Oxidative stress biomarkers in the freshwater characid fish, Brycon cephalus, exposed to organophosphorus insecticide Folisuper 600 (methyl parathion)

Comparative Biochemistry and Physiology, Part C 143 (2006) 141 – 149 www.elsevier.com/locate/cbpc

Oxidative stress biomarkers in the freshwater characid fish, Brycon cephalus, exposed to organophosphorus insecticide Folisuper 600 (methyl parathion) Diana Amaral Monteiro a , Jeane Alves de Almeida b , Francisco Tadeu Rantin a , Ana Lúcia Kalinin a,⁎ a

Department of Physiological Sciences, Federal University of São Carlos, UFSCar, Via Washington Luís km 235, 13565-905, São Carlos, São Paulo, Brazil b Federal University of Tocantins, Arraias, Tocantins, Brazil Received 5 October 2005; received in revised form 22 December 2005; accepted 6 January 2006 Available online 20 March 2006

Abstract Methyl parathion (MP) is an organophosphorus insecticide used worldwide in agriculture and aquaculture due to its high activity against a broad spectrum of insect pests. The effect of a single exposure to 2 mg L− 1 of a commercial formulation of MP (MPc: Folisuper 600®, MP 600 g L− 1) on catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione S-transferase (GST), reduced glutathione (GSH) and lipid peroxidation (LPO) of the liver, white muscle and gills of Brycon cephalus was evaluated after 96 h of treatment. MPc exposure resulted in a significant induction of SOD, CAT and GST activity in all tissues. However, the GPx activity decreased significantly in white muscle and gills, whereas no alterations were observed in hepatic GPx activity. MPc also induced a significant increase in LPO values in the white muscle and gills, while hepatic LPO levels did not show any significant alteration. The current data suggest that MPc has oxidative-stress-inducing potential in fish, and that gills and white muscle are the most sensitive organs of B. cephalus, with poor antioxidant potentials. The various parameters studied in this investigation can also be used as biomarkers of exposure to MPc. © 2006 Elsevier Inc. All rights reserved. Keywords: Brycon cephalus; Characidae; Methyl parathion; Oxidative stress; Biomarkers; Antioxidant enzymes; Lipid peroxidation; Glutathione; Organophosphate

1. Introduction Changes in the chemical composition of natural aquatic environments can affect the non-target organisms, particularly fish. Fish have been largely used to evaluate the quality of aquatic systems as bioindicators for environmental pollutants (Adams and Greeley, 2000). In polluted areas, exposure of fish to xenobiotics leads to interactions between these chemicals and biological systems, which give rise to biochemical disturbances (Gül et al., 2004). Organophosphorus compounds (OPC) are widely used for agriculture and domestic purposes when controlling insect pests (Videira et al., 2001). Due to their rapid breakdown in water and their low environmental persistence, OPC have largely replaced the use of organochlorides in recent years (Li and Zhang, 2001). ⁎ Corresponding author. Tel.: +55 16 3351 8314; fax: +55 16 3351 8401. E-mail address: [email protected] (A.L. Kalinin). 1532-0456/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2006.01.004

However, there is evidence that OPC are sufficiently persistent in reaching the river environment at concentrations high enough to affect aquatic animals (Hai et al., 1997). In this regard, fish are particularly sensitive to OPC. Methyl parathion is listed in the HazDat database of chemicals detected in water surface or groundwater at National Priorities List (NPL) sites identified by the United States Environmental Protection Agency (EPA). The maximum limit established for MP in aquatic ecosystem by the Brazilian Environment National Council (CONAMA) is 0.04 μg L− 1. However there is no available data regarding the MP concentrations in Brazilian natural water bodies. The concentrations of some OPC (like methyl parathion and dichlorvos) in food regularly and substantially exceed the maximum permissible residue limits in Brazil (Caldas and Souza, 2000). Methyl parathion (MP) is one of several OPC developed to replace organochlorides (Machado and Fanta, 2003). MP is extensively applied as an insecticide in agriculture, food storage shelters and pest control programs due to its high activity against

