Chemosphere xxx (2014) xxx–xxx

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Chemosphere

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The neonicotinoid pesticide imidacloprid and the dithiocarbamatefungicide mancozeb disrupt the pituitary–thyroid axis of a wildlife bird

http://dx.doi.org/10.1016/j.chemosphere.2014.11.0610045-6535/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel./fax: +91 532 2460788 (Office).E-mail addresses: surya.zoology@gmail.com (S.P. Pandey), drbana_mohanty@

rediffmail.com, banalata.mohanty@gmail.com (B. Mohanty).1 Tel.: +91 8858883382; fax: +91 532 2460788 (Office).

Please cite this article in press as: Pandey, S.P., Mohanty, B. The neonicotinoid pesticide imidacloprid and the dithiocarbamate fungicide mancozebthe pituitary–thyroid axis of a wildlife bird. Chemosphere (2014), http://dx.doi.org/10.1016/j.chemosphere.2014.11.061

Surya Prakash Pandey 1, Banalata Mohanty ⇑Department of Zoology, University of Allahabad, Allahabad 211002, India

h i g h l i g h t s

� Imidacloprid and mancozeb disrupt the pituitary–thyroid axis of the bird, Red Munia.� Weight and histopathology of the thyroid gland reflected substantial thyrotoxicity.� Altered plasma TSH, T4 and T3 revealed disruption of the pituitary–thyroid axis.� Disruption was more in breeding phase than pre-breeding phase of reproductive cycle.� This wildlife avian species is more prone to imidacloprid toxicity than mancozeb.

a r t i c l e i n f o

Article history:Received 6 April 2014Received in revised form 21 November 2014Accepted 22 November 2014Available online xxxx

Handling Editor: Caroline Gaus

Keywords:WildlifeHPT axisTSHBreeding phaseNeonicotinoidDithiocarbamate

a b s t r a c t

Thyroid is an important homeostatic regulator of metabolic activities as well as endocrine mechanismsincluding those of reproduction. Present investigation elucidated the thyroid disrupting potential of aneonicotinoid imidacloprid and a dithiocarbamate mancozeb in a seasonally breeding wildlife bird,Red Munia (Amandava amandava) who is vulnerable to these two pesticides through diet (seed grainsand small insects). Adult male birds were exposed to 0.5% LD50 mg kg�1 bw d�1 of both the pesticidesthrough food for 30 days during the preparatory and breeding phases. Weight, volume and histopathol-ogy of thyroid gland were distinctly altered. Disruption of thyroid follicles reflected in nucleus-to-cyto-plasm ratio (N/C) in epithelial and stromal cells, epithelial cell hypertrophy and altered colloid volume.Impairment of thyroid axis was pesticide and phase specific as evident from the plasma levels of thyroid(T4 and T3) and pituitary (TSH) hormones. In preparatory phase, plasma TSH was increased in response todecrease of T4 on mancozeb exposure showing responsiveness of the hypothalamic–pituitary–thyroid(HPT) axis to feedback regulation. On imidacloprid exposure, however, plasma levels of both T4 andTSH were decreased indicating non-functioning of negative feedback mechanism. Increased plasma T3in response to both the pesticides exposure might be due to synthesis from non-thyroidal source(s) ina compensatory response to decrease level of T4. In breeding phase, impairment of HPT axis was morepronounced as plasma T4, T3 and TSH were significantly decreased in response to both mancozeb andimidacloprid. Thus, low dose pesticide exposure could affect the thyroid homeostasis and reproduction.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Neonicotinoids and dithiocarbamates are widely used world-wide as insecticides and fungicides respectively (World HealthOrganization, 1988; Jeschke et al., 2011). The pesticides (and theirmetabolites) of these two groups are thus prevalent in the environ-ment and can be ingested, inhaled and/or absorbed transdermally

by non-target organisms, making them susceptible to their toxiceffects. Toxicological informations on both the groups of pesticidesprovide neurotoxic (Kimura-Kuroda et al., 2012; Overgaard et al.,2013), immunotoxic (Corsini et al., 2006; Mondal et al., 2009),developmental and teratogenic (NTP, 1992) effects in both lowand high doses. Endocrine toxicity/disruptive effects of neonicoti-noids and dithiocarbamates also have been demonstrated. Dithio-carbamates (mancozeb, maneb, zineb etc.) are potent thyroiddisruptors. Maneb and zineb are reported to disrupt the hypotha-lamic–pituitary–thyroid (HPT) axis in rats by affecting the hypo-thalamic thyrotropin-releasing hormone/TRH and pituitarythyroid-stimulating hormone/TSH (Laisi et al., 1985). Mancozeb/

