neuroprotective effect of lutein against 3-nitropropionic acid–induced huntington's...

Neuroprotective Effect of Lutein Against3-Nitropropionic Acid–Induced Huntington’s Disease–Like Symptoms:

Possible Behavioral, Biochemical, and Cellular Alterations

Yogita Binawade and Aarti Jagtap

Department of Pharmacology, Bombay College of Pharmacy, Mumbai, India.

ABSTRACT 3-Nitropropionic acid (3-NP) induces cellular energy deficit and oxidative stress–related neurotoxicity via an

irreversible inhibition of mitochondrial complex II enzyme, succinate dehydrogenase. Huntington’s disease (HD) is a neurological

disorder characterized by cognitive and motor dysfunctions. Lutein is a well-known antioxidant used in the management of oxidative

stress related diseases. Clinical trials have supported the beneficial effect of lutein in Alzheimer’s disease. The present study was

designed to explore possible neuroprotective effects of lutein on 3-NP–induced mitochondrial dysfunction and oxidative stress.

Systemic administration of 3-NP (25 mg/kg intraperitoneally [i.p.] for 4 consecutive days) caused loss of body weight and neu-

robehavioral deficits by hind-limb impairment (Narrow Beam test), motor coordination (locomotor activity) and memory dys-

function (Morris water maze and Elevated Plus maze performance). Biochemical analysis revealed significant increase in lipid

peroxidation, nitrite concentration, reduced gutathione levels, and acetyl cholinesterase levels and depleted catalase activities in rat

brain. The activities of mitochondrial complexes (I, II, IV, and MTT assay) were found to be significantly lowered in brain

mitochondria. Daily lutein (50 or 100 mg/kg orally [p.o.]) administration for 14 days significantly improved body weight, neuro-

behavioral alterations and attenuated oxidative stress and improved mitochondrial enzymes complex activities of rat brain. His-

topathological examination further affirmed the neuroprotective effect of lutein on 3-NP induced pathological lesions. The present

study indicates that lutein is a promising candidate for the management of HD and related conditions.

KEY WORDS: � antioxidant activity � in vivo � mitochondria � neurodegenerative disease � oxidative stress

INTRODUCTION

Huntington’s disease (hd) is a neurological disordercharacterized by progressive cell death in the striatum

and cortex accompanied by declines in cognitive, motor andpsychiatric functions.1 Genetic mutation in the interestingtranscript 15 gene is an expanded trinucleotide repeat (CAG)in exon 1 of the Huntington (htt) gene, leading to the pro-duction of mutant htt with an abnormally long polyglutaminerepeat. The mechanism by which the expanded polyglutaminetract in htt causes cell death in HD remains elusive.2 Differentbiochemical studies have revealed the existence of majordefects in the energy metabolism of HD patients characterizedby mitochondrial dysfunction and oxidative stress.3 Bio-chemical studies in HD postmortem tissue have revealed se-lective dysfunction of components of the mitochondrialtricarboxylic acid cycle and electron transport chain (ETC) inthe affected brain regions, particularly complexes I, II, and IV.The activity of complex II (succinate ubiquinol oxidoreduc-tase) of the respiratory chain is severely decreased in affected

brain regions (caudate and putamen) of symptomatic HDpatients. Consequently, pharmacological inhibitors of mito-chondrial complex II have been found to induce striataldamage and motor phenotypes in animals, which closely re-sembles the symptoms seen in HD.4

3-Nitropropionic acid (3-NP), a mycotoxin produced bythe fungus Arthrinium sp., is a suicide inhibitor of respira-tory chain and Krebs cycle enzyme succinate dehydroge-nase (SDH). 3-NP interferes with ATP synthesis in brainmitochondria, leading to mitochondrial dysfunction, andalso produces selective striatal lesions and induces prolif-erative changes in the dendrites of spiny neurons, resultingin neuronal degeneration within basal ganglia and move-ment dysfunction as observed in HD patients.5 3-NP de-pletes antioxidant defense enzymes and increases levels ofreactive oxygen/nitrogen species in different areas of thebrain, leading to oxidative damage.6

Recent studies have shown that the antioxidants of plantorigin with free-radical scavenging properties can functionas therapeutic agents in several diseases caused by oxidativestress.7 As a result, an increasing number of researchershave been studying the effects of antioxidants in the centralnervous system to develop new therapies to treat theseconditions. Lutein is a xanthophyll and one of 600 knownnaturally-occurring carotenoids which are synthesized only

Manuscript received 8 December 2012. Revision accepted 14 June 2013.

