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Aquatic Toxicology 128– 129 (2013) 183– 189

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Aquatic Toxicology

jou rn al h om epa ge: www.elsev ier .com/ locate /aquatox

Copper induced upregulation of apoptosis related genesin zebrafish (Danio rerio) gill

Ana Luzioa,∗, Sandra M. Monteiroa, António A. Fontaínhas-Fernandesa,Olinda Pinto-Carnideb, Manuela Matosb, Ana M. Coimbraa,∗

a Centro de Investigac ão de Tecnologias Agro-Ambientais e Biológicas (CITAB), Departamento de Biologia e Ambiente (DeBA), Escola de Ciências da Vida e Ambiente (ECVA), Universidadede Trás-os-Montes e Alto Douro (UTAD), Quinta de Prados, Apartado 1013, 5001-801 Vila Real, Portugalb Instituto de Biotecnologia e Bioengenharia, Centro de Genómica e Biotecnologia (IBB/CGB), Departamento de Genética e Biotecnologia (DGB), Escola de Ciências da Vida e Ambiente(ECVA), Universidade de Trás-os-Montes e Alto Douro (UTAD), Quinta de Prados, Apartado 1013, 5001-801 Vila Real, Portugal

a r t i c l e i n f o

Article history:Received 20 September 2012Received in revised form11 December 2012Accepted 19 December 2012

Keywords:AIFApoptosisCaspaseCopperp53RT-PCRTUNELZebrafish

a b s t r a c t

Copper (Cu) is an essential micronutrient that, when present in high concentrations, becomes toxic toaquatic organisms. It is known that Cu toxicity may induce apoptotic cell death. However, the precisemechanism and the pathways that are activated, in fish, are still unclear. Thus, this study aimed to assesswhich apoptotic pathways are triggered by Cu, in zebrafish (Danio rerio) gill, the main target of waterbornepollutants. Fish where exposed to 12.5 and 100 �g/L of Cu during 6, 12, 24 and 48 h. Fish gills werecollected to TUNEL assay and mRNA expression analysis of selected genes by real time PCR. An approachto different apoptosis pathways was done selecting p53, caspase-8, caspase-9 and apoptosis inducingfactor (AIF) genes. The higher incidence of TUNEL-positive cells, in gill epithelia of the exposed fish,proved that Cu induced apoptosis. The results suggest that different apoptosis pathways are triggered byCu at different time points of the exposure period, as the increase in transcripts was sequential, instead ofsimultaneous. Apoptosis seems to be initiated via intrinsic pathway (caspase-9), through p53 activation;then followed by the extrinsic pathway (caspase-8) and finally by the caspase-independent pathway(AIF). A possible model for Cu-induce apoptosis pathways is proposed.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Copper (Cu) is an essential micronutrient present in multipleproteins and involved in several biological processes (Linder, 2001;De Romana et al., 2011). However, when high concentrations arepresent in water, Cu becomes toxic to aquatic organisms (Monteiroet al., 2009; Main et al., 2010; Ebrahimpour et al., 2011; Tellis et al.,2012).

Naturally occurring levels of Cu, in unpolluted freshwaters,range from 0.2 to 30 �g/L (Craig et al., 2007; Us-Epa, 2007).However, in areas with high agriculture and mining activities, man-ufacturing industries and municipal effluents, Cu can be present intoxic concentrations that vary from 50 up to 560 �g/L (WHO, 1998;Us-Epa, 2007).

