Chemico-Biological Interactions 193 (2011) 216–224

Contents lists available at SciVerse ScienceDirect

Chemico-Biological Interactions

journal homepage: www.elsevier .com/locate /chembioint

The interactive effect of elevated temperature on deltamethrin-inducedbiochemical stress responses in Channa punctata Bloch

Manpreet Kaur a, Fahim Atif b, Rizwan A. Ansari a, Firoz Ahmad a, Sheikh Raisuddin a,⇑a Department of Medical Elementology and Toxicology, Hamdard University (Jamia Hamdard), New Delhi 110062, Indiab Brain Research Laboratory, Department of Emergency Medicine, Emory University, Atlanta, GA, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 11 February 2011Received in revised form 3 June 2011Accepted 30 June 2011Available online 23 July 2011

Keywords:DeltamethrinHeat stressHSPAntioxidant enzymesOxidative stress

0009-2797/$ - see front matter � 2011 Elsevier Irelandoi:10.1016/j.cbi.2011.06.011

Abbreviations: CAT, catalase; BOD, biological oxyoxygen demand; DO, dissolved oxygen; GSH, reducedS-transferase; GR, glutathione reductase; GPx, glutatshock protein; LPO, lipid peroxidation; T-SH, totalsolids; NP-SH, non-protein thiols; P-SH, protein thiols⇑ Corresponding author. Tel.: +91 11 26059688; fax

E-mail addresses: sheikhraisuddin@yahoo.com, sra(S. Raisuddin).

There are reports showing interactive effect of environmental factors with the toxic outcome of chemi-cals. We studied the interactive effect of elevated temperature as an abiotic stressor on deltamethrin-induced biochemical stress responses in a freshwater fish, Channa punctata Bloch. Heat stress (�12 �Cabove ambient temperature for 3 h) and pesticide exposure (deltamethrin 0.75 ppb for 48 h) showed sig-nificant induction of heat shock protein-70 (HSP70) in liver, kidney and gills of fishes. Elevated temper-ature when followed by deltamethrin exposure showed synergistic effect showing a high level of HSP70in liver and gills whereas response in the kidney was opposite. On the contrary, when deltamethrin expo-sure followed the heat stress, no significant difference was observed. Protein carbonylation was found tobe more pronounced in heat-stressed group compared with control fish group. A significant increase inlipid peroxidation (LPO) was observed in different tissues of fish exposed to either of the stressors. Inthe kidney of fish exposed to heat stress followed by deltamethrin, LPO was relatively lower as comparedto other treatments. Thiols content such as reduced glutathione (GSH), total thiols (T-SH), non-protein thiols (NP-SH) and protein thiols (P-SH) showed no consistent pattern in different tissues. Indeltamethrin-exposed group that was subsequently exposed to heat stress, the GSH content was higherin liver and lower in both kidney and gills when compared with other groups. Alteration in the activitiesof antioxidant enzymes such as catalase (CAT), glutathione S-transferase (GST), glutathione reductase(GR) and glutathione peroxidase (GPx) was also observed when fish were exposed to heat stressand/or deltamethrin. Our study demonstrated that heat stress modulated biochemical stress responsesin fish showing a tissue specific pattern. This implies that fish has the capacity to elicit differentialresponse to exposure to abiotic stressors in order to reduce the systemic magnitude of stress whichmay otherwise lead to severe dysfunction of vital tissues.

� 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Temperature is an important environmental variable that hasreflective impact on aquatic ectotherms, such as fishes. Stressresponses including those induced by temperature variation, dic-tate whether the organism adapts, survives, or dies [1]. Higherwater temperature lowers the availability of dissolved oxygen(DO), accelerates the metabolism, alters the respiration rate andenhances the oxygen demand of fish. Fishes are poikilotherms

d Ltd. All rights reserved.

gen demand; COD, chemicalglutathione; GST, glutathionehione peroxidase; HSP, heatthiols; TSS, total suspended.: +91 11 26059663.isuddin@jamiahamdard.ac.in

and change in water temperature has a great impact on their phys-iology [2–5]. Gradual change in temperature can be physiologicallycompensated for, but a rapid change disturbs homeostasis and thusbecomes a stress.

Pesticides are chemical stressors, which have become an inte-gral part of the ecosystem and regarded as major environmentalpollutants over the years. When used in the vicinity of aquaticecosystems, they tend to mix with water and may exert adverse ef-fects on fish populations [6,7]. Pyrethroids including deltamethrinare being used as substitutes for organochlorines and organophos-phates. They are extensively used in agriculture, for controllingpests, insects and vectors of endemic diseases, protecting seedsduring storage and fighting household insects because of theirlow environmental persistence [8]. The potential hazard of pyre-throids is owed to their heavy use in many aquatic larvicidal andmalarial control programs. Deltamethrin and other pyrethroidsexposure have been found to be highly toxic to fish and its

M. Kaur et al. / Chemico-Biological Interactions 193 (2011) 216–224 217

oxidative stress-inducing effect has been demonstrated in fish un-der short-term exposure [9,10], thus warranting a thorough eco-toxicological risk assessment [11–14].

Fishes can exhibit a cumulative response to stressors [15].When stressors, singularly or in combination, are severe enoughto challenge the homeostatic mechanisms beyond the compensa-tory limits of fish or permanently alter them, physiological pro-cesses generally adapt to compensate for the stress [16]. Heatshock proteins (HSPs) are a family of proteins that are expressedin response to a diverse range of biotic and abiotic stressors. Theyare thus, also referred to as stress proteins. Stress protein inductionhas been attributed to many environmental pollutants in aquaticorganisms. However, there is a paucity of data concerning HSPsand pesticide stress, with the exception of a few recent studies ofparaquat [17], heptachlor exposure in Homarus americanus larvae[18] and rainbow trout exposed to deltamethrin [19].

The physiology of fish is strongly related to temperature whichis supported by studies which have shown their response to chem-ical exposures is influenced by ambient temperature [1,5,15]. Theinteractive effect may be additive, synergistic or antagonistic andthe nature of the interaction may differ for different compoundsor stressors. Temperature has also been suggested to play animportant role in the toxicity of aquatic metals [20]. Additionally,exposures to pesticides in combination with other agents (viz.,stressors) may exert effects different from those experienced withpesticides alone [21].

It is well established that stressors can elicit non-specific re-sponses in fish, which are considered adaptive and hence enablethe fish to cope with the disturbance and maintain its homeo-static state. Antioxidant defense systems with non-enzymaticand enzymatic components are altered by stresses and are welldocumented [22]. Aquatic organisms including fish are routinelyexposed to a mixture of aquatic pollutants; we were interestedin studying the effect of temperature and deltamethrin on stressresponses in fish.

In the last decade, deltamethrin use has exponentially increasedin some countries, like, India [23], where the seasons are distinct,leading to substantial variation in temperature. Such environmen-tal variation of temperature may considerably influence the toxic-ity of pesticides. While the effects of temperature andcontaminants on fish have been studied as individual stressors,there is no detailed information about the combined effects of suchstressors. With this background the present research was under-taken to study stress responses of Channa punctata Bloch on inter-action of elevated temperature and deltamethrin exposure.

2. Materials and methods

2.1. Fish

C. punctata Bloch (Spotted snake-head murrel, Order: Percifor-mes, Family: Channidae) weighing 50–75 g were commerciallyprocured and maintained in 60 l aquaria following standard proce-dure [24]. Aquarium water was kept oxygen saturated by continu-ous aeration and ambient aquarium temperature was maintainedat 20 ± 2 �C with a photoperiod of 12 h light and 12 h dark cycle.Fish were acclimatized for 2 weeks under the above mentionedconditions before the start of the experimental paradigm. Phys-ico-chemical characteristics of aquarium water were monitoredevery alternate day. The normal ranges of selected parameterswere as follows: DO = 7.1 ± 0.8 mg/l, biological oxygen demand(BOD) = 9.7 ± 0.2 mg/l, chemical oxygen demand (COD) = 14.9 ±1.1 mg/l, total suspended solid (TSS) = 8.0 ± 0.9 mg/l, turbidity (inNTU) = 5 ± 0.2 and pH 7.6 ± 0.08. Aquarium water was replacedevery 24 h to minimize contamination from metabolic wastes. Fish

were handled using the institutional animal ethical committee(IAEC) guidelines of animal usage.

