Manganese (Mn2+), an essential trace metal, is found in all tissues of bacteria, plants, humans, and fish because it is required for normal amino acid, lipid, protein, and carbohydrate metabolism in vivo (Erikson et al. 2004). This metal is one of the most abundant elements and is widely used in industry (Gerber et al. 2002), pesticide formulations (Belpoggi et al. 2002), glass and ceramic production, and manufacture of dry cell (Srivastava et al. 1991; Mergler et al. 1994; Bader et al. 1999). It is also present at very high concentrations in formation water (produced water or oil field brine) from oil and gas extraction (Baldisserotto et al. 2012). Whereas Mn2+ deficiency is extremely rare, toxicity due to Mn2+ overexposure is more prevalent (Crossgrove and Zheng 2004). Mn2+ undergoes oxi-reduction reactions and may have negative physiological effects owing to oxidative stress induction (Huang et al. 2011).

Oxidative stress occurs due to either the overproduction of reactive oxygen species (ROS) or a decrease in cellular antioxidant levels. As a metal ion, Mn2+ is toxic because it enhances ROS formation and catecholamine oxidation by products (Prabhakaran et al. 2008; Falfushynska et al. 2011). ROS generated in tissues and subcellular compartments are efficiently scavenged by the antioxidant defense system, which is composed of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST) and nonenzymatic antioxidants, such as reduced glutathione (GSH). These antioxidant defenses can protect cells from lipid peroxidation (LPO), protein oxidation, and DNA damage (Halliwell and Gutteridge 1999).

There are several studies on exposure to Mn2+ and other metals in different aquatic species. In general, these studies aimed to analyze mortality and metal bioaccumulation in tissues (Nath and Kumar 1987; Seymore et al. 2006; Crafford and Avenant-Oldewage 2011). Only a few investigations have evaluated possible oxidative damage involved in aquatic animals exposed to Mn2+ (Jena et al. 1998; Falfushynska et al. 2011).

Tambaqui (C. macropomum) is an abundant species in the Amazon basin and is very important to the local economy (Affonso et al. 2002). This species has great longevity and high tolerance to changes in dissolved oxygen levels and pH (Marcon and Wilhelm 1999; Milsom et al. 2002; Florindo et al. 2004). Such characteristics make of tambaqui a good model for the study of metals.

Experiments with metals and native fish have become essential to assess the risk of environmental contamination. Thus, the purpose of this study was to evaluate oxidative stress generated in several organs of tambaqui exposed to high waterborne Mn2+ levels for 96 hours.

Materials and Methods

Chemicals

All reagent-grade chemicals were purchased from Sigma (St. Louis, MO).

Fish

Juvenile tambaqui (100–300 g) were obtained from Fazenda Santo Antônio in Rio Preto da Eva, Amazonas, Brazil. Fish were transported to the Laboratory of Ecophysiology and Molecular Evolution, National Institute of Amazon, and maintained in aerated well water for at least 21 days and were fed commercial dry food pellets once a day. Water parameters were as follows: temperature 28 °C, pH 6.3, Ca2+ 11 μmol l−1, Na+ 34 μmol l−1, Cl- 28 μmol l−1, Mg2+ 0.8 μmol l−1, K+ 15 μmol l−1, dissolved organic matter 0.9 mg C l−1, background Cu2+ 1.7 μg l−1, and background Cd2+ 0.3 μg l−1. The experimental protocol was approved by the Animal Health Committee of Federal University of Santa Maria, Rio Grande do Sul, Brazil.

