Mechanisms underlying the protective effect of zincand selenium against cadmium-induced oxidative stressin zebrafish Danio rerio

Mohamed Banni • Lina Chouchene • Khaled Said •

Abdelhamid Kerkeni • Imed Messaoudi

Received: 29 July 2010 / Accepted: 20 April 2011

� Springer Science+Business Media, LLC. 2011

Abstract The present study was designed to eluci-

date the protective effect mechanism of Zinc (Zn) and

Selenium (Se) on cadmium (Cd)-induced oxidative

stress in zebrafish. For this purpose we investigate the

response of oxidative stress markers, metallothionein

accumulation and gene expression in liver and ovary of

female zebrafish exposed to 0,4 mg/l Cd in water and

supplemented with Zn (5 mg kg-1) and/or Se (2 mg

kg-1) for 21 days in their diet. Liver and ovary Cd

uptake was evaluated after the exposure period. Cd

exposure significantly inhibited the antioxidant

enzyme activities termed as catalase (CAT), superoxide

dismutase (SOD) and glutathione peroxydase (GPx)

and caused a pronounced malondialdehyde (MDA)

accumulation in both organs. Co-administration of Zn

and Se reversed the Cd-induced toxicity in liver and

ovary measured as MDA accumulation. Interestingly,

gene expression patterns of Cat, CuZnSod and Gpx were

up-regulated when related enzymatic activities were

altered. Zebrafish metallothionein transcripts (zMt)

significantly decreased in tissues of fish supplemented

with Zn and/or Se when compared to Cd-exposed fish.

Our data would suggest that Zn and Se protective

mechanism against Cd-induced oxidative stress is more

depending on the correction of the proteins biological

activities rather than on the transcriptional level of

related genes.

Keywords Oxidative stress � Gene expression � Cd �Zn � Se � Zebrafish � Protective effect

Introduction

Cadmium (Cd) is a highly toxic and widely spread

pollutant that may cause adverse harmful effects. It

has no known biological function, and prolonged

exposure causes long-term toxic effects to humans

and animals. Mainly because of its low rate of

excretion from the body, Cd has a long biological

half-life and accumulates over time in blood, kidney,

and liver (EPA Agency EP 2004) as well as in the

reproductive organs (Piasek et al. 2001; Bonda et al.

2004).

The molecular mechanism responsible for the

toxic effects of Cd is far from being completely

M. Banni (&)

Laboratoire de Biochimie et Toxicologie de

l’Environnement (UR04AGR05), ISA, Chott-Mariem,

4042 Sousse, Tunisia

e-mail: m_banni@yahoo.fr

L. Chouchene � K. Said � I. Messaoudi

Unite de Recherche: Genetique, Biodiversite et

Valorisation des Bioressources, Institut Superieure de

Biotechnologie de Monastir, Monastir, Tunisia

A. Kerkeni

Departement de Biophysique, Faculte de Medecine de

Monastir, Unite de Recherche: Elements Traces, Radicaux

Libres, Antioxydants, Pathologies Humaines et

Environnement, Monastir, Tunisia

123

Biometals

DOI 10.1007/s10534-011-9456-z

understood. However, various studies connect Cd

with oxidative stress since this metal can alter the

antioxidant defense system in several tissues of

several animals, causing a depletion in the levels of

reduced glutathione, as well as an alteration in the

activity of antioxidant enzymes, and a change in the

structure of the cellular membrane through a process

of lipid peroxidation (Cuypers et al. 2010). Therefore,

it is reasonable to assume that antioxidant agents

(enzymatic and non-enzymatic) may prevent or at

least reduce the Cd toxicity.

Se and Zn are well-established antioxidants. Se

was recognized as an essential trace element within a

relatively low concentration range and its physiolog-

ical role was established when it was shown to be one

of the glutathione peroxidase (GPx) components

(Rotruck et al. 1973). Zn can act as an antioxidant

since it is an essential component of Cu/Zn–SOD. Zn

can also indirectly function as an antioxidant by

inducing the synthesis of metallothionein (MT), a

thiol-rich protein which can act by binding metals

with pro-oxidant activity such as Cd and by providing

thiol groups which can scavenge hydroxyl radicals

and singlet oxygen (Dondero et al. 2005).

The treatment with Se or Zn during Cd exposure

has been demonstrated to have protective effects on

Cd-induced toxicity in various organs and tissues

such as liver, kidney, skeleton, and blood (Yiin et al.

1999; Hu et al. 2004). Using the rat as a model, our

group found that the combined treatment with Se and

Zn was more effective than that with either of them

alone in reversing Cd-induced oxidative stress in

kidney (Messaoudi et al. 2009), liver (Jihen et al.

2009; Banni et al. 2010) and erythrocytes (Messaoudi

et al. 2010a, b). However, the exact mechanism

behind these protective effects remains largely unex-

plored. On the other hand, molecular studies have

indicated that aberrant gene expression can be an

important factor in Cd-induced toxicity, but no

information about Zn and Se effects on Cd-induced

changes in the antioxidative enzymes genes expres-

sion, such as Sod, Cat and Gpx genes are available.

Fish are particularly sensitive to water contamina-

tion and pollutants may impair many physiological

and biochemical processes when assimilated by fish

tissue. Due to the genomic resources available for

zebrafish and the long experience with this organism

in toxicity testing, it is easily possible to establish

biochemical and molecular endpoints for effects

assessment (Liu and We 2007). Additionally, the

zebrafish model offers a number of technical advan-

tages including ease and cost of maintenance, rapid

development and high fecundity (Segner 2008).

Therefore, this study was conducted to provide

new insights into the mechanism of reversing

Cd-induced oxidative stress by Se and Zn. For this

purpose a toxicity test was carried out to investigate

Cd accumulation and metal-mediated oxidative stress

responses in liver and ovaries of mature female

zebrafish to chronic Cd exposure in presence of Zn

and/or Se. The activities of SOD, CAT, GST, and the

levels of MDA and MTs were used as oxidative stress

biomarkers and specific response to Cd exposure.

Moreover the transcriptional changes in zMt and a set

of antioxidant genes, including Cat, CuZn-Sod and

Gpx were investigated.

