e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 8 ( 2 0 1 4 ) 948–958

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Toxicological effects of clofibric acid and diclofenacon plasma thyroid hormones of an Indian majorcarp, Cirrhinus mrigala during short and long-termexposures

Manoharan Saravanana,b, Jang-Hyun Hurb, Narayanasamy Arul c,Mathan Ramesha,∗

a Unit of Toxicology, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore 641 046,Tamil Nadu, Indiab Bio-Regulatory Chemistry Lab, Department of Biological Environment, College of Agriculture and Life Sciences,Kangwon National University, Chuncheon 200-701, Republic of Koreac Department of Life Science, Research Center for Cell Homeostasis, Ewha Womens University, Seoul 120-750,Republic of Korea

a r t i c l e i n f o

Article history:

Received 7 July 2014

Received in revised form

16 October 2014

Accepted 18 October 2014

Available online 24 October 2014

Keywords:

Clofibric acid

Diclofenac

Cirrhinus mrigala

Thyroid stimulating hormone

Thyroxine

a b s t r a c t

In the present investigation, the toxicity of most commonly detected pharmaceuticals in

the aquatic environment namely clofibric acid (CA) and diclofenac (DCF) was investigated

in an Indian major carp Cirrhinus mrigala. Fingerlings of C. mrigala were exposed to different

concentrations (1, 10 and 100 �g L−1) of CA and DCF for a period of 96 h (short term) and 35

days (long term). The toxic effects of CA and DCF on thyroid hormones (THs) such as thyroid

stimulating hormone (TSH), thyroxine (T4) and triiodothyronine (T3) levels were evaluated.

During the short and long-term exposure period TSH level was found to be decreased at

all concentrations of CA (except at the end of 14th day in 1 and 10 �g L−l and 21st day in

1 �g L−l) whereas in DCF exposed fish TSH level was found to be increased when compared

to control groups. T4 level was found to be decreased at 1 and 100 �g L−l of CA exposure at

the end of 96 h. However, T4 level was decreased at all concentrations of CA and DCF during

long-term (35 days) exposure period. Fish exposed to all concentrations of CA and DCF had

lower level of T3 in both the treatments. These results suggest that both CA and DCF drugs

Triiodothyronine induced significant changes (P < 0.01 and P < 0.05) on thyroid hormonal levels of C. mrigala.

The alterations of these hormonal levels can be used as potential biomarkers in monitoring

of pharmaceutical drugs in aquatic organisms.

© 2014 Elsevier B.V. All rights reserved.

∗ Corresponding author. Tel.: +91 422 2428493; fax: +91 422 2422387.E-mail address: mathanramesh@yahoo.com (M. Ramesh).

http://dx.doi.org/10.1016/j.etap.2014.10.0131382-6689/© 2014 Elsevier B.V. All rights reserved.

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. Introduction

ypically, pharmaceutical drugs are produced and sold foruman and veterinary, agriculture and aquaculture practices

Cunningham et al., 2009; Saravanan et al., 2012; Guerra et al.,014). Due to over production, indiscriminate usage and care-ess disposal, these drugs are being released into aquaticnvironment as parent compounds, conjugates or metabo-ites (Khetan and Collins, 2007; Gros et al., 2009; Guerra et al.,014). The continuous discharge and occurrence of pharma-eutical drugs in the aquatic ecosystem has become a majorroblem due to their long persistent period in the environ-ent. Furthermore, they can persist in the environment in an

ctive form and affecting the life of various aquatic organismsCunningham et al., 2009; Fedorova et al., 2014). Globally, theccurrence, fate, and the residue levels of these pharmaceuti-al drugs in the aquatic environment and organisms have beeneported (Hignite and Azarnoff, 1977; Daughton and Ternes,999; Heberer, 2002; Koutsouba et al., 2003; Naidoo et al., 2009;amaswamy et al., 2011; Martin et al., 2012; Huerta et al., 2013;rnold et al., 2013; Fedorova et al., 2014).

