Ecotoxicology and Environmental Safety 107 (2014) 55–60

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Metal, metallothionein and glutathione levels in blue crab (Callinectes sp.) specimens from southeastern Brazil Raquel Teixeira Lavradas a, Rachel Ann Hauser-Davis a,n, Ricardo Cavalcanti Lavandier a, Rafael Christian Chávez Rocha a, Tatiana D. Saint’ Pierre a, Tércia Seixas a, Helena Amaral Kehrig b, Isabel Moreira a a Pontifícia Universidade Católica do Rio de Janeiro (PUC-Rio), Departamento de Química, Laboratório de Bioanalítica, Rua Marquês de São Vicente, 225, Gávea, CEP: 22453-900 Rio de Janeiro, RJ, Brazil b Universidade Estadual do Norte Fluminense, Laboratório de Ciências Ambientais, Campos dos Goytacazes, CEP: 28013-602 Rio de Janeiro, RJ, Brazil

art ic l e i nf o

a b s t r a c t

Article history: Received 18 February 2014 Received in revised form 3 April 2014 Accepted 9 April 2014

Metal concentrations (Cu, Pb, Zn and Cd) were determined in muscle, gills, soft tissues and eggs in male, non-ovigerous and ovigerous female Callinectes sp. specimens from a reference site in Southeastern Brazil. Metallothionein (MT) and reduced glutathione (GSH) levels were also determined. Results demonstrate that sex has a significant influence on metal, MT and GSH concentrations. Significant maternal transfer of Pb and Zn from ovigerous females to eggs was verified, while female crabs, both ovigerous and non-ovigerous, showed elevated GSH and MT in viscera when compared to males, indicating possible MT role in excreting metals to eggs in ovigerous females of this species. Several strong statistical correlations between metals and MT indicate MTs role in detoxification of both toxic and essential elements in different organs. Pb and Zn were significantly correlated to GSH, indicating oxidative stress caused by the former and a direct link between Zn and GSH in maintaining homeostasis. Regarding human consumption, metal concentrations were lower than the maximum permissible levels established by international and Brazilian regulatory agencies, indicating that this species is safe for human consumption concerning this parameter. The presence of metals in Callinectes sp., however, is still of importance considering that this is a key species within the studied ecosystem and, therefore, plays a major role in the transference of pollutants to higher trophic levels. In addition, the presence of significant metal concentrations found in eggs must be considered in this context, since crab eggs are eaten by several other species, such as shorebirds, seabirds, and fish. Also, to the best of our knowledge, this is the first study regarding both MT and GSH levels in Callinectes sp. eggs and is of interest in the investigation of molecular mechanisms regarding metal exposure in these crustaceans. Data reported in this study support the conclusions from previous reports, provide mechanistic insights regarding metal exposure, metallothionein and oxidative stress induction in this species and also present novel data regarding eggs. & 2014 Elsevier Inc. All rights reserved.

Keywords: Metals Metallothionein Oxidative Stress Blue crab Metal transfer

1. Introduction Contamination by metals has received much attention in the last decades, since these pollutants offer serious risks due to their toxicity, persistence and bioaccumulation in the aquatic food chain (Smith, 1993; Yilmaz et al., 2007). When exposed to metals, most organisms undergo increased synthesis of metallothioneins (MT), metalloproteins that bind to metals through cysteine bonds (Kagi, 1991). These proteins act in the cellular regulation of metabolically important metals, such as Cu and Zn, as well as in reducing the

n

Corresponding author. E-mail address: [email protected] (R.A. Hauser-Davis).

http://dx.doi.org/10.1016/j.ecoenv.2014.04.013 0147-6513/& 2014 Elsevier Inc. All rights reserved.

