Moderate hypoxia is able to minimize the manganese-induced toxicity in tissues of silver catfish (Rhamdia quelen)

Ecotoxicology and Environmental Safety 91 (2013) 103–109

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Moderate hypoxia is able to minimize the manganese-induced toxicity in tissues of silver catfish (Rhamdia quelen) G.S. Dolci a, V.T. Dias c, K. Roversi c, Kr. Roversi c, C.S. Pase c, H.J. Segat c, A.M. Teixeira a, D.M. Benvegnu´ a, F. Trevizol a, R.C.S. Barcelos a, A.P.K. Riffel a, M.A.G. Nunes b, V.L. Dressler b, E.M.M. Flores b, c,n ¨ B. Baldisserotto c, M.E. Burger a

~ em Farmacologia, Departamento de Fisiologia e Farmacologia, Universidade Federal de Santa Maria (UFSM), Rio Grande do Sul, Brazil ´s-Graduac- ao Programa de Po ´s-Graduac- a~ o em Quı´mica, Departamento de Quı´mica, Centro de Ciˆencias Naturais e Exatas (UFSM), Rio Grande do Sul, Brazil Programa de Po c ´ de (UFSM), Rio Grande do Sul, Brazil Departamento de Fisiologia e Farmacologia, Centro de Ciˆencias da Sau b

a r t i c l e i n f o

abstract

Article history: Received 14 October 2012 Received in revised form 14 January 2013 Accepted 17 January 2013 Available online 20 February 2013

The aim of this study was to compare the effects of manganese (Mn) on silver catfish exposed to different levels of dissolved oxygen. Silver catfish (Rhamdia quelen) were exposed to increasing concentrations of Mn (4.2, 8.4 or 16.2 mg L  1) under either normoxia (100 percent saturation) or moderate hypoxia (51.87 percent saturation) for 15 days. Under normoxia, Mn exposure increased lipid peroxidation (LP) in brain and kidney; it increased gluthatione (GSH) levels in brain and decreased catalase (CAT) activity in both tissues. Moderate hypoxia was able to prevent Mn-induced LP in brain and to reduce this oxidative parameter in kidney; GSH level was increased in brain, while CAT activity was reduced in both tissues. Activity of isolated mitochondria of liver and gills was reduced by Mn exposure under both levels of dissolved oxygen, but this effect was more prominent in normoxia. As expected, liver, kidney and gills showed an increase of Mn accumulation according to waterborne levels, and these parameters presented positive relationship. The highest waterborne Mn (8.4 and 16.2 mg L  1) resulted in greater accumulation under normoxia, indicating that moderate hypoxia can stimulate mechanisms capable of reducing Mn accumulation in tissues (though not in blood). Moderate hypoxia can be considered a stress factor and Mn an aquatic anthropogenic contaminant. Therefore we hypothesized that these two conditions together are able to invoke defense mechanisms in juvenile silver catfish, acting in a compensatory form, which may be related to adaptation and/or hormesis. & 2013 Elsevier Inc. All rights reserved.

Keywords: Moderate hypoxia Metal contamination Mitochondrial activity Lipid peroxidation Antioxidant system

1. Introduction Silver catfish (Rhamdia quelen) is a Brazilian native species widely used in fishponds, whose cultivation has been increasing steadily (Baldisserotto, 2009). Human interference in the aquatic environment has led to fish mortality due to inadequate sewage treatment and release of industrial waste into waters. In this context, manganese (Mn) is a mineral which occurs naturally in freshwaters at concentrations from 1 to 200 mg L  1 (Barceloux, 1999). High levels this metal already were detected in toxic waste sites in the United States (ATSDR, 2000) and its environmental contamination can be related to use of pesticides (Abou-Arab et al., 1996), coal extraction (Crossgrove and Zheng, 2004) and oil exploration (Hatje et al., 2008). In this sense, formation water from oil and gas production, rich in salts and metals, including Mn, may affect ions transport in

n

Corresponding author. Fax: þ55 55 3220 7686. ¨ E-mail address: [email protected] (M.E. Burger).

0147-6513/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.01.013

fishes and reduce rivers’ biodiversity (Baldisserotto et al., 2012). In this sense, Mn is a redox active metal that acts as a reactive oxygen species (ROS) generator (Zhang et al., 2003), facilitating the development of oxidative stress (OS) for fish. According to Luk and Culotta (2001), Mn is essentially accumulated in organelles like the mitochondria, whereas concentrations in the cytoplasm are relatively low. While such concentrations are maintained by metal transporters in the membrane, its elimination from mitochondria is a slow process, which involves Naþ efflux (Gavin et al., 1999). Excessive loads of dissolved organic matter lead to greater demand for oxygen, reducing dissolved oxygen levels in water streams (Winemiller et al., 2008). Low dissolved oxygen levels can be a stress factor to aquatic organisms (Oweson et al., 2010) and induce changes on behavior, growth and survival of fish, as the silver catfish (Braun et al., 2006). Considering that hypoxia may also favor the OS and thus lead to increased lipid peroxidation (LP) (Braun et al., 2008), literature lacks studies about OS, Mn toxicity and hypoxia (Nikinmaa and Rees, 2005; Oweson et al., 2010), mainly when these two last factors occur concurrently.

