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Effects of diphenyl diselenide on growth, oxidative damage, and antioxidant response in silver catfish
Science of the Total Environment 542 (2016) 231–237
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Effects of diphenyl diselenide on growth, oxidative damage, and antioxidant response in silver catfish Charlene Menezes a,d,e, Aline Marins a, Camila Murussi b,d, Alexandra Pretto c, Jossiele Leitemperger b,d, Vania Lucia Loro a,d a
Programa de Pós — Graduação em Biodiversidade Animal Programa de Pós — Graduação em Bioquímica Toxicológica Universidade Federal do Pampa, Campus Uruguaiana, Uruguaiana, RS, Brazil d Laboratório de Toxicologia Aquática, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil e Corresponding author at: Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Santa Maria, 97105.900 Santa Maria, RS, Brazil b c
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Selenium is element participates of various metabolic routes. • (PhSe)2 in different concentrations in diet were investigated on silver catfish. • 1.5 and 3.0 mg/kg of (PhSe)2 increased antioxidant defenses of fish. • The best results were obtained after 60 days of feeding with (PhSe)2.
a r t i c l e
i n f o
Article history: Received 27 April 2015 Received in revised form 20 October 2015 Accepted 21 October 2015 Available online 3 November 2015 Editor: D. Barcelo Keywords: Antioxidants Fish Selenium Oxidative parameters
a b s t r a c t The aim of this study was to evaluate the effects of dietary diphenyl diselenide [(PhSe)2] at different concentrations (1.5, 3.0, and 5.0 mg/kg) on growth, oxidative damage and antioxidant parameters in silver catfish after 30 and 60 days. Fish fed with 5.0 mg/kg of (PhSe)2 experienced a significant decrease in weight, length, and condition factor after 30 days and these parameters increased after 60 days. Thiobarbituric acid reactive substances (TBARS) and protein carbonyl (PC) decreased in the liver of silver catfish supplemented with (PhSe)2 after 30 days at all concentrations, while after 60 days these parameters decreased in liver, gills, brain, and muscle. Supplementation with (PhSe)2 induced a decrease in catalase (CAT) activity from liver only after 60 days of feeding. Superoxide dismutase (SOD) decreased at 5.0 mg/kg after 30 and 60 days and glutathione peroxidase (GPx) was enhanced at 1.5 and 3.0 mg/kg after 30 and 60 days. Silver catfish supplemented for 30 days showed a significant increase in liver glutathione S-transferase (GST) at 3.0 mg/kg, while after 60 days GST activity increased in liver at 1.5, 3.0, and 5.0 mg/kg and in gills at 3.0 and 5.0 mg/kg of (PhSe)2. After 30 days, non-protein thiols (NPSH) did not change, while after 60 days NPSH increased in liver, gills, brain, and muscle. In addition, ascorbic acid (AA) levels after 30 days increased in liver at three concentrations and in gills and muscle at 1.5 mg/kg, while after 60 days, AA increased at all concentrations in all and tissues tested. Thus, diet supplemented with (PhSe)2
E-mail address: [email protected] (C. Menezes).
http://dx.doi.org/10.1016/j.scitotenv.2015.10.110 0048-9697/© 2015 Elsevier B.V. All rights reserved.
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for 60 days could be more effective for silver catfish. Although the concentration of 5.0 mg/kg showed decreased growth parameters, concentrations of 1.5 and 3.0 mg/kg, in general, decreased oxidative damage and increased antioxidant defenses. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Selenium (Se) is an essential nutrient in the diet of a variety of organisms, because it is important for growth, development, and physiological function (Hamilton, 2004; Kim and Kang, 2014). Se is an antioxidant mineral present in various selenoproteins that contain one or more selenocysteine residues in their active sites. This element participates in various metabolic routes, especially those involved in the antioxidant system, as its presence in active sites of glutathione peroxidase (GPx) (Nogueira et al., 2004; Li et al., 2008; Monteiro et al., 2009). However, nutritional requirements and toxic levels of Se exhibit a fine partition, and this element may be either essential or toxic, depending on its concentration. In fact, at high cellular concentrations, Se may be utilized in place of sulfur, causing errors in protein synthesis and damage to other molecules (e.g. lipids and DNA) as well as reproductive impairment, larval deformities, and mortality (Muscatello et al., 2006). Diphenyl diselenide [(PhSe)2] is a synthetic organoselenium compound that has been considered a potential pharmacological and antioxidant agent in various experimental models, such as fish, rats, and mice (Menezes et al., 2012; Costa et al., 2013; Fiuza et al., 2015). The exposure of fish to xenobiotics is a main cause of oxidative damage due to increased production of reactive species oxygen (ROS) in exposed organisms (Murussi et al., 2014; Pretto et al., 2014). Thus, the use of antioxidants such as Se in the diet may permit animals to overcome, healthy and without damage, adverse conditions that may occur (Monteiro et al., 2007). Menezes et al. (2012) evaluated the effects of (PhSe)2 on oxidative damage and antioxidant profile in different tissues of Cyprinus carpio. The authors observed that fish fed a diet without (PhSe)2 and exposed to Facet® herbicide showed oxidative damage in different organs, while (PhSe)2 treatment reversed oxidative damage by increasing some antioxidant defenses. Thus, (PhSe)2 could be a powerful antioxidant. However, for other species of fish, the requirement in terms of concentration and form as well as the threshold dose for its opposing toxic properties has not yet been established. The silver catfish (Rhamdia quelen), is a fish species with an extensive geographic distribution, occurring from southern Mexico to central Argentina. Its features, such as tolerance to management, omnivorous feeding behavior, ability to grow throughout the winter, and high yield, place it in a prominent position among the native species of interest to aquaculture in the region of southern Brazil (Fracalossi et al., 2004; Baldisserotto, 2009). Studies have evidenced the possibility that dietary supplementation with certain vitamins and minerals can increase disease resistance, prevent negative effects of stress, and minimize the toxicity of contaminants in fish (Borba et al., 2007; Monteiro et al., 2009; Menezes et al., 2012, 2014a). The objective of this study was to evaluate the efficacy of dietary supplementation with different concentrations of (PhSe)2 for 30 and 60 days in silver catfish, in order to establish the concentration that warrants further exploration as a potential supplement in silver catfish nutrition. 2. Materials and methods 2.1. Chemicals The reagents chemicals used in this study were obtained from Sigma Chemical Co. (St. Louis, MO, USA) and Merck (Rio de Janeiro, Brazil). Diphenyl diselenide [(PhSe)2] was synthesized according with
Paulmier (1986). Analysis of the 1HNMR and 13CNMR spectra showed analytical and spectroscopic data in agreement with its assigned structure. 2.2. Fish Silver catfish (body length, 7.0 ± 1.0 cm; mean weight, 18.0 ± 1.0 g) of both sexes were acquired from a local fish farm (RS, Brazil). Before the experiment, the fish were acclimated to the laboratory conditions for 15 days, in 250 L fiberglass boxes. They were kept in continuously aerated dechlorinated tap water with a static system and with a natural photoperiod (12 h light/12 h dark). During the acclimation, water temperature was maintained at 22.5 ± 1.0 °C, the dissolved oxygen content was 7.21 ± 1.0 mg/L, pH between 7.3 and 7.7, non-ionized ammonia 0.3 ± 0.01 μg/L and nitrite 0.05 ± 0.01 mg/L. In this period of acclimation, fish were fed once a day with commercial fish pellets (Supra, Brazil). Feces and food residues were removed by syphon and filter system was used to keep the quality of water. 2.3. Diet preparation and experimental design During the experiment, silver catfish were divided in four groups (n = 20 per group): (1) control, (2) (PhSe)2 1.5 mg/kg, (3) (PhSe)2 3.0 mg/kg and (4) (PhSe)2 5.0 mg/kg. The fish of control group were supplemented with diet without (PhSe)2. All fish were fed during 30 and 60 days and (PhSe)2 was added to the control diet. Diet formulation was performed based in previous studies from our group (Menezes et al., 2012, 2014b). Diets were made into pellets (5 mm, diameter) and stored at 4 °C until fed. During the experiment, silver catfish were fed with 3% biomass per day. The daily ration was divided into two equal meals fed at 09:00 and 16:00 h. The feces and food residues were removed by syphoning. Temperature, dissolved oxygen, pH, ammonia and nitrite were evaluated daily and were maintained similar values to those recorded during the acclimation period. At end of the each experimental period (30 and 60 days), ten fish of each group (n = 10) were weighed and measured. Total length (TL) and body weight (W) for each fish were recorded to evaluate growth and condition factor K (K = (weight / length3) × 100). Afterwards, the fish were anesthetized with 50 mg/L clove oil and euthanized by punching the spinal cord behind the opercula. Liver, gills, brain, and muscle were quickly collected and stored at − 80 °C for posteriors analysis. The study was approved by the Committee on Ethics and Animal Welfare of Federal University of Santa Maria, under the number: 84/2009. 