toxicity of non-essential metals, i.e. Cd and Pb (Livingstone, 1993; Viarengo et al., 1997). The relationship between metal levels in the environment and metallothionein levels in animal tissues has led to the use of these proteins as biomarkers for monitoring the biological effects resulting from exposure to metals (Hylland et al., 1992; Livingstone, 1993). In aquatic invertebrates it has been demonstrated that MT also acts against cellular oxidative stress, being directly connected to the cellular antioxidant defense system (Cavaletto et al., 2002; Viarengo et al., 2000). Glutathione (GSH, c-glutamyl-cysteinyl-glycine) is a major intracellular antioxidant in living organisms and the first line of defense against oxidative stress. It is also a central component in the multifaceted cellular detoxification system that constitutes an important mechanism for cellular protection against agents, such

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as metals, that produce radical oxygen species. There have been many reports on glutathione-mediated alleviation of metal stress in animals but studies on glutathione synthesis and its role in metal stress in Crustacea are limited (Wang et al., 2003). The blue crab, Callinectes sp., is an important species in Southeastern Brazil, where it is heavily consumed by humans and other species such as shorebirds, seabirds, and fish. Because of this, this species plays an important role in the transfer of pollutants to other trophic levels. In this context, the aims of the present study were to investigate metal exposure (Cu, Zn, Cd, Pb) and oxidative stress in the form of MT and GSH in blue crab Callinectes sp. tissues and eggs. To the best of our knowledge, no studies regarding these proteins have been conducted in crusteacean eggs, making this study of interest also in the context of elucidating cellular defense mechanisms against environmental stressors such as metals.

2. Methodology 2.1. Study area The Ilha Grande Bay, located in the southern coast of the state of Rio de Janeiro, between latitudes 221 500 S and 231 200 S and longitudes 441 450 W and 441 000 W, has an area of 65,258 ha and approximately 350 km of water line perimeter and composes an estuarine complex with other local bays (Creed et al., 2007). Despite the presence of potential pollution sources, such as an oil terminal, a shipyard and two nuclear power plants, this area has been reported as a reference area due to low levels of metals present in water, biota and sediments (Cardoso et al., 2001; Lacerda et al., 1981). 2.2. Specimen sampling and dissection Adult (n¼ 26; 6 males and 20 females, of which 10 were ovigerous and 10 were non-ovigerous) Callinectes sp. individuals were sampled from Ilha Grande Bay by local fishermen. Individuals were weighed, identified, sexed and measured with a caliper ( 70.1 mm). The shell width was measured between the base of the spines and the lateral length by the vertical position of the abdomen (Andrade et al., 2011). Muscle, soft tissues (viscerae, including the hepatopancreas), gills and eggs (when present) were removed and freeze-dried. 2.3. Metal determination Samples were prepared for metal determination by weighing 0.250 g of each sample and adding 5 mL of sub-boiled bi-distilled nitric acid (Vetec, Rio de Janeiro). The samples were left overnight and then heated in a digestion block, at 100 1C for 5 h (Balcerzak, 2002). After cooling, the volume was adjusted to 20 mL with ultrapure water. 2 mL aliquot was removed and diluted with 2 mL of ultrapure water for subsequent mass spectrometry analysis using inductively coupled plasma mass spectrometry (ICP-MS). Metal determinations were conducted using an ICPMS in the standard mode, without the use of a reaction cell (ELAN DRC II model, Perkin-Elmer Sciex, Norwalk, CT, USA). The sample introduction system consisted of a Meinhard-type nebulizer with a twister cyclonic chamber. During the analysis, 103 Rh was used as internal standard at a concentration of 20 mg L  1. The following isotopes known to bind to metallothioneins were determined: 65Cu, 66Zn, 114Cd and 208 Pb. The method accuracy was assessed by the use of certified reference material, DORM-3 (Dogfish muscle, NRC, Canada) and TORT-2 (Lobster hepatopancreas, NRC, Canada). 2.4. Metallothionein extraction and quantification Approximately 100 mg of each crab tissue was extracted according to the thermal extraction procedure described by Erk et al. (2002). Samples were homogenized at a 1:3 v/v ratio with a buffer containing 0.02 mol L  1 Tris–HCl pH 8.6, 0.0005 mol L  1 PMFS as a protease inhibitor, and 0.01% β-mercaptoethanol, as a reducing agent. The samples were then centrifuged at 20,000g for 30 min at 4 1C and the supernatants were carefully removed and transferred to sterile 2 mL tubes. Heating was conducted at 70 1C for 10 min and a further centrifugation in the same conditions was conducted for 30 min. After this last centrifugation, the supernatants were again separated and samples were frozen at  80 1C until analysis. MT were quantified using a spectrophotometric method described previously (Ellman, 1959). Briefly, extracted samples were treated with 1 mol L  1 HCl containing 0.004 mol L  1 EDTA and 2 M NaCl containing 0.00043 mol L  1 5,50 -dithiobis-2nitrobenzoic acid (DTNB) buffered with 0.2 mol L  1 sodium phosphate, pH 8.0. The supernatant absorbance was then measured at 412 nm on a microplate reader

