Neurobiology of Aging 33 (2012) 404–415

Long term exposure to cypermethrin induces nigrostriatal dopaminergicneurodegeneration in adult rats: postnatal exposure enhances the

susceptibility during adulthoodAnand Kumar Singha, Manindra Nath Tiwaria, Ghanshyam Upadhyaya,

Devendra Kumar Patela, Dhirendra Singha, Om Prakashb, Mahendra Pratap Singha,*a Indian Institute of Toxicology Research (Council of Scientific and Industrial Research), Lucknow, India

b Banaras Hindu University, Varanasi, India

Received 12 October 2009; received in revised form 1 February 2010; accepted 22 February 2010

Abstract

The study aimed to investigate the effects of cypermethrin on biochemical, histopathological, and motor behavioral indices of thenigrostriatal dopaminergic system in adult rats treated with or without cypermethrin (1/10 adult dose) during postnatal days 5–19.Spontaneous locomotor activity (SLA) and rotarod tests were performed to assess motor behavior. Levels of dopamine, 3,4-dihydroxy-phenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum, and tyrosine hydroxylase (TH) immunoreactivity and 4=,6-diamidino-2-phenylindole (DAPI)/Fluoro-Jade B staining in the substantia nigra were measured to assess dopaminergic neurodegeneration.Postnatal treated animals did not exhibit significant changes in any measured parameters. The significant reduction in the time of stay onrotarod, spontaneous locomotor activity, dopamine, 3,4-dihydroxyphenylacetic acid, and tyrosine hydroxylase immunoreactivity while anincrease in homovanillic acid level and Fluoro-Jade B-positive cells were observed in cypermethrin treated adult rats. These changes weremore pronounced in the animals treated with cypermethrin during postnatal days followed by adulthood compared with adulthood alone.The results obtained thus demonstrate that exposure to cypermethrin during adulthood induces dopaminergic neurodegeneration in rats andpostnatal exposure enhances the susceptibility of animals to dopaminergic neurodegeneration if rechallenged during adulthood.© 2012 Elsevier Inc. All rights reserved.

Keywords: 3,4-Dihydroxyphenylacetic acid; Cypermethrin; Dopamine; Homovanillic acid; Tyrosine hydroxylase

www.elsevier.com/locate/neuaging

1. Introduction

Parkinson’s disease (PD), a common movement disor-der, is associated with the progressive degeneration of do-paminergic neurons in the substantia nigra pars compacta(Bernheimer et al., 1973; Hornykiewicz and Kish, 1987;Singh et al., 2007). The degeneration of dopaminergic neu-rons results in reduced level of dopamine in the basalganglia leading to the onset of rigidity, postural instability,resting tremor, and bradykinesia in the patients (Bernheimeret al., 1973; Hornykiewicz and Kish, 1987; Singh et al.,

* Corresponding author at: Indian Institute of Toxicology Research(IITR), Mahatma Gandhi Marg, Post Box 80, Lucknow 226 001, UP, India.Tel: �91 522 2620106 2614869 � 337.

E-mail address: singhmahendrapratap@rediffmail.com (M.P. Singh).

0197-4580/$ – see front matter © 2012 Elsevier Inc. All rights reserved.doi:10.1016/j.neurobiolaging.2010.02.018

2007). Environmental factors have been hypothesized as themajor cause of increasing incidences of PD (Cory-Slechta etal., 2008). Among the environmental factors, exposure topesticides, heavy metals, and hydrocarbons have been themajor concern (Klodowska-Duda et al., 2005). Epidemio-logical studies prompted investigators to look into the roleof commonly used pesticides on dopaminergic neurons(Gorell et al., 1998; Klodowska-Duda et al., 2005). Thistheory gained momentum after the discovery that 1-methyl4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) elicits PD-like symptoms in humans (Langston et al., 1983). Pesticideexposure has been associated with the enhanced progressiveand irreversible nigrostriatal dopaminergic neurodegenera-tion and dopamine depletion leading to PD (Patel et al.,

2006).

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405A.K. Singh et al. / Neurobiology of Aging 33 (2012) 404–415

Cypermethrin, a class II pyrethroid insecticide, is a con-stituent of natural pyrethrin, derived from chrysanthemumplant and is used to control many pests, including mothpests of cotton, fruits, and vegetable crops (Crawford et al.,1981). The repetitive and liberal use of pyrethroids leads totheir unintended exposure to humans and animals, therebyincreasing the risk of intoxication in nontarget organisms(Crawford et al., 1981; Malaviya et al., 1993). Cyperme-thrin readily crosses the blood-brain barrier and achievesconsiderable concentration in the brain and induces motorincoordination and modulates the levels of �-aminobutyricacid (GABA) and sodium channels (Anadon et al., 1996;Gilbert et al., 1989; Manna et al., 2005; Staatz et al., 1982).The main target site of cypermethrin has been identified tobe the sodium channels, which are kept open for unusuallylong periods, causing prolonged sodium current to flow,which in turn, leads to hyperexcitation of the nervous sys-tem. Although type II pyrethroids, including cypermethrin,have effects on sodium conductance, some studies reportedthat type II pyrethroids antagonize GABA. In contrast, afew studies did not observe any changes, while some studiesreported decreased brain GABA levels at very high concen-tration of cypermethrin, i.e., lethal dose (LD) 50 dose (Gil-bert et al., 1989; Manna et al., 2005; Staatz et al., 1982).Long term exposure to cypermethrin has been shown toincrease nontarget organ toxicity in almost all vital organsof test animals (Crawford et al., 1981; Malaviya et al.,1993).

The role of pyrethroids, in nigrostriatal dopaminergicneurodegeneration has been a matter of great debate fromthe last decade. The short term and acute toxicities ofcypermethrin and other pyrethroids are primarily mediatedthrough their interaction with sodium channels, leading todepolarization and hyperexcitation of the nervous system(Eells and Dubocovich, 1988; Kirby et al., 1999; Narahashiet al., 1992). Although some information about the neuro-toxicity of cypermethrin in adults as well as during criticalperiods of development is available, it is not yet knownwhether cypermethrin induces dopaminergic neurodegen-eration in adult animals after prolonged exposure or not. Ascypermethrin leads to the production of reactive oxygenspecies and free radicals, it could lead to oxidative andperoxidative damage to dopaminergic neurons (Giray et al.,2001; Kale et al., 1999; Sian et al., 1994). Various experi-mental and epidemiological evidence has consistently sup-ported that environmental factors or endogenous agentscausing free radical generation leads to the degeneration ofdopaminergic neurons (Barbeau et al., 1987; Bocchetta andCorsini, 1986; Koller et al., 1990). Although, some effortshave been made in the past to understand the mechanism ofcypermethrin-mediated neurotoxicity, the underlying mo-lecular mechanism and causes of cypermethrin-mediatedneurodegeneration have not yet been elucidated. The neu-rotoxic effects of cypermethrin on the dopaminergic system