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Considering OS, the generation of by-products of oxygen metabolism by aerobic organisms occurs naturally from energy production via oxidative phosphorylation (Storey, 2004). ROS include superoxide anion (O2  ), hydrogen peroxide (H2O2), hydroxyl radical (OH  ), among others (Halliwell and Gutteridge, 1999), which may damage cellular components, particularly proteins, lipids, and nucleic acids, often leading to cumulative injury that can lead to cell death. Normally, OS is a consequence of the predominance of ROS generation in detriment of their degradation (Sies, 1991), which is carried out by antioxidant systems specially developed to maintain cellular integrity. The antioxidant system consist of low-molecular weight compounds such as glutathione (GSH), ascorbic and uric acids, tocopherols and proteins, also including such enzymes as superoxide dismutase (SOD) and catalase (CAT), among others (Hermes-Lima, 2004). Whereas Mn toxicity in fish has been little studied (Falfushynska et al., 2011; Pinsino et al., 2010), the influence of low dissolved oxygen has received more attention (Braun et al., 2008; Nikinmaa and Rees, 2005; Oweson et al., 2010). However, few studies combine these two conditions able to generate OS and toxicity of aquatic animals. In addition, Mn presents high mobility in conditions of low levels of DO due to reductive dissolution of MnO2 to Mn2 þ . This relevant chemical property of Mn may exacerbate situations of environmental contamination in waters subjected to hypoxia, and thus compromise the aquatic biota (Limburg et al., 2011; Itai et al., 2012). Here we propose to evaluate the influence of different waterborne Mn levels in silver catfish (Rhamdia quelen), under normoxia and moderate hypoxia conditions, mimicking environmental variations of dissolved oxygen levels, on tissue Mn accumulation, and its influence on the oxidative status, which involves mitochondrial dysfunction, OS and antioxidant defenses.

2. Material and methods 2.1. Fish One-hundred and sixty silver catfish (35.00 7 7.24 g; 16.03 7 1.4 cm) obtained from fishponds at the Universidade Federal de Santa Maria, Southern Brazil, were acclimated in continuously aerated 250 L tanks with controlled temperature (24 1C) for at least one week prior to experiments. During this period, they were fed once a day (42 percent crude protein). Methodologies of the experiments were approved by the Ethical and Animal Welfare Committee of the Federal University of Santa Maria (Process No. 105/2010).

2.2. Reagents Manganese sulfate hexahydrate [MnSO4  6H2O] (Vetecs, Rio de Janeiro, RJ, Brazil) was used to increase waterborne Mn levels. All chemicals and solvents used were of HPLC grade purchased of Sigma Aldrichs, Brazil.

2.3. Treatments Manganese levels were selected according to two criteria: (i) in a pilot-study it was observed the toxicity of the Mn as from 5 mg L  1, (ii) Mn levels in situations of environmental pollution (Baldisserotto et al., 2012; Oweson et al., 2010). After acclimation, silver catfish were randomly transferred to 20 L tanks (four fish per tank, in a semi-static system) and exposed for 15 days to four waterborne Mn levels (in mg L  1): 0 (control), 5, 10 and 20, whose values detected by inductively coupled plasma optical emission spectrometry (described below in 2.5.2.3) were 0.001, 4.2, 8.4 and 16.2, respectively, in normoxia (DO: 7.4870.28 mg L  1; pH: 7.8870.05) or hypoxia (DO: 3.8870.41 mg L  1; pH: 7.4470.10). The experimental design totalized eight treatments in quadruplicate. Different dissolved oxygen levels were maintained through aeration with air (air pump AC 2000, 0.0014 MPa pressure) and/or nitrogen (White Martins). After 15 days of exposure, animals were euthanized by section of spinal cord. Blood was obtained by caudal puncture and brain, kidney, liver and gill were collected for further analysis.