(Hamilton 190 SpectraMax) and MT concentrations were estimated using a calibration curve plotted with GSH as an external standard. MT levels were then estimated assuming a ratio of 1 mol MT for 20 mol GSH (Kagi, 1991). 2.5. GSH extraction and quantification Approximately 200 mg of each tissue was homogenized in 2 mL of sodium phosphate buffer 0.1 mol L  1 pH 7 containing sucrose 0.25 mol L  1 (Pereira et al., 1998). The samples were then centrifuged at 13,500 rpm for 30 min at 4 1C. The supernatants were removed and transferred to other sterile tubes and treated with DTNB 0.1 mol L  1 in phosphate buffer pH 8.0 at a 1:1 ratio. The samples were incubated for 15 min in the dark and their absorbance was measured in a microplate reader (SpectraMax 190 Hamilton) at 412 nm. Concentrations were estimated using a calibration curve plotted with GSH (reduced glutathione) as an external standard. 2.6. Statistical analyses The significance level adopted throughout this study was 95% (α ¼ 0.05). The Kruskal–Wallis test was used to determine significant differences among the different organs for metal, GSH and MT concentrations. Correlation between metals in the different organs; between the metals and GSH levels and between the metals and MT levels were verified by the Spearman correlation test.

3. Results and discussion 3.1. Quality assurance in ICP-MS metal determinations No statistically significant differences were observed between found and certified values for each analyte for both certified reference materials, indicating the adequacy of the method for the selected elements, as shown in Table 1. 3.2. Correlations between metal concentrations, size, weight and sex No correlations were observed between the analyzed elements, size and weight, corroborating with other studies in other crustacean species (Simonetti et al., 2012). Sex, on the other hand, presented weak correlations with Cu (R ¼  0.40; p o0.05), Cd (R¼  0.35; p o0.05) and Zn (R¼ 0.44; p o0.05). Because of this, we analyzed each sex separately also, including comparisons between ovigerous and non-ovigerous females, in order to better analyze sex influence on metal, GSH and MT concentrations. 3.3. Metal concentrations in muscle, soft tissues, gills and eggs Regarding metal concentrations (Table 2), significant differences (p o0.05) were observed between males and females (both ovigerous and non-ovigerous) for muscle regarding Cu concentrations, which was significantly higher in males, for viscera and Cd (significantly higher in males) and Zn (significantly higher in females) concentrations. When comparing ovigerous females and males (viscera), significant differences (p o0.05) were observed for Cd and Zn concentrations in viscera, which were significantly higher in males and females, respectively. The only difference observed for muscle in this group was for Cu concentrations, which was also significantly higher in males. When comparing viscera from ovigerous females with eggs themselves, significant differences (p o0.05) were observed for Cu and Cd, which were higher in viscera, and Pb and Zn, which were significantly higher in eggs. For the same comparison with muscle, differences were observed for Cd, Pb and Zn, which were all higher in eggs, except for Cd, which was lower, and for the same comparison with gills, significant differences were observed for Cu, Cd and Pb, higher in gills, and Zn, higher in eggs. As a key species within the studied ecosystem, Callinectes sp. could play a major role in the transference of environmental contaminants to higher trophic levels and the presence of these

