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Effect of EGb761 supplementation on the content of copper in mousebrain in an animal model of Parkinson’s disease

Patricia Rojas, Ph.D.a,*, Sergio Montes, Ph.D.b, Norma Serrano-García, M.Sc.a, andJulio Rojas-Castañeda, M.Sc.c

a Laboratory of Neurotoxicology, National Institute of Neurology and Neurosurgery, “Manuel Velasco Suárez”, Mexico, D.F., Mexicob Department of Neurochemistry, National Institute of Neurology and Neurosurgery, “Manuel Velasco Suárez”, Mexico, D.F., Mexico

c Laboratory of Histomorphology, National Institute of Pediatrics, Mexico, D.F., Mexico

Manuscript received October 9, 2008; accepted October 15, 2008.

bstract Objective: EGb761 is a well-defined mixture of active compounds extracted from Ginkgo bilobaleaves with neuroprotective effects in an animal model of Parkinson’s disease induced by 1-methyl-4-phenylpyridinium (MPP�). Because copper has been implicated in Parkinson’s disease, weinvestigated whether the protective effect of EGb761 in MPP� neurotoxicity is related to theregulation of copper in the brain.Methods: C-57BL/6 mice were pretreated with EGb761 (10 mg/kg) daily for 17 d followed byadministration of MPP� (0.72 mg/kg); the mice were sacrificed 24 h later. The copper content ofthe striatum, midbrain, hippocampus, frontal cortex, and cerebellum was analyzed by graphitefurnace atomic absorption spectrophotometry. Copper content is expressed as �g of copper per gramof wet tissue.Results: Copper content was reduced in the corpus striatum (45%; P � 0.05), and increased in themidbrain (65%; P � 0.05) and hippocampus (116%; P � 0.001) after MPP� administration.EGb761 pretreatment of the MPP� group prevented changes in the copper content of the striatum,midbrain, and hippocampus. No significant changes were found in the copper content of thecerebellum and frontal cortex in all treatment groups.Conclusion: We showed that the protective effect of EGb761 against MPP� neurotoxicity may be duein part to the regulation of copper homeostasis in the brain. © 2009 Elsevier Inc. All rights reserved.

eywords: Copper content; 1-Methyl-4-phenylpyridinium; Animal model of Parkinson’s disease; Ginkgo biloba extract;

Nutrition 25 (2009) 482–485www.nutritionjrnl.com

Striatum

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ntroduction

Ginkgo biloba extracts are one of the most popular sup-lements used in herbal medicine. EGb761 is a patentedxtract obtained from leaves of the G. biloba tree [1].Gb761 contains two major active compounds, flavonoids

24%) and terpenoids (6%; known as ginkgolides A, B, C,, J, and bilobalide) [1], of low molecular weight that

ermit their penetration of the blood–brain barrier. Theyay therefore exhibit a broad spectrum of pharmacological

ctions in the central nervous system [2].

* Corresponding author. Tel.: (� 52-55)-5424-0808; fax: (� 52-55)-5424-808.

rE-mail address: prcastane@hotmail.com (P. Rojas).

899-9007/09/$ – see front matter © 2009 Elsevier Inc. All rights reserved.oi:10.1016/j.nut.2008.10.013

EGb761 is a potent antioxidant and free radical scaven-er [2] used clinically to treat dementia, and vaso-occlusivend cochleovestibular disorders [3]. We previously reportedhat EGb761 protects against nigrostriatal dopaminer-ic neurotoxicity induced by 1-methyl-4-phenyl-1,2,3,-tetrahydropiridine (MPTP), an animal model of Par-inson’s disease [4]. We also found that EGb761 pre-reatment blocks the neurotoxic action of 1-methyl-4-henylpyridinium (MPP�; MPTP’s active metabolite) byeducing oxidative stress, enhancing dopamine levels,nd regulating monoamine oxidase activity [5,6].

Copper is an essential element in the development andunctioning of the nervous system [7]. Copper is requiredor the catalytic activity of enzymes that play an essential

ole in neurobiology and disease, including, cytochrome c

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xidase for electron transport in the mitochondrial respi-atory chain, copper/zinc superoxide dismutase for anti-xidant defense to prevent oxidative stress, dopamine-hydroxylase for catecholamine synthesis, and cerulo-lasmin for brain iron homeostasis [7]. Copper levels areltered in Parkinson’s disease. The consequences of cop-er deficiency can be dramatic in that the reduced coppervailability can contribute to several pathologic and det-imental changes, mediated by oxidative stress [7].

It has been reported that animals treated with diethyldi-hiocarbamate, a copper-chelating agent, are more suscep-ible to the oxidative damage and neuronal death caused by

PTP [8]. For this reason, copper deficiency could lead todisadvantage for neurons in their defense against toxic

amage. We investigated whether the protective effect ofGb761 in MPP� neurotoxicity is related to the regulationf copper in the brain.

aterials and methods

Experiments were conducted on male C-57BL/6 miceHarlan, Mexico, D.F., Mexico) at 11 to 13 wk of age. Eachreatment group included six to eight mice (25–30 g). An-mals were maintained in standard conditions in accordanceith the National Institutes of Health (Bethesda, MD, USA)uide for the Care and Use of Laboratory Animals and theational Institutes of Health of Mexico (NOM-062-ZOO-999; Mexico, D.F., Mexico).