Materials and methods

Chemicals

Cadmium chloride (CdCl2) was obtained from Merck

(Darmstadt, Germany). Sodium selenite (Na2SeO3)

and Zn chloride (ZnCl2) were purchased from Sigma,

St. Louis, MO, USA. All other chemicals were of

analytical grade and were purchased from standard

commercial suppliers.

Experimental fish

Healthy 6-month adult female fish were selected and

kept in aquaria. In each aquarium, water was pumped

continuously over a biofilter column at the rate of

4 l/min. The water was continuously aerated through-

out the experiment. Prior to exposure experiments,

the fish were acclimatized in a tank filled with water

at ambient temperature (25 ± 1�C) for 1 week, with a

photoperiod consisting of 14-h light/10-h dark seg-

ments for each day. The fish were fed twice a day

with tetramin (free of Cd). Female (weight

0.92 ± 0.18 g and 4.1 ± 0.34 cm length) fish were

randomly selected for exposure experiments. There

were no statistically significant differences in body

weight or length at the beginning of exposure (data

not shown).

Biometals

123

Fish exposure and sample collection

Fish were divided into 5 groups (n = 40 animals).

The control group was maintained in clean water and

was fed twice a day with ‘‘tetramin’’ (Diet I). In the

second group, Cd was added in water at a concen-

tration of 0.4 mg l-1 as CdCl2 and animals were fed

using Diet I. In the third group fish were exposed to

Cd and fed with control diet supplemented with 5 mg

kg-1 Zn as (ZnCl2) (Diet II). In the fourth group fish

were exposed to Cd and fed with control diet

supplemented with 2 mg kg-1 Se as (Na2SeO3) (Diet

III). Finally, in the fifth group animals were exposed

to Cd and fed with control diet supplemented with

2 mg kg-1 Se as (Na2SeO3) and 5 mg kg-1 Zn as

(ZnCl2) (Diet IV). In all conditions fish were fed

twice a day to apparent satiation for 3 weeks. Water

and exposure solutions were renewed every day and

the proven exposure concentration for Cd was

verified.

The Cd tested concentration represented the 1/10

of the acute toxicity LC50 (for 96 h) (Canton and

Slooff 1982). Previous studies showed that Zn and Se

supplementation lower than 20 and 3 mg kg-1 diet

had positive effects on animal growth and feed

conversion rate and did not produce adverse effects in

fish (Watanabe et al. 1997; Hamilton 2003). Thus, in

the present study, the chosen Zn and Se levels (5 and

2 mg kg-1 diet respectively) would not cause toxic

effects.

Before dissection, the fish were anesthetized on

ice. The livers and ovaries excised from the fish in

each exposure aquarium were randomly divided into

three samples: at least four fish were collected as one

sample, resulting in four pooled samples for bio-

chemical analysis; and four other samples were

pooled for RNA extraction and finally other set of

samples was used for Cd determination. These

samples were kept on dry ice while being prepared

and then stored at -80�C until they were analyzed. No

fish died during the course of the exposure.

Cadmium analysis

Hepatic and ovary tissues for Cd analyses were oven-

dried (60�C) to constant weight. The dried tissues

(100 mg pool from 4 animals) were digested with

3 ml trace pure nitric acid at 90�C for 24-48 h. The

volume was then adjusted to 5 ml with deionized

water. These measures were implemented using a

Zeenit 700-Analytik-Jena, Germany (Graphite-Fur-

nace AAS), equipped with deuterium and Zeeman

background correction, as recommended by the

manufacturer. Detection limit was 0.002 lg/l for

Graphite-Furnace AAS. The accuracy and precision

of our analysis for tissue metals content were based

on the analysis of Cd in a standard reference fish

liver. Our results show that the analytical results of

this study are of satisfactory quality. Samples were

analyzed in triplicate. The variation coefficient was

usually less than 10%. Concentrations of the metal in

the liver and ovary were calculated on a dry weight

basis and expressed as lg per gram dry tissue.

Biochemical assays

The tissue homogenates were obtained in 0.1 M

sodium phosphate buffer pH 7.0 at a ratio of 1:10w/v.

Homogenizations were carried out at 4�C followed by

centrifugation at 12,0009g for 30 min at 4�C. The

supernatants were collected and used to evaluate

enzymatic (SOD, CAT, GPx) activities and MDA

accumulation. Total protein content in the homoge-

nate was measured following the Bradford method

(Bradford 1976), at 595 nm, using bovine serum

albumin as standard.

CAT activity was determined according to Aebi

(1974) by following the consumption of 15 mM

H2O2 at 240 nm in 50 mM KH2PO4/K2HPO4 buffer,

pH 7.0 and 50 ll supernatant. One unit of CAT

activity was defined as the amount of enzyme

required to consume 1 lmol H2O2 in 1mn and was

expressed as U/mg protein. The total SOD activity

measurement was determined based on the ability of

the enzyme to inhibit the reduction of nitro blue

tetrazolium (NBT) (Crouch et al. 1981), which was

generated by 37.5 mM hydroxylamine in alkaline

solution. The assay was performed in a 0.5 M sodium

carbonate buffer (pH 10.2) with 2 mM EDTA and

10ll aliquot of the supernatant. The reduction of

NBT by superoxide anion to blue formazan was

measured at 560 nm. The SOD activity was calcu-

lated as relative to its ability to inhibit 50% reduction

of NBT per 1mn and expressed as U/mg protein. The

Se-dependent GPx activity was analyzed according to

the method described by Hafeman et al. (1974). GPx

degrades H2O2 in the presence of GSH thereby

depleting it. The remaining GSH is then measured by

Biometals

123

using 5.50-dithiobis 2-nitrobenzoic acid (DTNB). The

reaction was carried out at 37�C in a medium

containing 80 mM sodium phosphate buffer (pH

7.0), 80 mM EDTA, 1 mM NaN3, 0.4 mM GSH

and 0.25 mM H2O2 and 10 ll supernatant of tissue

homogenates. Absorbance was recorded at 412 nm.

One unit of GPx enzyme activity was defined as 1

lmole of GSH consumed/min. The GPx activity was

expressed in U/mg of protein. Lipid peroxidation was

estimated in terms of thiobarbituric acid reactive

species with use of 1,1,3,3- treaethyloxypropane as a

standard. The reaction was determined at 532 nm

using thiobarbituric acid reagent as per the method of

Buege and Aust (1978). Malondialdehyde (MDA)

content was expressed as nmoles equivalent MDA

per milligram protein.