In the aquatic environment, the pharmaceutical com-ounds may occur in low concentrations (ng L−l to �g L−l) and

nterfere with the biological systems by posing a substantialhreat to water quality, aquatic ecosystem and organisms, anduman health (Daughton and Ternes, 1999; Kummerer, 2001a;ent et al., 2006; Gunnarsson et al., 2008; Benotti et al., 2009;orcoran et al., 2010; Ramaswamy et al., 2011; Boleda et al.,011). Environmental pollution caused by pharmaceuticals is

complex issue due to involvement of thousands of differ-nt active molecules which belongs to various therapeuticlasses, with different physico-chemical properties, chemicaltructures, environmental behavior and persistence (Zuccatot al., 2006; Fatta-Kassinos et al., 2011) and are now emerg-ng as a new environmental problem as micro-compounds (Het al., 2013). Bioaccumulation of some pharmaceuticals haseen observed in fish collected from effluent (Brooks et al.,005; Ramirez et al., 2009) and during laboratory exposuresSchwaiger et al., 2004), indicating that tissue concentrationsan reach higher levels than concentrations measured in envi-onmental samples (Bain and Anupama Kumar, 2014).

The most commonly detected pharmaceuticals in thequatic environment include antibiotics, anti-inflammatoryrugs, lipid-lowering agents and anticonvulsants (Fent et al.,006). Clofibric acid (CA), a hypolipidemic compound and anctive derivative substance of clofibrate has been reportedn the water bodies all over the world (Buser et al., 1998;ernes, 1998; Weigel et al., 2002; Koutsouba et al., 2003; Nunest al., 2004). Furthermore, in the aquatic environment CAay act as an endocrine disruptor (Pfluger and Dietrich,

001) and also produce a synergistic toxic effect in the pres-nce of other drugs, such as carbamazepine (Cleuvers, 2003).ikewise, diclofenac (DCF) a non-steroidal anti-inflammatoryrugs (NSAID) has also been detected in different water bod-

es throughout the world (Ternes, 1998; Farre et al., 2001; Jux

t al., 2002; Thomas and Hilton, 2004; Rabiet et al., 2006; Hongt al., 2007). In spite of the many literature on the potentialdverse effect of these drugs on many species such as rats,ultures and humans (Atchison et al., 2000; Hickey et al., 2001;

m a c o l o g y 3 8 ( 2 0 1 4 ) 948–958 949

Oaks et al., 2004), studies on the toxicological implications ofthese drugs on non-target species like fish are very limited(Saravanan et al., 2011c, 2012, 2013; Saravanan and Ramesh,2013). To assess the ecotoxicological effects of pharmaceuticaldrugs, data on different levels of the biological hierarchy arerequired (Fent et al., 2006).

Fish have been widely used as bioindicators for assessingthe effects of environmental pollution on aquatic ecosystem(Saravanan et al., 2012; Saravanan and Ramesh, 2013). Forthe evaluation of pharmaceutical development and its toxic-ity fish models are widely used (Powers, 1989). In addition,the genetic make-up of fish, development and physiologyis similar to mammalian system (Gunnarsson et al., 2008).Recently, the study on the impact of environmental contam-inants on endocrine systems has attracted growing interest(Li et al., 2014). In fish, endocrine responses may offer aspotential indicators through their integrative and early war-ning capacity in the recognition and evaluation of toxicstress caused by xenobiotics or fish exposed to polluted envi-ronments (Hontela et al., 1993). Moreover, measurement ofcirculating levels of hormones can offer additional infor-mation on the lethal effects of chemicals (Folmar et al.,1993). Thyroid hormones (THs) are produced upon activa-tion of the neuroendocrine hypothalamo–pituitary–thyroid(HPT) axis (Eales, 2006; Zoeller et al., 2007) and are activein almost every cell in the vertebrates (Heijlen et al., 2013).In fish, the thyroid endocrine system is controlled primar-ily by the hypothalamic–pituitary–thyroid (HPT) axis, whichplay an important role in regulation of differentiation, migra-tion, sexual maturation, growth, metabolism, osmoregulationand salinity adaptation (Matty, 1985; Griffin, 2000; Power et al.,2001; Liu and Chan, 2002; Crane et al., 2004; Eales, 2006; Juganet al., 2010; Yan et al., 2012; Wang et al., 2013; Murk et al., 2013).In fish, most of the plasma THs are bound to transthyretin(TTR), a specific TH transport protein and only free hormonescan enter into target cells to elicit a response (Power et al.,2000; Zhang et al., 2013).