Address correspondence to: Aarti Jagtap, PhD, Department of Pharmacology, BombayCollege of Pharmacy, Kalina, Santacruz East, Mumbai 400 098, India, E-mail: [email protected]

JOURNAL OF MEDICINAL FOODJ Med Food 16 (10) 2013, 934–943# Mary Ann Liebert, Inc., and Korean Society of Food Science and NutritionDOI: 10.1089/jmf.2012.2698

934

by plants. Lutein is showing antioxidant properties8 whichmay be attributed to its unique chemical structure; it notonly has conjugated double bonds but also has two hydroxylgroups on both ends making it a stronger antioxidant com-pared to other carotenoids.9 Lutein has been studied foramelioration of conditions such as diabetic neuropathy andage-related macular degeneration (AMD), which are mainlyassociated with oxidative stress.10 Astaxenthin, a stereo-isomer of lutein, has been studied for its beneficial effects inapoptosis.11 Clinical trials also supported the beneficial ef-fect of lutein in Alzheimer’s disease.12 The present studywas designed to elucidate the beneficial effects of lutein asan antioxidant in ameliorating neurobehavioral deficits,mitochondrial dysfunction, and oxidative stress by 3-NP–induced neurotoxicity in an animal model of HD.

MATERIALS AND METHODS

Chemicals

All of the chemicals used in the present study were ofanalytical grade and were purchased from Sigma ChemicalsCo., Sisco Research Laboratories, and SD Fine Chemicals.Lutein was supplied by OmniActive Health Technologies,Ltd., Mumbai, India.

Animals and treatment schedule

Female Sprague-Dawley rats (200–250 g) were used inthe study. Animals were acclimatized to laboratory condi-tions before experimentation. The animals were kept ingroups of four per cage under standard conditions of light–dark cycle, with food and water ad libitum in acrylic boxcages with soft bedding. All of the experiments were carriedout between 9:00 a.m. and 3:00 p.m. The protocol followedwas approved by the Institutional Animal Ethics Committeeand was in accordance with guidelines of Committee for thePurpose of Control and Supervision of Experiments onAnimals for use and care of laboratory animals.

3-NP (Sigma Chemical) was diluted with saline (adjustedto pH 7.4) and administered intraperitonealy to rats. Luteinwas suspended in 0.5% sodium carboxy-methyl cellulosesolution and administered orally in a constant volume of0.5 mL/200 g of body weight. Animals were randomly di-vided into six groups of eight animals.

Group I received vehicle for lutein (p.o.) and also normalsaline (i.p.) for 14 days. Group II received vehicle for 14days and 3-NP (25 mg/kg i.p.) for the last 4 days (days 11–14). Groups III and IV received lutein (50 or 100 mg/kg p.o.,respectively) for 14 days. Groups V and VI received lutein(50 or 100 mg/kg, p.o., respectively) for 14 days and 3-NP(25 mg/kg i.p.) for the last 4 days (days 11–14). 3-NP wasadministered 1 h after lutein administration.

Measurement of body weight

Body weights of animals were recorded on the first andlast day of the experiment. Percent change in body weightwas calculated as change in body weight (initial weight

minus final weight) divided by initial body weight, multi-plied by 100%.

Behavioral assessments

All animals were trained for 8 days before starting theexperiment.

Narrow Beam test

The Narrow Beam test was used to measure hind-limbimpairment as described previously.13 The narrow beamused for the present experiments was a 105 cm long and4 cm broad wooden beam. The beam was suspended 80 cmfrom the ground by wooden supports at either end. At thestart end of the beam, a line was drawn 20 cm from the endof the beam. During the test, the rat was placed entirelywithin this 20 cm starting zone facing its home cage and astopwatch started immediately upon release of the animal.The time taken to traverse the beam was recorded. Themaximum time allowed for the task was 2 min; a fall wasalso recorded as a maximum time. The test was performedon days 0 and 15 of the study.

Elevated Plus maze

The Elevated Plus maze was used to evaluate spatial long-term memory according to the method of Lee O-H et al.11

Briefly, the apparatus consisted of two open arms and twoclosed arms. The arms extended from a central platform, andthe maze was elevated to a height of 50 cm from the floor.On the first day, each animal was placed at the end of anopen arm. Transfer latency (TL) was recorded as the timetaken by the rat to move into one of the enclosed arms. If theanimal did not enter a closed arm within 90 s it was gentlypushed into one closed arm and the TL latency was assignedas 90 s. The rats were allowed to explore the maze for 20 s,and then return to the home cage. TL was recorded on days 0and 15 of the study.

Morris water maze test

The Morris water maze test was performed for assessmentof cognitive performance by discrete modification to themethod of Kumar et al.14 The maze consists of a circularplastic tank (160 cm in diameter and 35 cm in height). Thetank was divided by four fixed points on its perimeter intofour quadrants. Water was made opaque by adding milk. Itcontains an escape platform of the same color as the rest ofthe basin (to eliminate any false positive results due to vi-sion) placed in the same quadrant of the basin throughout thetrials, and was placed 2 cm below the water surface. Ratswere placed gently at a start point in the middle of the rim ofa quadrant not containing the escape area with their face tothe wall. If the rat did not escape on to the platform within90 s it was guided to the platform and allowed to stay on itfor 20 s. Animals received four trials per day separated by10 min for 5 successive days (acquisition trials) duringwhich the time required to reach the platform was calcu-lated. After the acquisition trials, time taken to find the

LUTEIN IN THE MANAGEMENT OF HUNTINGTON’S DISEASE 935

hidden platform was recorded on day 0 and the 15th dayafter start of drug administration.