Mice in group I were subjected to treatment with salineolution (intraperitoneal [IP]) plus saline solution (intrace-ebroventricular [ICV]); those in group II to EGb761 (IP)lus saline solution (ICV); those in group III to salineolution (IP) plus MPP� (ICV); and those in group IV toGb761 (IP) plus MPP� (ICV).

Animals in groups I and III received normal saline so-ution (IP) and those in groups II and IV received EGb76110 mg/kg, IP) daily for 17 d. This dose of EGb761 has beeneported by our group to produce protection in acute MPP�

eurotoxicity [5]. After pretreatment, animals in groups IIInd IV were anesthetized and given 3 �L of a solutionontaining 18 �g of MPP� (0.72 mg/kg), injected into theight lateral ventricle (ICV) as described previously [5]. Wereviously reported that this acute dose of MPP� producesamage to striatal dopaminergic neurons [5]. Mice fromroups I and II, injected with saline solution (ICV), serveds controls. Mice were sacrificed by cervical dislocation at4 h after MPP� administration, as previously reported [5].he striatum, midbrain, hippocampus, frontal cortex, anderebellum were removed and their copper content waseasured. Brain samples were added to 500 �L of HNO3

Suprapur, Merck) and then digested in a shaking water batht 60°C for 30 min. After digestion, copper content waseasured in a Perkin-Elmer 3110 atomic absorption spec-

rophotometer equipped with an HGA-600 graphite furnace

nd an AS-60 autosampler, as previously described [9]. All B

amples were run in triplicate. Results were expressed as �gf copper per gram of wet tissue. Data were analyzed usingne-way analysis of variance followed by Duncan’s postoc test.

esults

The MPP� depleted 45% of the striatal copper (P �.05) compared with the control (saline � saline) groupFig. 1A). The midbrain and hippocampus (Fig. 1B,C)howed increases of 65% (P � 0.05) and 116% (P �.001), respectively, of copper content after MPP� neuro-oxicity compared with the control group (saline � saline).here were no changes in the frontal cortex and cerebellum

Fig. 1D,1E). EGb761 pretreatment in the groups adminis-ered MPP� prevented changes (decrease or increase) in theopper levels of the striatum, midbrain, and hippocampushen compared with their respective saline � MPP�

roups. No changes were observed in the cerebellum androntal cortex when the EGb761 � MPP� group was com-ared with the saline � MPP� group.

iscussion

EGb761 is a well-known antioxidant that prevents oxi-ative stress in MPTP/MPP� neurotoxicity [4,5]. EGb761upplements enhance dopamine levels, tyrosine hydroxy-ase activity (dopamine metabolism–related marker), andegulate monoamine oxidase activity (enzyme that metabo-izes dopamine) during MPP� neurotoxicity [5,6]. EGb761s also a potent free radical scavenger and has superoxideismutase activity [10] that reduces oxidative stress.

The brain is particularly susceptible to a deficit or anxcess of copper because it is extremely sensitive to oxida-ive stress as a consequence of a variety of factors. Copperas a role in oxygen metabolism both in facilitating electronransfer in oxidative phosphorylation (cytochrome c oxi-ase) and in regulating the levels of superoxide radical andydrogen peroxide. There is evidence that copper partici-ates in the control of free radical overproduction as part ofhe prosthetic group of important enzymes such as super-xide dismutase.

Copper depletion was marked in the striatum after MPP�

eurotoxicity as previously reported by us [11], but copperevel prevented changes in animals pretreated with EGb761n the MPP� group, suggesting an involvement of this trace

etal in the neuroprotection of EGb761. However, MPP�

ncreased copper content in the midbrain and hippocampus,ut pretreatment with EGb761 in the MPP� group pre-ented this increase in both regions. The effect of EGb761n copper content in different brain regions in MPP�

eurotoxicity demonstrates the ability of EGb761 to reg-late the homeostasis of copper content in the brain.

ecause this metal is localized in synaptic terminals [12],

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484 P. Rojas et al. / Nutrition 25 (2009) 482–485

t and/or EGb761 could influence the uptake and efflux ofopamine from synaptic terminals. Copper homeostasisn mammals requires that its levels and distribution beppropriately balanced at the cellular, tissue, and sys-emic levels. Cells have evolved mechanisms to balancelobal copper content and to distribute copper to differentubcellular compartments.

On the other hand, metallothionein is an intracellularrotein with a high affinity for copper and is a free radicalcavenger that prevents oxidative stress. We previouslyemonstrated that EGb761 regulated metallothionein I � IIn MPP� neurotoxicity as a protective action [13]. It haseen reported that MPTP neurotoxicity is enhanced by ad-

ig. 1. Copper content in different brain regions at 24 h after a single intraceith EGb761 (10 mg/kg) daily for 17 d: (A) striatum, (B) midbrain, (C) hiroup; values are means � SEMs. Data were analyzed using one-way anaersus saline � saline group. Cu, copper; MPP�, 1-methyl-4-phenylpyrid

inistration of diethyldithiocarbamate, a copper chelator d

8], suggesting that decreased availability of this metalromotes the deleterious effects of MPTP. EGb761 couldegulate the levels of copper by activating the oxidativetress protectors with high affinity for this metal.

onclusion

The protective effect of EGb761 against MPP� neuro-oxicity may be due in part to the regulation of copperomeostasis in the brain. Further studies are needed tovaluate the protective effect of EGb761 in Parkinson’s

entricular administration of MPP� (0.72 mg/kg) to mice previously treatedmpus, (D) frontal cortex, (E) cerebellum. Six to eight mice were used perf variance followed by Duncan’s post hoc test. *P � 0.05, ***P � 0.001

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