2.2. Exposure groups

Acclimatized fish were divided into five groups each compriseda minimum of eight fish. The first group maintained at ambienttemperature 20 ± 2 �C served as control (C). The second group offish was exposed to 0.75 ppb deltamethrin (in acetone) in aquar-ium water for 48 h (DEL). Fish of the third group were exposedto pre-heated water (32 �C; �12 �C above the average ambienttemperature) for 3 h (HS). The fourth group comprised of the fishwhich were first exposed to elevated temperature for 3 h and thento deltamethrin (0.75 ppb for 48 h) (HS + DEL). Whereas, the fish ofthe fifth group were first exposed to deltamethrin and then to heatstress at the same concentration and temperature as mentionedabove (DEL + HS). Experiment was designed in such a manner thatall the fish were sacrificed at the same time. The dose concentra-tion of deltamethrin was selected on the basis of previously pub-lished reports on C. punctata [9,10].

2.3. HSP measurement

Stress protein HSP70 was analyzed in different tissues of C.punctata using indirect ELISA procedure [25]. SDS–PAGE was alsoperformed to study induction of HSP70 in different tissues [26].

2.3.1. ELISAEach well of 96-well microtiter plate (Nunc, USA) was coated

with sample (50 lg protein) in triplicate and incubated at 4 �Covernight. After incubation, each well was washed thrice withTween-20 in PBS (TPBS, pH 7.2). After washes, plate was blockedwith 2% bovine serum albumin (BSA, Sigma) for 1 h at 37 �C. Afterblocking, wells were again washed and 100 ll anti-HSP70 primaryantibody (1:4000, anti-HSP70 [5A5, ab2787] mouse monoclonalfrom Abcam Ltd., UK) was added to each well and incubated at37 �C. Peroxidase tagged anti mouse IgG (1:4000 dilution) wasused as secondary antibody (Bangalore Genei Pvt. Ltd.) and theantigen–antibody reaction was developed by adding substratesolution (0.1% tetramethylbenzidine in phosphate citrate buffer,pH 5.0) to each well. The plate was incubated at 37 �C for 2 minand yellow color developed was read at 450 nm in MicroplateReader (Benchmark, Biorad) after stopping the reaction with50 ll of 2 M H2SO4.

2.3.2. SDS–PAGETissue samples were homogenized after thawing in 66 mM Tris

buffer (pH 7.8, Hi-Media Labs, Mumbai, India) with 1% Igepal CA630, 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and proteaseinhibitor cocktail (Sigma) to inhibit proteases. Samples were trans-ferred to microcentrifuge tubes and SDS sample buffer (63 mMTris–HCl, pH 6.8, 10% glycerol, 2% SDS, 0.0025% bromophenol blue;Hi-Media Labs) was added to each sample tube and heated at 95–100 �C for 5 min. The proteins were separated by sodium dodecylsulphate–polyacrylamide gel electrophoresis (SDS–PAGE, BioradMini Protein II) using 12.5% sodium dodecyl sulfate–polyacryl-amide gel with a 5% stacking gel as described by Laemmli [27],at 100 V for 1 h. Samples in duplicate were then run simulta-neously on gels. One was stained using Coomassie brilliant bluestain (E-Merck, Germany) and other was used for blotting.

2.4. Preparation of homogenate and post-mitochondrial supernatant(PMS)

After end of the treatment schedule fish were sacrificed and dis-sected to remove liver, kidney and gills. Tissues were washed in

218 M. Kaur et al. / Chemico-Biological Interactions 193 (2011) 216–224

ice-cold saline (0.85% NaCl) and for gills, gill rakers were removedand lamellae were homogenized. A 10% (w/v) tissue homogenatewas prepared in chilled phosphate buffer (0.1 M, pH 7.4) contain-ing KCl (1.17%) using a Potter–Elvehjem homogenizer. The homog-enate was centrifuged in a refrigerated centrifuge (Hermle, ModelZ323 K) at 800g for 5 min at 4 �C to separate the nuclear debris. Thesupernatants were again centrifuged at 10,500g for 30 min at 4 �Cto obtain the PMS for various biochemical analyses.

2.5. Protein carbonyl assay

Protein carbonyl content was assayed by the procedure of Floorand Wetzel, [28]. Soluble protein (0.5 ml) reacted with 10 mM 2,4-dinitrophenylhydrazine (DNPH) in 2 M hydrochloric acid for 1 h atroom temperature and precipitated with 6% trichloroacetic acid(TCA). The pelleted protein was washed thrice by resuspension inethanol/ethyl acetate (1:1). Proteins were then solubilized in 6 Mguanidine hydrochloride, 50% formic acid and centrifuged at16,000 g for 5 min to remove any trace of insoluble material. Thecarbonyl content was measured spectrophotometrically (Shima-dzu UV–Vis spectrophotometer, Japan) at 366 nm. Assay was per-formed in triplicate and a tissue blank incubated with 2 M HClwithout DNPH was included for each sample. The results were ex-pressed as nmol of carbonyl/mg protein based on the molar extinc-tion coefficient of 21,000 M�1 cm�1[28].

2.6. LPO

LPO was measured by the procedure of Mihara and Uchiyama,[29] with some modifications. Briefly, 0.25 ml of homogenatewas mixed with 25 ll of 10 mM butylated hydroxytoluene (BHT,Sigma), 3 ml of 1% o-phosphoric acid (OPA, CDH Chemicals, Mum-bai, India) and 1 ml of 0.67% thiobarbituric acid (TBA, Hi-MediaLabs) were added, and mixture was incubated at 90 �C for45 min. The absorbance was measured at 535 nm. The rate ofLPO was expressed as nmol of TBA reactive substance (TBARS)formed/h/g of tissue using a molar extinction coefficient of1.56 � 105 M�1 cm�1.

2.7. Estimation of reduced glutathione and thiols

Reduced glutathione was determined in PMS by the method ofJollow et al. [30]. Sulfosalicylic acid 4% in the ratio of 1:1 was usedto precipitate PMS. The samples were kept at 4 �C for 1 h followedby centrifugation at 400g for 15 min at 4 �C. The assay mixtureconsisted of PMS, phosphate buffer (0.1 M, pH 7.4) and dithio-bis-2-nitrobenzoic acid, (DTNB, Sigma) in the total volume of3 ml. The optical density of reaction product was read immediatelyat 412 nm on a spectrophotometer. The GSH values were calcu-lated as lmol GSH/g tissue.

Total thiol (T-SH), protein bound thiol (P-SH) and non-proteinbound thiol (NP-SH) groups in the PMS were determined usingthe method of Sedlak and Lindsay [31], as adopted by Parvezet al. [32] in case of fish. For total thiols, 1.5 ml Tris buffer(0.2 M, pH 8.2), 0.1 ml 0.01 M DTNB, PMS and methanol weremixed in the total volume of 10 ml. After 10 min, mixture was cen-trifuged at 3000g at 4 �C for 10 min and the absorbance of thesupernatant was measured at 412 nm. For non-protein thiols,0.4 ml PMS was precipitated with 0.1 ml 40% TCA and 0.5 ml dis-tilled water. After 10 min, mixture was centrifuged at 3000g. Theabsorbance of supernatant with tris buffer (0.4 M, pH 8.9) and0.01 M DTNB in the total volume of 1.5 ml was read at 412 nm.The level of P-SH was calculated by subtracting the values of NP-SH from T-SH content. The molar extinction coefficient of13,000 M�1 cm�1 was used to measure various thiols. The valuesare expressed as lmol/g of wet tissue.

