The effects of selenium on oxidative stress biomarkers in the freshwater characid fish matrinxã, Brycon cephalus (Günther, 1869) exposed to organophosphate insecticide Folisuper 600 BR® (methyl parathion)

Comparative Biochemistry and Physiology, Part C 149 (2009) 40–49

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The effects of selenium on oxidative stress biomarkers in the freshwater characid fish matrinxã, Brycon cephalus ( Günther, 1869) exposed to organophosphate insecticide Folisuper 600 BR® (methyl parathion) Diana Amaral Monteiro, Francisco Tadeu Rantin, Ana Lúcia Kalinin ⁎ Department of Physiological Science, Federal University of São Carlos, Via Washington Luís km 235, PO BOX 676, 13565-905, São Carlos, São Paulo, Brazil

A R T I C L E

I N F O

Article history: Received 15 May 2008 Received in revised form 27 June 2008 Accepted 28 June 2008 Available online 4 July 2008 Keywords: Antioxidant enzymes Brycon cephalus Lipid peroxidation Matrinxã Methyl parathion Organophosphate Oxidative stress Reduced glutathione Selenium

A B S T R A C T Methyl parathion (MP), an organophosphate widely applied in agriculture and aquaculture, induces oxidative stress due to free radical generation and changes in the antioxidant defense system. The antioxidant roles of selenium (Se) were evaluated in Brycon cephalus exposed to 2 mg L− 1 of Folisuper 600 BR® (MP commercial formulation — MPc, 600 g L− 1) for 96 h. Catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione S-transferase (GST), reduced glutathione (GSH) and lipid peroxidation (LPO) levels in the gills, white muscle and liver were evaluated in fish fed on diets containing 0 or 1.5 mg Se kg− 1 for 8 weeks. In fish treated with a Se-free diet, the MPc exposure increased SOD and CAT activities in all tissues. However, the GPx activity decreased in white muscle and gills whereas no alterations were observed in the liver. MPc also increased GST activity in all tissues with a concurrent decrease in GSH levels. LPO values increased in white muscle and gills and did not change in liver after MPc exposure. A Se-supplemented diet reversed these findings, preventing increases in LPO levels and concurrent decreases in GPx activity in gills and white muscle. Similarly, GSH levels were maintained in all tissue after MPc exposure. These results suggest that dietary Se supplementation protects cells against MPc-induced oxidative stress. © 2008 Elsevier Inc. All rights reserved.

1. Introduction The production of reactive oxygen species (ROS) can induce oxidative damage and may be a mechanism of toxicity for aquatic organisms living in environments receiving water-borne contaminants (Livingstone et al., 1990; Livingstone, 2001; Ferreira et al., 2005; Valavanidis et al., 2006). There is strong evidence indicating that xenobiotics can generate ROS U such as superoxide anion (O2−), hydrogen peroxide (H2O2), hydroxyl U radical ( OH), and singlet oxygen (O12), which can induce cell and tissue damage associated with different pathological processes (Adams et al., 1989; Wilhelm-Filho et al., 2001). Fish are particularly sensitive to water contamination and pollutants may impair many physiological and biochemical processes when assimilated by fish tissue (Durmaz et al., 2006). When abnormal or xenobiotic-induced ROS production exceeds the endogenous protection, damage to cellular components can be often observed. This process is known as oxidative stress (Oakes and Van der Kraak, 2003). The antioxidant defense system includes enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione S-transferase (GST) and other low molecular weight scavengers such as reduced glutathione (GSH) (Storey, 1996; Droge, 2002).

⁎ Corresponding author. Tel.: +55 16 3351 8314; fax: +55 16 3351 8401. E-mail address: [email protected] (A.L. Kalinin). 1532-0456/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2008.06.012

Pesticides are widely used in agriculture adversely affecting nontarget organisms wherein fish are major components of the aquatic biota. Methyl parathion (MP) is one of several organophosphate pesticides (OPs) widely applied as an insecticide in agriculture and pest control programs (Aguiar et al., 2004) due to its efficiency against a large spectrum of insect pests. This OP is also used to control larval stages of predatory insects that threaten fish larvae in culture tanks (Silva et al., 1993). In Brazil, MP is one of the most used OP for this purpose, in concentrations varying from 0.25 to 3 mg L− 1 (Figueiredo and Senhorini, 1990; Senhorini et al., 1991). Besides its use in aquaculture, the MP applied in agriculture can get into aquatic habitats, typically through unintentional and/or unavoidable overspray and/or carried by runoff. Indeed, Vinatea-Arana (1997) found an MP concentration of 2.3 mg L− 1 in water bodies close to rice fields. However, MP is a highly toxic insecticide, a United States EPA toxic class I, and all formulations of methyl parathion may be classified as Restricted Use Pesticides (RUPs). RUPs may be purchased and used only by certified applicators (EPA, 1999). The World Health Organization (WHO) classifies methyl parathion as an extremely hazardous pesticide. Like other organophosphate insecticides, methyl parathion is a cholinesterase inhibitor in mammals and fish (Boone and Chambers, 1996; Hai et al., 1997; Aguiar et al., 2004). Moreover, in a previous study we demonstrated that 2 mg L− 1 of MPc (1/3 of 96 h-LC50) has a high oxidative-stress-inducing potential in the neotropical fish matrinxã, Brycon cephalus, generating free radicals and inducing changes to the

