Research Article Carbofuran Modulating Functions of ...downloads.hindawi.com/archive/2016/3760967.pdf · monly known as Furadan, is a broad spectrum carbamate pesticide which is used

Research ArticleCarbofuran Modulating Functions of Acetylcholinesterase fromRat Brain In Vitro

Vivek Kumar Gupta1 Ashutosh Pathak1 Nikhat Jamal Siddiqi2 and Bechan Sharma1

1Department of Biochemistry Faculty of Science University of Allahabad Allahabad 211002 India2Department of Biochemistry College of Science King Saud University PO Box 22452 Riyadh 11495 Saudi Arabia

Correspondence should be addressed to Bechan Sharma sharmabiyahoocom

Received 16 August 2015 Revised 20 December 2015 Accepted 16 February 2016

Academic Editor Vianney Pichereau

Copyright copy 2016 Vivek Kumar Gupta et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Carbofuran a potential environmental xenobiotic has the ability to cross blood brain barrier and to adversely influence brainfunctions In the present study the impact of carbofuran on the biophysical and biochemical properties of rat brain AChE hasbeen evaluated in vitro This enzyme was membrane-bound which could be solubilised using Triton-X100 (02 vv) a nonionicdetergent in the extraction buffer (50mMphosphate pH 74)The enzymewas highly stable up to onemonth when stored at minus20∘Cand exhibited optimum activity at pH 74 and 37∘C AChE displayed a direct relationship between activity and varying substrateconcentrations (acetylthiocholine iodide (ATI)) by following Michaelis-Menten curve The119870

119898

and 119881max values as computed fromthe Lineweaver-Burk double reciprocal plot of the data were found to be 007mM and 0066 120583molemLmin respectively Theenzyme exhibited IC

50

value for carbofuran equal to 60 nM The steady-state kinetic studies to determine mode of action ofcarbofuran on rat brain AChE displayed it to be noncompetitive in nature with119870

119894

value equal to 5 nmThese experiments suggestedthat rat brain AChE was very sensitive to carbofuran and this enzymemight serve as a significant biomarker of carbofuran inducedneurotoxicity

1 Introduction

Carbofuran (C12H15NO3 23-dihydro-22-dimethyl-7-ben-

zofuranol methylcarbamate molecular weight 22125) com-monly known as Furadan is a broad spectrum carbamatepesticide which is used in various farm practices in orderto increase crop productivity Due to its broad spectrumaction and short half-life in the environment it is alsoused as an insecticide nematicide and acaricide [1 2] Thepresence of carbofuran is also reported in the nontargetmammalian systems such as maternal plasma umbilicalcord and blood of African-American women and new bornbabies respectively [3] It has been shown to be generallyaccumulated in the fat depots and exerts adverse effects ondifferent vital organs such as brain liver skeletal muscles andheart [1 4 5]The neuronal injury of mammalian system dueto carbofuran is due to the establishment of oxidative stress[6]

The underlying mechanism of pesticide toxicity relieson generation of oxidative stress [7] The administration of

carbofuran has been found to induce reactive oxygen andnitrogen species in skeletalmuscles [8] which are responsiblefor the peroxidation of membrane phospholipids whichdisrupt the normal function of lipid bilayer and help developseveral pathological conditions

The in vivo studies carried out by administering a sin-gle sublethal dose of seven N-methylcarbamate pesticides(carbaryl carbofuran formetanate methiocarb methomyloxamyl or propoxur) indicated pesticide mediated AChEinhibition in the brain and red blood cells which recoveredwithin 24 h after the withdrawal of the pesticide [9] Theearlier experimental results from our and other laboratorieshave established that treatment of rats with carbofuran invivo was able to inhibit AChE activity and also to produceoxidative stress in liver [4] brain [6 10] kidney [11] anderythrocytes [5 12]

Acetyl cholinesterase (AChE EC 3117) or acetyl hydro-lase is a serine protease that hydrolyzes the neurotrans-mitter acetylcholine to be acetyl-S-CoA and choline TheAChE molecule composed of two different protein domains

Hindawi Publishing CorporationAdvances in BiologyVolume 2016 Article ID 3760967 7 pageshttpdxdoiorg10115520163760967

