Shafiq-ur-Rehman1,2, Shaheen Rehman1

& M. I. S. Waliullah2

1Laboratory of Environmental Health and Toxicology, Division ofEnvironmental Sciences2Division of Entomology, S. K. University of Agricultural Sciencesand Technology of Kashmir, Srinagar, J&K, IndiaCorrespondence and requests for materials should be addressedto Shafiq-ur-Rehman (shafiqurrehman11@yahoo.com)

Received 27 October 2011 / Received in revised form 28 December 2011Accepted 2 March 2012DOI 10.1007/s13530-012-0114-9©The Korean Society of Environmental Risk Assessment and Health Science and Springer 2012

Abstract

Complaints regarding excessive use of chlorpyrifosand consequent poisoning to non target pollinatorshave increased throughout the world. Loss of honey-bee has been observed in the Kashmir valley of India,too. The lipid peroxidation, known to cause oxidativestress/damage, was found to be increased in nervoussystem of Apis mellifera exposed to chlorpyrifos. Fur-ther exacerbation of chlorpyrifos-induced oxidativedamage was observed in∙OH-generated H2O2 sys-tem. The∙OH radical scavenger, DMSO, mitigatedthe initiation of lipid peroxidation mediated by eitherH2O2 or CPF. The DMSO also repressed the com-bined oxidative effect of H2O2 and chlorpyrifos onthe nervous system. Findings suggest that oxidativestress/damage caused by chlorpyrifos in honeybeenervous system accomplished the toxic∙OH buildup, which successively provides a possible mechani-sm for chlorpyrifos neurotoxicity and its mitigationby∙OH scavenging biomolecules. Elevated malondi-aldehyde may possibly serve as an indicator of neuro-oxidative stress in bees and their loss due to chlor-pyrifos-contaminated environment.

Keywords: Apis mellifera, Chlorpyriphos neurotoxicity, Di-methylsulfoxide; Honeybee, Hydrogen peroxide, Hydroxyl ra-dical, Lipid peroxidation, Malondialdehyde, Oxidative damage

Introduction

In modern agriculture, pesticides play an importantrole by providing dependable, persistent and relatively

complete control against harmful pests with less ex-pense and efforts. They have, no doubt, increased cropyields by killing different types of pests that are knownto cause substantial or total crop damages. At the sametime, these chemicals are considerably potent environ-mental pollutants, and they produce undesirable toxiceffects on non-target organisms, including humans1-3.Bees can be also suffered with serious adverse effectsfrom toxic chemicals in their environments. In recentyears, indiscriminating evidences have shown enor-mous decline in bee’s population across U.S., France,Germany, and other parts of the world. Researchers inGermany and France have indicated over 30 percentdecline of honeybee population due to insecticide ap-plication on crops4. These investigations highlightedthat 80 per cent of fruits and vegetables, requiring pol-lination, may not make it to the market. The reportsfurther emphasized that decline of pollinators wouldupset the world economy by loss around 350 millionU.S. dollars per annum. Organophosphate (OP) insec-ticides, accounting for up to 50% of the global insecti-cidal use, were predominately related to incidents ofpoisoning bees5-7. The emerging evidences revealedthat CPF was highly toxic to honeybees, besides othernon target species such as birds, fish and even aquaticinvertebrates1,8,9.

Chlorpyrifos [CPF, Molecular formula: C9H11Cl3

NO3PS, Molecular mass: 350.6, Chemical Name(IUPAC): diethoxy-sulfanylidene-(3, 5, 6-trichloropy-ridin-2-yl) oxy-phosphorane, or chlorpryphosethyl(O,O′-diethyl O-[3, 5, 6-trichloro-2-pyridyl] phos-phorothionate)], a broad spectrum chlorinated OP, isthe most widely used insecticide, largely due to itsgreater stability, persistence, and deep effectivenessagainst a wide range of plant-eating insect pests. How-ever, the broad-spectrum insecticides common use (orabuse) were often, as toxic to beneficial insects as,they are to the target species10. CPF was metabolicallyactivated by oxidative desulfuration to a short-livedmetabolite CPF oxon, which inhibited the acetyl-cholinesterase (AChE) through phosphorylation of itsserine site11. Virtually, all types of OPs were consi-dered to have a common mechanism of toxicity, where-by the initial step in a cascade of reactions was to inhi-bit AChE12-14. However, evidences against a commonmechanism of toxicity were mounting. Fundamentally,AChE inhibition was non-specific and affected thewhole body systems through the cholinergic, muscari-

