Are metallothioneins equally good biomarkers of metal and oxidative stress?

Ecotoxicology and Environmental Safety 84 (2012) 185–190

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Are metallothioneins equally good biomarkers of metal and oxidative stress? Etelvina Figueira a,n, Diana Branco b, Sara C. Antunes c,d, Fernando Gonc- alves b,c, Rosa Freitas b,c a

Universidade de Aveiro, Departamento de Biologia & CBC (Centre for Cell Biology), Campus Universitario de Santiago, 3810-193 Aveiro, Portugal Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro, Portugal CESAM-Centro de Estudos do Ambiente e do Mar, Universidade de Aveiro, 3810-193 Aveiro, Portugal d Departamento de Biologia da Faculdade de Ciˆencias da Universidade do Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal b c

a r t i c l e i n f o

abstract

Article history: Received 1 March 2012 Received in revised form 4 July 2012 Accepted 9 July 2012 Available online 31 July 2012

Several researchers investigated the induction of metallothioneins (MTs) in the presence of metals, namely Cadmium (Cd). Fewer studies observed the induction of MTs due to oxidizing agents, and literature comparing the sensitivity of MTs to different stressors is even more scarce or even nonexistent. The role of MTs in metal and oxidative stress and thus their use as a stress biomarker, remains to be clearly elucidated. To better understand the role of MTs as a biomarker in Cerastoderma edule, a bivalve widely used as bioindicator, a laboratory assay was conducted aiming to assess the sensitivity of MTs to metal and oxidative stressors. For this purpose, Cd was used to induce metal stress, whereas hydrogen peroxide (H2O2), being an oxidizing compound, was used to impose oxidative stress. Results showed that induction of MTs occurred at very different levels in metal and oxidative stress. In the presence of the oxidizing agent (H2O2), MTs only increased significantly when the degree of oxidative stress was very high, and mortality rates were higher than 50 percent. On the contrary, C. edule survived to all Cd concentrations used and significant MTs increases, compared to the control, were observed in all Cd exposures. The present work also revealed that the number of ions and the metal bound to MTs varied with the exposure conditions. In the absence of disturbance, MTs bound most (60–70 percent) of the essential metals (Zn and Cu) in solution. In stressful situations, such as the exposure to Cd and H2O2, MTs did not bind to Cu and bound less to Zn. When organisms were exposed to Cd, the total number of ions bound per MT molecule did not change, compared to control. However the sort of ions bound per MT molecule differed; part of the Zn and all Cu ions where displaced by Cd ions. For organisms exposed to H2O2, each MT molecule bound less than half of the ions compared to control and Cd conditions, which indicates a partial oxidation of thiol groups in the cysteine residues through ROS scavenging. The present results suggest that MTs are excellent markers of metal stress, but not of oxidative stress. & 2012 Elsevier Inc. All rights reserved.

Keywords: Bioindicator Cerastoderma edule Metal contamination Metallothioneins Oxidative stress

1. Introduction Among the most used biomarkers for pollution in the marine environment, metallothioneins (MTs) have been particularly useful as a monitoring device, namely as a contaminant-specific biochemical indicator of metal exposure (de Lafontaine et al., 2000; Hamza-Chaffai et al., 2000; Mourgaud et al., 2002; Pavicˇicˇ et al., 1987; Roesijadi, 1992, 1994; Stegeman et al., 1992). Metallothioneins have been implicated in the homeostasis of essential metals such as Cu and Zn, and in the detoxification of excess levels of essential and non-essential metals in marine invertebrates (Roesijadi, 1992, 2000; Viarengo and Nott, 1993). Several researchers demonstrated that MTs protect organisms from metal toxicity due to the ability of MTs to bind metals (e.g.