2.4. Biochemical analysis At end of the each experimental period (30 and 60 days), ten fish of each group (n = 10) were used to biochemical analysis. In liver, gills, brain, and muscle the lipid peroxidation (LPO) levels was estimated using the thiobarbituric acid reactive substances (TBARS) assay of according to Buege and Aust (1978) and the protein carbonyl (PC) determination was measured by the method described by Yan et al. (1995). In liver, the superoxide dismutase (SOD) activity was determined as described by Misra and Fridovich (1972) based on inhibition of the radical superoxide reaction with adrenalin. Catalase (CAT) activity in liver was determined as reported by Nelson and Kiesow (1972). Glutathione peroxidase (GPx) activity in liver was measured by following the rate of NADPH oxidation at 340 nm by the coupled reaction with glutathione reductase as described by Paglia and Valentine (1987). The glutathione
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S-transferase (GST) activity was assayed in liver, gills, brain, and muscle of according to Habig et al. (1974). In liver, gills, brain, and muscle the non-protein thiols (NPSH) and ascorbic acid (AA) content was evaluated by the method of Ellman (1959) and Roe (1954) respectively. The protein content was measured as described by Bradford (1976) using bovine serum albumin as standard. All biochemical analyses were described in details in previous publications of our research group (Menezes et al., 2012, 2013). 2.5. Statistical analysis The data are presented as mean ± standard error of mean (S.E.M). Statistical analyses were realized using a two-way analyses of variance (ANOVA) followed by Newman–Keuls post hoc comparison. Analyses were performed using Graph Prism (Version 6.0) and the significance level was set at p b 0.05. 3. Results Mean values of W, TL, and K were recorded for two periods (30 and 60 days) (Table 1). At 30 days, the diet supplemented with (PhSe)2 at a concentration of 5.0 mg/kg demonstrated a reduction in W, while concentrations of 1.5 and 3.0 mg/kg did not show differences, relative to the control group. However, at 60 days, none of the dietary (PhSe)2 concentrations were associated with differences in W when compared to control (Table 1). Values for TL decreased at 5.0 mg/kg after 30 days, while those at 1.5 and 3.0 mg/kg did not change relative to the control group. At 60 days, no differences were observed at any concentration of (PhSe)2 when compared to the control group. The value of TL increased between 30 and 60 days in the 1.5 mg/kg group (Table 1). Values of K after 30 and 60 days for fish fed a diet containing (PhSe)2 at 1.5 and 3.0 mg/kg did not present differences, relative to the control group. At a concentration of 5.0 mg/kg, after 30 days, the K value showed a significant decrease in relation to the control, while no difference was detected after 60 days. At a concentration of 1.5 mg/kg, the value of K increased between 30 and 60 days (Table 1). 3.1. TBARS levels and PC content In the liver, supplementation with 1.5, 3.0, and 5.0 mg/kg (PhSe)2 decreased lipid peroxidation, as measured by TBARS levels after 30 and 60 days of supplementation, relative to the control group. However, after 60 days of supplementation, the decrease in TBARS levels was significantly greater at all concentrations in comparison with 30 days of supplementation (Fig. 1A). In gills, TBARS levels did not change at any concentration used, relative to the control group after 30 days. At 60 days, TBARS levels decreased in gills at 1.5 and 5.0 mg/kg when compared to control. Furthermore, after 60 days, TBARS levels in 1.5 and 5.0 mg/kg groups were lower compared with those detected after
Table 1 W, TL and K of silver catfish fed with (PhSe)2 (mg/kg) diets during 30 and 60 days. 0.0
1.5
3.0
5.0
W (g) 30 days 60 days
22.21 ± 0.62ab 22.21 ± 0.62ab
22.67 ± 1.06ab 25.67 ± 1.56a
23.57 ± 0.78a 23.84 ± 1.25a
18.02 ± 0.75c 19.59 ± 0.93bc
TL (cm) 30 days 60 days
6.72 ± 0.09ab 6.72 ± 0.09ab
6.57 ± 0.16a 7.05 ± 0.15b
6.70 ± 0.09ab 6.85 ± 0.12ab
6.02 ± 0.11c 6.31 ± 0.11ac
Fulton (K) 30 days 60 days
1.50 ± 0.05ab 1.50 ± 0.05ab
1.50 ± 0.10a 1.82 ± 0.15b
1.58 ± 0.07ab 1.63 ± 0.12ab
1.09 ± 0.06c 1.24 ± 0.08ac
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30 days fed with the same concentrations (Fig. 1B). At 30 days, no change was observed in brain at any concentration, while at 60 days, a reduction occurred in TBARS levels at 1.5, 3.0, and 5.0 mg/kg in relation to the control group. In addition, the group fed with 3.0 and 5.0 mg/kg presented a significant decrease in TBARS levels at 60 days, relative to 30 days (Fig. 1C). In the muscle, a decrease was observed in TBARS levels at 3.0 and 5.0 mg/kg at 30 days, while at 60 days a decrease occurred at all concentrations, relative to the control group. At 1.5 mg/kg, TBARS levels decreased at 60 days when compared to 30 days and no change was shown between days 30 and 60 at other concentrations (Fig. 1D). The PC content in the liver at 30 days was decreased at all concentrations. However, after 60 days, only fish supplement with 1.5 and 3.0 mg/kg (PhSe)2 presented a decrease in PC when compared to control group. At 60 days, PC content showed higher values at concentrations of 1.5 and 5.0 mg/kg, compared with values for these same concentrations after 30 days (Table 2). In gills, PC was unchanged after 30 days. On the other hand, after 60 days, PC was decreased in the 1.5 and 3.0 mg/kg groups when compared to control group. Comparing time periods, lower values were observed at 60 days than at 30 days in the 1.5 mg/kg group (Table 2). In brain, after 30 days, the PC did not change, however, at 60 days, it was decreased at all concentrations when compared to control group. Furthermore, we observed that PC showed lower values in the brain for all concentrations used at 60 days, compared with 30 days (Table 2). In muscle, no significant change was observed at 30 days, while at 60 days, PC content was decreased in the 3.0 and 5.0 mg/kg groups, relative to the control group. When the 30 and 60 day time points were compared, no significant changes were observed in the muscle (Table 2). 3.2. Hepatic antioxidant enzymes CAT activity at 30 days was reduced only at a (PhSe)2 concentration of 3.0 mg/kg, while at 60 days, all concentrations showed significant decreases in relation to the control group. In addition, we did not observe differences in CAT activity between 30 and 60 days (Fig. 2A). SOD activity was decreased at 30 days in the 1.5 and 5.0 mg/kg groups, relative to the control group. At 60 days, SOD activity increased at 1.5 mg/kg and decreased at 3.0 and 5.0 mg/kg, relative to the control. Furthermore, we observed an increase in SOD activity in the 1.5 mg/kg group at 60 days and a decrease in the 3.0 mg/kg when compared at 30 days (Fig. 2B). GPx activity showed an increased at 30 and 60 days in the 1.5 and 3.0 mg/kg groups when compared to control group, while in the 5.0 mg/kg no changes were observed during either period of feeding. Moreover, at 3.0 mg/kg, we observed a higher value of GPx activity at 60 days than at 30 days (Fig. 2C). 3.3. GST activity In the liver at 30 days, the GST activity showed an increase only at a concentration 3.0 mg/kg, while at 60 days, an increase occurred at all concentrations of the (PhSe)2, in relation to the control group. In addition, GST activity in the liver increased at concentrations of 1.5 and 5.0 mg/kg and decreased in the 3.0 mg/kg group after 60 days, relative to values detected after 30 days (Table 3). In gills, at 30 days, no difference was observed between the groups. At 60 days, the GST activity in gills increased at concentrations of 3.0 and 5.0 mg/kg. Furthermore, at these same concentrations, the GST activity presented higher values at 60 days than at 30 days (Table 3). In brain and muscle, no significant change was observed at 30 and 60 days or between time periods (Table 3). 3.4. Non-antioxidant enzymes
The values are the mean ± S.E.M (n = 10). Different letters indicate differences between groups (ANOVA/Newman–Keuls, p b 0.05).