Exposure to Mn

Stock solutions were prepared by dissolving manganese chloride (MnCl2) in water and added it to the experimental aquarium after the acclimation period. Juvenile fish were randomly separated into two 72–l aquaria (n = 10 each aquarium), one exposed to 3.88 ± 0.239 mg l−1 Mn+2 (measured level) and another nonexposed to the metal (control) for 96 hours. The water was not renewed during the experimental period. Then the fish were placed in containers filled with water and ice for 5 minutes for anaesthetization, after which blood was sampled from the caudal vein with heparinized syringes; the fish were killed by section of the spinal cord. The gills, liver, brain, and kidneys were removed and immediately frozen in liquid nitrogen. The tissues were stored at −70 °C for measurement of oxidative stress parameters or at −20 °C for posterior digestion with concentrated nitric acid (HNO3, 1 N; Merck). Mn concentrations in digested tissues and water samples were analyzed using graphite furnace–atomic absorption spectrophotometry (inductively coupled plasma–mass spectrometry (Elan DRCII Perkin Elmer SCIEX–Canada). Certified standards provided by the manufacturer were used throughout this study. Mn activity was calculated using the speciation program Visual MINTEQ version 3.0 (Gustafsson 2012).

Water Parameters

Water samples were collected from each aquarium to determine water-quality parameters at the beginning and at the end of the experiment. Water alkalinity (10.83 ± 0.48 mg l−1 CaCO3) was determined by the sulfuric acid method (Eaton et al. 2005). Measurements of dissolved oxygen (YSI model Y5512 oxygen meter) and water pH (7.1 ± 0.04) (Quimix 400A pH meter) were performed daily. Water hardness (13.22 ± 0.66 mg l−1 CaCO3) was determined by the ethylene diamine tetraacetic acid titrimetric method, and total ammonia (NH3 + NH4 +, final value 1.23 ± 0.05 mg l−1) was determined by the direct nesslerization method (Eaton et al. 2005).

Oxidative Stress Parameters

The tissues were homogenized as described previously by Azambuja et al. (2011). The homogenates were centrifuged at 1000 × g for 10 minutes at 4 °C to discard nuclei and cell debris, and the supernatant fraction obtained was frozen at –70 °C for analyses of oxidative stress parameters.

Lipid peroxidation (LPO) was measured by TBARS assay (Buege and Aust 1978). Results were expressed as nmol mg protein−1. Commercially available malonaldehyde was used as a standard. Protein content was measured by the method of Lowry et al. (1951) using bovine serum albumin as standard.

Total SOD activity was based on the inhibition rate of autocatalytic adenocrome generation at 480 nm in a reaction medium containing epinephrine and glycine/NaOH (pH 10.2). The enzyme activity was expressed as USOD mg protein−1. One SOD unit was defined as the amount of enzyme needed for 50 % inhibition of adenochrome formation as described by Misra and Fridovich (1972). CAT activity was evaluated according to the decrease in the 240 nm absorption in a reaction medium consisting of phosphate buffer (pH 7.4) and hydrogen peroxide (H2O2), thereby determining the pseudo–first order reaction constant (k’) of the decrease in H2O2 absorption. This was reported as nmol mg protein−1 (Boveris and Chance 1973). GST activity, expressed as μmol min−1 mg protein−1, was determined according to Habig et al. (1974). The assay was performed using potassium phosphate buffer (pH 6.5) with reduced glutathione (GSH) and 1-chloro-2,4-dinitrobenzene. Activity was calculated from the changes in absorbance at 340 nm (ε340 nm = 9.6 mM−1 cm−1). One unit of GST activity was defined as the amount of enzyme catalyzing the conjugation of CDNB with GSH/min at 25 °C. Tissue sulfhydryl groups, an indirect measure of GSH, were evaluated at 412 nm after reaction with 5,5’-dithiobis-(2-nitrobenzoic acid). Proteins were eliminated through the addition of perchloric acid. The final product formed is the yellow 2-nitro-5-mercapto-benzoic acid. The results were reported as nmol protein−1 using ε412 nm = 13.6 mM−1 cm−1 (Ellman 1959).

Statistical Analysis

The results are expressed as the means ± SEs. Levene’s test was performed to evaluate the homogeneity of variances. Unpaired Student t test was used for comparison of means. All analyses were executed by using GraphPad Instat software (San Diego, CA). Differences were considered significant at p < 0.05.