2.4. Water parameters Dissolved oxygen (DO) and temperature were measured with an YSI oxygen meter (Model Y5512; YSI Inc., Yellow Springs, OH, USA). The pH was determined with DMPH-2 pH meter (Digimed, Sa~ o Paulo, SP, Brazil) and total ammonia nitrogen (TAN) levels according to Eaton et al. (2005). Un-ionized ammonia (NH3) levels were calculated according to Colt (2002).Water hardness was analyzed by the EDTA titrimetric method. Alkalinity was determined according to Boyd and Tucker (1992). Water tanks were cleaned daily by siphoning and replaced with pre-adjusted Mn levels [MnSO4.6H2O]. At the end of each exposure, Mn wastes were chemically treated and properly disposed. 2.5. Analysis 2.5.1. Hematocrit To obtain the percentage of packed red cells, microhematocrit capillary tubes were filled with blood obtained from caudal vein and centrifuged at 1310  g for 15 min, and the results were obtained by means of a hematocrit card reader, according to Goldenfarb et al. (1971). 2.5.2. Sample Mn determination 2.5.2.1. Water samples. Aquaria water samples (5 mL) were collected every two day of the experiment and stored for further analysis. 2.5.2.2. Plasma and tissues samples. Plasma (0.5 mL) was collected after blood centrifugation (1310  g, 15 min). Digestion of plasma, liver, kidney, brain and gill was performed by conventional heating block (Velp Scientifica, Model DK, Italy) with open glass vessels. The procedure was carried out with concentrate nitric acid (14 mol L  1) (Merck, Darmstadt, Germany). H2O2 was added and the digests were heated up to 80 1C for 1 h. After cooling, digests were diluted with purified water (Milli-Q system, Millipore Corp., Bedford, USA) for further analysis. 2.5.2.3. Mn samples determination. Waterborne Mn levels and Mn accumulation in plasma and tissues were determined by inductively coupled plasma optical emission spectrometry (ICP OES, Model Spectro Ciros CCD, Spectro Analytical Instruments, Kleve, Germany), which was equipped with axial view configuration and a cross flow nebulizer coupled to a Scott type double pass nebulization spray chamber. The wavelength for Mn determination was 257.611 nm and the radiofrequency power was 1400 W. The flow rate for plasma generation, auxiliary and nebulization gas were 20.1 and 0.9 L  1 min  1, respectively. Argon (99.996 percent, White Martins–Praxair, Sa~ o Paulo, SP, Brazil) was used for plasma generation, for nebulization and as auxiliary gas. For accuracy evaluation, a certified reference material (CRM) of dogfish muscle tissue (DORM-2) from The National Research Council, Canada, was used. 2.5.3. Oxidative stress (OS) parameters determination While several studies support the vulnerability of the brain as a target for metals in the aquatic environment (Mieiro et al., 2010; Lushchak et al., 2009), the kidney has been related to homeostatic functions in fish, mainly due to its antioxidant role, making these organs important targets for studies of antioxidant defenses (Vieira et al., 2012). Brain and kidney were homogenized (1:5 w/v) in buffer Tris-HCl 10 mM (pH: 7.4) and centrifuged (3640  g, 15 min). The supernatants were used for estimation of the lipid peroxidation (LP), which was estimated by thiobarbituric acid reactive substances (TBARS) as described by Ohkawa et al. (1979). This measure is based on reaction of MDA (malondialdehyde) resulting from oxidative damage in the lipid membranes with thiobarbituric acid, whose pink chromogen generated can be spectrophotometrically measured at 532 nm. The TBARS levels were expressed as nmol of MDA g tissue  1. Glutathione levels (GSH) were determined in supernatant after reaction with DTNB [5,50 -bis-dithio-(2-nitrobenzoic acid)], according to Ellman (1959) with modifications (Jacques-Silva et al., 2001). Catalase (CAT) activity was spectrophotometrically quantified by the method of Aebi (1984), which monitors the disappearance of H2O2 in the presence of tissue at 240 nm. The enzymatic activity was expressed as mmol H2O2 g tissue  1 min  1 (1 U decomposes 1 mmol H2O2/min at pH 7 at 25 1C). 2.5.4. Mitochondrial isolation/activity Gill and liver were selected for mitochondria isolation following functional criteria, which made them preferential targets of xenobiotic uptake and metabolism, respectively. Silver catfish were fasted overnight prior to euthanasia by decapitation. Mitochondria were isolated as previously described by Brustovetsky and Dubinsky (2000), with some modifications: gill and liver were rapidly removed (within 1 min) and immersed in ice-cold ‘isolation buffer I’ containing 225 mM mannitol, 75 mM sucrose, 1 mM K þ -EGTA, 0.1 percent bovine serum albumin (BSA) and 10 mM K þ HEPES, pH 7.2. The tissues were minced using surgical scissors and then extensively washed. The tissue was then homogenized in a power-driven, tight-fitting Potter-

G.S. Dolci et al. / Ecotoxicology and Environmental Safety 91 (2013) 103–109 Elvehjem homogenizer with Teflon pestle. The resulting suspension was centrifuged for 7 min at 2000  g in a refrigerate ultracentrifuge. After centrifugation the supernatant was re-centrifuged for 10 min at 12,000  g. The pellet was re-suspended in ‘isolation buffer II’ containing 225 mM mannitol, 75 mM sucrose, 1 mM K þ -EGTA, and 10 mM K þ -HEPES pH 7.2 and recentrifuged at 12,000  g for 10 min. The supernatant was decanted and the final pellet gently washed and re-suspended in 600 mL of ‘isolation buffer III’ containing 10 mM sucrose, 60 mM KCl, 10 mM K þ HEPES buffer (pH 7.2), 50 mM EGTA, 5 mM glutamate and 5 mM succinate. Mitochondria activity was developed by colorimetric reduction of [3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]-MTT whose color can be spectrophotometrically measured (l ¼ 570–630 nm). This assay quantifies mitochondrial activity by measuring the formation of a dark violet formazan product formed by reduction of tetrazolium ring of MTT (yellow) (Mosmann, 1983). The MTT reduction mainly occur in the mitochondria through the action of succinate dehydrogenase, therefore providing a measure of mitochondrial function (Slater et al., 1963). For MTT assay, mitochondria suspension was prior diluted to a protein concentration of 30– 40 mg/mL (Lowry et al., 1951). Data were expressed as a percentage of the untreated control group.

2.6. Statistics All statistics were performed using Statistica (Statsoft Inc., Tulsa, USA). Data were analyzed by two-way ANOVA (Mn  dissolved oxygen levels) followed by Duncan’s multiple range test, when appropriate. p o 0.05 was regarded as statistically significant.

