Neurochemistry International 57 (2010) 637–646

2-Phenylethylamine, a constituent of chocolate and wine, causes mitochondrialcomplex-I inhibition, generation of hydroxyl radicals and depletion of striatalbiogenic amines leading to psycho-motor dysfunctions in Balb/c mice

T. Sengupta, K.P. Mohanakumar *

Laboratory of Clinical and Experimental Neurosciences, Division of Cell Biology and Physiology, Indian Institute of Chemical Biology (CSIR, Govt. of India), 4, Raja S C Mullick Road,

Jadavpur, Kolkata 700 032, India

A R T I C L E I N F O

Article history:

Received 25 February 2010

Received in revised form 29 June 2010

Accepted 24 July 2010

Available online 4 August 2010

Keywords:

Oxidative stress

NADH-unbiquinone oxidoreductase

Forced swim test

Elevated plus maze

Anxiety

Depression

Parkinson’s disease

Endogenous amines contained in food

Chocolate

Wine

A B S T R A C T

Behavioral and neurochemical effects of chronic administration of high doses of 2-phenylethylamine

(PEA; 25–75 mg/kg, i.p. for up to 7 days) have been investigated in Balb/c mice. Depression and anxiety,

as demonstrated respectively by increased floating time in forced swim test, and reduction in number of

entries and the time spent in the open arms in an elevated plus maze were observed in these animals.

General motor disabilities in terms of akinesia, catalepsy and decreased swimming ability were also

observed in these animals. Acute and sub-acute administration of PEA caused significant, dose-

dependent depletion of striatal dopamine, and its metabolites levels. PEA caused dose-dependent

generation of hydroxyl radicals in vitro in Fenton’s reaction in test tubes, in isolated mitochondrial

fraction, and in vivo in the striatum of mice. A significant inhibition of NADH-ubiquinone oxidoreductase

(complex-I; EC: 1.6.5.3) activity suggests the inhibition in oxidative phosphorylation in the

mitochondria resulting in hydroxyl radical generation. Nissl staining and TH immnunohistochemistry

in brain sections failed to show any morphological aberrations in dopaminergic neurons or nerve

terminals. Long-term over-consumption of PEA containing food items could be a neurological risk factor

having significant pathological relevance to disease conditions such as depression or motor dysfunction.

However, per-oral administration of higher doses of PEA (75–125 mg/kg; 7 days) failed to cause such

overt neurochemical effects in rats, which suggested safe consumption of food items rich in this trace

amine by normal population.

� 2010 Elsevier Ltd. All rights reserved.

Contents lists available at ScienceDirect

Neurochemistry International

journa l homepage: www.e lsev ier .com/ locate /neuint

1. Introduction

2-Phenylethylamine (PEA) is an endogenous trace amine,identified in the brain of several mammalian species includinghuman (Durden et al., 1973; Philips et al., 1978; Reynolds et al.,1978). PEA has also been detected in several food items such as cocoaproducts (Hurst and Toomey, 1981; Ziegleder et al., 1992; Granvoglet al., 2006), cheese (Bonetta et al., 2008) and wines (Garcia et al.,2006; Moreno-Arribas and Polo, 2008; Simo et al., 2008). While thephysiological function of PEA has never been fully established(Berry, 2004), several studies in the past have focused attention onits amphetamine like action (O’Reilly and Davis, 1994; Premontet al., 2001; Branchek and Blackburn, 2003). However, the discoveryof G protein-coupled receptors for PEA in rodent and human tissues

* Corresponding author. Tel.: +91 33 2413 3223; fax: +91 33 2473 5197/2472 3967.

E-mail addresses: mohankumar@iicb.res.in, kpmohanakumar@yahoo.com

(K.P. Mohanakumar).

0197-0186/$ – see front matter � 2010 Elsevier Ltd. All rights reserved.

doi:10.1016/j.neuint.2010.07.013

(Borowsky et al., 2001) generated great deal of interest on thephysiological and pathological role of this trace amine.

Zhou et al. (1997) have demonstrated negative correlation withthe concentration of PEA in cerebrospinal fluid of Parkinson’sdisease (PD) to that of Hoehn and Yahr stage of the disease. Adecreased level of plasma PEA in PD patients has been linked tohigh platelet monoamine oxidase-B (MAO-B) activity (Zhou et al.,2001), a finding that supported high levels of this amine inpostmortem brains of PD patients on prolonged L-deprenyltherapy (Reynolds et al., 1978). It has been shown that destructionof dopaminergic neurons of the substantia nigra (SN) reducedlevels of PEA and dopamine (DA) in the striatum (Greenshaw et al.,1986). This was shown to be specific for dopaminergic neurons,since lesioning serotoninergic neurons of raphe did not cause anychange in brain PEA level (Greenshaw et al., 1986). These clinicalreports and findings in experimental animal suggested thecomplicity of this amine in the nigrostriatal neuronal death, andparkinsonism.

High doses of PEA induce behavioral responses in rodentssimilar to those observed following amphetamine administration

T. Sengupta, K.P. Mohanakumar / Neurochemistry International 57 (2010) 637–646638

(Moja et al., 1976; Dourish, 1982; Ortmann et al., 1984; Lapin,1996). Amphetamine and its derivatives are known to causeincreased production of hydroxyl radical (�OH) in the brain (Huanget al., 1997; Wan et al., 2000a) resulting in acute striatal DA andserotonin depletion (Kita et al., 1999; Shankaran et al., 1999). It hasalso been shown to induce neurodegeneration in animals (Wanet al., 2000b; Jeng et al., 2006). In a solitary study, PEA itself hasbeen shown to generate �OH in tobacco suspension culture(Kawano et al., 2000). At a time when acute amphetamine-likeneurochemical and behavioral effects of PEA have been demon-strated and reviewed in the literature (Baker et al., 1987; Patersonet al., 1990; Sato et al., 1997), no long-term effects of this biogenicamine has ever been attempted except for demonstration ofhypersensitivity to DA flux in the striatum following PEAwithdrawal (Kuroki et al., 1990). Since PEA is a major constituentof several food items routinely consumed by humans, we thoughtit important to assess effects, if any, of long-term administration ofPEA on striatal and nigral morphology, biogenic amine levels in thebrain that are related to motor activity, anxiety and depression.Mitochondrial electron transport chain function and oxidativestress in the behavioral and neurochemical abnormalities inducedby PEA are also investigated. Since intraperitoneal injection of PEAcaused significant alterations in the neurotransmitters investigat-ed, we thought it prudent to check whether similar effects are alsoconceivable following per oral administration of PEA, which couldbe relevant to real life conditions.

2. Materials and methods

2.1. Animals

Adult male Balb/c mice weighing 20–25 g and male adult Sprague–Dawley rats

(250 g; for the mitochondria preparation) were obtained from the animal house of

the Institute and housed under standard conditions of temperature (22 � 1 8C),

humidity (60 � 5%) and illumination (12-h light–dark cycle). They were provided with

food and water ad libitum. The experimental protocol met the National Guidelines

(CPCSCEA) on the ‘Proper Care and Use of Animals in Laboratory Research’ (Indian

National Science Academy, 2000) and was approved by the Animal Ethics Committee of

the Institute. Proper care was taken to minimize the number of animals used for

generating statistically significant data.

