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Ecotoxicology and Environmental Safety 70 (2008) 99–105

www.elsevier.com/locate/ecoenv

Evaluation of biochemical responses in Palearctic and Lake Baikalendemic amphipod species exposed to CdCl2

M.A. Timofeyeva,b,�, Z.M. Shatilinaa,b, D.S. Bedulinaa, M.V. Protopopovaa,V.V. Pavlichenkoa, O.I. Grabelnychc, A.V. Kolesnichenkob,d

aIrkutsk State University, Karl-Marx 1, 664003 Irkutsk, RussiabBaikalean Research Center, Karl-Marx 5-10, 664003 Irkutsk, Russia

cSiberian Institute of Plant Physiology and Biochemistry SB RAS, Lermontova 132, 664033 Irkutsk, RussiadInstitute of Plant Physiology RAS, Botanicheskaya, 35 Moscow, Russia

Received 14 October 2006; received in revised form 10 June 2007; accepted 18 June 2007

Available online 24 October 2007

Abstract

This study evaluated small heat shock proteins (sHSP) (related to a-crystallin) and antioxidant enzymes (POD, peroxidase and CAT,

catalase) as possible biomarkers for use in toxicological studies. Biochemical responses to cadmium chloride in two Lake Baikal endemic

amphipods (Eulimnogammarus verrucosus, Eulimnogammarus cyaneus) and Palearctic species (Gammarus lacustris) were compared. Our

findings showed that cadmium chloride toxicity directly influenced POD activity and sHSP synthesis in all amphipod species. The

Baikalean endemic and the Palearctic amphipod species responded by decreasing activity of POD and they exhibited a dose-dependent

activation of sHSP synthesis. All measured parameters differed among species and depended on the species’ ability to resist cadmium

chloride toxicity. CAT activity in the Palearctic species responded significantly to cadmium chloride exposure; however, responses were

negligible for both Baikalean species. We suggest that synthesis of sHSP, together with changes in POD activity, could be used as

biomarkers for further studies of amphipod species including endemics from Lake Baikal.

r 2007 Elsevier Inc. All rights reserved.

Keywords: Lake Baikal; Amphipods; Endemics; Cadmium chloride; Peroxidase; Catalase; sHSP; a-Crystallin

1. Introduction

Heat shock proteins (HSP) and antioxidant enzymes arewidely used as biomarkers of stress in aquatic organisms.Narrowly adapted, endemic species may have specificmechanisms for stress adaptation that differ from thoseof common model organisms. In addition, numerouspublished reports show that in some species even highlyconservative stress markers (e.g., HSP synthesis) may varyor not be detected (Brennecke et al., 1998; Hofmann et al.,2000; Tomanek, 2002; Buckley et al., 2004). Alternatively,antioxidant status can depend on environmental character-istics experienced by species populations (Walker et al.,

e front matter r 2007 Elsevier Inc. All rights reserved.

oenv.2007.06.014

ing author. Irkutsk State University, Karl-Marx 1, 664003

. Fax: +73952 201219.

ess: mtim@irk.ru (M.A. Timofeyev).

2000; Niyogi et al., 2001; Borgeraas and Hessen, 2002). Thepossibility of such inter-specific differences in biochemicalstress responses, however, is often not considered.Such differences are especially important for developing

evaluation methods for the fauna of ancient ecosystemssuch as that found in Lake Baikal. More than 80%of the animal species in Lake Baikal are endemic; theirevolution occurred over a long time period in stable,clear conditions with high level of oxygen saturation(Kozhova and Izmest’eva, 1998). We predict that most ofthese narrowly adapted Baikalean species may have lowadaptations to stress. In some Baikalean endemic species,we have found a reduced ability of HSP70 induction(Timofeyev et al., 2004, 2006a) which is widely recom-mended for use as a significant biomarker for toxicologicalstudies (Eckwert et al., 1997; Mukhopadhyay et al.,2003). We also found that some Baikalean species

ARTICLE IN PRESSM.A. Timofeyev et al. / Ecotoxicology and Environmental Safety 70 (2008) 99–105100

have specific modes of antioxidant activity which aredifferent from Palearctic species (Timofeyev et al., 2006b).Therefore, the application of the common methods ofstress evaluation (which are mainly based on Palearcticmodel species) to Lake Baikal animals may result inerroneous conclusions.