2 Advances in Biology

a large catalytic domain of about 500 residues and a smallC-terminal peptide of less than 50 resides [13] is a keyenzyme of nerve impulse transmission and is reported tobe negatively modulated by carbofuran The AChE occurswith high specific activity in the brain and nervous tissuesas well as in the membranes of muscles and erythrocytes[14] AChE has been the focus of much attention since it wasfirst suggested that it plays an important role in the rapiddestruction of the neurotransmitter acetylcholine (ACh) ina living system [15] The catalytic properties occurrence ofAChE its histochemical localization catalytic functions andmolecular heterogeneity in different tissues of various animalspecies have been extensively studied [16ndash20]

However the systematic and detailed studies concerningthe localization as well as biochemical properties of AChEwith special reference to its interaction in vitro have notbeen properly carried out In the present paper the aforesaidproperties of rat brain AChE have been determined in vitrowith special attention to its steady-state kinetics explainingsensitivity of enzyme to carbofuran and its mode of actioninfluencing the enzyme behavior which have been illustratedThe results indicated that rat brain AChE could be exploitedas a suitable biomarker of pesticide induced neurotoxicity

2 Materials and Methods

21 Chemicals S-Acetylthiocholine iodide (ATI) was ob-tained from Tokyo Chemical Industry Co Ltd Japan and5 51015840-dithiobis (2-nitro benzoic acid) (DTNB) was purchasedfrom SRL Pvt Ltd India Triton X-100 was purchasedfrom Merck India The bovine serum albumin (BSA)sodiumdihydrogen orthophosphate and disodiumhydrogenphosphate were procured from Fisher Scientific Folin andCiocalteursquos phenol reagent was from Spectrochem Pvt LtdIndia All other chemicals used were of analytical grade

22 Animals Three male albino rats of same age groupweighing 180ndash210 g were selected for all these experimentsAnimals obtained from CDRI Lucknow India were housedin propylene cages at 30 plusmn 5∘C and 45 plusmn 5 relative humiditywith 12 h of light and dark cycle Animals were fed withstandard rat feed available commercially with free accessto water Protocols for care and maintenance of the ratswere strictly followed as per the guidelines approved by theinstitutional ethical committee

23 Collection of Brain Tissues and Preparation of Homo-genates The healthy rats were sacrificed using mild chlo-roform anesthesia and cervical dislocation causing minimalpain The whole brain was quickly excised washed withisotonic ice cold NaCl (09 wv) solution blotted todryness and weighed The brain tissue homogenate (10wv) was made in 50mM sodium phosphate buffer (pH74) with and without Triton X-100 (02 vv) using Potter-Elvehjem homogenizer fitted with a Teflon coated pestleunder ice cold condition (4ndash6∘C) The homogenates werecentrifuged at 9000timesg for 30min using REMI refrigeratedcentrifuge The supernatants were removed and the pellets

were reconstituted in equal volume of homogenizing bufferBoth the supernatants and the pellets suspensions were usedfor protein estimation and determination of AChE activityThe soluble fraction was assayed for detailed characterizationof enzyme including its interaction with carbofuran in vitro

24 Determination of Protein The protein was estimated indifferent fractions of rat brain using Folin and Ciocalteursquosphenol reagent [21] The bovine serum albumin was usedas an standard The absorbance of blue color complex wasmonitored at 620 nm

25 Assay of Rat Brain Acetylcholinesterase Activity Theactivity of rat brain AChE was determined by the methodof Ellman [22]The 3mL reaction mixture contained 05mMATI 05mM DTNB and 50mM phosphate buffer (pH 74)The change in absorbance of light wasmeasured at 412 nm for3min at regular intervals of 30 sec using UV-Visible doublebeam spectrophotometer (Thermo Scientific SpectroscanUV 2700) The extinction coefficient (136 times 103Mminus1 cmminus1)was used for calculation of the enzyme activity It wasexpressed as units (U) that is 120583moles of acetylthiocholine(ATI) hydrolyzed mLminus1minminus1 The substrate or enzymeblanks respectively were without substrate or enzyme Theexperimental observations were corrected by subtracting theabsorbance minminus1 recorded in controls

26 Effect of Substrate on Enzyme Activity To determine theeffect of substrate concentration on the activity of enzymethe enzyme (100 120583g) was assayed at varying substrate (ATI)concentrations at the room temperature (26 plusmn 2∘C) andthe Michaelis-Menten constant (119870