3. Results 3.1. Water parameters All of the assessed parameters (pH; DO; hardness, alkalinity; NH4; NH3; nitrite) showed similar values, with no variations throughout the experiment (data not shown). 3.2. Hematocrit and final body weight Silver catfish exposed to Mn and either normoxia or hypoxia did not show significant differences in body weight at the end of the treatments. Duncan’s test showed a reduction of hematocrit value in fish under normoxia exposed to Mn 8.4 mg L  1 and Mn 16.2 mg L  1 relative to both groups control and Mn 4.2 mg L  1. Under moderate hypoxia, different concentrations of Mn did not change the hematocrit (Table 1). 3.3. Mn accumulation The increase of waterborne Mn concentration increased its accumulation in the plasma and tissues analyzed irrespective of dissolved oxygen level (Fig. 1). Significantly higher Mn accumulation in the liver was observed in silver catfish maintained in normoxia at Mn

Table 1 Hematocrit (% cell volume) of silver catfish exposed to waterborne manganese (Mn) levels under normoxia or hypoxia for 15 days. Mn (mgL  1)

0.001 4.2 8.4 16.2

Hematocrit (%) Normoxia

Hypoxia

28.75 7 3.83a 27.007 1.76a 19.307 1.81b 20.707 1.73b

27.90 7 1.79 27.80 7 1.32 29.50 7 2.53n 26.90 7 2.10n

Values are expressed as mean 7 S.E.M. n

Indicates significant difference from normoxia in the same waterborne Mn level (po 0.05). Different lowercase in the columns indicates significant difference among waterborne Mn level in the same dissolved oxygen (DO) level (p o 0.05).

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16.2 mg L  1 (Fig. 1A), while hypoxia increased this accumulation in the plasma of those exposed to the same Mn levels (Fig. 1D). After exposure to Mn 8.4 mg L  1, normoxia was able to significantly increase Mn accumulation in the kidney of silver catfish in relation to moderate hypoxia at the same Mn concentration (Fig. 1B). Normoxia was able to significantly increase Mn accumulation in silver catfish gills at all Mn concentrations in relation to hypoxia (Fig. 1C). 3.4. Oxidative stress parameters 3.4.1. Brain Post hoc test showed a significant increase of LP with the highest Mn concentration (Mn 16.2 mg L  1) in brain of silver catfish under normoxia in relation to all other groups at the same oxygen level. Under moderate hypoxia, no differences between the different concentrations of Mn were observed. In fact, normoxia increased LP in brain of silver catfish exposed to Mn 16.2 mg L  1 as compared to moderate hypoxia at the same Mn concentration (Fig. 2A). Post hoc test showed increased GSH levels in brain of silver catfish under normoxia exposed to Mn 8.4 mg L  1 and Mn 16.2 mg L  1 in relation to Mn 4.2 mg L  1 and control. Under hypoxia, Mn concentrations of 0.001–8.4 mg L  1 showed no differences on GSH levels, while the highest Mn concentration (Mn 16.2 mg L  1) increased GSH levels as compared to other groups. Moderate hypoxia increased GSH levels in brain of silver catfish exposed to Mn 0.001 and Mn 4.2 mg L  1, but not in those exposed to Mn 8.4 and Mn 16.2 mg L  1 in relation to fish kept at the same Mn concentrations under normoxia (Fig. 2B). Under normoxia, catalase (CAT) activity reduced in brain of silver catfish exposed to Mn 8.4 mg L  1 and Mn 16.2 mg L  1 when compared to controls. Under moderate hypoxia, enzyme activity was not modified by Mn exposure. Irrespective of Mn concentrations hypoxia did not affect CAT activity (Fig. 2C). 3.4.2. Kidney Under normoxia, increased LP was observed in kidney of silver catfish exposed to all Mn concentrations as compared to controls. Under moderate hypoxia, only the highest Mn concentration increased LP in relation to other concentrations (Mn 4.2 and 8.4 mg L  1). Moderate hypoxia was able to reduce LP of fish exposed to Mn 4.2 mg L  1and Mn 16.2 mg L  1 in relation to fish kept on normoxia at the same Mn concentrations (Fig. 2D). No significant effects of dissolved oxygen level, Mn concentration or their interaction on GSH levels were observed in the kidney of silver catfish (Fig. 2E). Under normoxia, post hoc test showed that all fish exposed to Mn concentrations presented reduced kidney CAT activity in relation to controls, and the highest Mn concentration (Mn 16.2 mg L  1) also reduced CAT activity in relation to Mn 4.2 mg L  1, but not to Mn 8.4 mg L. Under moderate hypoxia, silver catfish exposed to Mn 16.2 mg L  1 showed lower kidney catalase activity when compared to controls and those exposed Mn 4.2 mg L  1. In fact, as compared to normoxia, hypoxia decreased CAT activity in kidney of control silver catfish and those exposed to Mn 4.2 mg L  1 (Fig. 2F). 3.5. Mitochondrial activity Under normoxia and hypoxia, all silver catfish exposed to Mn concentrations decreased liver mitochondrial activity in relation to controls. In control fish moderate hypoxia was able to increase liver mitochondrial activity in relation to normoxia (Fig. 3A).