Thyroid stimulating hormone (TSH) is a member of the ver-tebrate glycoprotein hormone family, which also comprisesthe pituitary and chorionic gonadotropins. TSH stimulates thethyroid gland to produce the THs namely thyroxine (T4) andtri-iodothyronine (T3). T4 is produced by the thyroid gland ina large amount than T3 and the deiodination of T4 in extra-thyroidal tissues is the main source of the biological moreactive hormone, T3 (McNabb, 1992; Sapin and Schlienger, 2003;Larsen et al., 2011; Villanger et al., 2011). TSH, T4 and T3 arethe principal hormones and have a wide range of biologicaleffects in physiological processes of vertebrates (Power et al.,2001; Schnitzler et al., 2008; Subhash Peter, 2011). These threeendpoints are routinely used for evaluating TH homeostasisin fish (O’Connor et al., 2000; Yamasaki et al., 2002) and alsoroutinely used as potential biomarkers (Zaccaroni et al., 2009)in fish (Sayed et al., 2012). However, information on effects ofenvironmental pollutants on the thyroid system in fish is stillscarce (Schmidt et al., 2012).

In Indian subcontinent, there is a possible risk of envi-ronmental contamination with the pharmaceutical residues

due to a rapid growth of pharmaceutical manufacturing com-panies (Rehman et al., 2013) and also over consumption ofpharmaceuticals in densely populated regions. In addition,

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about 75% of the sewage from industries and domestic wastesis still discharged into local waterways without any treat-ment and the water quality of rivers has been affected due topresence of pharmaceutical drugs and personal care products(Ramaswamy et al., 2011). Non-steroidal anti-inflammatorydrugs such as diclofenac, ketoprofen, naproxen, ibuprofen,and acetylsalicylic acid were detected in surface waters of theSouthern India (Shanmugam et al., 2014). Furthermore, it hasbeen suggested that the focus of future research on ecotoxi-cology of various types of pharmaceuticals (Brooks et al., 2009)and the effects of low concentrations of pharmaceuticals insusceptible aquatic species need to be better identified (Bainand Anupama Kumar, 2014). However, there is a noticeablelack of investigation of short and long-term effects on non-target organisms (Santos et al., 2010; Ramaswamy et al., 2011;Saravanan and Ramesh, 2013; Saravanan et al., 2013) and alsothe potential risk assessment of both pharmaceutical drugsCA and DCF concentrations on THs are meager on fish espe-cially in an Indian major carps.

Therefore, the present study was aimed to examine theshort and long-term ecotoxicological effects of most com-monly used pharmaceutical drugs CA and DCF on THs (TSH,T4 and T3) level in an Indian major carp Cirrhinus mrigala. Thefish C. mrigala is the most important among Indian major carpswhich is an intensively cultivated species. To our knowledge,this is the first study depicting the effects of CA and DCF onTHs (TSH, T4 and T3) in an Indian major carp, C. mrigala undershort and long-term exposures.

2. Materials and methods

The Department of Zoology, School of Life Sciences, BharathiarUniversity, Coimbatore 641046 has been registered with theCommittee for the Purpose of Control and Supervision ofExperiments on Animals (CPCSEA), Government of India. Theexperiments and handling of organisms were carried out asper the guidelines of CPCSEA.

2.1. Chemicals

Analytical grade clofibric acid (a-(p-chlorophenoxy) isobutyricacid, 97% pure, CAS No. 882-09-7) and diclofenac (2-[(2,6-dichlorophenyl) amino] benzene acetic acid sodium salt, 99.9%pure, CAS No. 15307-79-6) were purchased from Sigma–AldrichChemie GmbH, Germany. Dimethyl sulfoxide (DMSO) (CAS No.67-68-5) was purchased from Fischer Scientific India Pvt. Ltd.,India and 0.2 mL L−l used to prepare the stock solution at dif-ferent concentrations (1, 10, and 100 �g L−l).