MT protein levels in liver and ovary were deter-

mined using a spectrophotometric assay for MT using

Ellman’s reagent (0.4 mM 5,5’ Dithio-Nitro-Benzoate

(DTNB) in 100 mM KH2PO4) at pH 8.5 in a solution

containing 2 M NaCl and 1 mM EDTA (Viarengo

et al. 1997). In brief, aliquots were homogenized in

three volumes of 0.5 M sucrose, 20 mM Tris–HCl

buffer, pH 8.6, with added 0.006 mM leupeptine,

0.5 mM PMSF (phenylmethylsulphonyl_fluoride) as

antiproteolitic and 0.01% 2-mercaptoethanol as reducing

agent. The homogenate was then centrifuged at

15,0009g for 30mn at 4�C. The obtained supernatant

was treated with ethanol/chloroform as described by

Viarengo et al. (1997) in order to obtain the MT

enriched pellet. The obtained MT pellet was resus-

pended in HCl/EDTA in order to remove metal cations

still bound to the MT. Finally, 2 M NaCl was added to

the solution to facilitate thiol interactions with DTNB

by reducing the interaction of divalent metals with the

apothionein.

Gene expression analysis

RNA isolation and cDNA synthesis

Total RNA was extracted from about 10 mg frozen

liver or ovary tissues using the Trizol reagent (Sigma-

Aldrich, St. Louis, USA) according to the manufacture

instruction. The RNA purity was verified by the

OD260/OD280 absorption ratio ([1.8). RNA quality

was verified by comparing 18S and 28S peaks on

electropherograms for each samples tested. Only intact

RNA was used for further analysis. A total amount of

1.5–2 lg of total RNA was reverse transcribed in a

20 ll reaction mixture using random hexamers primers

(Roche) and 200 U of M-MuLV H- RT (Fermentas,

Vilnius, LI), 0.5 mM dNTPs (Roche), 19 M-MulV RT

buffer as described in Dondero et al. (2005).

Briefly, the RNA was denatured by heating for

5 min at 70�C, cooled on ice, and incubated with

reverse transcriptase reaction mixture. For reverse

transcription, tubes were incubated at 42�C for

60 min, followed by rapid cooling. The volume of

the RT mixture was raised to 100 ll with nuclease-

free distilled water, and 6 ll was used for amplifica-

tion of the gene targets.

Real-time quantitative PCR

Real-time quantitative PCR (RT-qPCR) was per-

formed in a real time apparatus (iCycler, Bio-Rad

Laboratories), in the presence of 19 QuantiTect Sybr

Green PCR Master Mix (Qiagen), 10 nM fluorescein,

0.2 lM of each gene Q-PCR primers (Table 1).

Relative expression data were geometrically normal-

ized on 18S rRNA and a beta Actin gene RNA. 18S

and beta Actin were chosen as internal reference

genes based on their good average expression stability

as previously reported by Tang et al. (2007) and

McCurley and Callard (2008) in zebrafish tissues.

The relative expression stability of the two reference

genes was calculated in our experimental conditions

using geNorm (Vandesompele et al. 2002). Our data

showed expression stability values of 0.32 and 0.44,

respectively for beta actin and 18S targets. The

thermal protocol was as follows: 10 min at 95�C,

followed by 40 cycles (10 s at 95�C, 20 s at 60�C,

30 s at 72�C where the signal was acquired). All

primers were confirmed to produce only one gene

product based on a single peak in the melting curve

(60–90�C) and a single band of the predicted size

detected on agarose gels in preliminary studies. All

amplifications had a PCR efficiency value between

1.92 and 2.10. RT-qPCR reaction was performed in

triplicate for each sample and a mean value used to

calculate mRNA levels. Five biological replicates

were measured for each group.

Statistics

To calculate the normalised relative gene expression

levels (fold induction), data were analysed using the

Biometals

123

Relative expression software tool (REST), in which

the mathematical model used is based on mean

threshold cycle differences between the sample and

the control group (Pfaffl et al. 2002). For each

analysed target it has been used the median PCR

efficiency value obtained from at least 4 different

experiments (3 replicate per experiments). REST was

also utilized to perform a randomisation test with a

pair-wise reallocation in order to assess the statistical

significance of the differences in expression between

the control and treated samples.

For metal accumulation and biochemical data, data

were analyzed by calculating mean and the standard

error of the mean (SEM), and Mann–Whitney’s test

was applied after a Bonferroni correction to find the

statistical significance. Data were considered statis-

tically significant at P \ 0.05 level.

Results

Liver and ovary Cd content

The liver and ovaries Cd contents after 3 weeks

exposure to 0,4 mg/l Cd in the water are reported in

Fig. 1. Our data indicated a significant (P \ 0.01)

accumulation of Cd in the liver (Fig. 1a) in comparison

to control fish. While Se supply was not effective in

changing Cd accumulation pattern in liver, the Zn

supply induced a significant increase in the levels of Cd

uptake (14.26 ± 2.22 lg/g dry weight) when compared

with the levels of the Cd group (8.06 ± 0.99 lg/g dry

weight). The simultaneous administration of Se and

Zn resulted in a important accumulation of Cd (15.93

± 1.94 lg/g dry weight) when compared to Cd-treated

animals. The accumulation pattern in ovaries (Fig. 1b)

was completely different from that observed in liver.

Indeed, Zn supply rendered a significant decrease in the

levels of Cd uptake (0.84 ± 0.12 lg/g dry weight)

when compared to the Cd group (2.24 ± 0.41 lg/g dry

weight). Moreover, the concomitant supply of Zn and

Se resulted in a more pronounced decreased in ovaries

Cd uptake (0.61 ±

0.081 lg/g dry weight).

