Involvement of dopaminergic and serotonergic systems in the neurobehavioral toxicity of lambda-cyhalothrin in developing rats

Toxicology Letters 211 (2012) 1–9

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Involvement of dopaminergic and serotonergic systems in the neurobehavioral toxicity of lambda-cyhalothrin in developing rats Reyaz W. Ansari a , Rajendra K. Shukla a , Rajesh S. Yadav a , Kavita Seth a , Aditya B. Pant a , Dhirendra Singh a , Ashok K. Agrawal a , Fakhrul Islam b , Vinay K. Khanna a,∗ a b

CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Post Box 80, MG Marg, Lucknow 226001, India Neurotoxicology Laboratory, Department of Medical Elementology and Toxicology, Jamia Hamdard, New Delhi 110062, India

a r t i c l e

i n f o

Article history: Received 5 July 2011 Received in revised form 13 January 2012 Accepted 15 February 2012 Available online 24 February 2012 Keywords: Lambda-cyhalothrin Rat brain Dopamine-D2 receptor Serotonin-2A receptor TH immunoreactivity

a b s t r a c t In view of extensive uses of lambda-cyhalothrin, a new generation type II synthetic pyrethroid, human exposure is quite imminent. The present study has therefore been carried out to investigate effect of lambda-cyhalothrin on brain dopaminergic and serotonergic systems and functional alterations associated with them. Post-lactational exposure to lambda-cyhalothrin (1.0 mg/kg or 3.0 mg/kg body weight, p.o.) from PD22 to PD49 caused a significant decrease in the motor activity and rota-rod performance in rats on PD50 as compared to controls. Decrease in motor activity in lambda-cyhalothrin treated rats was found to persist 15 days after withdrawal of exposure on PD65 while a trend of recovery in rota-rod performance was observed. A decrease in the binding of 3 H-Spiperone, known to label dopamine-D2 receptors in corpus striatum associated with decreased expression of tyrosine hydroxylase (TH)-immunoreactivity and TH protein was observed in lambda-cyhalothrin treated rats on PD50 and PD65 compared to controls. Increase in the binding of 3 H-Ketanserin, known to label serotonin-2A receptors in frontal cortex was observed in lambda-cyhalothrin exposed rats on PD50 and PD65 as compared to respective controls. The changes were more marked in rats exposed to lambda-cyhalothrin at a higher dose (3.0 mg/kg) and persisted even 15 days after withdrawal of exposure. The results exhibit vulnerability of developing rats to lambda-cyhalothrin and suggest that striatal dopaminergic system is a target of lambda-cyhalothrin. Involvement of serotonin-2A receptors in the neurotoxicity of lambda-cyhalothrin is also suggested. The results further indicate that neurobehavioral changes may be more intense in case exposure to lambda-cyhalothrin continues. © 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Synthetic pyrethroids are widely used insecticides in agriculture and home formulations because of their high bioefficacy (Casida and Quistad, 1998; Fetoui et al., 2009; Jurisic et al., 2010). It has been estimated that the use of synthetic pyrethroids is several times more as compared to other class of insecticides due to their wide safety margin and environmental compatibility (Shafer et al., 2005). Pyrethroids chemically are the structural derivatives of naturally occurring pyrethrins and have greater potency. Based on the structural differences, the pyrethroids are broadly classified into two types. Type I class of pyrethroids do not contain ␣-cyano group while type II class of pyrethroids have a cyano group at ␣ position and thus their clinical symptoms also vary (Verschoyle

∗ Corresponding author at: Developmental Toxicology, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Post Box 80, MG Marg, Lucknow 226 001, India. Tel.: +91 522 2620207/2620107; fax: +91 522 2628227. E-mail address: [email protected] (V.K. Khanna). 0378-4274/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2012.02.012

and Aldridge, 1980; Soderlund et al., 2002; Ray and Fry, 2006). Risk of human exposure to pyrethroids has significantly enhanced in recent times due to their increased consumption and extensive usage. Although pesticides are intended to be used to control pests, their excessive and injudicious use may affect the non-target species including humans. Further, a link between insecticide exposure with increasing incidences of Parkinson’s disease, as revealed by a number of epidemiological studies, has caused a great concern among the health scientists (Semchuk et al., 1992; Butterfield et al., 1993; Brown et al., 2006). Besides, exposure to pesticides in humans may also cause subtle to severe neurophysiological and neurobehavioral abnormalities (Shafer et al., 2005; Wolansky et al., 2006; Wolansky and Harrill, 2008). Population studies on biomonitoring of pyrethroids have been carried out to assess the risk of human exposure (Heudorf and Angerer, 2001; Schettgen et al., 2004; Barr et al., 2010). Children have been found to have higher exposure than adolescent and adults as indicated by the presence of their metabolites in the urine (Heudorf et al., 2004; Barr et al., 2010). Metabolites of pyrethroids have also been found in the urine of pre-school and elementary age