disrupt

2 S.P. Pandey, B. Mohanty / Chemosphere xxx (2014) xxx–xxx

MCZ specifically inhibits enzyme thyroid peroxidase, affects theweight and histopathology of thyroid gland as well as cause thehypothyroidism on acute high dose exposures to laboratoryrodents (Ksheerasagar and Kaliwal, 2003; Axelstad et al., 2011).Ethylene thiourea (ETU) is the principal metabolic by-product ofdithiocarbamate pesticides which exerts various toxic effectsincluding thyroid disruption (Axelstad et al., 2011). Neonicotinoidsspecifically act as insect-nicotinic acetylcholine receptor (nAChR)inhibitor (Palmer et al., 2013). Imidacloprid/IMI, one of the neoni-cotinoid insecticides, was shown to have affinity for mammaliannAChR and has been elucidated toxic to mammals (Kimura-Kuroda et al., 2012). IMI is reported to cause the thyroid lesionson acute high dose exposure in laboratory rodents (Zaror et al.,2010) however thyroid disrupting potential of neonicotinoids hasnot been explored thoroughly. Thyroid gland, through its hor-mones (THs: thyroxine/T4 and triiodothyronine/T3), not only reg-ulates metabolic activities but also maintains reproductivehomeostasis (McNabb, 2007; Nakao et al., 2008; Wagner et al.,2008) in a variety of animals. Circulating concentration of THs isregulated by HPT axis through a negative feedback response. THsup regulate the gonadal growth/development and reproductiveaxis (Nakao et al., 2008; Wagner et al., 2008) in mammals. In birds,positive regulation though has been reported for temperate zonebirds, such as Japanese quail (Yoshimura et al., 2003); there arevaried reports on thyroid regulation of reproductive axis in tropicalzone birds, such as estrildid finches (Dawson and Thapliyal, 2001).Both dithiocarbamates and neonicotinoids are reported to disruptthe reproductive (Anway et al., 2005; Bal et al., 2012) and meta-bolic (Bhaskar and Mohanty, 2014) functions in mammals underlaboratory conditions through disruption of thyroid axis.

The present investigation demonstrated the effect of dithiocar-bamate MCZ and neonicotinoid IMI, two popularly used dithiocar-bamates and neonicotinoids respectively, on pituitary–thyroid axisof a seasonally breeding wildlife avian species, Red Munia (Amand-ava amandava). Specific action of MCZ and IMI, as fungicide andinsecticide respectively, demands their simultaneous use in agri-cultural fields making the wildlife birds vulnerable to their expo-sure. The bird of the present investigation feeds on seed grains aswell as small insects, and thus, is susceptible to both MCZ andIMI through dietary exposure. In view of lack of informations onMCZ and IMI induced disruption of HPT axis of wildlife avian spe-cies, a comparative study of dithiocarbamate and neonicotinoidpesticides was conducted during two of the important stages ofreproductive cycle i.e. pre-breeding/preparatory and breedingstages using environmentally relevant/low dose. The disruptionof thyroid physiology was evaluated through assessment of thevarious end points/biomarkers of thyroid health (thyroid weight& volume, follicles & colloids, epithelial cell height & nucleus sizeand their N/C) and alterations in circulating concentration of hor-mones of pituitary–thyroid axis (TSH, T4 and T3).