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a broad spectrum of insect pests. It is also widely used in fish culture tanks to eliminate aquatic larvae of predator insects that threaten fish larvae (Silva et al., 1993) and is currently used on crops established on floodplains of the Amazon River (Soumis et al., 2003). MP is a highly toxic insecticide ranked by the EPA as a class I toxicant. Due to rain or soil drainage, it often contaminates water bodies, affecting non-target organisms such as fish. In the Amazon River, Soumis et al. (2003) detected residues of MP in eight Amazonian fish species. This demonstrates that pesticides widely used in agriculture are being leached through rains, contaminating waters and fish of the region, as well as other foods. The primary effect of MP and other OPC on both invertebrate and vertebrate organisms, including humans, is the inhibition of the enzyme acetylcholinesterase (AChE). However, the effects of OPC are not restricted to AChE inhibition. It has been reported that OPC, besides its inhibitory effect on AChE, also induce oxidative stress (Hai et al., 1997; Yarsan et al., 1999; Peña-Llopis et al., 2003; Mohammad et al., 2004). Oxidative stress occurs when the critical balance between oxidants and antioxidants is disrupted due to the depletion of antioxidants or excessive accumulation of the reactive oxygen species (ROS), or both, leading to damage (Scandalios, 2005). Many xenobiotics, such as pesticides, may cause oxidative stress leading to the generation of ROS and alterations in antioxidants or free oxygen radicals scavenging enzyme systems in aquatic organisms (Livingstone, 2001). ROS, such as superoxide anion radical (O2•−), hydrogen peroxide (H2O2) and highly reactive hydroxyl radical (•OH) can react with susceptible biological macromolecules and produce lipid peroxidation (LPO), DNA damage and protein oxidation, resulting in oxidative stress (Livingstone et al., 1993; Nordberg and Arnér, 2001; Shi et al., 2005). Contaminant-stimulated ROS are associated with different pathologic processes involved in the etiology of many fish diseases and may also be a mechanism of toxicity in aquatic organisms exposed to pollutants (Kehrer, 1993; Banerjee et al., 1999). Despite the potential danger of ROS, cells present a variety of defense mechanisms to neutralize the harmful effects of free radicals. The antioxidant defense system includes enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), glutathione S-transferase (GST) and other low molecular weight scavengers such as glutathione (GSH) (Droge, 2002; Storey, 1996). Superoxide dismutase (EC 1.15.1.1), such as the cytosolic Cu, Zn-SOD or the mitochondrial Mn-SOD, metabolizes superoxide anion into a less reactive species, molecular oxygen and hydrogen peroxide (H2O2) (Sies, 1986; Nordberg and Arnér, 2001). H2O2 is decomposed to water and molecular oxygen by the peroxisomal and mitochondrial catalase (EC 1.11.1.16) (Oost et al., 2003). H2O2 and organic hydroperoxides may be destroyed by cytosolic and mitochondrial glutathione peroxidase (EC 1.11.1.9) in the presence of tripeptide GSH (Gaté et al., 1999). This compound also contributes to the removal of electrophilic components by reacting with glutathione S-transferase (EC 2.1.1.41) (Sies, 1986; Scandalios, 2005). GST is a group of multifunctional enzymes that catalyze the conjugation of GSH with a variety of elec-