2.2. Chemicals and consumables

PEA (99% purity) and acetonitrile were obtained from SISCO Research

Laboratories (Mumbai, India). DA, homovanillic acid (HVA), 3,4-dihydroxypheny-

lacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA), 5-hydroxytryptamine

(5-HT), norepinephrine (NE), ethylenediaminetetraacetic acid disodium salt

(EDTA), 3,3-diaminobenzidine, paraformaldehyde, sodium salicylate, ferrous

citrate, citric acid, heptane sulfonic acid, 2,3- and 2,5-dihydroxybenzoic acid

(DHBA), NADH, coenzyme Q0 (2,3-dimethoxy-5-methyl-1,4-benzoquinone) and

rotenone were purchased from Sigma–Aldrich Co., St. Louis, MO, USA. Rabbit

tyrosine hydroxylase (TH) polyclonal antibody and horseradish peroxidase (HRP)

conjugated goat anti-rabbit antibodies were obtained from Chemicon International,

Temecula, USA. Chloral hydrate was obtained from Fluka, Germany. Other reagents

and chemicals of analytical grade were procured from E. Merck (India) Ltd., SRL

(Mumbai, India) and Glaxo (Mumbai, India).

2.3. Experimental design

For studying the effects of prolonged administration of PEA, the animals were

divided into four groups, having 10 animals each, except group IV, which contained 8

animals. PEA was dissolved in 5% ethanol in saline and administered (i.p.) once daily

for 7 days at doses of 25 and 50 mg/kg in groups I and II, and 3 days a dose of 75 mg/kg

in group III. Group IV animals received the vehicle for 7 days. One day after the last PEA

injection 6 animals each from each group (group IV, n = 4) were sacrificed and brain

neurotransmitter levels were analyzed. Three behavioral parameters viz. akinesia,

catalepsy and swim score were checked 4 h before sacrifice. For the forced swim and

plus maze tests, 4 animals each from the groups were used. The forced swim and the

plus maze test scores were noted 24 h after the last dose of PEA. These animals were

used for histological studies. Behavioral assessments were made by two independent

investigators who were blind to treatments.

In studies involving per-oral administration of PEA, four groups of mice (n = 6–8)

were given respectively vehicle, 75, 100 and 125 mg/kg doses of PEA by gavation

once daily for 7 days. They were sacrificed on day 8, 24 h following last dose of PEA.

Striatal dopamine and serotonin levels were evaluated.

For studying acute effect of PEA on brain neurotransmitters and in vivo

generation of �OH four groups consisting of 9–11 animals each were used. A fifth

group containing 5 animals exclusively used for �OH generation studies received

100 mg/kg PEA. Groups I–III animals received single injection of 25, 50 and 75 mg/

kg (i.p.) doses of PEA respectively, and the fourth group (control; n = 9) received 5%

ethanol in saline. Animals from I–IV groups (control = 4; treated = 6) were sacrificed

30 min after the injection, and brain neurotransmitter levels were analyzed. For the

estimation of �OH generation in vivo, the animals injected with PEA (25–100 mg/kg,

i.p.; n = 5) were administered after 30 min with sodium salicylate (250 mg/kg, i.p.).

They were sacrificed 30 min following salicylate, and analyzed for striatal 2,3-DHBA

content.

2.4. Analysis of behavioral parameters

2.4.1. Akinesia

Akinesia was measured by noting the latency in seconds (s) of animals to move

all the four limbs. The test was terminated if the latency exceeded 180 s (Mitra et al.,

1992). Each animal was initially acclimatized for 5 min on an elevated wooden

platform (40 cm � 40 cm � 30 cm), and thereafter the latency was examined. The

exercise was repeated five times for each animal.

2.4.2. Catalepsy

Catalepsy is defined as the inability of an animal to correct an externally imposed

posture. Catalepsy was measured by placing an animal on a flat horizontal surface

with both hind limbs placed on a 3 cm high square wooden block and noting the

latency in seconds to move both the limbs from the block to the horizontal surface

(Weihmuller et al., 1989; Mitra et al., 1992).

2.4.3. Swim test

Swimming ability test (Haobam et al., 2005) was carried out in tubs

(40 cm � 25 cm � 16 cm) with 12 cm high water maintained at 27 � 2 8C. They

were placed in water and the swimming ability for a period of 10 min was scored every

min as follows: 3—continuous swimming, 2—swimming with occasional floating, 1—

more floating with occasional swimming with hind limbs, and 0—hind part sinks with

only the head floating.

2.4.4. Forced swim test

Forced swim test was intended to obtain an estimate of animal’s despair in a

difficult circumstance (Kulkarni and Dhir, 2007). The mice were individually made

to swim in a glass cylinder (25 cm height; 12 cm diameter), containing 12 cm

column of water (27 � 2 8C). Two minutes acclimatization time was given, after which

for a period of 6 min the total immobility time was noted. Animal was considered

immobile when it remained floating passively in water in a slightly hunched position

with its nose above the surface of water.

2.4.5. Elevated plus maze

Elevated plus maze test was conducted on a wooden maze elevated 60 cm from

the floor, having two open or uncovered arms, 50 cm � 10 cm, crossed at right

angles by two arms of the same dimensions enclosed by 50 cm high walls (closed

arms). One centimeter high edge surrounded the open arms to prevent the animals

from falling. Animal was placed at the center of the arms, facing the open arm, and

allowed to explore the plus maze for 5 min. The number of entries and the time

spent in each arm during this period was noted. The tendency to stay in the closed

arms of the maze is enhanced by anxiogenics, but anxiolytics promotes greater

exploration of the open arms (Hogg, 1996).

2.4.6. PEA on in vitro generation of �OH

Sodium salicylate can trap �OH generated to form its hydroxylation products of

2,3- and 2,5-DHBA providing a direct index of �OH generation. An in vitro system

containing ferrous citrate complex for free radical generation was tested for PEA

effect on �OH production (Mohanakumar et al., 1994). The sample and control tubes

were respectively kept under incandescent light or on ice and in the dark, at room

temperature for 20 min. One milliliter reaction mixture contained 210 nmol ferrous

citrate, 1 mM sodium salicylate and 1–100 mM PEA. The mixture was acidified

(0.1 M HClO4, 0.01% EDTA) and 10 ml samples were injected into HPLC for the

estimation of 2,3- and 2,5-DHBA.

2.4.7. PEA on ex vivo generation of �OH

PEA on ex vivo generation of �OH adducts of salicylate was tested in

mitochondrial P2 fraction, prepared from cerebral cortex of normal rats (n = 3).

Cortices were homogenized in cold 0.32 M sucrose in 10 mM potassium phosphate

buffer (pH 7.2) with a glass-Teflon homogenizer. The pellet obtained after

centrifugation at 10,000 � g for 10 min at 4 8C was discarded. The pellet obtained

from the resulting supernatant centrifuged at 20,000 � g for 30 min at 4 8C was re-

suspended in cold 1:1, 50 mM Tris:10 mM potassium phosphate buffer (pH 7.2).

This was centrifuged at 20,000 � g for 30 min at 4 8C. The resulting pellet was re-

suspended in 10 mM potassium phosphate buffer (pH 7.2), briefly vortexed for

uniform dispersion and was incubated with PEA (25–100 mM) and salicylic acid

[(Fig._1)TD$FIG]

Fig. 1. Effect of long-term administration of PEA on motor activity in mice: akinesia

(A) and catalepsy (B) were measured at 20 h, and swim-test (C) was conducted

following 21 h following the last dose of the drug. The animals receiving the highest

dose of PEA showed significantly more latency to move all four limbs as compared

to the lowest dose (A), or to correct an externally imposed posture as compared to

the lower two doses (B). Swim score was recorded on the basis of a performance

intensity scale of 0–3 for 10 min (1–11 min; C). The data represented are

mean � SEM. *p � 0.05 as compared to the control group, yp � 0.05 as compared to

the lower dose treated group, n = treated 6; control 4.