The main toxic action of copper to freshwater fish is impair-ment of the ionoregulatory function (Lauren and Mcdonald, 1985,1987; Grosell et al., 2001). Among the already known molecu-lar effects of Cu exposure are the reactive oxygen species (ROS)

∗ Corresponding authors. Tel.: +351 259 350 108.E-mail addresses: aluzio@utad.pt (A. Luzio), acoimbra@utad.pt (A.M. Coimbra).

upregulation (Chen and Chan, 2011), DNA cleavage (Kawakamiet al., 2008), Bax upregulation (Zhai et al., 2000; Kawakami et al.,2008) and the induction of p53 expression (Narayanan et al.,2001; Tassabehji et al., 2005). All of these can result in apopto-sis, a genetically controlled and evolutionary conserved form ofactive cell death. Generally, apoptosis occurs through caspase-dependent or caspase-independent pathways. In mammals, thecaspase-dependent pathway, depending on the stimulus, may trig-ger two different paths that converge on the activation of effectorcaspases (Ashkenazi and Dixit, 1998; Jin and El-Deiry, 2005). Theintrinsic pathway involves cell death signals derived directly frommitochondria (Zhang et al., 2003; Jin and El-Deiry, 2005). The acti-vation by extrinsic pathway starts in the tumor necrosis factor (TNF)family receptors (Green and Reed, 1998; Fan et al., 2005). Caspase-8is involved in the extrinsic pathway, while caspase-9 participates inthe intrinsic pathway (Thorburn, 2004; Jin and El-Deiry, 2005). Thecaspase-independent pathway is mediated by the apoptosis induc-ing factor (AIF), a mitochondrial protein that can be translocated tothe cytosol, as well as into the nucleus (Daugas et al., 2000; Nagataet al., 2003). Furthermore, DNA damage and the subsequent p53activation may also induce apoptotic cell death through a nuclearpathway (Tassabehji et al., 2005). Several lines of evidence suggest

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that, in mammals, metals preferentially induce apoptosis throughthe mitochondrial pathway (Pulido and Parrish, 2003; Kawakamiet al., 2008).

In fish, the available data suggest the existence of equivalentapoptotic pathways to the ones present in mammals (Pyati et al.,2007; Dos Santos et al., 2008; Krumschnabel and Podrabsky, 2009),although the genes involved have not been assessed in the gills onexposure to pollutants. To our knowledge, their expression afterCu exposure is only studied to small extent in gill, a key site of Cuuptake and the first target organ to its toxicity (Bury et al., 1998,2003; Grosell et al., 2004; Peles et al., 2012). In a previous study,with Nile tilapia (Oreochromis niloticus), no Cu effects in gill apopto-sis induction were observed (Monteiro et al., 2009), corroboratingprevious works (Bury et al., 1998). However, other studies showedthat Cu induces morphological and cytochemical changes, consis-tent with apoptosis occurrence in fish (Li et al., 1998; Mazon et al.,2002). In addition, the activation of caspase-3 was detected in trouthepatocytes exposed to Cu, while in gill cell lines a complete lack ofcaspase activation was observed (Krumschnabel et al., 2005, 2010).As in mammals, this suggests some variability amongst species,organs, or even cell types in the apoptosis pathways induced by Cuexposure (Pyati et al., 2007; Krumschnabel and Podrabsky, 2009).

Zebrafish (Danio rerio) has long being used as a model intoxicological studies, regarding the effects of different classes ofenvironmental contaminants (Chan and Cheng, 2003; Craig et al.,2007; Sassi-Messai et al., 2009; Soares et al., 2009; Hernandez et al.,2011). In order to evaluate if copper overload induces an apop-totic response in fish gill and to clarify which pathways might beinduced, in the present work, adult zebrafish were exposed to 12.5and 100 �g/L of Cu for 6, 12, 24 and 48 h. Four genes were selectedto explore the copper activated apoptosis pathway(s) in zebrafishgills: caspase-8 (extrinsic pathway), caspase-9 (intrinsic pathway),apoptosis inducing factor (AIF; caspase-independent) and p53.

2. Materials and methods

2.1. Exposure conditions

Adult wild-type D. rerio were obtained from local suppliers andkept, during a 30-day period of acclimatization, in 120 L aquaria,in a recirculated system with dechlorinated and aerated water.Standardized conditions were maintained: water was continuouslymechanically and biologically filtered (thermo-controlled filter,Eheim, Germany), aerated and kept at 28 ± 1 ◦C; photoperiod waskept at 14:10 (light:dark cycle). Fish were daily fed with a standarddiet (Westerfield, 2000) three times a day.