In liver, no significant changes were observed in NPSH levels after 30 days, while at 60 days, an increase occurred only in fish supplemented with 3.0 mg/kg when compared to control group. In addition, NPSH
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Fig. 1. TBARS levels in liver (A), gills (B), brain (C) and muscle (D) of silver catfish fed with (PhSe)2 (mg/kg) during 30 and 60 days. The values are the mean ± S.E.M (n = 10). Different letters indicate differences between groups (ANOVA/Newman–Keuls, p b 0.05).
levels in the liver at 3.0 mg/kg presented higher values after 60 days, compared to those obtained after 30 days (Fig. 3A). In gills, NPSH levels at 30 days did not show significant changes, and after 60 days were increased only in the concentration 3.0 mg/kg group compared with the control. Moreover, NPSH levels in gills presented higher values at all concentrations after 60 days compared with those measured at 30 days (Fig. 3B). In brain, after 30 days, NPSH levels were increased at 3.0 mg/kg and at 60 days NPSH levels were enhanced in all concentrations in relation to the control group. Furthermore, NPSH levels in the brain presented higher values at all concentrations after 60 days compared with those measured after 30 days (Fig. 3C). In muscle, at 30 days, no change was observed, while at 60 days, NPSH levels were increased only in the concentration 3.0 mg/kg relative to the control group. NPSH levels in muscle showed higher values in 1.5 and 3.0 mg/kg groups at 60 days compared with those seen at 30 days (Fig. 3D).
AA levels in the liver at 30 and 60 days were enhanced at 1.5, 3.0, and 5.0 mg/kg in comparison to the control group. Furthermore, in liver of the 5.0 mg/kg treatment group, AA levels increased in 60 days when compared with 30 days (Fig. 4A). In gills the AA levels at 30 days were enhanced only at a concentration 1.5 mg/kg, while those at 60 days were increased at all concentrations relative to the control group. However, in gills the AA levels after 60 days at 3.0 and 5.0 mg/kg presented higher values than at 30 days (Fig. 4B). In brain, at 30 days, AA levels were increased only at 5.0 mg/kg, while after 60 days, brain AA levels were increased at 1.5, 3.0, and 5.0 mg/kg when compared to control group. Furthermore, AA levels in the brain presented higher values at 1.5 and 3.0 mg/kg after 60 days of supplementation compared with 30 days (Fig. 4C). In muscle, after 30 days, AA levels were increased at 1.5 mg/kg, while after 60 days, AA levels were increased at 1.5 and 5.0 mg/kg in relation to control group. AA levels in muscle at 60 days showed higher values in the concentration 5.0 mg/kg than at 30 days (Fig. 4D). 4. Discussion
Table 2 PC content in tissues of silver catfish fed with (PhSe)2 (mg/kg) during 30 and 60 days. 0.0
1.5
3.0
5.0
Liver 30 days 60 days
5.90 ± 0.20ac 6.19 ± 0.18a
4.97 ± 0.16b 5.63 ± 0.04c
4.81 ± 0.14b 5.10 ± 0.15b
3.98 ± 0.14d 6.05 ± 0.08ac
Gills 30 days 60 days
6.92 ± 0.23ab 7.61 ± 0.27a
6.71 ± 0.23b 5.32 ± 0.22c
6.71 ± 0.19b 6.48 ± 0.14b
5.99 ± 0.25b 6.96 ± 0.18a
Brain 30 days 60 days
6.09 ± 0.05a 6.98 ± 0.09b
6.18 ± 0.20a 4.24 ± 0.20c
5.68 ± 0.18a 3.10 ± 0.09d
6.07 ± 0.22a 3.90 ± 0.16c
Muscle 30 days 60 days
5.15 ± 0.27ab 5.84 ± 0.16a
4.73 ± 0.22bc 5.35 ± 0.16ac
5.41 ± 0.13ac 4.51 ± 0.19bc
4.39 ± 0.26b 4.97 ± 0.27bc
Carbonyl protein was expressed nmol carbonyl/mg protein. The values are the mean ± S.E.M (n = 10). Different letters indicate differences between groups (ANOVA/Newman–Keuls, p b 0.05).
The use of food supplemented with various concentrations of Se for fish nutrition could be economical and have beneficial effects that increase the aquaculture potential of fish species, such as carp and silver catfish. Aside from the knowledge concerning beneficial properties of selenium in fish food, additional evidence was needed to address certain questions. To this end, our study demonstrated the effect of dietary (PhSe)2 supplementation comparing two durations of feeding (30 and 60 days) and three concentrations of (PhSe)2. Overall, after 30 days of feeding with supplemental (PhSe)2, few changes were seen in tissues of silver catfish that improved the antioxidant capacity of the fish. However, after 60 days of feeding, we observed a decrease in lipid peroxidation and protein carbonylation and an increase in the NPSH and AA levels. Increases in length and factor condition were observed at 1.5 mg/kg of (PhSe)2 after 60 days of feeding relative to 30 days. K is a good indicator of fish health when it presents values higher than 1.4 (Pacini et al., 2012). Data showed that carp supplemented with 1.5 mg/kg of
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Fig. 2. Hepatic activity of CAT (A), SOD (B), and GPx (C) in silver catfish fed with (PhSe)2 (mg/kg) during 30 and 60 days. The values are the mean ± S.E.M (n = 10). Different letters indicate differences between groups (ANOVA/Newman–Keuls, p b 0.05).
(PhSe)2 did not experience changes in growth parameters relative to the control group, but carp supplemented with 3.0 mg/kg of (PhSe)2 showed a significant increase in weight and length (Menezes et al.,
Table 3 GST activity in tissues of silver catfish fed with (PhSe)2 (mg/kg) during 30 and 60 days. 0.0
1.5
3.0
5.0
Liver 30 days 60 days
0.34 ± 0.01ac 0.31 ± 0.01a
0.33 ± 0.01ac 0.45 ± 0.01b
0.45 ± 0.02b 0.37 ± 0.01c
0.30 ± 0.01a 0.42 ± 0.01b
Gills 30 days 60 days
0.27 ± 0.01a 0.27 ± 0.01a
0.27 ± 0.02a 0.29 ± 0.01a
0.26 ± 0.01a 0.36 ± 0.02b
0.25 ± 0.01a 0.34 ± 0.01b
Brain 30 days 60 days
0.16 ± 0.01 0.17 ± 0.01
0.17 ± 0.01 0.14 ± 0.01
0.16 ± 0.01 0.17 ± 0.01
0.18 ± 0.02 0.15 ± 0.01
Muscle 30 days 60 days
0.15 ± 0.01 0.15 ± 0.01
0.16 ± 0.01 0.17 ± 0.01
0.18 ± 0.01 0.16 ± 0.01
0.17 ± 0.01 0.17 ± 0.01
GST was expressed μmol GS-DNB/min/mg protein. The values are the mean ± S.E.M (n = 10). Different letters indicate differences between groups (ANOVA/Newman–Keuls, p b 0.05).