2.2. Experimental fish

Fingerlings of C. mrigala were obtained from a local fish farm ofTamil Nadu Fisheries Development Corporation Limited, Ali-yar, Tamil Nadu, India. Mean weight and length of the fish were

8.0 g and 7.0 cm, respectively. They were acclimatized to thelaboratory conditions for about 20 d in large tank (1000 L capac-ity) and fish were fed ad libitum with rice bran and groundnutoil cake in the form of dough one time a day. One third of the

a r m a c o l o g y 3 8 ( 2 0 1 4 ) 948–958

water was renewed daily and feeding was withheld 24 h beforethe commencement of the experiment.

2.3. Water quality parameters

The tap water free from chlorine was used for the exper-iments and the water had the following physicochemicalcharacteristics (APHA, 1998); temperature (26.0 ± 1.5 ◦C), pH(7.2 ± 0.08), dissolved oxygen (6.2 ± 0.04 mg L−1), total alka-linity (18.1 ± 7.0 mg L−1), total hardness (18.3 ± 0.5 mg L−1),salinity (0.3 ± 0.02‰), calcium(4.1 ± 0.3 mg L−1) and magne-sium (2.3 ± 0.6 mg L−1).

2.4. Short-term exposure

For short-term toxicity studies, healthy fish were taken fromthe stock and were maintained in the glass tank. Two daysprior to experiments and during the experimental periodfeeding was discontinued. Various concentrations of CA andDCF (1, 10 and 100 �g L−l) were added in each glass aquaria(120 cm × 80 cm × 40 cm) containing 60 L of water. Four repli-cates were maintained for each concentration and 30 fishof equal size and weight were introduced. The test waterwas renewed at the end of 24 h and freshly prepared solu-tion was added to maintain the concentration of CA and DCFat a constant level. A concurrent control of 30 fish in fourdifferent glass aquaria were also maintained under identicalconditions. No mortality was observed during the above studyperiod. At the end of 96 h period fish from the control and drugtreated groups were taken for further analysis.

2.5. Long-term exposure

For long-term toxicity studies, various concentrations of CAand DCF (1, 10 and 100 �g L−l) were added in each glass aquaria(160 L capacity) containing 150 L of water. Four replicates weremaintained for each concentration and 75 fish of equal sizeand weight were introduced. Fish were fed ad libitum everyday. Water was changed daily in order to avoid accumula-tion of faecal matter and excess feed and renewed with thetoxicant. A separate control was also maintained by stocking75 fish in a glass tank without adding toxicant. Four repli-cates were also maintained. Experiments were conducted fora period of 35 d with 7 d sampling frequency. No mortality wasobserved during the experimental period. Upon completion ofthe stipulated exposure period of 7, 14, 21, 28 and 35 d, 15 fishwere randomly selected from control and drug treated groupsand sacrificed without anesthetizing for further analysis. Afterremoval of fish at various intervals of time, the volume ofthe control and drug treated glass aquarium were adjusted tomaintain a constant density of fish per unit volume of water.

2.6. Blood sample collection

Blood samples were collected by heart puncture using plastic

disposable syringes fitted with 26 gauge needles. The syringeand needle were prechilled and coated with heparin an antico-agulant. The collected blood was transferred into small vials,which is previously rinsed with heparin. Whole blood sample

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as centrifuged at 9392 × g, at 4 ◦C for 20 min to separate thelasma, which was used for the estimation of TSH, T4 and T3.

.7. TSH assay

SH assay was performed by enzyme linked immunosorbentssay (ELISA) kit (ANOGEN, Canada) following the manufac-urer’s instructions.

.8. T4 assay

4 activity was estimated by using the method of ELISAescribed by Wistom (1976).

.9. T3 assay

3 level was estimated by using the method of ELISA describedy Walker (1977).

