1 Laboratory of Neurotoxicology, Instituto Nacional de Neurologı´ay Neurocirugía, “Manuel Velasco Suárez” SS, Av. Insurgentes SurNo. 3877, C.P. 14269, México, D.F., México.

2 Instituto de Investigaciones Biomédicas, Department of Physiol-ogy, Universidad Nacional Autónoma de México Apartado Postal70228 C.P. 04510, México, D.F., México.

3 Laboratory of Histomorphology, Instituto Nacional de Pediatría,Av. Insurgentes Sur No. 3700-C, Col. Insurgentes Cuicuilco, C.P.04530, México, D.F., México.

4 Department of Neurochemistry, Instituto Nacional de Neurologı´ay Neurocirugía, “Manuel Velasco Suárez” SS, Av. Insurgentes SurNo. 3877, C.P. 14269, México, D.F., México.

5 Address reprint request to: Patricia Rojas, Ph.D., Laboratory ofNeurotoxicology, Instituto Nacional de Neurología y Neuro-cirugía. Av. Insurgentes Sur No. 3877, Col. La Fama C.P. 14269,México D.F., México. Tel: 152 5 606 4040; Fax: 152 5 424 0808;

EGb761 Blocks MPP1-Induced Lipid Peroxidationin Mouse Corpus Striatum

Patricia Rojas,1,5 Belén Garduño,1 Carolina Rojas,2 Rosa Marı́a Vigueras,3

Julio Rojas-Castañeda,3 Camilo Ríos,4 and Norma Serrano-Garcia1

(Accepted September 28, 2001)

EGb761 has been suggested to be an antioxidant and free radical scavenger. Excess generationof free radicals, leading to lipid peroxidation (LP), has been proposed to play a role in the dam-age to striatal neurons induced by 1-methyl-4-phenylpyridinium (MPP1). We investigated theeffects of EGb761 pretreatment on MPP1 neurotoxicity. C-57 black mice were pretreated withEGb761 for 17 days at different doses (0.63, 1.25, 2.5, 5 or 10 mg/kg) followed by administra-tion of MPP1, (0.18, 0.36 or 0.72 mg/kg). LP was analyzed in corpus striatum at 30 min, 1 h, 2 hand 24 h after MPP1 administration. Striatal dopamine content was analyzed by HPLC at thehighest EGb761 dose at 2 h and 24 h after MPP1 administration. MPP1-induced LP was blocked(100%) by EGb761 (10 mg/kg). Pretreatment with EGb761 partially prevented (32%) thedopamine-depleting effect of MPP1 at 24 h. These results suggest that supplements of EGb761may be effective at preventing MPP1-induced oxidative stress.

KEY WORDS: MPP1; lipid peroxidation; neuroprotection; parkinson’s disease; EGb761.

Neurochemical Research, Vol. 26, No. 11, November 2001 (© 2002), pp. 1245–1251

12450364-3190/02/1100–1245/0 © 2002 Plenum Publishing Corporation

and death of the dopaminergic neurons of the nigros-triatal pathway in the brain (1). Death of these neuronsproduces a decrease in the striatal dopamine (DA) con-tent. As a result, bradykinesia, tremor, and rigidityappear (2). The cause of this neuronal loss is unclearbut there is increasing evidence that oxidative stress,via free radical production, plays an important role inthe process.

Free radicals are produced constitutively undernormal physiological conditions. Organisms have de-veloped various defense mechanisms to protect them-selves against injury from free radicals. Such defensemechanisms include antioxidant enzymes, free radicalscavengers, and metal chelating agents (3). The antioxi-dant enzymes are catalase, glutathione peroxidase, andsuperoxide dismutase (SOD). Normally, there is a bal-ance between generation of free radicals and antioxi-dant defense system activity in vivo. When this balanceis altered to favor production of free radicals due to de-pletion of antioxidant system components or increased

INTRODUCTION

The etiology of Parkinson’s disease remains un-known. Nonetheless, its pathology has been well-described. The disease results from the degeneration

generation of free radicals, oxidative stress occurs (4).Oxidative stress leads to damage of polyunsaturatedlipids by lipid peroxidation (LP), a chain-reaction thatresults in numerous degradation products (5).

Post-mortem studies on patients with Parkinson’sdisease have reported alterations in substantia nigra:the level of iron is elevated (6), there is a loss of glu-tathione (7), and increased LP (8).

The disease is routinely treated by administrationof the DA precursor, L-DOPA, resulting in increasedsynthesis of the neurotransmitter dopamine in brain(9). L-DOPA treatment has secondary effects, some ofthem severe, such as the production of uncontrolledmovements and dystonia (10). Therefore the search fornew therapeutic modalities is of considerable interest.

The therapeutic efficacy of new anti-parkinsoniandrugs is usually assessed in experimental models ofthe disease. Among them, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is regarded as the bestavailable experimental model of the neurochemical se-quelae of Parkinson’s disease (11).