119898) and maximum velocity

(119881max) were computed

27 Determination of Effect of Incubation Time on CarbofuranInduced Inhibition of Enzyme The 100 120583g enzyme was addedwith 2 nM carbofuran and assayed for its residual activityat different time intervals (0min to 120min) at room tem-perature (26 plusmn 2∘C) The enzyme activity recorded at thesetime points in absence of carbofuran served as controlsThe zero time reaction refers to the reaction rate measuredimmediately after mixing the enzyme with other reagents

28 Determination of IC50 Value for Enzyme Inhibition byCarbofuran The 100 120583g enzyme was used to monitor theresidual activity by assaying it in the presence of varying con-centrations of carbofuran The control set of reaction did notcontain carbofuran and the activity recorded was consideredas 100 The IC

50value was calculated by extrapolating the

data

29 Determination of Mechanism of Inhibition of AChE byCarbofuran The 119870

119894and 119881max values were determined by

assaying the enzyme (100120583g) at varying concentrations ofATI in the absence and presence of carbofuran (20 nM)and plotting the graph using inverse of rate of reaction andsubstrate concentrations on 119884- and 119883-axes respectively The

Advances in Biology 3

Table 1 Extraction of rat brain acetylcholinesterase

Extraction buffer Fractions Total activity (120583molemLmin) Total protein (mg)

Phosphate buffer (50mM pH 74) Supernatant 0165 775Pellet 0562 305

Phosphate buffer with Triton X-100 (02 vv) (50mM pH 74) Supernatant 0990 1575Pellet 0275 120

One unit of the activity of AChE has been defined as the micromoles of substrate hydrolyzed per min per mL

intersections by the straight line at the 119884- and at the 119883-axesrespectively of the Lineweaver-Burkrsquos double reciprocal plotwere used for calculations of the above parameters

210 Determination of 119870119894of Carbofuran The mechanism

of inhibition of enzyme by carbofuran was determinedby assaying the enzyme as described in Section 29 Thefollowing formulae were used 119881max + 119868 = 119881max minus 1198681 + [119868]119870119894or119870119898+119868 = 119870

1198981+[119868]119870

119894 where119881max+119868 and119881maxminus119868 are the

maximal velocities of reactions in the presence and absenceof carbofuran Similarly119870

119898+119868 and119870

119898denote the119870

119898values

in the presence and absence of carbofuran [119868] represents theconcentration of inhibitor used that is 20 nM The 119870

119894value

may also be calculated using 119870119894= IC501 + [119878]119870

119898

211 Statistical Analysis of Data Statistical analysis of datawas performed using Graph Pad Prism version 6 for win-dows All values were expressed as mean standard deviationof 3 observations

3 Results

31 Localization of AChE from Rat Brain In order to localizethe enzyme protein from the rat brain one part of the tissuewas treated with Triton X-100 (02 vv) a nonionic deter-gent in phosphate buffer (50mM pH 74) to solubilise themembrane-bound protein It was observed that the detergentsolubilised fraction containedmore enzyme activity than thatof without detergentThe results of this experiment indicatedthat AChE in rat brain was membrane-bound The quantityof protein was also found to be higher in the soluble fractionsthe values being 315mgmL protein as against 155mgmL inthe fraction not treated with the detergent The pellet withTriton X-100 contained 120mgmL against 305mgmL inthe fraction obtained without treatment with the detergent(Table 1)

32 Effect of Low Temperature (minus20∘C) on the Stability ofEnzyme Activity The effect of low temperature (minus20∘C) wasstudied on the activity of rat brain AChE by storing theenzyme at this temperature and assaying for its activity using100 120583g protein at the interval of 7 days The data summarizedin Table 2 demonstrated that the enzyme did not lose anyactivity up to 30 days

33 Impact of Optimum pH for the Rat Brain AChE Theenzyme was assayed at constant concentration of substrate(05mM) and enzyme (100 120583g) for the determination of pH

Table 2 Stability of rat brain AChE activity

Days AChE activity remaining AChE activity0 100 01320007 100 013200014 998 013173621 998 013173628 995 013134030 995 0131340Effect of storage time at minus20∘C on the activity of AChE from rat brain wasobserved by carrying out the enzyme assay employing 100120583g protein ondifferent days as described in Section 2The enzyme was stored in phosphatebuffer (50mM pH 74) containing 02 Triton X-100