trophilic metabolites, which are involved in the detoxification of both reactive intermediates and oxygen radicals (Di Giulio et al., 1995). GST is involved in the detoxification of xenobiotics and highly reactive electrophilic components can be removed before they covalently bind to tissue nucleophilic compounds, which would lead to toxic effects (Storey, 1996). Enzymatic and non-enzymatic antioxidants are essential to maintain the redox status of fish cells and serve as an important biological defense against oxidative stress. Antioxidants of fish may be useful biomarkers of exposure to aquatic pollutants (Bainy, 1996; Ahmad et al., 2000). Biochemical mechanisms involved in the cellular detoxification are particularly relevant in understanding the deleterious effects of several metals or other environmental pollutants (Lopez et al., 2001). The antioxidant enzyme activities, the glutathione redox status, the level of lipid peroxidation product and the specific induction of glutathione Stransferase are most frequently used as biomarkers in toxicological evaluations (Doyotte et al., 1997; Oruc et al., 2004). Tropical ecosystems are currently threatened by human activities and environmental degradation; however, little research has been done on the impact of contaminants on tropical ecosystems and tropical fish species (Almeida et al., 2005; Lacher and Goldstein, 1997). Fish can be exposed to MP, either accidentally or under treatment conditions. In fish culture, the treatment with OPC is a common method employed to control larval stages of predator insects that threaten fish larvae. Furthermore, OPC are also used to treat skin and gill infections caused by external parasites. Depending on the parasite and on the fish species, the concentration of OPC recommended for biological control ranges between 0.25 and 12.5 mg L− 1, reaching, or even exceeding, the LC50 (Williams and Jones, 1994). In Brazil, Folisuper 600® is one of the most utilized OP for this purpose, in concentrations varying from 0.25 to 3 ppm (Figueiredo and Senhorini, 1990; Senhorini et al., 1991). Although organophosphorus insecticide, which contains MP as the active substance, has been extensively used in Brazil, there are few reports in the literature on MP induced oxidative stress and its effect on fish antioxidants. Therefore, the goal of the present study was to determine if the exposure to insecticides containing MP results in oxidative stress in fish. The neotropical freshwater fish matrinxã, Brycon cephalus (Teleostei, Characidae), is a native species of the Amazon basin of great economic importance and potential among the commercially farmed fish in Brazil due to its excellent meet quality. The species presents desirable traits for fish culture, including high growth rate and appetite for commercial pellets (Scorvo-Filho et al., 1998). Matrinxã is moderately tolerant to ammonia (Carneiro and Urbinati, 2001) and very sensitive to nitrite (Avilez et al., 2004). The LC50 for matrinxã exposed to Folidol 600®, another trade marc for MP-600 g L− 1, was determined by Aguiar et al. (2004) as 6.54 ppm. These authors also reported changes in the intermediary metabolism at 2 ppm, indicating cell injuries. However, there is no information about MP-induced oxidative stress in B. cephalus. To elucidate this aspect, specimens of B. cephalus Günther, 1869 were exposed to a sublethal concentration of a commercial MP formulation (MPc) and its effects

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on enzymatic and non-enzymatic antioxidants, including SOD, CAT, GST, GPx, GSH and LPO, were investigated in liver, white muscle and gills. 2. Materials and methods

A commercial formulation of the organophosphorus insecticide methyl parathion (O,O-dimethyl O-4-nitrophenyl phosphorothioate)–Folisuper 600 BR® (methyl parathion 600 g L− 1, Agripec) was used. All chemicals and reagents were purchased from Sigma-Aldrich Chemical Co. and Merck. 2.2. Experimental design Juvenile B. cephalus (mass = 25.9 ± 5.1 g; length = 13.5 ± 1.0 cm) were obtained at the Águas Claras fish farm, Mococa, Sao Paulo State, Brazil. Fish were acclimated for 60 days prior to experimentation in 1000 L holding tanks equipped with a continuous supply of well-aerated and dechlorinated water at 24 ± 2 °C and under natural photoperiod (∼12 : 12 h). During this period, fish were fed ad libitum with commercial fish pellets (35% protein). The physical and chemical parameters were kept nearly constant: pH 6.7–7.5, DO2 6.0–7.5 mg L− 1, hardness 25–30 mg L− 1 (as CaCO3) and conductivity 65–72 μS cm− 1. After acclimation, twenty fish were divided into two experimental opaque plastic boxes (250 L): Control group (n = 10) and MPc group — animals treated with Folisuper 600®–MP 600 g L − 1 (n = 10). The fish were starved for 24 h prior to experimentation to avoid prandial effects and to prevent the deposition of feces in the course of the assay. After 24 h, the water was renewed and MPc group was submitted to a concentration of 2 mg L− 1 Folisuper 600® (1 / 3 of 96 h — LC50 previously established by Aguiar et al., 2004). Opaque experimental tanks were used to avoid external disturbances and they were sealed with a dark cover to prevent sample volatilization. Dissolved oxygen, temperature and photoperiod were maintained as described for the acclimation period. The fish remained under a semi-static system for 96 h where the experimental MPc solutions were renewed every 24 h to maintain water quality and adjust the concentration of MP. The control group was submitted to the same protocol but without adding MP. During this period, sublethal effects like level of activity, swimming performance and color changes were monitored. Table 1 Body mass, liver mass and hepatosomatic index of Brycon cephalus after 96 h of exposure to clean water (Control group) or to 2 mg L− 1 of methyl parathion (MP group)

