Inorganic mercury exposure: toxicological effects ... - Springer Link

Ecotoxicology (2010) 19:105–123 DOI 10.1007/s10646-009-0395-1

Inorganic mercury exposure: toxicological effects, oxidative stress biomarkers and bioaccumulation in the tropical freshwater fish matrinxa˜, Brycon amazonicus (Spix and Agassiz, 1829) Diana Amaral Monteiro Æ Francisco Tadeu Rantin Æ Ana Lućia Kalinin

Accepted: 16 July 2009 / Published online: 28 July 2009 Ó Springer Science+Business Media, LLC 2009

Abstract Alterations in the antioxidant cellular system have often been proposed as biomarkers of pollutantmediated toxicity. This study evaluated the effects of mercury on oxidative stress biomarkers and bioaccumulation in the liver, gills, white muscle and heart of the freshwater fish matrinxa˜, Brycon amazonicus, exposed to a nominal and sub-lethal concentration (*20% of 96 h-LC50) of 0.15 mg L-1 of mercury chloride (HgCl2) for 96 h in a static system. Increases in superoxide dismutase, catalase, glutathione peroxidase (GPx), glutathione S-transferase (GST) and glutathione reductase (GR) were observed in all tissues after HgCl2 exposure, except for white muscle GR activity and hepatic GPx. In the liver and gills, the exposure to HgCl2 also induced significant increases in reduced glutathione (GSH). Conversely, exposure to HgCl2 caused a significant decrease in the GSH levels and an increase in the oxidized glutathione (GSSG) content in the white muscle, while both GSH and GSSG levels increased significantly in the heart muscle. Metallothionein concentrations were significantly high after HgCl2 exposure in the liver, gills and heart, but remained at control values in the white muscle. HgCl2 exposure induced oxidative damage, increasing the lipid peroxidation and protein carbonyl content in all tissues. Mercury accumulated significantly in all the fish tissue. The pattern of accumulation follows the order gills [ liver heart [ white muscle. In conclusion, these data suggest that oxidative stress in response to inorganic mercury

D. A. Monteiro F. T. Rantin A. L. Kalinin (&) Department of Physiological Sciences, Federal University of Saõ Carlos, UFSCar, Via Washington Luı´s km 235, Saõ Carlos, SP, Brazil e-mail: [email protected]

exposure could be the main pathway of toxicity induced by this metal in fish. Keywords Inorganic mercury Antioxidant enzymes Lipid and protein oxidation Glutathione Metallothionein Bioaccumulation Neotropical teleost

Introduction Mercury (Hg) is recognized internationally as an important pollutant as Hg and its compounds are persistent, bioaccumulative and toxic. Consequently, mercury contaminations represent a serious risk to humans and ecosystems (Shastri and Diwekar 2008). In aquatic environments, mercury is found as a metallic or elemental form, inorganic compounds or organic compounds (Black et al. 2007). Each form has an individual toxicological profile, metabolic fate and biochemical effect. Although organic mercury is the most toxic form, inorganic mercury is the most common form of mercury released in the aquatic environment by industries, having a more significant effect on fish tissue (Oliveira Ribeiro et al. 1996). In Brazil, the main sources of Hg in aquatic ecosystems derive from industrial effluents, concentrated in the south and southeast regions, and gold mining, located mostly in the Amazon and Pantanal (Lacerda et al. 1991; Lacerda 1997). It is also important to mention that most of these water bodies are located over the Guarani Aquifer which is referred to as the largest groundwater reservoir of the planet. Although the industrial use of Hg has been reduced in recent years due to stricter regulations, high concentrations of Hg have been found in water samples at the sites with extensive industrial activities, mainly chloro-alkali,

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pulp and paper and ceramic industries. In these areas, total Hg concentrations ranged from 0.0005 to 0.23 mg L-1 (Hypolito et al. 2004; Alinnor 2005; Bollen et al. 2008). Shaw et al. (1989) found mercury levels ranging from 1.50 mg L-1 in effluents discharged by chloro-alkali industries. Mercury concentrations in streams and rivers near Hg deposits may contain up to 0.1 mg L-1 (Reeder et al. 1979). On the other hand, near gold mining areas, levels of the total Hg in surrounding water can reach values from 0.0001 to 19.82 mg L-1 (Pfeiffer et al. 1989; SerforArmah et al. 2005; Gammons et al. 2006). As a consequence, mercury levels in Brazilian freshwater fish, collected from areas with suspected mercury contamination, generally exceed the ANVISA food safety limit of the 0.5 mg kg-1 for non-predatory fish and 1.0 mg kg-1 for predatory fish (ANVISA 1998). The Hg levels were highest in fish from the Amazon and Pantanal, where the maximum concentrations in the muscles were 5.4 and 12.3 mg kg-1, respectively (Alho and Vieira 1997; Do´rea et al. 2006). Xenobiotics which are discharged daily into water bodies can induce reactive oxygen species (ROS) production, which are responsible for cell and tissue damage (Elia et al. 2003). Recent reports suggest that mercury toxicity involves the generation of ROS with marked alterations in the antioxidant defense systems and oxidative damage induction such as lipid peroxidation leading to cell death (Berntssen et al. 2003; Elia et al. 2003; Milaeva 2006; Verlecar et al. 2007; Larose et al. 2008; Verlecar et al. 2008) and different pathologic processes involved in the etiology of many fish diseases (Kehrer 1993; Banerjee et al. 1999). Defenses against ROS include scavenger compounds, for example, glutathione system (GSH/GSSG) and metallothionein (MT), and enzymes with antioxidant activities such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione reductase (GR), among others (Droge 2002; Storey 1996). Enzymatic and non-enzymatic antioxidants are essential in maintaining the redox status of fish cells and serve as an important biological defense against oxidative stress. Biochemical mechanisms involved in cellular detoxification are particularly relevant in understanding the deleterious effects of various metals or other environmental pollutants (Lopez et al. 2001) and useful biomarkers of exposure to aquatic pollutants (Bainy 1996; Ahmad et al. 2000). Most of the studies on mercury exposure in tropical fish investigated the kinetics of its uptake and distribution in organs and there is no available data on its effects on biochemical and survival in Brazilian fish species. Additionally, toxicological guidelines for mercury in most

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tropical countries are mainly derived from data collected in temperate ecosystems (Oliveira Ribeiro et al. 1996). Thus, the aim of this study was to determine the acute toxicity (LC50-96 h) of mercury chloride (HgCl2) and to evaluate the bioaccumulation and oxidative stress responses to short-term sublethal exposure (*20% of LC50-96 h). Endpoints included antioxidant enzyme activities (SOD, CAT, GST, GPx, GR), non-enzymatic antioxidants (GSH, GSSG and metallothionein contents), oxidative damage (lipid and protein oxidation levels) and total mercury content in the liver, gills, white muscle and heart of the freshwater fish matrinxa˜, Brycon amazonicus. Our hypothesis is that Hg, even in its inorganic form, is a potential hazard to fish at sublethal and environmentally relevant concentrations. B. amazonicus (Teleostei, Characidae) is a native species of the Amazon basin spread over the main Brazilian hydrographic basins (Margarido and Galetti 1996). The great economic importance and potential among commercially farmed fish in Brazil, mainly in Saõ Paulo State, are due to its excellent meat quality and desirable traits for fish culture, including a high growth rate and appetite for commercial food pellets (Scorvo-Filho et al. 1998).