When MPTP is administered to non-human pri-mates and mice it produces hypokinesia and neuronaldamage similar to that observed in idiopathic Parkin-son’s disease (11). These alterations are accompaniedby a reduction of striatal DA content. The model repro-duces the pathological and biochemical characteristicsof the human disease (11). 1-methyl-4-phenylpyri-dinium ion (MPP1) is the major metabolite of MPTP. Itis now accepted that MPP1 is responsible for the neu-rotoxic effects of MPTP (for a review see 11).

Biotransformation of MPTP to MPP1 is initiallyperformed by MAO-B. After this event, MPP1 is ac-tively accumulated in dopaminergic neurons by theDA uptake system and is accumulated in mitochondria(12). The high concentration of MPP1 inside mito-chondria blocks complex I activity in the respiratorychain (13). This process is reversible and leads to arapid loss of ATP (14).

MPTP oxidation to MPP1 by MAO-B in the braingenerates free radicals (15). Incubation of MPP1 withmitochondrial enzymes also induces free radical pro-duction (16) and the increased free radicals can furtherinhibit the function of complex I (17). These resultssuggest that neuronal death occurs not only due to theloss of energy, but also due to oxidative stress via theproduction of reactive oxygen species (18). Furthersupporting evidence includes the observation that pre-treatment of mice with diethyldithiocarbamate, a SODinhibitor, enhances MPTP-induced neurotoxicity (19)whereas transgenic mice that over-express human Cu-Zn-SOD, showing increased SOD activity, are re-

ported to be more resistant to MPTP than wild-typemice (20). With regard to oxidative stress hypothesiswe found enhanced LP, a process dependent on freeradicals overproduction, to be a consequence of MPP1

administration to mice (21).Manganese and copper constitute part of the ac-

tive sites of the SOD enzyme that catalyzes oxygenfree radicals metabolism. We found depletion of man-ganese and copper following MPTP administration(22). Pretreatment with these metals protected againstMPP1 neurotoxicity (23,24).

On the other hand, the Ginkgo bilobaextract hasbeen a part of traditional Chinese medicine for severalthousands of years. Recently, a leaf extract of Ginkgobiloba, termed EGb761, has become one of the mostwidely used medicinal plant products in Europe.EGb761 is a well-defined mixture of active com-pounds extracted from Ginkgo biloba leaves via apatented extraction process (25) and introduced intomedical practice. EGb761 is prepared as a dry powderand contains two groups of major substances,flavonoid glycosides (24%) and terpene lactones (6%)(25). The flavonoid fraction is composed of threeflavonols: quercetin, kaempferol and isorhamnetin,which are linked to a sugar (25). The terpenoid frac-tion is composed of ginkgolides and bilobalides (25).EGb761 has been used in the treatment of cerebrovas-cular insufficiency, degenerative dementia, peripheralvascular disturbances, and neurosensory disorders (fora review see 25).

The antioxidant and free radical scavenger actionof EGb761 has been claimed to be one of the molecu-lar mechanisms underlying the beneficial effects ofEGb761. It has been reported to scavenge superoxide,hydroxyl, and peroxyl radicals (26), and to interactwith nitric oxide (27); it also inhibits the enzymatic ac-tivity of inducible nitric oxide synthase (28). Severalstudies have demonstrated EGb761’s effects on DAneuronal metabolism (29).

In order to investigate the antioxidant effectsof EGb761 on the oxidative stress induced by MPP1,we studied the capacity of EGb761 to prevent MPP1-induced LP, using LP as an index of oxidative stress,as well as the effect of EGb761 pretreatment onMPP1-induced depletion of DA in striatum.

EXPERIMENTAL PROCEDURE

Experiments were conducted on male C-57 black mice (25–30 g)aged 11 to 13 weeks. Animals were maintained in standard condi-tions (12:12 h light/dark cycle, 21 6 2°C), and relative humidity

1246 Rojas et al.

(40%) and allowed access to food and water ad libitum. All animalswere treated humanely to minimize discomfort in accordance withthe ethical principles and regulations specified by Animal Care andUse Committee of our Institution and the standards of the NationalInstitutes of Health of Mexico.

MPP1 iodine was obtained from Research Biochemicals Incor-porated (Wayland, MA, USA). EGb761, prepared as a concentratedpowder, was kindly donated by Dr. Willmar Schwabe Pharmaceuti-cals (Karisruhe, Germany). The extract was dissolved in saline andthe pH adjusted to 7.4. Sodium octyl sulfate and DA were obtainedfrom Sigma Chemical Co, (St. Louis, MO), and methanol (HPLCgrade) from J. T. Baker (Mexico). All other reagents were obtainedfrom Merck (Mexico).

EGb761 Pretreatment and MPP1 Administration. For theseexperiments, there were four treatment groups: Group I (n 5 6)saline solution (ip) 1 saline solution (intracerebroventricular; icv);Group II (n 5 6) EGb761 (ip) 1 saline solution (icv); Group III(n 5 8) saline solution (ip) 1 MPP1 (icv); Group IV (n 5 8) EGb761(ip) 1 MPP1 (icv).