Results

The presence of Mn2+ in the water corresponded to increased levels of Mn2+ in the gill, brain, and kidney. The percentage of accumulation in liver was not significant. Mn2+ accumulation in the tissues occurred in the following sequence: gill > kidney > brain > liver (Fig. 1). Gills of tambaqui exposed to waterborne Mn2+ exhibited a significant increase in thiobarbituric acid reactive substances (TBARS) levels (40 %) in addition to a significant increase in SOD activity (80 %) and a significant decrease in GSH content (40 %). GST activity was unaffected, whereas CAT activity could not be detected (Fig. 2). Hepatic TBARS levels of the fish exposed to waterborne Mn2+ was decreased (35 %) compared with the control. This tissue also showed a significant increase in SOD (82 %) and GST activities (51 %), as well as GSH content (43 %), whereas no change in CAT activity was observed in animals exposed to this metal (Fig. 3). In brain, SOD and CAT activities were significantly decreased (32 and 65 %, respectively) in the group exposed to Mn2+ compared with control fish. Nonetheless, GST activity and TBARS levels were unaffected (Fig. 4). Moreover, TBARS levels increased significantly (53 %) in kidney of tambaqui exposed to waterborne Mn2+. SOD activity was significantly decreased (41 %) in renal tissue of these animals, whereas no change in CAT activity was observed (Fig. 5).

Fig. 1
figure 1

Mn2+ levels in gill, liver, brain, and kidney of C. macropomum exposed to 3.88 mg L−1 waterborne Mn for 96 hours. *Significantly different from control by unpaired Student t test (p < 0.05)

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Fig. 2
figure 2

TBARS levels (a), SOD (b), GST activities (c), and GSH content (d) in gill of tambaqui exposed to 3.88 mg l−1 Mn2+ for 96 hours. Data are reported as means ± SEs (n = 10). *Significantly different from control by unpaired Student t test (p < 0.05)

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Fig. 3
figure 3

TBARS levels (a), SOD (b), CAT (c), GST activities (d), and GSH content (e) in liver of tambaqui exposed to 3.88 mg l−1 Mn2+ for 96 hours. Data are reported as means ± SEs (n = 10). *Significantly different from control by unpaired Student t test (p < 0.05)

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Fig. 4
figure 4

TBARS levels (a), SOD (b), CAT (c), and GST activities (d) in brain of tambaqui exposed to 3.88 mg l−1 Mn2+ for 96 hours. Data are reported as means ± SEs (n = 10). *Significantly different from control by unpaired Student t test (p < 0.05)

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Fig. 5
figure 5

TBARS levels (a), SOD (b), and CAT activities (c) in kidneys of tambaqui exposed to 3.88 mg.L−1 Mn2+ for 96 hours. Data are reported as mean ± SE (n = 10). *Significantly different from control by unpaired Student t test (p < 0.05)

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Discussion

Because fish constitute an important link in the food chain, their contamination by toxic metals causes a direct threat not only to the entire aquatic environment but also to humans (Obasohan 2008). Toxicity of Mn2+ in fish, despite its highly variable levels in water (Linnik 2000) and dependence on complexation (Liccione and Maines 1988), has scarcely been studied (Falfushynska et al. 2011). In the present study, the calculated Mn speciation by Visual Minteq 3.0 showed that 97 % of total Mn existed mainly as the free ionic species, Mn2+.

The maximum allowed concentration of Mn2+ in Brazilian waters is 0.1 mg L−1 (Conselho Nacional do Meio Ambiente-CONAMA 2005), whereas this metal is present at 6.44 mg L−1 in the formation water from Urucu Reserve, Amazon (Baldisserotto et al. 2012). Therefore, the concentration used in the study is in between the maximum acceptable concentration and the concentration present in oilfield process water. Manganese is not distributed homogeneously throughout the organs in tambaqui. Most of the assessments on Mn2+ bioaccumulation are in accord with the measurement of trace metals in certain habitats rather than in a controlled exposure setting (Bharti and Banerjee, 2011; Alhashemi et al. 2012). Despite the lack of correlation between the data on bioaccumulation and oxidative stress parameters, the greater accumulation of the metal in tambaqui gills is in accordance with a preceding work with Esox lucius and Abramis brama (Rajkowska and Protasowicki 2012).