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Fig. 1. Multiple regressions between Mn bioaccumulation and Mn waterborne levels in silver catfish liver (A), kidney (B), gills (C) and plasma (D) co-exposed to Mn 0, 4.2, 8.4 and 16.2 mg L  1 for 15 days under normoxia or hypoxia. The r2 values for liver, kidney, gills and plasma were: 0.71 and 0.69; 0.70 and 0.84; 0.83 and 0.78; 0.80 and 0.68 in normoxia and hypoxia, respectively (p o0.001 for all).

Under normoxia, all exposure to Mn concentrations decreased gills mitochondrial activity in relation to controls, while in hypoxia such activity was reduced only in those kept at Mn 8.4 mg L  1 and 16.2 mg L  1, when compared to controls and Mn 4.2 mg L  1, respectively. Moderate hypoxia was able to increase gills mitochondrial activity of silver catfish exposed to Mn 4.2 mg L  1, in relation to normoxia (Fig. 3B).

4. Discussion Silver catfish exposed to Mn and normoxia showed higher LP in brain and kidney; hematocrit was reduced when the two highest Mn concentrations were associated with normoxia, while moderate hypoxia did not change this hematological parameter at the same concentrations of this metal. The effects of Mn on hematologic parameters in fish were reported (Barnhoorn and van Vuuren, 2001), reinforcing the toxicity of this metal on aquatic organisms. On the other hand, low oxygen levels increased hematocrit values and induced erythrocyte swelling and new red blood cell formation (Nikinmaa and Tervonen, 2004). Moreover, anaerobic metabolism developed during fish hypoxia seems to be related to increase of hematocrit and higher levels of red blood cells (Affonso et al., 2002). Although these studies showed compensatory mechanisms to overcome the lower rate of bioavailable oxygen in the aquatic environment, in the present study moderate hypoxia did not increase hematocrit in catfish, but was capable of preventing damages of Mn exposition under

normoxia, representing a protective factor of the lack of oxygen on hematopoiesis. In addition, groups treated with Mn and normoxia also showed higher Mn uptake, reinforcing a causal relationship. Mn accumulation was higher under normoxia in liver, kidney and gills, but not in plasma of silver catfish. In fact, plasma corresponds to a passage to deposit tissues and has no storage function like the ¨ other evaluated tissues (Van Der Putte and Part, 1982). In addition, as expected, positive relationships between waterborne Mn levels and its accumulation in tissues were also observed, confirming Mn toxicity. The mechanism of Mn toxicity is not fully understood, but its accumulation in the mitochondrial matrix showed inhibitory effects on oxidative phosphorylation (Gavin et al., 1992), reflecting its toxicity on organelle activity. As observed in the present study, Mn showed its toxic effects on mitochondria of liver and gills in silver catfish because there was of loss activity under normoxia. Several mechanisms may be involved in Mn cell toxicity, including substitution of Fe2 þ in cytochromes of the cellular respiratory chain (Missy et al., 2000), which lead to ROS generation, as well as a direct inhibitory effect of metal on mitochondrial enzymes (Singh et al., 1979). Two hypotheses can be proposed to explain the lower toxicity of Mn when silver catfish were maintained in moderate hypoxia: (i) development of tolerance and cellular adaptation of fish to reduced availability of oxygen; (ii) development of hormesis due to environmental stressors. Indeed, an adaptation process was described as the ability of cells to adapt to low oxygen levels through activation of HIF (hypoxia-inducible factor) (Oweson

G.S. Dolci et al. / Ecotoxicology and Environmental Safety 91 (2013) 103–109

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Fig. 2. Levels of lipid peroxidation, GSH and and catalase activity in brain (A, B and C, respectively) and kidney (D, E and F, respectively) of silver catfish co-exposed to manganese and either normoxia or hypoxia for 15 days. Data are expressed as mean 7 S.E.M. Lowercase indicates significant difference between the different concentrations of Mn and the same dissolved oxygen level (p o 0.05); *Indicates difference between normoxia and hypoxia in the same Mn concentration (po 0.05).

et al., 2010), whose expression was demonstrated in different organisms throughout evolution (Wu, 2002; Nikinmaa and Rees, 2005). Hypoxia may also induce fish to compensate oxygen shortage by increasing ventilation and blood flow in the gills (Sundin, 1999; Hattink et al., 2005, 2006), which could lead to higher Mn uptake. However, in spite of hyperventilation in hypoxia, accumulation of Cd and Zn in common carp, Cyprinus carpio, was not affected (Hattink et al., 2005, 2006). In addition, silver catfish exposed for four days to moderate hypoxia (3.5 and 4.5 mg L  1) did not show any significant change on net ion fluxes (Rosso et al., 2006). Therefore, it is unlikely that changes in ion transport (and consequently, Mn) in the gills are related to the decrease on Mn bioaccumulation in this species. The second hypothesis suggests that fish exposed to environmental stressors are able to trigger defense mechanisms, which would act much more efficiently than in fish not previously exposed to stressors. This hypothesis has been used to explain the antioxidant defenses in mammals, but can also be used for

fish, as previously reported by Bengtsson (1979). This author proposed the development of hormesis during exposure of fishes to low concentrations of environmental pollutants. Another study showed an enhanced growth rate of crustacean larvae exposed to petroleum pollutants for short periods as compared to unexposed ones, whose response was considered by the authors as hormesis (Laughlin et al., 1981). Our findings also point towards the development of hormesis, since oxidative damages and metal accumulation were lower in silver catfish exposed to Mn and moderate hypoxia than in those exposed to Mn and normoxia. In fact, our data are consistent with development of hormesis during exposure of fish to moderate hypoxia together with an environmental pollutant as Mn, allowing physiological changes counteract the damages caused by the metal. Furthermore, low levels of stress also showed positive effects on fish reproduction against greater stress, which presented negative effects (Schreck, 2010). These data also led the authors to the concept of hormesis, which is a useful way to think about the effect of stressors on fishes. So, hormesis is not an innovative term employed in environmental