T. Sengupta, K.P. Mohanakumar / Neurochemistry International 57 (2010) 637–646 639

(3.3 mM) in 1 ml aliquots. The reaction was stopped with ice-cold 0.1 M HClO4

containing 0.01% EDTA and centrifuged at 10,000 � g for 5 min. The supernatant

was directly injected into the HPLC for the estimation of 2,3- and 2,5-DHBA

(Thomas et al., 2008). The readings were normalized to total protein content in the

P2 fraction, measured by the method of Lowry et al. (1951).

2.4.8. PEA on in vivo formation of �OH

Effect of PEA on in vivo generation of �OH adducts: Mice were injected with PEA

and after 30 min 250 mg/kg (i.p.) dose of sodium salicylate was administered.

Animals were sacrificed 30 min later and striatal level of 2,3-DHBA, the more

reliable indicator of �OH generation in vivo (Halliwell et al., 1991), was analyzed

using HPLC (Thomas et al., 2000). The control animals received vehicle and

salicylate.

Analysis of biogenic amines were carried out employing HPLC equipped with an

electrochemical detector (Muralikrishnan and Mohanakumar, 1998). Animals were

sacrificed 30 min following the acute or 24 h following sub-acute administration of

PEA by cervical dislocation. The striata were quickly dissected out, weighed and

sonicated in chilled HClO4 (0.1 M) containing 0.05% EDTA. The homogenates were

kept on ice for 10 min, centrifuged at 10,000 � g for 5 min and the resulting

supernatants were injected into the HPLC system consisting of an isocratic pump

(Bioanalytical Systems, West Lafayette, USA), an amperometric detector (Epsilon,

Bioanalytical Systems) and C18, ion pair, analytical column (4.6 mm � 250 mm,

Ultrasphere IP, Beckman, USA), with a particle size of 5 mm and pore size of 80 A.

The mobile phase contained 8.65 mM heptane sulfonic acid, 0.27 mM EDTA, 13%

acetonitrile (HPLC grade), 0.45% triethylamine and 0.25% phosphoric acid (v/v). The

flow rate was 0.7 ml/min and the working electrode was kept at 0.74 V. The values

were calculated against standards containing pmol levels of the biogenic amines

and their metabolites. The HPLC conditions employed for the analysis of biogenic

amines were the same used for the estimation of �OH adducts of salicylate, 2,3- and

2,5-DHBA.

2.4.9. Mitochondrial complex-I assay

Spectrophotometric measurement of mitochondrial NADH-ubiquinone oxido-

reductase (complex-I) activity was carried out at 340 nm for 3 min at 37 8C in a

solution containing 90–100 mg protein of rat brain mitochondrial P2 fraction (see

above), 35 mM potassium phosphate buffer (pH 7.2), 2.65 mM NaN3, 1 mM EDTA,

5 mM MgCl2, 200 mM NADH and 100 mM coenzyme Q0 (Pandey et al., 2008). The

assay was carried out both in the presence and absence of 5 mM rotenone to obtain

the rotenone sensitive complex-I activity, which was expressed as nmol NADH

oxidized/min/mg protein (e340 = 6.23 � 10�3 M). In order to check the effect of PEA

on complex-I activity, mitochondrial P2 fraction was pre-incubated with different

concentrations of PEA (1–100 mM) for 10 min at 37 8C.

2.4.10. Brain histology and histochemistry

In order to carry out the histological analyses on brain sections TH

immunohistochemical reactions and Nissl staining were carried out as reported

earlier (Pandey et al., 2008). Following transcardial perfusion in anesthetized mice

with 20 ml of cold 100 mmol/l potassium phosphate buffer, pH 7.4, and 20 ml of 4%

(w/v) paraformaldehyde, brains were fixed overnight in the same fixative and

cryoprotected in 30% (w/v) sucrose. Twenty microns thick sections passing through

the striatum and substantia nigra were taken using a cryotome. The sections were

collected on gelatin-coated slides for cresyl violet staining. For immunostaining,

free-floating sections in phosphate buffered saline (PBS) were incubated with 1%

H2O2 for 5 min and blocked using 8% BSA and 0.02% Triton X-100 in PBS. The

sections were subsequently incubated with primary antibody for 16 h at 4 8C (anti-

rabbit TH polyclonal 1:1000). After washing, HRP-conjugated secondary antibody

(goat anti-rabbit IgG 1:300) was added (2 h), washed and incubated with 3,3-

diaminobenzidine for 5 min for color development. After mounting, the sections

were viewed under microscope and photographed. For Nissl staining, sections were

taken on slides, dried and dehydrated in decreasing concentrations of ethanol (70–

0%) for 1 min each followed by incubation in cresyl violet stain for 1.5 min. The

sections were dehydrated in increasing concentrations of ethanol (30–100%) for

1 min each, cleared in xylene, mounted in DPX mountant, observed under

microscope and photographed.

2.5. Statistics

The data were computed for significance employing Student’s t-test for

biochemical parameters. For the nonparametric behavioral assessments, one-

way ANOVA followed by Dunnet test was performed.

3. Results

3.1. General behavior

Decrease in body weight on the 4th day following 75 mg/kgdose of PEA for 3 days was statistically insignificant (control:26.5 � 1.9 g; treated: 23.8 � 1.7 g). Acutely PEA treated (25–75 mg/

kg) animals exhibited aggressive behavior and hyperactivity within5 min (Data not shown) that lasted for 40 min, after which theybecame akinetic. Animals treated with 25 or 50 mg/kg PEA for 7 dayssurvived, whereas animals that received the highest doses (75 mg/kg,i.p. or 125 mg/kg by gavation) had high mortality rate beyond 4 days.Long-term administration of PEA caused the animals to be lethargic asobserved in diminished exploratory activity, and became markedlyhypokinetic.

3.2. Motor behaviors

Akinesia was measured 20 h after the last dose of PEA. Animalsthat received the higher doses of PEA showed significantly morelatency to move all four limbs as compared to the lowest dose(Fig. 1A). Sub-acute PEA administration made the animalscataleptic as observed by an increase in the latency to correctan externally imposed posture, which was dose-dependent andsignificantly differed amongst the doses administered (Fig. 1B). Allthe animals treated with PEA displayed significantly poorerswimming ability as compared to the control animals. The swimscores at the 1st min were 3.00, 3.00, 2.50 and 2.25, whereas at the11th min these were 2.00, 1.00, 1.00 and 0.00 for 0, 25, 50 and75 mg/kg doses respectively. This effect was dose-dependentduring the initial 5 min period, comparable between the lower twodoses for the next 3 min, and was significantly lesser for theanimals that were receiving 75 mg/kg dose of PEA for 3 days(Fig. 1C).

3.3. Psychometric assessment

In the forced swim test (which demonstrates animal despairunlike the swim ability test, where general motor activity isrevealed), a significant increase in the duration of immobility wasobserved for the animals that received the two higher doses of PEA

[(Fig._2)TD$FIG]

Fig. 2. Long-term administration of PEA causes desperation-induced immobility in

mice: effect of long-term administration of PEA on immobility caused by forced

swim was evaluated 24 h after the last dose of the drug. After an initial

acclimatization for 2 min, the total immobility time in min was noted for a

period of 6 min. Animals that received the higher doses of PEA (50 and 75 mg/kg)

exhibited significantly greater immobility time. The data represented are as

mean � SEM. *p � 0.05 as compared to the control group, n = 4.