.10. Statistical analysis

ll values were expressed as means and analyzed by analysisf variance (ANOVA), followed by a Duncan multiple range test

DMRT) to determine the significant differences (P < 0.01 and < 0.05) among the concentrations, between the drugs, andhe difference between the concentrations and drugs on eacharameter.

. Results

.1. Plasma TSH, T4, and T3 assay: short-termxposure

he hormones viz., TSH, T4 and T3 in C. mrigala exposedo various concentrations (1, 10 and 100 �g L−l) of CA andCF for 96 h exposure showed significant alterations when

Table 1 – Alterations in the hormonal (plasma TSH, T4 and T3) lnominal concentrations of CA and DCF (1, 10 and 100 �g L−1 ) fo

96 h exp

Drug concentrations (�g L−1) TSH (�IU/mL)

CAControl 4.562a

1 2.096b

10 1.144c

100 0.41c

DCFControl 4.562d

1 59.12b

10 46.96c

100 92.64a

F – statistics**

Concentration (C, F3,32) 6046.74**

Drug (D, F1,32) 47,664.28**

C × D (F3,32) 7255.29**

Means in a column followed by common letters for the drug are not signifi∗∗ Significant at P < 0.01.

m a c o l o g y 3 8 ( 2 0 1 4 ) 948–958 951

compared to control groups (Table 1). TSH exhibited a lowerlevel in fish exposed to all concentrations of CA and higherlevel in all concentrations of DCF exposures. T4 was found tobe increased in all concentrations of CA treated fish (exceptin 10 �g L−l), whereas, a significant decrease was observedin all DCF concentrations. Likewise, a significant increase inT3 level was noticed in 10 and 100 �g L−l of CA treatments.In contrast, a significant decrease in T3 level was observedin all concentrations of DCF exposure. A significant (P < 0.01)difference was observed in TSH, T4 and T3 among the concen-trations of both CA and DCF, between drugs and also betweenthe concentrations and drugs.

3.2. Plasma TSH response: long-term exposure

Plasma TSH level was found to be decreased in CA treatmentsthroughout the study period (except 1 and 10 �g L−l at theend of 14th day and 1 �g L−l at 21st day) when compared tothat of their control groups. However, in DCF treatments, itwas increased throughout the study period. In the presentinvestigation, a significant (P < 0.01) difference was observedin TSH among the concentrations of both CA and DCF,between drugs and also between the concentrations and drugs(Fig. 1).

3.3. Plasma T4 response: long-term exposure

The level of T4 in plasma significantly decreased in all concen-trations of CA up to 28th day (except on 7th day in 10 �g L−l and28th day in 10 and 100 �g L−l). On 35th day, T4 level was foundto be increased in comparison to the control groups. But in DCFconcentrations it was decreased throughout the study period(except 100 �g L−l at the end of 35th day). A significant (P < 0.01)

difference was observed in T4 level among the concentrationsof both CA and DCF, between drugs and also between theconcentrations and drugs (except at the end of 28th day). Onday 28th there was no significant (P < 0.01) difference in T4

evels of an Indian major carp C. mrigala treated withr the period of 96 h.

osure

T4 (�g/dL) T3 (ng/mL)

6.3c 172c

7.04b 130d

5.92d 186b

9.1a 292a

6.3a 172a

1.24bc 31c

1.42b 42b

0.92c 19d

212.37** 7029.87**

3082.64** 68,552.61**

458.83** 13,217.30**

cantly different (P < 0.05) according to DMRT.

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TSH

(μIU

/mL

)

Exposure period (days) Exposu re period (days)

CA DCF

Control 1 μg/L 10 μg/L 100 μg/L

Fig. 1 – Changes in the plasma TSH level of an Indian major carp C. mrigala treated with nominal concentrations of CA andDCF (1, 10 and 100 �g L−1; 35 days). Means in the bars followed by common letters for the drug are not significantly different

(P < 0.05) according to DMRT.

level among the concentrations of both CA and DCF, betweendrugs and also between the concentrations and drugs(Fig. 2).