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Cd, Cu, Zn, and Hg), and the ability to increase the MTs expression with the raise of metal concentrations in tissues (Amiard et al., 2006; Machreki-Ajmi and Hamza-Chaffai, 2008; Martı´n-Dı´az et al., 2008; Monserrat et al., 2007; Paul-Pont et al., 2010a; 2010b; Perceval et al., 2006). Furthermore, different studies suggested that MTs can be a useful surrogate for the toxicity assessment of metals in aquatic organisms before they experience sublethal and lethal damage (Pavicˇicˇ et al., 1987; Paek et al., 1999). Besides metals, MTs are also induced by compounds able to cause oxidative stress, suggesting that MTs may protect cells from oxidative stress (Amiard et al., 2006; Roesijadi, 1996; Viarengo et al., 2000). Bauman et al. (1991, 1992) and Cai et al. (1999) showed that the cellular levels of MTs can be increased by different oxidant chemicals (e.g. paraquat, diquat, menadione, metronidazole, adriamycin, cisplatin, diamide, and diethyl maleate). The role of MTs as an oxidative stress protector can be achieved in several ways, distributing Zn, as this metal plays a

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role in protecting against lipid peroxidation, maintaining the adequate level of MTs, being an essential component of superoxide dismutase (SOD), preventing the free radical formation caused by the interaction between chemical groups with iron, stabilizing DNA and reacting directly with reactive oxygen species (ROS), as demonstrated in some in vitro studies (Cai et al., 2000; Chvapil et al., 1972; Kumari et al., 1998; Maret, 2008; Thomas et al., 1986; Thornalley and Vasa´k 1985). Although several studies reported the ability of MTs to sequester ROS, most of them used vertebrates as test species (Atif et al., 2006; Bauman et al., 1991; Cai et al., 1999) and few researchers focused on the antioxidant activity of MTs in aquatic invertebrates. Viarengo et al. (1999) suggested an antioxidant role for MTs in the mussel Mytillus galloprovincialis, which seemed to occur through oxyradical scavenging. The work of Leung and Furness (2001) in dogwhelks (Nucella lapillus) showed that MTs were induced by metals and also by oxidative stress. These results challenge the use of MTs as specific indicators of metal stress. However, to use a certain parameter as a stress biomarker, this parameter must be sensitive to a specific stress factor and present a proportional response to the level of stress (contamination) induced in organisms. Although numerous studies exist regarding the induction of MTs by metals, namely Cd (e.g. Bebiano and Langston, 1995; Bebiano and Serafim, 2003; Ge´ret et al., 2002; Hamza-Chaffai et al., 2000), and less regarding the induction of MTs by oxidizing agents (e.g. Cai et al., 1999), studies comparing the sensitivity of MTs to different stressors are very scarce. Furthermore, the number of metal ions bound to MTs, when organisms are submitted to oxidative and metal stress is not assessed yet, and the role of MTs in both stressors, together with their use as stress biomarkers, remains to be elucidated. To better understand the role of MTs as a biomarker in aquatic invertebrates, a laboratory assay was conducted aiming to assess the sensitivity of MTs to metal and oxidative stressors. For this purpose, under experimental conditions, Cd was used to induce metal stress, whereas hydrogen peroxide (H2O2), being an oxidizing compound, was used to impose oxidative stress. In the present study Cerastoderma edule (Bivalvia: Cardiidae), was selected since it is widely distributed, inhabiting the majority of estuarine areas from the Barents Sea to Morocco (Gam et al., 2010), and is one of the most economically relevant bivalves in Ria de Aveiro (Portugal). This species is highly tolerant to environmental variations of physical and chemical parameters such as sediment grain size and salinity, and may thus be used as an indicator organism along an estuarine gradient. Furthermore, the levels of MTs in this bivalve species revealed to be a good biomarker of metal stress (Freitas et al., 2012a).