Materials and methods Chemicals Mercury chloride (HgCl2 [ 99.5% purity) was obtained from Sigma–Aldrich (St. Louis, MO, USA). All other reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA) and Merck (Darmstadt, Germany). Fish Juvenile matrinxa˜, Brycon amazonicus, (Wt = 17.94 ± ´ guas 3.13 g; Lt = 11.15 ± 0.83 cm) were obtained at the A Claras fish farm, Mococa, Saõ Paulo State, Brazil. Fish were acclimated for 30 days prior to experimentation in 1,000 L holding tanks equipped with a continuous supply of well-aerated and dechlorinated water at 24 ± 2°C and under natural photoperiod (*12:12 L:D). During this period, fish were fed ad libitum with commercial fish pellets (40% of protein). The water quality parameters were kept nearly constant: dissolved oxygen (7.0–7.5 mg L-1), pH (7.1–7.4), conductivity (125–130 lS cm-1), alkalinity (35–43 mg L-1 as CaCO3), and total hardness (39–50 mg L-1 as CaCO3). Acute toxicity test (LC50-96 h) To decide the sublethal HgCl2 concentration to be used in the acute exposure, acute toxicity tests were performed.

Inorganic mercury exposure

The static method of acute toxicity test was used. The experimental procedure for this test was conducted in accordance with OECD 203 guidelines for testing chemicals (OECD 1992). A preliminary series of static toxicity tests (0.001, 0.01, 0.1, 1.0 and 10.0 mg L-1 HgCl2) were performed to determine the appropriate range of Hg toxicity for B. amazonicus. Based on these preliminary tests, fish were randomly divided into six groups (n = 10 for each group) and kept in 180 L experimental boxes with a fish/water ratio of 1 g L-1. One group was considered the control and the others were exposed to HgCl2 at nominal concentrations of 0.57, 0.70, 0.84, 1.00 and 1.20 mg L-1. To preserve the water quality, food was withdrawn 24 h before and during the trials. The survival at the end of every 6, 24, 48, 72 and 96 h was recorded. Dead fish were removed immediately. The acute toxicity tests were conducted in three replicates. Test solutions of the chosen concentrations were prepared by diluting a 100 or 1,000 mg L-1 HgCl2 stock solution. The LC50-96 h of HgCl2 was calculated by the trimmed Spearman–Karber method with 95% confidence limits (Hamilton et al. 1977). Water quality parameters were monitored daily and were kept nearly constant as described for the acclimation period. Mercury was removed from wastewater by precipitation with sodium sulfite and pH adjustment according the protocol described by Micaroni et al. (2000). After filtration, the precipitate was destined to the Residue Center of the Federal University of Saõ Carlos. The final effluent (after dilution) was discharged and it did not exceed the effluent maximum permissible Hg level of 0.01 mg L-1 established by the Brazilian National Environment Council (CONAMA 357/2005). Experimental design In order to evaluate mercury effects, fish were exposed to a sublethal HgCl2 concentration corresponding to 20% of the 96 h LC50. After acclimation, twenty fish were divided into two experimental opaque boxes (180 L): The control group (n = 10) and HgCl2 group—fish exposed to HgCl2 (n = 10). All controls and experiments were conducted in quadruplicate. Fish were starved for 24 h prior to experimentation to avoid prandial effects and to prevent the deposition of feces in the course of the assay. After 24 h, the water was renewed and the HgCl2 group was submitted to a nominal concentration of 150 lg L-1 of HgCl2 (20% of LC50-96 h). Concentrations of total mercury ranging from 0.001 to 0.230 mg L-1 are usually detected in water bodies near industrialized areas (Hypolito et al. 2004; Alinnor 2005; Bollen et al. 2008). Opaque experimental tanks were used to avoid external disturbances and they were sealed with a transparent cover to prevent sample volatilization and to guarantee the

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photoperiod. Dissolved oxygen, temperature and photoperiod were maintained as described for the acclimation period. The fish remained in the static system for 96 h. During this period, sublethal effects such as the level of activity, loss of equilibrium, abnormal swimming and color changes in the skin were observed. The water chemistry and physical parameters were also monitored and water samples for total Hg analysis were collected. Tissue samples At the end of 96 h, fish of both experimental groups were killed by transecting the spinal cord. After biometry, the gills, liver, heart and white muscle were carefully excised and washed with cold physiological saline (0.9% NaCl). Samples were taken and immediately frozen into liquid nitrogen. Frozen samples were stored at -80°C until the biochemical determinations were carried out. The hepatic somatic index (HSI) was calculated according to the follow equation: HSI = (liver weight/fish weight 9 100), where the weight was expressed in grams. Antioxidant enzymes All enzyme activities were measured spectrophotometrically (Spectronic Genesys 5, Milton Roy Co., Rochester, NY, USA) at 25°C. Samples of frozen tissue were quickly weighed and then homogenized at 18,000 rpm in a Turratec TE 102 (Tecnal, Piracicaba, SP, Brazil). The tissues were homogenized in a 0.1 M Na?/K? phosphate buffer pH 7.0 at a ratio of 1: 5 w/v Homogenates were centrifuged at 12,000 rpm for 30 min at 4°C and the supernatants were used for SOD, CAT, GST, GPx and GR activity assays. The SOD activity was measured according to the ¨ tting (1984), based on the method described by Flohe´ and O determination of the reduction rate of cytochrome c by superoxide anions, monitored at 550 nm, utilizing the xanthine–xanthine oxidase system as the source of superoxide radicals. One unit of SOD was defined as the amount of enzyme which inhibits the rate of cytochrome c reduction, under the specified conditions, by 50%. SOD values were expressed as units per mg of protein. The CAT activity was measured by decreasing the H2O2 concentration at 240 nm (Aebi 1974). One unit of CAT (according to Bergmeyer) is the amount of enzyme, which liberates half the peroxide oxygen from the H2O2 solution of any concentration in 100 s at 25°C. CAT values were expressed as Bergmeyer units (B.U.) per mg of protein. The GPx activity was measured by a coupled assay with GR catalyzed oxidation of NADPH (Nakamura et al. 1974) at 340 nm. The activity of GPx was expressed as mU mg protein-1 and 1 mU was defined as 1 nmol of NADPH

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consumed min-1 mL-1 of the sample, using a molar extinction coefficient of e340 = 6.2 mM-1 cm-1. The GST activity was measured according to Habig et al. (1974) using 1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate. The activity was measured as the amount of enzyme catalyzing the formation of 1 nmol of the product min-1 mg-1 of protein, using a molar extinction coefficient of e340 = 9.6 mM-1 cm-1. The GR activity was measured according to the method described by Carlberg and Mannervik (1975). The oxidation of 1 lmol of NADPH min-1 at 340 nm was used as a unit of GR activity. The GR values were expressed as units per mg of protein. Determination of reduced glutathione (GSH) and oxidized glutathione (GSSG) GSH and GSSG amounts were determined using the DTNB–GSSG reductase recycling assay described by Anderson (1985). Frozen tissue samples were homogenized in five volumes of cold 5% w/v sulfosalicylic acid and then centrifuged at 12,000 rpm for 5 min and supernatants used as a sample. Glutathione reductase reduced all GSSG to GSH, which in turn reacted with DTNB to produce thionitrobenzoic acid (TNB). The rate of TNB formation was followed at 412 nm indicated amounts of total glutathione (GSH plus GSSG), or GSSG alone when 5 lL of vinylpyridine was added to 100 lL of samples to DTNB in order to sequester GSH, as described by Cunha Bastos et al. (2007). The total GSH and GSSG contents were presented as lmol per gram of wet weight of tissue. Reduced and oxidized glutathione ratios were calculated as follows: GSH:GSSG = (GSH - 2.GSSG)/GSSG and total glutathione contents were expressed as GSH equivalents (GSHeq = GSH ? 2 GSSG).