3.4. Plasma T3 response: long-term exposure

Plasma T3 level was significantly decreased in 1 and 10 �g L−l

of CA exposed fish throughout the study period (except10 �g L−l on 35th day) when compared with their con-trol groups. However, T3 level was found to be higherin 10 �g L−l on 35th day and in 100 �g L−l of CA exposedfish (except on 14th day). In DCF treatment, a significant

decrease in T3 level was observed throughout the study period.A significant (P < 0.01) difference was observed in plasmaT3 level among the concentrations of both CA and DCF,between drugs and also between the concentrations and drugs(Fig. 3).

b a a ab db

cb b

ca

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ab b

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0

2

4

6

8

10

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7 14 21 28 35

T4

(μg/

dL)

Exposure period (days)

CA

Control 1 μg/ L

Fig. 2 – Changes in the plasma T4 level of an Indian major carp CDCF (1, 10 and 100 �g L−1; 35 days). Means in the bars followed b(P < 0.05) according to DMRT.

4. Discussion

THs are crucial for many biological processes (Bentley, 1998;Chan and Kilby, 2000). Environmental contaminants arethought to affect the synthesis, transport and metabolism ofTHs by disrupting the thyroid system in vertebrates (Zhouet al., 2000; Coimbra et al., 2005; Morgado et al., 2009). Ade-nohypophysis of teleost fish produces TSH, a glycoproteinhormone that stimulates the thyroid gland to synthesize andsecrete THs (Eales, 1979). TSH secretion in teleost fish appearsto be primarily under hypothalamic inhibition (MacKenzieet al., 2009). The level of plasma TSH is used as indices of itstranslation and secretion (Larsen et al., 2011) which is respon-sible for regulating the synthesis and release of T4 and T3 in

vertebrates (Zoeller et al., 2007). The assay of TSH may helpfulto determine the impact of chemicals on thyroid gland and itsstatus (Zoeller and Tan, 2007a).

a a a a b

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Exposure period (days)

DCF

10 μg/ L 100 μg/ L

. mrigala treated with nominal concentrations of CA andy common letters for the drug are not significantly different

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 8 ( 2 0 1 4 ) 948–958 953

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50

100

150

200

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300

7 14 21 28 35 7 14 21 28 35

T3

(ng/

mL

)

Exposure period (days) Exposure period (days)

CA DCF

Control 1 μg/ L 10 μg/ L 100 μg/ L

Fig. 3 – Changes in the plasma T3 level of an Indian major carp C. mrigala treated with nominal concentrations of CA andDCF (1, 10 and 100 �g L−1; 35 days). Means in the bars followed by common letters for the drug are not significantly different(

lomic(aa1di2bDaadmwepa

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P < 0.05) according to DMRT.

In the present study, different responses of plasma TSHevel was observed upon exposure to various concentrationsf CA (decreased at maximum level) and DCF (increased ataximum level) during short and long-term treatments. An

ncreased level of TSH has been reported in fish Liza aurataollected from contaminated sites of Ria de Aveiro, PortugalOliveira et al., 2011), zebra fish (Danio rerio) exposed to tri-dimefon (Liu et al., 2011) and perchlorate (Patino et al., 2003)nd in Fundulus heteroclitus exposed to melatonin (Grau et al.,985). TSH increase would be expected upon a plasma T4

ecrease and/or lower level of TH production by the pitu-tary (Patino et al., 2003; Teles et al., 2005; Oliveira et al.,011). A similar mechanism may be operated in this studyecause lower level of T4 level was noticed in both CA andCF treatments. Moreover, both CA and DCF might have prob-bly disrupted the synthesis or secretion of the circulating THnd the conversion of T4 to T3 in treated fish. The observedecrease in TSH level both in short and long-term exposureight have resulted from a down-regulation of CRH and TSHhich resulted in lesser concentrations of T3 and T4 (Laier

t al., 2006). A decrease in TSH level was also observed in Euro-ean eel Anguilla anguilla treated with Cu (Oliveira et al., 2008)nd �-naphthoflavone (Teles et al., 2005).

