Ecotoxicology and Environmental Safety 74 (2011) 270–278

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Ecotoxicology and Environmental Safety

0147-65

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Metabolic and osmoregulatory changes and cell proliferation in gilthead seabream (Sparus aurata) exposed to cadmium$

Sofia Garcia-Santos a,n, L. Vargas-Chacoff b, I. Ruiz-Jarabo c, J.L. Varela c, J.M. Mancera c,A. Fontaınhas-Fernandes a, J.M. Wilson d

a Universidade de Tras-os-Montes e Alto Douro e Centro de Investigac- ~ao e de Tecnologias Agro-Ambientais e Biologicas (CITAB), Vila Real, Portugalb Instituto de Zoologıa, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chilec Departamento de Biologıa, Facultad de Ciencias del Mar y Ambientales, Universidad de Cadiz, Espanad Centro Interdisciplinar de Investigac- ~ao Marinha e Ambiental (CIIMAR), Porto, Portugal

a r t i c l e i n f o

Article history:

Received 21 December 2009

Received in revised form

13 August 2010

Accepted 18 August 2010Available online 8 October 2010

Keywords:

Cadmium

Metabolism

Osmoregulation

Salinity

Sparus aurata

13/$ - see front matter & 2010 Elsevier Inc. A

016/j.ecoenv.2010.08.023

mal welfare: we hereby declare that this stud

imal welfare laws, guidelines and policies

nce with the institutional guidelines for the p

mal welfare.

esponding author. Fax: +351 259 350 245.

ail address: ssantos@utad.pt (S. Garcia-Santos

a b s t r a c t

The impact of cadmium on metabolism and osmoregulation was assessed in gilthead sea bream

(Sparus aurata). Seawater acclimated fish were injected intraperitoneally with a sublethal dose of

cadmium (1.25 mg Cd/kg body wt). After 7 days, half of the injected fish were sampled. The remaining

fish were transferred to hypersaline water and sampled 4 days later. Gill and kidney Na+/K+-ATPase

activities, plasma levels of cortisol, several metabolites and osmolytes, as well as osmolality were

measured. Hepatosomatic index and condition factor were calculated. The expression levels of

Na+/K+-ATPase, heat shock proteins (HSP70, HSP90) and proliferating cell nuclear antigen was assessed

by western blotting. Cadmium treatment adversely affected the Na+/K+-ATPase activity, although,

there was no perturbation in ion homeostasis and the animals were not compromised following

transfer to hypersaline water. Increased cell proliferation and Hsp90 expression likely contributed to

the attenuation of the deleterious effects of cadmium exposure.

& 2010 Elsevier Inc. All rights reserved.

1. Introduction

Heavy metal contamination in the marine environment is asevere problem, particularly in estuaries and coastal areas.Cadmium (Cd) is a non-essential metal that is a cause for concernbecause of its toxicity to both marine organisms and humans. Thismetal can enter the environment from various anthropogenicsources, such as by-products from zinc refining, coal combustion,mine wastes, electroplating processes, iron and steel production,pigments, fertilizers and pesticides (USEPA, 2001).

In fish, Cd can exert a wide range of pathological effects likeskeletal deformities (Muramoto, 1981), damage to organ structure(Pratap and Wendelaar Bonga, 1993; Thophon et al., 2003;Giari et al., 2007), changes in some plasma stress parameters(i.e. cortisol and glucose) (Fu et al., 1990; Chowdhury et al., 2004),disruption in whole-body or plasma ion regulation (Pratap et al.,1989; McGeer et al., 2000) and alterations in enzyme activity(Vaglio and Landriscina, 1999; Lionetto et al., 2000).

ll rights reserved.

y complies with all relevant

, and it was conducted in

rotection of human subjects

).

Gilthead seabream (Sparus aurata) is a marine teleost withhigh economic importance that inhabits coastal areas of theMediterranean and Southern Europe. This very euryhaline speciesis reared in farms situated in waters with extreme salinity levelsor which suffer salinity alterations (e.g. bays, natural ponds)(Laiz-Carrion et al., 2005; Sangiao-Alvarellos et al., 2005).Additionally, it is expected that human activities in these areasmay result in relatively high concentrations of Cd or othercontaminants. These areas receive a wide variety of industrial andmunicipal wastes. The wastes generated from industries includefertilizer, paints and pigments, dye manufacturing units andelectroplating units, etc. These industrial effluents contaminatethe water with a variety of heavy metals acting as point sources.According to Sadiq (1992), Cd enters the marine environment viaatmospheric deposition and through effluent discharges frompoint sources in near-shore areas.

Some studies have focused on the adaptability of giltheadseabream to considerable changes in environmental salinitiesthrough osmoregulatory and metabolic changes (Sangiao-Alvarelloset al., 2003, 2005; Laiz-Carrion et al., 2005). Others have studied thetoxicological responses of different fish species to heavy-metals,at biochemical, physiological and histopathological levels, includ-ing Cd (Almeida et al., 2001; Migliarini et al., 2005; Garcia-Santoset al., 2006, 2007). However, investigations of metal toxicity inmarine species lag far behind those in freshwater species and few

S. Garcia-Santos et al. / Ecotoxicology and Environmental Safety 74 (2011) 270–278 271

studies have examined the interaction of salinity and metaltoxicity. Recent studies focused on toxicological effect of Cd on S.

aurata at different levels (Vaglio and Landriscina, 1999; Bouraouiet al., 2008, Isani et al., 2009; Kalman et al., 2010; Ghedira et al.,2010), although, to our knowledge, no information is availableabout the effect of this metal on the ability of this, or other fishspecies, to acclimate to the physiologically demanding conditionsof hypersalinity.