Assessment of gross behavioral activity (locomotor activity)

The locomotor activity was monitored using an actophot-ometer on the first and last days of study. Each interruption ofa beam on the x- or y-axis generated an electric impulse,which was presented on a digital counter. The apparatus wasplaced in a darkened, light- and sound-attenuated, and ven-tilated testing room. Each animal was observed over a periodof 5 min and values are expressed as counts per 5 min.15

Mitochondrial respiratory chain enzymes activities

Animals were sacrificed using a carbon dioxide chamberon day 15. Mitochondria were isolated by a modifiedmethod of Sandhir et al.16 Briefly, the whole brain wasdissected, rinsed in ice-cold isotonic saline, and homoge-nized with 10 volumes (w/v) of ice-cold extraction buffer(10 mM Tris-HCl, pH 7.4, 0.44 M sucrose, 10 mM ethyl-enediaminetetraacetic acid [EDTA], and 0.1% bovine serumalbumin [BSA]). The homogenate was centrifuged using aRemi cooling centrifugor (RH 106) at 5000 rpm for 30 minat 4�C. The pellet was discarded and the supernatant re-centrifuged at 10,000 rpm for 30 min at 4�C. Supernatantobtained before recentrifugation was used for the estimationof acetyl cholinesterase (AChE) and nitrite levels and oxi-dative stress parameters. After recentrifugation, the crudemitochondrial pellet was separated, washed with extractionbuffer and centrifuged at 10,000 rpm for 30 min at 4�C. Thefinal mitochondrial pellet was resuspended in buffer con-taining 0.44 M sucrose in 10 mM Tris-HCl, pH 7.4. Sus-pensions of the mitochondrial pellet were used for thefollowing mitochondrial enzymes estimations.

Nicotinamide adenine dinucleotide (NADH) dehydroge-nase activity was measured by the method of King and Ho-ward,17 which involves catalytic oxidation of NADH withsubsequent reduction of cytochrome c. Briefly, mitochondrialfraction (0.05 mL) was added to 3.0 mL of reaction mixture,which contained 0.35 mL (0.2 M) glycyl glycine pH 8.5,0.10 mL (6 mM) NADH, 0.1 mL (1 mM) oxidized cytochromec and 0.05 mL (0.02 M) NaHCO3 and 2.40 mL water. Theincrease in absorbance was read at 550 nm for 3 min. Resultswere expressed as nmol NADH oxidized/min/mg protein.

SDH activity in the mitochondrial fraction was estimatedaccording to the method of King.18 The reaction mixtureconsisted of 1.5 mL of phosphate buffer (0.2 M, pH 7.8),0.2 mL of succinic acid (0.6 M, pH 7.8), 0.3 mL of BSA(1%, w/v), 0.1 mL of 0.03 M potassium ferricyanide and thereaction was started by addition of mitochondrial fraction(0.05 mL). The decrease in absorbance was recorded at420 nm for 3 min. Results were expressed as nmol succinateoxidized/min/mg protein.

Cytochrome oxidase was assayed in the mitochondrialfraction according to the method of Sottocasa et al.19

Briefly, cytochrome c (3 mM) was reduced first by the ad-dition of a few crystals of sodium borohydride, and thenneutralized to pH 7.0 by 0.1 M HCl. Reduced cytochrome c

(0.3 mM) was added to 0.075 M phosphate buffer (pH 7.4),and the reaction was initiated by addition of mitochondrialsuspension (50 lL). The decrease in absorbance was mea-sured at 550 nm for 3 min. Results were expressed as nmolcytochrome c oxidized/min/mg protein, using molar ex-tinction coefficient of cytochrome c (19.6/mM/cm).

MTT reduction was used to assess the mitochondrialfunction by the method described by Kamboj et al.20 Thereaction mixture containing mitochondrial fraction (500 lL)and MTT (500 lL, 0.1 mg/mL) was incubated at 37�C for120 min, and then centrifuged to obtain formazan pellet. Thepellets were dissolved in 1 mL of absolute ethanol and themixture was recentrifuged. The absorbance of the superna-tant was measured at 595 nm. Results were expressed as mgformazan formed/min/mg protein.