2.8. Antioxidant enzymes

Catalase (CAT) activity was measured by the method of Clai-borne, [33]. The assay mixture consisted of 1.95 ml phosphate buf-fer (0.05 M, pH 7.0), 1 ml hydrogen peroxide (0.019 M H2O2, CDHchemicals) and 0.05 ml PMS in a final volume of 3 ml. CAT activitywas measured at 240 nm and calculated as nmol H2O2 consumed/min/mg protein.

Glutathione S-transferase (GST) activity was measured by themethod of Habig et al. [34]. The reaction mixture consisted of1.65 ml phosphate buffer (0.1 M pH 6.5), 0.1 ml GSH (1 mM,Sigma), 0.05 ml 1-chloro-2,4-dinitrobenzene (1 mM CDNB, Sigma)and 0.2 ml PMS in a total volume of 2 ml. The enzyme activitywas measured at 340 nm and calculated as nmol CDNB conju-gates/min/mg protein using a molar extinction coefficient of9.6 � 103 M�1 cm�1.

Glutathione peroxidase (GPx) activity was assayed according tothe method described by Mohandas et al. [35] with some modifica-tions. The assay mixture consisted of 1.44 ml phosphate buffer(0.1 M, pH 7.6), 0.1 ml ethylene diamine tetra-acetic acid (1 mM,EDTA, Sigma), 0.1 ml sodium azide, 0.05 ml glutathione reductase1 IU/ml (GR, Sigma), 0.1 ml GSH (1 mM), 0.1 ml nicotinamide ade-nine dinucleotide phosphate reduced (0.2 mM, NADPH, Sigma),0.01 ml H2O2 (0.25 mM) and 0.1 ml PMS in a total volume of2 ml. The enzyme activity was measured at 340 nm and calculatedas nmol NADPH oxidized/min/mg of protein, using a molar extinc-tion coefficient of 6.22 � 103 M�1 cm�1.

The glutathione reductase (GR) activity was measured by themethod of Pandey et al. [36]. The reaction mixture consisted of1.6 ml phosphate buffer (0.1 M, pH 7.4), 0.1 ml EDTA (0.5 mM),0.1 ml GSSG (1 mM, Sigma), 0.1 ml NADPH (0.1 mM) and 0.1 mlPMS in a total volume of 2 ml. The enzyme activity was quantitatedat 25 �C by measuring the disappearance of NADPH at 340 nm andcalculated as nmol NADPH oxidized/min/mg protein using a molarextinction coefficient of 6.22 � 103 M�1 cm�1.

All the enzymatic assays were monitored for 3 min and the dif-ference of OD per min was used to calculate the respective activi-ties against non-enzymatic blank consisting of all the reagentsexcept PMS and making up the volume using phosphate buffers.

2.9. Protein estimation

Protein content in various samples was estimated by the methodof Lowry et al. [37] using Folin reagent (Sigma) and BSA as standard.

2.10. Statistical Analysis

Analysis of variance (ANOVA) was applied to determine signif-icant differences between data of different groups when comparedwith control. p values <0.05 were considered significant. Subse-quently, Students–Newman–Keul’s test was applied for analyzingthe significant difference between different treatment groups.The values are expressed as means ± SE.

3. Results

3.1. Effect on HSP70

Deltamethrin at the dose of 0.75 ppb for 48 h and heat stress for3 h both induced HSP70 in all the tissues of C. punctata as mea-sured by ELISA (Fig. 1). A noticeable induction of amount of consti-tutive HSP70 was also noted in all the tissues analyzed. HSP levelsin liver increased significantly (p < 0.01) with more or less in samemanner in all the groups as compared to control but there were nosignificant differences recorded among combined exposures when

Fig. 1. HSP70 in fish Channa punctata liver (A), kidney (B) and gills (C) afterexposure to heat stress (3 h) and deltamethrin 0.75 ppb for 48 h only and incombined exposure followed one by the other. The values are expressed as in termsof relative optical density at 490 nm in ELISA (means ± SE, n = 5). Significantdifference is shown as bp < 0.01 when compared with controls; pp < 0.05 andqp < 0.01 when compared with heat stressed (3 h) group and yp < 0.01 whencompared with deltamethrin (0.75 ppb x 48 h) only exposed group.

A B E F

116.066.2

A B F

116.066.2

A B

C D

C D E

C D E F

116.066.2

A

B

C

Fig. 2. SDS–PAGE image showing protein profile of fish liver (A), kidney (B) and gills(C) after exposure to heat stress (3 h) and deltamethrin 0.75 ppb for 48 h only andin combined exposure followed one by the other. Lane A was loaded with molecularweight protein marker. Lane B is control, Lane C exposed to deltamethrin alone andLane D to heat stress only. Lanes E and F of combined exposure of deltamethrinfollowed by heat stress and vice versa, respectively. Arrow indicates HSP70 (stressproteins).

Table 1Protein carbonyl content in different tissues of Channa punctata Bloch followingexposure to deltamethrin and heat stress.

Groups Tissues

Liver Kidney Gills

Control 0.9 ± 0.06 1.4 ± 0.09 1.6 ± 0.09Heat Stress (HS) 2.3 ± 0.16b 2.2 ± 0.12b 2.9 ± 0.14b

Deltamethrin (DEL) 1.4 ± 0.02b 2.4 ± 0.13b 2.2 ± 0.07b

HS + DEL 1.7 ± 0.02b,q,x 2.4 ± .09b 2.0 ± 0.04a,q

DEL + HS 2.0 ± 0.03b, x 1.9 ± 0.06b,x 2.4 ± 0.10b

Values are expressed as nmol of carbonyl/mg protein (means ± SE, n = 5). Significantdifference is shown as ap < 0.05, bp < 0.01 when compared with controls; qp < 0.01when compared with heat stressed (3 h) group and xp < 0.05, when compared withdeltamethrin (0.75 ppb � 48 h) only exposed group.

M. Kaur et al. / Chemico-Biological Interactions 193 (2011) 216–224 219

compared to the deltamethrin exposed and heat stressed group. Inkidney, although the HSP levels were significantly (p < 0.05–0.01)higher in the groups with stress of deltamethrin or heat, the valuesof HSP70 in the combined stressed groups were relatively lowerthan those with either of the stressors. Induction of HSP in kidneywas found to be significantly (p < 0.01) lower in the group whereheat stress followed deltamethrin exposure when compared to del-tamethrin exposed and heat stressed groups. In gills of fish, heatstress induced significantly (p < 0.01) more HSP than deltamethrinand values were still significantly higher (p < 0.05–0.01) for groupsthat received combined exposure when compared to the groupsthat received exposure, either of heat stress or deltamethrin alone.The results were also supported by the SDS–PAGE profile of differ-ent tissue samples (Fig. 2).

3.2. Effect on protein carbonyl groups

A significant (p < 0.05–0.01) rise in the content of protein car-bonyls in liver, kidney and gills was observed in groups exposedto deltamethrin, heat exposure and combination of both the stress-ors as compared to that of controls (Table 1). Protein carbonylation

was more prominent in heat stressed group as compared to delta-methrin-exposed group. In the group of fish where heat stress fol-lowed the exposure by deltamethrin or vice versa showeddecreased carbonylation of proteins as compared to only heatstressed group.

3.3. Effect on LPO

Sequential exposures to deltamethrin for 48 h at the dose of0.75 ppb and heat stress for 3 h induced a significant (p < 0.05–0.01) increase in LPO in all the tissues when compared with controlfish (Fig. 3). Similar responses were observed with the combinedexposure of the two stresses in the study, one following the other.In liver, it increased LPO levels significantly (p < 0.01) in all cases ascompared to controls. In kidney also, the response was sameexcept in the group of fish where heat stress followed deltamethrinexposure, the significant increase (p < 0.05) in LPO was relativelylower as compared to other treatments. LPO response was note-worthy in gills where significant increase (p < 0.05) in the produc-tion of TBARS was observed for group exposed to deltamethrinonly and the group with deltamethrin exposure followed by heatstress when compared to fish with heat stress only.