For these reasons, the present work aims to determinethe impact of Cd (as cadmium chloride) on osmoregulatory andmetabolic performance of seawater (SW, 38%)-acclimatedS. aurata and in specimens challenged to hypersaline water(HSW, 55%) conditions. In this study a multivariable approachwas used: (i) osmoregulatory variables include gill and kidneyNa+/K+-ATPase expression (enzymatic activity and protein level),as well as plasma osmolality and ion levels; (ii) metabolicindicators such as plasma lactate, tryglicerides, glucose andprotein values; (iii) stress response indicators including plasmacortisol levels as well as branchial and renal heat shock proteins(HSPs) expression and (iv) cell turnover markers were alsoassessed in liver and kidney through the study of caspase3 (apoptosis) and proliferating cell nuclear antigen (PCNA;proliferation) expression levels.

Intraperitoneal injection was chosen not to mimic a realisticenvironmental concentration, but to investigate the direct effectsof cadmium in S. aurata (Vaglio and Landriscina, 1999; Bouraouiet al., 2008; Kalman et al., 2010; Ghedira et al., 2010). This methodcould be considered a preliminary study on the effects of Cdand ensures the precise knowledge of the dose received by theorganism, mainly because it is known that salinity reduces thecadmium uptake by forming complexes that are not available toaquatic organisms.

The sublethal concentration of Cd used in this study wasselected based on published works on S. aurata (2.5 mg Cd/kg;Vaglio and Landriscina, 1999) and Dicentrarchus labrax (LC503 mg/kg; Romeo et al., 2000).

2. Material and methods

2.1. Fish

Immature gilthead sea breams (S. aurata, 79.171.9 g body mass) were

obtained from Planta de Cultivos Marinos (C.A.S.E.M., University of Cadiz, Puerto

Real, Spain) and transferred to the laboratories at Faculty of Marine Science

(Puerto Real, Cadiz). The fish were randomly divided into two groups (16 per

group) and acclimated to seawater (SW, 38% salinity) in 300 L tanks in an open

system for 1 week prior to experimentation. The common water quality

parameters were monitored throughout the experiment and no major changes

were observed (pH: 8–8.2; DO: 8.2–8.6 mg L�1, hardness: 100–120 mg L�1 CaCO3,

ammonia: 0.001–0.0035 mg L�1, nitrite: 0.001–0.002 mg L�1 and suspended

solids). During the experiment (March, 2007), fish were maintained under natural

photoperiod and constant temperature (18 1C). Fish were fed daily with

commercial dry pellets at a ration of 1% of body weight (Dibaq-Diprotg SA,

Segovia, Spain) and were fasted for 24 h before the injection and sampling.

2.2. Experimental design

After acclimation, the fish from one group were anaesthetized with

2-phenoxyethanol (0.5 mL/L), weighed and injected intraperitoneally with

1.25 mg Cd/kg body mass in the form of CdCl2 in saline solution (9% NaCl) and

returned to the same SW system. The second group was injected with vehicle

alone and served as the sham control.

After 7 days, 8 fish from each group were sampled (see Section 2.3), while the

remaining fish from both groups were transferred to 50 L tanks (2 fish/tank)

containing hypersaline water (HSW, 55% salinity) and sampled 4 days later. The

HSW was obtained by mixing full SW with natural marine salts (Unionsal, Cadiz,

Spain) in a recirculating system. No mortality was observed. The described

experiment complies with the Guidelines of the European Union Council (86/609/

EU) and of the University of Cadiz (Spain) for the use of laboratory animals.

2.3. Sampling

Fish were anaesthetized with 2-phenoxyethanol (1 mL/L), weighed and

sampled. Blood was collected by caudal puncture using ammonia-heparinized

needles and syringes. Plasma was separated by centrifugation (3 min at 10,000g)

and aliquots were immediately frozen in liquid nitrogen and stored at �80 1C.

From each fish, small pieces from the posterior portion of the kidney and from

a gill arch were taken using fine-point scissors. The tissue were placed in SEI buffer

(300 mM sucrose/20 mM EDTA/50 mM imidazole, pH 7.5), and frozen at �80 1C

for later Na+/K+-ATPase activity measurement. Livers were excised and weighed

to calculate the hepatosomatic index (HSI). Larger samples of gill, kidney and liver

were also taken, but frozen directly in liquid nitrogen and stored at �80 1C for

immunoblotting and other future analysis.

2.4. Analytical techniques

2.4.1. Plasma analysis

Glucose, lactate, triglyceride, calcium and chloride levels were measured with

commercial kits from Spinreact (Sant Esteve de Bas, Spain) adapted to 96-well

microplates. Plasma protein was measured using the bicinchoninic acid method

with BCA protein kit (Pierce, Rockford, USA) for microplates, with bovine serum

albumin (BSA) as standard. All assays were performed with a Bio Kinetics EL-340i

Automated Microplate Reader (Bio-Tek Instruments, Winooski, VT, USA) using

DeltaSoft3 software for Macintosh (BioMetallics Inc., Princeton, NJ, USA).

Osmolality was measured with a vapor pressure osmometer (Fiske-One-Ten

Osmometer, Fiske, VT, USA) and expressed as mOsm/kg. Plasma Na+ levels were

measured using a flame atomic absorption spectrophotometer (UNICAM 938,

UNICAM, Cambridge, UK).