[(Fig._4)TD$FIG]

Fig. 4. Effect of PEA on striatal dopamine and metabolites: animals treated with

different doses of PEA acutely (A–C) or long-term (D–F) were sacrificed respectively

30 min or 24 h following the administration of the drug. Since there appeared no

significant variation in the levels of the neurochemical parameters studied between

the control groups, these data were pooled (n = 8). Striata were dissected out and

the supernatants resulting from homogenization in HClO4/EDTA were assayed

employing a sensitive HPLC-electrochemical procedure for the determination of

dopamine (DA) and its metabolites 3,4-dihydroxyplehanyl acetic acid (DOPAC) and

homovanillic acid (HVA). The data are represented as mean � SEM. *p � 0.05 as

compared to the control group, yp � 0.05 as compared to the lower dose administered

group and @p � 0.05 as compared to the 50 mg/kg dose treated group, n = 6.

T. Sengupta, K.P. Mohanakumar / Neurochemistry International 57 (2010) 637–646640

(Fig. 2). In the elevated plus maze test, the two lower doses did notcause any significant change in the number of entries into the openor closed arms, or in the time spent in the open arm (data notprovided). However, administration of the highest dose of PEA for 3days caused the animals to spend significantly lesser time the openarm (Fig. 3A), and lesser number of entries into the open arm(Fig. 3C). The number of entries to the closed arm was notstatistically different from the controls (Fig. 3B). The total numberof entries into both closed and open arms was significantly less inanimals receiving the highest dose of PEA (Fig. 3D).

3.4. Acute and sub-chronic effects of PEA on striatal biogenic amine

metabolism

Acute administration of PEA at different doses led to asignificant, dose-dependent loss in striatal DA (Fig. 4A) and DOPAC(Fig. 4B), but a dose-dependent increase in the level of HVA(Fig. 4C). Prolonged administration of PEA at the two higher does,but not the lowest dose in mice caused a significant decrease in thelevels of this catecholamine neurotransmitter (Fig. 4D). DOPAC(Fig. 4E) and HVA (Fig. 4F) levels were significantly lowered for allthe doses of PEA administered. The highest dose effects on DA andDOPAC levels were found to be significantly different from thelower two doses (Fig. 4D and E). The levels of 5-HT and 5-HIAA inthe striatum showed significant increase in animals that received asingle dose of 75 mg/kg PEA, but remained unchanged for the

[(Fig._3)TD$FIG]

Fig. 3. Exploratory activity of mice in an elevated plus maze: the animals were assessed fo

allowed to explore both open and closed arms of the maze for a period of 5 min, and the

arm (C) and the number of entries in both the arms (D) are depicted. The data provide

lower two doses (Fig. 5A and B). On the other hand, levels of 5-HTand 5-HIAA were found to be significantly decreased dose-dependently in animals administered PEA for long periods(Fig. 5C and D). The highest dose effect on 5-HT was statisticallydifferent from both the lower doses (Fig. 5C).

r exploratory behavior 24 h following the last dose of the drug (75 mg/kg). Mice were

time spent on the open arm (A), the number of entries into the closed arm (B), open

d are mean � SEM. *p � 0.05 as compared to the control group, n = 4.

[(Fig._5)TD$FIG]

Fig. 5. Effect of PEA on striatal serotonin and its metabolite: animals treated with

different doses of PEA acutely (A, B) or long-term (C, D) were sacrificed 30 min or

24 h respectively following the administration of the drug. Striata were dissected

out and homogenized in 0.1 N HClO4 containing 0.01% EDTA. The deproteinated

supernatants were assayed employing a sensitive HPLC-electrochemical procedure

for the determination of serotonin (5-HT) and 5-hydroxyindole acetic acid (5-

HIAA). The data are represented as mean � SEM. *p � 0.05 as compared to the control

group, and yp � 0.05 as compared to the 50 mg/kg PEA administered group, n = 6–8.

[(Fig._7)TD$FIG]

Fig. 7. Effects of PEA on striatal norepinephrine content: acute administration of the

drug could deplete the levels of striatal norepinephrine (NE) for all the three doses

of PEA (A). Long-term administration of PEA depleted striatal NE for 25 and 50 mg/

kg doses only (B). The data are mean � SEM. *p � 0.05 as compared to the control

group, n = 6–8.

[(Fig._8)TD$FIG]

T. Sengupta, K.P. Mohanakumar / Neurochemistry International 57 (2010) 637–646 641

The turnover of DA, as deducted by the ratio of the metabolitesto the catecholamine was significantly lower for the two higherdoses of PEA, for both acute and long-term administration (Fig. 6Aand B). Serotonin turnover, deduced as the ratio of 5-HIAA to 5-HTwas not altered following acute treatment of PEA (Fig. 6C), but thehighest dose of the drug caused a significant increase in theindoleamine turnover after sub-acute administration of PEA

[(Fig._6)TD$FIG]

Fig. 6. Effects of PEA on dopamine and serotonin turnover: acute and long-term

administration of PEA on dopamine (A, B) and 5-HT (C, D) turnover were computed

as the ratio of the metabolites levels to the neurotransmitter [DA

turnover = (HVA + DOPAC):DA; 5-HT turnover = 5-HIAA:5-HT]. The data are

represented as mean � SEM. *p � 0.05 as compared to the control group, n = 6–8.

(Fig. 6D). The levels of NE in the striatum following acuteadministration of PEA were decreased significantly for all thethree doses of PEA (Fig. 7A). Long-term administration of PEAcaused a significant diminution of striatal NE levels only for 25 and50 mg/kg doses (Fig. 7B).

Fig. 8. Effect of PEA on in vitro generation of hydroxyl radical (�OH): ferrous citrate

can dose-dependently generate �OH adducts of salicylic acid 2,3- and 2,5-

dihydroxybenzoic acid (DHBA). Ferrous citrate (210 nmol/ml) generated

0.57 � 0.01, 1.13 � 0.04, and 1.69 � 0.04 nmol/ml respectively of 2,5-, 2,3- and total

DHBA per 20 min. PEA (1–100 mM) caused significant dose-dependent increase in

DHBA concentrations. The data represented are mean � SEM, for three independent

experiments, in duplicate. *p � 0.05 as compared to the control group, #p � 0.05 as

compared to 1 mM effect, yp � 0.05 as compared to the 25 mM effect, and @p � 0.05 as

compared to the 50 mM effect.

[(Fig._9)TD$FIG]

Fig. 9. Effect of PEA on �OH generation from isolated mitochondria: rat brain mitochondria generate 2,3- and 2,5-dihydroxybenzoic acid (DHBA) in an ex vivo environment

when incubated with sodium salicylate, by trapping �OH formed. Increasing concentrations of PEA (25–100 mM) caused an increase in the levels of �OH adducts of salicylate

as estimated using HPLC coupled with an electrochemical detector. The data presented are mean � SEM for 3 independent experiments, in duplicate. *p � 0.05 as compared to

the control; yp � 0.05 as compared to the 50 mM PEA treatment effect.

T. Sengupta, K.P. Mohanakumar / Neurochemistry International 57 (2010) 637–646642

3.5. Effects of PEA on the production of hydroxyl radical

The �OH adducts of salicylic acid, 2,3-, and 2,5-DHBA were dose-dependently increased in the test tubes where a Fenton-likereaction was undergoing in presence of PEA at different doses (1–100 mM; Fig. 8A–C). The increase was 2-, 5-, and 12-fold for 2,5-DHBA (Fig. 8A); 1.5-, 3- and 6-fold for 2,3-DHBA (Fig. 8B), and 2-,3.5- and 8-fold for the total DHBA (Fig. 8C). Mitochondria preparedfrom normal rat brain cortices produced 0.28 � 0.02, 0.11 � 0.004and 0.39 � 0.01 pmol/mg protein respectively of 2,3-, 2,5- and totalDHBA (Fig. 9). Treatment of the mitochondria with PEA caused 50 and400% increase in 2,3-DHBA concentrations for the two higher doses(50 and 100 mM), about 200% increase in 2,5-DHBA for all the threedoses tested (25, 50 and 100 mM), and 100 and 400% for total DHBAfor the higher two doses (Fig. 9). Mice administered PEA at fourdifferent doses (25–100 mg/kg, i.p.) caused significant increase in thelevels of 2,3-DHBA, which was not dose-dependent for the lowerthree doses. The increase was 3.2-, 3.6-, 3.4- and 4.5-folds,respectively for 25, 50, 75 and 100 mg/kg doses of PEA (Fig. 10).The highest dose effect was significantly more when compared to theother three doses.