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antioxidant enzymes and increasing lipid peroxidation. The gills and white muscle are the most sensitive organs (Monteiro et al., 2006). Selenium (Se) is an essential trace element in animal nutrition, including fish (Hamilton, 2004). This element is an integral part of the functional units of all selenoenzymes known so far, the most prominent being glutathione peroxidase, iodothyronine deiodinase, thioredoxin reductase and selenophosphatase synthetase (Zuberbuehler et al., 2006). Selenoenzymes are part of the antioxidant defense system and are involved in thyroid hormone metabolism, in spermatogenesis, and probably in other processes unidentified to date. The activity of these enzymes depends on adequate Se intake, defining this trace element as an essential nutrient (Zuberbuehler et al., 2006). Therefore, Se is involved in many functions such as moderation of the immune system and prevention of cancer, acting directly as a support for the organismal health (Hsu and Guo, 2002; Sarada et al., 2002; Chien et al., 2003). Se plays an important antioxidant role since it is a GPx cofactor. GPx scavenges H2O2 and lipid hydroperoxides, using reducing equivalents from glutathione and protecting membrane lipids and macromolecules from oxidative damage (Watanabe et al., 1997). The Se present in the active site of GPx contributes both to its catalytic activity and spatial conformation (Rotruck et al., 1973). Food, the main source of Se, presents a variable content of this element. Bioavailable forms include both inorganic (e.g. selenate, selenite) and organic (e.g. selenomethionine and selenocysteine) forms from dietary sources (Arteel and Sies, 2001). Considering this, it has been suggested that any significant modification of selenium status would lead to changes in GPx activity, having important consequences regarding the susceptibility of the tissues to oxidative stress (Tanguy et al., 1998; Toufektsian et al., 2000). In attempting to verify if Se has the same antioxidant roles in fish as observed in mammals, we evaluated the effect of pretreatment with dietary selenium in fish exposed to MP commercial formulation (MPc). The aim of this study was to determine the effects of supplementary Se (1.5 mg kg− 1 feed) on lipid peroxidation and antioxidant defense systems in gills, white muscle and liver of the freshwater fish matrinxã, B. cephalus, exposed to 2 mg L− 1 of Folisuper 600 BR® (commercial formulation of MPc, 600 g L− 1). The neotropical freshwater teleost B. cephalus (Teleostei, Characidae) is a native from the Amazon basin and an extremely important fishery resource in the Amazon region (Bittencourt and Cox-Fernandes, 1990). It is also considered as one of the main cultivated fish species in Brazil (Pereira Filho, 1994), and is of great economic importance and potentiality among commercially farmed fish due to its excellent meat, high growth rate and appetite for commercial pellets (ScorvoFilho et al., 1998). Moreover, matrinxã is threatened with extinction in some regions of the São Francisco basin (Sato and Bremmer, 1999). The matrinxã culture has been encouraged not only to supply fish markets but also to preserve its wild stocks (Goulding, 1980). The species-specific nutritional requirements are important to understand the resistance against adverse environmental conditions. Furthermore, the dietary manipulation can improve the health status and production quality of fish.

Paulo State, Brazil, where they were hatched and cultivated in artificial, open-air reservoirs with non-polluted water. Juvenile fish were acclimated for 60 days prior to experimentation in 1000 L holding tanks equipped with a continuous supply of well-aerated and dechlorinated water at 24 ± 2 °C and under natural photoperiod (~ 12 h:12 h). The physical and chemical parameters were kept nearly constant: pH 6.7–7.5, DO2 6.0–7.5 mg L− 1, hardness 25–30 mg L− 1 (as CaCO3) and conductivity 65–72 µS cm− 1. Fish were fed with a Se-free control diet (Diet I) or with a diet supplemented with 1.5 mg of Se kg− 1 of diet (Diet II). The control diet (Table 1), containing 35% crude protein and 3.07 kcal available energy/g diet, was formulated from practical ingredients to satisfy known nutrient requirements of B. cephalus, except the selenium requirement (Mendonça et al., 1993; Pereira Filho et al., 1995) according to the National Research Council guidelines (NRC, 1993). This control diet was supplemented with 1.5 mg of Se kg− 1 to formulate Diet II. Fish were fed ad libitum with Diet I or Diet II three times a day to apparent satiation for 8 weeks. Previous studies showed that Se supplementation lower than 3 mg kg− 1 diet had positive effects on animal growth and feed conversion rate and did not produce adverse effects in fish (Hilton et al., 1980; Maier and Knight, 1994; Usdoi, 1998; DeForest et al., 1999; Hamilton, 2003). Thus, in the present study, the chosen Se level (1.5 mg kg− 1 diet) would not cause toxic effects and it is nutritionally higher than levels normally used in the Brazilian fish commercial rations, which are based on recommended nutritional levels for nonnative species. Sodium selenite (Na2SeO3) was added to a Se-free vitamin–mineral premix and all the ingredients were completely mixed and pelletized by adding distilled water (40–60% of ingredient weight) before further homogenizations. Pellets of approximately 2 mm in diameter were formed by grinding the mixture through a meat grinder. The experimental diet pellets were stored at 4 °C until they were used.

0 mg Se kg− 1

1.5 mg Se kg− 1

Ingredients Fish flour (60%) Soybean meal (46%) Wheat middlings Corn Alginate Limestone (38% of calcium) Dicalcium phosphate Sodium chloride Vitamin C (35%) BHT (antioxidant) Soybean oil Vitamin and mineral premixa Sodium selenite (0.18%)

10.00 54.00 20.00 11.50 0.20 1.50 0.82 0.10 0.10 0.02 1.68 0.50 0.00

10.00 54.00 20.00 11.50 0.20 1.50 0.82 0.10 0.10 0.02 1.68 0.50 0.00845

2. Materials and methods

Proximate composition Crude protein (%) Dry matter (%) Ether extract (%) Ash (%) Crude fiber (%) Selenium (mg/kg)b

35.91 96.89 4.12 8.60 7.11 0.0024

35.77 96.67 4.10 8.42 7.57 1.42

2.1. Chemicals A commercial formulation of the OP methyl parathion (O, O-dimethyl O-4-nitrophenyl phosphorothioate) - Folisuper 600 BR® (methyl parathion 600 g L− 1, Agripec) was used. All the other chemicals and reagents were purchased from Sigma-Aldrich Chemical Co. and Merck. 2.2. Animals, diet preparation and experimental design Fish (B. cephalus, Günther, 1869 (Actinopterygii: Characidae) (mean mass ~ 10 g) were obtained at the Águas Claras fish farm, Mococa, São

Table 1 Composition of the experimental diets (% dry weight) and diet proximate composition Diets

a Vitamin and mineral premix contains the following amounts of vitamins and minerals/kg of mixture and it is purchased from Supremais Biochemical Products (Valinhos, São Paulo, Brazil): Folic acid 1200 mg; Calcium pantothenate 12 000 mg; Vitamin B1 4800 mg; Vitamin B2 4800 mg; Vitamin B6 4800 mg; Vitamin B12 4800 mg; Niacin 24 000 mg; Vitamin A 1200 000 000 UI; Vitamin K 2400 mg; Vitamin D3 200 000 000 UI; Vitamin C 48 000 mg; Vitamin E 12 000 mg; Cobalt 2 mg; Copper 600 mg; Iron 10 000 mg; Iodine 20 mg; Manganese 4000 mg; Zinc 6000 mg. b Selenium levels obtained by atomic absorption spectrometry.