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2014b). On the other hand, a diet containing 5.0 mg/kg of (PhSe)2 had negative effects on fish growth. In agreement with this result, Kim and Kang (2014), studying Pagrus major, showed that fish exposed to waterborne Se experienced decreased growth rates when Se exposure was increased, as noted after 2 weeks of exposure to 400 μg/L Se. Analysis of TBARS and PC are popular and commonly used methods to assess oxidative damage. In this study, after 30 days of feeding a diet supplemented with (PhSe)2, the observed response was a decrease in TBARS levels and PC in liver at all concentrations tested and a decrease in TBARS levels in muscle at 3.0 and 5.0 mg/kg. The absence of a response of other tissues could be due to the exposure time, considering that liver tissue was able to reduce pro-oxidative parameters. However, after 60 days, TBARS levels and PC content were decreased in liver, gills, brain, and muscle at different concentrations of (PhSe)2. Therefore, the diet containing (PhSe)2 per se did not cause oxidative damage in fish tissues as verified by TBARS and PC results. Previously, dietary Se has been shown to reduce TBARS levels and PC in fish under normal conditions (Menezes et al., 2014b; Hao et al., 2014; Saleh et al., 2014) or in the presence of oxidative stress (Menezes et al., 2012, 2013; Özkan-Yilmaz et al., 2014). Nevertheless, the mechanisms by which Se exerts antioxidant protection in fish are not clear. In fact, some authors have shown that carp exhibits a different response, in which increased (PhSe)2 causes oxidative damage; additionally, there is variation between tissues of the same fish species (Menezes et al., 2014b). The enzymes, CAT and SOD play important roles in organisms due to their antioxidant function. In the present study, we observed in the liver of silver catfish a decreased of CAT activity only after 60 days of feeding. In addition, SOD activity decreased at a concentration of 5.0 mg/kg after 30 and 60 days. A similar study with methyl parathion poisoning in Brycon cephalus showed a decrease in CAT and SOD activities in fish fed a diet containing 1.5 mg/kg of sodium selenite, relative to fish fed a control diet (Monteiro et al., 2009). The lower SOD activity could indicate reduced protection from the toxic effects of the superoxide anion radical. Thus, (PhSe)2 was not able to increase these antioxidants defenses, suggesting that the effect of this Se compound, at least in this study, appears not to involve the enzymes CAT and SOD. GPx can be used as a biomarker of Se in vertebrates and responds to alterations in Se status (Sunde et al., 2009). This enzyme protects tissues from oxidative damage because it reduces H2O2 and lipid peroxides. The induction of GPx activity at concentrations of 1.5 and 3.0 mg/kg after 30 and 60 days represents a valuable defense response against the prooxidant effect of reactive oxygen species (ROS). Thus, this increase in GPx can reinforce the detoxification capacity against H2O2. Accordingly, Liu et al. (2010) observed an increase in GPx activity in liver of Rachycentron canadun fed selenomethionine diets for 10 weeks. We showed in the present study that the organic selenium compound, (PhSe)2 in fish diet could be an important antioxidant protecting fish tissues against oxidative damage. Another enzyme important in the detoxification process and involved in the glutathione system is GST. In our experiment, silver catfish supplemented for 30 days presented an increase in GST activity only in liver at a concentration of 3.0 mg/kg. On the other hand, after 60 days, GST activity increased in liver at all concentrations and in gills at 3.0 and 5.0 mg/kg of (PhSe)2. Considering the detoxification process, the increase in GST activity induced by (PhSe)2 represents an important additional line of defense in fish. This enzyme has an antioxidant function because it protects cells from toxic products resulting from lipid peroxidation. Diets containing (PhSe)2 improved the levels of NPSH and AA. However, the increase in these non-enzymatic antioxidant defenses presented the best results after 60 days of feeding with (PhSe)2. After 30 days, levels of NPSH did not change in any of the organs analyzed, while after 60 days, they increased in liver, gills, brain, and muscle. In addition, AA levels were increased after 30 days in liver, gills, brain, and muscle at some concentrations of (PhSe)2, while after 60 days, they were increased at all concentrations and tissues tested. In agreement with our results, Menezes et al. (2014b) observed that NPSH and AA levels in
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Fig. 3. NPSH levels in liver (A), gills (B), brain (C) and muscle (D) of silver catfish fed with (PhSe)2 (mg/kg) during 30 and 60 days. The values are the mean ± S.E.M (n = 10). Different letters indicate differences between groups (ANOVA/Newman–Keuls, p b 0.05).
liver, gills, and muscle were significantly increased in carp supplemented for 60 days with (PhSe)2 at a concentration of 3.0 mg/kg. Sufficient amounts of minerals can be provided to animals through feed additives. Se is one of these minerals capable of preserving health and increasing animal productivity. Studies have shown that increased productivity and improved fish health are some of the results of adding
Se to the diet of fish of commercial interest (Monteiro et al., 2007; Menezes et al., 2012, 2013). In this study, the most significant results were observed after 60 days of feeding with (PhSe)2. Furthermore, concentrations of 1.5 and 3.0 mg/kg decreased oxidative damage and increased antioxidant defenses, while 5.0 mg/kg decreased growth parameters. These results suggest that dietary supplementation with
Fig. 4. AA levels in liver (A), gills (B), brain (C) and muscle (D) of silver catfish fed with (PhSe)2 (mg/kg) during 30 and 60 days. The values are the mean ± S.E.M (n = 10). Different letters indicate differences between groups (ANOVA/Newman–Keuls, p b 0.05).
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(PhSe)2 at concentrations ranging from 1.5 to 3.0 mg/kg enhanced cellular protection against oxidative damage, ameliorating antioxidant defenses in this fish species. References Baldisserotto, B., 2009. Piscicultura continental no Rio Grande do Sul: situação atual, problemas e perspectivas Para o futuro. Ciênc Rural 39, 291–299. Bradford, M.M.A., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Borba, M.R., Fracalossi, D.M., Freitas, F.A., 2007. Efeito da suplementação de vitamina C na dieta sobre a susceptibilidade de alevinos de jundiá, Rhamdia quelen, ao Ichthyophthirius multifiliis. Acta Sci Anim Sci 29, 93–99. Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. Methods Enzymol. 52, 302–309. Costa, M.D., de Freitas, M.L., Dalmolin, L., Oliveira, L.P., Fleck, M.A., Pagliari, P., Acker, C., Roman, S.S., Brandão, R., 2013. Diphenyl diselenide prevents hepatic alterations induced by paraquat in rats. Environ. Toxicol. Pharmacol. 36, 750–758. Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. 82, 70–77. Fiuza, T.L., Oliveira, C.S., da Costa, M., Oliveira, V.A., Zeni, G., Pereira, M.E., 2015. Effectiveness of (PhSe)2 in protect against the HgCl2 toxicity. J. Trace Elem. Med. Biol. 29, 255–262. Fracalossi, D.M., Meyer, G., Santamaria, F.M., Weingartner, M., Filho, E.Z., 2004. Desempenho do jundiá, Rhamdia quelen, e do dourado, Salminus brasiliensis, em viveiros de terra na região sul do Brasil. Acta Sci Anim Sci 26, 345–352. Habig, W.H., Pabst, M.J., Jacoby, W.B., 1974. Glutathione S-transferase, the first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130–7139. Hamilton, S.J., 2004. Review of selenium toxicity in the aquatic food chain. Sci. Total Environ. 326, 1–21. Hao, X., Ling, Q., Hong, F., 2014. Effects of dietary selenium on the pathological changes and oxidative stress in loach (Paramisgurnus dabryanus). Fish Physiol. Biochem. 40, 1313–1323. Kim, J.H., Kang, J.C., 2014. The selenium accumulation and its effects on growth, and hematological parameters in Red Sea bream, Pagrus major, exposed to waterborne selenium. Ecotoxicol. Environ. Saf. 104, 96–102. Li, H., Zhang, J., Wang, T., Luo, W., Zhou, Q., Jiang, G., 2008. Elemental selenium particles at nano-size (Nano-Se) are more toxic to Medaka (Oryzias latipes) as a consequence of hyper-accumulation of selenium: a comparison with sodium selenite. Aquat. Toxicol. 89, 251–256. Liu, K., Wang, X.J., Ai, Q., Mai, K., Zhang, W., 2010. Dietary selenium requirement for juvenile cobia, Rachycentron canadum L. Aquacult. Res. 41, 594–601. Menezes, C.C., Leitemperger, J., Santi, A., Lópes, T., Veiverberg, C.A., Peixoto, S., Adaime, M.B., Zanella, R., Barbosa, N.B.V., Loro, V.L., 2012. The effects of diphenyl diselenide on oxidative stress biomarkers in Cyprinus carpio exposed to herbicide quinclorac (Facet). Ecotoxicol. Environ. Saf. 81, 91–97. Menezes, C., Leitemperger, J., Toni, C., Santi, A., Lópes, T., Barbosa, N.B.V., Neto, J.R., Loro, V.L., 2013. Comparative study on effects of dietary with diphenyl diselenide on oxidative stress in carp (Cyprinus carpio) and silver catfish (Rhamdia sp.) exposed to herbicide clomazone. Environ. Toxicol. Pharmacol. 36, 706–714.