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children as a result of residential exposure (Morgan et al., 2007; Lu et al., 2006, 2009). Detection of pyrethroid metabolites in the urine of pregnant women has further raised health concern to the risk of exposure of fetus and developing children (Whyatt et al., 2002; Berkowitz et al., 2003). Numerous experimental studies have been carried out to understand the mechanism of neurobehavioral toxicity of both type I and type II pyrethroids (Ahlbom et al., 1994; Aziz et al., 2001; Eriksson and Nordberg, 1990; Eriksson and Fredriksson, 1991; Gupta et al., 1999a,b; Husain et al., 1992, 1994; Talts et al., 1998; Malaviya et al., 1993; Patro et al., 1997; Tsuji et al., 2002). Delay in development of physical milestone and reflexes, impairment in motor activity, grip strength and other behaviors associated with neurochemical alterations has been reported following pyrethroid exposure (Husain et al., 1992; Malaviya et al., 1993; McDaniel and Moser, 1993; Hassouna et al., 1996; Imamura et al., 2000; Dayal et al., 2003; Shafer et al., 2005; Wolansky et al., 2006). However, consistent changes have not been observed and could be due to variation in route, dose and duration of treatment and time of exposure. As pyrethroids have differential neurobehavioral effects, there is a need of well designed and concerted studies for investigating the mechanism of their developmental neurobehavioral toxicity. Lambda-cyhalothrin, a new generation type II synthetic pyrethroid has extensive uses as an agro-pesticide (Mathirajan et al., 2000; Gu et al., 2007; Fetoui et al., 2009). It is widely used to protect food and non-food crops (Mathirajan et al., 2000; Gu et al., 2007; Seenivasan and Muraleedharan, 2009). Besides, lambdacyhalothrin has veterinary application on farm animals to prevent and control ectoparasites (Davies et al., 2000; Abbud Righi and Palermo-Neto, 2003; Lawler et al., 2007) and in public health program (Fetoui et al., 2008, 2009). Residues of lambda-cyhalothrin have been reported in vegetables and fruits (Amoah et al., 2006; Mohapatra and Ahuja, 2010; Turgut et al., 2011), milk and blood of dairy cows (Bissacot and Vassilieff, 1997) and also in cattle meat (Muhammad et al., 2010). Placental transfer of lambda-cyhalothrin has been observed in goats (Oliveira et al., 2000). Persistence of lambda-cyhalothrin on different indoor surfaces especially used against malaria vector in malaria epidemic prone areas has been reported (Mulambalah et al., 2010). Although levels of lambdacyhalothrin in natural conditions are low, intense and continuous usage of pesticides may build up high levels in the environment and thus enhance the risk of human exposure including growing children. The primary effect of pyrethroids on the voltage sensitive sodium channels disrupting the functioning of the nervous system both in insects and mammals is well established (Narahashi, 1996; Hossain and Richardson, 2011). A number of other mechanisms including their action on voltage sensitive calcium channels, voltage sensitive chloride channels, GABAA receptors and modulation in the release of neurotransmitters especially acetylcholine, dopamine (DA) and serotonin have also been suggested in their neurotoxicity (Narahashi, 1996; Martinez-Larranaga et al., 2003; Hossain et al., 2004; Shafer et al., 2005; Ray and Fry, 2006; Brown et al., 2006; Nasuti et al., 2007; Breckenridge et al., 2009). Studies on lambda-cyhalothrin and cyhalothrin (similar structural analog) have been carried out to assess their toxic potential on experimental models (DeMicco et al., 2010). Lambda-cyhalothrin has been found to be genotoxic and cause toxicopathological alterations (Campana et al., 1999; Celik et al., 2003, 2005; Basir et al., 2011). Besides, it has been found to affect the functioning of the brain involving dopaminergic, cholinergic and serotonergic systems (Hossain et al., 2004, 2005, 2006; Martinez-Larranaga et al., 2003). Recently, we found that post-lactational exposure of rats to lambda-cyhalothrin affected their learning and decreased the muscarinic-cholinergic receptors in brain. These changes were found to be associated with increased brain oxidative stress (Ansari