R.T. Lavradas et al. / Ecotoxicology and Environmental Safety 107 (2014) 55–60

elements may pose concern to the local biota. As Ilha Grande Bay is considered a reference site, we expected to find low metal levels in the subjects. This was seemingly corroborated when comparing the values observed in the present study with other studies (Table 3), such as the study from Santos et al. (2007) in Macéio, Alagoas, Brazil, which is considered as a moderately contaminated area, all elements were lower in soft tissue compared to the hepatopancreas analyzed by those authors (we may compare these organs because soft tissues from the present study include hepatopancreas). This was not the case, however, for Cd, Pb and Zn in muscle, which were higher than other non-contaminated sites (Bordon et al., 2012) and for all elements in gills, which were significantly higher than non-contaminated lagoons in the Mediterranean (Mutlu et al., 2011). This is also observed for Cd in soft tissues, which were higher than some non-contaminated estuaries in the Unites States (Jop et al., 1997). This may indicate potential Cd, Cu and Zn contamination in the area from an unknown source. Table 3 compares the metal values observed in the present study with values observed in the literature. Regarding egg metal concentrations, Cu and Cd concentrations were significantly lower (p o0.05) in eggs when compared to ovigerous female viscera, while Zn and Pb concentrations were significantly higher (p o0.05). This seems to indicate transfer from the ovigerous females to the eggs. Since metals are sequestered in eggs in other species, such as several types of birds and crocodilians (Burger and Gochfeld, 1993; Burger et al., 1999; Delany et al., 1988), it is not surprising that they are also sequestered in invertebrate eggs (Burger and Gochfeld, 1995; Fimreite et al., 1982). Literature, however, is extremely scarce regarding metals in crustacean eggs. In one study, Pb, Hg, Cd, Cr and Mn concentrations were measured in the eggs of horseshoe crabs (Limulus polyphemus) (Burger, 1997). The metal concentrations in those eggs represented levels derived from females, which could come from two sources, according to the authors: the first from sequestration from the female during egg formation and the second from the surrounding water. Horseshoe crab eggs,

Table 1 Trace element concentrations (mg kg  1 dry wt) in certified reference materials (DORM-2 and DOLT-3). Values are displayed as mean 7 SD (n¼ 20 for DORM-3 and n¼ 10 for TORT-2). Element

Zn Cd Pb Cu

DORM-3 (Dogfish liver)

TORT-2 (Dogfish muscle)

Certified value

Found value

Certified value

Found value

51.3 7 3.1 0.290 7 0.020 0.395 7 0.050 15.50 7 0.63

52.6 73.0 0.310 70.010 0.340 70.020 15.80 70.59

1807 6 26.7 7 0.6 0.357 0.13 106 7 10

1837 4 26.8 7 0.8 0.337 0.10 1017 9

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however, are laid beneath the sand at the high tide line, where they are inundated with water continuously (Botton et al., 1988), being in balance with the surrounding seawater, presumably taking in some water, and reflecting local levels of pollutants in the water. Callinectes sp. eggs, on the other hand, are carried in the females' abdomen until it is their time to hatch, when the females leave the estuaries and return to the sea. Thus, it is more likely that the metal levels observed in Callinectes sp. eggs in the present study reflect metal levels found in females. This is of importance, since high metal levels may potentially impair developmental processes in the young due to the fact that metal accumulation is occurring before the hatch and crab's eggs are ingested by several other species, such as several, shorebirds and seabirds. Unfortunately, there is no published data on metals in eggs of this species but only in T. tridentatus and L. polyphemus which are not very taxonomically close to the blue crab. This impairs discussion on metal levels and comparisons with the literature, however highlights the importance of the present study. Regarding human risk assessment, according to several government health agencies (ANVISA, USFDA, USEPA, WHO), 90% of contaminant ingestion in humans occurs through food intake via fish and sea food. As muscle tissue is the main edible part of crabs and can directly influence human health, it is important to investigate metal content in this tissue. Brazil has an average daily intake of 24.74 g of fish and seafood per capita, corresponding to an average of 173.18 g per week, however, in the metropolitan region of Rio de Janeiro, this consumption doubles to 355.00 g per week (Brasil, 2009). Taking this into account and using the body weight of 70 kg individual, metal ingestion was calculated for the weekly intake of crab per body weight. Values observed in edible muscle tissue were of 75.97 mg g  1 dry weight for Cu, 0.25 mg g  1 dry weight for Cd, 0.02 mg g  1 dry weight for Pb and 183.48 mg g  1 dry weight for Zn, all below the maximum permissible limits for human ingestion stipulated by the US EPA, FAO/JECFA and ANVISA (Brazil) regulatory agencies. Therefore, this species is safe regarding human ingestion and may continue to be part of the diet of the human population.