Table 3 Effect of pH on the rat brain AChE activity

pH Activity (120583molemLmin)36 00650 00763 00774 01392 011100 008Effect of pH at room temperature (26 plusmn 2∘C) on the activity of AChE fromrat brain was observed by carrying out the enzyme assay employing 100120583gprotein at different pH of different buffers (sodium acetate buffer for pH 36and 50 sodium phosphate buffer for pH 637 and 74 and carbonate bufferfor pH 92 and 100) as described in Section 2 The enzyme was stored inphosphate buffer (50mM pH 74) containing 02 Triton X-100

optima at room temperature (26 plusmn 2∘C)The different buffersused were sodium acetate buffer for pH 36 and 50 sodiumphosphate buffer for pH 637 and 74 carbonate buffer for pH92 and 100 as described in Section 2 The results presentedin Table 3 demonstrated that the enzyme exhibitedmaximumactivity at pH 74The enzyme assayed in buffers of higher pHvalues displayed inhibitory effect

34 Effect of Optimum Temperature for the Rat Brain AChEThe enzyme was assayed at constant concentration of sub-strate (05mM) and enzyme (100 120583g) at pH 74 by incubatingthe enzyme at different temperatures for 5min for the deter-mination of temperature optima as described in Section 2AChE from rat brain was found to exhibit optimum activityat 37∘CThe increase in temperature however caused gradualloss in its activity of enzyme (Table 4)

4 Advances in Biology

Table 4 Effect of temperature on the rat brain AChE activity

Temperature (∘C) AChE activity (120583molemLmin)4 005710 007220 007237 009640 008750 006160 000875 0005Effect of varying temperatures on the activity of AChE from rat brain wasobserved by carrying out the enzyme assay employing 100120583g protein at pH74 of phosphate buffer as described in Section 2 The enzyme was stored inphosphate buffer (50mM pH 74) containing 02 Triton X-100

35 Effect of Substrate Concentration on the Activity of RatBrain AChE In order to evaluate the effect of substrate (ATI)concentration on the activity of the enzyme 100 120583g of proteinwas usedwith varying concentrations of the substrate (ATI) atroom temperature (26 plusmn 2∘C) The enzyme activity indicateda direct correlation with the substrate displaying a hyperboliccurve (Figure 2(a)) When the data was extrapolated usinginverse of rate of reaction (V) and the substrate concentration[119878] a straight line was obtained which intersected at 119884- andat the negative abscissa of 119883-axes The calculated 119881max and119870

119898values were found to be 0066120583molesmLminus1minminus1 and

007mM respectively

36 Effect of Carbofuran on the Rate of Reaction Catalyzedby Rat Brain AChE The effect of carbofuran on the rateof reaction catalyzed by rat brain AChE was determinedby assaying the activity in the presence of different con-centrations of carbofuran (0ndash200 nM) The results indicatedconsistent decrease in enzyme activity After extrapolation ofthe data using percent residual activity and the carbofuranconcentrations on 119884- and119883-axes respectively the IC

50value

of this pesticide for rat brain enzyme was found to be 60 nM(Figure 3 and Table 5)

37 Time Dependent Impact of Carbofuran on the Rate ofAChE Catalyzed Reaction In order to evaluate the effect ofincubation time on the carbofuran induced inhibition of theactivity AChE from rat brain at room temperature (26 plusmn 2∘C)the enzyme (100120583g)was assayed at different durations in boththe absence and the presence of the carbofuran (20 nM)The enzyme activity in the absence of pesticide acted asa control The results presented in Figure 1 demonstratedthat the enzyme activity did not change with increasingduration of incubation in the presence of carbofuran whencompared at zero time point The results also demonstratedthat carbofuran at this concentration (20 nM) caused sharpinhibition of the enzyme However in the absence of thepesticide the enzyme activity was fund to be highly stable atroom temperature as it did not show any decrease in activityat increasing incubation time up to 60min under similarassay conditions (Figure 1)

000018

000020

000022

000024

000026

000028

AChE

activ

ity (120583

mol

esm

inm

L)