2. Materials and method

2.1. Experimental design

Male birds were captured from around Allahabad (25�270N81�440E), UP, India, from a particular forest area away from agricul-tural croplands (to avoid background exposures to pesticides) inthe beginning of preparatory (first week of July) and breeding (firstweek of September) phases of the reproductive cycle. Preparatoryphase of the reproductive cycle is the pre-breeding transition stagein which a severe cellular/tissue differentiation, remodeling anddevelopment take place in gonads before entering into activebreeding phase. The preparatory phase is characterized by a com-plex of developing secondary sexual characters as well as sexually

Please cite this article in press as: Pandey, S.P., Mohanty, B. The neonicotinoid pthe pituitary–thyroid axis of a wildlife bird. Chemosphere (2014), http://dx.do

motivated behaviors (Sharp, 1996). Birds were acclimatized inopen air aviaries under natural conditions of temperature, humid-ity and photoperiod for 10 days (d). Food (grinded wheat grains,grown without any background chemical exposure/organic; avail-able commertially) and water were given ad libitum. Food intake ofindividual bird was maintained.

Acclimatized male birds (body weight/bw 8.5 ± 0.5 gm) weredivided randomly and maintained in three groups (n = 8/group):MCZ-exposed group, IMI-exposed-group and control. Low dose(0.5% of median LD50) of commercial pesticides MCZ (75%w/w,Uthane M-45) and IMI (17.80% w/w, confidor) were given to expo-sure groups through diet using soy oil as vehicle. Control birdswere given food with vehicle. Food was mixed (coated) with pesti-cides using vehicle and kept overnight. Two sets of the experimentwere executed in preparatory (mid July-mid August) and breeding(phase of active mating and courtship; mid September-mid Octo-ber) phases respectively and were exposed for 30 d in both sets.All the birds (pesticides-exposed as well as control) in each set ofexperiment were euthanized at the end of experiment. Dietarymedian LD50 of MCZ for bird (860 mg kg�1 bw d�1) was taken asthe reference dose (Health and Consumer Protection Directorate-General, European Commission, 2009). For IMI, chronic medianLD50 of Japanese quail (31 mg kg�1 bw d�1) was taken as the refer-ence dose (Lopez-Antia et al., 2012). Body weight was recordedevery alternate day. Precision of pesticide-dose intake by each birdwas maintained by exposing them to the decided dose through cal-culated amount of food taken by birds during first 2 h of feeding(7:00–9:00 A.M.) each day. The test dose (0.5% of LD50) was consid-ered as environmentally relevant. Studies have reported the envi-ronmental concentrations of imidacloprid (Blacquie‘re et al.,2012) and mancozeb (Koppad and Umarbhadsha, 2006; Adamskiet al., 2009) in invertebrates, seeds/grains and crop fields whichis equivalent to our test dose, however, the precise biomonitoringdata on test compounds are not available for birds.

2.2. Plasma sampling and hormonal assay

Birds were terminated by decapitation, blood was collected bycardiac puncture in 0.1% EDTA treated vials, centrifuged at 2500r.p.m. for 15 min for separation of plasma and pooled. ELISA assayfor TSH, T4 and T3 hormones was carried out immediately withoutstoring/freezing the plasma. ELISA kits were used for measuringplasma conc. of TSH (SmarTest Diagnostics, Israel), T3 and T4(LDN GmbH & Co. KG, Germany). Interassay and intraassay coeffi-cient of variations (%) were <10% for T3 (7.6 & 7.0), T4 (8.1 & 3.9)and TSH (7.6 & 4.6) respectively. Samples were run in duplicateand optical density was measured by Bio-Rad iMark microplatereader (USA).

2.3. Thyroid histopathology

Thyroids were quickly dissected out, blotted and weighedbefore fixation in Bouin’s fixative for overnight followed by wash-ing and paraffin embedding according the standard protocol of ourlaboratory (Mishra and Mohanty, 2010). Thyroid sections were cutserially (5–6 lm), stretched on sterilized glass slides and stainedwith eosin-haematoxylene stain. Microphotography was doneusing Leica DM 2500 (Germany) light microscope. Every fifth serialsection out of total 120–150 sections (per bird) was analyzed forhistomorphometric analysis of epithelial cell heights & their nuclei,number of follicles filled with colloids and volume of thyroid gland(mentioned below) using ImageJ 1.32j (Image analysis softwarepackage, NIH, Bethesda, MD). Each of the histology measures forthe thyroid gland was conducted in replicates and 32 replicateswere conducted per bird.

esticide imidacloprid and the dithiocarbamate fungicide mancozeb disrupti.org/10.1016/j.chemosphere.2014.11.061

S.P. Pandey, B. Mohanty / Chemosphere xxx (2014) xxx–xxx 3

2.4. Volume of the thyroid gland

Thyroid volume quantification is an important biomarker ofiodine uptake, and therefore, it is an important parameter of thy-roid health assessment. Total volume of thyroid gland was deter-mined according to Kendeigh et al. (1966). Sections wereinvestigated at magnification 10�. Major and minor axis (perpen-dicular to each other; passing through center) of each serial section(spheroid in shape) were measured and volume was then calcu-lated according to the formula of prolate spheroid: Vg ¼ 4

3 pab2

(Vg: total volume of the gland; a: radius of major axis/long arm;b: radius of minor axis/short arm passing through the center ofthe thyroid gland).