4. MT and GSH 4.1. MT The trend observed for MT in females (all) and non-ovigerous females was muscle o gills oviscera, while males showed the trend gills o muscle –viscera and ovigerous females showed the trend gills omuscle oeggs oviscera. To the best of our knowledge, no studies regarding MT levels in crustacean eggs are available, making this the first study to present this data.

Table 2 Metal concentrations in Callinectes sp. organs from the Ilha Grande Bay, RJ, Brazil. Sex

Organ

Cu

Cd

Pb

Zn

Females (all, with and without eggs)

Muscle Gills Viscera Muscle Gills Viscera Muscle Gills Viscera Eggs Muscle Gills Viscera

46.26 7 8.86 225.36 7 41.90 78.377 34.39 51.217 5.32 207.34 7 42.27 64.547 28.03 42.95 7 9.56 249.39 7 32.75 87.60 7 37.45 44.107 11.33 59.08 7 5.37 246.947 41.25 111.99 7 38.50

0.157 0.07 0.747 0.21 0.727 0.28 0.177 0.05 0.767 0.28 0.747 0.09 0.147 0.08 0.727 0.14 0.707 0.37 0.05 7 0.01 0.22 7 0.09 0.99 7 0.48 1.32 7 0.27

0.14 70.03 14.45 711.53 0.63 70.16 0.15 70.05 16.46 714.82 0.68 70.12 0.14 70.02 11.7777.14 0.60 70.18 1.38 70.43 0.11 70.03 32.98 734.66 0.49 70.18

116.34 7 19.50 81.62 7 6.71 161.45 7 32.46 122.79 7 22.45 78.20 7 6.10 179.36 7 10.57 112.047 18.08 86.197 4.95 149.517 37.44 290.217 58.17 131.25 7 32.17 100.94 7 27.51 86.42 7 15.23

Females (no eggs)

Females (with eggs)

Males

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Table 3 Metal concentrations in different Callinectes species from around the world. Species Callinectes sp.

Sampling site

Tissue

Ilha Grande Bay, Rio de Janeiro, Brazil Muscle Gills Soft tissue Callinectes Mediterranean Lagoons Muscle Sapidus Gills Callinectes danae Santos Estuarine System of São Paulo Muscle state, Brazil Callinectes sp. River Cubatão Basin, Brazil Soft tissue Callinectes Connecticut Estuaries, USA Muscle sapidus Hepatopancreas Callinectes Maceió, Alagoas, Brazil Hepatopancreas bocourti

Cu

Zn

Pb

Cd

Reference

31.6–64.1 149.9–287.1 35.5–167.7 5.4–11.7 7.7–24.5 3.5–20.1

82.7–179.2 69.9–140.6 69.7–208.6 13.9–20.1 7.7–11.5 20.1–33.8

0.07–0.22 4.7–77.8 0.33–0.87 n.a. n.a. 0.003–1.725

0.05–0.32 0.45–1.67 0.24–1.55 0.03–0.08 0.04–0.10 0.012–0.022

Present study

dez/20 31.2–32.8 27.2–28.3 149–170

0.61–1.51 0.01–0.12 0.003–0.30 0.92–1.15

0.12–0.22 0.05–0.40 0.93–1.18 0.43–0.59

14–18 15.9–16.2 20.7–94.8 99.7–125

Mutlu et al. (2011) Bordon et al. (2012) Virga & Geraldo (2008) Jop et al. (1997) Santos et al. (2007)

Fig. 1. Metallothionein (MT) concentrations (mmol mg-1) in Callinectes sp. muscle (1A), gills (1B) and viscera and eggs (1C) are shown in box and whiskers plots, where the horizontal line inside the box represents the median value, the box represents 25–75% data range and the whiskers are the minimum and maximum values. The outliers are represented by asterisks.

