RESEARCH ARTICLE

Alterations in Plasma and Tissue Acetylcholinesterase ActivityFollowing Repeated Oral Exposure of Chlorpyrifos Aloneand in Conjunction with Fluoride in Wistar Rats

Naseer Ahmad Baba • Rajinder Raina •

Pawan Kumar Verma • Mudasir Sultana

Received: 17 April 2013 / Revised: 14 October 2013 / Accepted: 16 December 2013

� The National Academy of Sciences, India 2014

Abstract Concurrent exposures of more than one envi-

ronmental contaminants are commonly encountered by

human beings and animals. This study investigated the

effect of chlorpyrifos alone and in conjunction with fluo-

ride on plasma and tissue acetylcholinesterase (AChE)

activity in wistar rats. Fluoride at 1 or 10 ppm in drinking

water produced significant (P \ 0.05) inhibition of AChE

activity in a dose dependant manner and inhibition was

maximum in brain (26.39 %) and least in heart (10.23 %).

Similarly repeated oral administration of chlorpyrifos at 1

or 10 mg/kg produced a significant dose dependant inhi-

bition of AChE activity (P \ 0.05) which was observed

maximum in brain (28.93 %) and least in heart (12.86 %).

Co-exposure of chlorpyrifos and fluoride at higher doses

produces more pronounced inhibition as compared to

chlorpyrifos or fluoride exposed groups. Co-exposure

produces maximum inhibition in erythrocyte (37.54 %)

and least in plasma (18.26 %) of exposed animals as

compared to control group. Observations from the present

study suggested that co-exposure of fluoride and chlor-

pyrifos produces more pronounced inhibition of AChE than

the individual exposure of either toxicant. Therefore the

applications of chlorpyrifos as a pesticide should be

reduced in areas where fluoride level in soil or ground

water is high to avoid AChE inhibition.

Keywords Acetylcholinesterase � Chlorpyrifos �Fluoride � Toxicity � Wistar rats

Introduction

Acetylcholinesterase (AChE) is a hydrolytic enzyme

present in neuromuscular junctions, erythrocytes, brain,

lung, liver, kidney, plasma, etc. for the hydrolysis of cho-

line esters of exogenous or endogenous origin. Acetyl-

choline (ACh) is an endogenous choline ester acting as a

classical neurotransmitter in central and peripheral nervous

system. ACh primarily regulates neuromuscular coordina-

tion and is involved in learning, memory and mood of

mammals. A number of therapeutic agents and toxicants

cause alterations in the activity of AChE for their beneficial

and toxic effects respectively [1, 2]. Chlorpyrifos (O–O-

diethyl-O-{3,5,6 trichloro-2-pyridyl}-phosphorothioate)

(CPF) is a common organophosphorus pesticide used

worldwide for the management of agricultural and house-

hold pests [3, 4]. Chlorpyrifos elicits toxicity by inhibiting

activity of AChE which leads to accumulation of the ACh

in neuronal synapses and neuromuscular junctions leading

to excessive stimulation of cholinergic receptors resulting

to neurotoxicity [1, 2]. Besides AChE inhibition, CPF is

also known to alter several cholinergic neurotransmission

parameters that could potentially modify cholinergic tox-

icity, including cholinergic receptor density, high affinity

choline uptake, muscarinic and nicotinic auto-receptor

function and acetylcholine synthesis [5–10]. Direct inter-

action of CPF or its active metabolite chlorpyrifos oxon

with pre- or postsynaptic cholinergic neurotransmission

processes could also influence the expression of cholinergic

toxicity [11–15].

Fluorosis is a crippling disease of human beings and

animals resulting from excessive fluoride (F) ingestion

through food and water. Excess F content in groundwater is

an important public health problem all over the world and

reports of endemic fluorosis occur in all inhabited

N. A. Baba � R. Raina � P. K. Verma (&) � M. Sultana

Division of Veterinary Pharmacology and Toxicology, Faculty

of Veterinary Sciences and Animal Husbandry, Sher-e-Kashmir

University of Agricultural Sciences and Technology of Jammu,

R S Pura, Jammu 181102, India

e-mail: vermapk1975@gmail.com

123

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.

DOI 10.1007/s40011-013-0286-3

continents [16]. It produces multiple actions by different

mechanisms, but the exact one is not clear. Fluoride

exposure decreases the activity of erythrocyte sodium

potassium ATPase and glucose-6-phosphate dehydroge-

nase [17]. It also causes disturbance in plasma Na, K, Mg

and Ca ion levels [18–20] and these ions are essential for

normal electrical conduction, depolarization and repolari-

zation of the cardiac and nerve cells resulting in neuro-

logical disorders. Therefore the present study was aimed to

investigate the effect of repeated oral exposure of CPF

alone and in conjunction with F on plasma and tissue

AChE activity in wistar rats.

Material and Methods

Wistar rats of either sex weighing 150–200 g were pro-

cured from the Indian Institute of Integrative Medicine

(Council of Scientific and Industrial Research) Jammu.

The animals were maintained under standard experimental

conditions with ad libitum feed and drinking water. The

experimental design was approved by the University

Animal Ethical Committee vide No. AU/FVSc/C-11/

2,456-68 on ethical standards in animal experimentation.