Experiments complied with European Directive (2010/63/EU)for the correct use of laboratorial animals. Seventy-two sexuallymature male and female zebrafish (average weight 0.9 ± 0.35 gand length 4.3 ± 0.45 cm) were randomly distributed through 5 Laquaria and allowed to acclimatize for 24 h prior to the addition ofthe Cu solutions (CuSO4; Merck, Darmstadt, Germany). Zebrafishwere then exposed to 12.5 �g/L and 100 �g/L (nominal) Cu con-centrations for 48 h, in triplicate. A third group was maintainedwithout Cu added to water and used as control. The Cu concentra-tion of 100 �g/L was chosen based on the 96 h LC50 (94 �g/L) forthis species (De Oliveira-Filho et al., 2004). The lower concentra-tion is one eighth (12.5%) of the 96 h LC50 and was selected basedon the range of Cu (0.2–30 �g/L) observed in unpolluted freshwa-ters (Craig et al., 2007; Us-Epa, 2007). At 6, 12, 24 and 48 h, sixzebrafish per treatment were randomly sampled, stunned in icedwater, measured, weighted and immediately decapitated. Theirgills were collected to Buffered Formaldehyde (Panreac, Barcelona,Spain), for histological examination, and to “RNAlater” (Sigma, St.Louis, USA), for gene expression studies.

2.2. Detection of DNA fragmentation by Terminaldeoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling(TUNEL assay)

TUNEL staining was performed according to the manufacturer’sprotocol (“In situ cell death detection kit”, Ap-Roche, Mannheim,Germany). Briefly, paraffin-embedded gill tissue from five fish pergroup was sectioned and mounted in glass slides. After deparaf-finization and rehydration, slides were rinsed in distilled waterand pre-treated, for 5 min, with 0.1 M Citrate buffer (pH 6) under350 W microwave irradiation. Afterwards, sections were rinsed(3 × 2 min) with Phosphate Buffer Saline (PBS-10 mM, pH 7.2) andincubated at 37 ◦C for 60 min (in humidified chamber) with TUNELreaction mixture (50 �g/L of enzyme solution and 450 �g/L of labelsolution). Negative controls were incubated with label solution.After PBS washes (3 × 5 min), sections were incubated with alka-line phosphatase-converter during 30 min at 37 ◦C, rinsed with PBS(3 × 2 min) and then incubated with the substrate solution (Fast-Red, Sigma) for 10 min in the dark. Finally, sections were rinsedin PBS (3 × 5 min), counterstained with Gill’s hematoxylin for 30 sand mounted under glass coverslip with PBS/glycerol (1:9) andobserved in an Olympus IX 51 microscope (Japan).

2.3. Stereological analysis

To determine the volume density (VV) of the apoptotic cells(marked with TUNEL) in gill filaments, a stereological approachaccording to Monteiro et al. (2009) was used. The volume den-sity (VV) is defined as the percentage of the total volume of a welldefined reference space occupied by any given component within it.According to the definition, the VV estimate of the different identi-fied cellular elements within the gill filament (the reference space)was made by a classical stereological technique based on pointcounting (Freere and Weibel, 1967) using the following formula:

VV(structure, reference) = P(s) × 100P(r)

where P(s) is the number of test points within each structuralcomponent (apoptotic cells) and P(r) is the total number of testpoints lying over the reference space. Counting was done using amicroscope (Olympus IX 51, Japan) equipped with a CCD camera(Olympus U-CMDA3, Color-View soft imaging system) connectedto a 17 in. PC monitor (Dell). The first field of vision was ran-domly selected. Following fields were systematically sampledby stepwise movements of the stage in the x- and y-directions(220 �m × 170 �m). A counting frame with two sub-sets of points(ratio 1:12) was super-imposed on the live image of the monitor.To estimate the VV of the reference space (gill filament) the firstsubset, made of eight equidistant points was used, whereas for theVV of cells, the main subset of 96 points was utilized. Counting wasmade at a final magnification of 400×, with analysis of an averageof 30 fields per fish.