Body mass (g) Liver mass (g) Hepatosomatic index (%)

Table 2 Antioxidant enzyme activities in the liver of Brycon cephalus after 96 h of exposure to clean water (Control group) or to 2 mg L− 1 of methyl parathion (MPc group) SOD (U mg protein− 1) CAT (B.U. mg protein− 1) GPx (U mg protein− 1) GST (nmol min− 1 mg protein− 1)

2.1. Chemicals

Control group

MPc group

25.40 ± 1.95 0.27 ± 0.02 0.96 ± 0.04

25.83 ± 2.04 0.19 ± 0.01a 0.84 ± 0.03a

Values are mean ± S.E.M., n = 10. a Indicates significant difference in relation to control group (P b 0.05).

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Control group

MP group

12.62 ± 0.72 3.17 ± 0.44 29.51 ± 1.82 88.16 ± 6.86

16.87 ± 1.58a 6.4 ± 1.25a 32.74 ± 1.70 118.40 ± 6.42a

Values are mean ± S.E.M., n = 10. a Indicates significant difference in relation to control group (P b 0.05).

2.3. Tissue samples At the end of 96 h of exposure to MPc, fish of both experimental groups were killed by transecting the spinal cord and the mass and total length of fish were measured. Following the biometry, organs were carefully excised and washed with cold physiological saline (0.9% NaCl). Gills, liver and white muscle were excised in this sequence and, subsequently, samples were taken and immediately frozen into liquid nitrogen. Frozen samples were stored at − 80 °C until the biochemical determinations were carried out. The hepatosomatic index was calculated according to the follow equation: ½HIS ¼ ð liver weight= fish weightÞ  100:

2.4. 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 in a Turratec TE 102 (Tecnal, Piracicaba, SP, Brazil) homogenizer at 18,000 rpm. The buffers for homogenization of tissues were: 0.1 M sodium phosphate buffer pH 7.0 at a ratio of 1 : 10 w/v for SOD, CAT and GPx activity assays; 0.1 M potassium phosphate buffer pH 7.0 keeping the proportion 1 : 5 w/v for GST activity assay. Samples were centrifuged at 12,000 ×g for 30 min at 4 °C. SOD activity was determined based on the ability of the enzyme to inhibit the reduction of nitro blue tetrazolium (NBT) (Crouch et al., 1981), which was generated by 37.5 mM hydroxylamine in alkaline solution (Otero et al., 1983). The assay was performed in 0.5 M sodium carbonate buffer (pH 10.2) with 2 mM EDTA. The reduction of NBT by superoxide anion to blue formazan was measured at 560 nm. The rate of NBT reduction in the absence of tissue was used as the reference rate. One unit of SOD was defined as the amount of protein needed to decrease the reference rate to 50% of maximum inhibition. The SOD activity was expressed in units per mg protein. The CAT activity was measured by decreasing the H2O2 concentration at 240 nm (Aebi, 1974). Decays in absorbance were recorded during 17 s in 50 mM sodium phosphate buffer (pH 7.0) containing 15 mM H2O2 and the enzyme extract. CAT