2. Material and methods 2.1. Sampling and experimental conditions Due to the significant role in ecosystem functioning and high socioeconomic value, in the present study we used the species C. edule, collected in an intertidal zone of the Ria de Aveiro, a shallow lagoon in the Northwest Atlantic coast of Portugal. C. edule ranks the first position in Portugal (4062t) (FAO, 2009) and in Ria de Aveiro, cockles comprise 98 percent of shellfish harvesting, being the most important activity related to the fisheries exploitation, supporting the economic activity of more than 250 licensed professionals. Specimens of similar size (26.6 71.0 mm) and weight (7.8 71.0 g) were selected to minimize differences in the results. Organisms were acclimatized to laboratory conditions during one week (temperature 20 72 1C, photoperiod 12 hL: 12 hD) in seawater (salinity of 28 7 1) under continuous aeration. Cultures were fed daily with algae produced in the laboratory. Two independent ecotoxicological assays were conducted with a concentration range of metal (Cd, in the form of CdCl2: C1–0.01, C2–0.1, C3–5.0, and C4–10.0 mM) and oxidizing agent (H2O2, 30 wt. percent in water: C1–0.02, C2–0.2, C3–10.0, and C4–20.0 mM). Assays consisted in seven organisms  three replicates  four

concentrations for each contaminant (Cd or H2O2), and seven organisms  three replicates for control conditions (C0–0 mM Cd and H2O2). Each replicate contained 600 g of autoclaved sediment and 1000 ml of clean seawater (salinity 2871). Daily, aeration was monitored and animals were checked for mortality. Water was renewed every day to maintain the Cd and H2O2 levels during the experiment. After a 96 h exposure period, cockles were collected and immediately frozen at  801 C for further analysis. 2.2. Lipid peroxidation For lipid peroxidation (LPO) measurements, frozen organisms (soft tissues, three organisms per replicate) were thawed, weighted, and homogenized in ice-cold phosphate buffer (50 mM, pH¼ 7.0 with 0.1 percent TRITON X-100). Homogenates were centrifuged at 10,000 g for 10 min and supernatants were divided into two aliquots: thiobarbituric acid reactive substances (TBARS) and protein analysis. The extent of LPO was measured by the quantification of TBARS, according to the protocol described by Buege and Aust (1978). This methodology is based on the reaction of LPO by-products, such as malondialdehyde (MDA), with 2-thiobarbituric acid (TBA), forming TBARS. The amount of TBARS was measured spectrophotometrically, at a wavelength of 535 nm (e¼ 156 mM  1 cm  1), and results were expressed as nanomoles of MDA equivalents per milligram of protein. Protein concentration was determined according to the spectrophotometric (wavelength 595 nm) method of Bradford (1976) adapted to microplate, using BSA as standard. 2.3. Methallotioneins and metal quantification (Cd, Zn, and Cu) Frozen organisms (soft tissues, three organisms per replicate) were thawed, weighted, and homogenized with liquid nitrogen using a mortar and a pestle. The buffer used was 100 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (pH 8.6), 1 mM phenylmethylsulfonyl fluoride and 0.2 percent Tween 20 (v/v). The extract was centrifuged at 10,000g during 10 min at 4 1C and the supernatant collected, corresponding to the soluble fraction. The cell debris was washed two more times with the same buffer, followed by centrifugations at 10,000 g during 10 min at 4 1C and the supernatants were pooled out. In C1, C2 and C3 concentrations of both treatments two sub-samples were taken, for the protein determination and for Cu, Zn and Cd quantification. From C0 and C4 concentrations three sub-samples were taken: for the protein determination, for Cu, Zn and Cd quantification and for the size exclusion chromatography. Protein concentration was determined as described above. For the total metal concentration, present in the soluble fraction, samples were made up to 5 ml with 1 M HNO3. The concentration of Cd, Zn and Cu was determined by ICP–MS. Regarding the quality controls, the calibration of the apparatus was made with IV (Inorganic Venture, Christiansburg, Virginia, USA) standards (IV–ICP–MS 71A) and they were verified with reference material (NIST 1643e). During metal analysis, the accuracy observed, ranged between 90 percent and 110 percent. All samples below this accuracy level were rejected and the analysis repeated. Determinations were performed using three replicates. For size exclusion chromatography only C0 and C4 concentrations were tested: control (0 mM Cd and H2O2), 10 mM Cd and 20 mM H2O2. According to preliminary assays, the concentrations of H2O2 and Cd selected were those inducing similar amounts of MTs. The size exclusion chromatrography was performed in a Sephacryl S-100 (25  0.5 cm2 i.d., Amershan Biosciences) column. The column was equilibrated with degassed elution buffer 10 mM HEPES and 300 mM KCl. Samples (500 ml) were eluted at a flow rate of 1 ml min  1 at room temperature. The absorbance was monitored at 254 nm and the fractions collected every 3 min intervals during 1 h and 20 min. All fractions were sub-sampled for subsequent Cd, Cu and Zn quantification (by ICP–MS, as described previously) and for analysis of MTs by HPLC. For each treatment and control, at least three replicates were performed. Metallothioneins (MTs) were extracted according to Alhama et al. (2006), with some adaptations (Freitas et al., 2012a). Each chromatographic fraction and total soluble fraction (300 ml) was denatured for 10 min at 75 1C and centrifuged at 12,000g for 10 min at 4 1C to precipitate denatured proteins. The resulting supernatant (200 ml) was neutralized with 0.1 M NaOH, after the addition of 200 ml of 0.1 M Tris–HCl buffer (pH 8.0), 25 ml of 2 mM DTE (dithioerythritol) and 50 ml of 0.1 M EDTA (ethylenediamine tetraacetic acid). The mixture was incubated during 30 min at room temperature and 75 ml of 10 mM mBBr (monobromobimane–Calbiochem) was added. Derivatization was performed in the dark for 40 min at 35 1C. The reaction was stopped by the addition of acetic acid 5 percent (v/v) up to a total volume of 1.5 ml. MTs were quantified by Reverse Phase–High Pressure Liquid Chromatography with fluorescence detection. Derivatized proteins (20 ml aliquots) were separated in a Supelco C-18 column (0.46 cm  25 cm, 5 mm). The solvents used were as follows: A—0.1 percent TFA (Trifluoroacetic Acid) in H2O, and B—0.1 percent TFA in acetonitrile. Separation was achieved through a linear gradient up to 70 percent of solvent B, during 20 min with a 1 ml min  1 flow. Fluorescence of mBBr-labeled molecules was monitored with excitation at 382 nm and emission at 470 nm. MT identification and quantification was determined with purified rabbit liver MT1 and MT2 standards. MTs were expressed as microgram MTs per milligram protein.