reference standard and expressed as lg –SH groups per mg of protein. Lipid peroxidation (LPO) Tissue homogenates were prepared as described above for antioxidant enzymes assays. The xylenol orange assay for lipid hydroperoxide (FOX—ferrous oxidation-xylenol orange) was performed as described by Jiang et al. (1992). The lipid hydroperoxide was determined with 100 lL of sample (previously deproteinised with 10% trichloroacetic acid—TCA) and 900 lL of reaction mixture containing 0.25 mM FeSO4, 25 mM H2SO4, 0.1 mM xylenol orange and 4 mM butylated hydroxytoluene in 90% (v/v) methanol. Blanks contained all components without supernatant. The mixtures were incubated for 60 min at room temperature prior to measurements at 560 nm. Cumene hydroperoxide (CHP) was used as a standard. Lipid hydroperoxide levels were expressed as nmoles of CHP per mg protein. The decomposition of lipid hydroperoxides produces low molecular weight products, including malondialdehyde, which can be measured by the TBARS assay (Satoh 1978; Wilhelm Filho et al. 2005). Samples previously deproteinised with 20% TCA were combined with the same volume of 0.67% (w/v) 2-thiobarbituric acid (TBA) in 0.3% NaOH. The mixture was then boiled for 60 min and the resulting chromogen was extracted with 1 mL of butanol by vigorous shaking. Samples were centrifuged for 10 min at 5,000 rpm and the absorbance of the butanol phase was determined at 530 nm. Malondialdehyde (MDA) was employed as a standard. The values were expressed as nmoles of MDA per mg protein. Protein oxidation

Metallothionein (MT) concentration (–SH groups) MT concentration in the tissue was quantified by evaluating the –SH residue content by a spectrophotometric method described by Viarengo et al. (1997). Tissue was homogenized in three volumes of 0.5 M sucrose, 20 mM Tris–HCl buffer, pH 8.6, containing 0.5 mM PMFS and b-mercaptoethanol (0.01%). Supernatants were collected and an ethanol/chloroform solution was used to obtain partially purified MT-fraction. The samples were dried in a speed vac (SC210A plus, Thermo Savant) for approximately 4–6 h and re-suspended in a solution of 0.25 M NaCl, HCl 1 N and 4 mM EDTA. The concentration of MT was quantified using Ellman’s reagent containing NaCl 2 M, DTNB 0.43 M in a phosphate buffer 0.2 M (pH 8.0), measured spectrophotometrically at 412 nm. The metallothionein concentration was estimated using GSH as a

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The protein carbonyl (PC) content was assayed according to the method described by Reznick and Packer (1994). Frozen tissue samples were homogenized (1:5 w/v) in 50 mM potassium phosphate buffer containing 1 mM EDTA and 40 lg mL-1 phenylmethylsulfonyl fluoride (PMSF) and then centrifuged at 10,000 rpm for 10 min. Supernatants were reacted with 10 mM DNPH in 2.5 M hydrochloric acid for 1 h at room temperature and precipitated with 50% TCA. After centrifugation at 10,000 rpm for 10 min, the supernatants were discarded and the pellets were washed three times with 1 mL of ethanol/ethyl acetate (1:1 v/v) mixture. Pellets were then dissolved in 6 M guanidine hydrochloride and centrifuged to pellet insoluble particles. The carbonyl content was measured spectrophotometrically at 370 nm. A tissue blank incubated with 2 M HCl without DNPH was included for each sample. The results were


expressed as nmol of carbonyl per mg protein based on the molar extinction coefficient of 22,000 M-1 cm-1.

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Results Acute toxicity test (96 h LC50)

Protein content Protein determination was carried out according to the method of Bradford (1976) with Coomassie Brilliant Blue G-250 adapted to a microplate reader as described by Kruger (1994) using bovine serum albumin as a standard. The absorbance of samples was measured at 595 nm.

The cumulative mortality of matrinxa˜, B. amazonicus, at different concentrations of HgCl2 after exposure for 1, 6, 24, 48, 72 and 96 h is depicted in Table 2. As shown in Fig. 1, the calculated 96 h LC50 value of HgCl2, using a static bioassay system was found as 0.71 mg L-1 (0.67–0.75 mg L-1, 95% confidence limit).

Determination of total mercury in water and tissue

Exposure to a sublethal concentration of HgCl2

The total mercury in water samples was determined by Cold Vapor Atomic Fluorescence Spectrometry (CV-AFS) according to the USEPA 1631 detection technique, which is based on the fluorescence of excited elemental mercury (Hg0) atoms in an inert gas stream at a wavelength of 254 nm (USEPA 2002). The quantification limit for the total mercury was 0.1 lg L-1. The total mercury in the tissue was analyzed by a Cold Vapor Atomic Absorption Spectrometry (CV-AAS) at a wavelength of 254 nm using a Varian Spectra AA 240 FS coupled with a Varian Vapor Generation (VGA) following the method recommended by the United States Pharmacopeia (USP 2000). The quantification limit for the total mercury was 0.05 mg kg-1. The bioconcentration factors (BCF = Cb/Cwt) were calculated using the ratio of total mercury content in tissue (Cb) to the dissolved mercury concentration in the water (Cwt) (Gobas and Morrison 2000). The units for tissue samples of mg kg-1 divided by mg L-1 in water samples produces the BCF units of L kg-1 (Mackay and Fraser 2000). The BCF for the total Hg was calculated using the results obtained of fish tissue and water analysis described above.

Water quality parameters for the control group were: 24.98 ± 0.25°C; pH 7.32 ± 0.03; 7.46 ± 0.32 mgO2 L-1; 1.10 ± 0.24 mg L-1 total ammonia; 38.99 ± 1.94 mg L-1 chloride; 0.54 ± 0.23 mg L-1 nitrite; alkalinity of 39.48 ± 2.44 mg CaCO3 L-1; total hardness of 44.06 ± 3.16 CaCO3 L-1; conductivity of 122.53 ± 3.27 lS cm-1. The total amount of mercury was below the limit of quantification (LOQ = 0.1 lg L-1). These values did not change significantly in HgCl2 groups, except for the total Hg concentration of 106.00 ± 21.2 lg L-1. No mortality was observed in the controls and in the group exposed to 0.15 mg L-1 of HgCl2 (20% of the 96 h LC50). However, fish exposed to HgCl2 showed hyperactivity and aggressiveness, loss of equilibrium, dark skin pigmentation and loss of scales. Moreover, exposed fish displayed abnormal swimming behavior near the surface of the water and hemorrhagic areas in the gills. When compared to the controls, HgCl2 exposure caused significant increases in liver mass (0.20 ± 0.07–0.29 ± 0.06 g) and, consequently, in HIS values (1.10 ± 0.08– 1.64 ± 0.04%). However, no alterations were observed in 120 100

The values in all determinations are presented as means ± SD. For comparisons between two groups, t-tests (parametric) or Mann–Whitney U-tests (non-parametric) were applied. The Kolmogorov and Smirnov method was applied to evaluate normality of the samples and the F test was applied for homogeneity of variances (GraphPad Instat version 3.00, GraphPad Software, USA). Differences between means at a 5% (P \ 0.05) level were considered significant. Survival data was statistically analyzed using the Trimmed Spearman–Karber point estimate test to determine the lethal concentration to fifty percent (50%) of the test population (LC50) and associated 95% confidence limits (GWBasic version 3.10, Phoenix Software Associates Ltd).