The purpose of this study was to evaluate heat shockproteins (related to a-crystalline) and antioxidant enzymes(POD, peroxidase and CAT, catalase) as possible biomar-kers in toxicological studies. In this present study, twoLake Baikal endemic amphipods were compared withPalearctic species. To induce toxic stress, we used CdCl2 asCd is a significant pollutant because of its high toxicity toorganisms and its wide distribution in aquatic ecosystems(Reddy, 1999; Vaglio and Landriscina, 1999; Basha andRani, 2003).

Due to the reported oxidative effect of cadmium toxicity(Almeida et al., 2002; Geret et al., 2002; Basha and Rani,2003; Hansen et al., 2006), we chose main antioxidantelements as potential biomarkers in this present study. Keyantioxidant enzymes, POD and CAT, were chosen forresearch. As mentioned above, a reduction of HSP70synthesis was previously found in Baikalean amphipods(Timofeyev et al., 2004, 2006a). Consequently, we chose tofocus on small heat shock proteins (sHSP) (immunochemi-cally related to the a-crystalline family). Small HSPs, whichexhibit chaperon functions, are also reported to be animportant elements of antioxidant defense at the cellularlevel (Arrigo, 1998; Papp et al., 2003). Their presence waspreviously detected in two crustaceans groups: mainly inArtemia species (Clegg et al., 1999, 2000; Macrae, 2003;Collins and Clegg, 2004) and very recently in copepods(Seo et al., 2006). Due to problems with HSP70 usage inBaikalean species, we suspected sHSPs might serve aspossible biomarkers.

2. Materials and methods

2.1. Animals

Two Baikalean endemic amphipod species (Eulimnogammarus verruco-

sus (Gerstf.) and Eulimnogammarus cyaneus (Dyb.)), and one Palearctic

(Gammarus lacustris Sars) species were used in experiments. These species

have similar ecologic characteristics because they are inhabitants of the

upper littoral zone (Bazikalova, 1951; Beckman, 1954). E. verrucosus and

E. cyaneus were collected from Lake Baikal’s shoreline close to Irkutsk

State University’s biological station at the settlement Bolshie Koty

(Southern Baikal). G. lacustris was collected at small shallow lakes close

to Bolshie Koty.

The selection of species was determined by their known ability to resist

CdCl2 toxicity. Among these species, the Palearctic amphipod G. lacustris

was least sensitive to CdCl2 with a 24 h LC50 equal to 4.5� 10�5molL�1

(Timofeyev et al., 2000). Baikalean endemic species were less resistant to

this toxicant—E. verrucosus exhibited a 24 h LC50 of 1.8� 10�5molL�1

while the most sensitive species, E. cyaneus, showed a 24 h LC50 of

1.2� 10�5molL�1 (Timofeyev et al., 2000).

Amphipods were reared for 1–2 days prior to experiments in aerated

5L tanks containing Baikalean water. Tanks were maintained at 771 1C

and no food was provided to avoid any possible effects of feeding and

temperature modifications on enzyme activity or stress protein synthesis.

Actively moving amphipods were used in experiments.

2.2. Exposure experiments

Exposure experiments were conducted in triplicate. In each experiment,

three independent groups of 10 individuals each were placed in CdCl2solutions (Baikalean water) in well-aerated 500mL tanks at 771 1C. We

used inversion voltamperometry (Zakharova et al., 2001) to verify exact

concentration of Cd2+ ions in exposure solutions of CdCl2. For

experiments investigating sHSP levels, we exposed amphipods for 24 h

in three concentrations of CdCl2: nominal concentration 5mgL�1 (exact

Cd2+ concentration 1.6mgL�1), 0.5mgL�1 (Cd2+ 0.16mgL�1), and

0.05mgL�1 (Cd2+ 0.02mgL�1). At the highest concentration, which

usually caused the death of most of the animals, we analyzed only

surviving individuals. In experiments examining the activity of antioxidant

enzymes, we exposed amphipods at the lowest CdCl2 concentration

0.05mgL�1 (Cd2+ 0.02mgL�1) for 2, 6, 24, and 3 days (with daily change

of solutions). After exposure, amphipods were fixed in liquid nitrogen.