Plasma cortisol levels were quantified by enzyme-linked immunosorbent

assay (ELISA) adapting the method described by Rodrıguez et al. (2000) for

testosterone. Cortisol was extracted from 5 ml plasma in 1.5 mL methanol. Cortisol

standard, mouse anti-rabbit IgG monoclonal antibody, and specific anti-steroid

express antibody and enzymatic tracer (steroid acetylcholinesterase conjugate)

were purchased from Cayman Chemical Company (Michigan, USA). Microtiter

plates (MaxiSorpTM) were purchased from Nunc (Roskilde, Denmark). Standards

and extracted plasma samples were run in duplicate. The lower limit of detection

(90% of binding, ED90) was 0.37 ng mL�1 plasma. The inter-assay coefficient of

variation at 50% of binding was 8.2% (n¼3), while the mean intra-assay coefficient

of variation (calculated from the samples duplicates) was 5.4%. The mean

percentage of recovery was 95% (n¼4). Main cross-reactivity (41%; given by

the supplier) for anti-cortisol express antibody was detected with prednisolone

(22%), cortexolone (6.1%), cortisone (2.0%) and corticosterone (1.3%). As the

circulating levels of Cd decline significantly to very low days after a bolus

intraperitoneal injection (Kalman et al., 2010), and since solvent extraction of

plasma was performed, it was unlikely that Cd would interfere with the cortisol

assay.

2.4.2. ATPase assay

Gill and kidney Na+/K+-ATPase activities were measured using a microplate

technique developed by McCormick (1993) and adapted for non-salmonid fish. Gill

and kidney tissues were homogenized (Kontes pellet pestle motor 0.5 mL; Fisher

Scientific, Pittsburgh, USA) in 125 ml of SEID buffer (SEI buffer with 0.1%

deoxicholic acid; Sigma) and centrifuged at 5000g for 30 s. Ten microliters of

the homogenates in quadruplicate were pipetted into a 96-well plate. Each sample

had two wells containing an assay mixture with ouabain (0.5 mM) and two wells

containing an assay mixture without ouabain. The kinetic assay was read at a

wavelength of 340 nm at 25 1C for 10 min with intermittent mixing. Ouabain-

sensitive ATPase activity was detected by the enzymatic coupling of ATP

dephosphorylation to NADH oxidation. Protein concentrations were determined

in triplicate using the bicinchoninic acid (BCA) Protein Assay (Pierce, BCA Protein

kit, Rockford, USA), with bovine serum albumin as standard. Both assays were

performed in a microplate reader (EL-340i, Bio-Tek Instruments) using DeltaSoft3

software for Macintosh (BioMetallics Inc.).

2.4.3. Immunoblotting

For western analysis, proteins were prepared from gill, kidney and liver tissue

samples. Tissues were homogenized as described for the ATPase assay. Total

protein concentrations were assessed by the Bradford method using BSA as the

standard. Then, the homogenates were diluted with an equal volume of 2x

Laemmli’s buffer (Laemmli, 1970), vortexed, heated for 15 min at 70 1C and stored

at �20 1C. Before loading onto gels, samples were thawed, the protein

concentrations were adjusted to 1 mg/mL, vortexed and centrifuged at 10,000g

for 5 min. Samples and prestained molecular weight standards (Precision Blue

Plus, BioRad, Hercules, CA, USA) were loaded at 30 mL per well on miniature

vertical polyacrylamide gels (10–15% T resolving gels with 4% T stacking gels) and

run at 150 V using a Bio-Rad MiniProtean III system. To avoid complications with

inter-membrane variation, samples were randomized and run on multiple

membranes. Inter-membrane variation was checked and when present deviant

Table 1Hepatosomatic index (HSI), and plasma osmolality, sodium, calcium, chloride,

cortisol, glucose, lactate, triglycerides, protein concentrations in S. aurata control

and treated with Cd (1.25 mg Cd/kg body wt), acclimated to SW and transferred to

HSW for 4 days.

Salinity Treatment

Control Cd

HSI (%) SW 1.1270.05 A 1.5370.07 BHSW 1.0670.05 A 1.3770.13 B

Osmolality (mOsm/kg) SW 360.6372.96 (a) 358.3372.32 (a)HSW 380.1376.28 (b) 382.0076.31 (b)

Sodium (mmol/l) SW 172.6474.12 165.1776.74

HSW 165.1476.04 164.9374.17

Calcium (mmol/l) SW 2.6570.27 2.4570.15

HSW 2.4970.20 2.3170.09

Chloride (mmol/l) SW 136.6977.08 126.1074.57

HSW 134.4374.16 136.0175.77

Cortisol (ng/ml) SW 0.4670.07 A 6.5671.82 BHSW 1.9171.20 A 4.7971.42 B

Glucose (mmol/l) SW 3.5270.15 (a) 3.0770.14

HSW 4.5170.34 A(b) 3.4270.16 B

Lactate (mmol/l) SW 1.6470.07 1.1970.09

HSW 1.6370.29 1.2970.15

Triglycerides (mmol/l) SW 1.4970.09 1.6570.16

HSW 1.3070.09 1.3770.10

Protein (mg/ml) SW 33.1670.81 35.2071.97

HSW 34.6770.94 34.8271.54

Data are presented as mean7SEM (n¼8). Values with different capital letters

within the same salinity group (on the same line) are significantly different.