20 40 60 800Time (min)

minusI

+I

Figure 1 Effect of carbofuran (2 nM) on the activity of AChEfrom rat brain with respect to the varying incubation time atroom temperature (26 plusmn 2∘C) The enzyme assay was carried outemploying 100 120583g protein using the procedure as described inSection 2 The results indicate the average values of three indepen-dent experiments The enzyme in the absence of carbofuran servedas a control and did not show any decrease in activity +119868 and minus119868demonstrate the activity of AChE in the presence and absence ofcarbofuran

000

002

004

006

008

AChE

activ

ity (120583

mol

esm

inm

L)

05 10 15 20 2500[ATI] (mM)

(a)

(b)Lineweaver-Burkrsquos plot

10

20

30

40

1[V

]

30minus10 10 20minus20 0

1[S]

Figure 2 Effect of substrate (ATI) on the activity of AChEfrom rat brain was observed by assaying the enzyme at varyingconcentration ATI at room temperature (26 plusmn 2∘C) as described inSection 2 employing 100 120583g protein (a)The119870

119898

and119881max values werecalculated using the intersection of the straight line at 119884-axis andat the negative abscissa on 119883-axis respectively (b) [119881] is rate ofreaction and [119878] is substrate concentration

38 Determination of Mechanism of Inhibition of Enzymeby Carbofuran The results from the previous experimentsindicated that carbofuran was causing significant inhibitionof rat brainAChE It was therefore envisaged to determine themechanism of enzyme inhibition by carbofuran by assayingthe enzyme (100 120583g) using varying substrate concentrations

Advances in Biology 5

Table 5 Determination of IC50

value of carbofuran for rat brainAChE

Carbofuran(120583M) AChE activity remaining Activity

(120583molemLmin)000 10000 01085001 457841 004963002 406919 004411005 37131 004025010 32048 003474020 213192 002311Effect of varying concentrations of carbofuran on the activity of AChE fromrat brain was observed by carrying out the enzyme assay employing 100120583gprotein at pH 74 of phosphate buffer as described in Section 2 The enzymewas stored in phosphate buffer (50mM pH 74) containing 02 Triton X-100

005 010 015 020 025000Carbofuran concentration (120583M)

000

005

010

015

AChE

activ

ity (120583

mol

esm

inm

L)

Figure 3 Determination of IC50

value of carbofuran for rat brainAChE using the data from Table 5

in the absence and presence of the pesticide (20 nM) TheLineweaver-Burk double reciprocal plot of the data displayedtwo straight lines parallel to each other intersecting atdifferent points on 119884- and 119883-axes respectively Using theformula as shown in Section 210 119881max + 119868 (119881max value inthe presence of carbofuran) and 119870

119894were calculated with

the values being 005120583molmLmin and 5 nM respectively(Figure 4)

4 Discussion

The hydrolysis of the neurotransmitter acetylcholine (Ach)to acetic acid and choline by AChE is essential for thetransmission of normal nerve impulses at the synapse Theadverse effects of carbofuran in the occupationally exposedpeople while working in carbofuran infested environmentsmake it imperative to study the impact of carbofuran usingthe activity of AChE as a marker AChE is known to be

minus10minus20

20

40

60

1[V

]

minusI

+I

0 10 20 30

1[S]

Figure 4 Determination of inhibitory action mechanism of car-bofuran of AChE The enzyme (100 120583g) was assayed at varyingconcentration ATI at room temperature (26 plusmn 2∘C) in the absence(e) and presence (◼) of carbofuran (2 nM) as described in Section 2The 119870

119894

and 119881max values were calculated using the intersectionsof the straight line at 119884- and at the negative abscissa on 119883-axesrespectively

responsible for regulating the cholinergic functions andphysiological activities of living systems

The results of the present study indicated that AChE inrat brain was membrane-bound and it could be solubilisedby a nonionic detergent Triton X-100 The extraction andsolubilisation of AChE have been made by many workersusing various detergents as solubilising agents in both thepresence (014M) [23] and the absence of low concentrationof NaCl [24] It has been reported that mainly the hydropho-bic and electrostatic forces are responsible for binding ofthe AChE with the biological membrane Possibly the effectof NaCl is to weaken the electrostatic interactions withmembrane proteins that shields some part of the enzymefrom the action of the detergent Thus NaCl facilitates amore effective attack of the detergent on the membranehelping in release of more membrane-bound enzyme inthe soluble fraction There are some reports which suggestthat even by this treatment solubilisation of AChE is notcompletely achieved while using a combination of Triton X-100 and NaCl These findings suggest more tightly boundnature of protein to the membrane Also there is a pos-sibility that the enzyme would be shielded by such layersof macromolecules which make actions of Triton X-100and NaCl ineffective Other explanations to the resistanceoffered by enzyme towards solubilisation could be due tothe presence of different molecular forms of enzyme withdifferent modes of membrane anchorage However someenzyme activity was observed in the fraction without usingTritonX-100 in the extractionmediumThis could be becauseof release of some amount of membrane-bound enzyme dueto grinding of the tissues during homogenization process[25] Recently acetylcholinesterase has been shown to existonly as membrane-bound form in the human erythrocytes[26]