Animals of groups I and III received normal saline solution (ip)and groups II and IV received EGb761 at different doses (0.63, 1.25,2.5, 5 or 10 mg/kg, ip) for 17 days. These doses had been used in otherreports (30). After pretreatment, animals of groups III and IV wereanesthesized with ether and given 3 ml of solution containing 18 mgof MPP1 (0.72 mg/kg), injected into the right lateral ventricle (icv) asdescribed previously (21). This dose has been shown to produce spe-cific damage to dopaminergic neurons in mice (31). Mice from groupsI and II injected with saline solution (icv), served as controls. Micewere sacrificed by cervical dislocation 2 h after MPP1 administrationas we previously described (21). Brains were immediately removedand striatum was dissected out as described by Glowinski and Iversen(32) and assayed for LP by measuring the levels of lipid fluorescenceproducts (LFP) formation. Additional groups of mice pretreatedwith EGb761 (10 mg/kg, ip) were sacrificed for LFP formation assay30 min, 1 h and 24 h after MPP1 administration (0.72 mg/kg; 18mg/3 ml). Also, MPP1 dose-response (0.18, 0.36 or 0.72 mg/kg) was eval-uated 2 h after administration of MPP1 to animals previously treatedwith EGb761 (10 mg/kg, ip). Further groups of mice pretreated withEGb761 (10 mg/kg) followed by MPP1 administration (0.72 mg/kg,18 mg/3 ml) were included to analyze DA content in striatum at 2 hand 24 h after MPP1 administration.

Lipid Peroxidation Analysis.LFP formation was monitored inthe corpora striata of mice using the technique described by Triggsand Willmore (33) and previously modified by us (21). Striatal tis-sue was carefully weighed and homogenized in 2.5 ml of phosphatebuffer (pH 7.0). One milliliter aliquots of the homogenate were sep-arated in glass tubes and added to 3 ml of chloroform-methanol mix-ture (2:1 v/v). The tubes were capped, gently mixed and placed onice for 30 min. Aqueous phase was discarded and 1 ml of the chlo-roform layer was transferred into a quartz cuvette, and 0.1 ml ofmethanol was added. Fluorescence was measured in a Perkin-ElmerLS50B Luminescence spectrophotometer at 370 nm excitation and430 nm emission wavelenghts. Prior to measurement of the samples,sensitivity of the spectrophotometer was adjusted to 140 fluores-cence units with a 0.1 mg/l quinine standard prepared in 0.05 Maqueous sulfuric acid solution. Results were expressed as fluores-cence units per gram of wet tissue per ml extraction read. All sam-ples were run in duplicate.

Determination of Dopamine.We analyzed DA only in mousegroups treated with EGb761 (10 mg/kg, ip) for 17 days and the high-est dose of MPP1 (0.72 mg/kg, 18 mg/3 ml), with respective controlgroups.

Mouse brains were removed quickly and their corpora striatawere dissected out as described above. An aliquot (500 ml) of per-chloric acid-sodium metabisulfite solution (0.1% w/v) was added tothe tissue and sonicated with a Lab-line ultratip labsonic system(Lab-line instruments, Melrose Park, IL). Samples were then cen-trifuged at 4,000 g for 10 min and the supernatants were kept at270°C until analyzed.

Striatal content of DA was analyzed using HPLC system (LC250 Perkin-Elmer) with an electrochemical detector (Methrom 656)and a Hewlett-Packard 3396-II integrator as described previously(21). Calibration curves were constructed for DA. Concentration wasobtained by interpolation of the respective standard curve. An AlltechAssociates, Inc. (Deerfield, IL), adsorbosphere catecholamine analyt-ical column of 100 3 4.8 mm with 3 mm particle diameter was used.The mobile phase consisted of aqueous phosphate buffer (pH 3.1)which contained 0.2 mM sodium octyl sulfate, 0.1 mM EDTA, and15% v/v of methanol. The detector potential was adjusted to 0.8 V vs.Ag/AgCl reference electrode. The results were expressed as mg ofcompound per gram of tissue.

Data are expressed as mean 6 SEM. Results were analyzed sta-tistically using one-way analysis of variance (ANOVA), followedby Tukey’s test. Values of p , 0.05 and p , 0.01 were consideredto be statistically significant and highly significant, respectively.