2.4. Apoptosis gene transcription in gills of zebrafish exposed tocopper

To evaluate mRNA expression of caspase-8, caspase-9, AIF andp53 genes, total RNA was isolated from gills of control and Cuexposed fish, using the “Illustra RNAspin Mini kit” (GE Healthcare,Munich, Germany) according to the manufacturer’s protocol. TotalRNA was eluted in 40 �L of RNase-free water. The RNA concentra-tion was estimated using the absorbance values at 260 nm, whilethe purity of each sample was determined calculating the 260/280ratio. RNA integrity was assessed by inspection of the 28 S and 18S ribosomal RNA bands in a 1% agarose gel. Total RNA, 500 ng, was

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Table 1Gene, GenBank ID accession numbers and sequence of forward (Fw) and reverse(Rv) primers used in this study.

Gene/GenBank ID Primers (5′–3′)

p53 (NM131327)Chakraborty et al. (2009)

Fw ACC ACT GGG ACC AAA CGT AG

Rv CAG AGT CGC TTC TTC CTT CG

Caspase-9 (NM001007404) Fw CTG AGG CAA GCC ATA ATC GRv AGA GGA CAT GGG AAT AGC GT

Caspase-8(AY518734/AY518735)Sakata et al. (2007)

Fw CCA GAC AAT CTG GAT GAA CTT TAC

Rv TGC AAA CTG CTT TAT CTC ATC T

AIF (AY423007)Miller et al. (2012)

Fw AAA GTC CGG AAA GAG GGT GTRv GCC TGG AGC TCA GCA TTA AC

�-Actin (NM181601)Soares et al. (2012)

Fw ACT GTA TTG TCT GGT GGT AC

Rv TAC TCC TGC TTG CTA ATC C

reverse transcribed to produce the first strain of cDNA (iScript cDNASynthesis Kit, BioRad, California, U.S.A.), according to the manufac-turer’s instructions.

The primers used for amplification and mRNA expression anal-ysis are presented in Table 1. The design of caspase-9 primers was

based on the alignment of caspase-9 gene sequences of differentspecies with Multalin (http://multalin.toulouse.inra.fr/multalin/),using the most conserved region as target. The amplified prod-uct band was isolated, purified (Wizard® SV Gel and PCR Clean-upSystem, Promega, Madison, WI, USA), and send for sequencing(STABVIDA, Portugal), presenting a 99% identity with the zebrafishcaspase-9 sequence (GenBank accession no. NM 001007404.2).

The real time-polymerase chain reaction (RT-PCR) was preparedto a final volume of 15 �L:1 �L of cDNA, 200 nM of primers and “iTaqFast SYBR® Green Supermix with ROX” (Bio-Rad). The followingprogram was run: 95 ◦C for 1 min (1 cycle) and 95 ◦C for 3 s, and 30 sat the annealing temperatures (60 ◦C for �-actin, p53 and caspase-8genes; 62 ◦C for caspase-9 and AIF genes), 72 ◦C for 20 s (40 cycles),followed by a melting curve analysis to determine the specificityof the reaction. Melting curves were analyzed after amplificationreactions and single amplification products were present in all reac-tions. Real-time PCR reactions were carried out in triplicate andonly comparative threshold cycle (Ct) values leading to a Ct meanwith a standard deviation below 0.2 were considered. A 5-fold dilu-tion series of cDNA, prepared from a mix of the samples, was usedas reference for construction of standard curves, reaction efficien-cies and r2 determination (Table 2). The ��CT method was usedto calculate relative mRNA expression levels of genes with �-actingene as a housekeeping gene for normalization and values werenormalized to the control average value. The values obtained inthe exposed groups were then statistically compared.

