Chemosphere 82 (2011) 370–376

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Chemosphere

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Transcriptome analysis provides insights for understanding the adverse effectsof endosulfan in Drosophila melanogaster

Anurag Sharma a,c, M. Mishra a,c, K. Ravi Ram a,c, R. Kumar b,c, M.Z. Abdin d, D. Kar Chowdhuri a,c,⇑a Embryotoxicology Section, Indian Institute of Toxicology Research, Lucknow 226 001, Indiab Analytical Chemistry Section, Indian Institute of Toxicology Research, Lucknow 226 001, Indiac Council of Scientific and Industrial Research, New Delhi, Indiad Department of Biotechnology, Jamia Hamdard, New Delhi 110 062, India

a r t i c l e i n f o

Article history:Received 5 April 2010Received in revised form 24 September 2010Accepted 3 October 2010Available online 30 October 2010

Keywords:EndosulfanDrosophila melanogasterGene expressionStress responseOrganismal effect

0045-6535/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.chemosphere.2010.10.002

⇑ Corresponding author at: Embryotoxicology SToxicology Research, Mahatma Gandhi Marg, LucknIndia. Tel.: +91 522 2963825 (lab: direct), +91 522 26fax: +91 522 2628227/2611547.

E-mail address: dkarchowdhuri@rediffmail.com (D

a b s t r a c t

Indiscriminate use of agrochemicals worldwide, particularly, persistent organic pollutants (POPs), is ofconcern. Endosulfan, a POP, is used by various developing/developed nations and is known to adverselyaffect the development and the hormonal profiles of humans and animals. However, little is known aboutthe molecular players/pathways underlying the adverse effects of endosulfan. We therefore analyzed theglobal gene expression changes and subsequent adverse effects of endosulfan using Drosophila. We usedDrosophila melanogaster keeping in view of its well annotated genome and the wealth of genetic/molec-ular reagents available for this model organism. We exposed third instar larvae of D. melanogaster toendosulfan (2.0 lg mL�1) for 24 h and using microarray, we identified differential expression of 256genes in exposed organisms compared to controls. These genes are associated with cellular processessuch as development, stress and immune response and metabolism. Microarray results were validatedthrough quantitative PCR and biochemical assay on a subset of genes/proteins. Taking cues from micro-array data, we analyzed the effect of endosulfan on development, emergence and survival of the organ-ism. In exposed organisms, we observed deformities in hind-legs, reminiscent of those observed in higherorganisms exposed to endosulfan. In addition, we observed delayed and/or reduced emergence inexposed organisms when compared to their respective controls. Together, our studies not only highlightthe adverse effects of endosulfan on the organism but also provide an insight into the possible geneticperturbations underlying these effects, which might have potential implications to higher organisms.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Endosulfan, an organochlorine pesticide (OCP), is widely useddue to its ability to control several pests of agro-products (Mosset al., 2009). The prolonged persistence of endosulfan in the envi-ronment and its bioaccumulation through the food chain [persis-tent organic pollutant (POP); WHO, 1999] is a major healthconcern to all nations. Despite its ban or restriction in more than30 developing/developed nations, several countries (includingEuropean Union, India, Indonesia, Australia, Canada, United States,Brazil, China, Thailand) still continue to produce and/or use endo-sulfan (Reviewed in Weber et al., 2009). Various concentrations ofendosulfan are reported in the environment and different matrices(water bodies: 1.7, soil: 0.3–34.86, fruits: 0.36–212.28, milk:

ll rights reserved.

ection, Indian Institute ofow 226 001, Uttar Pradesh,20107x218/219 (lab: PABX);

.K. Chowdhuri).

0.4–56.2, butter: 0.6–13.4, coconut oil: 6.2–10.8, human blood inKasargod district of Kerala, India: 0.69–176.21 lg mL�1) (Ernstet al., 1991; CSE, 2001; Singh et al., 2007).