RESULTS

Using the LFP formation assay, we demonstratedthat EGb761 administration to mice produced no sig-nificant increases in LP at the different doses tested(0.63, 1.25, 2.5, 5 or 10 mg/kg; data not shown) whencompared with control animals. As shown in Fig. 1, LPwas significantly increased at 2 h after MPP1 adminis-tration (0.72 mg/kg, 18 mg/3 ml) without EGb761 pre-treatment (72% higher than saline-treated controls,

EGb761 Blocks MPP1 Neurotoxicity 1247

Fig. 1. Lipid fluorescence products formation in striatum 2 h afterMPP1 administration (0.72 mg/kg, 18 mg/3 ml) to mice previouslytreated with different doses of EGb761 for 17 days. Results areexpressed as mean 6 SEM, n 5 6–8 independent experiments.**Statistically different from control (“saline 1 saline”) group p ,0.01, Tukey’s test. 1Statistically different from MPP1-treated(“saline 1 MPP1”) group, p , 0.05, Tukey’s test. 11Statisticallydifferent from MPP1-treated (“saline 1 MPP1”) group, p , 0.01,Tukey’s test. EGb761 5 Ginkgo bilobaextract. MPP1 5 1-methyl-4-phenylpyridinium ion.

p , 0.01) according to results previously reported by us(21). In contrast, pretreatment with EGb761 at 10 mg/kgcompletely blocked (100% protection) the MPP1-induced LP (LFP formation levels were not statisticallydifferent from those of saline-treated controls).

In the EGb761 dose-response study the degree ofprotection increased with increasing doses of EGb761(Fig. 1). For example, the degree of protection was65%, 93% and 100% for 2.5, 5 and 10 mg/kg ofEGb761, respectively, when compared with “saline 1MPP1” group.

Since the highest dose (10 mg/kg) of EGb761 wasthe most effective at 2 h after MPP1 administration(0.72 mg/kg, 18 mg/3 ml), we used this dose to analyzeLFP formation at different times as well as with dif-ferent MPP1 doses. As seen in Table I, EGb761 at10 mg/kg had no significant effect on LFP formation at30 and 60 min after MPP1 administration (0.72 mg/kg,18 mg/3 ml). At 2 h and 24 h after MPP1 administration(0.72 mg/kg, 18 mg/3 ml) there was significant increasein LFP formation, with LFP values being 72% and 25%

higher than control values, respectively. At both times,pretreatment with EGb761 (10 mg/kg) provided signif-icant protection against MPP1 neurotoxicity.

To analyze the dependence of LFP formation onthe dose of MPP1, LFP formation was measured 2 hafter administering various doses of MPP1 (0.18, 0.36or 0.72 mg/kg; 4.5, 9 or 18 mg/3 ml) to mice with orwithout EGb761 (10 mg/kg) pretreatment for 17 days.A dose-response pattern was observed with MPP1 ad-ministration (Fig. 2), as we described previously (21).At 0.18 mg/kg (4.5 mg/3 ml) of MPP1 there wasnot significative increase in LFP formation comparedto controls. At 0.36 mg/kg (9 mg/3 ml), LFP levelsincreased statistically by 22% above control values(p , 0.01). At the highest dose of MPP1 (0.72 mg/kg,18 mg/3 ml) LFP formation increased by 72% abovethat of the control group (p , 0.01). EGb761 pretreat-ment (10 mg/kg) protected against MPP1 neurotoxicityat its highest dose (0.72 mg/kg, 18 mg/3 ml) but appar-ently not at the median dose (0.36 mg/kg, 9 mg/3 ml)of MPP1.

Striatal DA content after MPP1 administration(0.72 mg/kg, 18 mg/3 ml) is shown in Fig. 3. Signifi-cant decreases in DA concentration were observed at24 h (40% versus control) but not at 2 h, indicating thatLP enhancement preceeded the DA depletory effect ofMPP1. EGb761 administration (10 mg/kg, ip) to con-trol mice did not produce significant alteration in DA

1248 Rojas et al.

Fig. 2. Lipid fluorescence products formation in striatum 2 h after asingle intracerebroventricular administration of variable doses ofMPP1 (0.18, 0.36 or 0.72 mg/kg; 4.5, 9 or 18 mg/3 ml) to micepreviously treated with EGb761 (10 mg/kg) for 17 days. Results areexpressed as mean 6 SEM, n 5 6–8 independent experiments.*Statistically different from control (“saline 1 saline”) group, p ,0.05, Tukey’s test. **Statistically different from control (“saline 1saline”), p , 0.01, Tukey’s test. 1Statistically different from MPP1-treated (“saline 1 MPP1”) group, p , 0.05, Tukey’s test.11Statistically different from MPP1-treated (“saline 1 MPP1”)group, p , 0.01, Tukey’s test. EGb761 5 Ginkgo bilobaextract.MPP1 5 1-methyl-4-phenylpyridinium ion.