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Fig. 3. Mitochondrial viability of silver catfish liver (A) and gills (B) co-exposed to manganese and either normoxia or hypoxia for 15 days. Data are expressed as mean 7 S.E.M. Lowercase indicates significant difference between the different Mn concentrations and the same dissolved oxygen level (p o 0.05); *Indicates difference between normoxia and hypoxia in the same Mn concentration (p o 0.05).

contaminants and fishes, and can be defined as a physiological response that helps the organism to react to the continuous presence of a small stimulus such as low concentrations of ROS or stress, which can induce an increase in the antioxidant defenses, promoting compensatory processes following an initial disruption in homeostasis (hormesis hypothesis) (Calabrese and Baldwin, 2002). In our study, the lower levels of LP, larger mitochondrial activity, and largely unchanged hematocrit observed in silver catfish under moderate hypoxia may all indicate the development of mechanisms of adaptation and tolerance to a hostile environment, as well as enhanced defense mechanisms (Hamdoun and Epel, 2007) resulting from hormesis. Inversely to our experiments, Lushchak et al. (2005) suggested that increased dissolved oxygen levels may be able to increase ROS generation due to leakage of electrons from the electrontransport chains to join with molecular oxygen. While different fish species showed increased oxidative stress under hyperoxia (Lushchak et al., 2005), our experimental protocol was developed with moderate hypoxia, which caused favorable physiological adaptations on oxidative processes as well as highest mitochondrial activity in the presence of contaminants as Mn. Considering the oxidative status, Braun et al. (2008) reported a relationship between hypoxia and development of OS, but this condition was accompanied by increase of enzymatic antioxidant defenses, and therefore it is consistent with the hormesis hypothesis proposed in our study. A recent study showed that Mn-induced toxicity was related to metal accumulation and misregulated homeostasis of sea urchin embryos, stimulating protective agents against apoptosis (Pinsino et al., 2010). Concerning antioxidant defenses, while catalase activity in brain and kidney was slightly modified by the treatments, GSH levels in brain were more clearly improved with increasing concentrations of Mn under hypoxia, reinforcing the idea that reduced oxygen levels can stimulate antioxidant defenses. We believe that this event may be closely related to

hormesis/adaptation hypothesis proposed here, which showed be able to minimize the damages caused by this metal.

5. Conclusion Our study shows for the first time that under conditions of moderate hypoxia and presence of Mn, silver catfish are capable of developing mechanisms of adaptation and/or hormesis, which were observed through of the lowest metal bioaccumulation as well as its reduced toxicity. While the moderate hypoxia was related to lower oxidative damage in the brain and kidney, an increased of the mitochondrial activity was observed in liver and gills of the same fish concurrently exposed to moderate hypoxia and Mn. Such favorable effects observed under moderate hypoxia can be related to development of hormesis-induced physiological adaptations. More studies on the cellular mechanisms involved in tolerance and adaptation of fish exposed to moderate and severe hypoxia, as well as the hormesis development should be conducted.

Acknowledgments The authors (M.E.B.; B.B.; F.E.M.M.; C.S.P. and G.S.D.) are grateful to Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), (K.R.) Fundac- a~ o de Amparo a Pesquisa do Estado do Rio Grande do Sul (FAPERGS), and Center for studies of Adaptations of Aquatic Biota of the Amazon (Adapta) for fellowships and financial support. References Abou-Arab, A.K., Ayesh, A.M., Amra, H.A., Naguib, K., 1996. Characteristics levels of some pesticides and heavy metals in imported fish. Food Chem. 57 (4), 487–492. Aebi, H., 1984. Catalase in vitro. Methods Enzymol 105, 121–126.