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After the experimental feeding period of 8 weeks, fish were divided into four experimental groups of ten fish each: — Control group (fish fed Se-free Diet I), MPc group (fish fed Se-free Diet I and exposed to 2 mg L− 1 of Folisuper 600® — MPc 600 g L− 1 during 96 h), Se group (fish fed Diet II supplemented with 1.5 mg of Se kg− 1), MPc + Se group (fish fed Diet II supplemented with 1.5 mg of Se kg− 1 and exposed to 2 mg L− 1 of Folisuper 600® — MPc 600 g L− 1 during 96 h). Each experimental group was tested in duplicates (10 fish each group, n = 20) and these duplicates were statistically equivalents. The fish were starved for 24 h prior to experimentation to avoid prandial effects and to prevent the deposition of feces in the course of the assay. After 24 h, the water was renewed and MPc and MPc + Se groups were submitted to a concentration of 2 mg L− 1 Folisuper 600® (1/3 of 96 h-LC50 previously established by Aguiar et al., 2004). Opaque experimental tanks were used to avoid external disturbances and they were sealed with a dark plastic cover to prevent sample volatilization. Dissolved oxygen, temperature and a photoperiod were maintained as described for the acclimation period. The fish remained under the semi-static system for 96 h where the experimental MPc solutions were renewed every 24 h to maintain water quality and adjust the MPc concentration. The Control and Se groups were submitted to the same protocol but without adding MPc. The toxicity test was conducted according to OECD (1992) 203 guidelines. 2.3. Quantification of MP MPc concentration was monitored spectrophotometrically at 275 nm (absorption peak obtained by multiple wavelength scans for the Folisuper 600 BR®) using a standard curve previously determined with Folisuper 600 BR®. 2.4. Sample preparations and biochemical assays At the end of 96 h of exposure to MPc, fish from the experimental groups were killed by spinal cord transection. After biometry, the gills, liver and white muscle were carefully excised and washed with cold saline (0.9% NaCl). Tissue samples were immediately frozen in liquid nitrogen. Frozen samples were stored at −80 °C until the biochemical determinations were carried out. The hepatic somatic index (HSI) was obtained by dividing the liver weight by the total body weight. The tissue homogenates were obtained in 0.1 M sodium phosphate buffer pH 7.0 at a ratio of 1:10 w/v. Homogenizations were carried out at 4 °C in a Turratec TE 102 homogenizer at 18 000 r.p.m., followed by centrifugation at 12 000 ×g for 30 min at 4 °C. The supernatants were collected and used to evaluate enzymatic (SOD, CAT, GPx and GST) and non-enzymatic (GSH) antioxidants and lipid peroxidation levels (LPO). The different parameters were analyzed spectrophotometrically at 25 °C according to the following procedures: 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 (Otero et al., 1983). The assay was performed in a 0.5 M sodium

Table 2 Body mass, liver mass and hepatic somatic index of Brycon cephalus fed during 8 weeks with diets containing 0 or 1.5 mg Se kg− 1 after 96 h of exposure to 2 mg L− 1 of Folisuper 600 BR® or to control conditions Groups

Body mass (g)

Liver mass (g)

Hepatic somatic index (%)

Control MPc Se MPc + Se

29.66 ± 4.57 30.72 ± 5.22 32.96 ± 2.83 29.93 ± 4.58

0.33 ± 0.05 0.27 ± 0.012a 0.35 ± 0.06c 0.34 ± 0.04c

1.36 ± 0.41 0.87 ± 0.12a 1.41 ± 0.29c 1.31 ± 0.12c

Values are mean ± S.D., n = 20. a vs. Control (P b 0.05). b vs. Se (P b 0.05). c vs. MPc (P b 0.05).

Table 3 Protein contents (mg mL− 1) in liver, white muscle and gills of Brycon cephalus fed during 8 weeks with diets containing 0 or 1.5 mg Se kg− 1 after 96 h of exposure to 2 mg L− 1 of Folisuper 600 BR® or to control conditions Groups

Liver

White muscle

Gills

Control MPc Se MPc + Se

7.86 ± 1.31 7.98 ± 1.08 8.09 ± 0.98 8.15 ± 1.03

4.15 ± 0.84 4.00 ± 0.35 4.03 ± 0.64 4.41 ± 0.44

5.49 ± 1.19 5.32 ± 0.81 5.55 ± 0.69 5.53 ± 0.79

Values are mean ± S.D., n = 20.