Fig. 3. LPO level in different tissues of Channa punctata after exposure to heat stress(3 h) and deltamethrin (0.75 ppb for 48 h) individually (HS and DEL, respectively) orin combination when heat treatment was given either before or after deltamethrinexposure (HS + DEL and DEL + HS, respectively). Values are expressed as nmol ofTBARS formed/h/g of tissue (means ± SE, n = 5). Significant difference in result isshown on bar as ap < 0.05 and bp < 0.01 when exposure group data were comparedwith respective controls.

Table 2Non-enzymatic antioxidants as modulated by heat stress and deltamethrin in theliver of Channa punctata Bloch.

Groups Parameters

Total thiols Non-protein thiols Protein thiols

Control 72.5 ± 3.8 1.3 ± 0.04 71.2 ± 3.8Heat Stress (HS) 86.8 ± 2.0a 1.1 ± 0.04a 85.6 ± 1.9a

Deltamethrin (DEL) 90.1 ± 5.4a 1.7 ± 0.1b 88.3 ± 5.3a

HS + DEL 119.8 ± 6.5b,q,x 1.2 ± 0.04a,y 118.7 ± 6.5b,q,x

DEL + HS 130.8 ± 4.2b,q,y 1.4 ± 0.04q,y 129.4 ± 4.2b,q,y

Values are expressed as lmol/g of wet tissue (means ± SE, n = 5). Significant dif-ference is shown as ap < 0.05, bp < 0.01 when compared with controls; qp < 0.01when compared with heat stressed (3 h) group and xp < 0.05, yp < 0.01 whencompared with deltamethrin (0.75 ppb � 48 h) only exposed group.

Table 3Non-enzymatic antioxidants as modulated by heat stress and deltamethrin in thekidney of Channa punctata Bloch.

Groups Parameters

Total thiols Non-protein thiols Protein thiols

Control 89.3 ± 1.9 1.6 ± 0.2 87.7 ± 1.8Heat Stress (HS) 76.8 ± 1.3b 1.2 ± 0.1a 75.7 ± 1.3Deltamethrin (DEL) 118.3 ± 8.7a 2.2 ± 0.1a 116.1 ± 8.6a

HS + DEL 106.6 ± 7.0a,q 1.7 ± 0.1p,x 104.9 ± 6.9a,q

DEL + HS 81.7 ± 1.8a,q,y 1.1 ± 0.1a,y 80.6 ± 1.7a,q,y

Values are expressed as lmol/g of wet tissue (means ± SE, n = 5). Significant dif-ference is shown as ap < 0.05, bp < 0.01 when compared with controls; pp < 0.05,qp < 0.01 when compared with heat stressed (3 h) group and xp < 0.05, yp < 0.01when compared with deltamethrin (0.75 ppb � 48 h) only exposed group.

Table 4Non-enzymatic antioxidants as modulated by heat stress and deltamethrin in the gillsof Channa punctata Bloch.

Groups Parameters

Total thiols Non-protein thiols Protein thiols

Control 64.1 ± 3.6 0.6 ± 0.04 63.5 ± 3.6a b a

220 M. Kaur et al. / Chemico-Biological Interactions 193 (2011) 216–224

3.4. Effect on thiol profile

3.4.1. Effect on GSHBoth the stresses under study (deltamethrin and heat stress) in

combination or alone significantly altered the GSH content in var-ious tissues (Fig. 4). Heat stress for 3 h significantly (p < 0.01) de-creased the GSH content in all the tissues, whereas deltamethrinexposure significantly (p < 0.05–0.01) increased the content ofGSH in liver, kidney and gills when compared to controls. Fish ex-posed to deltamethrin after heat stress showed significant(p < 0.01) increase and decrease in quantity of GSH in kidney andgills, respectively whereas no significant changes were observed

Fig. 4. GSH content in different tissues of fish Channa punctata after exposure toheat stress (3 h) and deltamethrin 0.75 ppb for 48 h only and in combined exposurefollowed one by the other. The values are expressed as lmol of GSH per g of tissue(means ± SE, n = 5). Significant difference is shown as ap < 0.05, bp < 0.01 whencompared with controls; pp < 0.05, qp < 0.01 when compared with heat stressedgroup and xp < 0.05, yp < 0.01 when compared with deltamethrin only exposedgroup.

Heat Stress (HS) 79.1 ± 1.8 0.4 ± 0.03 78.7 ± 1.7Deltamethrin (DEL) 50.6 ± 0.9a 0.8 ± 0.05a 49.8 ± 0.9a

HS + DEL 65.4 ± 2.2q,y 0.7 ± 0.09a,q,x 64.7 ± 2.3q,y

DEL + HS 69.2 ± 3.6p,y 0.7 ± 0.01b,q 68.5 ± 3.6p,y

Values are expressed as lmol/g of wet tissue (means ± SE, n = 5). Significant dif-ference is shown as ap < 0.05, bp < 0.01 when compared with controls; pp < 0.05,qp < 0.01 when compared with heat stressed (3 h) group and xp < 0.05, yp < 0.01when compared with deltamethrin (0.75 ppb � 48 h) only exposed group.

in liver. In deltamethrin-exposed group that was subsequentlystressed by heat, the GSH content was significantly (p < 0.01) high-er in liver and significantly (p < 0.05–0.01) lower in both kidneyand gills when compared to deltamethrin and heat stressed groupsalone along with the control group.

3.4.2. Effect on T-SH, NP-SH and P-SHLevels of T-SH, NP-SH and P-SH were altered significantly

(p < 0.05–0.01) in liver, kidney and gills of all the stressed fishgroups. T-SH content increased significantly (p < 0.05–0.01) in allthe tissues with exception in kidney where it decreased signifi-cantly (p < 0.05–0.01) with heat stress and in gills of fish with del-tamethrin exposure. Dual exposure of stressors (heat anddeltamethrin) in liver significantly (p < 0.05–0.01) increased T-SHcontent when compared with controls and single stresses of delta-methrin and heat whereas the contents were unchanged in gills.NP-SH almost shared the pattern similar to GSH in all the tissues,

M. Kaur et al. / Chemico-Biological Interactions 193 (2011) 216–224 221

where it decreased significantly (p < 0.05–0.01) with heat stressand increased significantly (p < 0.05–0.01) with deltamethrinexposure. Groups in which one stress followed the other, showedsignificant changes when compared to groups with their respectivestressor in alone. Pattern of P-SH modulation was similar to thatfound with T-SH (Tables 2–4).

3.5. Effect on antioxidant enzymes

Antioxidant enzyme activities (Tables 5–7) displayed differen-tial but significant (p < 0.05–0.01) responses to single and com-bined exposures of deltamethrin and heat stress. With theexception of significant (p < 0.01) increase in liver in response toheat stress, CAT activity was significantly (p < 0.05–0.01) decreasedin response to heat stress and deltamethrin in rest of the tissues ascompared to controls. The values in liver were still significantly(p < 0.05–0.01) lower in the groups where one stress was followedby another when compared to groups that received single stress. Inkidney, groups with combined stress showed no changes as com-

Table 5Enzymatic antioxidants as modulated by heat stress and deltamethrin in the liver of Chan

Groups Parameters

Catalase (nmol H2O2

consumed/min/mg protein)Glutathione S-transferase (nmol CDNBconjugates/min/mg protein)

Control 112.5 ± 10.2 298.5 ± 4.6Heat Stress

(HS)159.6 ± 12.6a 238.2 ± 14.3a

Deltamethrin(DEL)

69.2 ± 5.3a 357.1 ± 2.6b,q

HS + DEL 95.7 ± 3.1q,x 276.3 ± 26.9x

DEL + HS 34.3 ± 1.8b,q,y 337.3 ± 4.2b,q,x

Values are expressed as means ± SE, n = 5. Significant difference is shown as ap < 0.05, bpstressed (3 h) group and xp < 0.05, yp < 0.01 when compared with deltamethrin (0.75 pp

Table 6Activities of enzymatic antioxidants as modulated by heat stress and deltamethrin in the

Groups Parameters

Catalase (nmol H2O2

consumed/min/mg protein)Glutathione S-transferase (nmol CDNBconjugates/min/mg protein)