has been a matter of strong debate among the researchers (

working in this area worldwide, as several studies havecontradicted each other (Chugh et al., 1992; Mun et al.,2005). Some studies have shown that cypermethrin causesnigrostriatal dopaminergic neurodegeneration (Chugh et al.,1992; Nasuti et al., 2007), whereas others have shown thatit enhances neurodegeneration only when some other chem-ical or stimulus triggers the phenomenon (Mun et al., 2005).All these studies were conducted after short term adulthoodor developmental exposure at varying doses, therefore, pos-sibly led to different consequences. Several studies usinganimal models have shown that few pesticides, eitheralone or in combination, induce dopaminergic neurode-generation in animals and the extent of neurodegenera-tion was enhanced when the animals were prechallengedwith the same pesticides during postnatal periods of de-velopment (Patel et al., 2006; Thiruchelvam et al., 2002).The present study was therefore, undertaken to investi-gate the effect of cypermethrin on dopaminergic neuro-degeneration in adult animals after prolonged exposure.Additionally, the animals were treated with cypermethrinduring critical periods of development to assess whetherdevelopmentally-exposed animals are more susceptibleto rechallenge during adulthood or not.

2. Methods

2.1. Chemicals

Cypermethrin, antiglutamic acid decarboxylase monoclonalantibody, 3-hydroxytyramine hydrochloride (dopamine), 3,3=-iaminobenzidine (DAB), 3,4-dihydroxybenzylamine hydro-romide (DHBA), 3,4-dihydroxyphenylacetic acid (DOPAC),omovanillic acid (HVA), sodium chloride, and serotoninere purchased from Sigma-Aldrich (St. Louis, Missouri).ydrogen peroxide, potassium dihydrogen orthophosphate,

nd potassium permanganate were procured from MerckMumbai, India), and dimethyl sulfoxide (DMSO) fromualigens (Mumbai, India). Anti-tyrosine hydroxylase (TH)onoclonal antibody was procured from Santa Cruz Bio-

echnology (Santa Cruz, CA); anti-NeuN monoclonal anti-ody from Chemicon (Temecula, CA); acetic acid, ethanol,eptane sulfonic acid, and nitric acid from Sisco Researchaboratory (Mumbai, India), and frozen section mediumeg-50 from Richard Allen Scientific (Kalamazoo, MI).erchloric acid was purchased from Ranbaxy Private Lim-

ted (New Delhi, India); Vectastain universal elite kit and=,6-diamidino-2-phenylindole (DAPI) from Vector Labo-atories (Burlingame, CA); and Fluoro-Jade B from Milli-ore (Temecula, CA).

.2. Animal treatment

The Institutional ethics committee for the use of labora-ory animals approved the study. The postnatal and adultale Wistar rats (Rattus norvegicus) were obtained from the

nimal colony of Indian Institute of Toxicology Research

IITR), Lucknow, India. The animals were maintained in the

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406 A.K. Singh et al. / Neurobiology of Aging 33 (2012) 404–415

animal house under the standard conditions (temperature:22 °C � 2 °C; humidity: 45%–55%; light intensity: 300–400 lux). The postnatal animals were fed with mother milkand adult animals were fed standard pellet diet and water adlibitum. The postnatal male animals were divided into con-trols and cypermethrin-treated groups. The postnatal ani-mals were treated intraperitoneally for 5–19 days (postnataldays) with cypermethrin (1.5 mg/kg; twice a week, firstinjection on day 5 and last injection on day 19) while thecontrols were treated with an equal volume of corn oil.Further, the animals were left untreated under normal con-ditions for 2 months. The adults and the respective controlswere then rechallenged with cypermethrin (15 mg/kg; in-traperitoneally, twice a week) and an equal volume of cornoil respectively for 4, 8 and 12 weeks. In some sets ofexperiments, same-aged animals were treated during adult-hood alone without (equal volume of corn oil) or withcypermethrin (15 mg/kg; intraperitoneally, twice a week)for 4, 8 and 12 weeks. For optimization of treatment dosesand time schedule, initially the adult animals were treatedwith various doses of cypermethrin for varying time pointsalong with respective controls to measure dopamine level inthe striatum.

2.3. Behavioral studies

Behavioral studies were performed using rotarod andopto-varimax. In rotarod test, animals were trained for 3consecutive days before the day of final treatment at a fixedspeed (5 rpm) for 5 minutes. The time after which theanimals fell down was determined and the maximal obser-vation time was 5 minutes. The experimental readings weretaken 24 hours after the last treatment in all animals groups.At least 4 experimental readings were recorded and theresults were averaged to obtain a single value for eachanimal (Manna et al., 2006). The spontaneous locomotoractivity (SLA) was measured in an infrared beam-activatedmovement monitoring chamber (Opto-varimax-MiniA; Co-lumbus Instruments, Columbus, OH). The animals wereplaced in the chamber for 1 minute before recording thelocomotor activity and the SLA was recorded further for 5minutes. Four experimental readings were taken and theresults were averaged to obtain a single value (Nasuti et al.,2007). The experiments were performed with 5 animals (5observations) in all experimental groups and the averages ofdifferent groups were taken separately. Three sets of similarexperiments (3 experiments � 5 observations) were per-ormed and final values were calculated. The animals usedor the behavioral studies were not used in any other exper-ments, i.e., neurochemical and immunohistochemical.

.4. Measurements of dopamine, DOPAC, HVA, anderotonin

The animals were sacrificed via cervical dislocation;rain was dissected out, immediately kept in liquid nitrogen

nd preceded for biochemical analyses. The striatum was

isolated and the levels of dopamine, DOPAC, HVA, andserotonin were measured as described previously (Curzon etal., 1978; DeVito and Wagner, 1989; Singh et al., 2009). Inbrief, the striatum (10% wt/vol) was homogenized and son-icated in perchloric acid (0.45 N) containing 100 ng/mol3,4-dihydroxybenzylamine hydrobromide, an internal stan-dard. The homogenate was centrifuged at 15,000 g for 10minutes at 4 °C and the supernatant was filtered through asyringe filter (0.22 �m). Dopamine, DOPAC, HVA, anderotonin were separated and quantified in the filtrate. Fil-rate (20 �L) was manually injected into reverse Phase–18 high performance liquid chromatography (HPLC) col-mn coupled with electrochemical detector (Waters, Mil-ord, Massachusetts). The mobile phase consisted of potas-ium phosphate (0.1 mol/L; pH 4.0), methanol (10% vol/ol), and heptane sulfonic acid (1.0 mmol/L vol/vol). Theeparation was achieved at a flow rate of 1.0 mL/minute.he concentrations of dopamine, DOPAC, HVA, and sero-

onin were calculated as ng/mg tissue.