Control animals were kept in similar condition but without CdCl2additions, and they were fixed at the start and at the end of each

experiment. For control amphipods, the activity of all enzymes and

parameters of sHSP synthesis did not change significantly over several

days. Consequently, control data apply to the entire exposure period.

2.3. Sample preparations and biochemical assays

Enzyme extraction from animal tissues was done as described in detail

by Wiegand et al. (1999). Frozen animals tissues were homogenized in

sodium phosphate buffer 0.1M pH 6.5 and centrifuged at 10,000 g.

Enzyme activities were measured in the collected supernatant with the

spectrophotometer UVIKON XS. Enzyme activities were calculated in

nanokatal/mg protein. For protein measurement, the Bradford assay

procedure was used. Measurement of the POD in the soluble fraction was

performed with guaiacol as the substrate at 436 nm, pH 5 according to

Bergmeyer (1983). CAT activity was measured with hydrogen peroxide at

240 nm and a pH 7 according to Aebi (1984).

For sHSP analyses, proteins were isolated using standard methods

(Kolesnichenko et al., 1999). sHSP content was determined by SDS–

electrophoresis of equal protein amounts followed by Western blotting

using anti-a-crystallin primary antibodies (StressGen). SDS–electrophor-

esis was performed in the polyacrylamide gel blocks 70� 80� 1mm3 in

Laemmli system (Laemmli, 1970) using the Mini-PROTEAN II Electro-

phoretic Cell (BIO-RAD, USA) apparatus for electrophoresis. Relative

molecular weights of proteins were determined using a LMW kit of

markers (Pharmacia, Sweden). Western blotting was performed according

to Towbin et al. (1979) with modification as described previously

(Kolesnichenko et al., 1999). Protein concentrations were determined

using the Lowry method (Lowry et al., 1951).

2.4. Statistical analysis

At least 30 animals were used for each experiment. For each analysis, five

individuals were taken. All experiments were replicated at least three times.

All data were analyzed statistically using the Statistica 5.0 software

(StatSoft) with Student’s t-test. Results were compared with values for

control groups. Statistical significance was tested at the 95% level (po0.05).

In the figures, stars indicate significant differences from controls at po0.05.

3. Results

3.1. Antioxidant enzymes activity

Exposure to CdCl2 caused a decrease of POD activity inall three amphipod species (Fig. 1). For the Baikalean

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Gammarus lacustris

Eulimnogammarus verrucosus

Eulimnogammarus cyaneus

2 h

Fig. 1. Peroxidase (POD) activities in the three amphipod species exposed to CdCl2 solutions 0.05mgL�1 (Cd2+ 0.02mgL�1). (*) Indicates significant

difference (po0.05) from the control.

M.A. Timofeyev et al. / Ecotoxicology and Environmental Safety 70 (2008) 99–105 101

E. cyaneus, a significant decrease of POD activity wasdetected after 6 h; however, for E. verrucosus this differenceoccurred after 3 days of exposure. For PalearcticG. lacustris, a statistically significant decrease of PODactivity was also detected after 3 days of exposure.

For both Baikalean species, exposure to CdCl2 for upto 3 days did not cause significant changes in CAT activity(Fig. 2). However for Palearctic G. lacustris, a significantdecrease of CAT activity was detected after only 2 h ofexposure.