Fig. 1. Sagittal sections of zebrafish (D. rerio) gills showing TUNEL-positive cells (arrows). Control fish sampled at 6 h (A) and 48 h (B); gills of fish exposed to 12.5 �g/L during6 h (C) and 48 h (D); gills of fish exposed to 100 �g/L during 6 h (E) and 48 h (F). Scale bar corresponds to 20 �m.

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Table 2Primer annealing temperature (◦C), amplification efficiency (%) and r2 of standardcurves.

Gene Ann temp (◦C) Efficiency (%) r2

p53 60 100.3 0.996Caspase-9 62 82.0 0.999Caspase-8 60 97.8 0.996AIF 62 97.2 0.997�-Actin 60 92.2 0.998

Fig. 2. Zebrafish (D. rerio) gills TUNEL-positive cells relative volume in control(white column), exposed to 12.5 �g/L of copper (light gray columns) and exposed to100 �g/L of copper (dark gray columns) at 6, 12, 24 and 48 h of exposure. Differentupper case letters represent significant differences between Cu concentrations forthe same time of exposure (p < 0.05). Different lower case letters represent signifi-cant differences between time of exposure for the same Cu concentration (p < 0.05).

2.5. Statistical analysis

Data were analyzed using Sigma-Stat (SPSS Inc.) software. Aftertesting for ANOVA assumptions (homogeneity of variances andnormality of data), statistical differences in the relative volumeof TUNEL-positive cells and in mRNA expression values wereevaluated by a two-way factorial ANOVA, to test isolated andinteractive effects of copper concentration and exposure time.

Statistical differences between the groups were determined by thepost hoc Tukey test, for p < 0.05.

3. Results

3.1. Detection of DNA fragmentation by TUNEL

TUNEL-positive cells were identified by their red stained nucleiand were found in the gill filament and lamellar epithelia (Fig. 1).Fish exposed to Cu evidenced a higher incidence of TUNEL-positivecells, clearly demonstrating the apoptosis induction.

In fish, exposed to 12.5 �g/L, the relative volume of apop-totic cells (Fig. 2) progressively increased until 12 h post-exposure,slightly decreasing thereafter. At 12 h (p = 0.016), 24 h (p = 0.020)and 48 h (p = 0.048) of exposure, the relative volume of TUNEL-positive cells differed significantly from the ones observed forcontrol.

In fish exposed to 100 �g/L, the relative volume of TUNEL-positive cells was slightly increased at 6 h (p = 0.081), peaked at12 h of exposure (p = 0.003), followed by a decline (24 h, p = 0.091;48 h, p = 0.167), when compared to control.

3.2. mRNA expression of genes

The mRNA expressions of caspase-8, capase-9, AIF and p53genes from zebrafish gills were differently affected by Cu exposure(Fig. 3). The transcriptional upregulation of these genes was time-dependent occurring at different points of the exposure period.As differences in control groups among different times were notobserved, for graphical presentation the values were combined.

Exposure to Cu induced an early increase in mRNA expressionof p53 gene when compared to control group. This expression, inzebrafish exposed to 12.5 �g/L, was significantly increased at 6 h(p < 0.05) and progressively decreased. Although no significant dif-ferences were detected, the same expression pattern was observedin zebrafish exposed to 100 �g/L (p > 0.05).

The mRNA expression of caspase-9 gene increased until the 12 hof exposure (p < 0.05), progressively decreasing thereafter, reach-ing the control values at 48 h. The same expression pattern wasobserved in the 100 �g/L Cu exposed zebrafish.

Fig. 3. Relative mRNA expression of p53 (A), caspase-9 (B), caspase-8 (C) and AIF(D) genes in zebrafish (D. rerio) gills in control fish(white column), in fish exposed to 12.5 �g/Lof copper (light gray columns) and in fish exposed to 100 �g/L of copper (dark gray columns) at 6, 12, 24 and 48 h of exposure. Different upper case letters represent significantdifferences between Cu concentrations for the same time of exposure (p < 0.05). Different low case letters represent significant differences between time of exposure for thesame Cu concentration (p < 0.05).

