Pesticide Biochemistry and Physiology 93 (2009) 96–99

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Pesticide Biochemistry and Physiology

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k-cyhalothrin and cypermethrin induced in vivo alterations in the activityof acetylcholinesterase in a freshwater fish, Channa punctatus (Bloch)

Amit Kumar a, Devendra K. Rai b, B. Sharma b, Ravi S. Pandey a,*

a Biochemistry Laboratory, Department of Zoology, University of Allahabad, Allahabad 211002, Indiab Department of Biochemistry, University of Allahabad, Allahabad 211002, India

a r t i c l e i n f o

Article history:Received 26 January 2008Accepted 17 December 2008Available online 25 December 2008

Keywords:k-cyhalothrinCypermethrinAcetylcholinesteraseC. punctatus

0048-3575/$ - see front matter � 2008 Elsevier Inc. Adoi:10.1016/j.pestbp.2008.12.005

* Corresponding author. Fax: +91 532 2460788.E-mail address: rspandey2004@yahoo.com (R.S. Pa

a b s t r a c t

In the present study, the in vivo effects of k-cyhalothrin and cypermethrin on the activity of acetylcholin-esterase (AChE, EC 3.1.1.7) were evaluated for 96 h in brain, muscle and gills of Channa punctatus. Bothcompounds exhibited tissue specific as well as dose dependent decrease in the activity of AChE. The trea-ted fish showed a significant decrease in the activity of AChE in brain and a lesser inhibition in muscle andgills in response to the increasing concentrations of k-cyhalothrin as well as cypermethrin. Our resultsindicated that the brain was the main target organ for both insecticides, followed by muscle and gills,as determined by AChE inhibition study. However, these organs showed variations in the degree of AChEinhibition for separate treatments of both insecticides. The k-cyhalothrin was a more potent AChE inhib-itor as compared to cypermethrin. These findings indicated that apart from the established mechanism ofdelayed closure of sodium ion channels, these pyrethroids inhibit the activity of AChE in C. punctatuswhich could further aggravate their neurotoxic effects.

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1. Introduction

Globally, more than 520 tonnes of active ingredient of pyre-throids are annually used in vector control programmes alone [1]because of their relatively good photostability and broad spectruminsecticidal activity. Pyrethroids act primarily on the nervous sys-tem. The pyrethroids are categorised into two types. Type I pyre-throids have a basic cyclopropane carboxylic ester structure,whereas type II pyrethroids are derived by a cyano substitutionon the a-methylene group of its alcohol moiety. Type I pyrethroidsare best suited to small-scale personal applications whilst type IIare preferred for large scale uses due to their higher potency [2].Type II pyrethroids are believed to slow the activation (opening)and inactivation (closing) of sodium ion channels. The delayedopening and closure of sodium channels prolong the sodium cur-rent, thereby holding the channel open long enough so that theneuron becomes depolarized and no longer fires [3,4]. But interac-tion with sodium channels is not the only mechanism of actionproposed for pyrethroids in insects and vertebrates [5]. Their ef-fects on the central nervous system (CNS) have also been suggestedto be dependent upon the antagonism of GABA mediated inhibition[6,7], modulation of nicotinic cholinergic transmission [7],enhancement of noradrenalin release [7], action on calcium chan-nels [8] or inhibition of monoamine oxidase [9].

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ndey).

However, there are only few reports on their interactions withanother important cholinergic neurotransmitter acetylcholine(ACh). It is usually an excitatory neurotransmitter, which is formedin presynaptic terminal from acetyl-CoA and choline in presence ofcholine acetyltransferase. It acts on the membrane excitatoryreceptors present on postsynaptic membrane to increase the mem-brane’s permeability to Na+, resulting in the initiation of action po-tential in the neuron. When acetylcholine is released in packagedform in vesicles at the synapse, the enzyme acetylcholinesterase(AChE), present in the cleft in bound form in proteoglycan matrix,splits it into acetate and choline, and thereby inactivates acetyl-choline. This biochemical interaction is very important with regardto the normal functioning of nerve signal transmission as it pre-vents the continued action forever, thereafter, by regulating acetyl-choline concentration. A significant inhibition in the activity ofAChE results in rapid accumulation of acetylcholine at nerve end-ings, leading to disruption of nervous activity by over stimulatingthe postsynaptic acetylcholine receptors (AChR), exertingneurotoxicity.