Mortality (%)

Statistical analysis

80 60 40 20 -1

LC50 96h = 0.71 ± 0.04 mg L (95% confidence limits)

0 0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

-1

HgCl2 (mg L ) Fig. 1 Sigmoidal regression analysis and 96 h LC50 estimation of 0.71 ± 0.04 mg L-1 of HgCl2 with 95% confidence limits (0.67–0.75 mg L-1). N = 30, in three replicates

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the body mass and the total length between control and HgCl2 groups (18.20 ± 0.69 vs. 17.68 ± 0.63 g, and 10.91 ± 0.81 vs. 11.22 ± 0.84 cm, respectively).

Antioxidant enzymes The antioxidant enzyme activities in the liver, white muscle, gills and heart are shown in Figs. 2, 3, 4 and 5, respectively. The HgCl2 exposure induced significant increases in the hepatic SOD, CAT, GST and GR activities (77, 42, 43 and 23%, respectively). However, no alterations were observed in the hepatic GPx activity in this group (Fig. 2). In the gills, the exposure to HgCl2 induced significant increases in all the antioxidant enzyme activities. The increased values observed for SOD, CAT, GPx, GST and GR were 19, 45, 33, 198 and 29%, respectively (Fig. 3).

Glutathione system The results of the glutathione system in matrinxa˜ tissues of HgCl2 and control groups are shown in Table 1. In the liver and gills, the exposure to HgCl2 induced significant increases in GSH (42 and 121%, respectively) and GSHeq (40 and 89%, respectively) levels and GSH:GSSG ratios (36 and 196%, respectively). However, neither liver nor gill GSSG levels changed significantly.

SOD U.B. mg protein

-1

* 50

25

0

600

Control

GPx -1

mU mg protein

400

200

HgCl2

GST

200

-1

mU mg protein

4

HgCl2

0

* 100

0

Control

HgCl2

GR

-1

20

mU mg protein

*

8

0

Control

* 10

0

Control

123

CAT

12 -1

75

U mg protein

Fig. 2 Antioxidant enzymes activity in liver of matrinxa˜, B. amazonicus, after 96 h of exposure to 0.15 mg L-1 of HgCl2 or under control conditions. Values are mean ± SD n = 10. * Significant difference in relation to the control (P \ 0.05)

Fish exposed to HgCl2 showed significantly higher SOD, CAT, GPx and GST activities (14, 37, 35 and 145%, respectively) in the white muscle when compared to the control group. In contrast, no differences were detected in the GR activity of both experimental groups (Fig. 4). In the heart, the exposure to HgCl2 also induced significant increases in SOD (113%), CAT (77%), GPx (150%), GST (332%) and GR (205%), as observed in Fig. 5.

HgCl2

Control

HgCl2


SOD

1.2

*

5

Control

GPx

-1

20

HgCl2

GST

HgCl2

*

400

200

0

Control

HgCl2

GR

-1

20

Control

-1

*

Control

0.6

600

40

0

*

0

HgCl2

mU mg protein

60

mU mg protein

U.B. mg protein

-1

10

0

mU mg protein

CAT

-1

15

U mg protein

Fig. 3 Antioxidant enzymes activity in the gills of matrinxa˜, B. amazonicus, after 96 h of exposure to 0.15 mg L-1 of HgCl2 or under control conditions. Values are mean ± SD n = 10. * Significant difference in relation to the control (P \ 0.05)

111

* 10

0

Control

On the other hand, in the white muscle, the exposure to HgCl2 caused significant decreases in the GSH levels (15%) and GSH:GSSG ratio (31%) and increases in GSSG content (20%). In addition, no changes were observed in the GSHeq concentration in the white muscle after HgCl2 exposure. In the heart, the HgCl2 exposure induced significant increases in the GSH, GSHeq and GSSG levels (24, 26 and 38%, respectively) when compared with control values. No significant differences were observed in the cardiac GSH:GSSH ratios between HgCl2 and the control groups. Metallothionein concentration (–SH groups) Figure 6 shows the metallothionein levels quantified by evaluating the –SH residue content. HgCl2 exposure significantly augmented the MT concentration in the liver (22%), gills (17%) and heart (40%) but remained similar to controls in the white muscle.

HgCl2

Peroxidative damage The results on the effects of HgCl2 exposure on lipid peroxidation levels in all tissue measured by FOX and TBARS assays are shown in Fig. 7. The exposure to HgCl2 increased the LPO levels measured by the FOX-reactive lipid hydroperoxide assay (determined as CHP) in the liver (117%), gills (138%), white muscle (193%) and heart (91%). Significant increases in the TBARS content (determined as MDA) were also observed in the liver (127%), gills (116%), white muscle (50%) and heart (106%) in response to HgCl2 exposure. In all tissue taken from fish exposed to HgCl2, the levels of protein carbonyls were significantly increased when compared to the control group. After 96 h of exposure, the increased values observed in the liver, gills, white muscle and heart were 51, 55, 65 and 72%, respectively (Fig. 8).

123

112

SOD

-1

* 5

0

* 0.5

0

Control

HgCl 2

Control

GPx

100

2.5

0

HgCl 2

GST

* -1

-1

*

mU mg protein

5

mU mg protein

CAT

-1

1

U.B. mg protein

10

U mg protein

Fig. 4 Antioxidant enzymes activity in white muscle of matrinxa˜, B. amazonicus, after 96 h of exposure to 0.15 mg L-1 of HgCl2 or under control conditions. Values are mean ± SD n = 10. * Significant difference in relation to the control (P \ 0.05)


50

0

Control

HgCl 2

Control

HgCl 2

GR

mU mg protein

-1

4

2

0

Control

Total mercury levels in tissues As shown in Table 2, the total Hg concentrations in the liver, gills, white muscle and heart after 96 h of exposure to 0.15 mg L-1 of HgCl2 were significantly higher than those of the control group. No mercury was detected in any tissue of the control group and all Hg levels remained below the limit of quantification of 0.05 mg kg-1. The general Hg distribution pattern was gills [ liver heart & white muscle. The mean BCFs were about 168 in gills, 99 in liver, 7 in heart and 6 in white muscle resulting in BCFs ranging from 0.78 to 2.22 log units (Table 3).