When comparing MT concentrations (Fig. 1) between males and all females, males and ovigerous females and males and nonovigerous females, significantly higher MT values (p o0.05) were always observed for the females (all, ovigerous and non-ovigerous) in viscera. When comparing MT levels in the ovigerous females with the eggs themselves, the only significant differences observed were for viscera, which presented significantly higher MT levels (p o0.05) than the eggs. It has been reported that tissues directly involved in metal uptake, storage and excretion, such as liver and gills have a high capacity for MT synthesis (Amiard et al., 2006; Filipovi and Raspor, 2003; Roesijadi and Robinson, 1994) and inter-organ differences in MT concentrations are usually observed. These proteins have been studied in the hepatopancreas and gills of some crustacean species (Brown et al., 2004; Canli et al., 1997; Martins and Bianchini, 2009; Silvestre et al., 2004). Based on these studies, the hepatopancreas has been described as the main organ involved in metal storage, accumulating more trace metals than gills and presenting the highest MT concentrations (Amiard et al., 2006; Legras et al., 2000). As the soft tissues (viscera) analyzed in the present study include the hepatopancreas, we expected to find higher MT levels in these samples, which in fact did occur for all analyzed groups, corroborating with other studies with other crustaceans, such as Carcinus maenas, Pachygrapsus marmoratus and C. sapidus (Legras et al., 2000; Martins and Bianchini, 2009; Mouneyrac et al., 2001; Pedersen et al., 1997). Unfortunately, not many reports regarding MT are available in crustaceans and the only other study we could find conducted with C. sapidus does not express the data in MT concentrations and also does not indicate the exact value found in gills in the study (Martins and Bianchini, 2009).

According to Virga and Geraldo (2008) female blue crabs show a trend of higher metal concentrations in comparison to males, which could explain the higher MT levels in this sex. Metal concentrations, however, were only significantly higher for females for Zn in viscera when comparing all females and males. The ovigerous group, however, is diferential since maternal transfer of Pb and Zn could have occurred from viscera, lowering the concentrations of these elements in these tissues, but perhaps maintaining MT presence in higher levels in order to detoxify these elements from the body and excrete them into the eggs. Significant statistical correlations (p o0.05) were observed between Cd and MT in gills in females (all). In ovigerous females, significant correlations were observed between Cu and MT (ρ¼0.829) in muscle, and Zn and MT (ρ¼1) in gills. In nonovigerous females, significant correlations were observed in viscera between Cd and MT (ρ¼  1) and Zn and MT (ρ¼ 1). In this group, significant correlations were also observed for Cu and MT (ρ¼1) in gills. In males, significant correlations were observed in viscera between Pb and MT (ρ¼  1). In eggs, significant correlations were observed between Pb and MT (ρ¼0.943). These very strong correlations indicate MT role in detoxification of both toxic and essential elements in different organs. 4.2. GSH The trend observed for GSH in females (all) and non-ovigerous females was of muscle ogills oviscera, while males showed the trend gills omuscle –viscera, the same as for MT. Ovigerous females showed the trend muscle o eggsogills oviscera. To the best of our knowledge, no studies regarding GSH levels in

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Fig. 2. Reduced glutathione (GSH) concentrations (mmol.mg  1) in Callinectes sp. muscle (2A), gills (2B) and viscera and eggs (2C) are shown in the box and whiskers plots, where the horizontal line inside the box represents the median value, the box represents 25–75% data range and the whiskers are the minimum and maximum values. The outliers are represented by asterisks.