2.5. Number of follicles (containing colloids)

Follicles are the functional units of the thyroid gland which arein multiple numbers, variable sizes, formed by a single layer of epi-thelial cells and are filled with colloid. Colloid is principally a pro-teinaceous mass, containing glycoprotein thyroglobulin, whichserves as a reservoir of materials for THs synthesis (Fawcett andJensh, 2002). Number of follicles (filled with colloids) was deter-mined according to Hartoft-Nielsen et al. (2005). One countingframe of 500 � 500 lm2 was created and then all the photomicro-graphs were analyzed in that frame. All the follicles (filled with col-loid) present within the frame were counted. Sections wereinvestigated at magnification 40�.

2.6. Volume of colloids

Total colloid volume in thyroid gland was determined as illus-trated by Kot et al. (2013). All the follicles containing colloids (fromall the serial sections of each gland) were considered for colloidvolume measurement. In each follicle, major and minor axes of col-loids (perpendicular to each other) were measured by ImageJ andthe diameter of colloid (d) was obtained by using the formula:d =p

a � b (a and b are the major and minor axes of the colloidrespectively). To compensate for the effects of sectioning a sphere,the measured diameter (d), was corrected to provide an estimate ofthe mean diameter [D(m)] by using formula: DðmÞ ¼ d� 4

p. Volume of

the colloid [V(col)] was calculated using formula: V ðcolÞ ¼ p�DðmÞ36 .

2.7. Epithelial cell height, nucleus size and nucleus-to-cytoplasm ratio(N/C) in epithelial and stromal cells

Epithelial cell height is an important biomarker of activity of fol-licular epithelial cells which are the sites of iodine uptake and T4synthesis (Crane et al., 2005). Epithelial cell height (lm) andnucleus size (lm) was measured at magnification 100�. Cell heightand nucleus size were determined by analyzing at least 100 cells ineach section. N/C ratio of epithelial and stromal cells was also deter-mined at magnification 100� in each section (Kot et al., 2013). Ineach selected cell, area of the cytoplasm with nucleus was deter-mined by measuring total cell area (Anc). The areas of all the nuclei(An) were summed up and were subtracted from the total cell area(Anc) and obtained the resultant cytoplasmic area (Ac). The N/C wasthen determined by the equation: N

C ¼ AnAnc�An.

2.8. Statistical analysis

OriginPro 9.0 32Bit (Origin Lab, MA, USA) statistical softwarewas used for all analyses. Results with a p-value less than 0.05,0.01 and 0.001 were considered significant unless stated other-wise. Data were analyzed using two way analyses of variance(ANOVA), represented as mean ± SD (standard deviation) and were

Please cite this article in press as: Pandey, S.P., Mohanty, B. The neonicotinoid pthe pituitary–thyroid axis of a wildlife bird. Chemosphere (2014), http://dx.do

analyzed by normal distribution and homogeneity of variance fol-lowed by Bonferroni’s posthoc test. Since the measures on the thy-roid gland were conducted in replicates per bird, therefore, nestedANOVA have been conducted for histomorphometric analyses. Sta-tistical analyses of data were done using Fisher’s exact test.

3. Results

3.1. Body weight

At 30 d, no significant alteration in relative body weight wasobserved in exposure groups of preparatory phase (F = 14.81). Inbreeding phase, relative body weight was reduced by approx. 6%(P < 0.01; F = 14.24) in both the pesticides-exposed groups(Table 1).