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Table 3 Antioxidant enzyme activities in the white muscle of Brycon cephalus after 96 h of exposure to clean water (Control group) or to 2 mg L− 1 of methyl parathion (MPc group) SOD (U mg protein− 1) CAT (B.U. mg protein− 1) GPx (U mg protein− 1) GST (nmol min− 1 mg protein− 1)

Control group

MP group

28.00 ± 1.84 0.24 ± 0.03 55.00 ± 3.94 18.43 ± 1.50

38.85 ± 3.50a 0.61 ± 0.10a 35.31 ± 3.27a 29.03 ± 2.46a

Values are mean ± S.E.M., n = 10. a Indicates significant difference in relation to control group (P b 0.05).

values were expressed as Bergmeyer units (B.U.) per mg protein. 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. According to Wilhelm Filho et al. (1993), 1 nmol CAT is correspondent to 33 B.U. The GPx activity was analyzed by a modified Mills' procedure 2 (Mills, 1959) as described by Hafeman et al. (1974). GPx degrades H2O2 in the presence of GSH thereby depleting it. The remaining GSH is then measured by using 5,5′-dithiobis 2-nitrobenzoic acid (DTNB). The incubation mixture at 37 °C contained 80 mM sodium phosphate buffer (pH 7.0), 80 mM EDTA, 1 mM NaN3, 0.4 mM GSH and 0.25 mM H2O2 and tissue homogenates. After 3 min, aliquots of this solution were removed and treated with metaphosphoric acid precipitation solution. The GSH in the protein free filtrate was then determined using 0.4 M Na2HPO4 and 1 mM DTNB in 1% trisodium citrate solution. The absorbance of this solution was recorded at 412 nm. A blank was carried out through the incubation simultaneously with the samples, since non-enzymatic GSH oxidation by H2O2 occurs during incubation. One unit of GPx enzyme activity was defined as 1 μg of GSH consumed per minute (Latha and Pari, 2004). GPx activity was expressed in units per milligram protein. GST activity was measured according to Habig et al. (1974) using 1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate. The assay mixture contained 1 mM CDNB in ethanol, 1 mM GSH, 100 mM potassium phosphate buffer (pH 7.0) and tissue homogenates. The formation of adduct S-2,4-dinitrophenyl glutathione was monitored by the increase in absorbance at 340 nm against blank. The molar extinction coefficient used for CDNB was 9.6 mM− 1 cm− 1. The activity was expressed as the amount

Table 4 Antioxidant enzyme activities in the gills of Brycon cephalus after 96 h of exposure to clean water (Control group) or to 2 mg L− 1 of methyl parathion (MPc group) − 1

SOD (U mg protein ) CAT (B.U. mg protein− 1) GPx (U mg protein− 1) GST (nmol min− 1 mg protein− 1)

Control group

MP group

18.77 ± 1.16 0.55 ± 0.07 44.86 ± 3.80 68.16 ± 4.03

23.95 ± 1.77a 0.73 ± 0.04a 31.69 ± 3.34a 94.14 ± 5.47a

Values are mean ± S.E.M., n = 10. a Indicates significant difference in relation to control group (P b 0.05).