Epidemiological and toxicological data suggest that in mam-mals, exposure to endosulfan induces various adverse biological ef-fects that include endocrine disruption, hepatotoxicity (Paul et al.,1995) neurological- (Silva and Gammon, 2009), reproductive- andimmune-dysfunctions (Saiyed et al., 2003; Aggarwal et al., 2008).Studies on cell lines [adrenocortical cells of rainbow trout, chinesehamster ovary (CHO) cells] and yeast (Saccharomyces cerevisiae)exposed to endosulfan, have shown increased oxidative stress(Sohn et al., 2004), genotoxicity (Bajpayee et al., 2006) and celldeath (Kannan et al., 2000). Although these studies relied on spe-cific markers of exposure, global changes in gene expression levelare still poorly understood. Analysis of global gene expressionchanges that accompany chemical exposure helped environmentaltoxicologists to characterize the toxicant mediated perturbationsin gene transcription and to unravel the molecular pathways in-volved in toxic response thereby providing mechanistic insightinto chemical mediated cellular toxicity (Royland and Kodavanti,

A. Sharma et al. / Chemosphere 82 (2011) 370–376 371

2008). We, therefore, carried out a microarray based gene expres-sion profiling and subsequent biochemical/organismal assays toidentify genes and the biological pathways that are significantly al-tered by endosulfan using Drosophila melanogaster as a modelorganism. We used this organism considering its powerful geneticand genomic tools, fewer ethical concerns and homology to hu-man. Moreover, the use of Drosophila is within the recommenda-tions of the European Centre for the Validation of AlternativeMethods (ECVAM) (Festing et al., 1998). Previously, global geneexpression study in Drosophila has provided insights into altera-tions in various biological processes in response to chemical treat-ment (King-Jones et al., 2006).

Our results suggest that the observed transcriptional responsein Drosophila against endosulfan has the potential to provideimportant insights to the endosulfan-induced biological responsein higher organisms. To the best of our knowledge, this is the firstmicroarray data generated for an organism exposed to endosulfan.

2. Materials and methods

2.1. Fly strains

Wild type D. melanogaster (Oregon R+), w1118, P-element inser-tion line of methuselah (mth1) (Lin et al., 1998) and transgenicstrain for hsp70, Bg9 (hsp70-lacZ) (Lis et al., 1983) were used inthe study. We selected methuselah mutant (mth1) stock becauseof its role in stress response and longevity. As a control for strainbackground of this mutant stock, we used w1118. Flies and larvaewere reared at 24 ± 1 �C on standard Drosophila diet (Singh et al.,2009). Additional yeast suspension was provided for healthygrowth of organisms.

2.2. Treatment schedule

For microarray/biochemical assay, third instar larvae (84 ± 2 h)of Oregon R+ or Bg9 were allowed to feed on food containing2.0 lg mL�1 endosulfan for 24 h (Analytical grade, Sigma, USA;99.7% purity). This concentration was chosen on the basis of MRLvalue in fruits [mixed with final concentration of 0.3% DMSO (Naziret al., 2003)]. Larvae fed on 0.3% DMSO food were used as controls.

2.3. Quantification of endosulfan in Drosophila larvae by gas liquidchromatography (GLC)

Control and endosulfan exposed larvae (in three replicates)were homogenized in acetonitrile and extracted with n-hexane.Cleaned and concentrated extracts (using an eluting mixture of15% ethyl acetate, 15% dichlorometane, 70% n-hexane with fluorisilcolumn) were applied on Agilent GLC 7890A (USA) equipped withElectron transport detector (ECD) to identify and quantify theresidual level of endosulfan (isomers and metabolites).

2.4. Sample preparation for microarray experiment

Control or treated larvae (60 larvae/group) were dissected inde-pendently in Poels’ salt solution (PSS) (Nazir et al., 2003) and trans-ferred to Eppendorf tubes containing RNAlater (Ambion, USA).These tubes were stored at �70 �C till further extraction of RNA.For each group, two biological replicates were used.