Mitochondrial oxidative stress parameters

The amount of malondialdehyde (MDA), a measure oflipid peroxidation was quantified by reaction with thio-barbituric acid (TBA) according to the method described byOhkawa et al.21 The tissue supernatant (0.1 mL) was mixedwith 2.5 mL TBA (0.7% in 30% glacial acetic acid). Themixture was incubated at 95�C for 60 min; the mixture wascooled under tap water. After cooling, the mixture wascentrifuged at 4000 rpm for 10 min. The absorbance of thesupernatant was read at 532 nm. The MDA content of thetest tissue was calculated from the standard graph and re-sults were expressed as nmol MDA/mg protein.

Catalase activity was assayed by the method of Luck22

wherein the breakdown of hydrogen peroxide (H2O2) ismeasured at 240 nm. Briefly, assay mixture consists of 3 mLof H2O2 phosphate buffer and 0.05 mL of tissue supernatantand change in absorbance was recorded at 240 nm for 3 min.The results were expressed as micromole H2O2 decomposedper milligram of protein/min.

Reduced glutathione was estimated according to themethod described by Ellman.23 To 0.2 mL of tissue super-natant, 0.6 mL of 0.2 M Tris-EDTA, 50 lL of 0.01 M 5,50-dithiobsis(2-nitrobenzoic acid) (DTNB), and 2.5 mL meth-anol was added. The mixture was incubated at 37�C for30 min with occasional shaking. The mixture was thencentrifuged at 4000 rpm for 15 min. The supernatant wasseparated. The intensity of color developed was determinedat 412 nm against blank treated in the same way, replacingtissue supernatant with distilled water. The values of reducedglutathione (GSH) were obtained by interpolation of a stan-dard plot of glutathione and values were expressed as reducedglutathione lmole/mg protein.

Nitrite levels were determined by a colorimetric assayusing Greiss reagent (0.1% naphthylethylene diamine di-hydrochloric acid and 1% sulfanilamide in 5% phosphoricacid). Equal volumes (500 lL) of tissue supernatant andGreiss reagent were mixed, the mixture was incubated for10 min at room temperature in dark and the absorbance wasdetermined at 540 nm.24 The concentration of nitrite in su-pernatant was determined from a sodium nitrite standard andexpressed as lmole/mg protein.

936 BINAWADE AND JAGTAP

AChE is a marker of loss of cholinergic neurons in theforebrain. The AChE activity was assessed by Ellmanmethod.25 The assay mixture contained 0.05 mL of tissuesupernatant, 3 mL of sodium phosphate buffer (pH 8),01 mL of acetyl-thiocholine iodide and 0.1 mL DTNB. Thechange in absorbance was measured for 2 min at 412 nmusing a Perkin-Elmer Lambda 20 spectrophotometer. Theresults were expressed as lmole acetyl-thiocholine iodidehydrolyzed/min/mg protein.

The protein content was estimated according to themethod of Lowry et al.26 One hundred microliters of su-pernatant was added to 2 mL of Lowry reagent and incu-bated for 10 min at room temperature. Later, 0.2 mL Folinsciocalteaus reagent was added, mixed immediately and in-cubated for 30 min at room temp. Absorbance was measuredat 660 nm, and results were expressed as mg/mL using BSAas standard.

Histopathological examination

Brains were fixed in 10% formalin solution for 24 h usingthe Hartz technique.27 The fixed tissues were washed in tapwater, dehydrated in a series of alcohol, cleared in xylene,then embedded in paraffin blocks. Sections 5 lm thick wereobtained from the blocks and stained by hematoxylin andeosin. The tissue sections were then examined under a lightmicroscope at different magnifications and photographswere taken.

Statistical analysis

All values are expressed as mean – standard deviation ofeight animals per group. Data were analyzed using one wayanalysis of variance followed by Tukey’s test. Values withP < .05 were considered as statistically significant.

RESULTS

Effect of lutein on body weight in 3-NP–treated rats

3-NP treatment caused significant decrease in bodyweight on day 15 as compared to the vehicle-treated group.Further lutein treatment (50 or 100 mg/kg) attenuated thebody weight of 3-NP–treated rats compared to the group

treated with only 3-NP. There was no significant change inthe initial and final body weights of vehicle- and per se–treated animals (Fig. 1).

Effect of lutein on neurobehavioral deficits

Hind limb impairment was measured by the NarrowBeam test in terms of time taken by each animal to trans-verse the beam (Fig. 2). On day 0, time taken by the animalsranged between 3 and 10 s in all groups. On day 15, the timetaken significantly increased in 3-NP treated animals ascompared to the control group. 3-NP–challenged rats pre-treated with lutein (50 or 100 mg/kg) showed a significantdecrease in time compared to 3-NP treatment alone. Per setreatment of lutein (50 or 100 mg/kg) did not show anysignificant change compared with 3-NP treatment.

Spatial memory was assessed in terms of TL in anElevated Plus maze task. 3-NP treatment significantly de-layed TL as compared to the vehicle-treated group. Incontrast, pretreatment with lutein (50 or 100 mg/kg) to 3-NP–challenged rats shortened TLs compared to 3-NPtreatment. However, treatment with lutein only (50 or100 mg/kg) did not show any significant change in TL ascompared to the vehicle-treated group (Fig. 3).