Discussion Fish acute toxicity tests play an important role in environmental risk assessment and hazard classification as they are able to evaluate relative toxicity of a variety of

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HgCl2

chemicals in different species (Wedekind et al. 2007). The 96 h LC50 mean value of HgCl2 was calculated as 0.71 mg L-1, showing that HgCl2 is highly toxic to B. amazonicus. This value is compatible with the literature, where HgCl2 96 h-LC50 values carried out at temperatures between 20 and 28°C and pH 7.1–7.8 on juveniles of freshwater fish ranged between 0.4 and 0.9 mg L-1 (Eisler and Hennekey 1977; Ramamurthi et al. 1982; Duncan and Klaverkamp 1983; Gaikwad 1989; Alam and Maughan 1992; Sinha and Kumar 1992; Alam and Maughan 1995; Gu¨l et al. 2004). Nevertheless, the LC50 values may vary in different studies carried out on the same species as water hardness, temperature and fish size may influence the results (Bleau et al. 1996). Moreover, metal sensitivity is highly species-specific and dependent on the chemical form of the metal (Soares et al. 2008). Matrinxa˜s exposed to HgCl2 sub-lethal concentration (20% of the 96 h LC50) of 0.15 mg L-1 showed aggressiveness, loss of equilibrium, dark skin pigmentation and abnormal swimming. These altered responses could have a


SOD

CAT

9

-1

* 10

0

Control

3

Control

40

0

mU mg protein

*

80

HgCl 2

GST

-1

1200

-1

mU mg protein

*

6

0

HgCl 2

GPx

120

* 800

400

0

Control

HgCl 2

Control

HgCl 2

GR

60

*

-1

mU mg protein

U.B. mg protein

-1

20

U mg protein

Fig. 5 Antioxidant enzymes activity in the heart of matrinxa˜, B. amazonicus, after 96 h of exposure to 0.15 mg L-1 of HgCl2 or under control conditions. Values are mean ± SD n = 6. * Significant difference in relation to the control (P \ 0.05)

113

40

20

0

Control

negative impact on fitness and survival, resulting in adverse consequences at a population level (Bridges 1997; Kane et al. 2005). It is well known that mercury compounds cause brain histopathological lesions, neurotoxic effects and inhibition of key enzymes involved in the neurological function such as Na?/K?-ATPase, monoamine oxidase, and acetylcholinesterase (Verma et al. 1983; Jagoe et al. 1996; Rouleau et al. 1999; Berntssen et al. 2003). These mercury-induced changes in the brain can affect fish behavior and locomotion activity due to a possible action of this metal in altering the neurotransmission, a mechanism that could account for the observed behavioral disorders observed in the present study. Exposure to HgCl2 also induced morphological alterations in the gills and liver. The gills presented hemorrhagic areas, consistent with the gill damages found in the tropical catfish Trichomycterus zonatus (Oliveira Ribeiro et al. 2000) and in the European sea bass, Dicentrarchus labrax (Giari et al. 2008) after exposure to inorganic mercury. A

HgCl 2

significant increase in the liver mass and HIS after HgCl2 exposure was also observed. Increased HSI values were also reported in field and laboratory studies with other fish species from contaminated sites (Fabacher and Baumann 1985; Martin and Black 1998; Kirby et al. 1999; Mcfarland et al. 1999). HIS is a physiological biomarker reflecting irreversible damage in response to pollutants (Hinton et al. 1992). Indeed, fish collected from polluted sites usually show hepatic hypertrophy and hyperplasia which could represent an attempt to maximize the hepatic detoxification mechanisms such as xenobiotic-metabolizing enzymes (Porter and Janz 2003; Van der Oost et al. 2003; Figueiredo-Fernandes et al. 2006; Fernandes et al. 2007). Inorganic mercury had a high oxidative-stress-inducing potential in matrinxa˜. Short term (96 h) exposure to HgCl2, in static conditions was sufficient to induce significant alterations in antioxidant enzymes such as SOD, CAT, GST, GPx and GR as well as the GSH/GSSG system and MT content inducing oxidative damages in lipids and proteins and, consequently, oxidative stress. All changes

123

114


Glutathione system

Control

HgCl2

Liver GSH (lmol g tissue-1)

4.09 ± 0.78

5.81 ± 1.56*

GSSG (lmol g tissue-1) GSHeq

0.090 ± 0.02 4.27 ± 0.77

0.086 ± 0.02 5.98 ± 1.59*

GSH:GSSG

48.35 ± 18.79

65.75 ± 12.19*

GSH (lmol g tissue-1)

1.69 ± 0.23

3.74 ± 0.32*

GSSG (lmol g tissue-1)

0.25 ± 0.07

0.22 ± 0.07

GSHeq

2.19 ± 0.20

4.14 ± 0.33*

GSH:GSSG

5.45 ± 2.93

16.16 ± 5.54*

GSH (lmol g tissue )

0.58 ± 0.08

0.49 ± 0.07*

GSSG (lmol g tissue-1)

0.055 ± 0.006

0.065 ± 0.01*

GSHeq

0.70 ± 0.08

0.62 ± 0.07

GSH:GSSG

8.73 ± 1.90

5.99 ± 1.90*

2.72 ± 0.35

3.38 ± 0.22*

GSSG (lmol g tissue ) GSHeq

0.27 ± 0.03 3.26 ± 0.36

0.37 ± 0.05* 4.11 ± 0.25*

GSH:GSSG

8.27 ± 1.75

7.27 ± 1.43

(A) 6

*

GSHeq = (GSH ? 2.GSSG) GSH:GSSG = [(GSH - 2.GSSG)/GSSG] Values are mean ± SD (n = 10)

Liver

Gills

White Muscle

Heart

* 2

* *

1

0

-1

*

2

-1

Heart GSH (lmol g tissue-1)

*

(B) 3 nmol MDA mg protein

-1

4

0

Gills

White muscle

*

-1

nmol CHP mg protein

Table 1 Glutathione system in liver, gills, white muscle and heart of matrinxa˜, B. amazonicus, after 96 h of exposure to 0.15 mg L-1 of HgCl2 or to control conditions

*

Liver

Gills

White Muscle

Heart

Fig. 7 Lipid peroxidation levels measured as cumene hydroperoxide (CHP) equivalents (a) or as malondialdehyde (MDA) equivalents (b) in the liver (n = 10), gills (n = 10), white muscle (n = 10) and heart (n = 6) of matrinxa˜, B. amazonicus, after 96 h of exposure to 0.15 mg L-1 of HgCl2 or under control condition. Values are mean ± SD. * Significant difference in relation to the control (P \ 0.05)

* Significantly different from control (P \ 0.05) 2

* 12

*

*

6

nmol PC mg protein

-1

nmol SH mg protein

*

-1

18

* 1

0

0

Liver

Gills

White Muscle

Heart

Fig. 6 Metallothionein levels (–SH groups) in the liver (n = 10), gills (n = 10), white muscle (n = 10) and heart (n = 6) of matrinxa˜, B. amazonicus after 96 h of exposure to 0.15 mg L-1 of HgCl2 or under control condition. Values are mean ± SD. * Significant difference in relation to the control (P \ 0.05)

observed in the liver, gills, white muscle and heart of matrinxa˜s exposed to nominal and sublethal concentration of 0.15 mg L-1 of HgCl2 (*20% of the 96 h LC50) are schematized in Figs. 9 and 10. SOD and CAT activities increased after 96 h of exposure to HgCl2 in all tissue of matrinxa˜. SOD catalyses the

123

*

Liver

Gills

*

White Muscle

Heart

Fig. 8 Protein carbonyl (PC) contents in liver (n = 10), gills (n = 10), white muscle (n = 10) and heart (n = 6) of matrinxa˜, B. amazonicus, after 96 h of exposure to 0.15 mg L-1 of HgCl2 or under control condition. Values are mean ± SD. * Significant difference in relation to the control (P \ 0.05)

dismutation of the O2 to H2O and H2O2, which is detoxified by CAT. Due to the inhibitory effects on ROS formation, the SOD–CAT system provides the first defense line against oxygen toxicity (Pandey et al. 2003) and is usually used as a biomarker indicating ROS production (Van der Oost et al. 2003; Regoli et al. 2003). The induction of the SOD–CAT system indicates a fast