Within either control or Cd exposure groups, values with different lowercase

letters (in the same column) are significantly different. (Po0.05, two-way ANOVA,

SNK test). SW¼seawater acclimated fish; HSW¼fish transferred from SW to HSW.

S. Garcia-Santos et al. / Ecotoxicology and Environmental Safety 74 (2011) 270–278272

membranes were excluded from analysis. Following electrophoresis, gels were

equilibrated in transfer buffer (48 mM Tris, 39 mM glycine and 0.0375% SDS) and

the protein bands were transferred to polyvinylidenefluoride (PVDF) membranes,

using a semidry transfer apparatus, for 30 min at 13 V (Bio-Rad). The membranes

were rinsed in TTBS (0.05% Tween-20 in Tris buffered saline, pH 7.4) and blocked

with 5% powdered skim milk in TTBS for 1 h. Following rinsing in TTBS,

membranes were probed with the primary antibody diluted in 1% BSA in TTBS

overnight at room temperature After another rinse with TTBS, membranes were

incubated with a horseradish peroxidase (HRP; EC 1.11.1.7) conjugated secondary

antibody (goat, rabbit or rat anti-mouse) diluted in TTBS during 1 h at room

temperature. The membranes were washed again in TTBS, and then blots were

detected by ECL (GE Healthcare, Carnaxide, Portugal), using Pierce CL blue film.

The film was scanned, and images were imported as TIFF format into an image

analysis software program (SigmaScan Pro, Image analysis, Version 5.0.0, SPSS

Chicago, IL, USA), which was used to semiquantify band intensity.

2.5. Antibodies

Na+/K+-ATPase was detected using the panspecific a5 mouse monoclonal

antibody specific to the a-subunit and developed by Douglas Fambrough

(Department of Biology, Johns Hopkins University, Baltimore, MD, USA; Takeyasu

et al., 1988). The antibody was obtained as culture supernatant from the

Developmental Hybridoma Bank, University of Iowa, Iowa City, under contract

N01-HD-7-3263 from the National Institute of Child Health and Human

Development. We have already used this antibody in different studies of teleost

fishes (Wilson et al., 2004; Garcia-Santos et al., 2006).The proliferating cell nuclear

antigen (PCNA) was detected using a mouse monoclonal antibody (clone PC10;

Abcam, Cambridge, UK) that previously had shown to react with teleost fish PCNA

(Dang et al., 1999; Monteiro et al., 2009). Heat shock proteins (HSPs) 70 and 90

were detected with a mouse monoclonal antibody (clone BRM-22; Sigma Chemical

Co., St. Louis, MO, USA) and a rat monoclonal antibody (clone 16F1; Abcam),

respectively. Both isoforms of these HSPs are recognized by these monoclonal

antibodies and, both previously shown to react with teleost fish tissues

(Burkhardt-Holm et al., 1998; Hawkins et al., 2008). Metallothionein (MT) is a

low-molecular-weight, cysteine-rich, metal-binding protein found in all verte-

brates. The primary structure of the protein is evolutionary conserved, and the

antibody used here (rabbit anti-cod MT polyclonal antibody, KH-1, from Biosense

Laboratories, Bergen, Norway) was previously shown to cross-react with MT

from several fish species (Hylland et al., 1995). Caspase 3 was detected

with an affinity-purified rabbit anti-human/mouse Caspase 3 active form

(R&D Systems, Minneapolis, USA), which is known to detect active caspase 3 of

fish (Reis et al., 2007).

2.6. Statistical analysis

Data are expressed as means7SEM. Differences among groups were tested by

two-way ANOVA. When significant differences were obtained, multiple compar-

isons were carried out using the post hoc Student Neuman Keuls (SNK) test or the

non-parametric equivalent (Kruskal-Wallis two-way ANOVA on the ranks and

Dunn’s test (SigmaStat 3.0, SPSS). The fiducial limit was set at 0.05.

3. Results

3.1. Mortality and morphometric parameters

No mortality occurred in either control or Cd-treated groupsduring the experimental time course. Hepatosomatic index increasedsignificantly in response to Cd treatment, while salinity transfer hadno effect (Table 1). On the other hand, the condition factor did notchange significant with Cd or salinity treatment (data not shown).

3.2. Plasma parameters

Plasma osmolality increased significantly after transfer fromSW to HSW without any difference between control andCd-treated specimens. Also, the injection of Cd had no significanteffect on this parameter in SW-acclimated specimens (Table 1).Differences in plasma osmolality were not reflected in differencesin plasma levels of the osmolytes sodium, calcium and chloride.There were also no significant differences in plasma lactate,triglycerides or protein concentrations with either experimentalconditions (Cd treatment or salinity transfer) (Table 1).

Cd treatment significantly enhanced plasma cortisol levels inSW-acclimated specimens, although the transfer from SW to HSWdid not lead to any further differences (Table 1). Plasma glucosevalues increased in control group transferred to HSW (Table 1).Fish exposed to Cd had lower plasma glucose levels than controlfish, but this difference was only significant after HSW transfer.

3.3. Gill and kidney Na+/K+-ATPase activities

When compared to the control groups, fish exposed tocadmium had lower gill and kidney Na+/K+-ATPase activities;however these differences were only significant in specimenstransferred to HSW (Fig. 1A, B). Branchial Na+/K+-ATPase activitywas significantly elevated in response to the higher salinity inboth control and with less magnitude in Cd-injected groups.However, in kidney, this salinity response was seen in controlgroup, but not in specimens injected with Cd.