3.2. Small heat shock proteins synthesis

Immunochemically related a-crystallin proteins were notdetected in the protein spectra for the E. cyaneus (Fig. 3) andE. verrucosus (Fig. 3b) control groups. The protein spectra forthe G. lacustris control group (Fig. 3c) detected only a weakband of protein with a molecular weight close to 32kDa.Exposure to CdCl2 resulted in the visual appearance of

a-crystallin-related proteins in the protein spectra of allthree amphipod species (Fig. 3). For E. cyaneus (Fig. 3a)

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Eulimnogammarus verrucosus

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Control 2 h

Exposure time

Fig. 2. Catalase (CAT) activities in the three amphipod species exposed to CdCl2 solutions 0.05mgL�1 (Cd2+ 0.02mgL�1). (*) Indicates significant

difference (po0.05) from the control.

M.A. Timofeyev et al. / Ecotoxicology and Environmental Safety 70 (2008) 99–105102

and E. verrucosus (Fig. 3b) a protein with a molecularweight close to 32 kDa was detected. There is a dosedependency of a-crystallin protein content with theconcentrations of cadmium ions used in these experiments.Maximum protein content was detected in experiments inwhich the highest concentration of CdCl2 solution (nom-inal 5mgL�1; Cd2+ 1.6mgL�1) was used. Minimumprotein content in these species was detected in experimentsin which the lowest concentration of CdCl2 solution(nominal 0.05mgL�1; Cd2+ 0.02mgL�1) was used.

In addition to this protein, two additional proteins withmolecular weights close to 52 and 15 kDa were detected in

E. cyaneus. Both proteins were observed in animalsexposed to medium concentrations of CdCl2 (nominal0.5mgL�1; Cd2+ 0.16mgL�1).In G. lacustris (Fig. 3c), exposure to the lowest

concentration of the CdCl2 solution (nominal 0.05mgL�1;Cd2+ 0.02mgL�1) caused an increase of sHSP proteincontent. Exposure of amphipods to CdCl2 solution withthe highest concentration (nominal 5mgL�1; Cd2+

1.6mgL�1) caused a visible increase of 32 kDa proteincontent and an appearance of a strong band withmolecular weight ranging from 52 to 57 kDa in the highmolecular weight region.

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Fig. 3. Expression of sHSP related to a-crystallin protein in three

amphipod species: (A) E. cyaneus, (B) E. verrucosus, and (C) G. lacustris,

after 24 h exposition in different CdCl2 solutions: line 1, control; line 2,

0.05mgL�1 (Cd2+ 0.02mgL�1); line 3, 0.5mgL�1 (Cd2+ 0.16mgL�1);

and line 4, 5mgL�1 (Cd2+ 1.60mgL�1).

M.A. Timofeyev et al. / Ecotoxicology and Environmental Safety 70 (2008) 99–105 103

4. Discussion

As proposed initially, all biomarkers investigated in thispresent study can be modulated by cadmium toxicity. Onthe other hand, such modulation effects vary in different

species, which can be of great importance for toxicologicalevaluations.The decrease of peroxidase activity, which we detected in

our study, therefore may correlate with a total decrease ofmetabolic process rates, including respiration and energymetabolism in the whole organism as a result of oxidativestress. The inhibition of POD activity by cadmium waspreviously shown in a number of aquatic organisms(e.g., fishes (Vaglio and Landriscina, 1999; Thomas andWofford, 1993; Berntssen et al., 2000) and mollusks(Chandran et al., 2005; Company et al., 2004; de Almeidaet al., 2004)). In contrast to results for previously studiedcrustaceans, POD reactions were activated (Luqing andZhang, 2006; Griffitt et al., 2006). Significant inhibition ofPOD activity shows that the amphipod species used in thispresent study differ from those most used in previousinvestigations.However, periods when POD activities began to decrease

were correlated with known cadmium resistance abilities ofthe species. Thus, in less resistant E. cyaneus, we observedvery rapid POD inhibition after only 6 h of exposure, but inother species which are more resistant to cadmium, adecrease of POD activity began after 3 days of exposure.As mentioned previously, cadmium salts cause oxidativestress in exposed animals. Assuming this, we propose thatperoxidase is a key component of the antioxidant systemand its activity can be maintained at the same level for alonger period in both resistant amphipods. In sensitivespecies, however, POD cannot provide adequate activityand support for resistance to oxidative stress.From a number of publications on effects of cadmium