Table I. Protective Effect of EGb761 in MPP1 Neurotoxicity

Fluorescence units/g wet wt/mlTreatment Mean 6 SEM

30 minutesSaline 1 Saline 123.28 6 8.65EGb761 1 Saline 124.33 6 12.06Saline 1 MPP1 144.92 6 13.38EGb761 1 MPP1 149.71 6 12.06

60 minutesSaline 1 Saline 123.17 6 7.17EGb761 1 Saline 130.57 6 3.03Saline 1 MPP1 148.42 6 6.53EGb761 1 MPP1 148.38 6 9.88

2 hoursSaline 1 Saline 134.06 6 5.87EGb761 1 Saline 115.92 6 5.68Saline 1 MPP1 231.15 6 9.30**EGb761 1 MPP1 129.84 6 14.92

24 hoursSaline 1 Saline 136.96 6 5.28EGb761 1 Saline 136.29 6 3.18Saline 1 MPP1 171.38 6 11.89*EGb761 1 MPP1 145.58 6 1.95

Lipid fluorescence products formation 30 min, 60 min, 2 h and 24 hafter MPP1 administration (0.72 mg/kg, 18 mg/3 ml) to mice previ-ously treated with normal saline or EGb761 (10 mg/kg) for 17 days.*Statistical different from all groups, p , 0.05, Tukey’s test. **Sta-tistically different from all groups, p , 0.01, Tukey’s tests. Resultsare expressed as mean 6 SEM, n 5 5–8 independent experiments.EGb761: Ginkgo bilobaextract; MPP1 5 1-methyl-4-phenylpyri-dinium ion.

content when compared to control animals (saline 1saline). Mice in the “saline 1 MPP1” group at 24 h pre-sented markedly reduced (40%) DA levels as a resultof the neurotoxic action of the compound (Fig. 3). Ad-ministration of EGb761 (10 mg/kg) before treating an-imals with MPP1 partially protected (32%) the animalsat 24 h against the neurotoxic effect of MPP1 (Fig. 3).

DISCUSSION

This study shows that EGb761 (10 mg/kg) pro-tects mouse striatum against MPP1-induced oxidativedamage, as measured by formation of LFP. This pro-tection is dependent on the dose of EGb761.

Given that MPP1 administration to rodents en-hances production of cytotoxic free radicals (16) aswell as LP (21), the neuroprotective effect of EGb761observed in this study could be due to the putative ac-tion of EGb761, that is, its free-radical scavenging ac-tivity and its other antioxidant properties (26). ThusEGb761 could limit the LP produced by MPP1 eitherby scavenging hydroxyl radicals, superoxide anions(26) or by preventing the formation of lipid peroxylradicals (34), the major chain-propagation step in LP,thus suggesting that the extract is a potential thera-peutic agent for the control of oxidative stress-inducedtissue damage. In support of our findings, it hasbeen shown that EGb761 reduces LP in a free radical-generating system (35–37), prevents apoptosis inducedby hydroxyl radicals (36), and protects against oxidativestress in mitochondria (38).

In vitro studies in neuroblastoma SH-SY5Y cellshave shown that EGb761 was able to reduce LP inducedby Fe21 (35) as well as protect neurons against oxidativestress induced by hydrogen peroxide (39). Administra-tion of EGb761 has been associated with a significant re-duction in cerebral edema and brain LP (40).

Significative protection by EGb761 againstMPP1-induced neurotoxicity was observed in thisstudy only at the highest dose of MPP1 (0.72 mg/kg,18 mg/3 ml) and not at the lower dose of 0.36 mg/kg(9 mg/3 ml). Since LFP formation was not increased byadministration of 0.18 mg/kg (4.5 mg/3 ml) of MPP1,no protection by EGb761 is expected at this dose. Thismakes our current dose-response data difficult to in-terpret. Further investigation is needed to elucidate theeffect of level of oxidative stress on the effectivenessof the protection provided by EGb761.

EGb761 is a standardized mixture of differentcompounds containing two major groups of substances:flavonoids and terpenoids. Flavonoids have been re-ported to be effective scavengers of superoxide (36)and hydroxyl radicals (41), as well as inhibitors of LP(42). Our study was not designed to define which com-ponent of EGb761 is responsible for its protective ef-fect. EGb761’s activity could be due to a particularcomponent, or to the action of two or more compo-nents. The main chemical structures of flavonoids pos-sess an aromatic ring and double bond, so flavonoidsprefer to react with hydroxyl radicals and yield an ad-dition product, thus directly scavenging the hydroxylradicals. Flavonoids also possess phenolic hydroxylgroups which may chelate the Fe21 and indirectly in-hibit the formation of hydroxyl radicals (43). It hasbeen reported that only flavone presents an importanteffect against free radicals and LP; the presence of the2–3 double bonds of the central pyran ring and two freehydroxyl groups at the ortho position on the B ring areessential for the effect against free radicals and LP(44). The protective effect of EGb761 on LFP producedby MPP1 may be via the direct scavenging of free rad-icals and the indirect inhibition of LP.

It can be considered that EGb761 protects neuronsagainst oxidative stress induced by several types of re-active oxygen species such as superoxide anion, hy-drogen peroxide and hydroxyl radical. In the case ofMPP1 neurotoxicity, pretreatment with EGb761 waseffective in protecting striatum against oxidative stress.Therefore, EGb761 possesses a considerable potentialfor protection of neurons against oxidative injury.