et al., 2012). Dopaminergic system has been found to be a target for both type I and type II pyrethroids with differential effects on the extracellular dopamine (Hossain et al., 2004). Although, it has been found that DA release and DA uptake are inhibited by cyhalothrin, effect on tyrosine hydroxylase, a rate limiting enzyme in the dopaminergic pathway and DA-D2 receptors, intimately involved to regulate motor behavior and motor coordination, is not understood. While, it has been found that lambda-cyhalothrin like other type II pyrethroids affects the serotonergic neurotransmission by altering the 5-HT turnover (Martinez-Larranaga et al., 2003), its effect on serotonin-2A receptor is also not known. Alterations in the dopaminergic system following exposure to lambda-cyhalothrin have been reported at a very high dose to understand the acute effect (Hossain et al., 2004, 2006). The present study has therefore been carried out following post-lactational exposure of developing rats from postnatal day (PD)22 to PD49 to lambda-cyhalothrin and effect on brain dopamine and serotonin receptors and functional alterations associated with them studied on PD50. To further understand whether these changes are transient or persistent, effect on neurobehavioral endpoints was studied 15 days after the withdrawal of lambda-cyhalothrin exposure on PD65. Acute effects of cyhalothrin/lambda-cyhalothrin have been studied at doses ranging from 10 to 80 mg/kg body weight in experimental studies on rats (Hossain et al., 2004, 2005, 2006; Mate et al., 2010). Effect of repeated exposure to lambda-cyhalothrin on genotoxicity, neurotoxicity, developmental/reproductive toxicity has been studied at doses from 0.8 to 61.2 mg/kg body weight in rats (Celik et al., 2003, 2005; Martinez-Larranaga et al., 2003; Ratnasooriya et al., 2003; Fetoui et al., 2008). In the present study, selection of doses (1.0 mg/kg and 3.0 mg/kg body weight) which are 1/60th and 1/20th respectively of LD50 of lambda-cyhalothrin was based on the NOAEL reported to be 2.5 mg/kg body weight in a 90-day oral study on rats (WHO, 1990; Lambda-cyhalothrin; Pesticide Tolerances, 1998). 2. Materials and methods 2.1. Animals and treatment Female rats of Wistar strain (21 days old, PD21) weighing around 24 ± 2 g, obtained from the central animal house of CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Lucknow were used in the study. Rats were housed in an air conditioned room at a temperature 25 ± 2 ◦ C with a 12-h light/dark cycle under standard hygiene conditions and had free access to pellet diet, procured from national supplier and water. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) of CSIR-IITR, Lucknow and all the experimental procedures were carried out in accordance with the guidelines laid down by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forests (Government of India), New Delhi, India. Rats were randomly divided in to three groups on PD22. In two groups, rats were administered lambda-cyhalothrin (Syngenta, India, 5% EC, suspended in corn oil) daily at either of the doses (1.0 mg/kg or 3.0 mg/kg body weight, p.o.) till PD49. The third group of rats was administered corn oil in an identical manner and served as control. Effect on behavioral parameters – motor activity and motor coordination was studied on PD50. A separate set of rats was sacrificed on PD50 for neurochemical and immunohistochemical assays. To study whether neurobehavioral changes are transient or persistent, a set of rats was left as such till PD64 and monitored daily. Effect on selected behavioral, neurochemical and immunohistochemical endpoints was studied on PD65. 2.2. Behavioral studies 2.2.1. Spontaneous motor activity Spontaneous motor activity was assessed using computerized Actimot (TSE, Germany) following the standard procedure as described by Yadav et al. (2009). The activity monitor operates on light beam principle and is equipped with high number and density of infrared beams (32 × 32). Briefly, rats were acclimatized for half an hour at room temperature. Following this, they were individually placed in the cage (45 cm × 45 cm). As soon as the animals are placed in the cage, sensors become active and counting device starts. Activity was monitored for 5 min and parameters viz. distance traveled, resting time, time moving and stereotypic time were recorded simultaneously.

R.W. Ansari et al. / Toxicology Letters 211 (2012) 1–9 2.2.2. Rota-rod performance Effect on motor coordination was studied in control and lambda-cyhalothrin exposed rats using Rotamex (Columbus Instruments, USA) and time of fall from the rotating rod recorded following the standard protocol (Yadav et al., 2009). In short, a set of rats in each treatment group was conditioned to the accelerating rotating rod individually. Each animal received training on the rota-rod (rotating at a constant speed of 8 rpm) until it achieved a criterion of staying on the rod for 60 s. The rats then received a single baseline training on the rota-rod in which the speed was increased from 4 to 40 rpm over a period of 5 min. Finally, each rat received another trial on the rota-rod, 24 h after the pre-trial and scoring was carried out by a person blind to the treatment conditions. Motor coordination was quantified as the ability of the rat to remain balanced on the rotating rod.

2.3. Neurochemical studies For neurochemical studies, rats were sacrificed by cervical dislocation. Brains were removed rapidly, placed on ice and dissected into specific regions (corpus striatum and frontal cortex) following the standard procedure (Glowinski and Iversen, 1966). Brain regions were stored at −80 ◦ C for the assay of neurotransmitter receptors and other neurochemical parameters.

2.3.1. Assay of dopamine-D2 and serotonin-2A receptors in corpus striatum and frontal cortex of rat brain Assay of dopamine-D2 receptors in corpus striatum and serotonin-2A receptors in frontal cortex was carried out by the radioligand receptor binding assays following the standard procedure (Khanna et al., 1994). Briefly, crude synaptic membrane was prepared by homogenizing the brain regions (corpus striatum and frontal cortex) in 19 volumes of Tris–HCl buffer (5 mM, pH 7.4). The homogenate was centrifuged at 40,000 × g for 15 min at 4 ◦ C. The sedimented pellet was washed twice by resuspending in homogenization buffer and recentrifuged at the same speed for 15 min at 4 ◦ C. Finally, the pellet was suspended in Tris–HCl buffer (40 mM, pH 7.4) and stored at −20 ◦ C for binding assays. Binding incubations in a final volume of 1.0 ml were carried out in triplicate. Assay of dopamine-D2 receptor was performed using 3 H-Spiperone (18.5 Ci/mmol, 1 × 10−9 M) as the radioligand and haloperidol (1 × 10−6 M) as competitor. For serotonin (5HT)-2A receptors, 3 H-Ketanserin (85 Ci/mmol, 1 × 10−9 M) was used as a radioligand and cinanserin (1 × 10−5 M) as competitor. The reaction mixture containing Tris–HCl buffer (40 mM, pH 7.4), together with membrane protein (300–400 ␮g) and appropriate radioligand was incubated for 15 min at 37 ◦ C in the presence or absence of competitor to assess the non-specific and total binding respectively. At the end of incubation, contents of the binding tubes were immediately filtered on glass fiber discs (25 mm diameter, 0.3 ␮m pore size, Whatman GF/B) and washed twice rapidly with 5 ml chilled Tris–HCl buffer (40 mM, pH 7.4). Filters were dried and transferred into vials and scintillation mixture (2,5-diphenyl oxazole; 1,4-bis-5, phenylozazolyl-benzene; naphthalene; toluene; methanol and 1,4 dioxane) added to it. The radioactivity was counted on ␤-scintillation counter (Packard, USA) at an efficiency of 30–40% for 3 H to determine membrane bound radioactivity. Specific binding was determined by subtracting the non-specific binding from the total binding and has been expressed as pmoles ligand bound/g protein. Scatchard analysis was carried out at varying concentrations of radioligands (generally 1/10 to 10 times of the affinity) to ascertain whether change in the binding is due to alteration in the affinity (Kd ) or number of receptor binding sites (Bmax ).