T4 and T3 supports various physiological functions inquatic organisms (Griffin, 2000; Liu and Chan, 2002; Eales,006; Arjona et al., 2008). Decreased level of plasma T4 wasbserved in A. anguilla exposed to chromium and copper (Telest al., 2005), in crucian carp (Carassius auratus) exposed toxtracted microcystins (Li et al., 2008), in sea bream (Sparusurata) treated with diethylstilbestrol, ioxynil and propilthy-uracil (Morgado et al., 2009), in D. rerio exposed to variousoncentrations of potassium perchlorate (Schmidt et al., 2012),nd also pentachlorophenol exposure in zebrafish larvae (Guond Zhou, 2013). A lower level of T4 may be due to an increasedctivity of UDP-transferase which responsible for glucuronida-ion and clearance of T (Zhou et al., 2001; Hallgren and

4

arnerud, 2002). Yu et al. (2013) reported that a significantecrease in T4 level in D. rerio exposed to the fungicidesexaconazole and tebuconazole indicate thyroid endocrine

disruption. A significant decrease of T4 levels was observedin zebrafish larvae with decabromodiphenyl ether (BDE-209)exposure whereas T3 levels were significantly increased (Chenet al., 2012) indicating that polybrominated diphenyl ethers(PBDE) induce thyroid endocrine disruption by disturbing T4

levels, but the changes in T3 levels may depend on exposuretime, species or concentrations.

In contrast, an increase in plasma T4 was reported in Sene-galese sole (Arjona et al., 2008) and in juvenile rainbow troutexposed to acute and sub-chronic exposures of selenite (Milleret al., 2007) and in zebra fish (D. rerio) embryos exposed to envi-ronmentally relevant concentrations of PBDE (Yu et al., 2011).Significant increase in T3 and T4 levels has been observed inCaspian roach (Rutilus rutilus caspicus) exposed to waterbornemanganese (Hoseini et al., 2014) and in zebrafish exposed tobutachlor (Chang et al., 2013). Maclatchy and Eales (1990) sug-gest that an increase in T4 level could be a consequence ofthe decreased GH levels. In addition, the increased level ofT4 might be either due to increase in TSH secretion or sensi-tivity of thyroid follicles to TSH (Grau et al., 1985) or may bedue to inhibition of 5′-deiodinase (responsible for accelerat-ing peripheral deiodination of T4 to T3 in plasma) (Gupta andPremabati, 2002). Increase in T4 may be explained as increasedTSH (TSH-beta) (Liu et al., 2011). Exposure of juvenile rainbowtrout to HgCl2 or MeHg caused an increase in plasma T4 andT3 levels indicating that Hg activates the HPT axis (Bleau et al.,1996). In the present study, the increase in T4 levels followingCA treatment may be due to an increased secretion of TSH or adirect thyrotropic action of CA. In general, T4 is the main formof TH secretion in the thyroid gland and TSH increase wouldbe expected upon a plasma T4 decrease (Oliveira et al., 2008,2011).

Generally, in all vertebrates including fish, the bulk ofplasma T3 concentrations are derived from peripheral con-versions of T4 by 5′-deiodinases (Eales et al., 1999; Plohmanet al., 2002; Eales, 2006; Zoeller et al., 2007). T is the active

3

form of THs and it is largely produced by the peripheral enzy-matic monodeiodination of T4 mainly in the liver and othertissues (Van der Geyten et al., 1998). Most of the chemical