Free radicals could alter dopamine synthesis (45),peroxidize membrane lipids, and alter functions suchas DA uptake (46). One or more of these mechanisms

EGb761 Blocks MPP1 Neurotoxicity 1249

Fig. 3. Striatal dopamine content. Results are expressed as mean 6SEM of 6–8 independent experiments. **Statistically different fromcontrol (“saline 1 saline”), p , 0.01, Tukey’s test. 1Statisticallydifferent from MPP1-treated (“saline 1 MPP1”) group, p , 0.05,Tukey’s test. EGb761 5 Ginkgo bilobaextract. MPP1 5 1-methyl-4-phenylpyridinium ion.

might be responsible for MPP1 neurotoxicity sincefree radicals are produced by MPP1. Moreover, thedecrease in synaptosomal DA uptake occuring underperoxidative conditions, has been shown to be pre-vented by EGb761 (47).

Our results show that LP preceeded the DA de-pletory effect of MPP1 as we previously reported (21).EGb761 partially prevented the striatal DA depletioncaused by MPP1 administration, only at 24 h after itsadministration.

Also, it has been shown that this Ginkgo bilobaextract has a high affinity for striatum (48), whichmakes it a very good candidate drug for protectionagainst MPP1 neurotoxicity and Parkinson’s disease.

It has been reported that EGb761 protects againstMPTP-induced nigrostriatal dopaminergic neurotoxic-ity and that the inhibitory effect of EGb761 on brainMAO may be involved in its neuroprotective effect (49)since MAO-B is required for biotransformation ofMPTP to MPP1. Our results suggest clearly that this isnot the mechanism responsible for EGb761’s protectiveeffect in this study because it did not interact peripher-ally with the metabolism of MPTP or with its cerebralaccess; we administered MPP1 directly into the brainand still observed neuroprotection by EGb761.

The present study shows that EGb761 protects thestriatum against MPP1-induced LP and protects par-tially against DA depletion, possibly by acting as a po-tent antioxidant agent.

ACKNOWLEDGMENTS

The authors thank Dr. Sultan Habeebu for his comments. Par-tially supported by the National Council of Science and Technologyof Mexico (CONACyT) grant 28605-M. Belén Garduño is a recipi-ent of a scholarship from CONACyT (No. 898).

REFERENCES

1. Jenner, P. 1989. Clues to the mechanism underlying dopaminecell death in Parkinson’s disease. J. Neurol. Neurosurg. Psychi-atry (suppl):22–28.

2. Javoid-Agid F., Ruberg, M., Hirsch, E., Cash, R., Raisman, R.,Taquet, H., Epelbaum, J., Scatton, B., Duyckaerts, C., and Agid,Y. 1986. Recent progress in the neurochemistry of Parkinson’sdisease. Pages 67–83, in Recent Developments in Parkinson’sdisease. Raven Press, New York.

3. Reiter, R. J. 1995. Oxidative processes and antioxidative de-fense mechanisms in the aging brain. FASEB J. 9:526–533.

4. Halliwel, B. 1992. Oxygen radicals as key mediators in neuro-logical disease: fact or fiction? Ann. Neurol. 32:S10–S15.

5. Niki, E., Noguchi, N., and Gotoh, N. 1993. Dynamics of lipidperoxidation and its inhibition by antioxidants. Biochem. Soc.Trans. 21:313–317.

6. Youdim, M. B. H., Schachar, D. Ben., and Riederer, P. 1989. IsParkinson’s disease a progressive siderosis of substantia nigraresulting in increased iron and melanin induced neurodegenera-tion? Acta Neurol. Scand. 126:47–54.

7. Sian, J., Dexter, D. T., Lees, A. J., Daniel, S., Agid, Y., Javoy-Agid, F., Jenner, P. P., and Marsden, C. D. 1994. Alterations inglutathione levels in Parkinson’s disease and other neurode-generative disorders affecting the basal ganglia. Ann. Neurol.36:348–355.

8. Dexter, D. T., Carter, C. J., Wells, F. R., Javoy-Agid, F., Agid,Y., Lees, A., Jenner, P., and Marsden, C. D. 1989. Basal lipidperoxidation in substantia nigra is increased in Parkinson’s dis-ease. J. Neurochem. 52:381–389.

9. Quinn, N. P. 1990. Levodopa-based therapy. Pages 169–184, inTherapy of Parkinson’s Disease. Marcel Dekker, New York.

10. Kanazawa, I. 1986. Clinical pathophysiology of basal gangliadiseases. Vol. 49, pages 65–92, in Vinken, P. T., Bruyn, G. W.,Klawans, H. L. (eds): Handbook of Clinical Neurology. ElsevierScience Publishers, New York.

11. Gerlach, M., Riederer, P., Przuntek, H., and Youdim, M. B. H.1991. MPTP mechanisms of neurotoxicity and their implica-tions for Parkinson’s disease. Eur. J. Pharmacol. Mol. Pharma-col. 208:273–286.