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Fig. 4. Schematic representation of the proposed apoptotic pathways induced by copper exposure. The sequential gene upregulation is indicated by the numbers insidethe boxes (1-6 h; 2-12 h; 3-24 h and 4-48 h). The nuclear p53 activation, initially triggers the intrinsic (mitochondrial) pathway (red continuous line), with the consequentupregulation of caspase-9. Subsequently, p53 leads to extrinsic pathway activation (blue dashed line). Caspase-8 seems to exert a crosstalk with the mitochondrial pathway,leading to AIF release and the induction of the caspase cascade-independent path. The extrinsic pathway may also progress through caspase cascade (dotted blue line). AIF– apoptosis inducing factor; Apaf-1 – apoptotic protease activating factor 1; Bak – Bcl-2-antagonist killer; Bax – Bcl-2-associated x protein; Bcl 2 – B-cell lymphoma 2; BID –Bcl 2 homology 3-interacting death agonist; Cyt C – cytochrome C; DR – death receptor; FADD – Fas associated death domain; FLIP – FLICE-inhibitory protein; IAP – inhibitorof apoptosis proteins; t-BID – truncated BID.

The pattern of mRNA expression of caspase-8 gene was depend-ent of copper concentration. A progressive increase on expressionwas observed in fish exposed to 12.5 �g/L up to 24 h (p < 0.05), fol-lowed by a non significant decrease at 48 h (p > 0.05). In 100 �g/Lexposed fish, the expression increased up to 48 h, being signifi-cantly higher than the expression of control zebrafish at 24 and48 h (p < 0.05).

The mRNA expression of AIF gene was similar in the two Cuexposed groups. In the first 24 h of exposure, the expression valuesremained similar to control levels, being significantly increased at48 h.

4. Discussion

The present study shows that environmentally relevant Cuconcentrations can induce apoptosis in zebrafish gill. Indeed, theresults obtained with the TUNEL assay corroborate previous studiesin different fish species, where apoptotic gill cells were observedafter in vivo and in vitro exposures to this metal (Li et al., 1998;Mazon et al., 2002, 2004).

The mRNA expression analyses showed that p53 gene wasthe first gene to be transcriptionally upregulated, at 6 h, followedby caspase-9 gene at 12 h, caspase-8 gene at 24 h and AIF geneat 48 h. These results suggest that different apoptosis pathwaysmay be activated by Cu exposure, also suggesting the existenceof crosstalk interactions between them. A schematic representa-tion of hypothetical Cu apoptosis pathways activation, based onthe present results and the current knowledge, is presented inFig. 4.

The Cu toxicity is attributed, at least in part, to its ability toinduce ROS formation (Bopp et al., 2008; Sandrini et al., 2009; Guoet al., 2010) which, in turn, has previously been associated with theactivation of p53 (Li et al., 1998; Ostrakhovitch and Cherian, 2005;Tassabehji et al., 2005). In fact, the early increase in mRNA expres-sion of p53 gene, observed in the present study, is suggestive ofits involvement in apoptosis activation, since TUNEL-positive cellsbecame more evident soon after 6 h of Cu exposure (Fig. 4-Box 1).This hypothesis is corroborated by studies with human Hep G2 cellsexposed to Cu, which showed evidence of apoptosis induction byp53 activation (Narayanan et al., 2001; Tassabehji et al., 2005).

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The transcriptional upregulation of caspase-9 gene, observed at12 and 24 h of Cu exposure, suggests the subsequent activation ofthe intrinsic pathway (Fig. 4-Box 2). Indeed, several p53-dependentgenes have been implicated in promoting apoptosis by the intrin-sic pathway (Haupt et al., 2003; Ostrakhovitch and Cherian, 2005).For example, p53 can translocate to mitochondria and induce therelease and activation of Bax (Benchimol, 2001; Ostrakhovitch andCherian, 2005) which, in turn, induces a selective process of mem-brane permeabilization, and the release of cytochrome c and AIFfrom the intermembrane space to the cytoplasm (Riedl and Shi,2004; Ostrakhovitch and Cherian, 2005; Zhang et al., 2003; Fanet al., 2005). When cytochrome c is released from the mitochondria,it can activate caspase-9 (Kuida, 2000) and consequently increasethe expression of caspase-9, as seen in the present study. Suppor-ting this hypothesis, several studies have shown an increase in Baxexpression after Cu exposure (Zhai et al., 2000; Kawakami et al.,2008; Sandrini et al., 2009).