Cognitive impairment was assessed using the Morriswater maze test in terms of time required by animals tolocate the platform (escape latency). On day 15, escape la-tency was significantly increased in the 3-NP–treated groupas compared to the control group. Pretreatment with lutein(50 or 100 mg/kg p.o.) showed a significant improvement inescape latency compared with the 3-NP–treated group.Treatment with lutein only (50 or 100 mg/kg) did not affectescape latency time in comparison with control treatment(Fig. 4).

3-NP treatment caused a significant decrease in locomo-tor activity as compared to the vehicle-treated group. Incontrast, treatment with lutein (50 or 100 mg/kg) signifi-cantly improved locomotor activity in 3-NP–treated ratscompared with the animals treated with 3-NP only. Per setreatment of lutein (50 or 100 mg/kg) did not show anysignificant change (Fig. 5).

FIG. 1. Effect of lutein on body weight in 3-nitropropionic acid (3-NP)–treated rats. Values are expressed as mean – standard deviation(SD); n = 8. Significance was determined using one-way ANOVAfollowed by Tukey’s test. #Significantly different from control group(P < .05). *Significantly different from 3-NP–treated group (P <.05). **Significantly different from L-50 + 3-NP–treated group. ANOVA,analysis of variance; L-50, lutein 50 mg/kg.

FIG. 2. Effect of lutein administration in 3-NP–treated rats on theNarrow Beam test in terms of total time. Values are expressed asmean – SD; n = 8. Significance was determined using one-wayANOVA followed by Tukey’s test. #Significantly different from con-trol group (P < .05). *Significantly different from 3-NP treated group(P < .05). **Significantly different from L-50 + 3-NP–treated group.


Effect of lutein on mitochondrial respiratory chain enzymesin 3-NP–treated rats

The activities of various mitochondrial ETC enzymes arepresented in Table 1.

Mitochondrial dysfunction induced by 3-NP exhibitedsignificant (P < .05) decrease in activities of mitochondrialenzyme complexes (I, II, IV, and MTT activities) as com-pared to the vehicle-treated group. Treatment with lutein (50or 100 mg/kg) for 14 days dose-dependently counteractedthe deleterious effects of 3-NP by increasing the mito-chondrial enzyme complexes’ activities as compared to 3-NP treatment. However, treatment with lutein only (50 or100 mg/kg) attenuated complex I (NADH dehydrogenase)activity compared with vehicle treatment, but the activitiesof other enzymes (II, IV, and MTT reduction) were notaltered significantly.

Effect of lutein on brain lipid peroxidation, antioxidantenzyme (catalase), and GSH levels

3-NP–treated rats exhibited a significant decrease inactivities of catalase and reduced GSH as compared to

controls. Pretreatment with lutein (50 or 100 mg/kg) dose-dependently counteracted the deleterious effects of 3-NP byincreasing the levels of antioxidant parameters as comparedto 3-NP treatment. Antioxidant enzyme (Catalase) activityincreased significantly after the per se treatment of lutein(50 or 100 mg/kg) compared with control treatment,whereas levels of reduced GSH were not affected (Table 2).

There was a significant elevation in the level of liquid per-oxidation in 3-NP–treated rats compared with vehicle-treatedrats. However, treatment with lutein (50 or 100 mg/kg) for 14days to 3-NP–treated rats significantly decreased these levelsdose-dependently, compared to 3-NP–treated rats. Per setreatment of lutein (50 or 100 mg/kg) did not affect TBARSlevels in comparison with vehicle treated group (Table 2).

Effect of lutein on nitrite and AChE enzyme levels

As shown in Table 2, systemic administration of 3-NPcaused significant increases in levels of nitrite and AChE ascompared to the control group. However, lutein pretreat-ment (50 or 100 mg/kg) to 3-NP–challenged rats signifi-cantly decreased the elevated levels of nitrite and AChE

FIG. 3. Effect of lutein administration on transfer latency in theElevated Plus maze task in 3-NP–treated rats. Values are expressedas mean – SD; n = 8. Significance was determined using one-wayANOVA followed by Tukey’s test. #Significantly different from con-trol group (P < .05). *Significantly different from 3-NP–treated group(P < .05). **Significantly different from L-50 + 3-NP–treated group.

FIG. 4. Effect of lutein administration on cognitive impairmentin the Morris water maze in 3-NP–treated rats. Values are expressedas mean – SD; n = 8. Significance was determined using one-wayANOVA followed by Tukey’s test. #Significantly different fromcontrol group (P < .05). *Significantly different from 3-NP–treatedgroup (P < .05). **Significantly different from L-50 + 3-NP–treatedgroup.