3.2. Thyroid weight

In MCZ-exposed group, the relative weight of the thyroid glandwas increased by approx. 63% (P < 0.01) and approx. 33% (P < 0.05)in preparatory and breeding phases (F = 9.87 and F = 5.01) respec-tively from their controls. In IMI-exposed group, relative weight ofthyroid gland was decreased insignificantly by approx. 25% in pre-paratory phase and increased by approx. 17% in breeding phasefrom the control (Table 1).

3.3. Effect on volume of thyroid gland

Histomorphometry revealed distinct alterations in the volumeof the thyroid gland in both the phases on pesticide exposure. InMCZ-exposed group, volume of thyroid gland was increased byapprox. 178–290% (P < 0.001) in preparatory and breeding phases.In IMI-exposed group, thyroid volume increased insignificantly byapprox. 44–70% in preparatory and breeding phases (Table 2).

3.4. Effect on follicles and colloids

Thyroid follicles of control birds in both preparatory and breed-ing phases were with regular epithelium and filled with colloids.Colloids in many follicles were in active state in preparatory phasewith secretory droplets (Fig. 1A). Colloids in most of the follicles inbreeding control were intact without secretory droplets (Fig. 1D).In pesticides exposed groups of both the phases, irregular shapedfollicles with ruptured epithelium and lesions in stroma wereobserved. In both MCZ- and IMI-exposed groups, number of folli-cles (filled with colloids) were reduced by approx. 42% (P < 0.01;F = 88.37) in preparatory phase and 18–20% (P < 0.05; F = 140.06)in breeding phase from their controls (Table 2). Damaged colloidsdevoid of secretary droplets were observed in follicles of both pre-paratory and breeding phases (Fig. 1B, C, E and F). In MCZ-exposedgroup, total colloid volume was decreased insignificantly by 48% inpreparatory phase but increased by 114% (P < 0.01; F = 36.51) inbreeding phase (Table 2). Depletion, shrinkage of colloids and exfo-liation of epithelial cells were also observed in MCZ-exposed groupin both phases (Fig. 1B and E). In IMI-exposed group, total colloidvolume was increased by approx. 70% (P < 0.05) in preparatoryphase but decreased approx. 58% (insignificant) in breeding phase(F = 18.21) (Table 2). Exfoliation of epithelial cells was observed inIMI-exposed group in both the phases while shrinkage and miner-alization of colloids were observed only in breeding phase (Fig. 1F).

3.5. Effect on epithelial cell height, nucleus size and N/C in epithelialand stromal cells

Epithelial cell height and nucleus size was significantlydecreased in pesticide-exposed groups of both the phases (Table 2).

esticide imidacloprid and the dithiocarbamate fungicide mancozeb disrupti.org/10.1016/j.chemosphere.2014.11.061

Table 1Effect on body weight and thyroid weight of pesticides-exposed groups in preparatory and breeding phases of the reproductive cycle.

Day 1 Day 30

Control MCZ IMI Control MCZ IMI

PreparatoryBW (gm) 8.55 ± 0.13 8.49 ± 0.25 8.47 ± 0.29 8.32 ± 0.26 8.13 ± 0.73 9.03 ± 0.61TW (mg) 0.10 ± 0.0065 0.09 ± 0.0028 0.09 ± 0.0046 0.08 ± 0.0054 0.13 ± 0.0038b 0.06 ± 0.0014

BreedingBW (gm) 8.52 ± 0.50 8.57 ± 0.16 8.48 ± 0.49 9.06 ± 0.091 8.49 ± 0.307b 8.55 ± 0.07b

TW (mg) 0.08 ± 0.0015 0.08 ± 0.0012 0.08 ± 0.0020 0.06 ± 0.0075 0.08 ± 0.011a 0.07 ± 0.014

BW = body weight, TW = thyroid weight, significance from control a = P < 0.05, b = P < 0.01, n = 8 birds/group, mean ± SD, two way analysis of varience (ANOVA), degree offreedom = 31.

Table 2Histomorphometric analysis of different components of thyroid in preparatory and breeding phases of the reproductive cycle.