Chlorpyrifos-Induced Neuro-Oxidative Damage in Bee

nic and nicotinic receptor pathways. Nonetheless, theaffected systems were basically the central nervoussystem and the autonomic nervous system, as well asperipheral muscular pathways15. Furthermore, therewas worldwide concern regarding OP’s involvementin carcinogenesis, birth defects, malfunctioning im-mune system, neurobehavioural disorders, and neuro-toxicity15-18. It seemed that unknown OP targets couldexist, and therefore, the initiation of toxicity would notonly be the inhibition of AChE, but also interactionbetween OP and non-AChE targets19. These concernswere focusing on CPF’s independent neurotoxicityfrom cholinergic inhibition20-22.

The reactive oxygen species (ROS) or free radicals,which were generated during xenobiotics attack, couldindiscriminately interfere with cellular constituents andcould cause oxidative damage to lipids, proteins andnucleic acids, and eventually could result in cell death.The peroxidative damage to membrane lipids by freeradicals simultaneously produce thio-barbituric acidreactive substances, such as malondialdehyde, leadingto lipid peroxidation. The role of lipid peroxidation asindicator of oxidative damage in living tissues, recei-ved considerable attention for its potential pathophys-iological events and toxicological risks from certainmetals and pesticides exposure23-28. Increasing eviden-ces revealed association of oxidative damage in CPF-induced neurotoxicosis22,29-33. Moreover, the exacerba-tion of pro-oxidative manifestation of Fe2++ in nervoustissues of bee by CPF indicated a risk of highly toxic

radical damage30. Because, ferrous iron was known tointeract with endogenous H2O2 to generate extremelyreactive toxic hydroxyl radical (∙OH), which couldreact at high rate with most molecules in the cell includ-ing DNA, proteins, lipids, amino acids, and could da-mage them34. The ∙OH, as one of the most destructiveROS, could cause indiscriminate toxicity and cell de-ath. The ∙OH was also shown to cause significant re-duction in AChE activity in the rat brain, which maybe partly due to the induction of oxidative stress35,36.These highly reactive pro-oxidant species seemed toput forth its dynamic role in chlorpyrifos induced neu-rotoxicity in terms of the oxidative stress in exposedorganisms. For these reasons, the present investigationwas undertaken to assess whether the toxic∙OH couldoffer a mechanism in CPF-induced oxidative damageof nervous tissues and could provide an alternate mech-anism of CPF neurotoxicity (other than AChE) and itsmitigation as well.

Results

Effects of Increasing Exposure Levels of CPFon the Formation of the Malondialdehyde

Relationships between the impact of different levels

Neuro-Oxidative Damage of Chlorpyriphos 31

Figure 1. Effect of increasing concentrations of Chlorpyrifoson oxidative stress in nervous system. Different concentrationsof Chlorpyrifos (0.2, 0.4, 0.8, 1.2, 1.6 and 2.0μg) were testedfor malonaldialdehyde formation as the index of lipid peroxi-dation to evaluate the oxidative stress in the nervous tissuesof Apis mellifera. Data were expressed as mean±SE. TheOxidative injuries showed a linear pattern of regression test as,MDA==1.58985++1.88404 CPF with a multiple R-Sq==0.991with the significant level (P) less than 0.0001.

Figure 2. Chlorpyrifos response with hydroxyl radical ge-nerator in nervous tissue of honeybee. Oxidative toxicity ofChlorpyrifos (1.0μg) exposure was assessed alone or in pre-sence of hydroxyl radical generator H2O2 (10 mM) in the Apismellifera nervous tissue homogenates. Malonaldialdehydeformation was assayed to evaluate the oxidative injuries inthe nervous tissues. Data are presented as lipid peroxidation(MDA nmol formed (mean±SE) for each experiment com-prising six replicate per treatment group and analyzed withANOVA. Pair samples t-test between control (C) or corres-ponding control (CC, i.e. CPF group) and the treatment groupswas performed. Asterisk indicates statistically significant dif-ferences from the control (or corresponding control), *P⁄0.01and **P⁄0.001.