Table 1. Effects of Lutein Administration on the Activity of Mitochondrial Complexes

in the 3-Nitropropionic Acid–Treated Group

Groupsa

Complex-I(lmole NADH

oxidized/min/mg protein)

Complex-II(lmole SDH

oxidized/min/mg protein)

Complex-IV(mmole cytochrome

oxidized/min/mg protein)MTT reduction(% of control)

Control 10.94 – 1.92 65.99 – 10.49 7.81 – 2.86 100 – 0.008L-50 13.37 – 0.74* 61.89 – 13.58 5.44 – 1.25 88.83 – 0.19L-100 13.45 – 1.38* 64.63 – 17.47 5.74 – 1.93 94.41 – 0.0053-NP 4.37 – 0.586# 14.46 – 2.67# 1.72 – 1.30# 37.93 – 0.009#

L-50 + 3-NP 6.42 – 2.08* 34.74 – 8.95* 2.85 – 1.73* 49.42 – 0.007*L-100 + 3-NP 7.56 – 1.91* 55.05 – 4.53** 3.24 – 1.05** 54.02 – 0.006**

Values are expressed as mean – SD; n = 8 (one-way ANOVA followed by Tukey’s test).aGroups were treated as follows: control, normal vehicle treated rats; L-50, lutein (50 mg/kg); L-100, lutein (100 mg/kg); 3-NP, 3-nitropropionic acid (25 mg/kg).#Significantly different from control group (P < .05).

*Significantly different from 3-NP group (P < .05).

**Significantly different from (lutein 50 mg/kg + 3-NP) and 3-NP treated group (P < .05).

SDH, succinate dehydrogenase; SD, standard deviation; ANOVA, analysis of variance.


compared with 3-NP treatment alone. Per se treatment oflutein (50 or 100 mg/kg) did not alter these levels as com-pared to control group.

Effect of lutein on histological alterations in striatum

Brain tissue sections from rats of the control groupshowed normal histological structure (Fig. 6). 3-NP treat-ment produced severe degeneration of neurons with com-plete loss of cell detail and architecture, and with theappearance of basophilic nuclear remnants in the centralzone. Animals pretreated with lutein before 3-NP adminis-tration showed mild focal gliosis associated with swelling inthe endothelial lining of blood capillaries. Striata of lutein-only treated rats showed no histopathological alterationscompared to control animals.

DISCUSSION

This is the first extensive report of the effect of lutein in 3-NP–induced HD-like symptoms in rats. Lutein is a xan-thophyll and has antioxidant properties.8 The pathogenesisof HD is not yet fully understood, but studies on 3-NP–induced neurotoxicity provide good insight into its pathol-

ogy and clearly indicate the involvement of oxidative stressand mitochondrial dysfunction. Consequently, pharmaco-logical inhibitors of mitochondrial complex II (3-NP) havebeen found to induce striatal damage and motor phenotypesin animals, which closely resembles the symptoms seen inHD patients.4 HD-like symptoms include chorea, memoryimpairment, oxidative stress, and mitochondrial dysfunctionin the rat brain.1

3-NP is a suicide inhibitor of respiratory chain and Krebscycle enzyme SDH. Inhibition of these enzymes in the ETCleads to an increase in electron leakage from mitochondriaand production of reactive oxidative species (ROS), whichcauses oxidative stress.28 Roles of various antioxidants havebeen recently reported in the management of HD-likesymptons: lycopene and epigallocatechin-3-gallate,29

Withania somnifera root extract,30 flavonoid kameferol,31

hesperidin and naringenin,32 sartraline,33 S-allylcysteine,34

l-carnitine,35 taurine,36 and resveratrol.37 All of these re-ports indicate the role of antioxidants as promising candi-dates in the management of HD. On the basis of thesereports, the present study was designed to explore the po-tential of lutein in the animal models of HD.

Antioxidants are molecules that act against any form ofoxidative stress and its associated ill effects on cellularsystems. They neutralize ROS and other kinds of free rad-icals produced as a consequence of oxidative stress and haveattracted the attention of clinicians due to therapeutic po-tential.5 Lutein, a xanthophyll having antioxidant proper-ties,8 shows beneficial effects in AMD, cataracts, cancer,coronary heart disease, and stroke,3 and is under investiga-tion for treatment of Alzheimer’s disease.12 Supporting thepresent investigation, lutein is capable is terminating freeradical reactions and protecting our body from oxidativestress, which is the basis for neurodegenerative disease. Inour experiment, we found that lutein protected the brainfrom 3-NP–induced oxidative stress and mitochondrialdysfunction via favorably improved neurobehavioral, bio-chemical, and histopathological parameters, suggesting itsneuroprotective action.