Parameters Preparatory Breeding

Control MCZ IMI Control MCZ IMI

Total Thyroid Volume (�105 mm3) 2.30 ± 0.59 6.41 ± 1.47c 3.32 ± 0.68 1.3 ± 0.37 5.09 ± 0.72c 2.25 ± 0.33Total colloid volume (mm3) 17.9 ± 6.54 9.2 ± 2.02 30.5 ± 6.08a 17.15 ± 5.97 36.8 ± 8.28b 7.1 ± 3.96No. of Follicles (filled with colloids) 67.7 ± 2.83 38.3 ± 5.30b 39.0 ± 4.28b 59.1 ± 4.51 46.5 ± 5.78a 48.4 ± 4.45a

Epithelial Cell Height (lm) 4.74 ± 0.64 3.64 ± 0.42 2.92 ± 0.49b 5.69 ± 0.65 4.57 ± 0.38 3.24 ± 0.31b

Epithelial Nucleus Size (lm) 2.23 ± 0.32 1.37 ± 0.25b 1.09 ± 0.15c 4.25 ± 0.48 1.17 ± 0.30c 0.88 ± 0.18c

Nuclear-to-cytoplasmic ratio 1.48 ± 0.66 0.25 ± 0.08c 0.24 ± 0.09c 0.42 ± 0.07 0.14 ± 0.09c 0.29 ± 0.04b

Significance from control a = P < 0.05, b = P < 0.01, c = P < 0.001, n = 8 bird/group, mean ± SD, nested ANOVA, degree of freedom = 31.

4 S.P. Pandey, B. Mohanty / Chemosphere xxx (2014) xxx–xxx

In MCZ-exposed group, the epithelial cell height was decreasedinsignificantly by approx. 20–23% in both the phases. In IMI-exposed group, epithelial cell height was decreased by 38–43%(P < 0.01; F = 46.07 & 35.29) group both in both the phases. Epithe-lial nucleus size, in both the exposure groups, was decreased byapprox. 38% (P < 0.01) to 51% (P < 0.001; F = 67.22) in preparatoryphase and by approx. 72–79% (P < 0.001; F = 251.17) in breedingphase. N/C was decreased by approx. 83% (P < 0.001; F = 86.51) inboth MCZ- and IMI-exposed groups in preparatory phase; whereasit was approx. 67% (P < 0.001) and 31% (P < 0.01) (F = 63.88) inMCZ- and IMI-exposed groups in breeding phase.

3.6. Thyroid hormone assays: plasma T4 and T3 levels

In preparatory phase, plasma T4 level was decreased by approx.12–16% (P < 0.05; F = 10.81) in MCZ- and IMI-exposed groups(Fig. 2A). In breeding phase, decrease was approx. 18% (P < 0.05)to 27% (P < 0.01) (F = 15.47) in MCZ- and IMI-exposed groups(Fig. 2D).

In preparatory phase, plasma T3 level was increased by approx.36% (P < 0.001) and 23% (P < 0.01) (F = 16.04) in MCZ- and IMI-exposed groups (Fig. 2B). In breeding phase, plasma T3 level wasdecreased by 26% (P < 0.01) to 17% (P < 0.05) (F = 14.52) in MCZ-and IMI-exposed groups (Fig. 2E).

3.7. Pituitary hormone assay: plasma TSH level

In preparatory phase, plasma TSH level was increased by 31%(P < 0.01) in MCZ-exposed group but decreased by 40% (P < 0.01)(F = 25.22) in IMI-exposed group (Fig. 2C). In breeding phase,plasma TSH level was decreased by 38–45% in both MCZ- andIMI-exposed groups (P < 0.001; F = 22.80) (Fig. 2F).

4. Discussion

Thyroid hormones are important homeostatic regulators ofmetabolism and development including that of reproductivedevelopment/seasonal reproduction in vertebrates (Wagner et al.,

Please cite this article in press as: Pandey, S.P., Mohanty, B. The neonicotinoid pthe pituitary–thyroid axis of a wildlife bird. Chemosphere (2014), http://dx.do

2008; Yoshimura, 2013). However, TH regulation of reproductivedevelopment is highly species specific among seasonally breedingvertebrates (Flood et al., 2013). Present study demonstrated thepesticide-specific and phase-specific disruption of pituitary–thy-roid axis from environmentally equivalent low dose exposure todithiocarbamate MCZ and neonicotinoid IMI in a seasonally breed-ing wildlife avian species, Red Munia.