MD

A(m

ol f

orm

ed/3

0m

in)

5.5

4.5

3.5

2.5

1.5

0 1 2

Chlorpyrifos (μg)

Regression analysis: MDA versus CPFMDA==1.58985++1.88404 CPF

Multiple R-Sq==0.991

0.2

1.6

0.4

0.8

1.2

Lip

id p

erox

idat

ion

(MD

A, n

mol

for

med

/30

min

)

Control H2O2 CPF CPF++H2O2

Groups

5

4

3

2

*c

**cc

**cc

of CPF exposure (0.2, 0.4, 0.8, 1.2, 1.6 and 2.0μg) andthe formation of MDA in the nervous tissues showeda linear pattern of regression test as, MDA==1.58985++1.88404 CPF with a multiple R-Sq==0.991, and signi-ficant level less than 0.0001. The result of experimen-tal study, displaying a trend of oxidative damage overexposure levels with statistical topography, was re-presented in a regression chart (Figure 1). One-wayANOVA demonstrated statistically significant differ-ences in the malondialdehyde levels among differentexperimental groups (P⁄0.0001).

Enhancement of Malondialdehyde inNervous Tissues by CPF and Hydroxyl ROSGenerator H2O2

The results showed that the formation of malondi-aldehyde in nervous tissues of honeybees was signifi-cantly increased (P⁄0.01) by H2O2. The production ofmalondialdehyde was observed significantly higher(P⁄0.001) in the nervous tissues of honeybees, fol-lowing the exposure of CPF as compared with con-trols. The CPF-induced significant increase (P⁄0.001)of malondialdehyde formation was aggravated signifi-cantly (P⁄0.001) by the ∙OH radical generator H2O2

(Figure 2).

Reduction of CPP-Induced Formation ofMalondialdehyde in Nervous Tissues by ROSRadical Scavenger DMSO

DMSO, which reacted with lipid peroxidation pro-cess by ∙OH free radical scavenging mechanism, re-duced the formation of malondialdehyde in honeybeenervous tissues (P⁄0.01), in comparison with con-trols. The H2O2-mediated formation (P⁄0.01) of ma-londialdehyde in nervous tissues of bees was signifi-cantly alleviated (P⁄0.001) from addition of DMSO.Performing in similar fashion, CPF induced significantproduction (P⁄0.001) of malondialdehyde in honey-bee nervous tissues, and DMSO significantly sup-pressed it (P⁄0.01) (Figure 3).

Involvement of ∙∙OH Radical in CPF-InducedOxidative Damage of Apis mellifera NervousSystem

The results showed that the CPF-induced (P⁄0.001)malondialdehyde formation was aggravated significan-tly (P⁄0.001) by the∙OH radical generator H2O2 inApis mellifera nervous system. Further observationindicated that the∙OH radical scavenger, DMSO, sig-nificantly mitigated (P⁄0.001) the exacerbated for-mation of the lipid peroxidation, from the combinedaction of CPF and H2O2 (P⁄0.001) (Figure 4).

32 Toxicol. Environ. Health. Sci. Vol. 4(1), 30-36, 2012

Figure 3. Effect of hydroxyl radical scavenger on Chlorpyri-fos-induced oxidative stress in nervous tissue of honeybee.Oxidative toxicity of Chlorpyrifos (1.0 μg) exposure was as-sessed alone or in presence of hydroxyl radical scavengerDMSO (0.5%) in the Apis mellifera nervous system. Data arepresented as lipid peroxidation (MDA nmol formed (mean±SE) for each experiment comprising six replicate per treat-ment group and analyzed with ANOVA. Pair samples t-testbetween control (C) or corresponding control (CC referred asH2O2 for H2O2++DMSO or as CPF for CPF++DMSO) and thetreatment groups was performed. Histograms marked withasterisk indicate statistically significant differences from thecontrol (or corresponding control), *P⁄0.01 and **P⁄0.001.