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Menezes, C., Ruiz-Jarabo, I., Martos-Sitcha, J.A., Leitemperger, J., Baldisserotto, B., Mancera, J.M., Rosemberg, D.B., Loro, V.L., 2014a. Diet with diphenyl diselenide mitigates quinclorac toxicity in silver catfish (Rhamdia quelen). PLoS One 9 (12), e114233. Menezes, C., Leitemperger, J., Santi, A., Dias, G., Pedron, F.A., Neto, J.R., Salman, S.M., Barbosa, N.B.V., Loro, V.L., 2014b. Evaluation of the effects induced by dietary diphenyl diselenide on common carp Cyprinus carpio. Fish Physiol. Biochem. 40, 141–149. Misra, H.P., Fridovich, I., 1972. The role of superoxide anion in the auto-oxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247, 3170–3175. Monteiro, D.A., Rantin, F.T., Kalinin, A.L., 2007. Use of selenium in matrinxã feed, Brycon cephalus. Braz J Anim Health Prod 8, 32–47. Monteiro, D.A., Rantin, F.T., Kalinin, A.L., 2009. The effects of selenium on oxidative stress biomarkers in the freshwater characid fish matrinxã, Brycon cephalus (Gunther, 1869) exposed to organophosphate insecticide Folisuper 600 BR® (methyl parathion). Comp. Biochem. Physiol. C 149, 40–49. Murussi, C.R., Thorstenberg, M.L., Leitemperger, J., Costa, M., Clasen, B., Santi, A., Menezes, C., Engers, V.K., Loro, V.L., 2014. Toxic effects of penoxsulam herbicide in two fish species reared in southern Brazil. Bull. Environ. Contam. Toxicol. 92, 81–84. Muscatello, J.R., Bennett, P.M., Himbeault, A.M., Belknap, A.M., Janz, D.M., 2006. Larval deformities associated with selenium accumulation in northern pike (Esox lucius) exposed to metal mining effluent. Environ. Sci. Technol. 40, 6506–6512. Nelson, D.P., Kiesow, L.A., 1972. Enthalphy of decomposition of hydrogen peroxide by catalase at 25 °C (with molar extinction coefficients of H2O2 solution in the UV). Anal. Biochem. 49, 474–478. Nogueira, C.W., Zeni, G., Rocha, J.B.T., 2004. Organoselenium and organotellurium compounds: toxicology and pharmacology. Chem. Rev. 104, 6255–6286. Özkan-Yilmaz, F., Özluër-Hunt, A., Gündüz, S., Berköz, M., Yalin, S., 2014. Effects of dietary selenium of organic form against lead toxicity on the antioxidant system in Cyprinus carpio. Fish Physiol. Biochem. 40, 355–363. Pacini, N., Abete, M.C., Dörr, A.J.M., Prearo, M., Natali, M., Elia, A.C., 2012. Detoxifying response in juvenile tench fed by selenium diet. Environ. Toxicol. Pharmacol. 33, 46–52. Paglia, D.E., Valentine, W.N., 1987. Studies on the quantitative and qualitative characterization of glutathione peroxidase. J. Lab. Clin. Med. 70, 158–165. Paulmier, C., 1986. Selenoorganic functional groups. In: Paulmier, C. (Ed.), Selenium Reagents and Intermediates in Organic Synthesis. Oxford, Pergamon Press, pp. 25–51. Pretto, A., Loro, V.L., Silva, V.M.M., Salbego, S., Menezes, C.C., Souza, C.F., Gioda, C.R., Baldisserotto, B., 2014. Exposure to sublethal concentrations of copper changes biochemistry parameters in silver catfish, Rhamdia quelen, Quoy & Gaimard. Bull. Environ. Contam. Toxicol. 92, 399–403. Roe, J.H., 1954. In: Glick, D. (Ed.), Methods of Biochemical Analysis. Interscience Publishers, New York, pp. 115–139. Saleh, R., Betancor, M.B., Roo, J., Montero, D., Zamorano, M.J., Izquierdo, M.S., 2014. Selenium levels in early weaning diets for gilthead seabream larvae. Aquaculture 426-427, 256–263. Sunde, R.A., Raines, A.M., Barnes, K.M., Evenson, J.K., 2009. Selenium status highly regulates selenoprotein mRNA levels for only a subset of the selenoproteins in the selenoproteome. Biosci. Rep. 29, 329–338. Yan, L.J., Traber, M.G., Packer, L., 1995. Spectrophotometric method for determination of carbonyls in oxidatively modified apolipoprotein B of human low-density lipoproteins. Anal. Biochem. 228, 349–351.
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Effects of diphenyl diselenide on growth, oxidative damage, and antioxidant response in silver catfish Charlene Menezes a,d,e, Aline Marins a, Camila Murussi b,d, Alexandra Pretto c, Jossiele Leitemperger b,d, Vania Lucia Loro a,d a
Programa de Pós — Graduação em Biodiversidade Animal Programa de Pós — Graduação em Bioquímica Toxicológica Universidade Federal do Pampa, Campus Uruguaiana, Uruguaiana, RS, Brazil d Laboratório de Toxicologia Aquática, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil e Corresponding author at: Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Santa Maria, 97105.900 Santa Maria, RS, Brazil b c
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Selenium is element participates of various metabolic routes. • (PhSe)2 in different concentrations in diet were investigated on silver catfish. • 1.5 and 3.0 mg/kg of (PhSe)2 increased antioxidant defenses of fish. • The best results were obtained after 60 days of feeding with (PhSe)2.
a r t i c l e
i n f o
Article history: Received 27 April 2015 Received in revised form 20 October 2015 Accepted 21 October 2015 Available online 3 November 2015 Editor: D. Barcelo Keywords: Antioxidants Fish Selenium Oxidative parameters
a b s t r a c t The aim of this study was to evaluate the effects of dietary diphenyl diselenide [(PhSe)2] at different concentrations (1.5, 3.0, and 5.0 mg/kg) on growth, oxidative damage and antioxidant parameters in silver catfish after 30 and 60 days. Fish fed with 5.0 mg/kg of (PhSe)2 experienced a significant decrease in weight, length, and condition factor after 30 days and these parameters increased after 60 days. Thiobarbituric acid reactive substances (TBARS) and protein carbonyl (PC) decreased in the liver of silver catfish supplemented with (PhSe)2 after 30 days at all concentrations, while after 60 days these parameters decreased in liver, gills, brain, and muscle. Supplementation with (PhSe)2 induced a decrease in catalase (CAT) activity from liver only after 60 days of feeding. Superoxide dismutase (SOD) decreased at 5.0 mg/kg after 30 and 60 days and glutathione peroxidase (GPx) was enhanced at 1.5 and 3.0 mg/kg after 30 and 60 days. Silver catfish supplemented for 30 days showed a significant increase in liver glutathione S-transferase (GST) at 3.0 mg/kg, while after 60 days GST activity increased in liver at 1.5, 3.0, and 5.0 mg/kg and in gills at 3.0 and 5.0 mg/kg of (PhSe)2. After 30 days, non-protein thiols (NPSH) did not change, while after 60 days NPSH increased in liver, gills, brain, and muscle. In addition, ascorbic acid (AA) levels after 30 days increased in liver at three concentrations and in gills and muscle at 1.5 mg/kg, while after 60 days, AA increased at all concentrations in all and tissues tested. Thus, diet supplemented with (PhSe)2
E-mail address: [email protected] (C. Menezes).
http://dx.doi.org/10.1016/j.scitotenv.2015.10.110 0048-9697/© 2015 Elsevier B.V. All rights reserved.