HD patients often show gradual decreases in body weightdespite normal and increased energy intakes. Decrease inbody weight can be considered as an indicator of 3-NP

Table 2. Effects of Lutein Treatment Against 3-Nitropropionic Acid–Induced Biochemical Changes in the Rat Brain

Groups

Lipid peroxidation(nmole/mg

protein)

Reduced glutathione(lmole/mg

protein)

Catalase (lM of H2O2

decomposed/min/mg protein)

Nitrite levels(lmole/mg

protein)

Acetyl cholinesteraselevel (lmole of substrate

hydrolyzed/min/mg protein)

Control 3.16 – 0.57 256.33 – 47.53 7.51 – 1.03 0.1626 – 0.04 25.92 – 5.47L-50 3.79 – 0.93 254.73 – 16.22 9.008 – 1.97* 0.1865 – 0.05 26.01 – 13.49L-100 3.58 – 0.77 259.99 – 18.39 13.46 – 1.34* 0.209 – 0.032 24.63 – 7.633-NP 6.46 – 2.22# 156.66 – 16.93# 4.54 – 2.20# 0.2815 – 0.03# 49.17 – 31.4#

L-50 + 3-NP 5.66 – 0.95 222.88 – 9.08* 9.68 – 12.5* 0.2046 – 0.03* 31.08 – 20.2L-100 + 3-NP 4.36 – 0.614** 247.23 – 18.51** 17.31 – 11.7* 0.1936 – 0.08** 29.42 – 15.3*

Values are expressed as mean – SD; n = 8 (one-way ANOVA followed by Tukey’s test).#Significantly different from control group (P < .05).

*Significantly different from 3-NP group (P < .05).

**Significantly different from (lutein 50 mg/kg + 3-NP) and 3-NP treated group (P < .05).

FIG. 5. Effect of lutein administration on locomotor activity in3-NP–treated rats. Values are expressed as mean – SD; n = 8.Significance was determined using one-way ANOVA followed byTukey’s test. #Significantly different from control group(P < .05).*Significantly different from 3-NP–treated group (P < .05).**Significantly different from L-50 + 3-NP–treated group.


FIG. 6. Histopathological pictures from the sections through the striatum of the brain, showing the effects of lutein on 3-NP–inducedpathological lesions. Color images available online at www.liebertpub.com/jmf


neurotoxicity due to depressed energy metabolism.38 Sup-porting the above observation, over the course of our study,3-NP–treated animals showed decreases in body weightwith significantly lower feed intakes, which was signifi-cantly improved by 14 days pretreatment with lutein, sug-gesting its therapeutic potential.

Neurobehavioral studies are considered as the most im-portant initial tests for diagnosing chorea and cognitiveimpairment, which are major symptoms of HD. The mainfunction of basal ganglia is to control the overall coordi-nation of body movements. As the striatum is the centralcore area in the basal ganglia that controls coordination ofmotor movement, it is accepted that the 3-NP inducesstriatal degeneration leading to impaired coordination ofmotor movement.39,40 In the present study, 3-NP–inducedmotor dysfunction was assessed by locomotor and hind-limbimpairment, which was significantly improved by luteinpretreatment compared to the 3-NP–treated group.

The memory of HD patients often declines with degen-eration of neurons in the brain. Cognitive impairment wassuggested to reflect a combination of lower energy levelsresulting from SDH inhibition by 3-NP and consequent shortterm changes in neural processing. In the present sets ofexperiments, memory performance was tested through theMorris water maze and Elevated Plus maze paradigms.Lutein treatment significantly improved cognitive task per-formance in both tests, suggesting the potential effect oflutein against 3-NP–induced memory dysfunction.

Apart from neurobehavioral studies, several biochemicalparameters, including mitochondrial respiratory chain en-zymes and antioxidant parameters, were estimated as apredictor for pathological changes in HD. Oxidative stressand mitochondrial dysfunction induced by 3-NP is mainlyresponsible for HD-like symptoms seen in rats (chorea andcognitive impairment).5 Oxidative stress is one of the majordeleterious events which contribute to the pathogenesis ofseveral neurodegenerative diseases, including HD.33,41 3-NPbinds irreversibly to complex II (SDH) and leads to inhibi-tion of free fatty acid oxidation pathways; inhibition of thesepathways leads to the energy impairment that follows un-coupling of oxidative phosphorylation. Disruption of mito-chondrial activity is associated with the abnormal formationof ROS. Inhibition of enzymes in the ETC can lead to anincreases in electron leakage from the mitochondria andproduction of ROS such as the superoxide radical (O2

- �),H2O2, reactive nitrogen species (NO - ) and the hydroxylradical (OH - �), and consequently causes oxidative stress.28

According to previous reports42,43 and the clear suggestionof the data in the present study, 3-NP administration sig-nificantly increased oxidative stress, as evident by increasesin ROS production, nitrite levels, and elevated lipid perox-idation products, and depletion of reduced GSH in 3-NP–treated rats. Oral administration of lutein to 3-NP–treatedanimals for 14 days significantly attenuated the ROS gen-eration by reversing these parameters in a dose-dependentmanner. Catalase is an enzyme which is an endogenousdefense against ROS generation. The activity of this enzymewas found to be lowered in 3-NP–treated animals compared

with the vehicle-treated group. In contrast, pretreating 3-NP–challenged rats with lutein for 14 days significantlyincreased levels of catalase. Per se treatment of lutein alsoincreased catalase levels as compared to control treatment,demonstrating that the capability of lutein to increase anti-oxidant enzyme (catalase) levels might be responsible to agreater extent for its protective role against ROS.