115

Table 2 Total mercury concentration (mg kg-1) in tissues of matrinxa˜, B. amazonicus, after 96 h of exposure to 0.15 mg L-1 of HgCl2 or under control conditions Tissues

Control

(A)

Liver

\LQ

10.46 ± 1.47*

\LQ

17.80 ± 4.50*

White muscle Heart

\LQ \LQ

0.63 ± 0.13* 0.72 ± 0.10*

GR GSH-conjugate

GSH + 2GSSG

GSSG

electrophilic compounds or HgCl2

(GSH-2.GSSG)/GSH

GPx LOOH

LOH + H2O

Lipid peroxidation

* Significantly different from control (P \ 0.05)

HgCl2

Table 3 Bioconcentration factors (BCFs—expressed in L kg-1 and log units) for inorganic mercury for tissues of matrinxa˜ exposed to 0.15 mg L-1 of HgCl2

98.66 ± 5.68

1.99

Gills

167.95 ± 17.31

2.22

White muscle

5.94 ± 0.49

0.77

Heart

6.80 ± 0.05

0.83

BCF = Cb/Cwt, where, Cb is the total mercury content in tissue and Cwt is the dissolved mercury concentration in the water

O2-

HgCl2

[Hg]total

PC OH

SOD

HgCl2

H2O2

CAT susceptible proteins H2O + O2

MT-Hg

(B)

NADPH + H+

NADP+

GR GSHeq

GSH-conjugate

GSH + 2GSSG

GSSG

GSH GST

GSH:GSSG: electrophilic compounds or HgCl2

(GSH-2.GSSG)/GSH

GPx

adaptive response of the redox-defense system in the liver, gills, white muscle and heart of matrinxa˜s after exposure to mercury. These data are consistent with the increased SOD–CAT activities in the brain of Atlantic salmon (Salmo salar) after chronic dietary mercury exposure (Berntssen et al. 2003). Unfortunately, there is no available data regarding the effect of inorganic mercury on other fish tissue. Previous studies showed that Hg induces GST activity (Canesi et al. 1999; Ferrat et al. 2002; Larose et al. 2008). In the present study, matrinxa˜s exposed to HgCl2 also showed increases in GST activity in all the tissue analyzed. GST catalyzes the transformation of a wide variety of electrophilic compounds to less toxic substances by conjugating them to GSH (Wilce and Parker 1994; Van der Oost et al. 2003). This activity is useful in the detoxification of endogenous compounds such as peroxidative products of DNA and lipids, as well as the metabolism of xenobiotics (Fournier et al. 1992; Banerjee et al. 1999). Consequently, GST plays an important role in the oxidative damage defense. In addition, some GST isozymes display peroxidase activity with respect to lipid hydroperoxides (Bartling et al. 1993; Hayes and Pulford 1995). Our results also indicate an induction of GPx activity in gills, heart and white muscle, although hepatic GPx activity did not change after HgCl2 exposure. GPx is an antioxidant selenoenzyme protected from oxidative stress by catalyzing the reduction of H2O2 to water and lipid

Protein carbonylation

MT

Log BCFs

Liver

CHP (FOX) MDA (TBARS)

GST

Values are mean ± SD (n = 6)

BCFs

GSH GST

GSH:GSSG

\LQ—below the limit of quantification (LOQ = 0.05 mg kg-1)

Tissues

NADP+

GSHeq

HgCl2

Gills

NADPH + H+

LOOH

LOH + H2O


GST Lipid peroxidation

HgCl2 O2-

HgCl2

[Hg]total

HgCl2

PC OH

SOD


H2O2

CAT susceptible proteins

MT H2O + O2 MT-Hg

Fig. 9 Schematic representation of the major effects of inorganic mercury in liver (a) and gills (b) of matrinxa˜, B. amazonicus, after 96 h of exposure to 0.15 mg L-1 of HgCl2

hydroperoxides (LOOH) to the corresponding stable alcohols (LOH) involving a concomitant oxidation of reduced GSH (as a source of reducing equivalents) to its oxidized form GSSG (Nordberg and Arne´r 2001). In the reaction, two molecules of GSH are oxidized to GSSG that can be subsequently reduced by GR activity at the expense of NADPH. GPx is considered to play an especially important role in protecting membranes from damage due to the LPO as the major detoxification function of GPx is the termination of radical chain propagation by quick reduction to yield further radicals (Van der Oost et al. 2003). However, in the present study, increases in GPx activity in the gills, white

123

116


(A)

NADPH + H+

NADP+

GR GSHeq

GSH-conjugate

GSH + 2GSSG

GSSG

GSH GST

GSH:GSSG: electrophilic compounds or HgCl2

(GSH-2.GSSG)/GSH

GPx LOOH

LOH + H2O


GST Lipid peroxidation HgCl2 O2 -

HgCl2

[Hg]total

PC OH

SOD

HgCl2


H2O2


MT H2O + O2 MT-Hg

(B)

NADPH + H+

NADP+

GR GSHeq

GSH-conjugate

GSH + 2GSSG

GSSG

GSH GST

GSH:GSSGH electrophilic compounds or HgCl2

(GSH-2.GSSG)/GSH

GPx LOOH

LOH + H2O


GST Lipid peroxidation HgCl2 O2-

HgCl2

[Hg]total

HgCl2

PC OH

SOD


H2O2


MT H2O + O2 MT-Hg

Fig. 10 Schematic representation of the major effects of inorganic mercury in white muscle (a) and the heart (b) of matrinxa˜, B. amazonicus, after 96 h of exposure to 0.15 mg L-1 of HgCl2

muscle and heart of matrinxa˜s exposed to HgCl2 were not able to maintain a low rate of LPO. Thus, the HgCl2 exposure enhanced ROS generation which overwhelms antioxidant defense systems resulting in an oxidative stress condition. Environmental pollutants are known to increase the GPx activity (Almeida et al. 2002; Sayeed et al. 2003; Zhang et al. 2004). Increases in the hepatic GPx activity were also observed in the Atlantic salmon (Salmo salar) and black bullhead (Ictalurus melas) after HgCl2 exposure via diet (100 mg kg-1 for 4 months) or water (35 lg L-1for 4 days), respectively (Berntssen et al. 2003; Elia et al. 2003).