12. Javitch, J. A., D’Amato, R. J., Strittmatter, S. M., and Snyder,S. H. 1985. Parkinsonism-inducing neurotoxin, N-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine: Uptake of the metaboliteN-methyl-4-phenylpyridine by dopamine neurons explains se-lective toxicity. Proc. Natl. Acad. Sci. USA 82:2173–2177.

13. Nicklas, W. J., Vyas, I., and Heikkila, R. E. 1985. Inhibitionof NADH-linked oxidation in brain mitochondria by 1-methyl-4-phenylpyridinium, a metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Life Sci. 36:2503–2508.

14. Chan, P., DeLanney, L. E., Irwin, I., Langston, J. W., andDiMonte, D. 1991. Rapid ATP loss caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mouse. J. Neurochem. 57:348–351.

15. Zang, L. Y. and Misra, H. P. 1993. Generation of reactive oxy-gen species during the monoamine oxidase-catalyzed oxidationof the neurotoxicant, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri-dine. J. Biol. Chem. 268:16504–16512.

16. Adams, J. D., Klaidman, L. K., and Leung, A. 1993. MPP1 andMPDP1 induced oxygen radical formation with mitochondrialenzymes. Free Radic. Biol. Med. 15:181–186.

17. Zhang, Y., Marcillat, O., Giulivi, C., Ernster, L., and Davies,K. J. 1990. The oxidative inactivation of mitochondrial eletrontransport chain components and ATPase. J. Biol. Chem. 265:16330–16336.

18. Adams, J. D. and Odunze, I. N. 1991. Biochemical mechanisms of1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity. Biochem.Pharmacol. 41:1099–1105.

19. Yurek, D. M., Deutch, A. Y., Roth, R. H., and Sladek, J. R. Jr.1989. Morphological, neurochemical, and behavioral characteri-zations associated with the combined treatment of diethyldithio-carbamate and 1-methyl-4-phenyl-1,2,3, 6- tetrahydropyridine inmice. Brain Res. 497:250–259.

20. Przedborski, S., Kostic, V., Jackson-Lewis, V., Naini, A. B., Si-monetti, S., Fahn, S., Carlson, E., Epstein, C. J., and Cadet, J. L.1992. Transgenic mice with increased Cu/Zn-superoxide dis-mutase activity are resistant to N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity. J. Neurosci. 12:1658–1667.

21. Rojas, P. and Ríos, C. 1993. Increased striatal lipid peroxidationafter intracerebroventricular MPP1 administration to mice.Pharmacol. Toxicol. 72:364–368.

22. Ríos, C., Alvarez-Vega, R., and Rojas, P. 1995. Depletion ofcopper and manganese in brain after MPTP treatment of mice.Pharmacol. Toxicol. 76:348–352.

23. Rojas, P. and Ríos, C. 1995. Short-term manganese pretreat-ment partially protects against 1-methyl-4-phenyl-1, 2, 3,

1250 Rojas et al.

6-tetrahydropyridine neurotoxicity. Neurochem. Res. 20:1217–1223.

24. Alcaraz-Zubeldia, M., Rojas, P., Boll, C., and Ríos, C. 2001.Neuroprotective effect of acute and chronic administration ofcopper (II) sulfate against MPP1 neurotoxicity in mice. Neuro-chem. Res. 26:59–64.

25. DeFeudis, F. V. 1998. Ginkgo bilobaextract (EGb761): fromChemistry to the Clinic. Pages 401. Ullstein Medical, Wies-baden.

26. Marcocci, L., Packer, L., Droy-Lefaix, M., Sekaki, A., andGardes-Albert, M. 1994. Antioxidant action of Ginkgo bilobaextract EGb761. Methods Enzymol. 234:462–475.

27. Marcocci, L., Maguire, J. J., Droy-Lefaix, M. T., and Packer, L.1994. The nitric oxide-scavenging properties of Ginkgo bilobaextract EGb761. Biochem. Biophys. Res. Commun. 201:748–755.

28. Kobuchi, H., Droy-Lefaix, M. T., Christen, Y., and Packer, L.1997. Ginkgo bilobaextract (EGb761): Inhibitory effect on ni-tric oxide production in the macrophage cell line RAW 264.7.Biochem. Pharmacol. 53:897–903.

29. Ramassamy, C., Clostre, F., Christen, Y., and Costentin, J. 1990.Prevention by a Ginkgo bilobaextract (GBE 761) of the dopa-minergic neurotoxicity of MPTP. J. Pharm. Pharmacol. 42:785–789.

30. Brailowsky, S. and Montiel, T. 1997. Motor function in youngand aged hemiplegic rats: effects of a Ginkgo bilobaextract.Neurobiol. Aging 18:219–227.

31. Mihatsch, W., Russ, H., and Przuntek, H. 1988. Intracerebroven-tricular administration of MPP1 in mice: Effects of simultane-ously administered nomifensine, deprenyl and 1-t-butyl-4,4-diphenylpiperidine. J. Neural. Transm. 71:177–188.

