicates that part of the damage may bedue to oxidative stress produced by free radicals as a consequenceof 3-NP administration.

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1142 E. Rodríguez-Martínez et al.

Vitamin C and E treatments were not as effective as taurine inpreventing the 3-NP-induced morphologic damage in survivor cells(Figure 3E and F). Thus, although taurine, vitamin C, and vitamin Eall exhibited antioxidant effects, seen as decreased lipid peroxidation,they did not provide equal neuroprotective effects against cellulardeath. This suggests that although part of the damage may be medi-ated by oxidative stress, other factors may contribute to the damageand death of cells.

Although the oxidative stress could start a cell damage processafter 3-NP administration, the ROS generated not only act on bio-molecules caused oxidation. They also induce metabolic changesthat lead to activation of oxidative pathways (Jae et al., 2003). Thepro-oxidant metabolites are produced and can interfere with enzy-matic antioxidant defense systems. It increases oxide reduction im-balance, raising the excitatory neurotransmitters release (John et al.,2000) and decreasing the glutamate recapture by the astrocytic cells.Conversely, oxidative signals induce activation of pathways that leadto cell death (caspases). The ROS also activate the nuclear factortranscription as Nuclear Factor κ-B, which induced transcription ofinflammatory mediators (Wulf, 2002). These factors contribute toincreased damage. Finally osmotic changes are produced by lipidand protein oxidation of cellular membranes. This would explainwhy taurine provided more neuroprotection than vitamins C and E.In addition to its effects as an antioxidant, taurine may also provideprotection from neuronal damage via its osmoregulatory properties(Pasantes-Morales & Schousboe, 1988). Taurine is released by thecell during damage and acts to prevent the rapid influx of sodiumand water, thereby helping to maintain osmotic equilibrium anddiminishing cellular edema. Additionally, it also has modulatoryeffects on calcium, preventing the massive influx of calcium thatoccurs during oxidative stress. This avoids the formation of themitochondrial transition pore, decreases ATP production and energydepletion that lead to cell death.

This study showed that although all treatments demonstrated antioxidant effects, this effect, in and by itself, was insufficient toprotect astrocytes totally from damage and cellular death. One in-terpretation of this finding is that once oxidative metabolic cascadeshave been activated, cells enter a vicious cycle in which the oxide-

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Antioxidants Ameliorate 3-NP Damage to Hippocampus 1143

reduction imbalance continues, leading to a loss in the efficiency ofendogenous antioxidant defenses.

The lost of oxide-reduction balance activates cellular signals, whichunchain an event cascade (such as osmotic changes, inflammatoryprocesses, gene modulation and protein transcription). Once theseevents have been activated, they will not depend on redox balance.But equilibrium of oxide-reduction reactions prevents oxidative stressstate in neighbor cells.

However, why do some cells survive? What mechanisms allowthese cells to recuperate from damage in the presence of taurine?Future studies are needed to resolve these questions.

In conclusion, this study found that taurine, vitamin C, and vitaminE exhibited antioxidant effects, preventing lipid peroxidation causedby 3-NP, indicating that 3-NP-induced hippocampal neurotoxicity ismediated, in part, by oxidative stress. Although all substances hadantioxidant effects, they did not protect cell death. However, onlytaurine was able to protect hippocampal survivor astrocytes and itwas possibly due to its effects at different levels.

As neurodegenerative diseases are complex systems in which damagemay occur by diverse mechanisms, treatments such as taurine thatact at different levels (e.g., antioxidant, energetic, etc.) may providemore optimal therapeutic strategies than antioxidants alone.

The symptomatology of many neurodegenerative diseases dis-plays when a great number of cells have died. The possibility ofsurvivor cells protection during diagnosis is essential to detain dis-ease progress and prevent future deterioration of patients.

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