As observed by Sarkar et al. (2011), copper exposure increasedmRNA expression of caspase-8 gene in the present study alsosuggesting the involvement of extrinsic pathway in Cu inducedapoptosis (Fig. 4-Box 3). Like transcription of caspase-9 gene, theincrease in caspase-8 gene transcription may result from upregu-lated expression of p53 target genes, such as DR5 and Bid genes(Fei et al., 2002; Fridman and Lowe, 2003; Haupt et al., 2003).The upregulation of caspase-8 can subsequently trigger the down-stream caspase cascade, by direct activation of caspase-3 (Greenand Reed, 1998; Fan et al., 2005). On the other hand, the acti-vated caspase-8 may cleave the pro-apoptotic protein Bid, whichis transported from the cytoplasm to the mitochondria (Wanget al., 1996; Zhang et al., 2003), triggering the cytosolic release ofmitochondrial proteins (Kruidering and Evan, 2000; Riedl and Shi,2004). Subsequently, these proteins can either lead to activation ofcaspase-dependent or-independent pathways (Gross et al., 1999;Zamzami and Kroemer, 2001).

The increase in mRNA expression of AIF gene, observed at 48 hof Cu exposure suggests the activation of the caspase-independentpathway (Fig. 4-Box 4). When activated, AIF is translocated tothe nucleus where it promotes chromatin condensation and DNAfragmentation, resulting in apoptosis (Daugas et al., 2000; Nagataet al., 2003). Moreover, several lines of evidence suggest that AIFis released from mitochondria during the p53-induced cell death,as a consequence of mitochondrial depolarization during exposureto metals (Ostrakhovitch and Cherian, 2005). Furthermore, as therise in AIF expression followed caspase-8 expression increase, theseresults suggest that Cu may induce crosstalk interactions betweenthe two major apoptotic pathways.

In zebrafish exposed to 100 �g/L, apoptotic cells were observedin high relative volumes shortly after the beginning of the expo-sure (6 and 12 h), with a lower transcriptional response at thetime points evaluated in this study. In fish exposed to the lowerCu concentration (12.5 �g/L), the increase in the relative volume ofapoptotic cells was only observed after 12 h and remained constantuntil the end of the exposure. These results suggest that the highand low Cu concentrations induce apoptotic responses at differenttime points, which was also seen in the pattern of transcription.Combining the results for the relative volume of apoptotic cellsand for the gene transcription along the analyzed time points, theseindicate that probably the transcriptional upregulation of apoptosisgenes by 100 �g/L of copper occurred prior to the first time pointanalyzed (6 h).

5. Conclusion

In conclusion, in zebrafish gills, the exposure to Cu induced thetranscriptional upregulation of apoptosis related genes, indicating

the activation of the different pathways. In addition, each geneupregulation occurred at different time point of the exposureperiod and in a sequential way, indicating a timeline of apoptosisevents. In this way, gill apoptosis induced by copper exposureinitiated via the intrinsic pathway through p53 activation, wasthen followed by the extrinsic pathway and finally by the caspase-independent pathway. Furthermore, this study suggests that thegill apoptotic response to Cu exposure is concentration dependent,with lower concentrations inducing later effects.

Acknowledgments

The authors thank to Ana Fraga and Donzilia Costa, for the his-tological support and training. This work was supported by thePortuguese Foundation for Science and Technology through theproject grant: PTCD/CTV/102453/2008 (FCOMP-01-0124 FEDER-009527) and Luzio A. PhD grant SFRH/BD/44794/2008.

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