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For the calculation of the number of ions bound to a MT molecule, metals and MTs concentrations were calculated as nmol per mg protein, and the resulting values for each ion were divided by the MTs concentration. For the expression of MTs concentration in nmol it was used the concentration of MTs determined by HPLC as microgram per milligram protein and taking into account the molecular weight of cockles MT (6.941 KDa, Paul-Pont et al., 2009).

2.4. Data analysis Data mortality, LPO levels, MT concentrations and number of ions/MT molecule were submitted to hypothesis testing using permutation multivariate analysis of variance with the PERMANOVA þadd-on in PRIMER v6 (Anderson et al., 2008), following the calculation of Euclidean distance matrices among samples. A one-way hierarchical design was followed, with the exposure concentrations as the main fixed factor. When the main test revealed statistical significant differences (Po 0.05), pairwise comparisons were performed. The t-statistic in the pairwise comparisons was evaluated in terms of significance among different treatments and concentrations. Significant differences were tested: (a) when considering mortality data: for each exposure concentration, between treatments, for each treatment, among concentrations; (b) when analyzing LPO levels: for each exposure concentration, between treatments for each treatment, among concentrations; (c) when considering MT concentrations, for each exposure concentration, between treatments, for each treatment, among concentrations; (d) when analyzing the number of ions bound to a MT molecule, for total number of ions between treatments, for Zn between treatments; (e) for each ion (Cd, Zn and Cu), when analyzing the total concentration and the concentration bound to MTs between treatments.

Fig. 2. Lipid peroxidation (nmol MDA mg  1 protein), in Cerastoderma edule when exposed to increasing concentration of Cd (mM) (C0 ¼ 0, C1¼0.01, C2¼0.1, C3¼5.0, C4¼10.0) and H2O2 (mM) (C0 ¼ 0, C1¼0.02, C2¼ 0.2, C3¼ 10.0, C4¼20.0). Asterisks represent significant differences between treatments, at each concentration level. Significance values: Po 0.05; Po 0.01. Lowercase letters represent, for each treatment, significant differences among concentrations.