Control 210.6 ± 8.2 314.3 ± 25.4Heat Stress

(HS)159.3 ± 11.7a 257.0 ± 13.2a

Deltamethrin(DEL)

126.6 ± 14.4b 427.4 ± 15.6a

HS + DEL 189.3 ± 7.0y 409.5 ± 23.1a,q

DEL + HS 198.8 ± 6.4p,y 615.7 ± 81.9a,q,x

Values are expressed as means ± SE, n = 5. Significant difference is shown as ap < 0.05, bpstressed (3 h) group and xp < 0.05, yp < 0.01 when compared with deltamethrin (0.75 pp

Table 7Activities of enzymatic antioxidants as modulated by heat stress and deltamethrin in the

Groups Parameters

Catalase (nmol H2O2

consumed/min/mg protein)Glutathione S-transferase (nmol CDNBconjugates/min/mg protein)

Control 211.7 ± 11.0 288.4 ± 3.0Heat Stress

(HS)140.4 ± 7.7b 199.6 ± 5.2b

Deltamethrin(DEL)

153.4 ± 9.7b 221.3 ± 15.8b

HS + DEL 186.6 ± 4.4b,q,x 205.2 ± 9.8b

DEL + HS 152.8 ± 2.9b 164.6 ± 9.0b,p,x

Values are expressed as means ± SE, n = 5. Significant difference is shown as bp < 0.01 wh(3 h) group and xp < 0.05, yp < 0.01 when compared with deltamethrin (0.75 ppb � 48 h)

pared to controls but the values were significantly (p < 0.05–0.01)higher to their respective stresses. In gills the group that was ex-posed to deltamethrin after heat stress showed significantly(p < 0.05–0.01) higher CAT activity than the group that receiveddeltamethrin and heat stress exposure only.

GST activities with heat stressed fish were significantly(p < 0.05) decreased whereas in deltamethrin exposure the activi-ties were significantly (p < 0.05–0.01) higher in liver and kidneybut lower (p < 0.01) in the gills in all the exposures (Tables 5–7).When heat stress followed deltamethrin stress the activity of GSTin liver and kidney were significantly (p < 0.05–0.01) increased incontrast to gills where it significantly (p < 0.05–0.01) decreasedwith their respective exposures.

GPx activities decreased significantly (p < 0.05–0.01) in all thetissues with 3 h heat stress and increased in liver and kidney(p < 0.05–0.01) with 48 h deltamethrin stress when compared tocontrol group. Heat stressed fish exposed to deltamethrin exhib-ited significantly (p < 0.05–0.01) lower and higher activitiesrespectively in liver and kidney as compared to controls along

na punctata Bloch.

Glutathione peroxidase (nmolNADPH oxidised/min/mg protein)

Glutathione reductase (nmol NADPHoxidized/min/mg protein)

241.8 ± 4.0 203.8 ± 13.0175.7 ± 11.6b 163.9 ± 7.2a

328.1 ± 12.5b 349.0 ± 16.7b

201.5 ± 10.2a,y 240.9 ± 11.8a,q,y

281.6 ± 6.4b,q,x 305.1 ± 7.1b,q,x

< 0.01 when compared with controls; pp < 0.05, qp < 0.01 when compared with heatb � 48 h) only exposed group.

kidney of Channa punctata Bloch.

Glutathione peroxidase (nmolNADPH oxidised/min/mg protein)

Glutathione reductase (nmol NADPHoxidized/min/mg protein)

274.5 ± 18.1 220.8 ± 10.3170.9 ± 12.1b 184.8 ± 7.6b

428.1 ± 15.7b 280.6 ± 10.7b

473.0 ± 12.2b,p,x 336.1 ± 32.1a,y

287.6 ± 43.3 293.0 ± 31.9a,p

< 0.01 when compared with controls; pp < 0.05, qp < 0.01 when compared with heatb � 48 h) only exposed group.

gills of Channa punctata Bloch.

Glutathione peroxidase (nmolNADPH oxidised/min/mg protein)

Glutathione reductase (nmol NADPHoxidized/min/mg protein)

392.6 ± 17.4 242.9 ± 11.5304.7 ± 16.9b 212.9 ± 14.8b

293.5 ± 14.9b 340.1 ± 15.2b

277.6 ± 20.8b,q 273.6 ± 15.2b,q,y

261.9 ± 46.9b 342.3 ± 9.2b,q

en compared with controls; pp < 0.05, qp < 0.01 when compared with heat stressedonly exposed group.

222 M. Kaur et al. / Chemico-Biological Interactions 193 (2011) 216–224

with single exposure groups. When deltamethrin exposed fishwere subsequently given heat stress the activity of GPx was sig-nificantly (p < 0.05–0.01) higher as compared to heat stressedgroup and lower than deltamethrin-exposed group. In gills, allthe exposures significantly decreased (p < 0.05–0.01) GPx activity(Tables 5–7).

GR followed the pattern of GPx in liver and kidney, but in gillsthe response was different. The GR activity was significantly(p < 0.01) higher in deltamethrin-exposed group and in the dualexposure groups as compared to controls. The activities in thegroups of combined exposures were significantly (p < 0.01) higher,when compared with control, than their respective exposures (Ta-bles 5–7).

4. Discussion

In the present study, deltamethrin-induced stress response in C.punctata, was manifested as induction of HSP70 and alterations ofvarious biochemical parameters. Additionally, heat stress aug-mented toxic effects of deltamethrin on certain biochemicalparameters, more importantly the antioxidants in most of the tis-sues. Deltamethrin and other pyrethroids possess a low degree oftoxicity and are considered to be environmentally less persistent[38]. Since their introduction the toxicity of pyrethroids has beenwidely studied in different animal models including fish. Pyre-throids have been shown to be lethal to fish at concentrations10–1000 times lower than corresponding values for mammalsand birds [39,40].

An interactive effect of contaminants has been reported in fish[41,42]. In the present study with deltamethrin and elevated tem-perature as stress, the combined exposure revealed differential re-sults in all the parameters studied when compared to theirindividual respective exposures. According to Sekine et al. [43]increasing or decreasing temperature can influence toxicity of pol-lutants. The synergistic effect of water temperature and herbicidehas been reported. Tarja et al. [44] observed significant decreasein activities of ethoxyresorufin-O-deethylase (EROD) with increas-ing water temperature in Oncorhynchus mykiss. Therefore, the pres-ence of pyrethroids in the aquatic environment and watertemperature may modulate the toxic impact on fish [45].

In addition to heat stress, deltamethrin at the concentration of0.75 ppb for 48 h induced HSP70 in different tissues. A noticeableamount of constitutive HSP70 was also found in all the tissues.Yu et al. [46] and Fader et al. [47] have demonstrated that fishand other aquatic organisms maintain detectable concentrationsof constitutive HSP70. The combined exposure of both stressorsshowed synergistic response in the induction of HSP70. In liver,where heat stress followed the deltamethrin exposure, HSP70 val-ues were lower than that induced by individual stressors. Levels ofHSP in kidney were significantly lower in the group where heatstress followed the deltamethrin exposure when compared to del-tamethrin exposed and heat stressed groups. In gills, HSP70 valuesin the heat stressed group were higher than deltamethrin exposedgroup as compared to controls and stressors in combination exhib-ited added response showing HSP levels higher than their individ-ual responses. All these observations infer that conditions at thetime of a pesticide challenge may modify the magnitude of re-sponse. The induction of hepatic HSP70 is reported to be differentagainst various interacting abiotic factors in fish exposed to envi-ronmental pollutants [48]. The decrease in the levels of HSP70could be supported by a study in brown trout (Salmo trutta f. fario)and stone loach (Barbatula barbatula) exposed to mixtures of envi-ronmental pollutants in laboratory, semi-field and in field studies.It was revealed that the stress response follows an optimum curve,resulting in a maximum HSP70 level under stress but rather low

HSP70 levels when stressors (chemicals, high temperature) be-come too severe. It has also been reported that the expression ofHSP in fishes may be subjected to seasonal variation [47].