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compounds such as polychlorinated biphenyls and pesticidescan lead to increase of T3 in blood by altering 5′-deiodinaseactivity in fish (Besselink et al., 1996; Adams et al., 2000;Coimbra et al., 2005; Picard-Aitken et al., 2007; Peter et al.,2009; Yu et al., 2011; Guo and Zhou, 2013; Yu et al., 2013). Inthis study, the observed increase in plasma T3 level may bea response to maintenance of the basal metabolic rate of thefish to ensure its survival. Significant increase in plasma T3 hasbeen reported in both female and male zebrafish exposed tobutachlor (Chang et al., 2013). On the other hand, the observedT3 decrease may have a serious impact in fish metabolismsince T3 is the biologically active TH (Brown et al., 1998).Peter et al. (2007) reported a decrease in the T3 level in theair-breathing perch (Anabas testudineus) exposed to monocro-tophos for 48 h. Likewise decrease in plasma T3 level wasnoticed in adult male goldfish (C. auratus) exposed to thepesticide monocrotophos. Leatherland and Farbridge (1992)suggested that lower level of plasma T3 may be a result ofan inability of the organism to produce plasma T3 optimumlevel or it may have been the result of chemical interactionalong the hypothalamic–pituitary–ovarian axis (Ruby et al.,1993). A decrease in plasma T3 levels is generally due to adrop in thyroidal T4 production and secretion and/or changesin peripheral THs metabolism (Li et al., 2008) or changes inperipheral TH deiodination or metabolism (Zhang et al., 2013).Iodothyronine deiodinases play vital roles in the mechanismof TH biotransformation in extra-thyroidal tissues (Zhanget al., 2013). Furthermore, in fish, deiodinase mRNA levels arehighly sensitive to numerous xenobiotics (Li et al., 2009, 2011;Picard-Aitken et al., 2007; Yu et al., 2010). The reduction inthe levels of circulating T3 in the monocrotophos (0.01 and0.10 mg L−l) exposed fish may also be due to higher stimulatedmetabolism of T3 (Zhang et al., 2013). In general, a declinein plasma T3 levels is mostly due to a drop in thyroidal T4

production and secretion.The alterations of THs in fish collected from contaminated

sites may be due to reduced thyroid levels resulting frominduced hepatic biotransformation enzymes (UDPGT and sul-fatase) and excretion (Kohn et al., 1996; Crofton, 2008), and/orchanges in expression of genes involved in TH production(Pocar et al., 2006; Brar et al., 2010). Moreover, thyroid disrup-tion due to chemical toxicity may also result in the alterationsof T4 and T3 (Li et al., 2014). Disruption of thyroid function bychemicals may affect the maintenance of a normal physiolog-ical status in vertebrates (Schnitzler et al., 2011). The previousliterature indicates that the alterations of THs (TSH, T4 andT3) levels may be a result of a variety of mechanisms, suchas tissue-specific alternations and THs related genes expres-sions (Li et al., 2009), changes in thyroid status and alterationsin leasing its hormones (Oliveira et al., 2008), in process ofbiosynthesis and secretion of T4 and T3 (Capen, 1997), changesin hypothalamus or in pituitary status (Alkindi et al., 1996)and changes in hormone catabolism and clearance rates (Saitoet al., 1991; Hontela et al., 1995). Moreover, these alterationsmay also depend upon the different exposure regime (Yu et al.,2011). Even though many studies reported, the mechanisms

of chemical interference with thyroid function in fishes areless known (Peter and Peter, 2007; Movahedinia et al., 2011). Inour previous studies, we revealed the biochemical, enzymo-logical and ionoregulatory responses of C. mrigala exposed to

a r m a c o l o g y 3 8 ( 2 0 1 4 ) 948–958

various concentrations of CA and DCF under short and long-term exposures (Saravanan et al., 2011c, 2013; Saravanan andRamesh, 2013).

5. Conclusion

The results of the present investigation concludes that differ-ent concentrations (1, 10, and 100 �g L−l) of CA and DCF have aprofound influence on the THs (TSH, T4 and T3) in the plasmaof an Indian major carp C. mrigala during short and long-termexposures. The alterations of these parameters could be usedas potential biomarkers for monitoring the impacts of phar-maceutical drugs and its residues in aquatic environment andorganisms. Since the mechanisms involved in disturbing THsin fish exposed to CA and DCF are not well known, furtherstudies on molecular and genetic mechanisms of action of CAand DCF need to be investigated in the future.

Conflict of interest

The authors declare that there are no conflicts of interest.

Transparency Document

The Transparency document associated with this article canbe found in the online version.

Acknowledgments

The author (Manoharan Saravanan) would like to thank andacknowledge the Council of Scientific and Industrial Research(CSIR), New Delhi, Government of India for providing finan-cial support in the form of Senior Research Fellowship for thisstudy (Fellowship Award Letter No.: 09/472(0141)/2009-EMR-I,dated: 01.05.2009).

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