3. Results 3.1. Organisms mortality Fig. 1 presents the mortality of C. edule, in percentage, when organisms were subjected to increasing concentrations of H2O2 and Cd. All organisms exposed to different Cd concentrations survived, but under H2O2 contamination only at the lowest concentrations (0.02 and 0.2 mM) 100 percent of the organisms survived, showing high mortality (62 percent and 81 percent) at the two highest concentrations (10 and 20 mM, respectively), with significant differences between concentrations (cf. Fig. 1). 3.2. Lipid peroxidation Fig. 2 presents the level of lipid peroxidation (LPO) as an indication of oxidative damage, when C. edule was subjected to

Fig. 3. Metallothioneins (mg MTs mg  1 protein), in Cerastoderma edule when exposed to increasing concentration of Cd (mM) (C0 ¼ 0, C1¼0.01, C2¼0.1, C3¼5.0, C4¼10.0) and H2O2 (mM) (C0 ¼ 0, C1¼0.02, C2¼ 0.2, C3¼ 10.0, C4¼20.0). Asterisks represent significant differences between treatments, at each concentration level. Significance values: Po 0.05; Po 0.01. Lowercase letters represent, for each treatment, significant differences among concentrations.

increasing concentrations of H2O2 and Cd. At the two lowest H2O2 concentrations (0.02 and 0.2 mM) LPO slightly increased compared to the control. However, at the highest H2O2 concentrations (10 and 20 mM) a strong LPO increase was noticed (cf. Fig. 2), evidencing the significant oxidative stress differences that organisms experienced between the two lowest (0.02 and 0.2 mM) and the two highest (10 and 20 mM) H2O2 concentrations. When C. edule was subjected to Cd, LPO levels remained low (0.30– 0.35 nmol mg  1 protein), only revealing significant differences (P o0.05) between the control and the highest Cd concentration (10 mM). When comparing LPO levels from H2O2 and Cd contamination, the more noticeable differences were identified at the highest concentrations (Po0.01, 5 and 10 mM of Cd and 10 and 20 mM of H2O2) being much higher for H2O2 than for Cd treatments (cf. Fig. 2). 3.3. Metallothioneins Fig. 1. Mortality (%) of Cerastoderma edule, when exposed to increasing concentration of Cd (mM) (C0 ¼0, C1¼ 0.01, C2¼ 0.1, C3¼ 5.0, C4¼10.0) and H2O2 (mM) (C0 ¼0, C1¼0.02, C2¼ 0.2, C3¼10.0, C4¼ 20.0). C0 represents control conditions. Asterisks represent significant differences between treatments, at each concentration level. Significance values: Po 0.05; P o0.01. Lowercase letters represent, for each treatment, significant differences among concentrations.

Fig. 3 presents the levels of MTs in C. edule organisms exposed to H2O2 and Cd contamination. In both treatments the synthesis of MTs increased with the exposure concentration. However, in organisms exposed to Cd, MTs increased significantly in all exposure concentrations, while in organisms exposed to H2O2

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the MTs increase was significantly higher than the control at C3 and C4 (10 and 20 mM) but not significant up to 0.2 mM (cf. Fig. 3). 3.4. Cd, Zn and Cu concentrations Table 1 shows the concentrations of Cd, Zn and Cu found in solution (total concentration and bound to MTs) when organisms were under control conditions, H2O2 and Cd contamination. In control conditions no Cd was found (neither bound nor in the free form, cf. Table 1). When the organisms were exposed to Cd, nearly all of the soluble Cd was bound to MTs, while under H2O2 contamination the small amount of Cd found in solution was free and not bound to MTs (cf. Table 1). As an essential metal, Zn was found in large amounts in the three conditions tested, although under Cd treatment the concentration was significantly lower than in the other two conditions. The majority of Zn ions were, in the three conditions, bound to MTs, ca 70 percent in the control and Cd treatments, and ca 80 percent in H2O2 (cf. Table 1). Although not significant, the levels of Cu decreased in H2O2 and Cd treatments compared to the control. In this condition (C0) most of Cu in solution was bound to MTs (60 percent), but when organisms were subjected to H2O2 and Cd contamination, the Cu ions were not bound to MTs, thus remaining free in the cytosol (cf. Table 1). 3.5. Number of metal ions/MT molecule Fig. 4 presents the number of ions (Cd, Zn and Cu) bound to an MT molecule when organisms were subjected to different conditions: control 0 mM, 20 mM H2O2 and 10 mM Cd. Under control conditions, only Cu and Zn ions were bound to MTs, being Zn the metal with higher amount of ions bound per MT molecule (2.93). When organisms were exposed to H2O2, Zn was the only ion bound to MTs, although in lower number than under control conditions (1.84). Under Cd contamination most of the ions bound to MTs were Cd (3.27), with less Zn (1.48) and no Cu ions (cf. Fig. 4).