After acclimatization animals were randomly allocated to

seven groups of six wistar rats each. Animals in the group

I served as control and received only normal tap water for

drinking. The animals of group II and III were provided

with drinking water containing F at 1 and 10 ppm of

water respectively whereas rats in Group IV and V were

administered CPF through oral gavage at 1 and 10 mg/kg

body weight respectively. The animals of group VI were

provided both water containing F at 1 ppm and CPF at

1 mg/kg through oral gavage whereas animals of group

VII received both F at 10 ppm in drinking water and CPF

at 10 mg/kg body weight daily through oral gavage. The

duration of repeated exposure of all the toxicants was

28 days. In order to minimize the possible instability,

both toxicants were prepared fresh in drinking water. All

the rats were weighed weekly to make necessary correc-

tions in the CPF dosage as per body weight. After

28 days of daily treatment blood samples were collected

from retro-orbital fossa using capillary tubes in heparin-

ised test tubes (5–10 IU/ml of blood). After cervical

dislocation tissue samples of different visceral organs

were collected in ice cooled phosphate buffer (pH 7.4).

Ten percent tissue homogenate of different visceral

organs were prepared by homogenizing 1 g of tissue in

10 ml of phosphate buffer (pH 7.4) using Teflon coated

homogenizer at 4 �C. AChE activity was determined in

erythrocytes (1 %), plasma and tissue homogenate (10 %)

of different visceral organs as per the method described

by Voss and Sachsse [21].

Statistical Analysis

The results were subjected to analysis of variance

(ANOVA) in completely randomized design with statistical

significance being tested using the Duncan multiple range

test [22].

Results and Discussion

Concurrent exposure of more than one chemical from

various sources such as food, air and water is common in

human beings and animals. The assessment of deleterious

or toxic effects produced by concurrent exposure to com-

monly encountered chemicals is of great significance in

order to find out toxicological consequences arising as the

result of their interactions. Such understanding will help in

comprehensive management of untoward effects induced

by these chemicals [23]. Various studies have reported that

repeated exposure of different agrochemicals like bifenth-

rin, fironil and CPF produce alterations in biochemical and

stress parameters in experimental animals [24–27].

The AChE activity in plasma and different visceral

organs of wistar rats in control and treated groups are

presented in Table 1. During the entire study period ani-

mals were closely observed for the clinical signs of toxicity

but no signs of overt toxicity were observed in treated

animals except reduction in feed and water intake. Repe-

ated oral administration of CPF at 1 or 10 mg/kg produce

dose dependent significant (P \ 0.05) inhibition of AChE

activity which was observed to be maximum in brain

(28.93 %) followed by lung (25.50 %), plasma (24.18 %),

erythrocytes (20.70 %), kidney (19.70 %), liver (17.59 %)

and least in heart (12.86 %). Chlorpyrifos dosing inhibits

brain AChE following the metabolism of CPF to its oxon

analog and intact CPF is much less potent as an inhibitor of

this function. Intensity of toxicity depends on the extent of

AChE inhibition or accumulation of acetylcholine [16, 23].

The resulting excess buildup of ACh causes a cholinergic

crisis at critical doses, but may also have other modulation

functions in the developing brain [11]. Similarly CPF

treatment has also been reported to produce significant

reduction of AChE activity in different regions of rat brain

[23]. Although the physiological significance of erythro-

cyte and plasma AChE in mammalian blood is uncertain,

the inhibition of these enzymes by organophosphorus

compounds (OPC) is considered as a better index to

ascertain exposure to OPC pesticides to animals.

Similarly F at 1 or 10 ppm in drinking water produced

significant (P \ 0.05) inhibition of AChE activity in a dose

dependant manner and inhibition was maximum in brain

(26.39 %) followed by erythrocyte (22.61 %) and lung

(19.43 %). The least inhibition was observed in heart

N. A. Baba et al.

123

(10.23 %). Exposure to F produces dose dependant inhibition

of AChE activity in different visceral organs. Maximum

inhibition was observed in brain. Various studies also sug-

gested that areas where F level in ground water is high have

more incidence of neurological disorders from least devel-

opment of nervous tissue in terms of memory, learning and

other disorders associated with nervous system [28, 29].

Exposure to F has been reported to decrease in activity of

AChE and BuChE in the brain of rats [30]. Similar decrease in

female mice has also been observed by Bhatnagar et al. [31].

Individual exposures of F and CPF in animals produce

AChE inhibition in all visceral organs while maximum inhi-

bition was observed in brain whereas least inhibition occurred

in heart. Co-exposure of CPF and F at higher doses produced

significant (P \0.05) and more pronounced AChE inhibition

as compared to individual CPF or F exposed groups. The co-

exposure of both toxicants at higher doses produced maximum

AChE activity inhibition in erythrocyte (37.54 %) followed by

lung (35.30 %), brain (33.29 %), liver (23.42 %), kidney

(21.86 %), heart (20.08 %) and least in plasma (18.26 %) of

exposed animal groups as compared to control group. Inhibi-

tion resulted in the accumulation of ACh leads to prolonged

excitatory postsynaptic potentials resulting in repeated,

uncontrolled firing of neurons and cessation of nerve impulses.

Chlorpyrifos also modifies the pre- and postsynaptic cholin-

ergic receptor density and transporter proteins required for

synthesis and transport of ACh, which can be assessed to

determine the extent of functions altered by exposure of CPF

[32–34]. There is increased prevalence of neurological disor-

ders in persons of all ages and people may experience the onset

of a neurological disorder at any time in their lives. In most of

the conditions the specific causes of disorders are unknown.

They are believed to be due to increasing contamination of

environment by metals and vast varieties of agrochemicals.

Conclusion

Observations from the present study suggested that repe-

ated animal exposure to F or CPF alone significantly

inhibits AChE activity in plasma and different visceral

organs. Co-exposure produces more pronounced inhibition

of AChE as compared to the individual exposure of either

toxicant. Therefore the application of CPF as pesticides

should be reduced in areas where fluoride levels in soil or

ground water is higher to avoid AChE inhibition.

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