Cognitive impairment is one of the major symptoms ofHD. Reduced activity of acetylcholine and choline acetyl-transferase has been observed in the brain of HD patients,and is responsible for cognitive deficits.44 It is reported that3-NP treatment of rats results in increased activity of theAChE enzyme, which decreases the level of acetylcholine.45

In the present study, lutein treatment ameliorated the 3-NP–induced increases in AChE activity, which is mainly re-sponsible for cognitive deficits. In addition, AchE-inhibitoryactivity of lutein is also supported by a clinical study.12 Allof these reports suggest that the AchE-inhibitory property oflutein might be responsible for cognitive improvement of-fered by lutein as seen in neurobehavioral studies.

Mitochondria of HD patients are affected by alterations inETC function, in which mitochondrial respiratory chainenzymes (complexes I, II, and IV) are affected.41 Complex I(NADH dehydrogenase) is the entry enzyme of the mito-chondrial respiratory chain and as such plays a crucial rolein ATP production and mitochondrial function in general.Complex I impairment leads to increased ROS production insub mitochondrial particles and cell systems, and is the mainsite for superoxide formation, while complex IV (cyto-chrome oxidase) is the last enzyme in the respiratory electontransport chain and responsible for hydroxyl radical for-mation. In our study, the activity of complex II (SDH de-hydrogenase) was decreased to a greater extent as comparedto other complexes, and this might be because of irreversibleinhibition of complex II by 3-NP. It is the only enzyme thatparticipates in both the citric acid cycle and the ETC; thus,inhibition of this enzyme is responsible for effectively de-creasing production of ATP. Based on the assumption thatmitochondrial SDH contributes to MTT reduction, the de-creased MTT reduction confirms inhibition of SDH.46 MTTreduction takes place only when reductase enzymes areactive, and hence, conversion was used to measure viablecells. This process of conversion mainly affects the timingof when oxidative damage impairs metabolic function, in-cluding the ability of mitochondria to make ATP.47

According to several reports, 3-NP depleted several re-spiratory chain enzymes (I, II, and IV) and declined thenumber of viable cells (MTT reduction). 3-NP disturbedmitochondrial enzyme functions, induced damage to thecell-signaling pathway in the brain, and impaired ATP inmitochondria.34,48 In the present study, these alterations inmitochondrial enzyme complex activities and viable cellcounts induced by 3-NP were significantly restored by luteinpretreatment. However, per se treatment of lutein attenuatedcomplex I (NADH dehydrogenase) activity significantlycompared with vehicle treatment, but activities of other en-zymes (II, IV, and MTT reduction) were not altered signifi-cantly, showing lutein’s greater propensity towards complex I


enzymes than others. In conclusion, lutein restores mito-chondrial complex activity, thus preserving normal ATPfunction and halting cell death induced by 3-NP.

To further investigate the neuroprotective action of luteinon 3-NP–induced pathological lesions, histopathologicalexaminations were performed. As per the reports of histo-pathological examination, 3-NP treatment produced severedegeneration of neurons, with complete loss of cell detailand architecture and with the appearance of basophilic nu-clear remnants in the central zone. Lutein pretreatment(50 mg/kg) showed mild focal gliosis accompanied by mildneuronal degeneration, whereas (100 mg/kg) treatmentshowed only mild focal gliosis. Thus, the histopathologicalfindings have reconfirmed the protective action of lutein.Brain striatum of the lutein per se treated group showednormal histopathological structure. This indicates that lu-tein does not possess any adverse effects under normalconditions.

In summary, the neurobehavioral, biochemical and his-topathological investigations demonstrate that lutein is ableto ameliorate 3-NP induced neurotoxicity by its capability toprotect against behavioral changes, restores antioxidantdefense enzyme in brain and improves mitochondrial en-zymes levels.

Conclusion

Results of the present study demonstrate that chronictreatment with lutein can attenuate HD-like symptoms inanimals. Lutein treatment mitigates behavioral, biochemicaland histological abnormalities caused by mitochondrialtoxin 3-NP in rats by attenuating free radical damage, andtherefore could be employed in the management of HD.Future research should therefore be directed to investigatewhether our findings using an experimental animal model ofHD would have clinical implications in management of HDin human patients.

ACKNOWLEDGMENT

The authors gratefully acknowledge the financial supportof AICTE, New Delhi, India.

AUTHOR DISCLOSURE STATEMENT

No competing financial interests exist.

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