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for 60 days could be more effective for silver catfish. Although the concentration of 5.0 mg/kg showed decreased growth parameters, concentrations of 1.5 and 3.0 mg/kg, in general, decreased oxidative damage and increased antioxidant defenses. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Selenium (Se) is an essential nutrient in the diet of a variety of organisms, because it is important for growth, development, and physiological function (Hamilton, 2004; Kim and Kang, 2014). Se is an antioxidant mineral present in various selenoproteins that contain one or more selenocysteine residues in their active sites. This element participates in various metabolic routes, especially those involved in the antioxidant system, as its presence in active sites of glutathione peroxidase (GPx) (Nogueira et al., 2004; Li et al., 2008; Monteiro et al., 2009). However, nutritional requirements and toxic levels of Se exhibit a fine partition, and this element may be either essential or toxic, depending on its concentration. In fact, at high cellular concentrations, Se may be utilized in place of sulfur, causing errors in protein synthesis and damage to other molecules (e.g. lipids and DNA) as well as reproductive impairment, larval deformities, and mortality (Muscatello et al., 2006). Diphenyl diselenide [(PhSe)2] is a synthetic organoselenium compound that has been considered a potential pharmacological and antioxidant agent in various experimental models, such as fish, rats, and mice (Menezes et al., 2012; Costa et al., 2013; Fiuza et al., 2015). The exposure of fish to xenobiotics is a main cause of oxidative damage due to increased production of reactive species oxygen (ROS) in exposed organisms (Murussi et al., 2014; Pretto et al., 2014). Thus, the use of antioxidants such as Se in the diet may permit animals to overcome, healthy and without damage, adverse conditions that may occur (Monteiro et al., 2007). Menezes et al. (2012) evaluated the effects of (PhSe)2 on oxidative damage and antioxidant profile in different tissues of Cyprinus carpio. The authors observed that fish fed a diet without (PhSe)2 and exposed to Facet® herbicide showed oxidative damage in different organs, while (PhSe)2 treatment reversed oxidative damage by increasing some antioxidant defenses. Thus, (PhSe)2 could be a powerful antioxidant. However, for other species of fish, the requirement in terms of concentration and form as well as the threshold dose for its opposing toxic properties has not yet been established. The silver catfish (Rhamdia quelen), is a fish species with an extensive geographic distribution, occurring from southern Mexico to central Argentina. Its features, such as tolerance to management, omnivorous feeding behavior, ability to grow throughout the winter, and high yield, place it in a prominent position among the native species of interest to aquaculture in the region of southern Brazil (Fracalossi et al., 2004; Baldisserotto, 2009). Studies have evidenced the possibility that dietary supplementation with certain vitamins and minerals can increase disease resistance, prevent negative effects of stress, and minimize the toxicity of contaminants in fish (Borba et al., 2007; Monteiro et al., 2009; Menezes et al., 2012, 2014a). The objective of this study was to evaluate the efficacy of dietary supplementation with different concentrations of (PhSe)2 for 30 and 60 days in silver catfish, in order to establish the concentration that warrants further exploration as a potential supplement in silver catfish nutrition. 2. Materials and methods 2.1. Chemicals The reagents chemicals used in this study were obtained from Sigma Chemical Co. (St. Louis, MO, USA) and Merck (Rio de Janeiro, Brazil). Diphenyl diselenide [(PhSe)2] was synthesized according with
Paulmier (1986). Analysis of the 1HNMR and 13CNMR spectra showed analytical and spectroscopic data in agreement with its assigned structure. 2.2. Fish Silver catfish (body length, 7.0 ± 1.0 cm; mean weight, 18.0 ± 1.0 g) of both sexes were acquired from a local fish farm (RS, Brazil). Before the experiment, the fish were acclimated to the laboratory conditions for 15 days, in 250 L fiberglass boxes. They were kept in continuously aerated dechlorinated tap water with a static system and with a natural photoperiod (12 h light/12 h dark). During the acclimation, water temperature was maintained at 22.5 ± 1.0 °C, the dissolved oxygen content was 7.21 ± 1.0 mg/L, pH between 7.3 and 7.7, non-ionized ammonia 0.3 ± 0.01 μg/L and nitrite 0.05 ± 0.01 mg/L. In this period of acclimation, fish were fed once a day with commercial fish pellets (Supra, Brazil). Feces and food residues were removed by syphon and filter system was used to keep the quality of water. 2.3. Diet preparation and experimental design During the experiment, silver catfish were divided in four groups (n = 20 per group): (1) control, (2) (PhSe)2 1.5 mg/kg, (3) (PhSe)2 3.0 mg/kg and (4) (PhSe)2 5.0 mg/kg. The fish of control group were supplemented with diet without (PhSe)2. All fish were fed during 30 and 60 days and (PhSe)2 was added to the control diet. Diet formulation was performed based in previous studies from our group (Menezes et al., 2012, 2014b). Diets were made into pellets (5 mm, diameter) and stored at 4 °C until fed. During the experiment, silver catfish were fed with 3% biomass per day. The daily ration was divided into two equal meals fed at 09:00 and 16:00 h. The feces and food residues were removed by syphoning. Temperature, dissolved oxygen, pH, ammonia and nitrite were evaluated daily and were maintained similar values to those recorded during the acclimation period. At end of the each experimental period (30 and 60 days), ten fish of each group (n = 10) were weighed and measured. Total length (TL) and body weight (W) for each fish were recorded to evaluate growth and condition factor K (K = (weight / length3) × 100). Afterwards, the fish were anesthetized with 50 mg/L clove oil and euthanized by punching the spinal cord behind the opercula. Liver, gills, brain, and muscle were quickly collected and stored at − 80 °C for posteriors analysis. The study was approved by the Committee on Ethics and Animal Welfare of Federal University of Santa Maria, under the number: 84/2009. 2.4. Biochemical analysis At end of the each experimental period (30 and 60 days), ten fish of each group (n = 10) were used to biochemical analysis. In liver, gills, brain, and muscle the lipid peroxidation (LPO) levels was estimated using the thiobarbituric acid reactive substances (TBARS) assay of according to Buege and Aust (1978) and the protein carbonyl (PC) determination was measured by the method described by Yan et al. (1995). In liver, the superoxide dismutase (SOD) activity was determined as described by Misra and Fridovich (1972) based on inhibition of the radical superoxide reaction with adrenalin. Catalase (CAT) activity in liver was determined as reported by Nelson and Kiesow (1972). Glutathione peroxidase (GPx) activity in liver was measured by following the rate of NADPH oxidation at 340 nm by the coupled reaction with glutathione reductase as described by Paglia and Valentine (1987). The glutathione
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S-transferase (GST) activity was assayed in liver, gills, brain, and muscle of according to Habig et al. (1974). In liver, gills, brain, and muscle the non-protein thiols (NPSH) and ascorbic acid (AA) content was evaluated by the method of Ellman (1959) and Roe (1954) respectively. The protein content was measured as described by Bradford (1976) using bovine serum albumin as standard. All biochemical analyses were described in details in previous publications of our research group (Menezes et al., 2012, 2013). 2.5. Statistical analysis The data are presented as mean ± standard error of mean (S.E.M). Statistical analyses were realized using a two-way analyses of variance (ANOVA) followed by Newman–Keuls post hoc comparison. Analyses were performed using Graph Prism (Version 6.0) and the significance level was set at p b 0.05. 3. Results Mean values of W, TL, and K were recorded for two periods (30 and 60 days) (Table 1). At 30 days, the diet supplemented with (PhSe)2 at a concentration of 5.0 mg/kg demonstrated a reduction in W, while concentrations of 1.5 and 3.0 mg/kg did not show differences, relative to the control group. However, at 60 days, none of the dietary (PhSe)2 concentrations were associated with differences in W when compared to control (Table 1). Values for TL decreased at 5.0 mg/kg after 30 days, while those at 1.5 and 3.0 mg/kg did not change relative to the control group. At 60 days, no differences were observed at any concentration of (PhSe)2 when compared to the control group. The value of TL increased between 30 and 60 days in the 1.5 mg/kg group (Table 1). Values of K after 30 and 60 days for fish fed a diet containing (PhSe)2 at 1.5 and 3.0 mg/kg did not present differences, relative to the control group. At a concentration of 5.0 mg/kg, after 30 days, the K value showed a significant decrease in relation to the control, while no difference was detected after 60 days. At a concentration of 1.5 mg/kg, the value of K increased between 30 and 60 days (Table 1). 3.1. TBARS levels and PC content In the liver, supplementation with 1.5, 3.0, and 5.0 mg/kg (PhSe)2 decreased lipid peroxidation, as measured by TBARS levels after 30 and 60 days of supplementation, relative to the control group. However, after 60 days of supplementation, the decrease in TBARS levels was significantly greater at all concentrations in comparison with 30 days of supplementation (Fig. 1A). In gills, TBARS levels did not change at any concentration used, relative to the control group after 30 days. At 60 days, TBARS levels decreased in gills at 1.5 and 5.0 mg/kg when compared to control. Furthermore, after 60 days, TBARS levels in 1.5 and 5.0 mg/kg groups were lower compared with those detected after