2.4.1. RNA extraction, labeling, hybridization and scanningTotal RNA was isolated using Qiagen RNAEasy Mini kit

(Valencia, CA) essentially following the manufacturer’s instruc-tions. After confirming RNA quality, double stranded cDNA wassynthesized. Subsequently, Cy3 CTP labeled cRNA was synthesized

through in vitro transcription. These cRNA samples were frag-mented and hybridized onto a Drosophila Gene Expression4 � 44k chip (AMADID:16705) using Gene Expression Hybridiza-tion kit (Agilent, USA). The hybridized slides were washed andscanned by Agilent Microarray Scanner G Model G2565BA at5 lm resolution (Please see Supplementary material for detailedmethodology).

2.4.2. Microarray data analysisFeature extracted raw data was analyzed using GeneSpring GX v

7.3.1 software (Agilent). To select significantly mis-regulated genes,we first filtered out probes with low intensities. Normalization ofthe data was done by GeneSpring GX using the recommended onecolor Per Chip and Per Gene Data Transformation (set measure-ments 0.01–0.01; Per Chip; normalized to 50th percentile; PerGene: normalized to specific samples). Subsequently, pair wisesample comparisons were made by calculating log2 ratio of normal-ized data. The correlation coefficients were within a range of 0.960–0.981 for the control group and 0.978–0.990 for the endosulfantreated group suggesting that the gene expression data obtainedin the study are highly reproducible. To select differentially ex-pressed genes, we used the following cutoff: 2.0-fold changes (upand down) in gene expression when compared to control and Pvalue of 60.05. Differentially regulated genes were clustered usinghierarchical clustering to identify significant gene expression pat-terns. Genes identified in the present study were assigned to biolog-ical pathways using the Database for Annotation, Visualization andIntegrated Discovery (DAVID) (Kasuya et al., 2009).

2.5. Quantitative real-time reverse transcriptase PCR (qPCR)

Expression profiles of seven genes in third instar Drosophila lar-vae exposed to 2.0 lg mL�1 endosulfan for 24 h were validated by7900 HT Fast Real time PCR of Applied BioSystems (CA, USA) fol-lowing Singh et al. (2010). Briefly, total RNA was reverse tran-scribed with MMLV reverse transcriptase (Fermentas, USA) andoligo-dT (Fermentas, USA). qPCR was performed in 96 well PCRplates using gene specific primers (see Supplementary Table S1)and power SYBR Green master mix (Applied BioSystems CA,USA). Relative quantification of gene expression was achieved byconcurrent amplification of Glyceraldehyde 3-phosphate dehydro-genase (GAPDH) as an endogenous control.

2.6. Soluble O-Nitrophenyl-b-D-galactopyranoside (ONPG assay)

ONPG assay was carried out following Stringham and Candido(1994). Briefly, larvae of Bg9, after washing, were placed in a micro-centrifuge tube (20 larvae per tube, three replicates per group),permeabilized for 10 min with acetone, incubated for 12 ± 1 h at37 �C in 600 lL of ONPG staining buffer and the reaction wasstopped by adding 300 lL of 1 M Na2CO3. b-galactosidase activitywas quantified by measuring the absorbance at 420 nm on UVspectrophotometer (Cintra 20 GBC, Australia).

2.7. Emergence assay

The emergence of flies in control and treated groups was eval-uated as in (Nazir et al., 2003). Larvae of Oregon R+ were trans-ferred to control food or food containing endosulfan (50 larvae/vial and 10 vials/group). The number of flies emerging from controland treated groups was recorded until all flies are emerged.

2.8. Cuticular mounting of legs

Cuticular mounting was carried out following Emerald and Roy(1998). (Please see Supplementary material for details.)

Fig. 1. Differentially expressed genes of third instar larvae of D. melanogaster exposed to endosulfan for 24 h (A). Cluster analysis of total number of genes differentiallyexpressed in endosulfan-exposed third instar D. melanogaster larvae as compared to those of DMSO group. The heat map is a visual demonstration of average intensity ofgenes in replicates; each colored line represents a single gene with the highest single intensity in red (up-regulated) and the lowest in green (down-regulated). The analysisdemonstrates distinct patterns for the DMSO and endosulfan exposed groups (B).