3.4. Immunoblotting

3.4.1. Na+/K+-ATPase

In gill and kidney western blots, the Na+/K+-ATPase a5antibody strongly immunoreacted with a pair of bands atapproximately 100 kDa (Fig. 2A, B). In gill, Cd exposure reducedexpression; however the difference was only statistical significantin specimens transferred to HSW. In contrast, salinity had nosignificant effect on branchial Na+/K+-ATPase abundance

0

5

10

15

20

25

30

35

40

45

Gill

NK

ATP

ase

activ

ity(µ

mol

AD

P/m

gpro

t/h)

0

2

4

6

8

10

12

14

Ctrl Cd

Ctrl Cd

Kid

ney

NK

ATP

ase

activ

ity(µ

mol

AD

P/m

gpro

t/h)

Fig. 1. Effect of Cd treatment and water salinity change on branchial (A) and renal

(B) Na+/K+-ATPase activity in S. aurata. Data are presented as mean7SEM (n¼8).

Different letters within the same treatment group indicate significant differences

(Po0.05) between seawater acclimated fish (SW) and fish transferred to high

salinity water (HSW). The pound sign (]) indicates statistically significant

differences (Po0.05) from the control (Ctrl) within the same salinity group.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Bra

nchi

al N

KA

� s

ubun

it ex

pres

sion

(n

orm

aliz

ed to

SW

Ctr

l)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Cd

Cd

Ren

al N

KA

� s

ubun

it ex

pres

sion

(n

orm

aliz

ed to

SW

Ctr

l)

Ctrl

Ctrl

Fig. 2. Effect of Cd treatment and water salinity change on branchial (A) and renal

(B) Na+/K+-ATPase a subunit expression determined by immunobotting, in

S. aurata and representative bands of c. 100 kDa. Data are presented as mean7SEM

(n¼8), relative to the SW control group. Further details as in legend of Fig. 1.

S. Garcia-Santos et al. / Ecotoxicology and Environmental Safety 74 (2011) 270–278 273

(Fig. 2A). Significant differences were not found in renal a subunitexpression with either cadmium or salinity (Fig. 2B).

3.4.2. Proliferating cell nuclear antigen (PCNA)

The PCNA antibody revealed a single cross-reactive band in theregion of 30 kDa in kidney (Fig. 3A) and liver (Fig. 3B) tissues.However, in gill expression was not detected. Quantification ofimmunoreactive bands generally showed an increase in expressionwith cadmium treatment in both tissues (Fig. 3A, B); however, inkidney, the increase was only statistically significant in SW fish.At the renal level, the effect of salinity was dependent on thetreatment to which the fish were exposed (Fig. 3A). In control fish,transference to HSW slightly increased PCNA expression althoughnot significantly. However, SW-acclimated specimens exposed toCd enhanced significantly PCNA levels, but transfer to HSWconditions significantly decreased this parameter. At the hepaticlevel PCNA expression was enhanced by Cd treatment, which didnot differ significantly with salinity transfer (Fig. 3B).

3.4.3. Caspase 3

The polyclonal rabbit anti-human caspase 3 (CPP32) recog-nized in kidney, a strong immunopositive band at approximately100 kDa (Fig. 4A) and in liver, a single band at approximately

32 kDa, corresponding to inactive proenzyme of caspase 3 (Fig. 4B);however no bands were detected at 17 kDa (active caspase 3).In kidney, band intensity increased significantly with Cd exposurewith no significant salinity dependence (Fig. 4A). In liver neithersalinity nor Cd treatment resulted in significant differencesbetween groups (Fig. 4B).

3.4.4. Heat shock proteins (HSP70 and HSP90)

HSP70 antibody, strongly immunoreacted with a single bandof approximately 70 kDa, in renal (Fig. 5A) and branchial (data notshown) tissues. In kidney, Cd treatment significantly increasedHSP70 expression in SW-acclimated fish, but this increase was notobserved in specimens transferred to HSW (Fig. 5A). In gill therewere no significant differences between groups (Table 2).

At the hepatic level, a doublet was detected with the HSP70antibody, probably due to the presence of both constitutive HSP73(hsc 70) and inducible HSP72 isoforms (hsp70). However, whenanalyzed both separately and together there were no significantdifferences with either salinity or Cd treatment (Table 2).

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Kid

ney

PCN

A e

xpre

ssio

n (n

orm

aliz

ed to

SWC

trl)

0

2

4

6

8

10

12

14

Cd

Cd

Hep

atic

PC

NA

exp

ress

ion

(nor

mal

ized

to S

WC

trl)

Ctrl

Ctrl

Fig. 3. Effect of Cd treatment and water salinity alteration on kidney (A) and liver

(B) PCNA expression determined by immunoblotting, in S. aurata and representa-

tive immunoblots showing bands of c. 30 kDa. Data are presented as mean7SEM

(n¼8), relative to the SW control group. Further details as in legend of Fig. 1.

0

2

4

6

8

10

12

Kid

ney

Cas

pase

3 ex

pres

sion

(nor

mal

ized

to S

WC

trl)

0.0

0.5

1.0

1.5

2.0

2.5

CdCtrl

Ctrl Cd

Hep

atic

Cas

pase

3 ex

pres

sion

(nor

mal

ized

to S

WC

trl)

Fig. 4. Effect of Cd treatment and water salinity transfer on kidney (A) and hepatic

(B) Caspase3 expression determined by immunobotting, in S. aurata and

representative bands of 100 and 32 kDa, respectively. Data are presented as

mean7SEM (n¼8), relative to the SW control group. Further details as in legend

of Fig. 1.