Fish gills represent a thin and extensive surface (≤90 % of total body surface) in intimate contact with water. They carry out three main functions: gas exchange, ion regulation, and excretion of metabolic waste products. Due to constant contact with the external environment, gills are the first target of waterborne pollutants (Perry and Laurent 1993) and are susceptible to damage caused by heavy metals. Metals induce oxidative stress by the overproduction of ROS; thus, a strong antioxidant defense is essential to neutralize the impact of these species (Ahmad et al. 2000; Kochhann et al. 2009). The increase in SOD activity observed in gills of tambaqui exposed to Mn2+ could represent a tissue response to compensate for the increased LPO.

SOD is a key antioxidant enzyme in the metabolism of ROS because it removes superoxide anion (O •−2 ) and prevents the formation of other ROS, such as hydroxyl radicals (OH•) (Enghild et al. 1999). O •−2 is the first species in the cascade of univalent decrease of molecular oxygen and therefore is the first indicator of increased generation of ROS. Steady-state concentrations of O •−2 are directly proportional to its rate of production and inversely proportional to the activity of scavenging enzymes, such as SOD (Ferreira et al. 2004). If there is an increase in SOD activity, there will be a decrease in O •−2 and an increase in H2O2 production. H2O2 is removed by two enzymes: CAT and glutathione peroxidase (GPx). The latter uses GSH as a cofactor to remove the H2O2. The present study data also showed a decrease in GSH levels. Mn2+ toxicity is related to the depletion of GSH in different animal phyla, including aquatic animals (Madejczyk et al. 2009). The depletion of GSH can enhance Mn2+ toxicity, albeit to a lesser extent than that registered for Cu2+ (Maracine and Segner 1998; Bozocaarmutlu and Arinc Bozcaarmutlu and Arinc 2004).

There is no pattern of antioxidant behavior in gills of fish exposed to metals. Chromium (Cr) exposure (10 mg l−1 Cr3+ or Cr6+) for 96 hours did not change GSSG and total GSH ratio, GST and glutathione reductase (GR) activities, and LPO levels in gills of C. auratus. However, Cr6+ treatment resulted in decrease of carbonyl proteins levels, whereas exposure to both concentrations led to a decrease in CAT activity (Kubrak et al. 2010). In turn, gills of C. auratus gibelio exposed to 1.7 mg l−1 Mn2+ for 14 days showed increased SOD activity in addition to decreases in LPO and GSH levels (Falfushynska et al. 2011). Moreover, exposure of Channa punctatus to different cadmium (Cd2+) levels did not modify the amount of LPO and CAT activity in the gills, although Cd2+ induced a significant increase in the activity of the other enzymes, such as SOD, GPx, and GST as well as GSH content. Finally, Arabi and Alaeddini (2005) showed that supplementation of 5.5 mg l−1 Mn2+ reverted the deleterious effects of mercury (Hg2+) and copper (Cu2+) to Oncorhynchus mykiss exposed because its application inhibited LPO levels, decreased GST activity, and increased GSH content in the gill samples.

Liver is the main organ of various key metabolic pathways and the most frequently studied tissue regarding oxidative stress. Our data showed that liver LPO levels were decreased in tambaqui exposed to Mn2+. In turn, the activity of antioxidant enzymes, such as SOD and GST, and the content of nonenzymatic antioxidant GSH presented an opposite pattern, whereas CAT activity was unaffected. The increased formation of GSH in liver of tambaqui exposed to Mn2+ suggests a role in the defense of cells against oxidative stress. Furthermore, our study also showed that GST plays an important role in the detoxification of the end products of LPO.