372 A. Sharma et al. / Chemosphere 82 (2011) 370–376

2.9. Survivorship assay

To determine the stress tolerance of organisms exposed toendosulfan, third instar larvae (84 ± 2 h) of w1118 and mth1 (60 lar-vae/vial, five replicates) were exposed, independently, to 2.0, 3.0,4.0 or 5.0 lg mL�1 of endosulfan for 24 h. After the treatment,number of larvae survived in each treatment group was scored.

2.10. Statistical analysis

Statistical analyses were carried out using Prism software (ver-sion 5.0, GraphPad Software, USA). Data from ONPG and emer-gence assays were analyzed by one way analysis of variance(ANOVA) and significance was ascribed at P < 0.01. The statisticalsignificance of data from survivorship assay was analyzed usingTwo-Way ANOVA followed by Bonferroni’s test for multiple com-parisons. The post hoc analysis was carried out to determine thesignificance at 5% level.

3. Results and discussion

In the present study, we focused on identifying the adverse ef-fects of endosulfan at the gene expression level and the organismallevels. Before we proceed with the experiments, we analyzed the

uptake and metabolism of endosulfan in the exposed organismsusing GLC.

3.1. Detection of endosulfan in Drosophila larvae

GLC analysis showed presence of endosulfan sulfate (1.9 lggm�1), the major metabolite of endosulfan and its isomer, b-endo-sulfan (0.3 lg gm�1), in the exposed organisms. The detected levelsof endosulfan and its metabolite suggest that the selected concen-tration is metabolized in a 24 h exposure period. Interestingly, thismetabolism of endosulfan is similar to that observed in higherorganisms as far as the presence of endosulfan sulfate is concerned(ATSDR, 2000). Subsequently, we carried out microarray to deter-mine the effect of endosulfan at the gene expression level.

3.2. Transcriptomic analysis of the D. melanogaster larvae in responseto endosulfan

3.2.1. Genes falling into three different functional classes are mis-regulated in response to endosulfan

Following microarray data analysis, we identified 256 genes,with 163 and 93 genes being up- and down-regulated, respec-tively, in the exposed organisms (Fig. 1A, heat map depicted inFig. 1B; also please see Supplementary Tables S2 and S3 for de-tails). To validate microarray data, qPCR was carried out on seven

Fig. 2. Expression of selected stress responsive genes in third instar D. melanogasterlarvae exposed to endosulfan for 24 h (A) showing the fold change in microarray,comparative qPCR analysis and (B) b-galactosidase activity in endosulfan-exposedBg9 (hsp70-lacZ) larvae. Data represent mean ± SD of three independent replicates.

A. Sharma et al. / Chemosphere 82 (2011) 370–376 373

genes, including heat shock proteins (hsps), turandot family mem-bers, thor, mth and mthl6. Expression of all the seven genes showeda trend similar to that observed in microarray experiments(Fig. 2A). We assigned the biological pathways to the identified256 genes using DAVID functional annotation tool, which anno-tates each gene and identifies the most relevant biological termsassociated with a given gene. However, these biological terms of-ten overlap due to inherent redundancy in annotations (Kasuyaet al., 2009). To improve the usefulness of the functional annota-tion analysis, we carried out DAVID clustering analysis and threeclusters met the statistical criteria (P < 0.05; enrichment scoresP1.3; (Huang da et al., 2009). These three classes are: (1) develop-mental process, (2) stress and immune response and (3) metabo-lism (Table 1).

Table 1Mis-regulated genes of endosulfan exposed larvae of D. melanogaster classified underdifferent functional classes using DAVID functional clustering analysis.