S. Garcia-Santos et al. / Ecotoxicology and Environmental Safety 74 (2011) 270–278274

The HSP90 antibody revealed a single cross-reactive band in theregion of 90 kDa in liver (Fig. 5B). HSP90 expression was stronglyinduced by Cd in SW- and HSW-acclimated specimens (Fig. 5B).

Neither the cadmium nor salinity changed significantly theexpression of this protein in kidney (Table 2).

3.4.5. Metallothionein (MT)

In liver western blots, the polyclonal antibody KH-1 stronglyreacted with a single band at approximately 15 kDa (data notshown). However, significant differences were not found inhepatic MT expression with either Cd or salinity treatment(Table 2). Corresponding bands in kidney and gill were notdetected.

4. Discussion

Cadmium treatment clearly inhibited the activity and expres-sion of Na+/K+-ATPase in the major osmoregulatory organs, gilland kidney, of S. aurata, yet had only a minor effect on overallosmoregulatory status even during a hypersaline challenge.

4.1. Salinity effect

Control fish exhibited changes in osmoregulatory parameterscomparable to those previously observed in this species whentransferred from SW to HSW (Sangiao-Alvarellos et al., 2003,2005; Laiz-Carrion et al., 2005). Na+/K+-ATPase is an importantenergizer of ion transport in epithelial tissue and is located mainlyin mitochondrion-rich cells (MRC) of gill epithelia (Wilson andLaurent, 2002) and kidney tubules (Ura et al., 1996). At thebranchial level S. aurata belongs to a group that presents aU-shape branchial Na+/K+-ATPase activity—environmental salinityrelationship, i.e. lower values of activity occur at intermediatesalinities and higher values at the hypo- and hypersalinity extremes(Laiz-Carrion et al., 2005).

Control fish showed an increase in Na+/K+-ATPase activityfollowing transfer from SW to HSW. Gill Na+/K+-ATPase activity islinked to the capacity for extrusion of excess ions in hyperosmoticenvironment (Evans et al., 2005) and these results agree with thephysiological role of this ion pump and with previous reports forthis species (Sangiao-Alvarellos et al., 2003, 2005; Laiz-Carrionet al., 2005). On the other hand, the activation of kidney

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Kid

ney

HSP

70 e

xpre

ssio

n (n

orm

aliz

ed to

SW

Ctr

l)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

CdCtrl

CdCtrl

Hep

atic

HSP

90 e

xpre

ssio

n (n

orm

aliz

ed to

SW

Ctr

l)

Fig. 5. Effect of Cd treatment and water salinity transfer on kidney HSP70 (A) and

hepatic HSP90 (B) expression determined by immunobotting, in S. aurata and

representative bands of 70 and 90 kDa, respectively. Data are presented as mean7SEM

(n¼8), relative to the SW control group. Further details as in legend of Fig. 1.

Table 2HSP 70 and 90, and metallothionein (MT) expression determined by immunoblot-

ting in gill, liver and kidney of S. aurata control and treated with Cd (1.25 mg Cd/kg

body wt), acclimated to SW and transferred to HSW for 4 days.

Salinity Treatment

Control Cd

Branchial HSP70 SW 1.0070.14 0.9771.15

HSW 1.5370.40 1.1470.357

Hepatic HSP70 SW 1.0070.22 1.4270.30

HSW 1.5170.26 1.4570.21

Renal HSP90 SW 1.0070.46 7.2773.37

HSW 4.4672.50 3.9772.32

Hepatic MT SW 1.0070.06 0.6770.08

HSW 0.7870.12 0.5170.13

Data are presented as mean7SEM (n¼8). SW¼seawater acclimated fish;

HSW¼fish transferred from SW to HSW; HSP¼heat shock proteins; MT¼

Metallothionein. Values are presented relative to the SW Control group.

S. Garcia-Santos et al. / Ecotoxicology and Environmental Safety 74 (2011) 270–278 275

Na+/K+-ATPase activity could be attributed to renal modificationsin urine production and/or ion transport due to hyperosmoticacclimation.

Changes in Na+/K+-ATPase activity, mainly associated withenvironmental salinity, have been positively correlated withmodifications in its a-subunit protein and/or mRNA expression(Hwang et al., 1998; Lee et al., 1998; Wilson et al., 2004),suggesting that alterations in activity are due to variationsin the relative abundance of the enzyme. In this study, theNa+/K+-ATPase protein level expression did not change withsalinity; however, its activity increased significantly. Similarresults were obtained by Hiroi and McCormick (2007) in threesalmonid species transferred from freshwater to seawater.According to them, this difference may arise from severalsources, including a more activated form of Na+/K+-ATPase in SWand/or measurement of inactive forms of Na+/K+-ATPase bywestern blotting.

Plasma glucose was significantly higher in control fish exposedto HSW. In a previous study by Sangiao-Alvarellos et al. (2003)glucose levels were elevated in sea bream juveniles acclimated toHSW and suggested that the hyperglycemia observed couldindicate a mobilization of glucose to satisfy the increased energeticdemand observed as result of the higher gill Na+/K+-ATPaseactivity. Gill ionocytes have been shown to use glucose preferen-tially for their energetic needs (Perry and Walsh, 1989).