carbonate buffer (pH 10.2) with 2 mM EDTA and 10 10 μL aliquot of the supernatant. The reduction of NBT by superoxide anion to blue formazan was measured at 560 nm. The rate of NBT reduction in the absence of tissue was used as the reference rate. One unit of SOD was defined as the amount of protein needed to decrease the reference rate to 50% of maximum inhibition. The SOD activity was expressed in units/mg of protein. CAT activity was determined according to Aebi (1974) by following the consumption of 15 mM H2O2 at 240 nm in 50 mM KH2PO4/ Na2HPO4 buffer, pH 7.0 and 50 μL supernatant. CAT values were expressed as Bergmeyer units (B.U.)/mg of protein. One unit of CAT is the amount of enzyme which liberates half the peroxide oxygen from the H2O2 solution of any concentration in 100 s at 25 °C. 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 using 5.5′-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 μL supernatant of tissue homogenates. Absorbance was recorded at 412 nm. One unit of GPx enzyme activity was defined as 1 μg of GSH consumed/min (Latha and Pari, 2004). The GPx activity was expressed in units/mg of protein. The GST activity was measured according to Habig et al. (1974) using 1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate. The assay mixture contained 1 mM CDNB in ethanol, 1 mM GSH, 100 mM potassium phosphate buffer (pH 7.0) and 20–50 μL supernatant of tissue homogenates. The formation of adduct S-2, 4-dinitrophenyl glutathione was monitored by the increase in absorbance at 340 nm against a blank. The activity was expressed as the amount of enzyme catalyzing the formation of 1 nmol of the product formed/min/mg of protein. Reduced glutathione (GSH) levels were measured according to Beutler et al. (1963), using Elmann's reagent (DTNB). Supernatants of the acid extracts (1:1 v/v with 12% TCA) were added to 0.25 mM DTNB in 0.1 sodium phosphate buffer, pH 8.0, and thiolate anion formation was determined at 412 nm against a GSH standard curve. Lipid peroxidation (LPO) was determined by measuring the lipid hydroperoxide (HP) levels using the FOX method (Ferrous OxidationXylenol orange) as described by Jiang et al. (1992). After deproteinisation with 10% TCA (1:1 v/v) and the removal of particles by centrifugation, samples were incubated for 30 min at room temperature with a reaction mixture containing 0.25 mM FeSO4, 25 mM H2SO4, 0.1 mM xylenol orange and 4 mM butylated hydroxytoluene in 90% (v/v) methanol. HP levels were detected spectrophotometrically at 560 nm and expressed as nmol/mg of protein using the molar extinction coefficient of 4.3 × 104 M− 1 cm− 1 for cumene hydroperoxide (Jiang et al., 1991). Protein determination was carried out according to the method of Bradford (1976) with Coomassie Brilliant Blue G-250 adapted to a microplate reader as described by Kruger (1994) using bovine serum albumin as a standard. Absorbance of samples was measured at 595 nm. 2.5. Selenium content The tissue and diet Se content were determined by an atomic absorption spectrometry as described by Welz et al. (1992) and Rosa et al.

D.A. Monteiro et al. / Comparative Biochemistry and Physiology, Part C 149 (2009) 40–49 Table 4 Selenium concentration in liver, white muscle and gills of Brycon cephalus fed during 8 weeks with diets containing 0 (Diet I) or 1.5 mg Se kg− 1 (Diet II) Selenium concentration (μg g− 1)

Tissues

Diet I Liver White muscle Gills

Diet II

Table 6 Antioxidant enzyme activities in the white muscle of Brycon cephalus fed during 8 weeks with diets containing 0 or 1.5 mg Se kg− 1 after 96 h of exposure to 2 mg L− 1 of Folisuper 600 BR® or to control conditions Groups

SOD (U mg protein− 1)

CAT (B.U. mg protein− 1)

GPx (U mg protein− 1)

GST (nmol min− 1 mg protein− 1)

Control MPc Se MPc + Se

13.77 ± 1.00 21.24 ± 2.70a 5.81 ± 2.44a,c 10.15 ± 3.24a,b,c

0.31 ± 0.09 0.56 ± 0.13a 0.42 ± 0.15 0.38 ± 0.18

53.70 ± 7.00 31.73 ± 3.46a 46.33 ± 3.26c 48.94 ± 5.16c

13.35 ± 1.62 24.67 ± 9.36a 16.30 ± 2.75 26.11 ± 4.25a,b

a

11.40 ± 0.02 3.35 ± 0.07 3.44 ± 0.06

13.54 ± 1.36 3.23 ± 0.35 6.00 ± 0.22a

Values are mean ± S.D., n = 6. a vs. Diet I (P b 0.05).

(2002). The fish and diet samples were mineralized in triplicate with nitric acid in a closed-vessel microwave-assisted acid-digestion system. Briefly, a volume of 2.5 mL HNO3 and 0.5 mL of 30% v/v H2O2 was added to 100 mg of dried powdered materials placed into PTFE tubes. Thereafter, the following power/time program was run: step 1, 200 W/1 min; step 2, 0 W/1 min; step 3, 400 W/3 min; step 4, 500 W/3 min; step 5, 700 W/ 3 min; step 6, ventilation. After cooling, the resulting acid digestates were diluted to 10 mL with distilled water and 10 μl + 2 μl of 100 mg L− 1 palladium nitrate were transferred to the auto-sampler cup of an atomic absorption spectrometer equipped with pyrolytic graphite coated graphite tubes. Absorbance signals were measured in a peak area mode. The Argon gas flow-rate was maintained at 1 L min− 1 during all the temperature steps, except during atomization in which the gas flow was stopped. The Se content was expressed in mg/kg of tissues or diet.

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Values are mean ± S.D., n = 20. a vs. Control (P b 0.05). b vs. Se (P b 0.05). c vs. MPc (P b 0.05).

The exposure to MPc caused a significant decrease in liver mass and HSI when compared to control (30% and 16% respectively). This MPc effect was abolished in the Se + MPc group, which presented values similar to those presented by control and Se groups (Table 2). Tissue protein contents are shown in Table 3. None of the experimental treatments showed significant alterations in protein levels in liver, gills and white muscle.

In fish fed on Diet II (1.5 mg Se kg− 1), the selenium concentrations in liver and gills were significantly higher than those of fish fed on Diet I (Se-free diet) after the experimental period of 8 weeks (19% and 74% respectively), but no alterations were observed in the Se content of white muscle (Table 4). The antioxidant enzyme activities in the liver, white muscle and gills are shown in Tables 5 to 7, respectively. In the MPc group, significant increases in the SOD, CAT and GST activities were observed in all tissues when compared to the control. However, the GPx activity decreased significantly in the white muscle (41%) and gills (15%), whereas no alterations were observed in hepatic GPx activity in this group. The Se supplementation reversed the MPc effects of decreased GPx activity in white muscle and gills, as the MPc + Se group recovered the values presented by the control and Se groups (Tables 6 and 7). The MPc exposure induced increases in the hepatic SOD, CAT and GST activities even after Se supplementation (MPc + Se group). In MPc + Se group the hepatic GPx activity was higher than that observed in MPc group showing that Se supplementation reversed the decreased in GPx activity induced by MPc (Table 5). The MPc exposure caused increases in SOD and GST activities in spite of the diet (with or without Se) in white muscle. With Se supplementation, SOD activity was lower than in the control and MPc treatments. The CAT activity of the MPc + Se group was maintained at the same levels of control and Se groups. Additionally, the SOD activity in the Se group was lower than that measured for the control. In the gills, the Se supplementation prevented the increased SOD activity shown by the MPc group while the GST and CAT activities were increased irrespective of diet. Fig. 1 shows the levels of lipid peroxidation in liver, white muscle and gills of experimental groups. The hepatic HP levels were maintained unchanged in all experimental groups. In the MPc group, HP levels in the white muscle and gills increased significantly when compared to the control group. Selenium supplementation completely inhibited the MPc-induced increases in HP in gills and white muscle of MPc + Se group (Fig. 1).