Some earlier studies have shown relation between cypermeth-rin and activity of acetylcholinesterase (AChE) in Cyprinus carpio[10,11]. The in vitro incubation of k-cyhalothrin with rabbit eryth-rocytes has been reported to cause significant decrease in theactivity of acetylcholinesterase (AChE) [12]. Keeping these factsin view and our earlier observations, which exhibited significanteffects of k-cyhalothrin and cypermethrin on the nucleic acidsand protein contents of C. punctatus [13], we have envisaged tostudy the in-vivo effect of k–cyhalothrin and cypermethrin (type

A. Kumar et al. / Pesticide Biochemistry and Physiology 93 (2009) 96–99 97

II synthetic pyrethroids) on the activity of AChE in different specifictissues such as brain, muscle, and gills of C. punctatus and com-pared overall effects of the two insecticides on AChE activity aswell as their tissue specific effects in order to establish the targettissue and their relative efficacy.

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Brain Muscle GillsConcentrations of λ-cyhalothrin (μg/l)

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Fig. 1. Changes in the specific activity of acetylcholinesterase in brain, muscle andgills of C. punctatus exposed to k-cyhalothrin for 96 h. Each bar represents thevalues as means ± SEM of five replicates of experiments. One-way ANOVA withDunnett post-test was used to compare the changes at each of the insecticideconcentrations from that of control. * and ** represent the values significant atp < 0.05 and p < 0.01, respectively.

2. Materials and methods

C. punctatus, the freshwater teleosts (average length 11–13 cm,weight 23 ± 2 g) were purchased from the local fish market. Fishwere treated with potassium permanganate solution (0.5% w/v)for 1 min to remove dermal adherents. The identification key ofDay [14] was used for their identification. The fish were acclima-tized for 2 weeks in a rectangular glass aquarium (capacity ofaquarium: 100 l; dimension of aquarium: 60 cm � 30 cm � 30 cm)having 70 l of dechlorinated aerated tap water at room tempera-ture (27 ± 1 �C) under standard laboratory conditions with 12 heach of light and dark periods. They were provided with food adlibitum (freeze-dried Daphnia worms obtained from Everyday�’s,Taiwan containing 52% crude fat, 12% crude protein, 2% crude fiber,2% moisture, and 12% ash) twice a day. Formula-grade insecticidesused in the study, namely Colt�25 (25% effective concentration ofcypermethrin) and REEVA-5 (5% effective concentration of k-cyhal-othrin) were procured from PI Industries Ltd., Division: PesticidesIndia, Ankleshwar, India and Rallis India Ltd. Factory, Patancheru,India, respectively.

2.1. Sublethal bioassays for cypermethrin and k-cyhalothrin

The fish were exposed to insecticides for 96 h period. Fish keptin an insecticide-free medium served as control. The same volumeof acetone used in the dissolution of insecticides (3.3 ll acetone/lof aquatic medium for cypermethrin and 3.2 ll acetone/l of aquaticmedium for k-cyhalothrin) was maintained in the control. 0.4 mg/l(95% confidence limits = 0.05–0.01) and 7.92 lg/l (95% confidencelimits = 0.05–0.01) were the obtained concentrations of cyper-methrin and k-cyhalothrin for the 96 h LC50 in acute bioassays withC. punctatus [15], and thus experimental concentrations at 10%,15%, and 20% of LC50 determined based on the active ingredientconcentrations of insecticides were used in the present study.The stock and test solutions of both pyrethroids were preparedby dissolving the insecticides in acetone. Each static-renewal testwas performed in five replicates with each aquarium (dimension:30 cm � 30 cm � 30 cm) containing four fish in 40 l of water in or-der to ensure the reproducibility of the results. The water qualitywas tested before treatment as per American Public Health Associ-ation (APHA) guidelines [16]. The mean values were: temperature28 ± 1 �C, pH 7.1 ± 0.7, DO 6.9 ± 0.6 mg/l, and total hardness113.3 ± 2 mg/l. Water and insecticides were replenished at 24 hintervals along with proper provisioning of aeration through airbubblers to maintain water quality and to prevent degradation ofthe insecticides. The fish were fed during the experiment twice(morning and evening) a day, ceasing 24 h prior to sampling.

2.2. Biochemical assays

2.2.1. Preparation of tissue extractsThe fish were sacrificed by decapitation after exposure to each

of the insecticides, and dissected. Brain, muscle, and gills were sur-gically removed, thoroughly rinsed in cold saline (4–6 �C), blotteddry, weighed and homogenized (10%, w/v) in 50 mM sodium phos-phate buffer (pH 8.0) containing 0.1% Triton X-100 using Potter–Elvehjam homogenizer fitted with a Teflon-coated pestle underice cold condition (4–6 �C). The homogenates were kept for30 min in cold with intermittent stirring and centrifuged at 4 �C

for 30 min at 10,000g in a refrigerated centrifuge (Sigma, model-3K30). The corresponding supernatants were either used afreshfor determination of protein contents or for assay of AChE.