of enzyme catalyzing the formation of 1 nmol of the product formed per min per milligram protein. 2.5. Lipid peroxidation (LPO) The xylenol orange assay for lipid hydroperoxide (FOX — ferrous oxidation–xylenol orange) was performed as described by Jiang et al. (1992). Tissue homogenates were prepared as described above for SOD, CAT and GPx assays. Lipid hydroperoxide was determined with 100 μL of sample (previously deproteinised with 10% TCA) and 900 μL 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. The mixtures were incubated for 30 min at room temperature prior to measurements at 560 nm. The molar extinction coefficient of 4.3 104 M− 1 cm− 1 for cumene hydroperoxide (Jiang et al., 1991) was used. Lipid hydroperoxide levels were expressed as nmol per milligram protein. 2.6. Reduced glutathione (GSH) GSH content in tissue homogenates were analyzed by HPLC (Waters 464 Pulsed Electrochemical Detector, Milford, MA, USA) system (Hiraku et al., 2002). Tissue samples were homogenized (1 : 10 w/v) in 10 mM sodium acetate buffer (pH 6.5) containing 10 μM DPTA and 0.5% Tween 20. The tissue homogenates were deproteinised with 1 volume of 10% TCA and centrifuged at 11,000 ×g for 10 min at 4 °C. Samples of 25 μL were injected in the HPLC system and analyzed with an electrochemical detector 247 Waters equipped with a gold electrode. Chromatographic separation was carried out using a X-Terra (4.5 × 250 mm, 5 μm) column and 100 mM potassium phosphate buffer pH 2.5 containing 200 mg L− 1 sodium heptanossulfonate and 5 mg L− 1 EDTA in 1% methanol (v/v) as the mobile phase (flow 0.5 mL min− 1). GSH was identified and quantified by electrochemical detection (oxidation potential 600 mV) and compared with known concentrations of standard run under the same chromatographic

2.5

lipid hydroperoxide nmol mg protein-1

144

2

*

Control group MPc group

1.5 1

*

0.5 0

Liver

White muscle

Gills

Fig. 1. Levels of lipid peroxidation in liver, white muscle and gills of Brycon cephalus after 96 h of exposure to clean water (Control group) or to 2 mg L− 1 of methyl parathion (MPc group). Values are mean ± S.E.M., n = 10. ⁎ indicates significant difference in relation to control group (P b 0.05).

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conditions. The GSH content was expressed as nmol of GSH per milligram protein. 2.7. Protein estimation The homogenate protein concentration was determined for all tissues by the classical Bradford method with Coomassie Brilliant Blue G-250 (Bradford, 1976) adapted to a microplate reader (Dynex Technologies Ltd., MRXTC, UK) as described by Kruger (1994), using bovine serum albumin as a standard. Absorbance of samples was measured at 595 nm. 2.8. Statistical analysis The values in all determinations are presented as means ± S.E. M. Significance was assessed with t test. The method of Kolmogorov and Smirnov was applied to evaluate normality of the samples and the F test was applied to homogeneity of variances (GraphPad Instat version 3.00, GraphPad Software, USA). P-values below 0.05 were regarded as significant. 3. Results When compared to the control group, fish exposed to MP (MPc group) showed lethargic movements with a partial lack of reflexes. Macroscopic findings such as yellow discoloration, hemorrhages and loose of firmness were suggestive of degenerative lesions and necrosis. Significant differences in liver weight and in the HIS were also observed. In MPc group, liver weight and HSI were significantly lower when compared to group I (30% and 10% respectively). However, the body weight mean values of both groups did not change (Table 1). The activities of antioxidant enzymes in the liver, white muscle and gill tissues are shown in Tables 2–4. The exposure to MPc caused a significant increase in the activities of SOD, CAT and GST in all tissues of MPc group when compared to the control. Increases of 34%, 101% and 34% in liver, 38%, 154% and 57% in white muscle and 28%, 34% and 38% in gills were

GSH nmol mg protein-1

100

Control group MPc group

80 60

* 40

*

*

20 0

Liver

White muscle

Gills

Fig. 2. Levels of reduced glutathione in liver, white muscle and gills of Brycon cephalus after 96 h of exposure to clean water (Control group) or to 2 mg L− 1 of methyl parathion (MPc group). Values are mean ± S.E.M., n = 10. ⁎ indicates significant difference in relation to control group (P b 0.05).