.5. Cryosectioning

The cryosectioning was performed as described previ-usly (Singh et al., 2009). In brief, the animals were anes-hetized under ether anesthesia and intracardiac perfusionas performed with normal saline, followed by 4% para-

ormaldehyde in phosphate-buffered saline at a flow rate of0 mL/minute for 4 minutes each. The brain was dissectedoronally through the median eminence; caudal block wasostfixed in paraformaldehyde solution (10% wt/vol) anderially cryoprotected in sucrose (10%, 20% and 30% wt/ol each) in phosphate-buffered saline. The sections forH-immunoreactivity (20 �m) and for Fluoro-Jade B stain-

ng (10 �m) were cut using a cryostat.

2.6. TH-immunoreactivity

By incubating the sections in blocking buffer (1.5%normal goat serum and 0.1% Triton x-100 in phosphate-buffered saline) for 2 hours, the nonspecific labeling wasprevented (Singh et al., 2009). Similarly, by incubating thesections in hydrogen peroxide (0.5% vol/vol in methanol),the endogenous peroxidase activity was minimized. Thesections were incubated with monoclonal anti-TH antibody(1 : 500) in blocking buffer at 4 °C for 48 hours, followedby 3 washings with phosphate-buffered saline (15 minuteseach). The sections were subsequently incubated with sec-ondary antibody for 1 hour and further in streptavidin-peroxidase complex for 30 minutes. The color was devel-oped with 3,3=-diaminobenzidine; sections were dehydratedin graded ethanol and permanently mounted with dibutylphthalate xylene (DPX). The sections were visualized underthe light microscope (Nikon ECLIPSE TE2000-S, Melville,NY) and the images were captured (Mochizuki et al., 2001).The TH-positive cells were counted as described previously(Singh et al., 2009) with slight modifications.

An experimenter coded all slides and another experi-

407A.K. Singh et al. / Neurobiology of Aging 33 (2012) 404–415

menter who was not aware of the animal treatments per-formed unbiased cell counting of all the coded slides. Ini-tially, each section was viewed at low magnification (4�)and the substantia nigra pars compacta and ventral tegmen-tal area were outlined using atlas and a set of anatomicallandmarks. The first section for counting was selected fromeach animal at a fixed distance from the bregma. Every thirdsection was then selected and TH-positive cells in the sub-stantia nigra were counted bilaterally in 6 sections peranimal. A minimum of 3 animals were used for such anal-yses in each group. Images were captured with a charge-coupled device (CCD) camera and the number of TH-positive neurons in the substantia nigra was counted at 20�magnification (Leica Microscope DM6000 B, Ernst-Leitz-Strasse, Wetzlar, Germany) using a computerized imageanalysis system (QWin Pro, Leica, Germany). The criterionfor counting TH-positive neurons was the presence of de-fined nucleus, cytoplasm, and nucleolus and its localizationwithin the selected zone, or touching the top margins of theselected zone, but not touching the bottom margins. Withineach tracing, the number of sampling sites was 6 (dimen-sions: 100 � 100 �m each) on average. For each animal,average neuronal survival in the substantia nigra was thenexpressed as the percentage of TH-positive neurons in con-trol.

2.7. NeuN/TH-immunoreactivity

For a more appropriate determination of TH-positivecells, NeuN/TH-immunoreactive cell bodies in the substan-tia nigra were counted. NeuN/TH-immunoreactivity wasperformed as described previously (McCoy et al., 2006)with slight modifications. In brief, the incubation, blockingof nonspecific labeling, and minimization of endoperoxi-dase activity were performed as described above for TH-immunoreactivity. The sections were further incubated witha cocktail of monoclonal anti-TH (1 : 500) and NeuN (1 :500) antibodies in blocking buffer at 4 °C for 48 hours,followed by subsequent washing and incubation with sec-ondary antibody. Visualization and imaging of sectionswere performed as described above for TH-immunoreac-tivty. TH-positive cells were counted using Leica QWin Proimage analysis software (version 3.5.1, Leica Microsys-tems, Heerbrugg, Switzerland), as stated under the headingTH-immunoreactivity.

2.8. Fluoro-Jade B and DAPI staining

Fluoro-Jade B and DAPI staining, as described else-where, further assessed the neurodegeneration (Saint-Pierreet al., 2006). In brief, the sections were dehydrated withascending grades of ethanol, hydrated with descendinggrades of ethanol, and finally rinsed with deionized water.The sections were dipped in potassium permanganate solu-tion (0.06% wt/vol) for 10 minutes, washed with deionizedwater, and further incubated with Fluoro-Jade B (0.0004%

vol/vol) and 0.1% vol/vol glacial acetic acid for 20 minutes

in the dark. The sections were washed with water, air-dried,dipped in xylene, and cover-slipped with dibutyl phthalatexylene containing DAPI (0.0002% vol/vol). The sectionswere visualized under the fluorescent light microscope at40� magnification (Leica DM6000 B).

2.9. Glutamic acid decarboxylase (GAD)immunoreactivity

The GAD immunostaining was performed as describedelsewhere (Hebb and Robertson, 2000) with some minormodifications. In brief, the incubation, blocking of nonspe-cific labeling, and minimization of endoperoxidase activitywere performed as described above for TH-immunoreactiv-ity. The sections were incubated with monoclonal antiglu-tamic acid decarboxylase antibody (1 : 500) in blockingbuffer at 4 °C for 48 hours, followed by 3 washings withphosphate-buffered saline (15 minutes each). Further incu-bation with secondary antibody, visualization, and imagingof sections were performed as described above for TH-immunoreactivity.

2.10. Statistical analysis

One-way (for dose and time response studies) and 2-wayanalysis of variance was used for comparisons betweengroups using Newman-Keuls and Bonferroni post-tests, re-spectively. The data are expressed as mean � standard errorof the mean (SEM). The differences were considered sta-tistically significant when the p value was less than 0.05.