Substantial thyrotoxicity was induced on exposure to both MCZand IMI as evident by damage to thyroid follicles and lesions instroma, more prominent in breeding phase. MCZ is reported tocause thyroid lesions on acute high dose exposures (Axelstadet al., 2011). MCZ-metabolite ETU is also reported to cause thyroidlesions on exposure to doses comparable to and lower than LOAEL/NOAEL in rats (Maranghi et al., 2013). IMI and their metabolites arereported to cause thyroid lesions in rodents on acute high doseexposures (Zaror et al., 2010). Hypertrophy and hyperplasia of epi-thelial and stromal cells, as observed in MCZ and IMI exposedgroups, might have contributed to changes in the thyroid gland,specifically weight and volume. High dose exposures to MCZ havebeen reported to cause hypertrophy and hyperplasia in follicularcells in rodents (Baligar and Kaliwal, 2001; Axelstad et al., 2011).Decreased N/C in epithelial and stromal cells might be due to pes-ticides-induced toxicity. Genotoxicity and DNA damaging effects ofMCZ (Calviello et al., 2006) and IMI (Costa et al., 2009) have beenshown in mammalian in vitro systems.

Thyroid disruption also reflected form impaired plasma levels ofthe hormones of pituitary–thyroid axis i.e. TSH, T4 and T3. PlasmaT4 level was reduced in both the phases from exposure to bothMCZ and IMI. MCZ and ETU are reported to reduce the synthesisand release of T4 and their storage in the colloids (Axelstad et al.,2011; Maranghi et al., 2013) by inhibiting the iodide uptake andthyroid peroxidase activity in the epithelial cells (Doerge andTakazawa, 1990; Miller et al., 2009). Increased plasma T3 levels inpreparatory phase in both MCZ and IMI exposed groups might bedue to the increased conversion of T4 to T3 in epithelial cells as wellas increased T3 synthesis from non-thyroidal sources, such as brainand pituitary in a compensatory response to the decrease in T4 con-centrations. Being in inactive state, epithelial cells of breedingphase thyroid might have less ability for a compensatory response

esticide imidacloprid and the dithiocarbamate fungicide mancozeb disrupti.org/10.1016/j.chemosphere.2014.11.061

Fig. 1. Cross-sections of thyroid of control and pesticides-exposed birds of preparatory (A: control, B: MCZ and C: IMI) and breeding (D: control, E: MCZ and F: IMI) phases.Note the disruption of follicles and stroma ( ) in pesticide-exposed groups. Secretory droplets (%) of colloids (c) of preparatory are less in breeding control but absent in allexposed groups where shrinkage (⁄) is evident. Elliptical epithelial cells ( ) in preparatory, flattened in breeding control and MCZ-exposed group; showing exfoliation intocolloids ( ) in all exposed groups. Mineralization of colloids (�) noticed in breeding phase IMI-exposed group. Bar 50 lm.

S.P. Pandey, B. Mohanty / Chemosphere xxx (2014) xxx–xxx 5

and therefore plasma T3 level was decreased in all exposure groups.Hypothyroidism can induce mineralization of colloids which wasevident in IMI-exposed thyroids in breeding phase; neonicotinoidshave been reported to induce mineralization of colloids as aresponse of hypothyroidism (Rose, 2012). Plasma TSH level wasincreased in preparatory phase of MCZ exposed birds indicatingthe normal negative feedback response of the HPT axis to lowplasma T4 concentration. However in breeding phase, plasma TSHlevel did not increase in response to decreased T4 and T3, indicatingimpairment of HPT axis. In vitro exposure (10–120 lM) of MCZ hasbeen reported to induce neurotoxicity and neuroendocrine disrup-tion in hypothalamus due to oxidative stress in primary mesence-phalic cells isolated from Sprague–Dawley rat embryos (Domicoet al., 2006). Plasma TSH level was decreased in IMI exposed birdsin both preparatory and breeding phases indicating lack of respon-siveness of HPT axis to the negative feedback against decreasedplasma T4 level. IMI can directly induce neurotoxicity due to cho-linergic receptor inhibition in neurons (Kimura-Kuroda et al.,2012) and result in disruption of HPT axis.