Effect of Zn and Se supply on antioxidant

enzymes

The antioxidant enzymes activities in the liver and

ovaries of zebrafish exposed to Cd and supplemented

with Zn and Se are reported in Fig. 2. Our results

indicated a significant decrease in CAT, SOD and

GPx activities in liver and ovaries of Cd-exposed

fishes. Zn supply was effective in recovering CAT

and SOD activities to control values in the investi-

gated tissues of Cd-exposed animals. Se supply was

only effective in recovering GPx activities to control

values in Cd exposed fishes. The concomitant supply

of Zn and Se resulted in a normalization of CAT,

SOD and GPx activities in liver and ovaries.

Effect of Zn and Se supply on lipid peroxidation

The liver and ovaries TBA-reactive metabolites

contents after 3 weeks exposure to 0.4 mg/l Cd in

the water are reported in Fig. 3. Our data suggest a

Table 1 Nucleotide

sequences of gene-specific

primers for real-time PCR

with their corresponding

PCR product size of b actin,

18S, zMt, Zn-Sod, Cat and

Gpx in zebrafish

Gene Accession number Primers(50-30) Amplicon size (bp)

b actin AF057040 ATGGATGAGGAAATCGCTGCC

CTCCCTGATGTCTGGGTCGTC

106

18S BX296557 CGGAGGTTCGAAGACGATCA

TCGCTAGTTGGCATCGTTTATG

150

zMt NM_194273.1 GCCAAGACTGGAACTTGCAAC

CGCAGCCAGAGGCACACT

130

Zn-Sod Y12236 GTCGTCTGGCTTGTGGAGTG

TGTCAGCGGGCTAGTGCTT

113

Cat AF170069 AGGGCAACTGGGATCTTACA

TTTATGGGACCAGACCTTGG

499

Gpx AW232474 AGATGTCATTCCTGCACACG

AAGGAGAAGCTTCCTCAGCC

94

Biometals

123

strong increase of TBA-reactive metabolites accumu-

lation in Cd-exposed fishes with respectively

1.84 ± 0.09 nmole/mg proteins and 1.14 ± 0.12

nmole/mg protein in liver and ovaries when compared

with control animals (0.82 ± 0.07 nmole/mg proteins

and 0.59 ± 0.06 nmole/mg proteins respectively in

liver and ovaries). Zn or Se single supply, only partially

reversed this increase. In fact, TBA-reactive metabo-

lites concentrations in the Cd ? Zn and Cd ? Se

groups was lower than in the Cd-exposed group

(P \ 0.01) but still significantly higher than in the

control animals (P \ 0.01). However, co-supply of Zn

and Se was effective in reversing Cd-induced increase

in liver and ovaries TBA-reactive metabolites

concentrations.

Effect of Zn and Se supply on total

metallothionein accumulation

Total metallothionein protein content was evaluated

in the liver and ovaries of zebrafish exposed to Cd

and supplemented with Zn and Se (Fig. 4). A

significant increase in MT levels in comparison with

to the control animals was registered in Cd-exposed

fish with up to 97.26 ± 6.79 ng/mg proteins (2.76

fold increase) in liver and 46.71 ± 4.37 ng/mg

proteins (2.23 fold increase) in ovaries. The increase

of the MT content in animals supplemented with Zn,

Se and their mixture was less pronounced than that of

Cd (1.87, 2.07 and 1.62 fold respect to control

animals) in the liver. The same pattern was observed

in the ovaries of fishes supplemented with Zn or Se

(1.42 and 1.52 fold increase respect to control

animals). No significant variation of the MTs accu-

mulation was registered in the ovaries of zebrafish

exposed to Cd ? Zn ? Se when compared to control

animals.

Effect of Zn and Se supply on mRNA expression

Expression analysis of various genes (Cat, CuZn–

SOD, Gpx, Mt) encoding antioxidant proteins and

metallothionein was performed by real time quanti-

tative PCR on liver and ovaries transcripts using 18S

and beta actin as reference genes (Fig. 5). A signif-

icant increase in Mts (13.61 folds), Cat (6.35 folds),

CuZn-Sod (5.41 folds) and Gpx (3.64 folds) tran-

scription was observed in liver of Cd-exposed fishes

when compared to control animals. The same pattern,

but with lesser extend was observed in ovaries with

an induction of 4, 2.70, 2.38 and 1.92 folds,

respectively for Mts, Cat, CuZn–SOD and Gpx.

Single Zn supply resulted in a decrease in CuZn-Sod

and zMt in liver and ovaries when compared to Cd-

exposed fishes. Interestingly, the transcription of the

antioxidant targets manifests values similar to control

when fishes are supplemented with Zn and Se in both

investigated organs. In deed no significant differences

in genes expression was recorder in that condition.

Concerning zMt mRNA abundance in liver and

ovaries, our data indicate a decreasing trend of gene

expression in presence of Zn or Se and a return

to control values when the two elements are

co-supplemented.

Discussion

In this study we have presented data concerning a set

of antioxidant enzyme activities, metallothionein

0

2

4

6

8

10

12

14

16

18

20

Control Cd Cd/Zn Cd/Se Cd/Zn/Se

µg

/g d

ry w

eig

ht

a

a b

a

a bA

0

0,5

1

1,5

2

2,5

3

Control Cd Cd/Zn Cd/Se Cd/Zn/Se

µg

/g d

ry w

eig

ht

a

a b

a

a b

B

Fig. 1 Cadmium concentrations in the liver (a) and ovary

(b) of female zebra fish exposed to Cd (0.4 mg/l) and

supplemented with Zn and/or Se in their diet, during 3 weeks.