Cortisol is a multifunctional hormone implicated in both hyper-and hypo-osmotic acclimation (McCormick, 2001). In the acclima-tion to SW, cortisol promotes salinity tolerance due to thedevelopment and proliferation of MRC, and enhanced gill Na+/K+-ATPase activity and expression/abundance of Na+/K+-ATPasea-subunit (McCormick, 1995). For example, Arjona et al. (2007)observed an increase in plasma cortisol levels of Solea senegalensis,one day after transfer to hypersaline water. However, in the presentstudy, since fish were sampled four days after the salinity transfer, itis probable that cortisol returned to basal levels. This would be inagreement with cortisol pulses observed previously in differentspecies (Morgan et al., 1997; Wilson et al., 2002; Scott et al., 2004),including S. aurata (Laiz-Carrion et al., 2005).

No significant changes were observed in ion concentrations;however, plasma osmolality increased with the transferenceof fish to high salinity. The observed rise (�20 m Osmol/kg) canpossibly be due to increased concentrations of HCO�3 with minorcontributions from K+ and Mg2 + ions or other osmolytes, such astrimethylamine oxide, (Kelly and Yancey, 1999; Pillans et al.,2005) that were not here evaluated.

4.2. Cadmium and cadmium versus salinity effects

Previous studies, mostly in freshwater, have demonstratedthat Cd inhibits Na+/K+-ATPase activity in different species(Oreochromis mossambicus, Pratap and Wendelaar Bonga, 1993;Anguilla anguilla, Lemaire-Gony and Mayer-Gostan, 1994; Lionettoet al., 2000). Although, no significant changes in Na+/K+-ATPaseactivity were seen in O. niloticus acutely exposed to sublethal Cdlevels (5, 15 and 25 mg L�1 of CdCl2, 24, 48 and 96 h of exposure,Garcia-Santos et al., 2006) or Cyprinus carpio exposed to 1.6 mg/LCd for 14 days (De la Torre et al., 2000). In this study, only afterHSW transfer, when the osmoregulatory system was challengedfurther is possible to see clearly the negative effect of Cd. Cd mayhave had a negative influence on the synthesis of new Na+/K+-ATPase pumps or inactivated preexistent ones.

Even though Cd had a negative effect on Na+/K+-ATPaseactivity, there was no indication that cadmium adverselyimpacted the osmotic balance of S. aurata in SW or after HSWtransfer since plasma osmoregulatory indicators were unaffected.The gill Na+/K+-ATPase activity decrease was reflected in therelative abundance, since Na+/K+-ATPase a subunit protein levelexpression showed the same trend. Overall, these results suggest

S. Garcia-Santos et al. / Ecotoxicology and Environmental Safety 74 (2011) 270–278276

that other physiological mechanisms have been triggered tocounteract the weakening of the osmoregulation system.

Usually, increases in liver size are commonly seen in fish thathave been exposed for long periods of time to contaminants(Heath, 1995). However, in our short duration experience, weobserved an increase in hepatic somatic index (HSI) with Cdtreatment. According to Heath (1995), this enhancement can beexplained by hyperplasia (increased cell number) and/or hyper-trophy (increase in size) and it may be associated with highercapacity to metabolize xenobiotics. In support, one of the findingsof this work was increased liver (and kidney) cell proliferationwith Cd treatment, as indicated by the PCNA protein levelexpression. However, while Cd induced hepatocyte proliferationremained unaffected after salinity transfer, the same was not truein kidney. In fact, there was no Cd induced proliferation in kidneyduring HSW challenge.

In the kidney, in parallel with an increase in cell proliferationwith Cd under SW conditions, we observed an increased caspase3expression. Although, the bands found with the CPP32 antibody inkidney and liver did not have the characteristic molecular weightof the active or inactive form of caspase3, they were veryconsistent and reproducible. Thus, we maintained the analysesand hypothesized that the band could be the result of caspasecleavage products, indicative of the apoptotic process. Furtherstudies must be conducted to confirm this assumption. Renalcaspase3 expression was also significantly elevated during HSWchallenge in contrast to PCNA.

In some studies apoptosis and cell proliferation markers havebeen proposed as cellular biomarkers of Cd or other contaminantexposure in aquatic organisms (Ortego et al., 1995; Piechottaet al., 1999; Sweet et al., 1999). In fact, Berntssen et al. (2001)found regulated cell death and proliferation in the intestineof salmon (S. salar) fed dietary cadmium. In fish gills, Lee et al.(1996) and Wong and Wong (2000) found an increase in chloridecells proliferation after Cd exposure. Rangsayatorn et al. (2004)observed increased cell necrosis and proliferation in both the liverand kidney of P. gonionotus fed cadmium-enriched cyanobacteria.Under toxicant exposure, apoptosis is suggested to removecritically damaged cells followed by compensatory cell regenera-tion to maintain tissue structure and function (Habeebu et al.,1998). So, it seems that in S. aurata exposed to Cd, the equilibriumbetween proliferation and apoptosis in renal tissue could bedisrupted after salinity transfer, i.e. the cell death was notcompensated by the proliferation mechanism. Although, sincethe kidney has a reduced importance at higher salinity (reducedglomerular filatration rate) a negative impact on osmotic balancewas not observed. In liver, where no significant changes inapoptosis were observed, increase in cell proliferation results inan overall increase in liver mass (suggested by HSI).