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Affonso, E.G., Polez, V.L.P., Corrˆea, C.F., Mazon, A.F., Arau´jo, M.R.R., Moraes, G., Rantin, F.T., 2002. Blood parameters and metabolites in the teleost fish Colossoma macropomum exposed to sulfide or hypoxia. Comp. Biochem. Physiol. C: Toxicol. Pharmacol 133, 375–382. ATSDR, 2000. Toxicological Profile for Manganese. United States Department of Health and Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry, Atlanta, GA. Baldisserotto, B., 2009. Freshwater fish culture in Rio Grande do Sul State: actual situation, problems and future perspectives. Ciˆenc. Rural 39 (1), 291–299. Baldisserotto, B., Garcia, L.O., Benaduce, A.P., Duarte, R.M., Nascimento, T.L., Gomes, L.C., Chippari Gomes, A.R., Val, A.L., 2012. Sodium fluxes in tamoata´, Hoplosternum litoralle, exposed to formation water from Urucu Reserve (Amazon, Brazil). Arch. Environ. Contam. Toxicol 62 (1), 78–84. Barceloux, D.G., 1999. Manganese. Clin.Toxicol. 37, 293–307. Barnhoorn, I., van Vuuren, J.H.J., 2001. Sublethal effects of manganese on the haematology and osmoregulation of Oreochromis mossambicus after acute exposure. Afr. J. Aquat. Sci 26 (1), 1–7. Bengtsson, B.E., 1979. Increased growth in minnows exposed to PCBs. Ambio 8 (4), 169–170. Boyd, C.E., Tucker, C.S., 1992. Water Quality and Pond Soil Analyses for Aquaculture. Alabama Agricultural Experiment Station, Auburn University, AL, USA. Braun, N., Lima, R.L., Moraes, B., Loro, V.L., 2006. Survival, growth and biochemical parameters of silver catfish, Rhamdia quelen (Quoy & Gaimard, 1824), juveniles exposed to different dissolved oxygen levels. Aquacult. Res. 37, 1524–1531. Braun, N., Lima, R.L., Dalla Flora, F., Lang, M.E., Bauermann, L.F., Loro, V.L., Baldisserotto, B., 2008. Lipid peroxidation and superoxide dismutase activity in silver catfish (Rhamdia quelen) juveniles exposed to different dissolved oxygen levels. Ciˆenc. Anim. Bras. 9 (3), 811–814. Brustovetsky, N., Dubinsky, J.M., 2000. Dual responses of CNS mitochondria to elevated calcium. J. Neurosci. 20, 103–113. Calabrese, E.J., Baldwin, L.A., 2002. Defining hormesis. Hum. Exp. Toxicol. 21, 91–97. Colt, J., 2002. List of spreadsheets prepared as a complement (Available in /http:// www.fisheries.org/afs/hatchery htmlS). In: Wedemeyer G.A. (ed.), Fish Hatchery Management, second ed., Amer Fish Soc Pub. Crossgrove, J., Zheng, W., 2004. Manganese toxicity upon overexposure NMR. Biomed 17, 544–553. Eaton, A.D., Clesceri, L.S., Rice, E.W., Greenberg, A.E., 2005. Standard Methods for the Examination of Water and Wastewater, 21st ed. Amer Public Health Assn, USA. Ellman, G.L., 1959. Tissue sulphydryl groups. Arch. Biochem. Biophys. 82, 70–77. Falfushynska, H.I., Gnatyshyna, L.L., Stoliar, O.B., Nam, Y.K., 2011. Various responses to copper and manganese exposure of Carassius auratus gibelio from two populations. Comp. Biochem. Physiol. C: Toxicol. Pharmacol 154 (3), 242–253. Gavin, C.E., Gunter, K.K., Gunter, T.E., 1992. Manganese sequestration by mitochondria and inhibition of oxidative phosphorylation. Toxicol. Appl. Pharmacol 115, 1–5. Gavin, C.E., Gunter, K.K., Gunter, T.E., 1999. Manganese and calcium transport inmitochondria: implications for manganese toxicity. Neurotoxicology 20 (2–3), 445–453. Goldenfarb, P.B., Bowyer, F.P., Hall, E., Brosious, E., 1971. Reproducibility in the hematology laboratory: the microhematocrit determination. Am. J. Clin. Path. 56, 35–39. Halliwell, B., Gutteridge, J.M.C., 1999. Free Radicals in Biology and Medicine, third ed. Clarendon Press, Oxford. Hamdoun, A., Epel, D., 2007. Embryo stability and vulnerability in an always changing world. Proc. Nat. Acad. Sci. USA 104, 1745–1750. Hatje, V., Barros, F., Magalha~ es, W., Riatto, V.B., Amorim, F.N., Figueiredo, M.B., Spano´, S., Cirano, M., 2008. Trace metals and benthic macrofauna distributions in Camamu Bay, Brazil: sediment quality prior oil and gas exploration. Mar. Pollut. Bull. 56, 348–379. Hattink, J., De Boeck, G., Blust, R., 2005. The toxicokinetics of cadmium in carp under normoxic and hypoxic conditions. Aquat. Toxicol. 75, 1–15. Hattink, J., De Boeck, G., Blust, R., 2006. Toxicity, accumulation, and retention of zinc by carp under normoxic and hypoxic conditions. Environ. Toxicol. Chem 25 (1), 87–96. Hermes-Lima, M., 2004. Oxygen in biology and biochemistry: role of free radicals. In: Storey, K.B. (Ed.), Functional Metabolism: Regulation and Adaptation. Wiley-Liss, Hoboken, pp. 319–368. Itai, T., Kumagai, M., Hyobu, Y., Hayase, D., Horai, S., Kuwae, M., Tanabe, S., 2012. Apparent increase in Mn and As accumulation in the surface of sediments in Lake Biwa, Japan, from 1977 to 2009. Geochem. J. 46, e47–e52.