Table 5 Antioxidant enzyme activities in the liver of Brycon cephalus fed during 8 weeks with diets containing 0 or 1.5 mg Se kg− 1 after 96 h of exposure to 2 mg L− 1 of Folisuper 600 BR® or to control conditions

Table 7 Antioxidant enzyme activities in the gills of Brycon cephalus fed during 8 weeks with diets containing 0 or 1.5 mg Se kg− 1 after 96 h of exposure to 2 mg L− 1 of Folisuper 600 BR® or to control conditions

2.6. Statistical analysis The values in all determinations are presented as means± S.D. Differences between means were analyzed by using one-way ANOVA followed by Tukey's test (GraphPad Instat version 3.00, GraphPad Software, USA). Two-way analysis of variance ANOVA (SigmaStat version 2.0 Jandel SigmaStat Statistical Software, USA) was used to determine the interaction between the two factors MPc exposure and Se diet levels. Assumptions of normality and homoscedasticity were in accordance for ANOVA requirements. By analyzing this, possible interactions between pretreatment with Se and methyl parathion exposure could be detected. The t-test was used to analyze the effects of the 2 different diets in the Se concentration in different tissues. Differences between means were considered significant at P b 0.05 for all tests used. 3. Results

Groups

SOD (U mg protein− 1)

CAT (B.U. mg protein− 1)

GPx (U mg protein− 1)

GST (nmol min− 1 mg protein− 1)

Groups

SOD (U mg protein− 1)

CAT (B.U. mg protein− 1)

GPx (U mg protein− 1)

GST (nmol min− 1 mg protein− 1)

Control MPc Se MPc + Se

11.41 ± 1.24 14.53 ± 2.17a 9.52 ± 1.76c 12.63 ± 1.04b

4.06 ± 1.24 6.20 ± 1.18a 4.28 ± 0.44 6.17 ± 0.95a,b

33.60 ± 2.70 31.43 ± 4.36 35.56 ± 5.15 38.31 ± 2.63c

86.65 ± 15.80 118.22 ± 32.52a 58.85 ± 7.09c 109.67 ± 23.05b

Control MPc Se MPc + Se

14.98 ± 2.26 18.26 ± 2.43a 13.58 ± 1.76c 13.57 ± 2.72c

0.51 ± 0.17 0.92 ± 0.14a 0.69 ± 0.12b 0.88 ± 0.12a,b

37.50 ± 7.15 31.74 ± 3.46a 41.98 ± 3.12a,c 39.48 ± 4.79c

79.76 ± 19.52 138.77 ± 20.35a 85.60 ± 28.59 191.73 ± 56.70a,b

Values are mean ± S.D., n = 20. a vs. Control (P b 0.05). b vs. Se (P b 0.05). c vs. MPc (P b 0.05).

Values are mean ± S.D., n = 20. a vs. Control (P b 0.05). b vs. Se (P b 0.05). c vs. MPc (P b 0.05).

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Selenium has relevant roles in the organism for growth, cell membranes normal conditions, immunity and carcinogenic inhibition (Combs and Combs, 1986). One of the most important functions of Se is related to its antioxidant role and participation in the antioxidant defense system (Köhrle et al., 2005). Oxidative stress can be induced by a large variety of conditions, including nutritional imbalance, exposure to chemical and physical agents in the environment, strenuous physical activities, injury, and hereditary disorders (Chow, 1991). The present study examined an attractive hypothesis concerning the antioxidant potential of Se in fish exposed to Folisuper 600 BR® (commercial formulation of methyl parathion). In mammals, selenium dietary supplementation has been demonstrated to reduce oxidative stress (Atroshi et al., 1999; Kowluru et al., 2000; Rizzo et al., 1994).

Fig. 1. Lipid hydroperoxide (HP) levels in liver (A), white muscle (B) and gills (C) of Brycon cephalus fed during 8 weeks with diets containing 0 or 1.5 mg Se kg− 1 after 96 h of exposure to 2 mg L− 1 of Folisuper 600 BR® or to control conditions. Values are mean ± S.D., n = 20. aIndicates significant difference in relation to Control (P b 0.05). b Indicates significant difference in relation to the Se group (P b 0.05). cIndicates significant difference in relation to the MPc group (P b 0.05).

The MPc group presented significantly lower GSH levels in all the analyzed tissues when compared to the control group. These effects were completely inhibited in the MPc + Se group (Fig. 2). Significant interactions between MPc exposure and Se supplementation were observed for the gills and white muscle SOD, CAT, GPx activities and GSH and HP levels, and for the hepatic GSH levels (Table 8). 4. Discussion Micronutrients, including vitamins and minerals, besides the crucial role in the normal growth, reproduction and health of fish (Kim et al., 2003), are also essential in the antioxidant defense system (MartinezAlvarez et al., 2005).

Fig. 2. Reduced glutathione (GSH) levels in liver (A), white muscle (B) and gills (C) of Brycon cephalus fed during 8 weeks with diets containing 0 or 1.5 mg Se kg− 1 after 96 h of exposure to 2 mg L− 1 Folisuper 600 BR® or to control conditions. Values are mean ± S.D., n = 20. aIndicates significant difference in relation to Control (P b 0.05). b Indicates significant difference in relation to the Se group (P b 0.05). cIndicates significant difference in relation to the MPc group (P b 0.05).

D.A. Monteiro et al. / Comparative Biochemistry and Physiology, Part C 149 (2009) 40–49 Table 8 The two-way analysis of variance performed on the observations Parameters

Two-way ANOVA (selenium × methyl parathion)

Liver SOD CAT GPx GST GSH HP HSI

ns ns ns ns * ns ns

White muscle SOD CAT GPx GST GSH HP

* * * ns * *

Gills SOD CAT GPx GST GSH HP

* * * ns * *

*P ≤ 0.05 and ns P N 0.05.