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2.3.2. Protein estimation Protein concentration in sample homogenates was measured following the method of Lowry et al. (1951) using bovine serum albumin (BSA) as the reference. 2.4. Western blotting Expression of tyrosine hydroxylase (TH) protein in the corpus striatum was assayed following the method of Jamal et al. (2007). Briefly, the samples (30 ␮g protein/lane) were electrophoresed on 12% SDS–PAGE, electroblotted to nitrocellulose membranes, blocked with blocking buffer and incubated with primary antibody (Anti-TH, Sigma, USA, 1:2000) followed by incubation with horseradish peroxidase-linked secondary antibody (anti-mouse IgG, 1:4000) at room temperature for 60 min. After the incubation, blots were washed and developed using an immobilon western chemiluminescent HRP substrate (Millipore, USA) following the recommended procedure. ␤-Actin was probed as an internal control and used to confirm that an equal amount of protein was loaded in each lane. A digital gel image analysis system (VersaDoc, Model 1000, Bio Rad) was used for semi-quantification of TH immunoreactivity. 2.5. Immunohistochemistry Immunohistochemical studies were carried out following the method of Goslin et al. (1990). Briefly, rats were anesthetized and perfused with 150 ml of phosphate-buffered saline (PBS, 0.1 M. pH 7.4) followed by 250 ml of ice-cold 4% paraformaldehyde in PBS for fixation of tissues. Brains were removed and post fixed in 10% paraformaldehyde in PBS and samples were kept in 10%, 20%, and 30% (w/v) sucrose in PBS. Serial coronal sections of 20-␮m thickness were cut on a cryomicrotome (Microm HM 520, Labcon, Germany), incubated with primary (anti-tyrosine hydroxylase, Sigma, USA, 1:200) and secondary antibodies (biotinylated peroxidase linked, Sigma USA, 1:400) and processed as per protocol. The intensity of tyrosine hydroxylase positive neurons in striatum of brain was determined using a computerized image analysis system (Leica Qwin 500 image analysis software) as described by Shingo et al. (2002). 2.6. Statistical analysis The data were analyzed using one way analysis of variance (ANOVA) followed by Newman–Keuls test for multiple pair wise comparisons among various groups. All values have been expressed as mean ± SEM. Value up to p < 0.05 has been considered significant.

3. Results 3.1. Post-lactational exposure to lambda-cyhalothrin and effect on body weight and brain to body weight ratio of rats Post-lactational exposure to lambda-cyhalothrin (1.0 mg/kg or 3.0 mg/kg body weight) from PD22 to PD49 caused a significant decrease in the body weight (F(2,21) = 16.3, p < 0.001, 18%, p < 0.001, 23%) of rats on PD50 as compared to controls (Fig. 1a). Although a trend of recovery was observed in the body weight 15 days after

Table 1 Effect on different parameters related with spontaneous motor activity following post-lactational exposure of rats to lambda-cyhalothrin for 28 days and 15 days after withdrawal of exposure. Parameter

Control

Treatment groups LCT I (1 mg/kg)

Treatment for 28 days Total distance Resting time Time moving Stereotypic time Time rearing 15 days after withdrawal of exposure Total distance Resting time Time moving Stereotypic time Time rearing

LCT II (3 mg/kg)

2290 198 101 139 19

± ± ± ± ±

133 3.05 3.05 21.2 4.14

1454 237 62 129 10.2

± ± ± ± ±

310* 14.0* 14.0* 4.50 2.97

1216 249 50 104 10

± ± ± ± ±

10* 9 5.42* 5.42* 11.7 1.30

3220 189 112 151 31.6

± ± ± ± ±

272 7.26 8.29 14.5 5.52

2493 206 93 139 21.8

± ± ± ± ±

86.3* 2.60 2.60* 7.67 2.56

1862 230 69 120 14.1

± ± ± ± ±

212* 9.00 9.00* 14.1 1.70*

Rats were exposed to lambda-cyhalothrin (1.0 mg/kg or 3.0 mg/kg body weight/day, p.o.) from post-lactational day (PD)22 to PD49. Effect on different parameters related with spontaneous motor activity studied 28 days after exposure of lambda-cyhalothrin on PD50. To assess whether changes are transient or persistent, effect on spontaneous motor activity also studied 15 days after withdrawal of lambda-cyhalothrin exposure on PD65. Data have been analyzed by one way analysis of variance followed by Newman–Keuls test. Values are mean ± SEM of five animals in each group. * Significantly differs from control group (p < 0.05). Values of total distance expressed in cm while resting time, time moving, stereotypic time and time rearing expressed in second.