Table 1 W, TL and K of silver catfish fed with (PhSe)2 (mg/kg) diets during 30 and 60 days. 0.0
1.5
3.0
5.0
W (g) 30 days 60 days
22.21 ± 0.62ab 22.21 ± 0.62ab
22.67 ± 1.06ab 25.67 ± 1.56a
23.57 ± 0.78a 23.84 ± 1.25a
18.02 ± 0.75c 19.59 ± 0.93bc
TL (cm) 30 days 60 days
6.72 ± 0.09ab 6.72 ± 0.09ab
6.57 ± 0.16a 7.05 ± 0.15b
6.70 ± 0.09ab 6.85 ± 0.12ab
6.02 ± 0.11c 6.31 ± 0.11ac
Fulton (K) 30 days 60 days
1.50 ± 0.05ab 1.50 ± 0.05ab
1.50 ± 0.10a 1.82 ± 0.15b
1.58 ± 0.07ab 1.63 ± 0.12ab
1.09 ± 0.06c 1.24 ± 0.08ac
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30 days fed with the same concentrations (Fig. 1B). At 30 days, no change was observed in brain at any concentration, while at 60 days, a reduction occurred in TBARS levels at 1.5, 3.0, and 5.0 mg/kg in relation to the control group. In addition, the group fed with 3.0 and 5.0 mg/kg presented a significant decrease in TBARS levels at 60 days, relative to 30 days (Fig. 1C). In the muscle, a decrease was observed in TBARS levels at 3.0 and 5.0 mg/kg at 30 days, while at 60 days a decrease occurred at all concentrations, relative to the control group. At 1.5 mg/kg, TBARS levels decreased at 60 days when compared to 30 days and no change was shown between days 30 and 60 at other concentrations (Fig. 1D). The PC content in the liver at 30 days was decreased at all concentrations. However, after 60 days, only fish supplement with 1.5 and 3.0 mg/kg (PhSe)2 presented a decrease in PC when compared to control group. At 60 days, PC content showed higher values at concentrations of 1.5 and 5.0 mg/kg, compared with values for these same concentrations after 30 days (Table 2). In gills, PC was unchanged after 30 days. On the other hand, after 60 days, PC was decreased in the 1.5 and 3.0 mg/kg groups when compared to control group. Comparing time periods, lower values were observed at 60 days than at 30 days in the 1.5 mg/kg group (Table 2). In brain, after 30 days, the PC did not change, however, at 60 days, it was decreased at all concentrations when compared to control group. Furthermore, we observed that PC showed lower values in the brain for all concentrations used at 60 days, compared with 30 days (Table 2). In muscle, no significant change was observed at 30 days, while at 60 days, PC content was decreased in the 3.0 and 5.0 mg/kg groups, relative to the control group. When the 30 and 60 day time points were compared, no significant changes were observed in the muscle (Table 2). 3.2. Hepatic antioxidant enzymes CAT activity at 30 days was reduced only at a (PhSe)2 concentration of 3.0 mg/kg, while at 60 days, all concentrations showed significant decreases in relation to the control group. In addition, we did not observe differences in CAT activity between 30 and 60 days (Fig. 2A). SOD activity was decreased at 30 days in the 1.5 and 5.0 mg/kg groups, relative to the control group. At 60 days, SOD activity increased at 1.5 mg/kg and decreased at 3.0 and 5.0 mg/kg, relative to the control. Furthermore, we observed an increase in SOD activity in the 1.5 mg/kg group at 60 days and a decrease in the 3.0 mg/kg when compared at 30 days (Fig. 2B). GPx activity showed an increased at 30 and 60 days in the 1.5 and 3.0 mg/kg groups when compared to control group, while in the 5.0 mg/kg no changes were observed during either period of feeding. Moreover, at 3.0 mg/kg, we observed a higher value of GPx activity at 60 days than at 30 days (Fig. 2C). 3.3. GST activity In the liver at 30 days, the GST activity showed an increase only at a concentration 3.0 mg/kg, while at 60 days, an increase occurred at all concentrations of the (PhSe)2, in relation to the control group. In addition, GST activity in the liver increased at concentrations of 1.5 and 5.0 mg/kg and decreased in the 3.0 mg/kg group after 60 days, relative to values detected after 30 days (Table 3). In gills, at 30 days, no difference was observed between the groups. At 60 days, the GST activity in gills increased at concentrations of 3.0 and 5.0 mg/kg. Furthermore, at these same concentrations, the GST activity presented higher values at 60 days than at 30 days (Table 3). In brain and muscle, no significant change was observed at 30 and 60 days or between time periods (Table 3). 3.4. Non-antioxidant enzymes
The values are the mean ± S.E.M (n = 10). Different letters indicate differences between groups (ANOVA/Newman–Keuls, p b 0.05).
In liver, no significant changes were observed in NPSH levels after 30 days, while at 60 days, an increase occurred only in fish supplemented with 3.0 mg/kg when compared to control group. In addition, NPSH
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Fig. 1. TBARS levels in liver (A), gills (B), brain (C) and muscle (D) of silver catfish fed with (PhSe)2 (mg/kg) during 30 and 60 days. The values are the mean ± S.E.M (n = 10). Different letters indicate differences between groups (ANOVA/Newman–Keuls, p b 0.05).
levels in the liver at 3.0 mg/kg presented higher values after 60 days, compared to those obtained after 30 days (Fig. 3A). In gills, NPSH levels at 30 days did not show significant changes, and after 60 days were increased only in the concentration 3.0 mg/kg group compared with the control. Moreover, NPSH levels in gills presented higher values at all concentrations after 60 days compared with those measured at 30 days (Fig. 3B). In brain, after 30 days, NPSH levels were increased at 3.0 mg/kg and at 60 days NPSH levels were enhanced in all concentrations in relation to the control group. Furthermore, NPSH levels in the brain presented higher values at all concentrations after 60 days compared with those measured after 30 days (Fig. 3C). In muscle, at 30 days, no change was observed, while at 60 days, NPSH levels were increased only in the concentration 3.0 mg/kg relative to the control group. NPSH levels in muscle showed higher values in 1.5 and 3.0 mg/kg groups at 60 days compared with those seen at 30 days (Fig. 3D).
AA levels in the liver at 30 and 60 days were enhanced at 1.5, 3.0, and 5.0 mg/kg in comparison to the control group. Furthermore, in liver of the 5.0 mg/kg treatment group, AA levels increased in 60 days when compared with 30 days (Fig. 4A). In gills the AA levels at 30 days were enhanced only at a concentration 1.5 mg/kg, while those at 60 days were increased at all concentrations relative to the control group. However, in gills the AA levels after 60 days at 3.0 and 5.0 mg/kg presented higher values than at 30 days (Fig. 4B). In brain, at 30 days, AA levels were increased only at 5.0 mg/kg, while after 60 days, brain AA levels were increased at 1.5, 3.0, and 5.0 mg/kg when compared to control group. Furthermore, AA levels in the brain presented higher values at 1.5 and 3.0 mg/kg after 60 days of supplementation compared with 30 days (Fig. 4C). In muscle, after 30 days, AA levels were increased at 1.5 mg/kg, while after 60 days, AA levels were increased at 1.5 and 5.0 mg/kg in relation to control group. AA levels in muscle at 60 days showed higher values in the concentration 5.0 mg/kg than at 30 days (Fig. 4D). 4. Discussion
Table 2 PC content in tissues of silver catfish fed with (PhSe)2 (mg/kg) during 30 and 60 days. 0.0
1.5
3.0
5.0
Liver 30 days 60 days
5.90 ± 0.20ac 6.19 ± 0.18a
4.97 ± 0.16b 5.63 ± 0.04c
4.81 ± 0.14b 5.10 ± 0.15b
3.98 ± 0.14d 6.05 ± 0.08ac
Gills 30 days 60 days
6.92 ± 0.23ab 7.61 ± 0.27a
6.71 ± 0.23b 5.32 ± 0.22c
6.71 ± 0.19b 6.48 ± 0.14b
5.99 ± 0.25b 6.96 ± 0.18a
Brain 30 days 60 days
6.09 ± 0.05a 6.98 ± 0.09b
6.18 ± 0.20a 4.24 ± 0.20c
5.68 ± 0.18a 3.10 ± 0.09d
6.07 ± 0.22a 3.90 ± 0.16c
Muscle 30 days 60 days
5.15 ± 0.27ab 5.84 ± 0.16a
4.73 ± 0.22bc 5.35 ± 0.16ac
5.41 ± 0.13ac 4.51 ± 0.19bc
4.39 ± 0.26b 4.97 ± 0.27bc
Carbonyl protein was expressed nmol carbonyl/mg protein. The values are the mean ± S.E.M (n = 10). Different letters indicate differences between groups (ANOVA/Newman–Keuls, p b 0.05).