However, few studies have been carried out regarding the antioxidant effect of Se in fish and its role on the impact of contaminants. Moreover, not one tropical fish species has been studied so far. The study of pesticide induced effects on various antioxidants in fish and other aquatic organisms can provide relevant information about the ecotoxicological consequences of pesticide use (Kavitha and Rao, 2007). Monteiro et al. (2006) demonstrated that the exposure to 2 mg L− 1 of Folisuper 600 BR® for 96 h induced significant increases in antioxidant enzymes such as SOD, CAT, GST and LPO levels with concomitant decreases in GPx activity, as well as the GSH content in the gills and white muscle, resulting in oxidative stress in B. cephalus. Moreover, the authors also described significant decreases in HSI in fish exposed to MPc. These results are compatible with the present study for fish fed with a Se-free diet and exposed to MPc. In the present study, the high levels of antioxidant enzymes (SOD, CAT and GST) demonstrate an MPc-induced adaptive response in attempting to neutralize the generated ROS. However, the enhanced lipid peroxidation in white muscle and gills of B. cephalus shows that insecticide-induced ROS are not totally scavenged by the antioxidant enzymes. This was aggravated by the decrease in GPx activity and GSH levels in these tissues. Organophosphate-induced oxidative stress was also observed after exposure to the OP diclorvos in other fish species such as the eel, Anguilla anguilla, carp, Cyprinus carpio and catfish, Ictalurus nebulosos (Hai et al., 1997; Peña-Llopis et al., 2003). However, Se supplementation showed a protective effect promoting the maintenance of a high steady state GSH level and a normal GPx activity after 96 h of exposure to 2 mg L− 1 of Folisuper 600 BR®. It also controlled lipid peroxidation in the gills and white muscle in fish exposed to this OP. These conditions showed that the intracellular redox balance and cellular integrity were maintained in fish fed with a Se-supplemented diet. In gills and white muscle, significant interactions between MPc exposure and Se supplementation were observed for SOD, CAT and GPx activities, GSH and HP levels. On the other hand, in the liver, the interaction among MPc exposure and Se supplementation was only observed for GSH levels. Therefore, the response of these parameters against Folisuper 600 BR® exposure depends on the Se level in the diet. These results show that Se supplementation is able to maintain the GPx activity and lipid peroxidation levels and the increases in GSH availability reducing ROS generation. Consequently, decreases in the

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SOD–CAT system activation were detected in response to Folisuper 600 BR® exposure, improving the antioxidant status of tissues susceptible to oxidative damage. The HSI is considered a general health indicator, reflecting both the metabolic energy demand and short-term changes in the nutritional status (Everaarts et al., 1993; Almeida et al., 2005). Decreases in HSI can indicate depletions in energy reserves stored as glycogen during stress responses (Wendelaar-Bonga, 1997). Rodrigues and Fanta (1998) and Machado and Fanta (2003) described that specimens of zebrafish, Brachydanio rerio, and spotted silver dollars, Metynnis roosevelt, exposed to MP presented decreased HIS due to hepatocyte necrosis. According to these authors, hepatocyte necrosis is a common effect in fish exposed to MP. Additionally, decreases in the HSI could result from a xenobioticinduced hepatic peroxisome proliferation, which could deplete the liver relative size through depletion of lipids by beta oxidation of fatty acids (Wolfrum et al., 2001; Wintz et al., 2006). The maintenance of liver weight and HSI in fish exposed to MPc and fed on 1.5 mg of Se kg− 1 suggests that Se supplementation contributes to hepatocyte proliferation, leading to hepatic regeneration, which is a critical step to prevent liver injury. Metabolism is controlled by the interactions of many hormones under different nutritional conditions, maintaining the energy reserves needed for the maintenance of the healthy organism (De Pedro et al., 2003). According to Sher (2001), Se status influences thyroid function and also affects other important functions. The author suggests that the effects of Se status on mood, behavior, and cognition may be partially mediated by changes in the thyroid function. In the present work, fish fed on a Se-free diet showed a decrease in swimming activity and growth, and increase in cannibalism after 50 days of treatment and color change as described by Monteiro et al. (2007). Taking this into account, specimens of spotted snakehead, Channa punctatus, and stinging catfish, Heteropneustes fossilis, treated with thyroxine (T4) by oral administration presented increased voluntary food intake and improved food consumption efficiency (Garg, 2007). Moreover, the radiothyroidectomy markedly reduced the rate of growth and development in juvenile rainbow trout (LaRoche et al., 1966). The Se hepatoprotective property probably results from its effect on the iodothyronine 5′-deiodinase activity, which makes the triiodothyronine (T3) thyroid hormone available, resulting in a generalized increase in the functional activity of the organism (Yamano, 2005). Selenium inorganic forms (selenate and selenite) are also efficiently absorbed from the gastrointestinal tract. Fractional absorption is greater than 50% and both selenate and selenite are delivered to the liver. A fraction of these forms are used by the hepatocytes and the rest are delivered via systemic circulation to the tissues of various organs. Within cells, these inorganic selenosalts are converted into hydrogen selenide (Dodig and Cepelak, 2004). Inorganic as well as organic Se forms can be metabolized to selenocysteine and incorporated into selenoenzymes (Dodig and Cepelak, 2004). Selenite, selenocysteine, and selenomethionine were equally effective in preventing the decreases in hepatic GPx activity as observed by El-Sayed et al. (2006) in weaning male rats after 9 weeks of treatment with a Se-deficient diet. Weiller et al. (2004) suggested that pro-oxidative reactions can be involved in the Se toxicity. When present in excessive amounts, selenium has been shown to interact with cellular sulphydryls, which leads to a depletion of intracellular glutathione and an increase in lipid peroxidation (Combs and Combs, 1986). On the other hand, our results indicate that pro-oxidative reactions did not occur in the metabolism of sodium selenite via a redox cycle between the Se compounds and reduced intracellular glutathione in fish treated with a Se-supplemented diet (1.5 mg Se kg− 1). Additionally, no alterations were observed in GSH and LPO levels in all tissues of the Se group and symptoms of selenosis were not observed, showing that the Se-supplemented diet was not toxic to matrinxã during the experimental period of 60 days. Monteiro et al. (2007) demonstrated that B. cephalus fed on a Sesupplemented diet (1.5 mg of Se kg− 1) for a period of 60 days