Each bar represents mean ± SE of 10 animals. Statistically

significant differences: aP \ 0.01 in comparison with control.bP \ 0.01 in comparison with Cd group

Biometals

123

0

20

40

60

80

100

120

CA

T U

/mg

pro

tein

s

aa b

a

a bA

0

5

10

15

20

25

30

35

40

Control Cd Cd/Zn Cd/Se Cd/Zn/Se

SO

D U

/mg

pro

tein

s

a

ba

a bB

0

5

10

15

20

25

30

35

40

45

Control Cd Cd/Zn Cd/Se Cd/Zn/Se

GP

x U

/mg

pro

tein

s

aa

bbC

0

20

40

60

80

100

120

140

Control Cd Cd/Zn Cd/Se Cd/Zn/SeControl Cd Cd/Zn Cd/Se Cd/Zn/Se

CA

T U

/mg

pro

tein

s

a b

a ba ba

D

0

5

10

15

20

25

30

35

40

Control Cd Cd/Zn Cd/Se Cd/Zn/Se

GP

x U

/mg

pro

tein

s b

a

a bF

0

5

10

15

20

25

30

Control Cd Cd/Zn Cd/Se Cd/Zn/Se

SO

D U

/mg

pro

tein

s

a

b b

a bE

Fig. 2 Activities of CAT (a, d), SOD (b, e) and GPx (c, f) in

the liver (a, b and c) and ovary (d, e and f) of female zebrafish

exposed to Cd (0.4 mg/l) and supplemented with Zn and/or Se

in their diet, during 3 weeks. Each bar represents mean ± SE

of 10 animals. Statistically significant differences: aP \ 0.01 in

comparison with control. bP \ 0.01 in comparison with Cd

group

0,0

0,5

1,0

1,5

2,0

Control Cd Cd/Zn Cd/Se Cd/Zn/Se

MD

A n

mo

le/m

g p

rote

ins

a

a b a bb

B

0,0

0,5

1,0

1,5

2,0

Control Cd Cd/Zn Cd/Se Cd/Zn/Se

MD

A n

mo

le/m

g p

rote

ins a

a ba b

b

A

Fig. 3 MDA contents in the liver (a) and ovary (b) of female

zebrafish exposed to Cd (0.4 mg/l) and supplemented with Zn

and/or Se in their diet, during 3 weeks. Each bar represents

mean ± SE of 10 animals. Statistically significant differences:aP \ 0.01 in comparison with control. bP \ 0.01 in compar-

ison with Cd group

Biometals

123

accumulation and their related gene expression in the

liver and ovaries tissues of female zebrafish exposed

to Cd and supplemented with Zn and Se. Several

environmental pollutants can become toxic through

the induction of oxidative stress. The effects of Cd on

aquatic organisms were largely documented (Giles

1988; Kraemer et al. 2005; Atli et al. 2006; Banni

et al. 2009) however, and to our knowledge, no

studies investigated the potential protective effects of

Zn and Se on Cd-induced toxicity in fish species and

the mechanism by which such protection occurs.

Many aquatic organisms have unique systems for

protecting themselves against reactive oxygen species

(ROS) damaging effects (Jin et al. 2010). The

antioxidant enzymes such as CAT, SOD and GPx

are among the most important components of this

defense mechanism (Atli et al. 2006; Ruas et al.

2008).

In this study, heavy metals analysis clearly

showed different degrees of Cd loads in the liver

and ovaries tissue of zebrafish from the different

experimental conditions. Our results indicated a

significant increase in Cd level in the liver. The

latter increase was more effective in presence of Zn.

Our results are in agreements with a large number of

studies indicating that Zn increases Cd concentration

in hepatic tissues but reduces it in other organs in

mammalian systems (Lamphere et al. 1984; Ueda

et al. 1987; Banni et al. 2010). This redistribution of

Cd in the organisms could be considered as a

protective mechanism against the Cd-cellular toxic-

ity and would explain the relatively lower effect of

Cd in ovaries when compared with that observed in

liver.

As expected, exposure to Cd, clearly decreased the

activities of CAT, SOD and GPx and rendered a

significant increase in MDA accumulation in both

liver and ovaries. Cd was also responsible of the

significant increase of MTs accumulation in the

investigated tissues. Similar effects were reported in

several aquatic biosystems (Banni et al. 2009; Isani

et al. 2009; Cao et al. 2010). It is well known that the

displacement of iron, Zn and copper from various

intracellular sites by Cd increases the concentration

of the ionic iron, Zn and copper (Casalino **et al.

1997). This causes oxidative stress through the

Fenton reaction, producing hydroxyl radical species

that are believed to initiate lipid peroxidation

(Jurczuk et al. 2004; Dondero et al. 2005) and

minimize the protective role of anti-oxidative stress

enzymes such as CAT, SOD and GPx (Bauer et al.

1980; Jihen et al. 2009). Interestingly, in Cd-exposed

animals, the gene expression analysis of the anti-

oxidative stress genes showed a marked up-regulation

pattern that could be attributed to the accumulation of

ROS due to the anti-oxidative stress enzymes inhi-

bition (Banni et al. 2010; Cuypers et al. 2010; Jihen

et al. 2009). Like other organisms, fish can combat

the increasing levels of ROS in their tissues produc-

ing protective ROS-scavenging enzymes such as

SOD and CAT, which convert superoxide anions

(O2-) into H2O2 and then into H2O and O2. Thus, it is

possible that an increase in the transcription of these

genes would contribute to the elimination of ROS

from the cell induced by Cd exposure.

Our data provided clues on the effects of dietary

Zn supplementation on the oxidative stress status of

the liver and ovaries tissues of zebrafish exposed to

0

20

40

60

80

100

120

Control Cd Cd/Zn Cd/Se Cd/Zn/Se

MT

ng

/mg

pro

tein

s

a b

a

a ba b

A

0

10

20

30

40

50

60

Control Cd Cd/Zn Cd/Se Cd/Zn/Se

MT

ng

/mg

pro

tein

s a

a b ba b

B

Fig. 4 Metallothionein (MT) accumulation in the liver (a) and

ovary (b) of female zebrafish exposed to Cd (0.4 mg/l) and

supplemented with Zn and/or Se in their diet, during 3 weeks.