Acute toxicity of deltamethrin on various aspects in fish hasbeen reported in Oreochromis niloticus L., Cyprinus carpio L. Carassiusauratus gibelio Bloch, and C. Punctata, [11,12,14,49]. Toxicity ofpyrethroids has also been reported in zooplankton communities,some beneficial aquatic arthropods, lobster and shrimp [50,51].

Besides the induction of HSP70, the stressors significantly in-creased the formation of protein carbonyls. Thus, protein oxidationappeared to be one of the apparent toxic effects of the stressors.When one stress followed the other, the response in the carbonyl-ation of proteins was different than that observed with individualstressor. Protein carbonyl formation can occur as a result of oxida-tive stress. An increase in the levels of carbonyl groups correlateswell with protein damage caused by oxidative stress [52]. Theinduction of protein carbonyl in fish was identified as a potentiallyuseful biomarker of oxidative damage in C. punctata and Zoarcesviviparus [53,54]. The level of protein carbonyl was consistent withthe induction of HSP70. When subjected to treatments causingprotein damage (proteotoxicity) stress-proteins are upregulatedproportionately to the degree of stress [55]. Under these adverseconditions stress-proteins are thought to counter proteotoxic ef-fects [56].

Exposure to deltamethrin and 3 h heat stress induced a percep-tible oxidative stress in fish. Oxidative stress-inducing effect of del-tamethrin has been established in fish [10]. Deltamethrin and heatstress significantly increased peroxidation of lipids in all the tis-sues Braguini et al. [8] attributed the toxicity of deltamethrin toperturbations in lipid–lipid, lipid–protein interactions and interfer-ence in transport mechanisms. Heat stress has also been reportedto cause lipid peroxidation [57,58] which is also evident with thefindings in the present study. In liver and kidney, extent of LPOin the combined exposure of deltamethrin and heat stress showedlower values than the two exposures individually. But in gills, thedifferential response of single and combined exposures was note-worthy where combined exposures of deltamethrin and heat stressdid not exhibit any additional effect in toxicity. The responses ofgills may be attributed to the high levels of HSP70 induction fol-lowing combined exposures. Su et al. [59] have reported a similarobservation of decreased LPO values involving HSP70.

Thiols, including GSH are also an integral part of antioxidantsystem [60]. In response to stress, thiols are reported to be modu-lated by the cells, as they are the first to be used in cellular defenseagainst stress [61]. Fish responded to deltamethrin exposure withincreased GSH but the response to heat shock for 3 h was oppositeto that of deltamethrin where GSH decreased (Fig. 4). In group offish which was exposed to deltamethrin after heat stress, no signif-icant increase was observed in GSH content. However, fish pre-ex-posed to deltamethrin followed with heat showed increase in GSHcontent in the liver and decrease in gills. Even when deltamethrinwas given after heat stress it was unable to lift the GSH content ofheat stress whereas heat stress to deltamethrin pre-exposed fur-ther increased the GSH in liver but decrease the same in gills show-ing that they are more vulnerable than the liver. Increase in GSHcontent has been described as one of the protective mechanismsthat fish adopt in the initial phases of exposure to aquatic pollu-tants [62,63]. Thiol profile was also modulated by deltamethrinand heat stress in combination or in their individual exposure.

Combined stress of deltamethrin and heat one after the othershowed differential response in different tissues. Increase in theactivities of antioxidants has been reported to be a general re-sponse of fish when exposed to environmental contaminants[63]. To neutralize the impact of reactive oxygen species (ROS),both enzymatic and non-enzymatic antioxidants are activated[64]. Similarly, deltamethrin exposure results in significant

M. Kaur et al. / Chemico-Biological Interactions 193 (2011) 216–224 223

increase in activities of glutathione-dependent antioxidant en-zymes. A significant increase was recorded in the activities ofGST, GR and GPx in liver and kidney, while there was a significantdecrease in the activities of GST and GPx in gills as in heat stresswhere all glutathione-dependent antioxidant enzymes were re-ported to decrease in gills besides in liver and kidney. The decreasein the activities of antioxidant enzymes may result when the stressis overwhelmed and cannot be compensated anymore [65]. Theexposure to deltamethrin and heat stress caused a significant de-crease in the activity of catalase in all the organs. This decreasein catalase activity could be attributed to the fluctuation of super-oxide radicals, which have been accounted to impede CAT activity[66]. In deltamethrin exposed fish, the levels of GPx, GST and GSHwere found elevated, apparently to provide protection against ROSdamage. Antioxidant responses and oxidative stress have beenused as biomarkers of exposure [22,67]. A high rate of absorptionof deltamethrin through gills also makes fish vulnerable to its tox-icity [50].

Once in the aquatic environment, pesticides can have deleteri-ous effects on aquatic organisms. Exposure to pesticides in combi-nation with other agents may exert effects different from thoseexperienced with pesticides alone. As the physiology of fish isstrongly related to temperature due to its ambient environment,their response to chemical exposures is also influenced by temper-ature. Although reports are available on the extent of oxidativedamage and antioxidant mechanisms in fish on exposure to delta-methrin, no such attempt has so far been made on the aspectregarding its response under other stressors, environmental orotherwise including heat stress. Seasonal temperature changeshave profound effects on the physiology of ectotherms, resultingin altered toxicity of chemicals. In the present study, we have dem-onstrated that modulation in one of the environmental variablesuch as temperature has significant effect on toxicity outcome ofpesticide deltamethrin. This study has an ecological relevance fromthe view point of effect of temperature on aquatic organisms. Italso provides an insight into the adaptation and survival of a fishcommunity in a habitat where temperature elevation is quite fre-quent in concurrence with exposure to environmental toxicants.

Conflict of interest statement

Authors declare that there are no conflicts of interest.

Acknowledgements

Financial support of Council of Scientific and Industrial Re-search (CSIR) Government of India to Manpreet Kaur in the formof Research Associateship is gratefully acknowledged.

References

[1] S. Peuranen, M. Keinanen, C. Tigerstedt, P.J. Vuorinen, Effects of temperature onthe recovery of juvenile grayling (Thymallus thymallus) from exposure toAl + Fe, Aquat. Toxicol. 65 (2003) 73–84.

[2] T.D. Clark, E. Sandblom, G.K. Cox, S.G. Hinch, A.P. Farrell, Circulatory limits tooxygen supply during an acute temperature increase in the Chinook salmon(Oncorhynchus tshawytscha), Am. J. Physiol. Regul. Integr. Comp. Physiol. 295(2008) R1631–R1639.

[3] J.C. Perez-Casanova, M.L. Rise, B. Dixon, L.O. Afonso, J.R. Hall, S.C. Johnson, A.K.Gamperl, The immune and stress responses of Atlantic cod to long-termincreases in water temperature, Fish Shellfish Immunol. 24 (2008) 600–609.

[4] R.S. Hattori, J.I. Fernandino, A. Kishii, H. Kimura, T. Kinno, M. Oura, G.M.Somoza, M. Yokota, C.A. Strussmann, S. Watanabe, Cortisol-inducedmasculinization: does thermal stress affect gonadal fate in pejerrey, a teleostfish with temperature-dependent sex determination? PLoS One 4 (2009)e6548.

[5] K. Said Ali, A. Ferencz, J. Nemcsok, E. Hermesz, Expressions of heat shock andmetallothionein genes in the heart of common carp (Cyprinus carpio): effects oftemperature shock and heavy metal exposure, Acta Biol. Hung. 61 (2010) 10–23.

[6] T. Smital, T. Luckenbach, R. Sauerborn, A.M. Hamdoun, R.L. Vega, D. Epel,Emerging contaminants – pesticides, PPCPs, microbial degradation productsand natural substances as inhibitors of multixenobiotic defense in aquaticorganisms, Mutat. Res. 552 (2004) 101–117.

[7] A. Slaninova, M. Smutna, H. Modra, Z. Svobodova, A review: oxidative stressin fish induced by pesticides, Neuro. Endocrinol. Lett. 30 (Suppl. 1) (2009)2–12.