Please cite this article in press as: Pandey, S.P., Mohanty, B. The neonicotinoid pthe pituitary–thyroid axis of a wildlife bird. Chemosphere (2014), http://dx.do

Disruption of THs synthesis/release in seasonally breeding birdsmay disturb neuroendocrine regulation of gonadal functions andimpair the reproductive cycle. THs imbalance and reproductiveabnormalities have been reported in some wildlife birds. In chicksof tree swallows (Tachycineta bicolor), bald eagles (Haliaeetus leuco-cephalus) and eastern bluebirds (Sialia sialis), exposure to p, p0-DDEinduced alterations in plasma THs level (Mayne et al., 2005; Ceshet al., 2010). Impaired plasma THs levels are associated with theimpairment of reproductive behavior and decreased reproductivesuccess because THs activation in the hypothalamus plays a criticalrole in the regulation of the neuroendocrine axis involved in sea-sonal reproduction in both birds and mammals (Nakao et al.,2008; Yoshimura, 2013). Impaired plasma T4 levels and changesin reproductive success and behavior have also been observed inbirds exposed to flame retardants (Marteinson et al., 2011, 2012).

THs metabolism play central role to regulate the body weighthomeostasis; hypothalamic thyroid hormone metabolism has beenreported to play a key role in the seasonal regulation of bodyweight homeostasis in Siberian hamsters (Barrett et al., 2007).

esticide imidacloprid and the dithiocarbamate fungicide mancozeb disrupti.org/10.1016/j.chemosphere.2014.11.061

Fig. 2. Graphs showing plasma T4 (A and D), plasma T3 (B and E) and plasma TSH (C and F) levels in the control and exposed groups during preparatory and breeding phasesrespectively. Data were represented as mean ± SD (standard deviation) and were analyzed by normal distribution and homogeneity of variance using two way analysis ofvariance (ANOVA) followed by Bonferroni’s posthoc test. ⁄p < 0.05, ⁄⁄p < 0.01 and ⁄⁄⁄p < 0.001 significantly different as compared to control.

6 S.P. Pandey, B. Mohanty / Chemosphere xxx (2014) xxx–xxx

Thus, pesticides-induced hypothyroidism may cause impairmentof body weight. Both MCZ and IMI have been reported to affectmetabolism adversely in high doses and reduce body weight inrodents (Ksheerasagar and Kaliwal, 2003; Bhardwaj et al., 2010).MCZ has also been reported to reduce the protein, glycogen andlipids level in mice which may be due to increased catabolism ofthe biomolecules to meet the enhanced energy demand of understress or reduced synthesis due to impaired cellular functions(Mahadevaswami et al., 2000). Birds keep on storing the food inform of glycogen and lipid in skeletal tissues and liver to fulfillthe energy requirements of producing and laying eggs. Increasedenergy demand as well as metabolic deregulation on thyroid

Please cite this article in press as: Pandey, S.P., Mohanty, B. The neonicotinoid pthe pituitary–thyroid axis of a wildlife bird. Chemosphere (2014), http://dx.do

disruption due to pesticides-exposure might have affected thebody weight in breeding phase.

This study led to conclude that pituitary–thyroid axis of a wild-life species, particularly avian population, is highly sensitive toMCZ and IMI exposure even in environmentally qeuivalent concen-trations. Sensitivity of the pituitary–thyroid axis to pesticides-exposure can cause alterations of reproductive and metabolichomeostasis. However, future studies on field-level exposures todithiocarbamate and neonicotinoid pesticides could throw morelight on how disruption of thyroid physiology in environmentallyrealistic situation can disturb the reproductive cycle andreproduction.

esticide imidacloprid and the dithiocarbamate fungicide mancozeb disrupti.org/10.1016/j.chemosphere.2014.11.061

S.P. Pandey, B. Mohanty / Chemosphere xxx (2014) xxx–xxx 7

Statement on ethics

Experimental protocols were approved by Institutional AnimalEthical Committee (IAEC) of the Department of Zoology, Universityof Allahabad. Guidelines of Committee for the Purpose of Controland Supervision of Experimental Animals (CPCSEA), Ministry ofEnvironment and Forests, Government of India were followed formaintenance and termination of the bird.

Acknowledgement

This work was financially supported by University Grant Com-mission (UGC), New Delhi, as a Major Research Project (No. 39-600/2010(SR) Dt. 10/01/2011 to Dr. B. Mohanty).

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