123

GPx and GST work together to counteract pro-oxidant processes utilizing GSH as a co-factor. GSH, an oxyradical scavenger, is important in the antioxidant defense and may be a very important indicator of the detoxification ability of an organism (Sun et al. 2006). This tripeptide also scavenges ROS directly and is involved in numerous processes essential to normal biological function, such as DNA and protein synthesis (Meister and Anderson 1983). When in contact with some pollutants, such as metals, fish cells usually try to remove them by direct conjugation with GSH. Increased fluxes of ROS can impose a drain on intracellular reducing equivalents with potentially profound consequences on a variety of metabolic processes (Van der Oost et al. 2003). The capacity of the GSH system depends on the activity of GPx, GR and pentose phosphate pathway enzymes, among others (Larose et al. 2008). The consumption of GSH due to the direct scavenging of ROS or as a co-factor for GST/GPx activities may decrease GSH levels and GSH:GSSG ratio leading to a disruption of redox homeostasis. The GSH:GSSG ratio or glutathione redox status is considered as an index of the cellular redox status and a biomarker of oxidative damage, because glutathione maintains the thiol-disulphide status of proteins, acting as a redox buffer (Penã-Llopis et al. 2003). The exposure to HgCl2 induced significant increases in the GSH content in the liver and gills of matrinxa˜s. However, no alterations were observed in the GSSG levels due to increases in GR activity resulting in a rise of GSH:GSSG ratios. Consequently, GSHeq levels were also enhanced by the increase of GSH levels in this tissue. Thus, concomitant increases in the levels of GSH and in the activities of GR and GPx/GST were observed. This increased hepatic GSH content is probably due to the enhanced hepatic uptake of amino acid substrates and the activities of biosynthetic enzymes in an attempt to protect the fish from oxidative stress. Indeed, liver is the most important site of GSH synthesis, which is subsequently exported to other tissue such as the kidney, brain and muscle (Penã et al. 2000; Atli and Canlia 2008). Increases in GSH levels in freshwater fish were previously observed in spotted snakehead, Channa punctatus, exposed to 5 lg L-1 HgCl2 during 30 days (Rana et al. 1995) and in golden grey mullet, Liza aurata, collected in a mercury contaminated area (Guilherme et al. 2008). On the other hand, the exposure of matrinxa˜ to HgCl2 caused a reduction in GSH levels and GSH:GSSG ratio and an increase in the GSSG content in white muscle. No alterations were detected in the GSHeq levels due to an increase in the GSSG with a simultaneous decrease in the GSH levels. Therefore, the GR activity did not show any significant alteration. This decline in GSH content may be attributed to a direct binding of Hg by the GSH or to an


enhanced oxidation of this thiol which, in such circumstances, would not be reconverted to GSH by GR activity. If the generation of GSSG is higher than the reduction back to GSH by GR, then GSSG accumulates and it is translocated outside to avoid NADPH exhaustion and, consequently, this is followed by depletion in the GSH pool (Penã-Llopis et al. 2001). Depletion in GSH levels in the white muscle can also be a sign of its exhaustion in phase II biotransformation as confirmed by the prominent increased GST activity in relation to liver and gill activity. White muscle has a low content of mitochondria and a low intensity oxidative metabolism (Lushchak et al. 2005). Consequently, the activities of all antioxidant enzymes and GSHeq levels were much lower in the matrinxa˜ white muscle than in other tissues. Additionally, one of the most important mechanisms for Hg-induced oxidative damage is its sulfidryl reactivity. Once absorbed in the cell, Hg compounds form covalent bonds with GSH causing irreversible excretion and depletion of GSH levels (Ercal et al. 2001). High affinity interaction of mercury with thiol groups can lead to oxidative stress due to the accumulation of ROS which had generally been eliminated by GSH. Unfortunately, there is no available data regarding the effects of mercury on GSH metabolism in fish muscle to compare. Nevertheless, the GSH depletion seems to enhance the risk of oxidative stress due to reduced cell protection ability, as a possible increased peroxidative overload may occur (Monteiro et al. 2006). Studying the effects of the organophosphate dichlorvos on glutathione metabolism of European eel, Anguilla Anguilla, Penã-Llopis et al. (2003) suggested that the depletion of GSH in the muscle could be a better biochemical marker of pollutant exposure than in liver as GSH replenishment in the muscle is less common than in the liver. This trend was also observed in the present study, where hepatic GSH levels augmented while the concentration of GSH in white muscle decreased. Inversely, exposure of matrinxa˜s to HgCl2 caused significant increases in GSH, GSSG and GSHeq concentrations in cardiac muscle while GR activity increased significantly and the GSH:GSSG ratio remained unchanged. Recent studies have suggested that heart muscle is highly vulnerable to intoxication by metals like vanadium and cadmium (Aureliano et al. 2002; Soares et al. 2006, 2007, 2008). However, the effects of mercury on fish heart tissue are unknown. Nevertheless, the oxidative stress and redox status loss plays a critical role in some cellular processes that are important in heart failure, including hypertrophy, impairment of excitation-contraction coupling and contractile dysfunction, reduced myofilament Ca2? responsiveness and programmed cell death (Choudhary and Dudley 2002; Luo et al. 2006; Hidaldo and Donoso 2008).

117

The present data suggest that cardiac muscle presented a higher antioxidant capacity than skeletal muscle, as demonstrated by the maintenance of redox homeostasis (GSH:GSSG) and increases in GSHeq in spite of the rise in GSSG levels. These differences in antioxidant profiles are probably related to their anatomic position determining the uptake, distribution and bioaccumulation of pollutants (Ahmad et al. 2006), as well as their different rates of ROS generation and different antioxidant potentials (Monteiro et al. 2006). Some studies also report tissue-specific oxidative stress responses and antioxidants in fish exposed to environmental contaminants (Oruç et al. 2004; Ahmad et al. 2006; Monteiro et al. 2009). One of the most important molecules scavenging and reducing the toxic effects of mercury is the metallothionein (MT), a low molecular weight (6–7 kDa) protein with high sulfhydryl content (up to 30% of total amino acid content) (Viarengo et al. 1999; Amiard et al. 2006). MTs are reported to play an important role in the detoxification of toxic metals such as Hg, and in the maintenance of the homeostasis of essential metals like zinc (Zn) and copper (Cu) (Huang et al. 2007). Due to its high sulfhydryl content, MT also displays ROS scavenger activity as part of the antioxidant defense system of the cells (Viarengo et al. 2000; Viarengo et al. 2007). Schlenk et al. (1995) observed a positive correlation between the expression of hepatic MT messenger RNA and Hg levels in fillets of largemouth bass (Micropterus salmoides) and channel catfish (Ictalurus punctatus). Fernandes et al. (2008) also described hepatic MT induction in specimens of four-spotted megrim (Lepidorhumbos boscii) and pouting (Trisopterus luscus) that had high amounts of Zn, Cd, Cr and Hg in their livers. Significant increases in MT concentrations were observed in the liver, gills and heart of matrinxa˜s exposed to HgCl2, whereas no changes were detected in the white muscle MT levels. The capacity of MT induction varies between tissues and its contribution in metal sequestration is more evident in the liver, gills, and kidney (Roesijadi and Robinson 1994; Hogstrand and Haux 1991; De Boeck et al. 2003). Tissues directly involved in metal uptake, storage and excretion have a high capacity to synthesize MTs (Amiard et al. 2006). MT induction during heavy metal exposure protects the organism against toxicity by rescuing the target ligands compromised by inappropriate metal binding (Roesijadi 1996). In contrast, it is plausible that the absence of MT induction in the white muscle of matrinxa˜ exposed to HgCl2 contributed to the disruption of the redox homeostasis, as observed by the reduction of GSH levels and GSH:GSSG ratio and enhancement of the GSSG content. The maintenance of white muscle MT levels after HgCl2 exposure may lead to higher mercury availability for