Each bar represents mean ± SE of 10 animals. Statistically

significant differences: aP \ 0.01 in comparison with control.bP \ 0.01 in comparison with Cd group

Biometals

123

Cd. Indeed, a significant recover of the CAT and

SOD activities to control values and a decrease in

MDA accumulation when compared to Cd-exposed

animals were observed in both tissues. Moreover, Zn

supply seems to affect the Cd distribution between

organs. A maximum liver Cd-uptake was observed in

D (Mt)

0

2

4

6

8

10

12

14

16

18

20

Cd Cd+Zn Cd+Se Cd+Zn+Se

Rel

ativ

e g

ene

exp

ress

ion

a

a ba b

b

A (Cat)

0

1

2

3

4

5

6

7

8

Cd Cd+Zn Cd+Se Cd+Zn+Se

Rel

ativ

e g

ene

exp

ress

ion

a

aa

b

B (Sod)

0

1

2

3

4

5

6

7

8

9

10

Cd Cd+Zn Cd+Se Cd+Zn+Se

Rel

ativ

e g

ene

exp

ress

ion

b

a b

a

a

C (Gpx)

0

1

2

3

4

5

6

Cd Cd+Zn Cd+Se Cd+Zn+Se

Rel

ativ

e g

ene

exp

ress

ion

a

aa

b

E (Cat)

0

0,5

1

1,5

2

2,5

3

3,5

Cd Cd+Zn Cd+Se Cd+Zn+Se

Rel

ativ

e g

ene

exp

ress

ion

aa a

b

F (Sod)

0

0,5

1

1,5

2

2,5

3

3,5

Cd Cd+Zn Cd+Se Cd+Zn+Se

Rel

ativ

e g

ene

exp

ress

ion

b

a b

aa b

G (Gpx)

0

0,5

1

1,5

2

2,5

Cd Cd+Zn Cd+Se Cd+Zn+Se

Rel

ativ

e g

ene

exp

ress

ion

a aa

b

H (Mt)

0

1

2

3

4

5

6

Cd Cd+Zn Cd+Se Cd+Zn+Se

Rel

ativ

e g

ene

exp

ress

ion

a

a b

a b

b

Fig. 5 Quantitative real time PCR expression-analysis of Cat

(a, e), Zn-Sod (b, f), Gpx (c, g) and Mt (d, h) genes in the liver

(a, b, c and d) and ovary (e, f, g and f) of female zebra fish

exposed to Cd (0.4 mg/l) and supplemented with Zn and/or Se

in their diet, during 3 weeks. Values were geometrically

normalized against b-actin and 18S (used as a house-keeping

genes), and represent the mean mRNA expression value ±

SEM (n = 5) relative to those of the controls. The asterisk

represents a statistically significant difference when compared

with the female controls; *at P \ 0.05

Biometals

123

presence of Zn, when the ovaries Cd-loads were low.

Zn has important roles in the organism for growth,

protein metabolism, energy production, gene regula-

tion, maintaining the health of cell membranes and

bones probably because it is a cofactor of numerous

enzymes (Watanabe et al. 1997; Yamaguchi 1998).

One of the most significant functions of Zn is related

to its antioxidant potential and its participation in the

antioxidant defense system (Powell 2000). Kucukbay

et al. (2006) reported that supplemental Zn in the diet

decreases serum and tissue lipid peroxidation in

rainbow trout.

The CuZn-Sod gene expression pattern decreased

markedly in Zn-supplemented fishes when compared

to Cd-exposed animals in liver and was similar to

control animals in ovaries. Chung et al. (2005)

demonstrated an apparent Zn dependency of

H2O2-induced expression of antioxidant genes in

rainbow trout gills cell culture, suggesting that Zn

might act as a physiological signal to mediate the

response to oxidative stress. Moreover, Zn stimulates

transcription of specific genes by binding to metal

regulatory Transcription Factor-1, which upon acti-

vation binds to metal-responsive elements of the

target genes (Andrews 2001). Gene regulation by Zn

is not restricted to those involved in Zn homeostasis

(Cousins et al. 2003; Egli et al. 2003). This may

explain the significant decrease in Mts mRNA

abundance as well as CuZn-Sod mRNA in liver and

ovaries of Zn-supplemented animals when compared

to Cd-exposed animals. Indeed in recent works

(Banni et al. 2010; Messaoudi et al. 2010a, b), Zn

amounts significantly decreased in testis and plasma

of rats exposed to Cd and to Cd ? Zn and increased

in liver tissues.

In our Study, Se supplementation alone decreased

the Cd-induced toxicity promoting the maintenance of

a normal steady state GPx activity in liver and ovaries.

Moreover, the CAT and SOD activities were recov-

ered to control values in the ovaries in

Se-supplemented animals. One of the most important

functions of Se is related to its antioxidant role and

participation in the antioxidant defense system since it

is a GPx cofactor (Kohrle et al. 2005). GPx scavenges

H2O2 and lipid hydroperoxides, using reducing equiv-

alents from glutathione and protecting membrane

lipids and macromolecules from oxidative damage

(Watanabe et al. 1997). Recently, the effects of Se on

oxidative stress biomarkers in the freshwater characid

fish Brycon cephalus exposed to the organophosphate

methyl parathion was investigated, suggesting that

dietary Se protects cells against the insecticide-

induced oxidative stress (Monteiro et al. 2009). The

gene expression patterns of the investigated targets did

not manifest any significant changes respect to

Cd-exposed animals except for Sod and Mt in ovaries

that showed a slight decrease where the CAT activity

was recovered to control values and the MTs protein

content significantly decreased in comparison with

Cd-Exposed animals. The latter could be attributed

to the decrease of the intracellular concentration of

free Cd.

In this work, we report for the first time the potential

protective effect of Zn and Se on Cd-induced toxicity

in fish species. In deed, our results show that the

co-supply of Zn and Se in the diet recovered the MDA

accumulation in Cd-exposed animals to control values.

Our data indicated also a significant improvement in

the response of the anti-oxidative stress enzymes when

compared to Cd-exposed and control animals. More-

over, the mRNA abundance of the Cat, Zn-Sod and

Gpx were maintained at control levels. Similar effects

were recently reported in mammalian biosystems

(Jihen et al. 2009; Messaoudi et al. 2009). Interest-

ingly, the up-regulation pattern of all investigated

genes observed in Cd-exposed animals was abolished,

except for Mts which maintained a slight up-regulation

trend when fishes are supplemented with Zn and Se.

Our data would suggest that the protective effect of Zn

and Se against Cd-induced toxicity passes through

non-MT gene expression mechanisms being more

depending of the oxidative stress status of the cell as it

has been recently proposed in rat tissues (Banni et al.

2010). Indeed, metallothionein induction was associ-

ated with the presence of some reactive oxygen species

and thus, with the oxidative stress status of the cell

(Dondero et al. 2005). It has been previously shown

that hydrogen peroxide and other oxidants can stim-

ulate Mt mRNA neosynthesis (Dalton et al. 1994), and

it is well known that Mts bear a high antioxidant

potential (Thornalley and Vasak 1985).