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presented better growth performance. For most fish, the requirement range is 0.25–0.70 mg Se kg− 1 diet (NRC, 1993) and the dietary toxic threshold is around 10 mg kg− 1 (Hamilton, 2003). DeForest et al. (1999) proposed a tissue Se toxic threshold of 11 µg g− 1 for cold-water fish and 10 µg g− 1 for warm-water fish. Our results, however, demonstrated that the diet supplemented with 1.5 mg of Se kg− 1 for a period of 60 days was not harmful for B. cephalus, even considering that the Se levels found in hepatic tissue were higher than this toxic level. Corroborating our results, Deng et al. (2007) found higher toxic Se thresholds for splittail (Pogonichthys macrolepidotus) than the previously described ones, ranging from 20 to 30 μg Se g− 1 in liver and from 11 to13 μg Se g− 1 in muscle. The Se-supplemented diet caused a significant Se accumulation in B. cephalus the liver and gills while no alterations were observed in white muscle. Similar Se tissue levels were obtained by Wang et al. (2007) for the allogynogenetic crucian carp (Carassius auratus gibelio) fed for 30 days on a diet supplemented with 0.5 mg Se kg− 1 (sodium selenite). Se tends to accumulate in tissues with intense metabolic activity such as liver and gills. Therefore, metal accumulation in these tissues occurs at higher levels when compared to others, like muscle, where metabolic activity is relatively lower (Heath, 1995; Roesijadi and Robinson, 1994; Canli et al., 1998; Saha et al., 2006). This tendency was observed in bluegill (Lepomis macrochirus), largemouth bass (Micropterus salmoides), white catfish (Ictalurus catus), and channel catfish (Ictalurus punctatus). In these species, Se was accumulated in higher concentrations in liver, followed by female reproductive and axial muscle tissues (Sager and Cofield, 1984). The Se concentration in different organs depends on the characteristics of the considered tissue, the amount and form of Se in the diet, the treatment duration and the studied species (Combs and Combs, 1986). Tissues ranked by Se concentration, generally follow the order: kidney N liver N pancreas and heart N skeletal muscle. This is remarkably similar among species. The greater portion of the absorbed selenium is stored in the liver (Underwood and Shutlle, 1999) and, in rainbow trout, liver and kidney are the primary Se storage sites (Hamilton, 2004). Wang et al. (2007) compared the effects of diets supplemented with different selenium sources on C. carassius gibelio and found that the Se concentration in muscle of a group treated with selenomethionine was higher than that of a group treated with sodium selenite. Similar results were observed in Atlantic salmon, Salmo salar, by Lorentzen et al. (1994) and in African catfish, Clarias gariepinus, by Schram et al. (2008). According to Waschulewski and Sunde (1988), selenomethionine is preferentially incorporated into muscle tissue of growing fish. In an attempt to dismutate superoxide anions and to decompose hydrogen peroxide, increases in SOD and CAT activities were detected in response to MPc exposure in fish fed on a Se-free diet. The increase in these enzymes was probably a response towards increased ROS generation in MPc toxicity (John et al., 2001). Similarly, exposure to MPc caused a significant increase in SOD, CAT activities in liver of fish fed on a Se-supplemented diet. However, CAT activity in white muscle of these animals was maintained at the same levels in the control group and increases in the SOD activity were less significant after exposure to MPc. When compared to the controls, SOD activity in the gills of the MPc + Se group did not change. Given that increases in the SOD activity were less significant or did not occur in white muscle and gills, it can be concluded that H2O2 formation declined in these tissues and that the CAT activity was sufficient to remove H2O2. Additionally, the lower SOD activity in the white muscle of the Se group could be U related to a decreased production and availability of O2− in response to the Se supplementation. These results are supported by the significant interactions found between Se and MPc for SOD and CAT activities in white muscle and gills. These interactions indicate that the response pattern of the SOD–CAT system resulting from MPc exposure is influenced by the presence of Se in diet.

The H2O2 level can also decrease due to its diffusion to the surrounding water. Wilhelm-Filho et al. (1994) demonstrated that an elimination of H2O2 by gill diffusion is an important physiological mechanism in freshwater fish Poecilia vellifera. If present, this mechanism does not seem to play an important role in the gills of B. cephalus since the MPc exposure induced increases in the CAT activity in spite of the diet (with or without Se). The increased CAT activity in the gills suggests that this organ is able to eliminate the blood H2O2 into the aquatic environment. This mechanism would explain the increased CAT activity in gills after exposure to MPc, despite the lack of change in the SOD activity in the MPc ± Se group. GST is a cytosolic or microsomal enzyme catalyzing the conjugation of electrophilic xenobiotics to GSH (Gadagbui and James, 2000) and plays a key role in protecting tissues from oxidative stress (Fournier et al., 1992). Despite the diet, the increased GST activity in all tissues after exposure to MPc indicates that this enzyme was induced either by the detoxification of hydroperoxides or by the GSH conjugation as part of Phase II of xenobiotic biotransformation. The selenium-dependent GPx is one of the enzymes protecting tissues from oxidative damage by reducing H2O2 and a wide range of organic hydroperoxides that form an important group of toxic compounds produced by oxygen metabolism (Helmy et al., 2000). The decreased GPx activity in gills and white muscle observed in the U present study after MPc exposure could be related to the O2− production, as suggested by Bagnasco et al. (1991) or to the direct action of this OP on the enzyme synthesis (Bainy et al., 1993). It is well known that GPx prevents lipid peroxidation in the membranes and acts as a ROS scavenger (Orbea et al., 2000). Our results showed that Se supplementation was able to maintain GPx activity close to control values in gills and white muscle after MPc exposure. Indeed, significant interactions between MPc and Se were verified for GPx activity in these tissues showing that Se supplementation prevented decreases in the GPx activity after MPc exposure. Selenium is fundamental for Se-dependent GPx activity, and dietary Se-deficiency depresses the activity of this enzyme in fish (Poston et al., 1976; Bell and Cowey, 1987; Watanabe et al., 1997). In the present study, a significant decrease in the GSH levels in all tissues was observed in fish exposed to MPc compared to the control group. GSH depletion decreases the reduced/oxidized glutathione ratio, which leads to the production of ROS, facilitating the production of lipid peroxidation (Nehru and Bansal, 1997; Sk and Bhattacharya, 2006). GSH protects cells from oxidative stress and plays a critical role in detoxification reactions by acting both as a nucleophilic scavenger of various undesired compounds and their toxic metabolites, and as a specific substrate for the enzyme glutathione peroxidase and glutathione S-transferase (Sk and Bhattacharya, 2006). Selenium enhanced the GSH availability, as demonstrated by the significant interactions between MPc and Se for GSH levels in all tissues. GSH is one of the most abundant intrinsic antioxidants preventing lipid peroxidation and, consequently, cell oxidative impairment (Hsu and Guo, 2002). In the present study, Se inhibited the GSH reduction in white muscle and gills after MPc exposure, showing its antioxidant effect. According to Sarada et al. (2002), Se facilitates GSH-dependent enzymatic antioxidants in the cellular system. The present results showed that Se enhanced the cell antioxidant capacity, protecting white muscle and gill against the MPc-induced damages, as shown by the maintenance of GPx activity and GSH content. GSH is an effective ROS scavenger, essential for GPx and GST activities, and is considered the first line of the antioxidant defense (Lima et al., 2006). The reduced GSH levels found after 96 h exposure to MPc can be considered an oxidative challenge for matrinxã. The overproduction of ROS, as indicated by the high level of lipid peroxidation, may be associated with the depletion in the GSH level (Arthur, 2000). The depletion of the glutathione level observed in the present study is one of the factors responsible for the enhanced lipid peroxidation.