2.3. Determination of total protein

Protein contents in supernatants of fish-tissue homogenateswere determined according to the method of Lowry et al. [17],using bovine serum albumin (BSA) as the standard.

2.3.1. Assay of acetylcholinesteraseThe activity of AChE was determined according to Ellman et al.

[18]. The reaction mixture (3 ml) contained 1.5 ml of 100 mM so-dium phosphate buffer (pH 8.0), 0.3 ml of 5 mM DTNB [5,50-dithi-obis-(nitrobenzoic acid) prepared in 10 mM sodium phosphatebuffer, pH 7.5 containing 15 mg sodium bicarbonate added per10 ml of solution], 0.3 ml of 5 mM acetylthiocholine iodide (ATI),0.1 ml of 10% homogenate, and 0.8 ml of distilled water. The in-crease in absorbance was monitored at 412 nm and 28 �C for3 min in a UV–visible double beam spectrophotometer (ELICO,model: SL-160) with quartz cuvettes (1 cm light path) against dis-tilled water as blank. Measurements were made in triplicate foreach tissue homogenate. Simultaneously, two blanks were alsoused. One blank contained phosphate buffer, DTNB, and ATI butnot enzyme protein to determine the spontaneous hydrolysis ofATI, and the second blank contained phosphate buffer, DTNB, andenzyme protein but no substrate (ATI) to correct for any non-AChE-dependent formation of thionitrobenzoic acid (TNB). Oneunit of AChE activity was expressed as nanomoles of substratehydrolyzed/min/mg protein under experimental conditions.

2.4. Statistical analysis

The values have been presented as means ± standard error ofmean (SEM) of observed data of five replicates. GraphPad prismversion 3.0 (GraphPad Prism Software Inc., San Diego, CA, USA)was used to analyze the data. One-way analysis of variance (ANO-VA) with Dunnett post-test was performed to determine the levelof significance.

3. Results and discussion

Both k-cyhalothrin and cypermethrin were observed to causedose dependent decrease in the activity of AChE in brain, muscle,and gills of C. punctatus. The results shown in Fig. 1 indicated that

98 A. Kumar et al. / Pesticide Biochemistry and Physiology 93 (2009) 96–99

k-cyhalothrin treatment at 10%, 15%, and 20% of the 96 h LC50 re-sulted in decrease in the activity of AChE by 18.8%, 35.8%, and46.2%, respectively, in brain (Specific activity ± SEM: 77 ± 4.36,62.56 ± 3.12, 49.42 ± 3.46, 41.4 ± 3.26 at control and 10%, 15%,and 20% of the 96 h LC50, respectively); 15.8%, 27.4%, and 37.3%in muscle (Specific activity ± SEM: 47.09 ± 2.76, 39.66 ± 2.79,34.17 ± 1.89, 29.54 ± 2.29 at control and 10%, 15%, and 20% of the96 h LC50, respectively); and 7.1%, 13.8%, and 18.9% in gills (Specificactivity ± SEM: 15.01 ± 0.61, 13.95 ± 0.78, 12.94 ± 0.25,12.18 ± 0.64 at control and 10%, 15%, and 20% of the 96 h LC50,respectively) as compared to the control. k-cyhalothrin treatmentat 10% of the 96 h LC50 caused only slight decrease (p < 0.05) inthe activity of AChE in brain while the decrease was quite signifi-cant (p < 0.01) at 15 and 20% of the 96 h LC50. The inhibition inthe activity of AChE in muscle was insignificant (p > 0.05) at 10%but the doses of 15% and 20% of the 96 h LC50 caused highly signif-icant inhibition in the activity of AChE (p < 0.01). Gills, however,were unaffected at low concentrations (10% and 15% of the 96 hLC50) and reflected slight decrease (p < 0.05) only at highest con-centration i.e. 20% of the 96 h LC50 of k-cyhalothrin.