MT include detoxification of both essential and non-essential metals and oxyradical scavenging. Viarengo et al. (2000) showed that MT synthesis is not only induced by metals, cytokines, hormones, but also by different oxidants and prooxidants. The authors also stated that MT overexpression increases the resistance of tissues and cells to oxidative stress. Our results showed that MTs induction occurred at very different levels of metal and oxidative stress. In the presence of the oxidizing agent (H2O2), MTs only increased significantly when the degree of oxidative stress, assessed by lipid peroxidation, was very high (10 and 20 mM H2O2), and when more than half of the organisms were dead. These results suggest that MTs are not good biomarkers of oxidative stress since, for the same survival level, mortality is a much easier and quicker parameter to evaluate stress. In fact, other studies (e.g. Cai et al., 1999) showed that MTs only increase significantly in organisms exposed to oxidizing agents when the degree of oxidative stress is high. On the contrary, our results showed that, for any concentration of the metal used, MTs increased significantly compared to the control, although all cockles survived in the range of Cd concentrations used and only at the highest concentration (10 mM) the level of lipid peroxidation was significantly higher than the control. These results confirmed that metals induce the expression of MTs, even when organisms apparently are not in stress, and that induction increased with the concentration of metal. Thus MTs may serve as a very sensitive biomarker when bivalves are exposed to Cd stress. In studies with C. edule, Paul-Pont et al. (2010b) demonstrated that the concentration of MTs increased with the progressive accumulation of Cd. Freitas et al. (2012a, 2012b), demonstrated the high sensitivity of MTs in C. edule exposed to different levels of environmental metal contamination (Cd, As, Hg, Pb, Zn, Cr, Ni, and Cu). Works performed by Smaoui-Damak et al. (2004),

4. Discussion The present work was performed to compare the sensitivity of MTs as a stress biomarker to metals and oxidizing agents. Our results showed that both types of stress induce the synthesis of MTs, as reported previously by several authors. Amiard et al. (2006) stated that MTs play roles both in the routine metabolic handling of essential Cu and Zn, but also in the detoxification of intracellular excess amounts of these metals and of non-essential Cd, Ag and Hg. Martı´n-Diaz et al. (2008) reported a significant induction of MTLPs in crabs and clams exposed to metal contaminated sediments. Perceval et al. (2006) showed that MTs induction was correlated to environment and organisms Cd levels. Monserrat et al. (2007) pointed out that biological functions of

Fig. 4. Number of metal ions (Cd, Zn and Cu) per MT molecule when Cerastoderma edule was exposed to different treatments: control (0 mM H2O2 and Cd), 20 mM H2O2 and 10 mM Cd. Capital letters represent, for the total number of ions/MT molecule, significant differences between treatments (control, H2O2 and Cd). Lowercase letters represent, for Zn ions, significant differences between treatments (control, H2O2 and Cd).

Table 1 Total concentration (ng/g FW) and concentration bound to MTs (ng/g FW) of Cd, Zn and Cu in the soluble fraction of C. edule exposed to 3 treatments: 10 mM Cd, 20 mM H2O2 and control (0 mM of Cd and H2O2). The percentage of bound metals in relation to the total concentration in solution is also presented. Lowercase letters represent, for each metal, significant differences between treatments.