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Therefore, decreases in the GSH content make the fish cells more susceptible to attack by toxic electrophilic compounds such as MPc. Increased lipid peroxidation is often a quick response to ROS generation (Halliwell and Gutteridge, 1999). Tissue-specific lipid peroxidation changes (measured as HP concentrations) in response to MPc exposition were observed in this study. The animals exposed to MPc showed significant increases in gills and white muscle HP levels while no alterations were observed in the liver. Differing from the majority of the Amazon basin fish species, high Vitamin E content was found in the liver of B. cephalus (Wilhelm-Filho and Marcon, 1996). These high Vitamin E levels in the liver of matrinxã probably provided an additional protecting effect against the LPO and, consequently, prevented increases in the hepatic LPO levels. This mechanism should explain the absence of significant interactions between MPc and Se for all the analyzed parameters in liver, except for GSH levels. The increased LPO in white muscle and gills suggests that the MPcmediated ROS production can be the cause of oxidative stress in these tissues. According to Ates et al. (2008), the lipid peroxidation is a complex, self-propagating and highly destructive process, increasing the rigidity of cellular membranes. According to Hermes-Lima (2004), LPO products may be involved in the CAT up-regulation and, therefore, higher CAT activities are observed when high HP contents are present. Moreover, GSH plays a critical role in the cellular redox maintenance. In the present study, the maintenance of LPO and GSH levels in white muscle by Se supplementation probably contributed to the maintenance of CAT activity similar to control values after MPc exposure. The increased HP contents found in the gills and white muscle of B. cephalus after 96 h MPc exposure suggests that this species was not able to compensate the ROS induced by this OP. Accordingly, the MPcinduced ROS formation and lipid peroxidation would play a role in its cytotoxicity (Monteiro et al., 2006). Likewise, the MPc-induced oxidative stress initiates an irreversible process resulting in the loss of an antioxidant compensatory response, reflected by reduced GPx activity and GSH levels in these tissues. These effects were counteracted by the Se supplementation. The significant interactions between MPc and Se reinforce the role of Se supplementation in preventing increases in the HP levels induced by MPc exposure in white muscle and gills. Nutritional studies have also revealed the crucial role of some minerals in preventing oxidative stress. According to MartinezAlvarez et al. (2005), selenium (Se), manganese (Mn), copper (Cu), and zinc (Zn) are nutritional factors involved in LPO prevention. Selenium compounds effectively decreased lipid peroxidation levels in mice treated with cadmium (Sk and Bhattacharya, 2006) and carbon tetrachloride (Das et al., 2004). Ates et al. (2008) demonstrated that selenium prevented the LPO induced by heavy metals such as lead and copper in rainbow trout (Onchorynchus mykiss). Taken together, our findings indicate that pretreatment with 1.5 mg of Se/kg of diet during 8 weeks prevents MPc-induced oxidative stress in B. cephalus. It seems that Se supplementation improves the antioxidant compensatory responses against deleterious MPc effects, which was shown by the maintenance of GSH and HP levels and GPx activity in gills and white muscle. Additionally, Se supplementation provides a hepatoprotective effect, as revealed by HSI values after MPc exposure. The ability of Se to restore the GPx activity and glutathione status involved in the antioxidant defense mechanisms may be crucial to biological protection from the toxic effects of MP. Moreover, Se has the ability to counteract ROS and protect the structure and function of proteins, lipids and DNA against oxidative damages (Yuan and Tang,1999). In conclusion, our results indicate that dietary intake of selenium can modulate some of the functional and structural damages of oxidative stress induced by MPc in the white muscle of B. cephalus. Different strategies have been proposed to inhibit xenobiotic-induced toxicity and to prevent the action of generated ROS and oxidative damage in response to pollutants such as MP. Taking this into account, natural antioxidants such as Se can be easily and safely increased in tissues using

47

diet supplementation. Zhu et al. (2008) suggested that the induction of oxidative stress may have an inhibitory effect on fish growth. Considering that MP is extensively used in aquaculture to control insect larvae and that small amounts of this OP can induce oxidative stress in B. cephalus, the introduction of antioxidants agents such as Se in the diet could be valuable to achieve the desirable production parameters of cultivated fish as health and growth performance. Acknowledgments The authors are thankful to CAPES (D.A. Monteiro fellowship) and Águas Claras fish farm, which provided the fish. They are also grateful to Supremais — Nutrição Animal for providing the vitamin/mineral premix and sodium selenite used to prepare the experimental diets. References Adams, S.M., Shepard, K.L., Greeley, M.S., Jimenez, B.D., Ryon, M.G., Shugart, L.R., McCarthy, J.F., Hinton, D.E., 1989. The use of bioindicators for assessing the effects of pollutant stress on fish. Mar. Environ. 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