32. Glowinski, J. and Iversen, L. L. 1966. Regional studies of cate-cholamines in the rat brain. Disposition of 3H-Norepinephrine,3H-dopamine and 3H-DOPA in various regions of the brain.J. Neurochem. 13:655–669.

33. Triggs, W. J. and Willmore, L. J. 1984. In vivo lipid peroxida-tion in rat brain following intracortical Fe21 injection. J. Neu-rochem. 42:976–979.

34. Maitra, I., Marcocci, L., Droy-Lefaix, M. T., and Packer, L.1995. Peroxyl Radical scavenging activity of Ginkgo bilobaextract EGb761. Biochem. Pharmacol. 49:1649–1655.

35. Sloley, B. D., Urichuk, L. J., Morley, P., Durkin, J., Shan, J. J.,Pang, P. K. T., and Coutts, R. T. 2000. Identification of kaemp-ferol as a monoamine oxidase inhibitor and potential neuropro-tectant in extracts of Ginkgo bilobaleaves. J. Pharm. Pharmacol.52:451–459.

36. Ni, Y., Zhao, B., Hou, J., and Xin, W. 1996. Preventive effectof Ginkgo bilobaextract on apoptosis in rat cerebellar neuronalcells induced by hydroxyl radicals. Neurosc. Lett. 214:115–118.

37. Ramassamy, C., Girbe, F., Christen, Y., and Costentin, J. 1993.Ginkgo bilobaextract EGb761 or trolox C prevent the ascorbicacid/Fe21 induced decrease in synaptosomal membrane fluidity.Free Radic. Res. Commun. 19:341–350.

38. Sastre, J., Millán, A., García, J., Plá, R., Juan, G., Pallardó,F. V., O’Connor, E., Martín, J. A., Droy-Lefaix, M. T., andViña, J. 1998. A ginkgo biloba extract (EGb761) prevents mi-tochondrial aging by protecting against oxidative strees. FreeRadic. Biol. Med. 24:298–304.

39. Oyama, Y., Chikahisa, L., Ueha, T., Kanemaru, K., and Noda,K. 1996. Ginkgo bilobaextract protects brain neurons againstoxidative strees induced by hydrogen peroxide. Brain Res.712:349–352.

40. Dorman, D. C., Coté, L. M., and Buck, W. B. 1992. Effects ofan extract of Ginkgo bilobaon bromethalin-induced cerebrallipid peroxidation and edema in rats. Am. J. Vet. Res. 53:138–142.

41. Bors, W., Heller, W., Michel, C., and Saran, M. 1990.Flavonoids as antioxidants: Determination of radical-scaveng-ing efficiencies. Methods Enzymol. 186:343–355.

42. Afanas’ev, I. B., Dorozhko, A. I., Brodskii, A. V., Kostyuk,V. A., and Potapovitch, A. I. 1989. Chelating and free radicalscavenging mechanisms of inhibitory action of rutin andquercetin in lipid peroxidation. Biochem. Pharmacol. 38:1763–1769.

43. Havsteen, B. 1983. Flavonoids, a class of natural high pharma-cological potency. Biochem. Pharmacol. 32:1141–1148.

44. Joyeux, M., Lobstein, A., Anton, R., and Mortier, F. 1995.Comparative antilipoperoxidant, antinecrotic and scavengingproperties of terpenes and biflavones from Ginkgo and someflavonoids. Planta. Med. 61:126–129.

45. Zaleska, M. M., Nagy, K., and Floyd, R. A. 1989. Iron-inducedlipid peroxidation and inhibition of dopamine synthesis in stria-tum synaptosomes. Neurochem. Res. 14:597–605.

46. Rafalowska, U., Liu, G. J., and Floyd, R. A. 1989. Peroxi-dation induced changes in synaptosomal transport of dopa-mine and gama-aminobutyric acid. Free Radical Biol. Med. 6:485–492.

47. Ramassamy, C., Naudin, B., Christen, Y., Clostre, F., and Cos-tentin, J. 1992. Prevention by Ginkgo bilobaextract (EGb761)and trolox C of the decrease in synaptosomal dopamine orserotonin uptake following incubation. Biochem. Pharmacol.44:2395–2401.

48. Moreau, J-P., Eck, J., McCabe, J., and Skinner, S. 1986. Ab-sorption, distribution et élimination de léxtrait marqué deGinkgo biloba chez le rat. Presse Méd. 4:2401–2402.

49. Wu, Wei-Ran and Zhu, Xing-Zu. 1999. Involvement of mono-amine oxidase inhibition in neuroprotective and restorative effectsof Ginkgo biloba extract against MPTP-induced nigrostriataldopaminergic toxicity in C-57 mice. Life Sci. 65:157–164.

EGb761 Blocks MPP1 Neurotoxicity 1251