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Fig. 1. Effect on body weight (a) and brain to body weight ratio (b) following post-lactational exposure of rats to lambda-cyhalothrin. Rats were exposed to lambda-cyhalothrin (1.0 mg/kg or 3.0 mg/kg body weight/day, p.o.) from post-lactational day (PD)22 to PD49. Effect on these parameters studied 28 days after exposure of lambda-cyhalothrin on PD50. To assess whether changes are transient or persistent, effect on body weight (a) and brain to body weight ratio (b) also studied 15 days after withdrawal of lambdacyhalothrin exposure on PD65. Data have been analyzed by one way analysis of variance followed by Newman–Keuls test. Values are mean ± SEM of 10 rats in case of body weight and five rats in case of brain to body weight ratio. *Significantly differs from control group (p < 0.05).

withdrawal of exposure, the body weight in lambda-cyhalothrin treated rats at a higher dose (3.0 mg/kg body weight) remained significantly decreased (F(2,19) = 4.42, p < 0.05, 10%) on PD65 as compared to respective controls (Fig. 1a). No significant change in the absolute brain weight was observed in rats exposed to lambda-cyhalothrin at either of the doses (1.0 mg/kg or 3.0 mg/kg body weight). However, the brain to body weight ratio was significantly increased (F(2,11) = 27.45, p < 0.001, 23%, p < 0.001, 23%) in rats exposed to lambda-cyhalothrin at both the doses (1.0 mg/kg or 3.0 mg/kg body weight). Increased brain to body weight ratio remained persisted (F(2,10) = 5.92, p < 0.05, 10%) in rats exposed to lambda-cyhalothrin at a higher dose (3.0 mg/kg body weight) 15 days after withdrawal of exposure on PD65 (Fig. 1b). 3.2. Behavioral studies 3.2.1. Post-lactational exposure to lambda-cyhalothrin and effect on spontaneous motor activity of rats: Effect of lambda-cyhalothrin exposure in rats on different parameters of motor activity was studied on PD50 and PD65 and results are summarized in Table 1. Post-lactational exposure to lambda-cyhalothrin (1.0 mg/kg or 3.0 mg/kg body weight) from PD22 to PD49 caused a decrease in distance traveled (F(2,12) = 7.58, p < 0.05, 136%, p < 0.01, 46%), moving time (F(2,12) = 9.00, p < 0.01, 38%, p < 0.01, 50%), stereotypic time (F(2,7) = 1.72, p > 0.05, 7%, p > 0.05, 25%) and rearing (F(2,12) = 2.99, p > 0.05, 47%, p > 0.05, 47%) of rats on PD50 as compared to controls. A significant increase (F(2,12) = 9.00, p < 0.01, 19%, p < 0.01, 25%) in resting time was also observed in rats exposed to lambda-cyhalothrin at both the doses in comparison to the respective controls (Table 1). The changes were more marked in rats exposed to lambda-cyhalothrin at a higher dose. Further, decrease in distance traveled (F(2,15) = 10.9, p < 0.05, 22%, p < 0.01, 42%), moving time (F(2,15) = 13.4, p < 0.05, 16%, p < 0.001, 38%), stereotypic time (F(2,8) = 1.55, p > 0.05, 7%, p > 0.05,

20%), rearing time (F(2,15) = 5.28, p > 0.05, 31%, p < 0.05, 54%) and increase in resting time (F(2,15) = 1.56, p > 0.05, 8%, p > 0.05, 30%) was found to persist in lambda-cyhalothrin exposed rats even 15 days after withdrawal of exposure on PD65 as compared to respective controls (Table 1). 3.2.2. Post-lactational exposure to lambda-cyhalothrin and effect on rota-rod performance of rats A significant impairment in the rota-rod performance was observed in rats on PD50 following post-lactational exposure to lambda-cyhalothrin (1.0 mg/kg or 3.0 mg/kg body weight) from PD22 to PD49. The time of fall from the rotating rod was significantly decreased (F(2,9) = 14.1, p < 0.01, 36%, p < 0.01, 55%) in rats exposed to lambda-cyhalothrin suggesting impairment in motor coordination (Fig. 2). Although, a trend of recovery in the rotarod performance was observed in lambda-cyhalothrin treated rats 15 days after withdrawal of exposure, the rota-rod performance remained impaired (F(2,15) = 0.40, p > 0.05, 8%, p > 0.05, 15%) at both the doses respectively (Fig. 2). 3.3. Neurochemical studies 3.3.1. Post-lactational exposure to lambda-cyhalothrin and effect on brain dopamine-D2 and serotonin-2A receptors of rats Post-lactational exposure to lambda-cyhalothrin (1.0 mg/kg or 3.0 mg/kg body weight) in rats from PD22 to PD49 caused a significant decrease (F(2,11) = 7.16, p < 0.05, 25%, p < 0.05, 29%) in the binding of 3 H-Spiperone to striatal membranes, known to label dopamine-D2 receptors as compared to controls (Fig. 3a). The decrease in the binding was found due to decrease in the number of binding sites (Bmax ) as assessed by the Scatchard analysis (Table 2). The decrease in the binding of striatal dopamine receptors in lambda-cyhalothrin treated rats was found to persist even 15 days after withdrawal of exposure as evident by a decrease (F(2,10) = 4.02, p > 0.05, 23%, p > 0.05, 24%) in the