3. Results

3.1. Selection of dose and time of exposure

As the reduced level of dopamine in the striatum isconsidered as one of the markers of dopaminergic neurode-generation, therefore, the level of dopamine was measuredin the animals treated with varying doses of cypermethrin(5–35 mg/kg body weight) for various time schedules inadult rats. Exposure to cypermethrin up to 10 mg/kg for 12weeks, showed no significant change in the level of dopa-mine in adult animals (Fig. 1a). Similarly, the animalsexposed to cypermethrin up to 10 mg/kg for 2–8 weeksduring adulthood did not exhibit significant change in thelevel of dopamine (data not shown). The animals treatedwith 15–35 mg/kg dose exhibited reductions in the level ofdopamine after 12 weeks of exposure (Fig. 1a). The animalsexposed to 15–35 mg/kg cypermethrin for 8 weeks alsoexhibited significant reduction in the dopamine level; thelevel was less than that of 12 weeks of exposure (data notshown). The reduction in dopamine level was higher at thedose of 20–35 mg/kg, the mortality was also observed in5%–10% of the animals, therefore, 15 mg/kg dose for 4–12weeks of exposure (Fig. 1b) was considered for furtherstudy. Increasing doses and long term exposure at moderatedoses led to the death of some animals in cypermethrin

treated groups. However, none of the animals treated with

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408 A.K. Singh et al. / Neurobiology of Aging 33 (2012) 404–415

low doses or short term exposure at moderate doses, alongwith their respective controls died. As all the animals wereobtained, stored, and sacrificed under similar conditions andprovided with similar food and water, therefore, higherdoses and long-term exposure to cypermethrin could be thecause of death among some of the treated animals ratherthan any other causes, such as sickness. The animals ex-posed during postnatal days did not exhibit any significantalteration in the level of dopamine compared with respec-tive controls when analyzed 2 months after the final treat-ment (Table 1).

3.2. Behavioral tests

The decrease in the time of stay on rotarod was sig-nificant in the animals treated with cypermethrin for 12weeks compared with that of controls during adulthood.In the animals treated with cypermethrin during the post-

Fig. 1. Dose- and time-dependent responses of cypermethrin on the dopa-mine level in the striatum of adult rats. (a) Dose-dependent response showsthe effect of varying doses of cypermethrin ranging from 5.0 to 35 mg/kgand (b) time of exposure-dependent response at a dose of 15 mg/kg in adultrats. The values are calculated as mean � standard error of the mean (n �

to 4). The data are expressed as ng/mg of tissue. Significant changes arexpressed as * (p � 0.05), ** (p � 0.01), and *** (p � 0.001) inomparison with controls.

natal period as well as well in adulthood, the decrease in

the time of stay on rotarod was more pronounced com-pared with that of animals exposed to cypermethrin onlyduring adulthood (Fig. 2a). Similarly, when SLA wasassessed, the distance traveled by cypermethrin-treatedadult animals exhibited a significant decrease in the caseof 8 and 12 weeks of exposure compared with controls.The locomotor impairment was more pronounced incypermethrin-treated postnatal animals re-exposed dur-ing adulthood compared with that in animals subjected toadulthood exposure only (Fig. 2b).

3.3. Levels of dopamine, its metabolites and serotonin intreated animals

The animals treated during postnatal days when rechal-lenged during adulthood for 4–12 weeks with cypermethrinshowed a more pronounced reduction in the level of dopa-mine compared with adulthood alone-exposed animals (Fig.3a). Level of DOPAC was significantly reduced in theanimals treated during adulthood in comparison with con-trols; the level of HVA was slightly increased. The animalstreated for 12 weeks exhibited a more pronounced changecompared with the animals treated for 4–8 weeks. Theeffects were higher in the animals treated with cypermethrinduring the postnatal period as well as in adulthood com-pared with the animals challenged during adulthood alone(Fig. 3b, c). No significant change in the level of serotoninwas observed in any treated groups compared with controls(Fig. 3d, Table 1).

3.4. TH-immunoreactivity

The number of TH-positive neurons was calculated andit was seen that the animals treated during adulthood for 12weeks exhibited a significant reduction in TH-immunoreac-tivity as well as the number of TH-positive neurons. Theanimals treated for 12 weeks exhibited a more pronouncedchange compared with animals treated for 4 – 8 weeks. Itwas observed that the extent of reduction in the number

Table 1Concentrations of dopamine, DOPAC, HVA, and serotonin in thestriatum and numbers of TH- and Fluoro-Jade B-positive cells in thesubstantia nigra of controls and animals treated postnatally withcypermethrin

Parameter (unit) Control Postnatal treated

Dopamine (ng/mg tissue) 10.63 � 1.02 9.62 � 0.82DOPAC (ng/mg tissue) 1.64 � 0.18 1.50 � 0.22HVA (ng/mg tissue) 0.93 � 0.10 0.96 � 0.06Serotonin (ng/mg tissue) 0.47 � 0.05 0.42 � 0.04TH-positive cells (percent of a control) 100 � 9 96 � 6Fluoro-Jade B positive cells (percent

of total cells)8.63 � 3.15 9.71 � 2.21

The data are expressed as mean � standard error of the mean (SEM). Therewere no significant changes in the postnatal alone-treated groups in any ofthe studied parameters compared with their respective controls.Key: DOPAC, 3,4-dihydroxyphenylacetic acid; HVA, homovanillic acid;TH, tyrosine hydroxylase.

of TH-positive neurons and immunoreactivity was

a

wadulthood alone-treated animals.

409A.K. Singh et al. / Neurobiology of Aging 33 (2012) 404–415

slightly higher in the animals treated postnatally as wellas in adulthood compared with the animals treated duringadulthood alone (Fig. 4a, b). The animals treated only forthe postnatal days did not exhibit a significant change inTH-immunoreactivity or in the number of TH-positiveneurons compared with respective controls (Table 1). Theeffects were more pronounced in the animals treated withcypermethrin both in postnatal and adulthood comparedwith the animals challenged during adulthood alone (Fig.4a, b). The ventral tegmental area of animals amongcontrol and the most severely affected set (12 weekscypermethrin-treated adult alone and postnatal � adult-treated) were compared (Fig. 4c) and no visual differencein the ventral tegmental area TH-immunoreactivity

Fig. 2. Cypermethrin induced changes on motor behavior in adult rats.Rotarod test of adult animals was performed at 15 mg/kg dose (a dose thatproduced significant reduction in dopamine level without any mortality inadult rats) in control and cypermethrin-exposed adult rats after 4, 8, and 12weeks of exposure. (a) The time of stay on rotarod was measured inseconds. (b) The spontaneous locomotor activity (SLA) in optovarimaxchamber was performed for controls and 4, 8, and 12 weeks for cyperme-thrin-exposed rats. The distance traveled by animals in the chamber wasrecorded in cm with an automated tracking device. The values are calcu-lated as mean � standard error of the mean (n � 3). The data are expressedas percentage of control. Significant changes are expressed as ** (p �0.01), *** (p � 0.001) in comparison with controls and # (p � 0.05), ##(p � 0.01), ### (p � 0.001) in comparison with adult alone-treatednimals.

among these groups was observed.