Conclusion

In conclusion, our results indicate that dietary intake

of Zn or Se can decrease the oxidative damages in

zebrafish exposed to Cd. Interestingly, co-supply of

Biometals

123

Zn and Se efficiently protected against Cd-induced

toxicity in two target organs; liver and ovaries. Our

study further demonstrated that the mRNA abun-

dances of genes, which encode antioxidant proteins

(Cat, Zn-Sod, and Gpx) were higher when related

enzymatic activities were altered. Finally, our data

would suggest that protective effect of Zn and Se

against Cd-induced toxicity passes through non-MT

gene expression mechanisms being more depending

of the oxidative stress status of the cell.

Acknowledgments This work was supported by founds from

‘‘Ministere de l’Enseignement Superieur et de la Recherche

Scientifique; UR «Biochimie et Toxicologie Environnemen-

tale» , ISA Chott-Mariem, Universite de Sousse «Tunisia» and

UR: «Genetique, Biodiversite et Valorisation des Bioressources,

Institut Superieure de Biotechnologie de Monastir» , Universite

de Monastir, «Tunisia» .Conflict of interest None.

References

Aebi H (1974) Catalase. In: Bergmeyer H-U (ed) Methods of

enzymatic analysis. Academic Press, New York, pp 671–684

Andrews GK (2001) Cellular zinc sensors: MTF-1 regulation

of gene expression. Biometals 14:223–237

Atli G, Alptekin O, Tukel S, Canli M (2006) Response of

catalase activity to Ag2? , Cd2? , Cr2? , Cu2? and

Zn2? in five tissues of freshwater fish Oreochromis nil-oticus. Comp Biochem Physiol C 143:218–224

Banni M, Bouraoui Z, Clerandeau C, Narbonne JF, Boussetta

H (2009) Mixture toxicity assessment of cadmium and

benzo[a]pyrene in the sea worm Hediste diversicolor.

Chemosphere 77:902–906

Banni M, Messaouidi I, Said L, El Heni J, Kerkeni A, Said K

(2010) metallothionein gene expression in liver of rats

exposed to cadmium and supplemented with zinc and

selenium. Arch Environ Contam Toxicol. doi: 10.1007/

s00244-010-9529-y

Bauer R, Demeter I, Hasemann V, Johansen JT (1980) Structural

properties of the zinc site in Cu, Zn-superoxide dismutase;

perturbed angular correlation of gamma rays pectroscopy

on the Cu, 111Cd-superoxide dismutase derivative. Bio-

chem Biophys Res Commun 94(4):1296–1302

Bonda E, Wlostowski T, Krasowska A (2004) Testicular tox-

icity induced by dietary cadmium is associated with

decreased testicular zinc and increased hepatic and renal

metallothionein and zinc in the bank vole (Clethrionomysglareolus). Biometals 17:615–624

Bradford M (1976) A rapid and sensitive method for the

quantification of microgram quantities of protein utilizing

the principle of protein-dye binding. Anal Biochem 72:

248–254

Buege JA, Aust SD (1978) Microsomal lipid peroxidation.

Methods Enzymol 52:302–310

Canton JH, Slooff W (1982) Toxicity and accumulation studies

of cadmium (Cd2?) with freshwater organisms of dif-

ferent trophic levels. Ecotoxicol Environ Saf 6(1):

113–128

Cao L, Huang W, Liu J, Yin X, Dou S (2010) Accumulation

and oxidative stress biomarkers in Japanese flounder lar-

vae and juveniles under chronic cadmium exposure.

Compar Biochem Physiol Part C 151:386–392

Chung MJ, Walker PA, Brown RW, Hogstrand C (2005)

ZINC-mediated gene expression offers protection against

H2O2-induced cytotoxicity. Toxicol Appl Pharmacol

205(3):225–236

Cousins RJ, Blanchard RK, Popp MP, Liu L, Cao J, Moore B,

Green CL (2003) A global view of the selectivity of zinc

deprivation and excess on genes expressed in human

THP-1 mononuclear cells. Proc Natl Acad Sci USA

100:6952–6957

Crouch RK, Gandy SC, Kinsey G (1981) The inhibition of islet

superoxide dismutase by diabetogenic drugs. Diabetes

30:235–241

Cuypers A, Plusquin M, Remans T, Jozefczak M, Keunen E,

Gielen H, Opdenakker K, Nair AR, Munters E, Artois TJ,

Nawrot T, Vangronsveld J, Smeets K (2010) Cadmium

stress: an oxidative challenge. Biometals 23(5):927–940

Dalton T, Palmiter RD, Andrews GK (1994) Transcriptional

induction of the mouse metallothionein-I gene in hydro-

gen peroxide-treated Hepa cells involves a composite

major late transcription factor/antioxidant response ele-

ment and metal response promoter elements. Nucleic Acid

Res 22:5016–5023

Dondero F, Piacentini L, Banni M, Rebelo M, Burlando B,

Viarengo A (2005) Quantitative PCR analysis of two

molluscan metallothionein genes unveils differential

expression and regulation. Gene 345:259–270

Egli D, Selvaraj A, Yepiskopsyan H, Zhang B, Hafen E,

Georgev O, Schaffner W (2003) Knockout of dmetal-

responsive transcription factorT MTF-1 in Drosophila by

homologous recombination reveals its central role in

heavy metal homeostasis. EMBO J 22:100–108

EPA Agency EP (2004) Ebdocrine Disruptor Screening Pro-

gram. Available online: http://www.epa.gov/scipoli/oscp

endo/edspoverview/index.htm

Giles MA (1988) Accumulation of cadmium by rainbow trout,

Salmo gairdneri, during extended exposure. Can J Fish

Aquat Sci 45:1045–1053

Hafeman DG, Sunde RA, Hoekstra WG (1974) Effect of die-

tary selenium on erythrocyte and liver glutathione per-

oxidase in the rat. J Nutr 104:580–587

Hamilton SJ (2003) Review of residue-based selenium toxicity

thresholds for freshwater fish. Ecotoxicol Environ Saf

56:201–210

Hu Y, Jin T, Zhou T, Pang B, Wang Y (2004) Effects of zinc

on gene expressions induced by cadmium in prostate and

testes of rats. Biometals 17:571–572

Isani G, Andreani G, Cocchioni F, Fedeli D, Carpene E, Fal-

cioni G (2009) Cadmium accumulation and biochemical

responses in Sparus aurata following sub-lethal Cd-

exposure. Ecotoxicol Environ Saf 72:224–230

Jihen EH, Imed M, Fatima H, Abdelhamid K (2009) Protective

effects of selenium (Se) and zinc (Zn) on cadmium (Cd)

Biometals

123

toxicity in the liver of the rat: effects on the oxidative

stress. Ecotoxicol Environ Saf 72:1559–1564

Jin Y, Zhang X, Shu L, Chen L, Sun L, Qian H, Liu W, Fu Z

(2010) Oxidative stress response and gene expression with

atrazine exposure in adult female zebrafish (Danio rerio).