145

recorded for SOD, CAT and GST activities, respectively. In contrast, MPc induced significant decreases of 36% and 30% in the activity of GPx of white muscle and gills, respectively, when compared to the control. However, significant differences were not detected in the hepatic GPx activity of both experimental groups. Fig. 1 shows the levels of lipid hydroperoxide in liver, white muscle and gills of both experimental groups. Fish in MPc group presented significant increases in the lipid hydroperoxide levels in the gills and white muscle (67% and 102%, respectively) when compared to control values. No significant differences were observed for the hepatic LPO levels between control group and MPc group. The exposure to MPc induced significant decreases in GSH content in liver (43%), white muscle (50%) and gills (50%) in relation to control group (Fig. 2). 4. Discussion The present study demonstrated that MPc has a high oxidative-stress-inducing potential in B. cephalus, and the gills and white muscle are the most sensitive organs. A period of 96 h of exposure to MPc was enough to induce significant alterations in antioxidant enzymes such as SOD, CAT, GST and GPx, as well as the GSH content and LPO levels, resulting in oxidative stress. The reduction of swimming performance observed in the fish exposed to MP could be attributed to the inhibition of AChE (Aguiar et al., 2004) or to a larger energy demand to support the detoxification processes (Rao and Rao, 1981; Heath, 1995) and/or to respond to stress stimuli (Wendelaar-Bonga, 1997). The MP toxicity results from the metabolic conversion processed in the endoplasmatic reticulum of the hepatocytes, directly affecting the morphology of these cells (Rodrigues and Fanta, 1998; Machado and Fanta, 2003) and contributing, in some extent, to the decreased HIS observed in the MPc group (see Table 1). Decreases in HIS can also reflect a depletion of energy reserves stored as liver glycogen related to stress response (Wendelaar-Bonga, 1997). This assumption is supported by the liver glycogen decrease of about 80% observed by Aguiar et al. (2004) in matrinxã exposed to 2 ppm of Folidol 600®. In this study SOD and CAT activities increased after 96 h of exposure to MPc in all tissues of B. cephalus. SOD and CAT enzymes have related functions. SOD catalyses the dismutation of the superoxide anion radical to H2O and H2O2, which is detoxified by both CAT and GPx activity. Due to the inhibitory effects on oxyradical formation, the SOD-CAT system provides the first defense line against oxygen toxicity (Pandey et al., 2003) and usually used as a biomarker indicating ROS production (Oost et al., 2003; Regoli et al., 2003). Increases in these enzyme activities are probably a response towards increased ROS generation in pesticide toxicity (John et al., 2001). This is corroborated by Dimitrova et al. (1994) that described a simultaneous induction response in SOD and CAT activities in carp, Cyprinus carpio, exposed to zinc. However, the activity of these enzymes in fish can also decrease after exposure to xenobiotics, as reported by Livingstone (2001).

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The increased SOD and CAT levels induced by MPc in B. cephalus indicate an elevated antioxidant status attempting to neutralize the impact of the ROS. These results corroborate the statement of Alves et al. (2002) that the exposure to pesticides can elicit pro-oxidant conditions that trigger adaptive responses such as increases in the activity of the antioxidant enzymes. On the other hand, the GPx activity in the gills and white muscles in MPc group showed a significantly lower activity than in group I. However, such a reduction was not observed in liver tissue. The decreased activity of GPx in gills and white muscle observed in the present study could be related to the O2•− production (Bagnasco et al., 1991) or to the direct action of pesticides on the enzyme synthesis (Bainy et al., 1993). Although environmental pollutants could increase the GPx activity (Almeida et al., 2002; Sayeed et al., 2003; Zhang et al., 2004), Fatima et al. (2000) reported a low activity of GPx in different fish tissues after exposure to paper mill effluent, indicating an inefficiency of these organs in neutralize the peroxide impacts. A similar decrease in GPx activity in rat liver was reported after 90 days of treatment with lindane, an organochlorine pesticide (Bainy et al., 1993). Similarly, the organophosphorus insecticide malathion reduced GPx activity in mice erythrocytes (Yarsan et al., 1999). GPx inhibition was reported after combined treatment with the pesticides 2,4-D and azinphosmethyl in the brain of carp, C. carpio (Oruc et al., 2004), and in the liver of Nile tilapia, Oreochromis niloticus (Oruc and Uner, 2000). Enzyme activity can be decreased by negative feedback from excess of substrate or damage by oxidative modification (Tabatabaie and Floyd, 1994). A reduced GPx activity could indicate that its antioxidant capacity was surpassed by the amount of hydroperoxide products of lipid peroxidation (Remacle et al., 1992). The GST activity is involved in xenobiotic detoxification and excretion of xenobiotics and their metabolites, including MP (Jokanovic, 2001). It plays an important role in protecting tissue from oxidative stress (Fournier et al., 1992; Banerjee et al., 1999). Increased GST activity in tissues may indicate the development of a defensive mechanism to counteract the effects of MP and may reflect the possibility of a more efficient protection against pesticide toxicity. The increased GST activity in all tissues observed in the present study after exposure to MP suggests that the detoxification processes were increased and corroborates these ascertains. GST has been reported as a biomarker for assessing the environmental impact of organic xenobiotics generating oxidative stress (Livingstone, 1998; Rodríguez-Ariza et al., 1991). The GST was more active in hepatic tissue than in white muscle and gill, which indicates the effective role of liver in xenobiotic detoxification (Basha and Rani, 2003). The increased GST activity was concomitant to the decreases in GSH content in all tissues analyzed. The GSH plays an important role in the detoxification of electrophiles and prevention of cellular oxidative stress (Hasspieler et al., 1994; Sies, 1999). The considerable decline in the GSH tissue content during exposure to MP may be due to an increased utilization of GSH, which can be converted into oxidized glutathione and an inefficient GSH regeneration.