Fig. 3. Effect of cypermethrin on dopamine, its metabolites, and serotoninlevels in the striatum of control and cypermethrin-treated rats after 4–12weeks (15 mg/kg) during adulthood and postnatal � adulthood. Highperformance liquid chromatography (HPLC) analyses of (a) dopamine, (b)3,4-dihydroxyphenylacetic acid (DOPAC), (c) homovanillic acid (HVA),and (d) serotonin were performed in the striatum of control and treated rats.The values are calculated as mean � standard error of the mean (n � 3 to4). The data are expressed as ng/mg of tissue. Significant changes areexpressed as * (p � 0.05), ** (p � 0.01), *** (p � 0.001) in comparison

ith controls, and # (p � 0.05), ### (p � 0.001) in comparison with

a reated a

410 A.K. Singh et al. / Neurobiology of Aging 33 (2012) 404–415

3.5. NeuN/TH-immunoreactivity

The number of NeuN/TH-immunoreactive cell bodieswere counted. The animals treated during adulthood for8–12 weeks exhibited a significant reduction in NeuN/TH-immunoreactivity as well as in the number of TH-positive

Fig. 4. Tyrosine hydroxylase (TH) immunoreactivity of dopaminergic neuras observed by bright-field microscopy at 10� and 4� magnifications, rescypermethrin adult alone-treated (B, E, and H), and cypermethrin-treatedcontrol, cypermethrin-treated adult alone, and cypermethrin-treated postna(A) and 12-week cypermethrin-treated adult rats without (B) and with (C)of dopaminergic neurons in the treated animals compared with their respe(n � 3). The data are expressed as percentage of control. Significant channd # (p � 0.05), ### (p � 0.001) in comparison with adulthood alone t

cell bodies. The animals treated for 12 weeks exhibited a

more pronounced change compared with animals treated for8 weeks (Fig. 5a, b).

3.6. Fluoro-Jade B and DAPI staining

Fluoro-Jade B staining, specific for degenerating neu-

he substantia nigra pars compacta and ventral tegmental area of the brain,ly. (a) The representative TH immunoreactivity in control (A, D, and G),al � adult rats (C, F, and I). (b) The counting of TH-positive neurons inult rats. (c) TH-immunoreactivity in the ventral tegmental area of controll treatment, showing that there were no significant changes in the stainingontrols. The values are calculated as mean � standard error of the meanexpressed as ** (p � 0.01), *** (p � 0.001) in comparison with controlnimals.

ons in tpectivepostnattal � adpostnatactive cges are

rons, and DAPI staining, specific for total cells, were mon-

ac(

ted ani

411A.K. Singh et al. / Neurobiology of Aging 33 (2012) 404–415

itored in the substantia nigra of controls and treated animals.The number of Fluoro-Jade B positive cells was increasedagainst DAPI positive cells in 8- and 12-week-treated adultanimals compared with controls. The response was higher in12-week treated animals compared with 8-week treated an-imals. However, the increase in Fluoro-Jade B positive cellswas more pronounced in the animals treated with cyperme-thrin during postnatal as well as adulthood periods com-pared with the animals treated during adulthood alone (Fig.6a, b).

3.7. GAD-immunoreactivity

The effect of cypermethrin on GAD positive neurons in

Fig. 5. NeuN/tyrosine hydroxylase (TH) immunoreactivity of dopaminergby bright-field microscopy at 10� magnification. (a) The representative Nlone (B, E, and H) and cypermethrin-treated postnatal � adult (C, F, anontrol, cypermethrin-treated adult alone and cypermethrin-treated postnatn � 3). The data are expressed as percentage of control. Significant chan

and # (p � 0.05), ## (p � 0.01) in comparison with adulthood alone-trea

the substantia nigra was assessed. The GAD imunoreactiv-

ity did not exhibit major changes among the controls andtreated groups (Fig. 7).

4. Discussion

The study was performed with the aim to investigate theeffect of cypermethrin on the nigrostriatal dopaminergicneurodegeneration, if any. If yes, whether it is selective andwhat is the impact of cypermethrin in the animals, in thosethat were already exposed to it during a critical period ofdevelopment. Although the cypermethrin-mediated neuro-toxicity in developmental and adult animals has been re-ported by a number of investigators (Wolansky and Harrill,2008), its effect on dopaminergic neurodegeneration is yet

ns in the substantia nigra pars compacta region of the brain, as observedH-immunoreactivity in control (A, D, and G), cypermethrin-treated adults. (b) Counting of TH-immunoreactive neurons of the substantia nigra inult rats. The values are calculated as mean � standard error of the meanexpressed as ** (p � 0.01), *** (p � 0.001) in comparison with controlsmals.

ic neuroeuN/T

d I) ratal � adges are

elusive. Some investigations have shown that cypermethrin

flt* 0.01) i

412 A.K. Singh et al. / Neurobiology of Aging 33 (2012) 404–415

induces dopaminergic neurodegeneration in rats (Chugh etal., 1992; Nasuti et al., 2007), however, others have shownthat it induces dopaminergic neurodegeneration responseonly when induced by any other chemical (Mun et al.,2005). During the last decade, paraquat and maneb arereported to induce dopaminergic neurodegeneration in miceand rats after prolonged exposure (Patel et al., 2006;Thiruchelvam et al., 2002). Because these are also pesti-cides, therefore, in this study the effect of cypermethrin wasevaluated after prolonged exposure. Secondly, animals ex-posed to maneb and paraquat (1/10 of adult dose) duringpostnatal days were more susceptible to neurodegenerationwhen rechallenged during adulthood (Thiruchelvam et al.,2002). The effect of cypermethrin was therefore evaluatedin adults as well as in the animals exposed during bothpostnatal days and adulthood. Cypermethrin did not pro-duce any significant alteration in motor behavioral tests andthe levels of dopamine, DOPAC, and HVA in postnatal-alone treated animals. This could be because of the use of a

Fig. 6. (a) The representative images of Fluoro-Jade B/4=,6-diamidino-2-phin the substantia nigra region of control and cypermethrin-treated rats with

uorescent microscope at 40� magnification. The representative Fluoro-Jreated adults animals with or without postnatal cypermethrin exposure** (p � 0.001) in comparison with controls, and # (p � 0.05), ## (p �

relatively low dose of cypermethrin in the study. Secondly,

these measurements in postnatal treated animals were per-formed along with adult animals, i.e., more than 3 (with4-week-treated group) to 5 months (with 12-week-treatedanimals) after discontinuation of cypermethrin treatment.