Lip

id p

erox

idat

ion

(MD

A, n

mol

for

med

/30

min

)

3.5

2.5

1.5

Groups

Control

DMSOH2O

2

H2O2++DMSO

CPF

CPF++DMSO

**cc

**c

*c

*c

*cc

Figuer 4. Function of hydroxyl radical initiator and scaven-ger in Chlorpyrifos neurotoxicity. Chlorpyrifos (1.0μg) expo-sure was evaluated for the oxidative toxicity in the Apis mel-lifera nervous tissue homogenates in presence of hydroxylradical generator H2O2 (10 mM) and scavenger DMSO (0.5%)alone or in combination. Data are presented as lipid peroxida-tion (MDA nmol formed (mean±SE) for each experimentcomprising six replicate per treatment group and analyzedwith ANOVA. Pair samples t-test between corresponding con-trol (CC referred as CPF for CPF++H2O2 or as CPF++H2O2 forCPF++H2O2++DMSO) and the treatment groups was performed.Histograms marked with asterisk indicate statistically signifi-cant differences from the corresponding control, **P⁄0.001.

Lip

id p

erox

idat

ion

(MD

A, n

mol

for

med

/30

min

)

4.9

4.4

3.9

3.4

CPF CPF++H2O2 CPF++H2O2++DMSO

Groups

**cc

**cc

Discussion

Among different pesticides, organophosphate insec-ticides were predominately related to incidents of poi-soning bee6. Chlorpyrifos for widespread agriculturalusage, was highly toxic to birds, fish, aquatic inverte-brates and honey bees1,8. The symptoms of dying beeswere typical for intoxication to CPF exposure7. Ourpreliminary investigation revealed for the first timethat CPF neurotoxicity increased malondialdehydeformation in honeybees30. Malondialdehyde, a thio-barbituric acid reactive substance (TBARS) and a mar-ker of lipid peroxidation of damaged membrane/cell,was the end product of oxidative stress in biologicalsystems, following pathophysiology or xenobioticstoxicity. Lipid peroxidation was linked with degrada-tion of phospholipids in the rat brain by toxic chemi-cals, like lead exposure23. In the present study, signifi-cant increases in malondialdehyde levels in honeybeenervous system by different levels of CPF exposuresshowed a dose dependent slope, signifying that appar-ent oxidative stress was taking place by the action ofthe insecticide. Earlier studies with pro-oxidant system(Fe II) also revealed the formation of lipid peroxidationproducts in CPF toxicity, suggesting oxidative damagein the honeybee nervous system30. Studies suggestedthat ROS may be involved in the oxidative stress in ratorgans, following CPF toxicity38,39. Other investiga-tions reported that toxicity caused by CPF may be dueto induction of oxidative stress in the central nervoussystem22,31,33,40-44. These studies also suggested thatCPF could induce ROS production in nervous tissuesof bees, resulting in neurotoxic effects through a highlytoxic∙OH radical mechanism instead of cholines-terase inhibition. For this reason, we examined∙OHradical generator (H2O2) and scavenger (DMSO) sys-tems to ascertain the role of∙OH radical in the neuro-oxidative damage caused by CPF neurotoxicity in thebees.

H2O2, a non radical derivative of oxygen, was a wellestablished reactive oxygen specie (ROS). It could crosscell membranes easily, and hence could attack thetarget directly45. Studies revealed that elevated forma-tion and build-up of H2O2 within the cells often couldcause DNA damage, although H2O2 did not directlyreacted with DNA or membrane lipids34. Nevertheless,H2O2 demonstrated its ability to be involve in the pro-cesses of oxidative stress by converting to the indiscri-minately reactive∙OH radical, in turn causing mole-cular damages and cell death46. The mode of action ofH2O2 in a biological system followed∙OH mechan-ism of lipid peroxidation process, whereas highly reac-tive∙OH radicals could frequently attack biologicalmolecules (including membrane lipids) by abstracting