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Fig. 2. Effect on rota-rod performance following post-lactational exposure of rats to lambda-cyhalothrin. Rats were exposed to lambda-cyhalothrin (1.0 mg/kg or 3.0 mg/kg body weight/day, p.o.) from post-lactational day (PD)22 to PD49. Effect on rota-rod performance and time of fall from rotating rod studied 28 days after exposure of lambdacyhalothrin on PD50.To assess whether changes are transient or persistent, effect on rota-rod performance also studied 15 days after withdrawal of lambda-cyhalothrin exposure on PD65. Data have been analyzed by one way analysis of variance followed by Newman–Keuls test. Values are mean ± SEM of five animals in each group. *Significantly differs from control group (p < 0.05).

binding of 3 H-Spiperone to striatal membranes at both the doses respectively (Fig. 3a). An increase in the binding of 3 H-Ketanserin to frontocortical membranes, known to label serotonin-2A receptors (F(2,7) = 5.23, p > 0.05,14%, p < 0.05, 26%) was observed in rats on PD50 following post-lactational exposure to lambda-cyhalothrin (1.0 mg/kg or 3.0 mg/kg body weight) from PD22 to PD49 as compared to controls (Fig. 3b). The increase in the binding was more pronounced in rats exposed to lambda-cyhalothrin at a higher dose. Scatchard analysis revealed that increase in binding of 3 H-Ketanserin to frontocortical membranes was due to increased number of receptor binding sites (Table 2). Although, the binding of serotonin-2A receptors in frontocortical membranes exhibited a trend of recovery in rats treated with lambda-cyhalothrin at a lower dose (1.0 mg/kg body weight) 15 days after withdrawal of exposure, increase (F(2,10) = 8.70, p < 0.01, 24%) in the binding of frontocortical serotonin-2A receptors in rats treated with lambda-cyhalothrin

at a higher dose (3.0 mg/kg body weight) remained persisted even 15 days after withdrawal of exposure in comparison to respective controls (Fig. 3b).

3.4. Western blot analysis 3.4.1. Post-lactational exposure to lambda-cyhalothrin and effect on the expression of tyrosine hydroxylase protein in striatum of rat brain Post-lactational exposure to lambda-cyhalothrin (1.0 mg/kg or 3.0 mg/kg body weight) from PD22 to PD49 in rats caused a significant decrease in the expression of TH protein in corpus striatum (2.1 fold, 3.6 fold) as compared to controls (Fig. 4). A trend of recovery was observed in the expression of TH (0.5 fold) protein in rats exposed to lambda-cyhalothrin at a low dose while expression of TH protein (2.8 fold) remained decreased in rats

Fig. 3. Effect on striatal dopamine-D2 (a) and frontocortical serotonin-2A (b) receptors following post-lactational exposure of rats to lambda-cyhalothrin. Rats were exposed to lambda-cyhalothrin (1.0 mg/kg or 3.0 mg/kg body weight/day, p.o.) from post-lactational day (PD)22 to PD49. Effect on striatal dopamine-D2 (a) and frontocortical serotonin2A (b) receptors studied 28 days after exposure of lambda-cyhalothrin on PD50. To assess whether changes are transient or persistent, effect on striatal dopamine-D2 receptors also studied 15 days after withdrawal of lambda-cyhalothrin exposure on PD65. Data have been analyzed by one way analysis of variance followed by Newman–Keuls test. Values are mean ± SEM of five animals in each group. *Significantly differs from control group (p < 0.05).

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Fig. 4. Effect on the expression of tyrosine hydroxylase protein in corpus striatum following post-lactational exposure of rats to lambda-cyhalothrin. Rats were exposed to lambda-cyhalothrin (1.0 mg/kg or 3.0 mg/kg body weight/day, p.o.) from post-lactational day (PD)22 to PD49. Effect on the expression of tyrosine hydroxylase protein studied 28 days after exposure of lambda-cyhalothrin on PD50. To assess whether changes are transient or persistent, effect on the expression of tyrosine hydroxylase protein also studied 15 days after withdrawal of lambda-cyhalothrin exposure on PD65. Data have been analyzed by one way analysis of variance followed by Newman–Keuls test. Values are mean ± SEM of three animals in each group. *Significantly differs from control group (p < 0.05).

exposed to lambda-cyhalothrin at a higher dose (3.0 mg/kg) 15 days after withdrawal of exposure (Fig. 4). 3.5. Immunohistochemical studies 3.5.1. Post-lactational exposure to lambda-cyhalothrin and effect on the expression of tyrosine hydroxylase in corpus striatum of rats Post-lactational exposure to lambda-cyhalothrin (1.0 mg/kg or 3.0 mg/kg body weight) from PD22 to PD49 in rats caused a significant decrease in the TH immunoreactivity in corpus striatum as compared to controls (Fig. 5). Quantification of immunoreactivity revealed that the percent area in TH expression was decreased (p < 0.05, 34%, p < 0.01, 88%) in rats exposed to lambda-cyhalothrin at both the doses respectively. A trend of recovery was observed in the TH immunoreactivity of rats exposed to lambda-cyhalothrin at a low dose (p > 0.05, 17%) while TH expression remained decreased (p < 0.01, 76%) in rats exposed to lambda-cyhalothrin at a higher dose 15 days after withdrawal of exposure (Fig. 5). 4. Discussion A large number of evidences have shown that dopamine, a major neurotransmitter is widely distributed in the CNS and regulates pharmacological and physiological functions (Husain et al., 1996; Missale et al., 1998; Cooper et al., 2003). Involvement of Table 2 Scatchard analysis of 3 H-Spiperone binding to striatal and 3 H-Ketanserin binding to frontocortical membranes of rats following post-lactational exposure to lambdacyhalothrin for 28 days. Brain region/binding