[(Fig._10)TD$FIG]

Fig. 10. Effect of PEA on �OH radical generation in vivo: mice injected with PEA (25–

100 mg/kg, i.p.) were administered after 30 min with 250 mg/kg dose of sodium

salicylate. After 30 min these animals were sacrificed and the striatal levels of 2,3-

dihydroxybenzoic acid (DHBA) were analyzed using HPLC coupled with

electrochemical detector. The data are represented as mean � SEM. *p � 0.05 as

compared to the control group, yp � 0.05 as compared to the 75 mg/kg treated group,

n = 4–8.

3.6. Mitochondrial complex-I activity

Mitochondrial P2 fractions when incubated with PEA (1–100 mM) caused a dose-dependent inhibition of the NADH-ubiquinone oxidoreductase activity. There was 17, 41 and 74%inhibition respectively for 1, 10 and 100 mM PEA (Fig. 11). Thehighest dose-effect was significantly different from the mediumdose (Fig. 11).

3.7. Histology

TH immnunohistochemistry did not reveal any microscopicchanges in dopaminergic neurons following long-term PEA(75 mg/kg) treatment (Fig. 12). The nerve terminal region, striatumshowed distinct striosomes (arrows) and nerve fibers (arrow-heads) in both control (Fig. 12A) and treated (Fig. 12B) brainsections. The perikarya of dopaminergic SN neurons were intactboth in control (Fig. 12C) and PEA treated (Fig. 12D) brains. Distinctneurons with their axons and dendrites (arrowheads) were visiblein both treated and control sections passing through SN. Distinctcell populations were seen in cresyl violet stained sections passingthrough the striatum in both the control (Fig. 13A) and treated(Fig. 13B) animals. Arrows show the striosmes. In the sectionspassing through SN, no difference in the cell morphology could be

[(Fig._11)TD$FIG]

Fig. 11. Effect of PEA on mitochondrial complex-I activity: mitochondrial P2 fraction

was pre-incubated with different concentrations of PEA (1–100 mM) for 10 min at

37 8C, and the rotenone sensitive NADH-ubiquinone oxidoreductase (complex-I)

activity was determined employing a spectrophotometric procedure. Complex-I

activity is expressed as nmol NADH oxidized/min/mg protein (e340 = 6.23� 10�3 M).

The data provided are mean� SEM. *p � 0.05 as compared to the control; yp � 0.05 as

compared to the 10 mM PEA treated set. n = 12.

[(Fig._12)TD$FIG]

Fig. 12. Tyrosine hydroxylase (TH) immnunohistochemistry: coronal sections (20 mm) passing through the striatum (A, B) and substantia nigra (C, D) were cut on a cryostat

and were incubated with primary antibody (anti-rabbit TH polyclonal 1:1000), followed by HRP-conjugated secondary antibody (goat anti-rabbit IgG 1:300). The sections

were washed and incubated with DAB for the color to develop. After mounting, the sections were viewed under microscope and photographed. (A) and (C) are sections from

control animals; (B) and (D) are sections from treated (75 mg/kg, i.p.) animals. No microscopic changes were observable in the neurons from treated sections. Arrows indicate

striosomes and the arrowheads the neuronal fibers (A–D), including axons and dendrites of the nigral dopaminergic neurons in (C) and (D).

T. Sengupta, K.P. Mohanakumar / Neurochemistry International 57 (2010) 637–646 643

discerned either in the PEA treated (Fig. 13D) as compared tocontrol (Fig. 13D).

3.8. Per-oral administration of PEA: effect on striatal biogenic amine

levels

Administration of 75, 100 and 125 mg/kg doses of PEA bygavation once daily for 7 days did not cause any significantalteration in the striatal DA (Fig. 14A) or 5-HT levels (Fig. 14B).

4. Discussion

This is the first time report on behavioral manifestationsindicative of severe motor dysfunction, anxiety or desperation inanimals following sub-acute systemic administration of PEA,caused by depletions in the levels of striatal dopamine andserotonin. We also report here that PEA administered by gavationdoes not produce such neurochemical alterations, indicating thepossibility that the biogenic amine is not absorbed from thegastrointestinal tract.

PEA, a breakdown product of phenylalanine, is present in thenormal brain in trace amounts (0.1–100 ng/g tissue) and has a highturnover rate (in seconds) under normal physiological conditions(Burchett and Hicks, 2006). It is a common constituent of variousfood items; cheese, chocolates and wines. Chocolates contain anaverage of 3.5–8.02 mg/g (Hurst and Toomey, 1981; Pastore et al.,2005), wines especially white wine about 0.1–1.8 mg/ml PEA, andbeers about 0.1–0.42 mg/ml of PEA (Landete et al., 2005, 2007;Ozdestan and Uren, 2009; Tang et al., 2009). It has been estimated

that on an average a chocolate addict consumes 720 g (containing2.5–58 mg of PEA) of chocolate per week (Hetherington andMacDiarmid, 1993), and might elevate PEA concentration in thebrain, especially in individuals with gastric ulcers or withabnormal gastrointestinal physiology and who are on antacids.Indications that chocolate possesses mood-enhancing effects(Rogers and Smit, 2000), and prolonged consumption could resultin addiction (Smit et al., 2004) strongly support the hypothesis thatat least in certain individuals PEA could be absorbed into the bloodstream and cross the blood brain barrier causing these effects.Interestingly chocolate addiction has been suggested to be due tothe presence of various pharmacologically active substances,including PEA (Smit et al., 2004).

Others (Kita et al., 1999; Shankaran et al., 1999) and ourfindings reveal significant increase in the striatal 5-HT levels in thebrain following acute administration of PEA. Conversely, long-termadministration of PEA caused significant, dose-dependent decreaseof striatal 5-HT levels. Our results showing the increasedimmobility in forced swim test, decreases in time spent or numberof entries into the open arm on the elevated plus maze along withsustained decrease in striatal 5-HT level suggest setting in ofdespair in these animals. Therefore, initial euphoria associatedwith chocolate consumption could be related to the immediateincrease in brain 5-HT levels, but low striatal 5-HT contentfollowing prolonged PEA administration explains moods ofdysphoria and depression amongst chocolate addicts. Alterationin PEA and other trace amine function is thought to be involved inthe etiology of a variety of neuropathological conditions, includinghallucinations, schizophrenia, major depression, anxiety states,

[(Fig._13)TD$FIG]

Fig. 13. Nissl staining: coronal sections (20 mm) passing through the striatum (A, B) and substantia nigra (C, D) were cut on a cryostat. Neurons in the striatum and substantia

nigra were stained with cresyl violet following vehicle (A, C) or sub-acute PEA treatment (75 mg/kg, i.p., BD). Arrows in (A) and (B) indicate the striosomes. No morphological

changes were seen in any neurons from the SN region.

T. Sengupta, K.P. Mohanakumar / Neurochemistry International 57 (2010) 637–646644

anorexia, obsessive-compulsive disorders, attention deficit hyper-activity disorder and bipolar disorders (Burchett and Hicks, 2006).