The adult animals exposed up to 4 weeks did not exhibitreductions in behavioral and biochemical indices. This re-sult demonstrates that the responses produced by cyperme-thrin were not acute but were progressive and delayed. Theneurotoxicity responses, such as motor behavior and bio-chemical analyses of cypermethrin are dependent on thedose, route, and time of exposure as well as the time takento measure the indices after exposure (Wolansky and Har-rill, 2008). It is not possible to assess the relevance of thisdose for the general population or pesticide applicators at thisstage, as limited epidemiological studies have been conductedthat have shown the effects of cypermethrin on humans (Choiet al., 2006; Sudakin, 2006). Secondly, the findings of suchepidemiological studies have been variable because of theapplication of variable assessment tools for measuring the

ole (DAPI) stained cells and (b) counting of Fluoro-Jade B positive cellshout postnatal treatment (n � 3). The images were taken under the uprightDAPI, and merged images of control and 4–12 weeks of cypermethrin-en in the figure. Significant changes are expressed as ** (p � 0.01),

n comparison with adulthood alone-treated animals.

enylindand witade B,are giv

level of cypermethrin exposure (Choi et al., 2006; Sudakin,

sesmms

T

3). The

413A.K. Singh et al. / Neurobiology of Aging 33 (2012) 404–415

2006). However, on the basis of epidemiological observa-tions and the current experimental findings, it may be statedthat the study could possibly be relevant for general popu-lations or pesticide applicators, if they are repeatedly ex-posed to moderate doses of cypermethrin for a longer time.The progressive and delayed degeneration of the dopami-nergic neurons have been reported in pesticide-induced PDphenotype (Patel et al., 2006; Thiruchelvam et al., 2002).After long term exposure to cypermethrin, the treated ani-mals exhibited a decrease in the time spent on rotarod anddistance traveled during SLA. This is in accordance withseveral reports that have shown the reduced locomotor ac-tivity following cypermethrin treatment. Wolansky andHarill (2008) reviewed the literature and found that most ofthe studies have shown decreased activity while a limitednumber of studies have demonstrated a slight increase in theactivity. Therefore, the decreased behavioral response is inaccordance with most of the reported observations (Wolan-sky and Harrill, 2008).

The pronounced decrease in the behavioral and biochem-ical responses in the animals treated during the postnatalperiod as well as in adulthood compared with adulthoodalone-treated animals showed that during postnatal expo-sure, possibly cypermethrin exerts some irreversible butinvisible changes that severely appear in the animals duringadulthood exposure. In the postnatal period, brain developsand animals acquire many new motor and sensory abilitiesassociated with numerous biochemical changes that trans-form the developmental brain into mature adult brain (Dob-bing, 1975). During the critical period of development, thesynthesis of brain lipids and the turnover of proteins are attheir highest levels (Lajtha and Dunlop, 1981). Therefore,the possibility of permanent and irreversible changes is not

Fig. 7. The representative images of glutamate decarboxylase stained cellsrats with (B, E and H) and without postnatal treatment (C, F and I) (n �

a new phenomenon. This finding indicates that the rats, c

which were pretreated during postnatal days, become moresusceptible to cypermethrin exposure in adulthood. Thereductions in motor behavior could be due to dopaminergicneurodegeneration and increased dopamine turnover in thestriatum, as dopamine is known to regulate the locomotorfunctions of the body (Nasuti et al., 2007). The decrease intime of stay on rotarod could be due to degeneration in thebasal ganglia especially the substantia nigra pars compacta.

An accelerated dopamine turnover leads to the formationof reactive species, such as hydrogen peroxide and reactivequinine and semiquinine species produced by dopamineauto-oxidation (Makes et al., 1981). Dopamine undergoescatabolism by monoamine oxidase and catechol-O-methyltransferase to DOPAC and HVA (Jinsmaa et al., 2009;Marchitti et al., 2007). The reduced levels of dopamine andDOPAC but an increased level of HVA was seen in thisstudy. This result agrees with previous reports in whichcypermethrin and its analogue permethrin have shown anincrease in HVA level in rats (Nasuti et al., 2007). Thedecreased level of DOPAC in cypermethrin-treated animalsmay be described on the basis of decreased level of dopa-mine in these animals. The decrease in dopamine level aftercypermethrin exposure could be due to either inhibition ofbiosynthesis of dopamine or decrease in TH or decreasein aromatic-L-amino-acid decarboxylase synthesis, as ob-erved with deltamethrin (an analogue of cypermethrin)xposure (Liu and Shi, 2006). No significant changes inerotonin were observed in the cypermethrin-treated ani-als, showing the degeneration was probably of the dopa-inergic neurons and the serotoninergic neurons are not

ignificantly affected.Behavioral and biochemical analyses indicated loss of

H-positive neurons in the animals at intermediate doses of

substantia nigra region of control (A, D and G) and cypermethrin treatedimages were taken under the upright microscope at 20� magnification.

in the

ypermethrin for long term exposure. Measuring the level

414 A.K. Singh et al. / Neurobiology of Aging 33 (2012) 404–415

of TH-immunoreactivity and number of TH-positive neu-rons did the confirmation. As seen in the control and themost severely affected set (12 weeks), there was no visualdifference in the ventral tegmental area TH-immunoreactiv-ity among these groups, which indicated that neurons of thesubstantia nigra are mostly affected. NeuN/TH-immunore-activity is usually performed to demonstrate the neurode-generation more accurately and to count cell bodies in thesubstantia nigra as reported (McCoy et al., 2006). There-fore, the same was performed in this study. The results weresimilar as observed with TH-immunoreactivity, clearly in-dicating that prolonged exposure to cypermethrin inducesneurodegeneration in rats. The results showed that sub-chronic exposure to cypermethrin targets TH-positive do-paminergic neurons. Cypermethrin is highly lipophilic andreadily crosses the blood-brain barrier and cell membraneswithout any specific or active transport (Mun et al., 2005)and therefore, could damage the dopaminergic neurons. Theincreased Fluoro-Jade B positive cells in DAPI positivecells further provided support that cypermethrin causes do-paminergic neurodegeneration because Fluoro-Jade B stainsdegenerating neurons and DAPI stains all the cells, regard-less of specific insult or mechanism of cell death (Schmuedet al., 2005). Lack of major difference in the GAD immu-noreactivity indicated that cypermethrin effectively degen-erates TH-positive neurons in the substantia nigra.