Control

Corpus striatum/ 3 H-Spiperone 1.12 ± Kd Bmax 880 ± 3 Frontal cortex/ H-Ketanserin 1.13 ± Kd 592 ± Bmax

Treatment groups LCT I (1.0 mg/kg)

LCT II (3.0 mg/kg)

0.12 79

1.21 ± 0.15 587 ± 51*

0.92 ± 0.18 561 ± 51*

0.12 54

1.01 ± 0.15 660 ± 69

1.27 ± 0.18 754 ± 75*

Values are mean ± SEM of five animals in each group. Kd : dissociation constant expressed in nM, Bmax : maximum number of binding sites expressed in pmoles. 3 H-Spiperone and pmoles. 3 H-Ketanserin bound/g protein. * Significantly differs from control group (p < 0.05).

the dopaminergic system in many psychiatric and neurological disorders including Parkinson’s disease has been reported (Shafer et al., 2005). As dopaminergic system has been found to be a target of many insecticides including pyrethroids (Karen et al., 2001; Nasuti et al., 2007), exposure to pyrethroids has been associated with environmentally induced Parkinson’s disease (Nasuti et al., 2007). Upregulation of dopamine transporter (Elwan et al., 2006), increased dopamine turnover (Brodie and Opacka, 1985; Karen et al., 2001; Gillette and Bloomquist, 2003) associated with decreased dopamine levels (Lazarini et al., 2001) have been suggested to be involved in the pyrethroid induced dopaminergic alterations. In an interesting study on conscious rats, Hossain et al. (2006) investigated the effect of pyrethroids on extracellular levels of dopamine and its metabolites in the striatum. Exposure to cyhalothrin, a type II pyrethroid inhibited dopamine release and its uptake while deltamethrin, another type II pyrethroid, in contrast, increased both dopamine release and uptake. Interestingly, exposure to allethrin, a type I pyrethroid had a dual effect on dopamine release as increase in extracellular dopamine levels was observed at a lower dose and decrease in extracellular dopamine levels at a higher dose. It was suggested that alterations in the extracellular dopamine in striatum could be due to the effect on the sodium and calcium channels on nigrostriatal dopaminergic terminals or GABAergic interneurons. Based on these findings, it was suggested that the activity of striatal dopaminergic neurotransmission is modulated differently by the three pyrethroids involving multiple mechanisms. Alterations in brain dopaminergic system following prenatal exposure to deltamethrin and bioallethrin associated with increased levels of DOPAC in the striatum of adult rats have also been reported (Lazarini et al., 2001; Shafer et al., 2005). Liu and Shi (2006) found that chronic deltamethrin treatment decreased dopamine levels in the striatum. The change was selective and associated with inhibition in dopamine synthesis and increased dopamine turnover in the striatum. Inhibition in the activity and mRNA/protein expression of TH in striatum was also observed in deltamethrin treated rats suggesting TH as a molecular target of pesticides. Decreased expression of TH protein and immunoreactivity in the striatum following exposure to lambda-cyhalothrin in the present study indicates vulnerability of dopaminergic neurons. As tyrosine hydroxylase is a rate limiting enzyme, decrease in its expression in the striatum could be due to auto feedback control as a result of high levels of dopamine. In the present study, decrease in the binding

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Fig. 5. Photomicrographs of rat corpus striatum sections illustrating effect on the tyrosine hydroxylase immunoreactivity following post-lactational exposure of rats to lambda-cyhalothrin. Rats were exposed to lambda-cyhalothrin (1.0 mg/kg or 3.0 mg/kg body weight/day, p.o.) from post-lactational day (PD)22 to PD49. Effect on tyrosine hydroxylase immunoreactivity studied 28 days after exposure of lambda-cyhalothrin on PD50. Exposed rats (b and c) showed decreased tyrosine hydroxylase immunoreactivity as compared to control (a). To assess whether changes are transient or persistent, effect on the tyrosine hydroxylase immunoreactivity also studied 15 days after withdrawal of lambda-cyhalothrin exposure on PD65. A significant impairment was evident in rats treated with lambda-cyhalothrin at a high dose 15 days after withdrawal of exposure (f) as compared to respective control (d) whereas rats exposed at 1.0 mg/kg dose (e) showed restoration in corpus striatum tyrosine hydroxylase expression. Arrow indicates immunoreactivity for tyrosine hydroxylase. Scale bar = 300 ␮m.