Long-term administration of PEA leads to motor disabilities asmanifested by the increase in latency to akinesia and catalepsy.Swimming is an ability, which remains latent and manifests only ina new environment. A high degree of correlation exists betweenstriatal dopamine levels and score in swim test which is a reliablemethod to assess motor deficits induced by a toxin (Haobam et al.,2005). The decrease in swimming ability and decrease in the totalnumber of entries into the closed and open arms of the plus mazeare also indications of reduced motor activity following long-termadministration of PEA. Therefore, our data clearly demonstrate thaton all fronts chronic administration PEA is detrimental to themotor abilities of the animals, which could be due to significantstriatal DA depletion.

The potent mitochondrial complex-I inhibitory and prooxidantactions of PEA are yet another important and interesting findingthat has never been reported in the literature. There exists acontinuous generation of �OH in the mitochondria (Dykens, 1994;Knaryan et al., 2006; Navneet et al., 2008), and effective methodsare available to detect production of this reactive oxygen species inthe brain (Thomas et al., 2000, 2008). The enhanced production ofthis biologically detrimental molecule within the biological systemcould be resulting from the inhibition of complex-I activity of the

mitochondrial electron transport chain by PEA. Interestingly adiminished NAD-linked mitochondrial O2 consumption due to PEAhas been demonstrated in the past (Gluck and Zeevalk, 2004).Therefore, inhibition of electron transport in the mitochondriacould lead to electron leakage resulting in the generation ofsuperoxide anion, which in turn is converted to the highly toxic�OH. In analogous situations inhibition of mitochondrial complex-Iby rotenone (Sousa et al., 2003; Saravanan et al., 2006), or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (Thomas et al., 2008;Thomas and Mohanakumar, 2004), or 3-nitropropionic acid(Pandey et al., 2008, 2009) have been shown to result in �OHgeneration in the mitochondria. Whereas the in vitro generation of�OH in cell free system suggests its pro-Fenton reactivity topotentiate redox potential of ferrous citrate, and prove the directeffect of PEA rather than its metabolites in this action. PEA’s sucheffects in the isolated mitochondria and �OH production in thestriatum following systemic administration of PEA have directrelevance to its neurotoxic potential. It may be presumed thatcomplex-I inhibition following extremely high doses of PEA (eitherby gavation or by via i.p.) could be high enough to affect cardiacfunctions resulting in death of the animals.

It has been believed that PEA is ‘‘body’s natural amphetamine’’.At a time when amphetamine and its derivatives are known tocause neurotoxicity and neurodegeneration following chronic

[(Fig._14)TD$FIG]

Fig. 14. Effect of long-term per-oral administration of PEA on striatal DA and 5-HT

levels: animals were treated by gavation with 75, 100 and 125 mg/kg doses of PEA

once daily for 7 days. These animals were sacrificed 24 h following the last drug

treatment and striatal dopamine (DA) (A) and serotonin (5-HT) (B) levels were

analyzed as employing HPLC-electrochemistry. n = 6–8.

T. Sengupta, K.P. Mohanakumar / Neurochemistry International 57 (2010) 637–646 645

administration, PEA has never been shown to cause neurotoxicityunder any situation. However, the present study brings to lightcertain similarities in molecular action of PEA with that ofamphetamine. As for example, amphetamine has also been shownto cause production of �OH in rat striatum (Huang et al., 1997; Wanet al., 2000a), mitochondrial electron transport chain dysfunction(Cunha-Oliveira et al., 2006; Mao et al., 2007; Ajjimaporn et al.,2008), and depletion of striatal DA which are all similar to ourresults. A number of neurotoxins such as rotenone, paraquat, 3-nitropropionic acid and 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine have shown to cause significant inhibition of complex-Iactivity and are known to cause striatal dopamine depletion andparkinsonian and huntingtonian like syndromes in animals. PEA-induced inhibition of mitochondrial complex-I activity andincrease in �OH as seen in the present study is comparable tomajor deficits which are well established pathophysiologicallandmarks in a number of neurodegenerative diseases, such asidiopathic PD (Dawson and Dawson, 2003; Mizuno et al., 1989),familial amyotrophic lateral sclerosis (Jung et al., 2002; Rizzardiniet al., 2006), Alzheimer’s (Manczak et al., 2004) and Huntington’sdiseases (Pandey et al., 2009). Interestingly, Sida cordifolia rootextract, one of the four ingredients used in the traditional Indianmedical system, Ayurveda for treating PD, (Nagashayana et al.,2000) contains PEA (Ghosal et al., 1975). Therefore the presentstudy highlights the need for complete biochemical characteriza-tion of traditional medicinal preparations in order to identify thepossible neurotoxic ingredients.

Neither neuronal staining with cresyl violet, nor TH immuno-histochemistry revealed any change in neuronal integrity follow-ing multiple administrations of 75 mg/kg of PEA. These findingssuggest that PEA may not cause any micro-level damage to theneurons, especially to dopaminergic neurons. However, electronmicroscopic assessment is essential to verify ultrastructural

changes, if any, to the neurons or the supporting cells. Traceamines such as PEA are known to have extremely rapid turnoverrates with an endogenous half-life of approximately 30 s (Berry,2004), which may explain why PEA failed to cause overtneurodegeneration either in the cell body (i.e. SN) or terminalregions (i.e. striatum) of dopaminergic neurons.

Acknowledgements

TS is a recipient of junior and senior research fellowships fromthe Council of Scientific & Industrial Research (CSIR), Govt. of India.

References

Ajjimaporn, A., Shavali, S., Ebadi, M., Govitrapong, P., 2008. Zinc rescues dopami-nergic SK-N-SH cell lines from methamphetamine induced toxicity. Brain Res.Bull. 77, 361–366.

Baker, G.B., Coutts, R.T., Rao, T.S., 1987. Neuropharmacological and neurochemicalproperties of N-(2-cyanoethyl)-2-phenylethylamine, a prodrug of 2-phenyleth-ylamine. Br. J. Pharmacol. 92, 243–255.

Berry, M.D., 2004. Mammalian central nervous system trace amines. Pharmacologicamphetamines, physiologic neuromodulators. J. Neurochem. 90, 257–271.

Bonetta, S., Bonetta, S., Carraro, E., Coısson, J.D., Travaglia, F., Arlorio, M., 2008.Detection of biogenic amine producer bacteria in a typical Italian goat cheese. J.Food Prot. 71, 205–209.

Borowsky, B., Adham, N., Jones, K.A., Raddatz, R., Artymyshyn, R., Ogozalek, K.L.,Durkin, M.M., Lakhlani, P.P., Bonini, J.A., Pathirana, S., Boyle, N., Pu, X., Kour-anova, E., Lichtblau, H., Ochoa, F.Y., Branchek, T.A., Gerald, C., 2001. Traceamines: identification of a family of mammalian G protein-coupled receptors.Proc. Natl. Acad. Sci. U.S.A. 98, 8966–8971.

Branchek, T.A., Blackburn, T.P., 2003. Trace amine receptors as targets for noveltherapeutics: legend, myth and fact. Curr. Opin. Pharmacol. 3, 90–97.

Burchett, S.A., Hicks, T.P., 2006. The mysterious trace amines: protean neuromo-dulators of synaptic transmission in mammalian brain. Prog. Neurobiol. 79,223–246.

Cunha-Oliveira, T., Rego, A.C., Cardoso, S.M., Borges, F., Swerdlow, R.H., Macedo,T., de Oliveira, C.R., 2006. Mitochondrial dysfunction and caspase activationin rat cortical neurons treated with cocaine or amphetamine. Brain Res. 1089,44–54.