The effect of sublethal concentrations of cypermethrin on theactivity of AChE in different fish organs is shown in Fig. 2. The resultsindicated that cypermethrin treatment at 10%, 15%, and 20% of the96 h LC50 resulted in 12.9%, 24.7%, and 32.7% decrease, respectively,in brain (Specific activity ± SEM: 77 ± 4.36, 67.1 ± 1.56, 58.02 ± 2.58,51.8 ± 4.25 at control and 10%, 15%, and 20% of the 96 h LC50, respec-tively) while 8.6%, 19.8%, and 23.7% decrease, respectively, in muscle(Specific activity ± SEM: 47.09 ± 2.76, 43.06 ± 2, 37.77 ± 0.85,35.94 ± 2.69 at control and 10%, 15%, and 20% of the 96 h LC50,respectively) and 4.4%, 12.5%, and 14.9% decrease, respectively, ingills (Specific activity ± SEM: 15.01 ± 0.61, 14.35 ± 0.61,13.14 ± 0.23, 12.78 ± 0.46 at control and 10%, 15%, and 20% of the96 h LC50, respectively) in comparison to the control. The results sug-gested that cypermethrin did not cause significant AChE inhibition(p > 0.05) in brain at 10%, but 15% and 20% of the 96 h LC50 of cyper-methrin resulted in highly significant AChE inhibition (p < 0.01). Inmuscle, the AChE inhibition was insignificant (p > 0.05) at 10%,slightly significant (p < 0.05) at 15% and highly significant(p < 0.01) at 20% of the 96 h LC50 of cypermethrin. The gills werefound to be least affected. The AChE inhibition in gills was insignifi-cant (p > 0.05) at 10 and 15% and only slight inhibition (p < 0.05) wasobserved at 20% of the 96 h LC50 of cypermethrin. AChE inhibitioncaused by k-cyhalothrin and cypermethrin might be due to hydro-phobic interaction of these pyrethroids (having an acid moiety, acentral ester bond, and an alcohol moiety) with hydrophobic aro-matic surface region of AChE [19]. The intensity of AChE inhibition

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Fig. 2. Changes in the specific activity of acetylcholinesterase in brain, muscle andgills of C. punctatus exposed to cypermethrin for 96 h. Each bar represents values asmean ± SEM of five replicates of experiments. One-way ANOVA with Dunnett post-test was used to compare the changes at each of the insecticide concentrations fromthat of control. * and ** represent the values significant at p < 0.05 and p < 0.01,respectively.

might also be correlated with the degree of accumulation of pyre-throids and/or their metabolites in different organs [20,21]. The dif-ferential distribution of different molecular forms of AChE as well astheir mode of interaction with pyrethoids could be another possiblereason for observed patterns of AChE inhibition in different organs[22]. In addition, earlier reports on AChE activity inhibition due topyrethoids exposure in Cyprinus carpio corroborate our findings[10,11,23].

The results indicated that in response to k-cyhalothrin treatment,AChE inhibition in the brain was quite high in comparison to the gillswhile it was moderate as compared to the muscle. However, level ofAChE inhibition in muscle was slightly greater than that of gills.These results clearly suggested the brain to be the main target organ,followed by muscle and gills, respectively, for both of the insecti-cides. Furthermore, these organs showed variations in the degreeof AChE inhibition for separate treatments of both of the insecticides.The results for cypermethrin treatment revealed approximatelysimilar levels of AChE inhibition in muscle and gills, which couldbe due to the fact that they belong to the same group of pyrethoids.The results are indicative of a clear correlation between AChE inhibi-tion and the degree of nerve innervations in these organs [24].k-cyhalothrin was found to be a potent AChE inhibitor in both brainand muscle, while the degree of AChE inhibition in gills was not aspronounced. This might be due to some differences in the type ofinteraction between k-cyhalothrin and/or its metabolites with AChEin various organs, as well as the relative coherence between AChEinhibition and the degree of nerve innervations in these organs. Inconclusion, k-cyhalothrin was found to be more potent AChE inhib-itor as compared to cypermethrin in the experimental conditionsused. As the pyrethroids delay and prolong sodium channel openingleading to membrane depolarization through its increased perme-ability to sodium ions [25], the observed inhibition in AChE activitycould aggravate it further by over stimulation of ACh receptors,which also increases membrane permeability to sodium ions. Thesymptomatic behavioral consequences could be seen in the formof fervent activity, loss of balance, rapid rate of swimming, convul-sions, etc. due to neurotoxicity caused by cypermethrin and k-cyhal-othrin at higher concentrations [15].

Acknowledgments

The authors express their gratitude to Professor (Dr.) PratimaGaur, Head, Department of Zoology, for providing Central Instru-mentation Facility developed with the financial assistance fromUGC-SAP and DST-FIST for carrying out this work. Financial assis-tance to one of the authors (Amit Kumar) from the Council of Sci-entific and Industrial Research (CSIR), India, in the form of CSIR-NET-SRF (F.No.9/1 (274)/2005-EMR-I) is gratefully acknowledged.

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