Functional class Genes

Developmentalprocess

bowl, broad, distal-less, ng1, ng2, ng3, dusky

Stress and immuneresponse

CG13551,dif, gnbp2, tot A, IP3K1,mth, thor, hsp22, hsp23,tot C, mthl6, attC,im10,im14, im3, lectin-galC1, l(3)mbn

Metabolism Detoxification (cyp18a1, cyp4c3, cyp4e3, cyp4g1, gstE7,gstD9, gstD1)Chitin metabolism (Cpr47Ec, Cpr47Eg, Cpr49Af, Cpr60D,Cpr65Ax1, Cpr67Fa1, Cpr67Fa2, Cpr78Cc, lcp9, lcp2, lcp1,lcp3, lcp4, lcp5, lcp8 obstructor-A, obstructor-E)General (adar, alkaline phosphatase, cp7Fb, CG10320,CG14946, CG18519, CG31016, CG4017, CG42335,CG42345, CG5342, CG8563, CG9521, G protein c30A,glucose dehydrogenase, henna, abrupt, hgo, PH4aMP,PH4aNE1, spn-E, CG17562, CG17560, CG14893, CG1441,aTub85E, alpha-amylase, act79B)

3.2.1.1. Transcripts involved in developmental processes. Adverse ef-fects on developmental genes may have a serious impact on popu-lation dynamics influencing growth, metamorphic traits, andreproductive performance. Recently, Brunelli et al. (2009) showedthat exposure of tadpole (Bufo bufo) to endosulfan impaired theirswimming activity, reduced larval growth, prolonged time tometamorphosis and an increased incidence of deformities. In thisstudy, we observed that transcript levels of seven genes (bowl,dusky, distal-less, broad, ng1, ng2 and ng3) associated with develop-ment were altered in exposed organisms. In Drosophila, dusky is in-volved in wing imaginal disc differentiation (Ren et al., 2005).While bowl plays an important role in leg morphogenesis (de CelisIbeas and Bray, 2003), distal-less is involved in the development ofmetatarsus (Cohen et al., 1989). broad is suggested to be associatedwith various regulatory networks specially during organ morpho-genesis (Spokony and Restifo, 2007) and also known to be a keyregulatory gene for induction of ecdysone (Gonzy et al., 2002).The family of new glue genes (ng1, ng2 and ng3) is predicted tobe involved in puparial adhesion (www.flybase.org). Interestingly,induction of these ng-genes was suggested to be under the controlof ecdysone response during early third instar stage (D’.Avino et al.,1995). The induction of these genes in the present study mightsuggest the adverse effects of endosulfan on morphogenesis and/or hormonal profile during the development of Drosophila. Basedon these observations, we hypothesized that exposure to endosul-fan may either delay or affect the fly development. To test this, weanalyzed the emergence pattern of exposed organisms and alsolooked for the morphological deformities in the emerged flies.3.2.1.1.1. Hind-leg deformities and delayed emergence of organismsexposed to endosulfan. In the emergence assay, as expected, we ob-served not only a delay of 2 d in the emergence but also a signifi-cant decrease (P < 0.01) in the number of flies that emerged in thetreated group as compared to control (Fig. 3A). In addition, we alsoobserved hind limb deformities such as truncation and/or fusion oftarsal segments (tarsomeres) in flies that emerged from the treat-ment group, indicating the teratogenic potential of endosulfan(Fig. 3B and Supplementary Fig. S1). Interestingly, these pheno-types are similar to the fusion of tarsomeres observed in bowl mu-tant clones of Drosophila (de Celis Ibeas and Bray, 2003). At present,we do not know if such deformities are a consequence of down-regulation of bowl in exposed organisms. Further, the hind limbdeformities observed in the present study are reminiscent of thoseobserved in vertebrates.

3.2.1.2. Transcripts involved in stress and immune response. Gener-ally, heat shock proteins are involved in maintaining cellularhomeostasis in stressful conditions by protecting denatured pro-teins (Sorensen et al., 2005). Among them, Hsp70 is a well knownfirst tier bio-indicator of chemical stress in response to the minuteassault (Mukhopadhyay et al., 2003). In the present study, how-ever, we did not observe hsp70 induction. This is further validatedin our fly based transgenic reporter gene assay wherein b-galacto-sidase activity in exposed third instar larvae of hsp70-LacZ (Bg9)was similar to controls (P > 0.05; Fig. 2B). This parallels the previ-ous observation of lack of hsp70 induction by endosulfan in HeLacells by Ait-Aissa et al. (2000). Alternatively, we observed theinduction of small heat shock proteins (sHsps) such as hsp22 andhsp23. These results are consistent with the suggestion of dePomerai (1996) that screening for specific stress proteins maynot provide a sufficiently sensitive bio-indicator for various pollu-tants due to differences in their potential to induce stress proteins.sHsps are involved in diverse functions such as basic chaperoningactivity, cytoskeleton protection and modulation of apoptoticprocess, representing means of cellular defence under stressconditions (Reviewed in Gupta et al., 2010). We also observedthe up-regulation of Drosophila 4E-BP (thor) and turandot-family