Dawson, T.M., Dawson, V.L., 2003. Molecular pathways of neurodegeneration inParkinson’s disease. Science 302, 819–822.

Dourish, C.T., 1982. A pharmacological analysis of the hyperactivity syndromeinduced by beta-phenylethylamine in the mouse. Br. J. Pharmacol. 77, 129–139.

Durden, D.A., Philips, S.R., Boulton, A.A., 1973. Identification and distribution ofbeta-phenylethylamine in the rat. Can. J. Biochem. 51, 995–1002.

Dykens, J.A., 1994. Isolated cerebral and cerebellar mitochondria produce freeradicals when exposed to elevated Ca2+ and Na+: implications for neurodegen-eration. J. Neurochem. 63, 584–591.

Garcia, V.N., Saurina, J., Hernandez-Cassou, S., 2006. High-performance liquidchromatographic determination of biogenic amines in wines with an experi-mental design optimization procedure. Anal. Chim. Acta 575, 97–105.

Ghosal, S., Rama, B., Chauhan, P.S., Mehta, R., 1975. Alkaloids of Sida Cordifolia.Phytochemistry 14, 830–832.

Gluck, M.R., Zeevalk, G.D., 2004. Inhibition of brain mitochondrial respiration bydopamine and its metabolites: implications for Parkinson’s disease and cate-cholamine-associated diseases. J. Neurochem. 91, 788–795.

Granvogl, M., Bugan, S., Schieberle, P., 2006. Formation of amines and aldehydesfrom parent amino acids during thermal processing of cocoa and modelsystems: new insights into pathways of the Strecker reaction. J. Agric. FoodChem. 54, 1730–1739.

Greenshaw, A.J., Juorio, A.V., Nguyen, T.V., 1986. Depletion of striatal beta-phenyl-ethylamine following dopamine but not 5-HT denervation. Brain Res. Bull. 17,477–484.

Halliwell, B., Kaur, H., Ingelman-Sundberg, M., 1991. Hydroxylation of salicylate asan assay for hydroxyl radicals: a cautionary note. J. Free Radic. Biol. Med. 10,439–441.

Haobam, R., Sindhu, K.M., Chandra, G., Mohanakumar, K.P., 2005. Swim-test as afunction of motor impairment in MPTP model of Parkinson’s disease: a com-parative study in two mouse strains. Behav. Brain Res. 163, 159–167.

Hetherington, M.M., MacDiarmid, J.I., 1993. ‘‘Chocolate addiction’’: a preliminarystudy of its description and its relationship to problem eating. Appetite 21, 233–246.

Hogg, S., 1996. A review of the validity and variability of the elevated plus-maze asan animal model of anxiety. Pharmacol. Biochem. Behav. 54, 21–30.

Huang, N.K., Wan, F.J., Tseng, C.J., Tung, C.S., 1997. Amphetamine induces hydroxylradical formation in the striatum of rats. Life Sci. 61, 2219–2229.

Hurst, W.J., Toomey, P.B., 1981. High-performance liquid chromatographic deter-mination of four biogenic amines in chocolate. Analyst 106, 394–402.

Jeng, W., Ramkissoon, A., Parman, T., Wells, P.G., 2006. Prostaglandin H synthase-catalyzed bioactivation of amphetamines to free radical intermediates thatcause CNS regional DNA oxidation and nerve terminal degeneration. FASEB J.20, 638–650.

T. Sengupta, K.P. Mohanakumar / Neurochemistry International 57 (2010) 637–646646

Jung, C., Higgins, C.M., Xu, Z., 2002. Mitochondrial electron transport chain complexdysfunction in a transgenic mouse model for amyotrophic lateral sclerosis. J.Neurochem. 83, 535–545.

Kawano, T., Pinontoan, R., Uozumi, N., Morimitsu, Y., Miyake, C., Asada, K., Muto, S.,2000. Phenylethylamine-induced generation of reactive oxygen species andascorbate free radicals in tobacco suspension culture: mechanism for oxidativeburst mediating Ca2+ influx. Plant Cell Physiol. 41, 1259–1266.

Kita, T., Takahashi, M., Kubo, K., Wagner, G.C., Nakashima, T., 1999. Hydroxyl radicalformation following methamphetamine administration to rats. Pharmacol.Toxicol. 85, 133–137.

Knaryan, V.H., Samantaray, S., Varghese, M., Srinivasan, A., Galoyan, A.A., Mohana-kumar, K.P., 2006. Synthetic bovine proline-rich-polypeptides generate hydrox-yl radicals and fail to protect dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurotoxicity inmice. Neuropeptides 40, 291–298.

Kulkarni, S.K., Dhir, A., 2007. Effect of various classes of antidepressants in behav-ioral paradigms of despair. Prog. Neuropsychopharmacol. Biol. Psychiatry 31,1248–1254.

Kuroki, T., Tsutsumi, T., Hirano, M., Matsumoto, T., Tatebayashi, Y., Nishiyama, K.,Uchimura, H., Shiraishi, A., Nakahara, T., Nakamura, K., 1990. Behavioral sensi-tization to beta-phenylethylamine (PEA): enduring modifications of specificdopaminergic neuron systems in the rat. Psychopharmacology 102, 5–10.

Landete, J.M., Ferrer, S., Polo, L., Pardo, I., 2005. Biogenic amines in wines from threeSpanish regions. J. Agric. Food Chem. 53, 1119–1124.

Landete, J.M., Pardo, I., Ferrer, S., 2007. Tyramine and phenylethylamine produc-tion among lactic acid bacteria isolated from wine. Int. J. Food Microbiol. 115,364–368.

Lapin, I.P., 1996. Antagonism by CPP (+/�)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid, of beta-phenylethylamine (PEA)-induced hypermotility inmice of different strains. Pharmacol. Biochem. Behav. 55, 175–178.

Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurementwith the Folin phenol reagent. J. Biol. Chem. 193, 265–275.

Manczak, M., Park, B.S., Jung, Y., Reddy, P.H., 2004. Differential expression ofoxidative phosphorylation genes in patients with Alzheimer’s disease: implica-tions for early mitochondrial dysfunction and oxidative damage. Neuromol.Med. 5, 147–162.

Mao, P., Ardeshiri, A., Jacks, R., Yang, S., Hurn, P.D., Alkayed, N.J., 2007. Mitochondrialmechanism of neuroprotection by CART. Eur. J. Neurosci. 26, 624–632.

Mitra, N., Mohanakumar, K.P., Ganguly, D.K., 1992. Dissociation of serotoninergicand dopaminergic components in acute effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice. Brain Res. Bull. 28, 355–364.

Mizuno, Y., Ohta, S., Tanaka, M., Takamiya, S., Suzuki, K., Sato, T., Oya, H., Ozawa, T.,Kagawa, Y., 1989. Deficiencies in complex I subunits of the respiratory chain inParkinson’s disease. Biochem. Biophys. Res. Commun. 163, 1450–1455.

Mohanakumar, K.P., Bartolomeis, A., Wu, R.M., Yeh, K.J., Sternberger, L.M., Peng, S.Y.,Murphy, D.L., Chiueh, C.C., 1994. Ferrous–citrate complex and nigral degenera-tion: evidence for free-radical formation and lipid peroxidation. Ann. N.Y. Acad.Sci. 738, 392–399.

Moja, E.A., Stoff, D.M., Gillin, J.C., Wyatt, R.J., 1976. Dose–response effects of beta-phenylethylamine on stereotyped behavior in pargyline-pretreated rats. Biol.Psychiatry 11, 731–742.

Moreno-Arribas, M.V., Polo, M.C., 2008. Occurrence of lactic acid bacteria andbiogenic amines in biologically aged wines. Food Microbiol. 25, 875–881.