109

Jacques-Silva, M.C., Nogueira, C.W., Broch, L.C., Flores, E.M.M., Rocha, J.B.T., 2001. Diphenyl diselenide and ascorbic acid changes deposition of selenium and ascorbic acid in liver and brain of mice. Pharmacol. Appl. Toxicol 88, 119–125. Laughlin Jr, R.B., Ng, J., Guard, H.E., 1981. Hormesis: a response to low environmental concentrations of petroleum hydrocarbons. Science 211, 705–707. Limburg, K.E., Olson, C., Walther, Y., Dale, D., Slomp, C.P., Høie, H., 2011. Tracking Baltic hypoxia and cod migration over millennia with natural tags. Environ. Sci 1, 1–6. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275. Luk, E.E., Culotta, V.C., 2001. Manganese superoxide dismutase in Saccaromyces cerevisiae acquires its metal co-factor through a pathway involving the Nramp metal transporter, Smf2p. J. Biol. Chem 276, 556–562. Lushchak, O.V., Kubrak, O.I., Torous, I.M., Nazarchuk, T.Y., Storey, K.B., Lushchak, V.I., 2009. Trivalent chromium induces oxidative stress in goldfish brain. Chemosphere 75, 56–62. Lushchak, V.I., Bagnyukova, T.V., Husak, V.V., Luzhna, L.I., Lushchak, O.V., Storey, K.B., 2005. Hyperoxia results in transient oxidative stress and an adaptative response by antioxidant enzymes in goldfish tissues. Int. J. Biochem. Cell Biol. 37, 1670–1680. Mieiro, C.L., Ahmad, I., Pereira, M.E., Duarte, A.C., Pacheco, M., 2010. Antioxidant system breakdown in brain of feral golden grey mullet (Liza aurata) as an effect of mercury exposure. Ecotoxicology 19, 1034–1045. Missy, P., Joyeux, M., Lanhers, M.C., Cunat, L., Burnel, D., 2000. Effects of subchronic exposure to manganese chloride on tissue distribution of three essential elements in rats. Int. J. Toxicol. 19, 313–321. Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Meth 65 (1–2), 55–63. Nikinmaa, M. Tervonen, V., 2004. Regulation of blood haemoglobin concentration in hypoxic fish. In: Rupp., G.L., White, M.D., Athens, G.A. (Eds.), Proceedings of the Seventh International Symposium on Fish Physiology, Toxicology, and Water Quality, U.S. Environmental Protection Agency, Ecosys. Res. Divis, pp. 243–252. Nikinmaa, M., Rees, B.B., 2005. Oxygen-dependent gene expression in fishes. Am. J. Physiol. Regul. Integr. Comp. Physiol 288, 1079–1090. Ohkawa, H., Ohishi, H., Yagi, K., 1979. Assay for lipid peroxide in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358. ¨ ¨ Oweson, C., Li, C., Soderh all, I., Hernroth, B., 2010. Effects of manganese and hypoxia on coelomocyte renewal in the echinoderm Asterias rubens (L.). Aquat. Toxicol. 100, 84–90. Pinsino, A., Matranga, V., Trinchella, F., Roccheri, M.C., 2010. Sea urchin embryos as an in vivo model for the assessment of manganese toxicity: developmental and stress response effects. Ecotoxicology 19, 555–562. Rosso, F.L., Bolner, K.C.S., Baldisserotto, B., 2006. Ion fluxes in silver catfish (Rhamdia quelen) juveniles exposed to different dissolved oxygen levels. Neotrop. Ichthyol. 4 (4), 435–440. Schreck, C.B., 2010. Stress and fish reproduction: the role of allostasis an hormesis. Gen. Comp. Endocrin 165 (3), 549–556. Sies, H., 1991. Oxidative stress: introduction. In: Sies, H. (Ed.), Oxidative Stress: Oxidants and Antioxidants. Academic Press, San Diego, pp. 21–48. Singh, S., Shukla, G.S., Srivastava, R.S., Chandra, S.V., 1979. The interaction between ethanol and manganese in rat brain. Arch. Toxicol. 41, 307–316. Slater, T.F., Sawyer, B., Strauli, U., 1963. Studies on succinate-tetrazolium reductase systems. III. Points of coupling of four different tetrazolium salts. Biochim. Biophys. Acta 77, 383–393. Storey, K.B., 2004. Functional Metabolism: Regulation and Adaptation. Wiley-Liss and Hoboken, New Jersey, pp. 319-368. Sundin, L., 1999. Hypoxia and blood flow control in fish gills. pp. 353–362. In: Val, A.L., Almeida-Val, V.M.F. (Eds.), Biology of Tropical Fishes. Manaus, INPA, pp. 460 p. ¨ P., 1982. Oxygen and chromium transfer in perfused gills of Van Der Putte, I., Part, rainbow trout (Salmo gairdneri) exposed to hexavalent chromium at two different pH levels. Aquatic. Toxicol 2, 31–45. Vieira, M.C., Torronteras, R., Co´rdoba, F., Canalejo, A., 2012. Acute toxicity of manganese in goldfish Carassius auratus is associated with oxidative stress and organ specific antioxidant responses. Ecotoxicol. Environ. Saf. 78, 212–217. Winemiller, K.O., Agostinho, A.A., Caramaschi, E.P., 2008. Fish ecology in tropical streams, Tropical Stream Ecology, first ed. Elsevier, London, pp. 107–140. Wu, R.S.S., 2002. Hypoxia: from molecular responses to ecosystem responses. Mar. Poll. Bull 45, 35–45. Zhang, S., Zhou, Z., Fu, J., 2003. Effect of manganese chloride exposure on liver and brain mitochondria function in rats. Environ. Res. 93, 149–157.