Ion Condition Total Concentration bound to MTs Percentage bound to MTs

Cd

Zn

Control a

0.00 0.00a 0.00

H2O2 b

0.38 0.00a 0.00

Cd

Cu

Control c

64.36 64.29b 99.90

a

40.28 30.96a 71.00

H2O2 a

32.18 25.74a 80.00

Cd

Control b

25.67 18.49b 72.00

a

4.16 2.50a 60.00

H2O2 a

3.28 0.00b 0.00

Cd 3.30a 0.00b 0.00

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demonstrated the potential use of MTs as a biomarker of Cd contamination in Ruditapes decussatus. Recently Serafim and Bebiano (2010) described the response of MTs to a mixture of sublethal Cd, Cu and Zn concentrations in R. decussatus. The potential of MTs, as a biomarker of metal exposure (Cd, Cu and Zn) was shown in R. decussatus from the environment (Hamza-Chaffai et al., 2000, 2003). Working with Ruditapes philippinarum, Ng and Wang (2004) demonstrated that MTs play an important role in the detoxification of Cd. The sensitivity of MTs was further demonstrated when R. phillipinarum was subjected to Cd (Wang et al., 2011). Figueira et al. (2012), while studying the responses of R. decussatus and R. philippinarum to Cd exposure, also showed the induction of MTs synthesis with increasing Cd levels. Our work revealed that the number of ions and the metal bound to MTs vary with the treatment applied. In the absence of disturbance (control) MTs bound most (60–70 percent) of the essential metals (Zn and Cu) in solution. In stressful situations, such as the exposure to Cd and H2O2, MTs did not bind Cu and bound less Zn. In organisms exposed to Cd the total number of ions bound per MT molecule did not vary in relation to control, being part of the Zn ions and all Cu ions displaced by Cd ions. In organisms exposed to H2O2, each MT molecule bound less than half of the ions compared to the control and Cd treatments, indicating that part of the thiol groups in the cysteine residues have been oxidized by the scavenging of ROS accumulated during oxidative stress. Works indicating the role of MTs as ROS scavengers are few. Bauman et al. (1991), working with mice, showed the increase in MTs by chemicals that induce oxidative stress. Kumari et al. (1998) examined the free radicals scavenging effects of hepatic MTs isoforms (I and II), and showed that both are able to scavenge free radicals. Leung and Furness (2001) studying dogwhelks (Nassarius lapillus) demonstrated that the synthesis of MTs increased in the presence of metals and also of oxidative agents. Works conducted by Anderson et al. (1999) with oysters (Crassostrea virginica) exposed to H2O2, showed that the interaction of ROS with MTs in hemocytes could play a role in protection of the cells and surrounding tissues from oxidants associated with antimicrobial responses. In fact, the results obtained suggested that MTs may be involved in immunoregulatory pathways in oyster hemocytes as a result of its ability to scavenge antimicrobial ROS. Viarengo et al. (1999) exposed M. galloprovincialis to Fe (oxidant inducer) after or not a preexposition to Cd. The results obtained showed that the treatment with Fe led to a significant increase in oxyradical production and malondialdehyde level only in mussels not preexposed to Cd. Furthermore, analyses on whole organisms showed that anoxic survival was lowered in mussels that had been treated with Fe, but such an effect was less pronounced in Cd-preexposed mussels compared with nonpreexposed ones. These data suggested an antioxidant role for MT, which seems to occur through oxyradical scavenging and is able to protect both isolated cells and the entire organism from oxidative stress. Overall, these researches point out the concern that must be taken into account when using MTs as exclusive biomarkers of metals, but none of them evaluates the ability of MTs as oxidative stress biomarker.

5. Conclusions The ability of MTs to act as ROS scavengers was shown by several authors, rendering the use of MTs as biomarkers of metal stress controversial. Therefore, the results obtained in our work contributed to clarify the sensitivity of MTs towards metal and oxidative stress. MTs are biomarkers of both Cd and H2O2, but their induction responses are better with metal than with oxidative stress.

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