Fig. 3. Effect of endosulfan on emergence (A), hind leg morphology (B) and percent survival (C) of D. melanogaster exposed to endosulfan. Leg morphology was analyzed usingD. melanogaster (Oregon R+) larvae fed on DMSO (3Ba) or endosulfan contaminated food (3B b-d) (please see Supplementary Fig. S1 for different types of hind-leg deformitiesobserved in this study), bar represents 50 lm. Survivorship assay was carried out using mth1 larvae with w1118 serving as a control for the strain background. Significance isascribed as *P < 0.01, **P < 0.001 vs. DMSO.

374 A. Sharma et al. / Chemosphere 82 (2011) 370–376

of genes in the exposed organisms. Drosophila 4E-BP, homologousto 4E-BPs from other species including mammals, is critical for sur-vival under various stress conditions (Sheikh and Fornace, 1999;Miron et al., 2001). The only transcription factor known to regulatethe transcription of d4E-BP is dFOXO (Junger et al., 2003). However,lack of significant change in dFoxo expression in our study suggeststhat an alternative signalling pathway might target d4E-BP in re-sponse to endosulfan stress in Drosophila.

Among turandot (tot) family of genes, we observed up-regulation of totA and totC in the exposed organisms. Inductionof totA is considered to protect flies against oxidative stress(Ekengren and Hultmark, 2001) and are known to be activatedin certain septic conditions (Brun et al., 2006). Interestingly,activators of tot and hsps are similar under conditions of stress,but induction of tot is slower and/or requires severe stress(Ekengren and Hultmark, 2001) than heat shock response andis persistent. The up-regulation of these genes in this study thussuggests a systemic response in the exposed organism to chem-ical stress.

3.2.1.2.1. mth1 flies show higher tolerance to endosulfan. Apart fromsHsps, we observed up-regulation of methuselah (mth) and mthl6(isoform of methuselah). Flies deficient for Mth (G-protein-coupledreceptor) were shown to have increased longevity and more toler-ance to oxidative stress (Lin et al., 1998). We therefore hypothesizedthat over-expression of mth observed in our study may be a downstream consequence of oxidative stress and if so, organisms deficientin mth (mth1) will have higher tolerance to endosulfan. To test thishypothesis, we exposed larvae of mth1 or their controls to 2.0–5.0 lg mL�1 endosulfan and analyzed their survival. Consistent withour hypothesis, we observed significantly higher survival (98% and87%) of mth1 larvae compared to their controls (59 and 26% inw1118, P < 0.001), when grown on food containing 4.0 and5.0 lg mL�1 endosulfan (Fig. 3C). We did not observe any differencein the survival of both w1118 and mth1 larvae grown on control food orfood containing 2.0 or 3.0 lg mL�1 endosulfan (97–100%; P > 0.05).

Taken together, altered expression of different classes of stressgenes indicates the multifaceted nature of stress response to main-tain cellular homeostasis against endosulfan exposure.

A. Sharma et al. / Chemosphere 82 (2011) 370–376 375

3.2.1.3. Transcripts involved in metabolism. Among the 52 mis-regulated genes, seven belong to detoxification process, 17 underchitin metabolism, and 28 under general metabolism.