Similar results were also shown in C. auratus gibelio exposed to 1.7 mg l−1 Mn2+ for 14 days. This species showed a decrease in levels of liver LPO associated with an increase in Mn-SOD activity compared with the respective control (Falfushynska et al. 2011). Moreover, Huang et al. (2011) also described a decrease in LPO levels in addition to an increase in GSH content in liver of rats exposed to Mn2+. However, in opposition to our data, Casalino et al. (2004) reported an increase in LPO levels in liver of rats 24 hours after administration of 2.0 mg kg−1 Mn2+. Nevertheless, these investigators found an increase in GST activity in this organ, thus corroborating our findings in tambaqui. Induction of GST activity depends on the type of tissue and nature of the inducer. In another experiment, C. punctatus exposed to sublethal concentrations of Cd2+ for 24, 48, 72, and 96 hours presented increased levels of liver LPO and modulated activities of SOD, CAT, GPx, GR, and GST as well as GSH content (Dabas et al. 2011). Thus, the current results suggest that the increase in both types of antioxidants (enzymatic and nonenzymatic) in liver of tambaqui exposed to Mn2+ is compensating for the decrease in LPO levels.

The brain is very susceptible to oxidative damage by ROS as it contains high amounts of unsaturated lipids and uses approximately 20 % of total the body’s oxygen demand (Stella and Lajtha 1987). Our data showed that LPO levels and GST activity remained unchanged, whilst SOD and CAT activities decreased in brain tissue of tambaqui exposed to Mn2+ compared with the respective control. This decrease observed in SOD and CAT activities indicates oxidative damage to organs in the presence of Mn2+.

Chtourou et al. (2010) described similar data because they verified a decrease in the antioxidant enzymes in cerebral cortex of rats that received Mn2+ in drinking water for 30 days. Mn2+ is an important cofactor for a variety of enzymes, including SOD (Hurley and Keen 1987). This metal scavenges O •−2 and OH• even when SOD activity is inhibited (Hussain and Ali 1999). However, the prooxidant effects of Mn2+ have been confirmed repeatedly in in vitro and in vivo studies (Ali et al. 1995; Zhang et al. 2004; Jiao et al. 2008). An in vitro analysis showed that 18.31 mg l−1 Mn2+ significantly inhibited CAT activity in brain of fish and lizards (Jena et al. 1998). Cr exposure (10 mg l−1 Cr3+ or Cr6+) of C. auratus for 96 hours resulted in increased brain content of carbonyl protein and no changes in SOD, CAT, and GST activities in this tissue (Kubrak et al. 2010). The same investigators published another study in 2011, in which they evaluated the effects of various concentrations of cobalt (Co2+) on brain of C. auratus. Exposure to 50 mg l−1 Co2+ for 96 hours did not affect LPO levels and GR activity; however, this induced a decrease in SOD, CAT, and glucose-6-phosphate dehydrogenase activities (Kubrak et al. 2011). These findings are in accordance with our data.

Finally, our results also showed that Mn+2 exposure of tambaqui may reflect the development of renal oxidative stress because it led to an increase in LPO levels associated with a decrease in SOD activity, although no change was observed in CAT activity. SOD, along with CAT, represents the first barrier against ROS and is essential to cell survival (Remacle et al. 1992; Mates et al. 1999; Halliwell 2001). Travacio and Llesuy (1996) reported that different models of oxidative stress involve a biphasic response of antioxidant enzyme activities. At first, enzymatic activities are markedly decreased, but with time the activity levels increase, probably as a consequence of a new synthesis and/or enzymatic activation.

C. punctatus exposed to Cd2+ (6.7, 13.4, and 20.1 mg l−1) for various time periods (24, 48, 72, and 96 hours) presented increased levels of LPO as well as SOD, GST, and GR activities, whereas CAT activity was decreased (Dabas et al. 2011). In turn, C. auratus exposed to various concentrations of Cr6+ for 96 hours showed increased renal hydroperoxide levels and SOD activity and no significant differences in CAT activity (Velma and Tchounwou 2010).

The results of the current research clearly show that there were changes in the balance of pro-oxidants and antioxidants in different organs of tambaqui. Such changes were more evident in liver and kidney. Furthermore, there was no correlation between the oxidative stress results and the bioaccumulation data. Present findings may contribute to the scarce literature regarding fish subchronic exposure to Mn2+.