6 Advances in Biology

This enzyme was highly stable until 30 days without anysignificant loss in activity when stored at minus20∘C carryingout the enzyme assay employing 100120583g protein on differentdays as shown in Table 2 Similar findings have also beenreported for the stability of acetylcholinesterase from humanerythrocytes [26]

The activity of rat brain AChE showed optimum activityat pH 74 and 37∘C Almost similar results have been pre-sented by Padilla et al [9] However the optimal pH andtemperature requirements vary from one living system toanother and also from one organ to another in the sameliving system AChE from rat brain possessed Michaelis-Menten constant (119870

119898) value for its substrate (ATI) equal to

007mM Khandkar et al [27] have reported 119870119898value for

humanAChE to be 098mM which is very high as comparedto that in rat brain as reported in the present study Roy andChaudhuri [28] have reported 119870

119898value for AChE isolated

from different parts of the rat brain the values were 263454 and 50mM for AChE of cerebrum hypothalamus andcerebellum respectively The present study has included the119870

119898value (70 120583M) for the total AChE isolated from all parts

of the rat brain However the119870119898values reported by Roy and

Chaudhuri [28] are about 37ndash70-fold higher the reasons forthat are not clear In another study Jadhav et al [29] haveshown119870

119898value for AChE from rat brain to be 59 120583M which

is quite close to that obtained in the present studyThe IC

50value of carbofuran for AChE from the rat brain

was found to be 60 nM Smulders et al [30] have reportedIC50

values for rat brain AChE using different pesticidesof organocarbamate group The values recorded indicatedthat the potency order of these carbamates to inhibit ratbrain acetylcholinesterase is bendiocarb gt propoxur aldicarbgt carbaryl ≫ S-ethyl NN-dipropylthiocarbamate (EPTC)fenoxycarb with IC

50values ranging from 1M for bendiocarb

to 17M for carbaryl and ≫1mM for EPTC and fenoxycarbThe IC

50value of carbofuran for AChE isolated from ery-

throcytes of rat was 33 nM [30] IC50

values for differentcarbamates vary depending on their molecular size Thevariations in the IC

50value from 33 to 307 120583M for different

carbamate inhibitors could be partly due to different tissuesand experimental conditions employed such as temperaturepH and incubation time [31] Analysis of agricultural prod-ucts in India showed contamination with pesticide residuesData for pesticide residues from selected fields are beinggenerated by various agricultural universities and IndianCouncil of Agricultural Research Institutions in the country(ICARs)The percent presence of pesticide residues observedin the samples was found to be 606 for carbofuran Forcarbofuran the pesticide residues was detected reported tobe in the range of 50ndash1200 nglit The pesticide residues havealso been reported to be present in nonagricultural productsfor public consumption like meat fish and milk

In the present investigation carbofuran displayed inhi-bition of rat brain AChE in noncompetitive manner with119870

119894value being 5 nM thereby reducing the 119881max value with

an unchanged substrate affinity to the enzyme that is 119870119898

value Since 119881max changes and 119870119898

remains constant theratio of 119870

119898119881max gives rise to straight lines corresponding to

the uninhibited and inhibited reactions in Lineweaver-Burk

double reciprocal plot This kind of inhibition of rat brainAChE suggested that carbofuran was binding somewhere onthe surface of the enzyme protein and thereby influencing thecatalytic pocket in a way to reduce the velocity of reactionwithout influencing the affinity The action of carbofuranin the biological systems is mediated through binding withthe hydroxyl group of serine residue located at the enzymersquosactive site The noncompetitive inhibition by carbofuransuggests that it has other interaction sites different from thecatalytic sites in the enzyme It could be further ascertainedby performing detailed investigation using suitable bioinfor-matics tools However the presence and role of serine residuein the active site pocket responsible for the enzyme catalysiscould be ascertained by the site directed mutagenesis