Chemosphere 78:846–852

Jurczuk M, Brzoska MM, Moniuszko-Jakoniuk J, Gaazyn-

Sidorczuk M, Kulikowska-Karpinska E (2004) Antioxi-

dant enzymes activity and lipid peroxidation in liver and

kidney of rats exposed to cadmium and ethanol. Food

Chem Toxicol 42(3):429–438

Kohrle J, Jakob F, Contempre B, Dumont JE (2005) Selenium,

the thyroid, and the endocrine system endocrine. Endocr

Rev 26:944–984

Kraemer LD, Campbell PGC, Hare L (2005) Dynamics of Cd,

Cu and Zn accumulation in organs and sub-cellular frac-

tions in field transplantated juvenile yellow perch (Percaflavescens). Environ Poll 138(2):324–337

Kucukbay Z, Yazlak H, Sahin N, Tuzcu M, Cakmak MN,

Gurdogan F, Juturu V, Sahin K (2006) Zinc picolinate

supplementation decreases oxidative stress in rainbow

trout (Oncorhynchus mykiss). Aquaculture 257:465–469

Lamphere DN, Dorn CR, Reddy CS, Meyer AW (1984)

Reduced cadmium body burden in cadmium-exposed

calves fed supplemental zinc. Environ Res 33(1):119–129

Liu L, We G (2007) Growth differentiation factor 9 and its

spatiotemporal expression and regulation in the zebrafish

ovary. Biol Reprod 76:294–301

McCurley AT, Callard GV (2008) Characterization of house-

keeping genes in zebrafish: male-female differences and

effects of tissue type, developmental stage and chemical

treatment. BMC Mol Biol 12:90–102

Messaoudi I, El Heni J, Hammouda F, Saıd K, Kerkeni A

(2009) Protective effects of selenium, zinc, or their

combination on cadmium-induced oxidative stress in rat

kidney. Biol Trace Elem Res 130:152–161

Messaoudi I, Hammouda F, El Heni J, Baati T, Saıd K, Kerkeni

A (2010a) Reversal of cadmium induced oxidative stress

in rat erythrocytes by selenium, zinc or their combination.

Exp Toxicol Pathol 62(3):281–288

Messaoudi I, Banni M, Saıd L, Saıd K, Kerkeni A (2010b)

Evaluation of involvement of testicular metallothionein

gene expression in the protective effect of zinc against

cadmium-induced testicular pathophysiology in rat. Re-

prod Toxicol 29(3):339–345

Monteiro DA, Rantin FT, Kalinin AL (2009) The effects of

selenium on oxidative stress biomarkers in the freshwater

characid fish matrinxa, Brycon cephalus (Gunther, 1869)

exposed to organophosphate insecticide Folisuper 600

BR� (methyl parathion). Comp Biochem Physiol C 149:

40–49

Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expres-

sion software tool (REST) for group-wise comparison and

statistical analysis of relative expression results in real-

time PCR. Nucleic Acid Res 30:e36

Piasek M, Blanua M, Kostial K, Laskey JW (2001) Placental

cadmium and progesterone concentrations in cigarette

smokers. Reprod Toxicol 15:673–681

Powell SR (2000) The antioxidant properties of zinc. J Nutr

130:1447S–1454S

Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hofeman DG,

Hoekstra WG (1973) Selenium: biochemical role as a com-

ponent of glutathione peroxidase. Science 179:578–590

Ruas CBG, Carvalho CdS, De Araujo HSS, Espindola ELG,

Fernandes MN (2008) Oxidative stress biomarkers of

exposure in the blood of cichlid species from a metal-

contaminated river. Ecotoxicol Environ Saf 71:86–93

Segner H (2008) Zebrafish (Danio rerio) as a model organism

for investigating endocrine disruption. Comp Biochem

Physiol C 149:187–195

Tang R, Dodd A, Lai D, McNabb WC, Love DR (2007) Val-

idation of zebrafish (Danio rerio) reference genes for

quantitative real-time RT-PCR normalization. Acta Bio-

chim Biophys Sin (Shanghai) 39(5):384–390

Thornalley PJ, Vasak M (1985) Possible role for metallothio-

nein in protection against radiation-induced oxidative

stress. Kinetics and mechanism of its reaction with

superoxide and hydroxyl radicals. Biochim Biophys Acta

827:36–44

Ueda F, Seki H, Fujiwara H, Ebara K, Minomiya S, Shimaki Y

(1987) Interacting effects of zinc and cadmium on the

cadmium distribution in the mouse. Vet Hum Toxicol

29(5):367–372

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N,

De Paepe A, Speleman F (2002) Accurate normalization

of real-time quantitative QPCR data by geometric aver-

aging of multiple internal controls. Genome Biol 3:34

Viarengo A, Ponzano E, Dondero F, Fabbri R (1997) A simple

spectrophotometric method for metallothionein evaluation

in marine organisms: an application to Mediterranean and

Antarctic molluscs. Mar Env Res 44:69–84

Watanabe T, Kiron V, Datoh S (1997) Trace minerals in fish

nutrition. Aquaculture 151:185–207

Yamaguchi M (1998) Role of zinc in bone formation and bone

resorption. J Trace Elem Exp Med 11:119–135

Yiin SJ, Cheru CL, Sheu JY, Lin TH (1999) Cadmium-induced

lipid peroxidation in rat testes and protection by selenium.

Biometals 12:353–359

Biometals

123