crustacean eggs are available either, making this also the first study to present this data. When comparing GSH concentrations between males and all females and males and non-ovigerous females, significantly higher GSH values (p o0.05) were only observed for both female groups in viscera. The same occurred when comparing males and ovigerous females. When comparing ovigerous and non-ovigerous females, significantly higher GSH values (p o0.05) were only observed in viscera from the ovigerous females. No significant differences for ovigerous females and eggs were observed. GSH data (mmol g-1) are presented in Fig. 2. In general, GSH plays a key role in cellular defense and serves as a reservoir for the aminoacid cysteine. GSH depletion leads to cell death and has been documented in many degenerative conditions (Kovacevic, 2008). GSH is involved in two mechanisms that combat metal stress: (i) by relieving the oxidative stress effects caused by heavy metals through the formation of oxidized glutathione (GSSG); and (ii) by forming metal complexes (Wang et al., 2008). GSH levels are induced and fluctuate in response to stimuli which produce oxidative stress (Swiergosz-Kowalewska et al., 2006). The data from the present study indicate that female crabs, both ovigerous and non-ovigerous, show elevated GSH in viscera compared to males, and that the ovigerous females show elevated GSH in viscera in comparison to the non-ovigerous females. This can be expected, since, as discussed previously, females crabs tend to show higher metal concentrations than males (Virga and Geraldo, 2008) which, besides increasing MT concentrations, can also be responsible for oxidative stress. As Zn and Pb were significantly higher in eggs, with possible maternal transfer from the viscera to the eggs, it is reasonable to assume that this can be the cause for the higher GSH values in the ovigerous females. Significant correlations (p o0.05) were observed for females (all) in viscera for Zn and GSH (ρ ¼  0.67), for ovigerous females between Pb and GSH (ρ¼0.829) in muscle and between Pb and GSH (ρ¼1) in gills. No significant correlations were observed in non-ovigerous females, males and eggs. As Pb is a toxic element and known to induce oxidative stress, these correlations are to be expected. Zn, however, is an essential element, and necessary for organism homeostasis. Thus, this significant correlation seems to indicate that Zn in viscera for all females is directly linked to GSH levels in maintaining homeostasis. 4.3. Associations between MT and GSH Very strong positive associations (p o0.05) were observed between MT and GSH in the following cases in the viscera of ovigerous females (ρ¼ 0.9), in the muscle of non-ovigerous female

muscle (ρ¼ 0.982), in male gills (ρ¼1) and in eggs (ρ¼ 0.829). These correlations are a definite sign that MT and GSH concentrations are influenced by each other as found in other animals. This is especially interesting in the case of ovigerous female viscera and eggs, further demonstrating that ovigerous female viscera and eggs are intrinsically connected and interdependent regarding molecular mechanisms of metal detoxification, homeostasis and oxidative stress regulation.

5. Conclusions Sex was shown to significantly influence the metal, MT and GSH results. Significant maternal transfer of Pb and Zn from ovigerous females to eggs was observed, while female crabs, both ovigerous and non-ovigerous, show elevated GSH and MT in viscera when compared to males, indicating a possible MT role in excreting metals to eggs in ovigerous females. Several strong statistical correlations between metals and MT indicate MT role in detoxification of both toxic and essential elements in different organs. Pb and Zn were significantly correlated to GSH, indicating oxidative stress caused by the former and a direct link between Zn and GSH in maintaining homeostasis. Regarding human consumption, metal concentrations were lower than the maximum permissible levels established by international and Brazilian regulatory agencies, indicating that this species is safe for human consumption concerning this parameter. The presence of metals in Callinectes sp., however, is still of importance considering that this is a key species within the studied ecosystem playing a major role in the transference of pollutants to higher trophic levels. In addition, the presence of significant metal concentrations found in eggs must be considered. Data reported in this study support the conclusions from previous reports, provide mechanistic insights regarding metal exposure, metallothionein and oxidative stress induction in this species and also present novel data regarding eggs.

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Metal, metallothionein and glutathione levels in blue crab (Callinectes sp.) specimens from southeastern Brazil.

Metal concentrations (Cu, Pb, Zn and Cd) were determined in muscle, gills, soft tissues and eggs in male, non-ovigerous and ovigerous female Callinect...
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