5 Conclusion

Rat brain AChE was found to be membrane-bound whichcould be solubilised by using Triton X-100 a nonionicdetergent The significant inhibition of AChE activity in ratbrain by carbofuran indicates that the compound even at verylow concentration (119870

1198945 nM) is extremely neurotoxic to the

mammals Interestingly carbofuran was able to significantlyreduce AChE activity with respect to increasing incubationtime for enzyme-inhibitor complex Similar to the resultsfrom other studies this investigation also suggests that ratbrainAChE contains at least one serine residue at the catalyticpocket which is carbamylated by carbofuran causing inhibi-tion of the enzyme in a noncompetitive manner This eventresults in reduction of the maximum velocity of reaction(119881max) but there is any impact of affinity of enzyme to thesubstrateThe results of this study clearly indicated that AChEfrom rat brain might serve as a biomarker of carbofuraninduced toxicity in those occupants staying in pesticideinfested environment

Competing Interests

The authors declare that they do not have any competinginterests

Acknowledgments

Vivek Kumar Gupta is grateful to the University Grant Com-mission New Delhi for providing research scholarship forthis work at the Department of Biochemistry University ofAllahabad India Nikhat Jamal Siddiqi gratefully thanks theResearch Center Female Center for Scientific and MedicalColleges King Saud University Riyadh for the support

References

[1] R C Gupta ldquoCarbofuran toxicityrdquo Journal of Toxicology andEnvironmental Health vol 43 no 4 pp 383ndash418 1994

[2] J Rendon-Von Osten A M V M Soares and L Guilher-mino ldquoBlack-bellied whistling duck (Dendrocygna autumnalis)brain cholinesterase characterization and diagnosis of anti-cholinesterase pesticide exposure in wild populations from

Advances in Biology 7

Mexicordquo Environmental Toxicology and Chemistry vol 24 no2 pp 313ndash317 2005

[3] R M Whyatt D B Barr D E Camann et al ldquoContemporary-use pesticide in personal air samples during pregnancy andblood samples at delivery among urban minority mothers andnewbornsrdquo Environmental Health Perspectives vol 111 no 5 pp749ndash756 2003

[4] M Kaur and R Sandhir ldquoComparative effects of acute andchronic carbofuran exposure on oxidative stress and drug-metabolizing enzymes in liverrdquo Drug and Chemical Toxicologyvol 29 no 4 pp 415ndash421 2006

[5] D K Rai P K Rai S I Rizvi G Watal and B SharmaldquoCarbofuran-induced toxicity in rats protective role of vitaminCrdquo Experimental and Toxicologic Pathology vol 61 no 6 pp531ndash535 2009

[6] D K Rai R K Sharma P K Rai G Watal and B SharmaldquoRole of aqueous extract of Cynodon dactylon in preventionof carbofuran-induced oxidative stress and acetylcholinesteraseinhibition in rat brainrdquo Cellular and Molecular Biology vol 57no 1 pp 135ndash142 2011

[7] A Agrawal and B Sharma ldquoPesticides induced oxidative stressin mammalian systems a reviewrdquo International Journal ofBiological and Medical Research vol 1 no 3 pp 90ndash104 2010

[8] D Milatovic R C Gupta and M Aschner ldquoAnticholinesterasetoxicity and oxidative stressrdquo TheScientificWorldJournal vol 6pp 295ndash310 2006

[9] S Padilla R S Marshall D L Hunter and A Lowit ldquoTimecourse of cholinesterase inhibition in adult rats treated acutelywith carbaryl carbofuran formetanate methomyl methiocarboxamyl or propoxurrdquoToxicology andApplied Pharmacology vol219 no 2-3 pp 202ndash209 2007

[10] D K Rai and B Sharma ldquoCarbofuran-induced oxidative stressinmammalian brainrdquoMolecular Biotechnology vol 37 no 1 pp66ndash71 2007

[11] B Kaur A Khera and R Sandhir ldquoAttenuation of cellularantioxidant defense mechanisms in kidney of rats intoxicatedwith carbofuranrdquo Journal of Biochemical andMolecular Toxicol-ogy vol 26 no 10 pp 393ndash398 2012