Dopaminergic neurodegeneration was more pronouncedin the animals treated during postnatal days followed byadulthood compared with adulthood-alone exposure. Thisfinding is in accordance with behavioral and biochemicalobservations of the study. The reduced behavioral manifes-tations are likely to be associated with the severe loss of thenigrostriatal neurons as indicated by TH-immunoreactivity,Fluoro-Jade B/DAPI staining, and dopamine level. The pro-nounced effect in the animals treated during postnatal daysfollowed by adult re-exposure could be due to the phenom-enon of imprinting.

Conclusively, the study indicates that although cyperme-thrin does not induce dopaminergic neurodegeneration inthe animals exposed to low doses and short term exposure,its moderate doses for long term exposure induce dopami-nergic neurodegeneration without requiring any other stim-uli to initiate the dopaminergic neurotoxicity. Animals ex-posed to very low dose of cypermethrin during the postnatalperiod, the most critical period of neuronal development,are more susceptible to dopaminergic neurodegenerationduring adulthood if rechallenged with cypermethrin. Thiscould be due to the involvement of some imprinted genes/proteins that need to be investigated.

Disclosure statement

There are no actual or potential conflicts of interest.

The appropriate approval and procedures were used con-

cerning animals. The Institutional ethics committee for theuse of laboratory animals approved the study.

Acknowledgements

The authors acknowledge CSIR for providing researchfellowships to Anand Kumar Singh, Manindra Nath Tiwari,and Ghanshyam Upadhyay. The IITR communication num-ber of this article is 2781. This work was supported byCouncil of Scientific and Industrial Research (CSIR), NewDelhi through suprainstitutional project ”Investigative Tox-icology: New Paradigms” (SIP-08).

References

Anadon, A., Martinez-Larranaga, M.R., Fernandez-Cruz, M.L., Diaz, M.J.,Fernandez, M.C., Martinez, M.A., 1996. Toxicokinetics of deltame-thrin and its 4V–HO-metabolite in the rat. Toxicol. Appl. Pharmacol.141, 8–16.

Barbeau, A., Roy, M., Bernier, G., Campanella, G., Paris, S., 1987. Eco-genetics of Parkinson’s disease: prevalence and environmental aspectsin rural areas. Can. J. Neurol. Sci. 14, 36–41.

Bernheimer, H., Birkmayer, W., Hornykiewicz, O., Jellinger, K., Seitel-berger, F., 1973. Brain dopamine and the syndromes of Parkinson andHuntington. Clinical, morphological and neurochemical correlations.J. Neurol. Sci. 20, 415–455.

Bocchetta, A., Corsini, G.U., 1986. Parkinson’s disease and pesticides.Lancet 2, 1163.

Choi, H., Moon, J.K., Liu, K.H., Park, H.W., Ihm, Y.B., Park, B.S., Kim,J.H., 2006. Risk Assessment of Human Exposure to CypermethrinDuring Treatment of Mandarin Fields. Arch. Environ. Contam. Toxi-col. 50, 437–442.

Chugh, Y., Sankaranarayanan, A., Sharma, P.L., 1992. MK-801 antago-nizes the lethal action of centrally and peripherally administered cyper-methrin in mice and rats. J. Pharm. Pharmacol. 44, 521–523.

Cory-Slechta, D.A., Weiss, B., Cranmer, J., 2008. The environmentaletiologies of neurobehavioral deficits and disorders: weaving complexoutcomes and risk modifiers into the equation. Neurotoxicology 29,759–760.

Crawford, M.J., Croucher, A., Hutson, D.H., 1981. Metabolism of cis- andtrans-cypermethrin in rats. Balance and tissue retention study. J. Agric.Food Chem. 29, 130–135.

Curzon, G., Fernando, J.C.R., Marsden, C.A., 1978. Hydroxytryptamine:The Effects of Impaired Synthesis on its Metabolism and Release inRat. Br. J. Pharmacol. 63, 627–634.

DeVito, M.J., Wagner, G.C., 1989. Functional consequences followingmethamphetamine-induced neuronal damage. Psychopharmacology 97,432–435.

Dobbing, J., 1975. Prenatal nutrition and neurological development. UCLAForum Med. Sci. 18, 401–420.

Eells, J.T., Dubocovich, M.L., 1988. Pyrethroid insecticides evoke neu-rotransmitter release from rabbit striatal slices. J. Pharmacol. Exp.Ther. 246, 514–521.

Gilbert, M.E., Mack, C.M., Crofton, K.M., 1989. Pyrethroids and enhancedinhibition in the hippocampus of the rats. Brain Res. 477, 314–321.

Giray, B., Gurbay, A., Hincal, F., 2001. Cypermethrin-induced oxidativestress in rat brain and liver is prevented by Vitamin E or allopurinol.Toxicol. Lett. 118, 139–146.

Gorell, J.M., Johnson, C.C., Rybicki, B.A., Peterson, E.L., Richardson,R.J., 1998. The risk of Parkinson’s disease with exposure to pesticides,

farming, well water, and rural living. Neurology 50, 1346–1350.

415A.K. Singh et al. / Neurobiology of Aging 33 (2012) 404–415

Hebb, M.O., Robertson, H.A., 2000. Identification of a subpopulation ofsubstantia nigra pars compacta gamma-aminobutyric acid neuron thatis regulated by basal ganglia activity. J. Comp. Neurol. 416, 30–44.

Hornykiewicz, O., Kish, S.J., 1987. Biochemical pathophysiology of Par-kinson’s disease. Adv. Neurol. 45, 19–34.

Jinsmaa, Y., Florang, V.R., Rees, J.N., Anderson, D.G., Strack, S., Doorn,J.A., 2009. Products of oxidative stress inhibit aldehyde oxidation andreduction pathways in dopamine catabolism yielding elevated levels ofa reactive intermediate. Chem. Res. Toxicol. 22, 835–841.

Kale, M., Rathore, N., John, S., Bhatnagar, D., 1999. Lipid peroxidativedamage on pyrethroid exposure and alterations in antioxidant status inrat erythrocytes: a possible involvement of reactive oxygen species.Toxicol. Lett. 105, 197–205.