Muralikrishnan, D., Mohanakumar, K.P., 1998. Neuroprotection by bromocriptineagainst 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicityin mice. FASEB J. 12, 905–912.

Nagashayana, N., Sankarankutty, P., Nampoothiri, M.R., Mohan, P.K., Mohanakumar,K.P., 2000. Association of L-DOPA with recovery following ayurveda medicationin Parkinson’s disease. J. Neurol. Sci. 176, 124–127.

Navneet, A.K., Appukuttan, T.A., Pandey, M., Mohanakumar, K.P., 2008. Taurine failsto protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-inducedstriatal dopamine depletion in mice. Amino Acids 35, 457–461.

O’Reilly, R.L., Davis, B.A., 1994. Phenylethylamine and schizophrenia. Prog. Neu-ropsychopharmacol. Biol. Psychiatry 18, 63–75.

Ortmann, R., Schaub, M., Felner, A., Lauber, J., Christen, P., Waldmeier, P.C., 1984.Phenylethylamine-induced stereotypes in the rat: a behavioral test system forassessment of MAO-B inhibitors. Psychopharmacology 84, 22–27.

Ozdestan, O., Uren, A., 2009. A method for benzoyl chloride derivatization of biogenicamines for high performance liquid chromatography. Talanta 78, 1321–1326.

Pandey, M., Varghese, M., Sindhu, K.M., Sreetama, S., Mohanakumar, K.P., Usha, R.,2008. Mitochondrial NAD+-linked state 3 respiration and complex-I activity arecompromised in the cerebral cortex of 3-nitropropionic acid-induced rat modelof Huntington’s disease. J. Neurochem. 104, 420–434.

Pandey, M., Borah, A., Varghese, M., Barman, P.K., Mohanakumar, K.P., Usha, R.,2009. Striatal dopamine level contributes to hydroxyl radical generation andsubsequent neurodegeneration in the striatum in 3-nitropropionic acid-in-duced Huntington’s disease in rats. Neurochem. Int. 55, 431–437.

Pastore, P., Favaro, G., Badocco, D., Tapparo, A., Cavalli, S., Saccani, G., 2005.Determination of biogenic amines in chocolate by ion chromatographic sepa-ration and pulsed integrated amperometric detection with implemented wave-form at Au disposable electrode. J. Chromatogr. A 1098, 111–115.

Paterson, I.A., Juorio, A.V., Boulton, A.A., 1990. Phenylethylamine: a modulator ofcatecholamine transmission in the mammalian central nervous system? J.Neurochem. 55, 1827–1837.

Philips, S.R., Rozdilsky, B., Boulton, A.A., 1978. Evidence for the presence of m-tyramine, p-tyramine, tryptamine, and phenylethylamine in the rat brain andseveral areas of the human brain. Biol. Psychiatry 13, 51–57.

Premont, R.T., Gainetdinov, R.R., Caron, M.G., 2001. Following the trace of elusiveamines. Proc. Natl. Acad. Sci. U.S.A 98, 9474–9475.

Reynolds, G.P., Riederer, P., Sandler, M., Jellinger, K., Seemann, D., 1978. Amphet-amine and 2-phenylethylamine in post-mortem Parkinsonian brain after(�)deprenyl administration. J. Neural Transm. 43, 271–277.

Rizzardini, M., Lupi, M., Mangolini, A., Babetto, E., Ubezio, P., Cantoni, L., 2006.Neurodegeneration induced by complex I inhibition in a cellular model offamilial amyotrophic lateral sclerosis. Brain Res. Bull. 69, 465–474.

Rogers, P.J., Smit, H.J., 2000. Food craving and food ‘‘addiction’’: a critical review ofthe evidence from a biopsychosocial perspective. Pharmacol. Biochem. Behav.66, 3–14.

Saravanan, K.S., Sindhu, K.M., Senthilkumar, K.S., Mohanakumar, K.P., 2006. L-deprenyl protects against rotenone-induced, oxidative stress-mediated dopa-minergic neurodegeneration in rats. Neurochem. Int. 49, 28–40.

Sato, S., Tamura, A., Kitagawa, S., Koshiro, A., 1997. A kinetic analysis of the effects ofbeta-phenylethylamine on the concentrations of dopamine and its metabolitesin the rat striatum. J. Pharm. Sci. 86, 487–496.

Shankaran, M., Yamamoto, B.K., Gudelsky, G.A., 1999. Involvement of the serotonintransporter in the formation of hydroxyl radicals induced by 3,4-methylene-dioxymethamphetamine. Eur. J. Pharmacol. 385, 103–110.

Simo, C., Moreno-Arribas, M.V., Cifuentes, A., 2008. Ion-trap versus time-of-flightmass spectrometry coupled to capillary electrophoresis to analyze biogenicamines in wine. J. Chromatogr. A 1195, 150–156.

Smit, H.J., Gaffan, E.A., Rogers, P.J., 2004. Methylxanthines are the psycho-pharma-cologically active constituents of chocolate. Psychopharmacology 176, 412–419.

Sousa, S.C., Marciel, E.N., Vercesi, A.E., Castilho, R.F., 2003. Ca2+-induced oxidativestress in brain mitochondria treated with the respiratory chain inhibitor rote-none. FEBS Lett. 543, 179–183.

Tang, T., Shi, T., Qian, K., Li, P., Li, J., Cao, Y., 2009. Determination of biogenic aminesin beer with pre-column derivatization by high performance liquid chroma-tography. J. Chromatogr. B 877, 507–512.

Thomas, B., Mohanakumar, K.P., 2004. Melatonin protects against oxidative stresscaused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in the mouse nigros-tiratum. J. Pineal Res. 36, 25–32.

Thomas, B., Muralikrishnan, D., Mohanakumar, K.P., 2000. In vivo hydroxyl radicalgeneration in the striatum following systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice. Brain Res. 852, 221–224.

Thomas, B., Saravanan, K.S., Mohanakumar, K.P., 2008. In vitro and in vivo evidencesthat antioxidant action contributes to the neuroprotective effects of the neuro-nal nitric oxide synthase and monoamine oxidase-B inhibitor, 7-nitroindazole.Neurochem. Int. 52, 990–1001.

Wan, F.J., Lin, H.C., Lin, Y.S., Tseng, C.J., 2000a. Intra-striatal infusion of D-amphet-amine induces hydroxyl radical formation: inhibition by MK-801 pretreatment.Neuropharmacology 39, 419–426.

Wan, F.J., Lin, H.C., Huang, K.L., Tseng, C.J., Wong, C.S., 2000b. Systemic administra-tion of D-amphetamine induces long-lasting oxidative stress in the rat striatum.Life Sci. 66, 205–212.

Weihmuller, F.B., Hadjiconstantinou, M., Bruno, J.P., 1989. Dissociation betweenbiochemical and behavioral recovery in MPTP-treated mice. Pharmacol. Bio-chem. Behav. 34, 113–117.

Zhou, G., Shoji, H., Yamada, S., Matsuishi, T., 1997. Decreased b-phenylethylaminein CSF in Parkinson’s disease. J. Neurol. Neurosurg. Psychiatry 63, 754–758.

Zhou, G., Miura, Y., Shoji, H., Yamada, S., Matsuishi, T., 2001. Platelet monoamineoxidase B and plasma beta-phenylethylamine in Parkinson’s disease. J. Neurol.Neurosurg. Psychiatry 70, 229–231.

Ziegleder, G., Stojacic, E., Stumpf, B., 1992. Occurrence of beta-phenylethylamineand its derivatives in cocoa and cocoa products. Zeit. Lebensmittel-Untersu-chung Forsch. 195, 235–238.