[12] R K Sharma and B Sharma ldquoIn-vitro carbofuran inducedgenotoxicity in human lymphocytes and its mitigation byvitamins C and Erdquo Disease Markers vol 32 no 3 pp 153ndash1632012

[13] M G Lionetto R Caricato A Calisi M E Giordano andT Schettino ldquoAcetylcholinesterase as a biomarker in environ-mental and occupational medicine new insights and futureperspectivesrdquo BioMed Research International vol 2013 ArticleID 321213 8 pages 2013

[14] A G E Pearse ldquoHistochemistry theoretical and appliedrdquoin Histochemistry vol 2 chapter 17 pp 761ndash807 Churchill-Livingstone London UK 3rd edition 1972

[15] HHDale ldquoTheaction of certain esters of cholme and then rela-tion to muscarmerdquo Journal of Pharmacology and ExperimentalTherapeutics vol 6 pp 147ndash190 1914

[16] A Silver The Biology of Cholinesterases North Holland Ams-terdam Netherlands 1974

[17] T L Rosenberry ldquoAcetylcholmesteraserdquo Advances in Enzymol-ogy vol 43 pp 103ndash218 1975

[18] A J Trevor M A Gordon K K Parker and S-L ChanldquoAcetylcholinesterasesrdquo Life Sciences vol 23 no 12 pp 1209ndash1220 1978

[19] J Massoulie and S Bon ldquoThemolecular forms of cholinesteraseand acetylcholinesterase in vertebratesrdquo Annual Review ofNeuroscience vol 5 pp 57ndash106 1982

[20] S Brimijoin ldquoMolecular forms of acetylcholinesterase in brainnerve and muscle nature localization and dynamicsrdquo Progressin Neurobiology vol 21 no 4 pp 291ndash322 1983

[21] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[22] G L Ellman ldquoTissue sulfhydryl groupsrdquo Archives of Biochem-istry and Biophysics vol 82 no 1 pp 70ndash77 1959

[23] G Civenni S T Test U Brodbeck and P Butikofer ldquoIn vitroincorporation of GPI anchored proteins and their fate in themembranerdquo Blood vol 91 pp 1784ndash1792 1998

[24] B Sharma ldquoSome properties of partially purified acetyl-cholinesterase from the adult female filarial parasite Setariacervirdquo Helminthologia vol 33 no 1 pp 13ndash20 1996

[25] J Massoulie L I Pezzementi S Bon E Krejci and F-MVallette ldquoMolecular and cellular biology of cholinesterasesrdquoProgress in Neurobiology vol 41 no 1 pp 31ndash91 1993

[26] V K Gupta R Pal N J Siddiqi and B SharmaldquoAcetylcholinesterase from human erythrocytes as a surrogatebiomarker of lead induced neurotoxicityrdquo Enzyme Researchvol 2015 Article ID 370705 7 pages 2015

[27] M A Khandkar E Mukherjee D V Parmar and S S KatyareldquoAlloxan-diabetes alters kinetic properties of the membrane-bound form but not of the soluble form of acetylcholinesterasein rat brainrdquo Biochemical Journal vol 307 no 3 pp 647ndash6491995

[28] R Roy and A N Chaudhuri ldquoDifferential acetylcholinesteraseactivity in rat cerebrum cerebellum and hypothalamusrdquo IndianJournal of Experimental Biology vol 44 no 5 pp 381ndash386 2006

[29] K B Jadhav S Shivaya H Prasad et al ldquoComparativeresponses of acetylcholinesterase (AChE) of rat brain andmodelinvertebrate Caenorhabditis elegans in vitrordquo Applied BiologicalResearch vol 16 no 2 pp 169ndash175 2014

[30] C J Smulders T J Bueters R G Van Kleef and H PVijverberg ldquoSelective effects of carbamate pesticides on ratneuronal nicotinic acetylcholine receptors and rat brain acetyl-cholinesteraserdquo Toxicology and Applied Pharmacology vol 193no 2 pp 139ndash146 2003

[31] D K Rai and B Sharma ldquoOn the neurotoxicity of carbofuran inmammalian systemrdquo in Proceedings of the 4th International Aca-demic Conference on Environmental andOccupationalMedicinepp 71ndash79 Shanghai China October 2006

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