[8] W.L. Braguini, S.M. Cadena, E.G. Carnieri, M.E. Rocha, M.B. de Oliveira, Effects ofdeltamethrin on functions of rat liver mitochondria and on native andsynthetic model membranes, Toxicol. Lett. 152 (2004) 191–202.

[9] I. Sayeed, S. Parvez, S. Pandey, B. Bin-Hafeez, R. Haque, S. Raisuddin, Oxidativestress biomarkers of exposure to deltamethrin in freshwater fish, Channapunctatus Bloch, Ecotoxicol. Environ. Saf. 56 (2003) 295–301.

[10] F. Atif, S. Parvez, S. Pandey, M. Ali, M. Kaur, H. Rehman, H.A. Khan, S. Raisuddin,Modulatory effect of cadmium exposure on deltamethrin-induced oxidativestress in Channa punctata Bloch, Arch. Environ. Contam. Toxicol. 49 (2005)371–377.

[11] M.Z. Yildirim, A.C. Benli, M. Selvi, A. Ozkul, F. Erkoc, O. Kocak, Acute toxicity,behavioral changes, and histopathological effects of deltamethrin on tissues(gills, liver, brain, spleen, kidney, muscle, skin) of Nile tilapia (Oreochromisniloticus L.) fingerlings, Environ. Toxicol. 21 (2006) 614–620.

[12] J. Velisek, R. Dobsikova, Z. Svobodova, H. Modra, V. Luskova, Effect ofdeltamethrin on the biochemical profile of common carp (Cyprinus carpio L.),Bull. Environ. Contam. Toxicol. 76 (2006) 992–998.

[13] M.A. Ansari, R.K. Razdan, Concurrent control of mosquitoes and domestic pestsby use of deltamethrin-treated curtains in the New Delhi MunicipalCommittee, India, J. Am. Mosquito Control Assoc. 17 (2001) 131–136.

[14] D. Dinu, D. Marinescu, M.C. Munteanu, A.C. Staicu, M. Costache, A. Dinischiotu,Modulatory effects of deltamethrin on antioxidant defense mechanisms andlipid peroxidation in Carassius auratus gibelio liver and intestine, Arch.Environ. Contam. Toxicol. 58 (2010) 757–764.

[15] A. Hegyi, T. Beres, L. Varadi, K.K. Lefler, B. Toth, B. Urbanyi, Investigation oflong-term stress induced by several stressors by determination of theconcentration of different blood plasma components in a model of Prussiancarp (Carassius auratus gibelio BLOCH, and Common carp (Cyprinus carpio L.,1758), Acta Biol. Hung. 57 (2006) (1783) 301–313.

[16] C.B. Schreck, Accumulation and long-term effects of stress in fish, in: G.P.Moberg, J.A. Mench (Eds.), The Biology of Animal Stress, CABI Publishing,Wallingford, UK, 2002, pp. 147–158.

[17] A. Kaetsu, T. Fukushima, S. Inoue, H. Lim, M. Moriyama, Role of heat shockprotein 60 (HSP60) on paraquat intoxication, J. Appl. Toxicol. 21 (2001) 425–430.

[18] M.J. Snyder, E.P. Mulder, Environmental endocrine disruption in decapodcrustacean larvae, hormone titers, cytochrome P450, and stress proteinresponses to heptachlor exposure, Aquat. Toxicol. 55 (2001) 177–190.

[19] S.B. Ceyhun, M. Senturk, D. Ekinci, O. Erdogan, A. Ciltas, E.M. Kocaman,Deltamethrin attenuates antioxidant defense system and induces theexpression of heat shock protein 70 in rainbow trout, Comp. Biochem.Physiol. C Toxicol. Pharmacol. 152 (2010) 215–223.

[20] H.N. Yang, H.C. Chen, Uptake and elimination of cadmium by Japanese eel,Anguilla japonica, at various temperatures, Bull. Environ. Contam. Toxicol. 56(1996) 670–676.

[21] M. Marinovich, F. Ghilardi, C.L. Galli, Effect of pesticide mixtures on in vitronervous cells, comparison with single pesticides, Toxicology 108 (1996) 201–206.

[22] A. Valavanidis, T. Vlahogianni, M. Dassenakis, M. Scoullos, Molecularbiomarkers of oxidative stress in aquatic organisms in relation to toxicenvironmental pollutants, Ecotoxicol. Environ. Saf. 64 (2006) 178–189(review).

[23] K. Wirtz, S. Bala, A. Amann, A. Elbert, A promise extended – future role ofpyrethroids in agriculture, Bayer Crop Sci. J. 62 (2009) 145–154.

[24] L.S. Clesceri, A.E. Greenberg, A.D. Eaton, Standard Methods for the Examinationof Water and Wastewater, 20th ed., American Public Health Association(APHA), Washington, DC, 1998.

[25] R.L. Anderson, C.Y. Wang, I.V. Kersen, K.J. Lee, W.J. Welch, P. Lavagnini, G.M.Hahn, An immunoassay for heat shock protein 73/72, use of the assay tocorrelate HSP73/72 levels in mammalian cells with heat response, Int. J.Hyperther. 9 (1993) 539–552.

[26] B.S. Washburn, J.J. Moreland, A.M. Slaughter, I. Werner, D.E. Hinton, B.M.Sanders, Effects of handling on heat shock protein expression in rainbow trout(Oncorhynchus mykiss), Environ. Toxicol. Chem. 2 (2002) 557–560.

[27] U.K. Laemmli, Cleavage of structural proteins during the assembly of the headof bacteriophage T4, Nature 227 (1970) 680–685.

[28] E. Floor, G. Wetzel, Increased protein oxidation in human substantial nigrapars compacta in comparison with basal ganglia and prefrontal cortexmeasured with an improved dinitrophenyl hydrazine, J. Neurochem. 70(1998) 268–275.

[29] M. Mihara, M. Uchiyama, Determination of malonaldehyde precursor intissues by thiobarbituric acid test, Anal. Biochem. 86 (1978) 271–278.

[30] D.W. Jollow, J.R. Mitchell, N. Zampagilone, J.R. Gilete, Bromobenzene inducedliver necrosis, protective role of glutathione and evidence for 3,4-bromobenzeneoxide as a hepatotoxic intermediate, Pharmacology 11 (1974)151–169.

[31] J. Sedlak, H.R. Lindsay, Estimation of total, protein-bound and non-proteinsulfhydryl groups in tissues with Ellman’s reagent, Anal. Biochem. 25 (1968)192–205.

224 M. Kaur et al. / Chemico-Biological Interactions 193 (2011) 216–224

[32] S. Parvez, I. Sayeed, S. Pandey, A. Ahmad, B. Bin-Hafeez, R. Haque, I. Ahmad, S.Raisuddin, Modulatory effect of copper on non-enzymatic antioxidants infreshwater fish Channa punctatus (Bloch), Biol. Trace Elem. Res. 93 (2003) 237–248.

[33] A. Claiborne, Catalase activity, in: R.A. Greenwald (Ed.), CRC HandBook ofMethods in Oxygen Radical Research, CRC Press Inc., Boca Raton, FL, 1985, pp.283–284.

[34] W.H. Habig, M.J. Pabst, W.B. Jokoby, Glutathione S-transferase: the firstenzymatic step in mercapturic acid formation, J. Biol. Chem. 249 (1974) 7130–7139.

[35] M. Mohandas, J.J. Marshall, G.G. Duggin, J.S. Horvath, D. Tiller, Differentialdistribution of glutathione and glutathione related enzymes in rabbit kidney,Cancer Res. 44 (1984) 5086–5091.

[36] S. Pandey, S. Parvez, I. Sayeed, R. Haque, B. Bin-Hafeez, S. Raisuddin,Biomarkers of oxidative stress: a comparative study of river Yamuna fishWallago attu (Bl. & Schn.), Sci. Tot. Environ. 309 (2003) 105–115.