of striatal dopamine-D2 receptors was observed in rats following lambda-cyhalothrin exposure for 28 days which persisted even after withdrawal of exposure. Availability of neurotransmitters including dopamine may influence the sensitivity of their receptors. At present it is difficult to explain the reason for decreased binding of dopamine-D2 receptors but it could be due to increase in the levels of dopamine in the striatum. As a number of pesticides have been found to interact with the receptors, direct interaction of lambda-cyhalothrin with dopamine receptors is quite likely and may be associated with decreased binding of dopamine receptors as observed in the present study. Role of dopamine and its receptors in modulating the motor behavior is well accepted. Exposure to pyrethroids has been found to affect the neurobehavioral performance in humans and experimental animals (Wolansky and Harrill, 2008). Alterations in open field behavior, motor activity, catalepsy and operant behavior have been reported following exposure to pyrethroids (Lazarini et al., 2001; Wolansky et al., 2006). Decrease in distance traveled associated with resting time in rats exposed to lambda-cyhalothrin indicates impaired motor performance which remained persistent even after withdrawal of exposure. Further, decrease in rotarod performance in rats following lambda-cyhalothrin exposure

indicates impaired motor coordination. Decrease in the sensitivity of dopamine receptors in corpus striatum in lambdacyhalothrin treated rats as observed in the present study may be associated with the impaired motor activity and motor coordination. Involvement of serotonin levels in modulating physiological and pharmacological functions and affective state is well accepted (Lucki, 1988; Lesch, 2004; Frederick and Stanwood, 2009). The mechanism of serotonergic neurotransmission is quite complicated and mediated by interacting with its receptors (Nakamura and Hasegawa, 2009). Exposure to environmental chemicals including pesticides has been found to affect the integrity of serotonergic system (Slotkin et al., 2009; Chen et al., 2011). Decrease in the brain serotonin levels associated with decrease in the synthesis of serotonin and loss of serotonergic neurons was found in rats exposed to type II pyrethroids–deltamethrin, cyfluthrin and cyhalothrin (Martinez-Larranaga et al., 2003). Although it is difficult to comment on the increase in the binding of frontocortical serotonin-2A receptors which remained persistent even after withdrawal of exposure, upregulation of brain serotonin-2A receptors could be linked with decreased levels of serotonin in the present study.

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Decrease in the body weight of rats following exposure to lambda-cyhalothrin as observed in the present study is consistent with earlier report (Ratnasooriya et al., 2002; Fetoui et al., 2008). However, the decreased body weight in lambda-cyhalothrin treated rats in earlier study by Fetoui et al. (2008) was associated with decreased food and water intake which is in contrast as no significant change in food and water intake in lambda-cyhalothrin exposed rats was observed in the present study. Interestingly, no significant effect on food and water intake was also observed in spite of decreased body weight in a number of experimental studies on rats (Yano et al., 2000; Farag et al., 2007; Fraga and Bertages, 2009; Sangha et al., 2011). It has been suggested that in spite of normal food and water consumption, the decreased body weight could also be associated with some other physiological variables including physiological urinary and fecal losses, intestinal transit time, metabolic features and nutritional absorption although we have not measured these parameters (Fraga and Bertages, 2009). Adverse effect on the body weight of rats in the present study could therefore be due to lambda-cyhalothrin exposure. The decrease in body weight of rats exposed to lambda-cyhalothrin at a higher dose remained persistent even after withdrawal of exposure indicates growth impairment and suggests that changes may be more severe if exposure continues. Decrease in dopamine-D2 receptors associated with decreased motor activity and motor co-ordination in developing rats indicate their vulnerability to lambda-cyhalothrin and suggest that striatal dopaminergic system is a target of lambda-cyhalothrin. The data also indicate involvement of serotonin-2A receptors in the neurotoxicity of lambda-cyhalothrin. The results further suggest that neurobehavioral changes may be more intense in case exposure to lambda-cyhalothrin continues. Conflict of interest statement None. Acknowledgments The authors thank the Director, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Lucknow for his keen interest in the present study. The authors also thank Mr. B.D. Bhattacharji, Senior Principal Scientist, CSIR-IITR, Lucknow for painstakingly going through the manuscript and making the necessary changes in the language. Financial support by Indian Council of Medical Research (ICMR), New Delhi for carrying out the study is also acknowledged. The authors also thank to Dr. Pramod Kumar and Mr. B.S. Pandey for their technical assistance in the study. The CSIR-IITR communication no. is 2960. References Abbud Righi, D., Palermo-Neto, J., 2003. Behavioral effects of type II pyrethroid cyhalothrin in rats. Toxicol. Appl. Pharmacol. 191, 167–176. Ahlbom, J., Fredriksson, A., Eriksson, P., 1994. Neonatal exposure to a type-I pyrethroid (bioallethrin) induces dose-response changes in brain muscarinic receptors and behaviour in neonatal and adult mice. Brain Res. 645, 318–324. Amoah, P., Drechsel, P., Abaidoo, R.C., Ntow, W.J., 2006. Pesticide and pathogen contamination of vegetables in Ghana’s urban markets. Arch. Environ. Contam. Toxicol. 50, 1–6. Ansari, R.W., Shukla, R.K., Yadav, R.S., Seth, K., Pant, A.B., Singh, D., Agrawal, A.K., Islam, F., Khanna, V.K., 2012. Cholinergic Dysfunctions and Enhanced Oxidative Stress in the Neurobehavioral Toxicity of Lambda-Cyhalothrin in Developing Rats. Neurotox Res. Feb 11. doi:10.1007/s12640-012-9313-z. Aziz, M.H., Agrawal, A.K., Adhami, V.M., Shukla, Y., Seth, P.K., 2001. Neurobehavioural consequences of gestational exposure (GD14–GD20) to low dose deltamethrin in rats. Neurosci. Lett. 300, 161–165. Barr, D.B., Olsson, A.O., Wong, L.Y., Udunka, S., Baker, S.E., Whitehead, R.D., Magsumbol, M.S., Williams, B.L., Needham, L.L., 2010. Urinary concentrations of metabolites of pyrethroid insecticides in the general U.S. Population: National

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