hydrogen from them. This represented a mechanismof initiating the process of lipid peroxidation. The oxi-dative stress in a system could lead to cell damage,which consequently could influence into lipid peroxi-dation. Therefore, lipid peroxidation appeared to be ahighly significant consequence of oxidative stress inpatho-toxicity events. Nervous tissues were particular-ly highly sensitive to oxidative damage from lipid per-oxidation, due to its high oxygen consumption cou-pled with low levels of anti-oxidant defence systemand high membrane constituents with polyunsaturatedlipids, prone to oxidation47. In the present study, ex-posure of H2O2 increased the generation of malondi-aldehyde molecules in the nervous tissues, hinting adependency on∙OH radicals in the lipid peroxidationprocess. CPF individually caused the significant for-mation of high level of MDA molecules in the beenervous tissues. Furthermore, we observed that CPFpotentiated TBARS production during H2O2 stimulatedlipid peroxidation in the nervous tissues. This showeda mediatory role of the∙OH radical in the oxidativedamage in the nervous system of honeybees fromCPF toxicosis. These evidences supported our earlierdemonstration of CPF-induced lipid peroxidation,which was aggravated by a pro-oxidant Fe II sys-tem30. The Fe2++ ions, which were present within thebiological system, reacted with H2O2 molecules togenerate a bunch of very active∙OH species that sub-sequently took part in the process of chain reactions ofthe lipid peroxidation.

It was acknowledged that one possible candidate fortriggering lipid peroxidation was the∙OH. Suppressionof the∙OH formation could alleviate oxidative pro-pagation of the critical chain reaction of the lipid per-oxidation with subsequent reductions in toxic build up,as end-products in the system. With this scheme, theproxidative chain reactions, resulting from xenobioticattack, could be inhibited by the scavenger of free radi-cals to avert the cell damage. So, the validation of∙OHparticipation of CPF-mediated oxidative damage in thehoneybee nervous system was reaffirmed in the pres-ence of DMSO, a well-established inhibitor of∙OHspecies. Our findings, therefore, indicated that DMSOpossed an ability of significantly reducing the CPF-induced formation of malondialdehyde in the nervoussystem. Moreover, the exaggerated production of themalondialdehyde caused by CPF action during H2O2-stimulated lipid peroxidation in nervous tissues of thebee was inhibited by the∙OH scavenger DMSO.Hence, the∙OH scavenger showed its capability ofalleviating the exaggerated TBARS production in thenervous tissues, caused by the oxidative stress poten-tiated by H2O2 and CPF toxicity. This apparently re-vealed the involvement of∙OH in CPF toxicity, as a

Neuro-Oxidative Damage of Chlorpyriphos 33

contributory factor to the oxidative stress in the beenervous system from the lipid peroxidation definitely.Therefore, biological scavengers, competently inhibi-ting the∙OH radicals, could mitigate the lipid peroxi-dative damage of the nervous system following CPFtoxicity.

Conclusions

The increased presence of highly poisonous CPFinsecticide in the agricultural and contiguous environ-ment placed entire pollinators’ population into surviv-ing edge. The observed oxidative stress in the honeybeenervous system by CPF toxicity was measured to beelevated from the increased levels of malondialdehyde.Further evidences obtained from the present observa-tions with ROS generator and inhibitor systems sup-ported the involvement of most damaging∙OH radi-cals by initiating the lipid peroxidation process in thenervous system of bee from CPF toxicity. The studysuggested that the elevation of malondialdehyde levelsin nervous tissues could be considered as an indicatorof CPF poisoning in honeybees and their populationloss. The anti-oxidative intervention strategies couldalso play a potential role for ameliorating the CPFneurotoxicity.

Materials and Methods

Chemicals Chlorpyrifos-ethyl [CPF; O,O′-diethyl O-(3, 5, 6-tri-

chloro-2-pyridyl) phosphorothionate] and 2-thiobarbi-turic acid were from Sigma (USA). Other chemicalsand reagents used in this study were of purified grade.

Dissection of Nervous System and TissuePreparation

Apis mellifera was ventrally dissected out for ner-vous system30. The nervous system tissues were pooledin a Petri dish on an ice bath. Nervous ganglion washomogenized in chilled 0.10 M KCl, using Teflon pes-tle to obtain a 3% w/v homogenate for in vitro studies.