123

118

irreversible binding with sensitive molecules such as GSH, resulting in a severe oxidative stress condition. The loss of thiol redox balance can elicit deleterious consequences for metabolic regulation, cellular integrity, and organ homeostasis. Furthermore, high levels of GSSG can exert deleterious effects through nonspecific reactions with the free sulfhydryl groups of proteins to form mixed disulfides, reactions that can lead to inactivation of enzymes possessing sulfhydryl groups at their active sites (Halliwell and Gutteridge 2000). If the initial increase in ROS is relatively small, the antioxidative response may be sufficient to compensate for ROS increases and to reset the original balance between the ROS production and ROS scavenging capacity (Droge 2002). However, our results clearly show that short-term exposure to HgCl2 induced oxidative damage in all tissues analyzed. The oxidative stress was confirmed by the increases in lipid peroxidation and protein oxidation in liver, gills, white muscle and heart of matrinxa˜ after exposure to HgCl2. Consequently, the data suggest that Hginduced oxidative stress can be greatly responsible for its toxic effects. The lipid peroxidation (LPO) has been reported as a major contributor to the loss of cell function under oxidative stress conditions (Storey 1996). Considering that LPO is a valuable indicator of oxidative damage of cellular components, our results suggest that exposure to HgCl2 enhanced ROS production in the liver, gills, white muscle and heart of matrinxa˜ and that antioxidant defenses were not totally able to effectively scavenge them, resulting in increases in the LPO process. In this study, the levels of LPO were evaluated in terms of MDA (TBARS method) and HP equivalents (FOX method). According to Hermes-Lima et al. (1995), there is a significant correlation for measurements of samples from various animal species using the TBARS and FOX methods. However, the TBARS method measures the end products of lipid peroxidation such as MDA, whereas the FOX assay has the advantage of direct detection of lipid hydroperoxides (HP). In HgCl2 exposed fish, higher FOX reactive substance values were found mainly in the gills and heart, while in the other tissues TBARS and FOX values were almost independent of the method used. This could reflect tissue-specific dynamics in the lipid oxidation chain propagation reactions. However, independently of the method used, these results indicate that HgCl2 can promote significant increases in LPO leading to oxidative stress in the liver, gills, white muscle and heart of matrinxa˜s. Similarly, after mercury exposure, increases in LPO levels were also detected in the brain and kidney of the Atlantic salmon (Berntssen et al. 2003) and in the liver of Russian sturgeon Acipenser gueldenstaedti (see Milaeva 2006).

123


Protein oxidation measured as protein carbonyl content can be also evaluated as a potential biomarker of Hg-mediated oxidative damage. Our results showed increases in the protein carbonylation in all the tissue of fish exposed to HgCl2, indicating that the proteins were subjected to oxidative damage. The formation of carbonyl groups results from the direct oxidation of amino acid side chains by metals or ROS and from the modification of proteins by oxidation-derived secondary products, such as lipid peroxidation products (Pantke et al. 1999; Grune 2000; Requena et al. 2003). The formation of carbonyl derivatives is non-reversible, causing conformational changes, decreased catalytic activity of enzymes and, eventually, resulting in a breakdown of proteins by proteases due to increased susceptibility (Almroth et al. 2005). The accumulation pattern of total mercury in organs was gills [ liver heart [ white muscle. The mean bioconcentration factors (BCFs) were 168 in gills, 99 in liver, 7 in heart and 6 in white muscle. The degree of mercury accumulation in matrinxa˜ was shown by the log BCF, which ranged from 0.78 to 2.22 log units, indicating the occurrence of Hg bioconcentration, especially in the gills and liver (log BCFs [ 1). These findings are corroborated by the data from Elia et al. (2003) for I. melas exposed to 35–140 lg L-1 of Hg for 10 days who found the following order of accumulation of inorganic mercury: gills [ kidney [ liver [ muscle. The bioaccumulation of toxic metals in fish tissue depends on their uptake and elimination rates, bioavailability and environmental conditions such as water temperature, age and size of the organisms. Different metals show different affinities to fish tissues and most of them accumulate mainly in the liver, kidney and gills (Huang et al. 2007; Fernandes et al. 2008). When compared to other tissues, fish muscles usually contain the lowest levels of metals (Jezierska and Witeska 2007). Cuvin-Aralar and Furness (1990) also detected lower Hg concentrations in muscle tissues in minnows, Phoxinus phoxinus, exposed to inorganic mercury. Carvalho et al. (2009) detected total Hg levels of 0.85 mg kg-1 in muscle, 10.9 mg kg-1 liver, 17.2 mg kg-1 in heart and 79.7 mg kg-1 in gills of Nile tilapia, Oreochromis niloticus, exposed to 0.08 mg L-1 of Hg2? for 14 days, similar to the present data. Inorganic mercury does not present a great affinity to muscle tissue and this property is characteristic of the organomercurial forms (WHO 2003; Kehrig et al. 2002). However, in the present study, the total Hg concentration exceeded the maximum limit of 0.5 mg kg-1 established by the ANVISA for human consumption. Muscle tissue is the part which is most often consumed and, consequently, mercury concentrations in fish muscle have important


consequences for consumer health and risk assessment (Balshaw et al. 2008). The gills, liver and kidney are commonly the primary target organs for many pollutants (Giari et al. 2008). The organs in the visceral region (liver, heart, intestine, and kidney) carry out the primary activities related to absorption, distribution and elimination (Rao et al. 2005). Similarly to the present data, the higher accumulation of Hg in fish visceral organs than in muscle tissue was also observed in Atlantic salmon, S. salar (Berntssen et al. 2004), cambeva, Trichomycterus zonatus, and arctic charr, Salvelinus alpinus (Oliveira Ribeiro et al. 1996, 2000). Tissue-specific responses related to antioxidant defenses were observed in the present study. The results indicate that white muscle and the heart are the most sensitive organs to oxidative stress, showing signs of redox homeostasis failure, detected by the increases in GSSG levels and/or decreases in GSH:GSSG ratios. These data pointed out the existence of different antioxidant capacities and rates of ROS generation in the different tissue analyzed. According to Bickham et al. (2000), pollutants might show their toxic effects at the molecular level, but also initiate a cascade of responses at higher levels including tissue, organismal health, reproduction, population demographics, population genetics, and finally, evolutionary processes including extinction. Consequently, the use of mercury must be carefully evaluated, mainly because this metal represents serious risks to the aquatic biota, biodiversity preservation and to the health of human populations depending on fish as the main protein source. The bioaccumulation of inorganic mercury in fish suggests its potential for biomagnification in humans. Vieira and Alho (2000) evaluated the biomagnification of mercury considering the levels of contamination in the sediments, clams, fish (meat and liver) and birds (feathers and liver) in the Brazilian Pantanal. These authors found high mercury levels in the birds (feathers and liver) and the piscivorous birds showed higher levels than those fed on clams. The results suggest that there is mercury biomagnification throughout the food chain as high mercury content is found in the organisms of high trophic levels. This study is the first to comprehensively examine multiple endpoints such as the LC50, bioaccumulation and enzymatic and non-enzymatic antioxidants as well as oxidative damages in various tissues after being exposed to mercury in a single fish species. Our results indicate that mercury, even in the inorganic form, at a sub-lethal concentration and in a short-term exposure (96 h), is a potent inductor of oxidative stress in matrinxa˜. This was sufficient to impair the protein and lipid membrane functions, essential to maintain the cell homeostasis. The adaptations of the antioxidant defense system of matrinxa˜ against the

119

HgCl2 exposure were not sufficient to counteract the ROSinduced damage. The data suggest that the mercury, at concentrations which are environmentally relevant, can have a negative impact on behavior, health status, performance and success of matrinxa˜, an Amazon native species, making their survival and/or population vulnerable.

Ethics in animal experimentation This study was conducted in accordance with the COBEA (Brazilian College of Animal Experimentation) and duly approved by the Committee of Ethics in Animal Experimentation and Committee of Environmental Ethics/Federal University of Saõ Carlos, Brazil. Acknowledgments This study was supported by Saõ Paulo State Research Foundation (FAPESP—Proc. 06/50772-6) and the National Council for the Development of Research and Technology (CNPq). ´ guas Claras fish farm, which provided The authors are thankful to A the fish.

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