[37] O.H. Lowry, N.J. Rosenbrough, A.L. Farr, R.J. Randall, Protein measurement withFolin phenol reagent, J. Biol. Chem. 193 (1951) 265–275.

[38] M. Villarini, M. Moretti, R. Pasquini, G. Scassellati-Sforzolini, C. Fatigoni, M.Marcarelli, S. Monarca, A.V. Rodrıguez, In vitro genotoxic effects of theinsecticide deltamethrin in human peripheral blood leukocytes: DNAdamage (‘comet’ assay) in relation to the induction of sister-chromatidexchanges and micronuclei, Toxicology 130 (1998) 129–139.

[39] S.P. Bradbury, J.R. Coats, Toxicological and toxicodynamics of pyrethroidinsecticide in fish, Environ. Toxicol. Chem. 8 (1989) 373–386.

[40] J.T. Eells, J.L. Rasmussen, P.A. Bandettini, J.M. Propp, Differences in theneuroexcitatory actions of pyrethroid insecticides and sodium channel-specific neurotoxins in rat and trout brain synaptosomes, Toxicol. Appl.Pharmacol. 123 (1993) 107–119.

[41] M. Calta, M.S. Ural, Acute toxicity of the synthetic pyrethroid deltamethrin toyoung mirror carp, Cyprinus carpio, Fresen. Environ. Bull. 13 (2004) 1179–1183.

[42] R. Viran, F.U. Erkoc, H. Polat, O. Kocak, Investigation of acute toxicity ofdeltamethrin on guppies (Poecilia reticulata), Ecotoxicol. Environ. Saf. 55(2003) 82–85.

[43] M. Sekine, H. Nakanishi, M. Ukita, Study on fish mortality caused by thecombined effects of pesticides and changes in environmental conditions, Ecol.Model. 86 (1996) 259–264.

[44] N. Tarja, E. Kirsti, L. Marja, E. Kari, Thermal and metabolic factors affectingbioaccumulation of triazine herbicides by rainbow trout (Oncorhynchusmykiss), Environ. Toxicol. 18 (2003) 219–226.

[45] A. Moore, C.P. Waring, The effects of a synthetic pyrethroid pesticide on someaspects of reproduction in Atlantic salmon (Salmo salar L.), Aquat. Toxicol. 52(2001) 1–12.

[46] Z. Yu, W.E. Magee, J.R. Spotila, Monoclonal antibody ELISA test indicates thatlarge amounts of constitutive hsp-70 are present in salamanders, turtle andfish, J. Therm. Biol. 19 (1994) 41–53.

[47] S.C. Fader, Z.M. Yu, J.R. Spotila, Seasonal variation in heat shock proteins(hsp70) in stream fish under natural conditions, J. Ther. Biol. 19 (1994) 335–341.

[48] H.R. Kohler, C. Bartussek, H. Eckwert, K. Farian, S. Granzer, T. Knigge, N. Kunz,The hepatic stress protein (hsp70) response to interacting abiotic parametersin fish exposed to various levels of pollution, J. Aquat. Ecosys. Stress Recov. 8(2001) 261–279.

[49] R.A. Ansari, M. Kaur, F. Ahmad, S. Rahman, H. Rashid, F. Islam, S. Raisuddin,Genotoxic and oxidative stress-inducing effects of deltamethrin in the

erythrocytes of a freshwater biomarker fish species, Channa punctata Bloch,Environ. Toxicol. 24 (2009) 429–436.

[50] A.K. Srivastav, S.K. Srivastava, S.K. Srivastav, Impact of deltamethrin on serumcalcium and inorganic phosphate of freshwater catsh, Heteropneustes fossilis,Bull. Environ. Contam. Toxicol. 59 (1997) 841–846.

[51] U. Friberg-Jensen, L. Wendt-Rasch, P. Woin, K. Christoffersen, Effects of thepyrethroid insecticide, cypermethrin, on a freshwater community studiedunder field conditions I. Direct and indirect effects on abundance measures oforganisms at different trophic levels, Aquat. Toxicol. 63 (2003) 357–371.

[52] I. Dalle-Donne, R. Rossi, D. Giustarini, A. Milzani, R. Colombo, Protein carbonylgroups as biomarkers of oxidative stress, Clin. Chim. Acta 329 (2003) 23–38.

[53] S. Parvez, S. Raisuddin, Protein carbonyls, novel biomarkers of exposure tooxidative stress-inducing pesticides in freshwater fish Channa punctata(Bloch), Environ. Toxicol. Pharmacol. 20 (2005) 112–117.

[54] B.C. Almroth, J. Sturve, A. Berglund, L. Forlin, Oxidative damage in eelpout(Zoarces viviparus), measured as protein carbonyls and TBARS, as biomarkers,Aquat. Toxicol. 73 (2005) 171–180.

[55] V. Dowling, P.C. Hoarau, M. Romeo, J. O’Halloran, F. van Pelt, N. O’Brien, D.Sheehan, Protein carbonylation and heat shock response in Ruditapesdecussatus following p,p0-dichlorodiphenyldichloroethylene (DDE) exposure:a proteomic approach reveals that DDE causes oxidative stress, Aquat. Toxicol.77 (2005) 11–18.

[56] L.E. Hightower, Heat shock, stress proteins, chaperones, and proteotoxicity,Cell 66 (1991) 191–197.

[57] M.S. Parihar, T. Javeri, T. Hemnani, A.K. Dubey, P. Prakash, Responses ofsuperoxide dismutase, glutathione peroxidase and reduced glutathioneantioxidant defenses in gills of the fresh water catfish (Heteropneustesfossilis) to short-term elevated temperature, J. Therm. Biol. 22 (1997) 151–156.

[58] L.T. Chien, D.F. Hwang, Effects of thermal stress and vitamin C on lipidperoxidation and fatty acid composition in the liver of thornfish Teraponjarbua, Comp. Biochem. Physiol. B 128 (2001) 91–97.

[59] C.Y. Su, K.Y. Chong, K. Edelstein, S. Lille, R. Khardori, C.C. Lai, ConstitutiveHSP70 attenuates hydrogen peroxide-induced membrane lipid peroxidation,Biochem. Biophys. Res. Commun. 265 (1999) 279–284.

[60] A. Pastore, G. Federici, E. Bertini, F. Piemonte, Analysis of glutathione:implication in redox and detoxification, Clin. Chim. Acta 333 (2003) 19–39.

[61] D.A. Dickinson, H.J. Forman, Glutathione in defense and signaling, lessons froma small thiol, Ann. NY Acad. Sci. 973 (2002) 488–504.

[62] B.M. Hasspieler, J.V. Behar, R.T. Di Giulio, Glutathione-dependent defense inchannel catfish (Ictalurus punctatus) and brown bullhead (Ameriurusnebulosus), Ecotoxicol. Environ. Saf. 28 (1994) 82–90.

[63] E. Stephensen, J. Sturve, L. Forlin, Effects of redox cycling compounds onglutathione content and activity of glutathione-related enzymes in rainbowtrout liver, Comp. Biochem. Physiol. C 133 (2002) 435–442.

[64] M. Lopez-Torres, R. Perez-Campo, S. Cadenas, C. Rojas, G. Barja, A comparativestudy of free radicals in vertebrates – II. Non-enzymatic antioxidants andoxidative stress, Comp. Biochem. Physiol. B 105 (1993) 757–763.

[65] M. Kaur, F. Atif, M. Ali, H. Rehman, S. Raisuddin, Heat stress-inducedalterations of antioxidants in the freshwater fish Channa punctata Bloch, J.Fish Biol. 67 (2005) 1653–1665.

[66] D.W. Filho, Fish antioxidant defenses – a comparative approach, Braz. J. Med.Biol. Res. 29 (1996) 1735–1742.

[67] L. Vigano, A. Arillo, C. Falugi, F. Melodia, S. Polesello, Biomarkers of exposureand effect in flounder (Platichthys flesus) exposed to sediments of the Adriaticsea, Mar. Poll. Bull. 42 (2001) 887–894.