Formation of Malondialdehyde in NervousTissues Homogenate Exposed to IncreasingConcentration of CPF

The observation of formation of malondialdehydein the nervous system of Apis mellifera was performedwith different exposure levels of CPF (ranging 0.2-2.0μg) in tissue homogenates. For the control, experimentwas performed without addition of the CPF.

Formation of Malondialdehyde in NervousTissues Exposed to CPF under ROS(H2O2 System)

The toxic competency of CPF, as provoker of oxida-tive stress, was evaluated under pro-oxidation environ-ment in the Apis mellifera nervous system. Dose ofCPF (1μg) was considered to be a minimal dose toreach the nervous ganglia30. For interaction studies, thetissue homogenates were treated with H2O2 (10 mM),CPF (1μg), CPF (1μg) plus H2O2 (10 mM), or withoutany addition of stimulator. The malondialdehyde for-mation was assayed, as described below.

Effect CPF-Induced Formation ofMalondialdehyde in Nervous Tissues underROS Radical Scavenger (DMSO System)

Effect of anti-oxidant DMSO was tested for malon-dialdehyde formation in the nervous tissues of Apismellifera exposed to CPF. The nervous ganglia homo-genates were incubated in presence of DMSO (0.5%)alone or in combination with CPF (1μg). The malon-dialdehyde formation was assayed, as described below.

Involvement of ∙∙OH Radical in CPF-InducedOxidative Damage of Apis mellifera NervousSystem

The validation of involvement of highly toxic ∙OHROS in the oxidative damage of the nervous systemof Apis mellifera was examined in the following ex-periments: the homogenates consisted H2O2 (10 mM)alone, or H2O2 (10 mM) and DMSO (0.5%) combina-tion, CPF (1 μg) and H2O2 (10 mM) combination, orCPF (1μg), H2O2 (10 mM) and DMSO (0.5%) combi-nation. Experimental groups [H2O2 (10 mM); CPF (1μg) plus H2O2 (10 mM)] were considered, as the cor-responding controls. The malondialdehyde formationwas assayed accordingly, as described below.

Lipid Peroxidation (Molecular Formation ofMalondialdehyde) Assay

The quantitative assay of malondialdehyde formationwas performed by the 2-thiobarbituric acid (TBA)reaction method, as described by Shafiq-ur-Rehman23

and Shafiq-ur-Rehman et al.24. For the purpose, 1 mLof nervous tissue homogenate of Apis mellifera in atest tube was aerobically incubated at 37�C in a waterbath-cum-metabolic shaker (180 strokes/min of 2 cmamplitude) for 2 h. The test tube was immediatelycooled with tap water, and 1 mL cold 10% w/v trichlo-roacetic acid was added. The solution was thoroughlymixed on vortex-mixer and centrifuged at 2000 rpmfor 10 min. One mL of supernatant was transferred toanother test tube and allowed to react with an equalvolume of 0.67% w/v TBA for 10 min in a boiling

34 Toxicol. Environ. Health. Sci. Vol. 4(1), 30-36, 2012

water bath. The tube was cooled with tap water, andthe mixture was diluted with 1 mL of double distilledwater. The TBA reactive substance (TBARS), malon-dialdehyde, thus formed was measured at 535 nm. Theresult was expressed as n mol of malondialdehydeformed per 30 min; the molecular extension coefficientof malondialdehyde expressed as E535==1.56×105 37.

Statistical AnalysisThe statistical analysis and presentation were per-

formed with statistical software (SPSS version 11 orMinitab version 13). Values were expressed as mean++standard error (SE). Relationships between CPF ex-posure and MDA formation levels were tested by linearregression. Significant differences among groups wereevaluated using one-way analysis of variance (ANOVA)followed by Turkey’s pair-wise multiple comparisonspost hoc test. Comparison between control/correspon-ding control and treatment groups were analysed byStudent’s paired t-test. Values of P less than 0.05 (orotherwise mentioned) were considered statisticallysignificant.

Acknowledgements

I am highly thankful to Prof. Anwar Alam, Vice-Chancellor, and Prof. A. R. Trag, Director Research(now Vice-Chancellor of the Islamic University, Srina-gar) for their continuous encouragement and support.The work was carried out from our own available re-sources and facilities at the university.

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