Kirby, M.L., Castagnoli, K., Bloomquist, J.R., 1999. In vivo effects ofdeltamethrin on dopamine neurochemistry and the role of augmentedneurotransmitter release. Pestic. Biochem. Physiol. 65, 160–168.

Klodowska-Duda, G., Jasinska-Myga, B., Safranow, K., Boczarska-Jedy-nak, M., Opala, G., 2005. The role of environmental factors in Parkin-son’s disease may depend on disease onset age. Neurol. Neurochir. Pol.39, 445–450.

Koller, W., Vetere-Overfield, B., Gray, C., Alexander, C., Chin, T., Dole-zal, J., Hassanein, R., Tanner, C., 1990. Environmental risk factors inParkinson’s disease. Neurology 40, 1218–1221.

Lajtha, A., Dunlop, D., 1981. Turnover of protein in the nervous system.Life Sci. 29, 755–767.

Langston, J.W., Ballard, P., Tetrud, J.W., Irwin, I., 1983. Chronic parkin-sonism in humans due to a product of meperidine analog synthesis.Science 219, 979–980.

Liu, G.P., Shi, N., 2006. The inhibitory effects of deltamethrin on dopa-mine biosynthesis in rat PC12 cells. Toxicol. Lett. 161, 195–199.

Makes, H.S., Weiss, C., Silides, D.J., Cohen, G., 1981. Coupling ofdopamine oxidation (monoamine oxidase activity) to glutathione oxi-dation via the generation of hydrogen peroxide in rat brain homoge-nates. J. Neurochem. 36, 589–593.

Malaviya, M., Husain, R., Seth, P.K., 1993. Perinatal effects of twopyrethroid insecticides on brain neurotransmitter function in the neo-natal rat. Vet. Hum. Toxicol. 35, 119–122.

McCoy, M.K., Martinez, T.N., Ruhn, K.A., Szymkowski, D.E., Smith,C.G., Botterman, B.R., Tansey, K.E., Tansey, M.G., 2006. Blockingsoluble tumor necrosis factor signaling with dominant-negative tumornecrosis factor inhibitor attenuates loss of dopaminergic neurons inmodels of Parkinson’s disease. J. Neurosci. 26, 9365–9375.

Manna, S., Bhattacharyya, D., Mandal, T.K., Dey, S., 2005. Neurophar-macological effects of alpha-cypermethrin in rats. Indian J. Pharmacol.37, 18–20.

Manna, S., Bhattacharyya, D., Mandal, T.K., Dey, S., 2006. Neurophar-macological effects of deltamethrin in rats. J. Vet. Sci. 7, 133–136.

Marchitti, S.A., Deitrich, R.A., Vasiliou, V., 2007. Neurotoxicity andmetabolism of the catecholamine-derived 3,4-dihydroxyphenylacetal-dehyde and 3,4-dihydroxyphenylglycolaldehyde: the role of aldehyde

dehydrogenase. Pharmacol. Rev. 59, 125–150.

Mochizuki, H., Hayakawa, H., Migita, M., Shibata, M., Tanaka, R., Su-zuki, A., Shimo-Nakanishi, Y., Urabe, T., Yamada, M., Tamayose, K.,Shimada, T., Miura, M., Mizuno, Y., 2001. An AAV-derived Apaf-1dominant negative inhibitor prevents MPTP toxicity as antiapoptoticgene therapy for Parkinson’s disease. Proc. Natl. Acad. Sci. U. S. A. 98,10918–10923.

Mun, J.Y., Lee, W.Y., Han, S.S., 2005. Effects of cypermethrin on thedopaminergic neurons in the progressive hemiparkinsonian rats. Toxi-col. Mech. Methods 15, 399–404.

Narahashi, T., Frey, J.M., Ginsburg, K.S., Roy, M.L., 1992. Sodium andGABA-activated channels as the targets of pyrethroids and cyclo-dienes. Toxicol. Lett. 64, 429–436.

Nasuti, C., Gabbianelli, R., Falcioni, M.L., Di Stefano, A., Sozio, P.,Cantalamessa, F., 2007. Dopaminergic system modulation, behavioralchanges and oxidative stress after neonatal administration of pyre-throids. Toxicology 229, 194–205.

Patel, S., Singh, V., Kumar, A., Gupta, Y.K., Singh, M.P., 2006. Status ofantioxidant defense system and expression of toxicant responsive genesin striatum of maneb and paraquat-induced Parkinson’s disease pheno-type in mouse: mechanism of neurodegeneration. Brain Res. 1081,9–18.

Saint-Pierre, M., Tremblay, M.E., Sik, A., Gross, R.E., Cicchetti, F., 2006.Temporal effects of paraquat/maneb on microglial activation and do-pamine neuronal loss in older rats. J. Neurochem. 98, 760–772.

Schmued, L.C., Stowers, C.C., Scallet, A.C., Xu, L., 2005. Fluoro-Jade Cresults in ultra high resolution and contrast labeling of degeneratingneurons. Brain Res. 1035, 24–31.

Sian, J., Dexter, D.T., Lees, A.J., Daniel, S., Jenner, P., Marsden, C.D.,1994. Glutathione-related enzymes in brain in Parkinson’s disease.Ann. Neurol. 36, 356–361.

Singh, C., Ahmad, I., Kumar, A., 2007. Pesticides and metals inducedParkinson’s disease: involvement of free radicals and oxidative stress.Cell. Mol. Biol. 53, 19–28.

Singh, S., Singh, K., Patel, D.K., Singh, C., Nath, C., Singh, V.K., Singh,R.K., Singh, M.P., 2009. The expression of CYP2D22, an ortholog ofhuman CYP2D6, in mouse striatum and its modulation in 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson’s disease phe-notype and nicotine-mediated neuroprotection. Rejuvenation Res. 12,185–197.

Sudakin, D.L., 2006. Pyrethroid insecticides: advances and challenges inbiomonitoring. Clin. Toxicol. 44, 31–37.

Staatz, C.G., Bloom, A.S., Lech, J.J., 1982. A pharmacological study ofpyrethroid neurotoxicity in mice. Pest. Biochem. Physiologist 17, 287–292.

Thiruchelvam, M., Richfield, E.K., Goodman, B.M., Baggs, R.B., Cory-Slechta, D.A., 2002. Developmental exposure to the pesticides paraquatand maneb and the Parkinson’s disease phenotype. Neurotoxicology23, 621–633.

Wolansky, M.J., Harrill, J.A., 2008. Neurobehavioral toxicology of pyre-throid insecticides in adult animals: A critical review. Neurotoxicol.

Teratol. 30, 55–78.