The use of food supplemented with various concentrations of Se for fish nutrition could be economical and have beneficial effects that increase the aquaculture potential of fish species, such as carp and silver catfish. Aside from the knowledge concerning beneficial properties of selenium in fish food, additional evidence was needed to address certain questions. To this end, our study demonstrated the effect of dietary (PhSe)2 supplementation comparing two durations of feeding (30 and 60 days) and three concentrations of (PhSe)2. Overall, after 30 days of feeding with supplemental (PhSe)2, few changes were seen in tissues of silver catfish that improved the antioxidant capacity of the fish. However, after 60 days of feeding, we observed a decrease in lipid peroxidation and protein carbonylation and an increase in the NPSH and AA levels. Increases in length and factor condition were observed at 1.5 mg/kg of (PhSe)2 after 60 days of feeding relative to 30 days. K is a good indicator of fish health when it presents values higher than 1.4 (Pacini et al., 2012). Data showed that carp supplemented with 1.5 mg/kg of
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Fig. 2. Hepatic activity of CAT (A), SOD (B), and GPx (C) in silver catfish fed with (PhSe)2 (mg/kg) during 30 and 60 days. The values are the mean ± S.E.M (n = 10). Different letters indicate differences between groups (ANOVA/Newman–Keuls, p b 0.05).
(PhSe)2 did not experience changes in growth parameters relative to the control group, but carp supplemented with 3.0 mg/kg of (PhSe)2 showed a significant increase in weight and length (Menezes et al.,
Table 3 GST activity in tissues of silver catfish fed with (PhSe)2 (mg/kg) during 30 and 60 days. 0.0
1.5
3.0
5.0
Liver 30 days 60 days
0.34 ± 0.01ac 0.31 ± 0.01a
0.33 ± 0.01ac 0.45 ± 0.01b
0.45 ± 0.02b 0.37 ± 0.01c
0.30 ± 0.01a 0.42 ± 0.01b
Gills 30 days 60 days
0.27 ± 0.01a 0.27 ± 0.01a
0.27 ± 0.02a 0.29 ± 0.01a
0.26 ± 0.01a 0.36 ± 0.02b
0.25 ± 0.01a 0.34 ± 0.01b
Brain 30 days 60 days
0.16 ± 0.01 0.17 ± 0.01
0.17 ± 0.01 0.14 ± 0.01
0.16 ± 0.01 0.17 ± 0.01
0.18 ± 0.02 0.15 ± 0.01
Muscle 30 days 60 days
0.15 ± 0.01 0.15 ± 0.01
0.16 ± 0.01 0.17 ± 0.01
0.18 ± 0.01 0.16 ± 0.01
0.17 ± 0.01 0.17 ± 0.01
GST was expressed μmol GS-DNB/min/mg protein. The values are the mean ± S.E.M (n = 10). Different letters indicate differences between groups (ANOVA/Newman–Keuls, p b 0.05).
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2014b). On the other hand, a diet containing 5.0 mg/kg of (PhSe)2 had negative effects on fish growth. In agreement with this result, Kim and Kang (2014), studying Pagrus major, showed that fish exposed to waterborne Se experienced decreased growth rates when Se exposure was increased, as noted after 2 weeks of exposure to 400 μg/L Se. Analysis of TBARS and PC are popular and commonly used methods to assess oxidative damage. In this study, after 30 days of feeding a diet supplemented with (PhSe)2, the observed response was a decrease in TBARS levels and PC in liver at all concentrations tested and a decrease in TBARS levels in muscle at 3.0 and 5.0 mg/kg. The absence of a response of other tissues could be due to the exposure time, considering that liver tissue was able to reduce pro-oxidative parameters. However, after 60 days, TBARS levels and PC content were decreased in liver, gills, brain, and muscle at different concentrations of (PhSe)2. Therefore, the diet containing (PhSe)2 per se did not cause oxidative damage in fish tissues as verified by TBARS and PC results. Previously, dietary Se has been shown to reduce TBARS levels and PC in fish under normal conditions (Menezes et al., 2014b; Hao et al., 2014; Saleh et al., 2014) or in the presence of oxidative stress (Menezes et al., 2012, 2013; Özkan-Yilmaz et al., 2014). Nevertheless, the mechanisms by which Se exerts antioxidant protection in fish are not clear. In fact, some authors have shown that carp exhibits a different response, in which increased (PhSe)2 causes oxidative damage; additionally, there is variation between tissues of the same fish species (Menezes et al., 2014b). The enzymes, CAT and SOD play important roles in organisms due to their antioxidant function. In the present study, we observed in the liver of silver catfish a decreased of CAT activity only after 60 days of feeding. In addition, SOD activity decreased at a concentration of 5.0 mg/kg after 30 and 60 days. A similar study with methyl parathion poisoning in Brycon cephalus showed a decrease in CAT and SOD activities in fish fed a diet containing 1.5 mg/kg of sodium selenite, relative to fish fed a control diet (Monteiro et al., 2009). The lower SOD activity could indicate reduced protection from the toxic effects of the superoxide anion radical. Thus, (PhSe)2 was not able to increase these antioxidants defenses, suggesting that the effect of this Se compound, at least in this study, appears not to involve the enzymes CAT and SOD. GPx can be used as a biomarker of Se in vertebrates and responds to alterations in Se status (Sunde et al., 2009). This enzyme protects tissues from oxidative damage because it reduces H2O2 and lipid peroxides. The induction of GPx activity at concentrations of 1.5 and 3.0 mg/kg after 30 and 60 days represents a valuable defense response against the prooxidant effect of reactive oxygen species (ROS). Thus, this increase in GPx can reinforce the detoxification capacity against H2O2. Accordingly, Liu et al. (2010) observed an increase in GPx activity in liver of Rachycentron canadun fed selenomethionine diets for 10 weeks. We showed in the present study that the organic selenium compound, (PhSe)2 in fish diet could be an important antioxidant protecting fish tissues against oxidative damage. Another enzyme important in the detoxification process and involved in the glutathione system is GST. In our experiment, silver catfish supplemented for 30 days presented an increase in GST activity only in liver at a concentration of 3.0 mg/kg. On the other hand, after 60 days, GST activity increased in liver at all concentrations and in gills at 3.0 and 5.0 mg/kg of (PhSe)2. Considering the detoxification process, the increase in GST activity induced by (PhSe)2 represents an important additional line of defense in fish. This enzyme has an antioxidant function because it protects cells from toxic products resulting from lipid peroxidation. Diets containing (PhSe)2 improved the levels of NPSH and AA. However, the increase in these non-enzymatic antioxidant defenses presented the best results after 60 days of feeding with (PhSe)2. After 30 days, levels of NPSH did not change in any of the organs analyzed, while after 60 days, they increased in liver, gills, brain, and muscle. In addition, AA levels were increased after 30 days in liver, gills, brain, and muscle at some concentrations of (PhSe)2, while after 60 days, they were increased at all concentrations and tissues tested. In agreement with our results, Menezes et al. (2014b) observed that NPSH and AA levels in
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Fig. 3. NPSH levels in liver (A), gills (B), brain (C) and muscle (D) of silver catfish fed with (PhSe)2 (mg/kg) during 30 and 60 days. The values are the mean ± S.E.M (n = 10). Different letters indicate differences between groups (ANOVA/Newman–Keuls, p b 0.05).
liver, gills, and muscle were significantly increased in carp supplemented for 60 days with (PhSe)2 at a concentration of 3.0 mg/kg. Sufficient amounts of minerals can be provided to animals through feed additives. Se is one of these minerals capable of preserving health and increasing animal productivity. Studies have shown that increased productivity and improved fish health are some of the results of adding
Se to the diet of fish of commercial interest (Monteiro et al., 2007; Menezes et al., 2012, 2013). In this study, the most significant results were observed after 60 days of feeding with (PhSe)2. Furthermore, concentrations of 1.5 and 3.0 mg/kg decreased oxidative damage and increased antioxidant defenses, while 5.0 mg/kg decreased growth parameters. These results suggest that dietary supplementation with
Fig. 4. AA levels in liver (A), gills (B), brain (C) and muscle (D) of silver catfish fed with (PhSe)2 (mg/kg) during 30 and 60 days. The values are the mean ± S.E.M (n = 10). Different letters indicate differences between groups (ANOVA/Newman–Keuls, p b 0.05).
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