salts on CAT, we can assume that in aquatic animals anenzyme activation (Hansen et al., 2006; Basha and Rani,2003; Luqing and Zhang, 2006; de Almeida et al., 2004) aswell as inhibition (Vaglio and Landriscina, 1999; Chandranet al., 2005; Company et al., 2004, 2006; Geret et al., 2002)or no reactions (Reddy, 1999) could be observed. The dataobtained from CAT measurements were unexpectedbecause for the most resistant Palearctic G. lacustris, weobserved very rapid inhibition of CAT, while in bothBaikalean species, CAT activity was unchanged. In livingcells, CAT is mostly confined to peroxisomes, whereasPOD has a wider distribution in cells and tissues. It can beproposed that such different CAT responses mostly dependon the inter-specific diversity of the antioxidant activationmode, but it is not associated with species’ adaptive abilityor toxicity resistance. The possibility of inter-specificdifferences in amphipos antioxidant activities or evenmode of reaction to stress was shown previously in ourprevious studies (Timofeyev, 2006; Timofeyev et al.,2006b).In all three species, we observed well-expressed induction

of sHSP, immunochemically related to the a-crystallinefamily. Assuming this protein has a chaperon function(Jakob et al, 1993; Wang, 1995), we suggest this increaseprevents damage to other cellular proteins. In such a case,this protein functions as a component of the cellular

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defense system protecting against toxic damage. It ispossible that this protein is involved in the maintenanceof correct protein conformations that are broken underoxidative stress caused by cadmium.

As it was mentioned above, the presence of a-crystallinprotein in crustaceans was previously shown only inArtemia species and the copepod Tigriopits japonicus. Bothspecies are limited mainly to special environmental condi-tions (Willsie and Clegg, 2001, 2002; Day et al., 2003;Macrae, 2003; Seo et al., 2006). In contrast, the amphipodG. lacustris is widely distributed in Palearctic waterbodiesand it can be used easily as a model species for toxicologyassessment in different aquatic habitats.

Our findings show that an oxidative stress caused bycadmium in all studied amphipod species directly influ-enced the main elements of their antioxidant systemsincluding POD and sHSPs. Such effect has dose responsedependence and is also associated with the species’ abilityto resist toxic impact of cadmium. In all amphipod species,we found induction of sHSPs related to a-crystalline whichseems to be a significant component of amphipodsantioxidant system and it deals with the prevention ofother cellular proteins from damage caused by oxidativecadmium impact. This parameter, together with peroxidaseactivity, could be used successfully in future studies asbiomarkers in amphipod species. However, we cannotrecommend using CAT as a biomarker for oxidativeimpacts in Baikalean species due to its decreased involve-ment in their antioxidant reactions.

5. Conclusions

In conclusion, we emphasize that cadmium causedsignificant effects of the main antioxidant componentsincluding enzymes and small heat chock proteins. TheBaikalean endemic and Palearctic amphipod speciesreacted by decreasing activity of peroxidase and showingan enhanced dose-dependent synthesis of sHSP related tothe a-crystalline group. All measured parameters varied indifferent species and depended on the ability of the speciesto resist cadmium toxicity. Catalase activity respondedsignificantly to cadmium exposure in the Palearctic species;however, reactions were negligible for the Baikaleanspecies. We suggest that synthesis of sHSP (related to a-crystalline), together with changes in peroxidase activity,could be successfully used as biomarkers for further studiesin amphipod species including endemics from Lake Baikal.

Acknowledgments

This research was partially supported by grants ofRussian Foundation for Basic Research Nos. 06-04-48099-a, 05-04-97239-baikal, 05-04-97263-baikal. This sup-port is gratefully acknowledged. Finally, we would like tocordially thank Dr. Marianne Moore (Wellesley College,Wellesley, MA) for the language check.

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