It has been argued that many of the effects of sublethalstressors on fish can be attributed to elevated blood cortisol. Forexample, the protective effect of cortisol against copper inductingapoptosis has been postulated by Bury et al. (1998) and confirmedby Mazon et al. (2004) in gill epithelium in vitro. In the presentstudy, Cd treatment increased plasma cortisol levels (7 days),which remained significantly elevated after transfer to HSW(11 days). These results were in general agreement with otherstudies. The elevation of serum cortisol was an indicator of theprimary response to Cd stress (Wu et al., 2007). Generally, acuteand sub-chronic Cd exposures increase plasma cortisol concen-trations, which subsequently (within a few days) return tobaseline levels (Pratap and Wendelaar Bonga, 1990; Fu et al.,1990; Wu et al., 2007), although the time frame for return to pre-exposure concentration varies widely.

Cortisol also affects carbohydrate metabolism and a rise incortisol levels is frequently followed by hyperglycemia in fishes

(Wendelaar Bonga, 1997). Indeed, increase in serum glucoselevels in fish under Cd stress was reported by Cicik and Engin(2005), Chowdhury et al. (2004) and Almeida et al. (2001).However, in S. aurata an increase in glucose levels with the Cdtreatment was not observed. Instead, fish exposed to cadmiumshowed similar values to their controls. On the other hand, fishexposed to Cd failed to respond equally when they were salinitychallenged (hypersalinity), showing a significant drop in plasmaglucose concentration when compared to the control at the samesalinity. The decline may not be due to reduced production, butrather to an enhanced utilization and clearance rate.

In addition to these physiological responses, there is ageneralized stress response at the cellular level that is in partmediated by the actions of a family of proteins known as heatshock proteins (HSP). These proteins are highly conserved, andhave been measured in nearly all organisms studied (see Iwamaet al., 1998 for review). Studies in fish have demonstrated thatseveral stressors, including pollutants and thermal stress, caninduce HSP expression (Iwama et al., 2004). The function of HSPduring stress is related to cytoprotection as these proteins can actto prevent and repair protein damage. Although, the HSP responseseems to vary with factors such as species, development stage,tissue examined, stressor and concentration and duration ofexposure (Hori et al., 2008), increased HSP levels have beenreported in fish exposed to Cd (Hermesz et al., 2001; Boone andVijayan, 2002; Migliarini et al., 2005; Fulladosa et al., 2006). Infact, SW-acclimated fish showed an increase in renal HSP70 whentreated with Cd. However, the lack of response obtained in liverand gill may indicate that the stress induced there does not reachthe level required to trigger a detectable response. HSP90 proteinlevel expression in liver of fish injected with Cd was significantlyhigher when compared with control fish at either salinity.A similar response to Cd has been observed in the freshwatercarp at the mRNA level (Hermesz et al., 2001), while renal HSP90alevels showed only a transient induction. Similar to our findings,no differences were observed in renal HSP90a levels with anexposure of 96 h in C. carpio (Hermesz et al., 2001) or in humanproximal tubule cells (Somji et al., 2002). In fishes, osmotic stressdoes not appear to induce HSP expression in vivo (Pan et al., 2000;Niu et al., 2008; Todgham et al., 2005), which is consistent withthe findings in S. aurata. However, a pre-thermal stress has beenshown to confer greater salinity tolerance to fish (DuBeau et al.,1998; Todgham et al., 2005). Thus Cd exposure, which inducesHSP expression may help in coping with the subsequent HSWchallenge by elevating HSP and thus compensating for its negativeeffects on Na+/K+-ATPase.

Metallothioneins are low-molecular-mass cysteine-rich metal-binding proteins with high affinity for heavy metal ions, playing acrucial role in their detoxification process. Cd is believed to be oneof the most important metal inducers of MT. Indeed, other studiesin S. aurata have shown that Cd injection stimulates the MTsynthesis in different tissues (Ghedira et al., 2010; Kalman et al.,2010). However, in the present study no significant changes werereported in liver MT levels after Cd treatment. This apparentdivergence may be due to the different methodologies used in MTevaluation. In fact, it is known that western blotting has somelimitations in MT detection. These may be related to the typicalstructural properties of MT, which can influence its interactionwith membrane surface (Mizzen et al., 1996). Moreover, despitethe use of reducing agents (e.g. b-mercaptoethanol), MT tend toform aggregates that are exacerbated with the metal binding.Thus, the determination of total MT in the sample could beimprecise (Krizkova et al., 2009).

In summary, Cd had a limited impact on the hypersalinityresponse and tolerance in S. aurata. Na+/K+-ATPase is animportant component in the osmoregulatory response of

S. Garcia-Santos et al. / Ecotoxicology and Environmental Safety 74 (2011) 270–278 277

euryhaline animals to HSW challenges, although, it appears thatan elevation in activity is not essential in the case of the S. aurata.Other physiological mechanisms are likely involved mitigatingthe adverse effects of Cd on the ability of this fish to adapt itsosmoregulatory system for hypoosmoregulation and to the HSWchallenge. This result can be partially explained by the compen-satory effects achieved through an increase in cell proliferationand expression of HSP90 in some affected organs.

Acknowledgments

We acknowledge the facilities provided by the Planta deCultivos Marinos (C.A.S.E.M) and the laboratories at Facultyof Marine Science, University of Cadiz, Puerto Real, Spain, theCenter for Interdisciplinary Marine and Environmental Research(CIMAR) and the Center for the Research and Technology of Agro-Environment and Biological Sciences (CITAB). This work waspartially supported by the Portuguese Foundation for Scienceand Technology (FCT) through a Ph.D. Grant to S Garcia-Santos(SFRH/BD/22750/2005).

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