3.2.1.3.1. Transcripts of xenobiotic metabolism genes are mis-regulated. Cytochrome P450s (cyps) or phase I and Glutathione-S-transferase (GSTs) or phase II are known to be involved in themetabolism of xenobiotics. In this study, we observed up-regula-tion of four cyps (cyp4g1, cyp4e3, cyp4c3, cyp18a1) and three gsts(gstD9, gstE7, gstD1). Three of the four cyps mis-regulated, belongto cyp4 family and members of cyp4 family of Drosophila havehomologs in other organisms including vertebrates (Tijet et al.,2001). Drosophila cyp18a1 is homologous to cyp2B6 in humanand cyp2B6 is associated with a-endosulfan metabolism in human.Therefore, up-regulation of cyp18a1 together with its homology tocyp2B6, probably suggests the involvement of cyp18a1 in endosul-fan metabolism in Drosophila. Interestingly, a different family ofcyps such as cyp6 family members (cyp6g1, cyp6a2) were inducedin Drosophila when exposed to another organochlorine compound,DDT (Daborn et al., 2002). Taken together, these observations sug-gest the cyp genes induced in the present study may probably beinvolved in endosulfan detoxification. The over all up-regulationof Phase I and Phase II metabolism system might reflect oxidativestress in the exposed larvae (Li et al., 2008).

3.2.1.3.2. Transcripts involved in chitin metabolism. Chitin, aneukaryotic biopolymer and a major carbohydrate source, formsthe peritrophic matrix which is an important structure withinthe gut (Lehane, 1997). Being an integral supportive componentin insects, chitin has been considered as a selective target for pes-ticide (Cohen, 2005). In our study, we observed up-regulation of 17genes (lcp9, lcp2, lcp1, lcp3, lcp4, lcp5, lcp8, obstructor-A, obstructor-E, Cpr47Ec, Cpr47Eg, Cpr49Af, Cpr60D, Cpr65Ax1, Cpr67Fa1,Cpr67Fa2, Cpr78Cc), predicted to have chitin binding properties(www.flybase.org). When sequence of these genes were subjectedto Rebers and Riddiford Consensus (R&R Consensus) verificationusing the R&R prediction tool at cuticle data base (cuticle DB)(http://bioinformatics2.biol.uoa.gr/cuticleDB/index.jsp; (Karouzouet al., 2007), we found that, all these proteins, with the exceptionof three, (obstructor-A, obstructor-E, Cpr60D), belong to RR1 consen-sus group. Proteins of the RR-1 form are associated with soft/flex-ible cuticle (Willis et al., 2005). Up-regulation of these chitinbinding genes may indicate the adverse effect of endosulfan onperitrophic matrix and the cellular response to replenish this dam-aged matrix.

In addition, we observed up-regulation of glucose dehydroge-nase and a-amylase (amyrel), which are involved in general carbo-hydrate metabolism (Mishra et al., 2002) and the elevated levels ofthese genes in the study may reflect increased energy require-ments of organism under stress conditions (Araujo et al., 2008).We also detected induction of two genes (act79B and aTub85E) pre-dicted to be involved in the cytoskeleton organization (www.flybase.org) in exposed larvae, suggesting the adverse effect ofendosulfan on cytoskeleton.

4. Conclusion

To conclude, we have shown the effect of endosulfan on theexpression of genes involved in cellular processes such as develop-ment, stress and immune response, and metabolism. Consistentwith our transcriptomic data, we observed adverse effect(s) ofendosulfan on longevity and development of hind leg in D. melano-gaster. The latter is reminiscent of hind limb deformities observedin vertebrates exposed to endosulfan. Our findings not only high-light the adverse effects of endosulfan on the organism but alsoprovide an insight into the possible genetic perturbations underly-ing these effects.

Conflict of interests

None declared.

Acknowledgements

We thank the Director, IITR for support, Genotypic Technology,Bangalore, India for assistance in microarray studies, Dr. WilliamJa, Caltech, for w1118 and mth1 stocks and Prof. S.C. Lakhotia BHU,Varanasi for Bg9 stock and Dr. D. Parmar, Developmental ToxicologyDivision, IITR for qPCR facility. DKC, MM and AS thank DBT (BT/PR5640/BCE/08/382/2004), CSIR (NWP-34), DBT-SRF (JRF/06-07/194) and ICMR-SRF (45/6/2009/BMS/CMB) for financial supportrespectively. IITR communication No. 2882.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.chemosphere.2010.10.002.

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