During a moderate oxidative stress, the GSH levels can increase as an adaptive mechanism by means of an increased synthesis. However, a severe oxidative stress may suppress GSH levels due to the impairment of the adaptive mechanisms (Zhang et al., 2004). According to Elia et al. (2003) GSH depletion may reduce the cellular ability to scavenge free radicals raising the general oxidative potential in the cells. When in contact with some pollutants, like MP, fish cells usually try to remove them by direct conjugation with GSH or by means of GST, which decreases GSH levels. GST utilizes GSH for the xenobiotic detoxification. The observed GSH decrease is probably an indication of its exhaustion in phase II biotransformation as confirmed by the increased GST activity. In this circumstance, the GSH depletion seems to enhance the risk of oxidative stress due to a reduced cell protection ability since a possible increased peroxidative overload could be induced by a high SOD activity, as shown in the present study. The correlation between the changes in the GST activity and the GSH levels could imply, to some extent, that there is a restriction of GST activity by the available levels of GSH. However, it is not clear to which extent the reductions in the GSH levels are responsible for the increases in the GST activity. Moreover, the decreased GSH levels were accompanied by decreases in GPx activity. Using GSH as a reducing agent, the GPx enzymes catalyze the reduction of H2O2 and organic peroxides to water and their corresponding stable alcohols. The GPx activity depends on the presence of GSH, which is oxidized in this process. Thus, GPx activity is likely to be influenced by GSH levels. The decreased GPx activity may also be related to the decreased availability of the GSH needed to reduce the ROS impact (Cheung et al., 2004). Hai et al. (1997) found decreased levels of GSH in carp liver and muscle after 24 h exposure to 1 and 5 mg L− 1 of dichlorvos, an organophosphorus insecticide known to induce oxidative damages. Dichlorvos also decreased glutathione levels and inhibited AChE and GPx activities in several tissues of rats (Julka et al., 1992). In human poisoning cases, lindane (organochlorine) and malathion (organophosphate) also decreased blood GSH content (Banerjee et al., 1999). LPO has been reported as a major contributor to the loss of cell function under oxidative stress conditions (Storey, 1996). Considering that the typical reaction during ROS-induced damage involves the peroxidation of unsaturated fatty acids, our results clearly showed that MP exposure for 96 h led to oxidative stress, with significant increases of LPO values in gill and white muscle when compared to the control group. The increased hydroperoxide lipid production in the present study suggests that ROS-induced oxidative damage can be one of the main toxic effects of MP. It has been reported that LPO may be induced by a variety of environmental pollutants (Ploch et al., 1999; Ahmad et al., 2000; Wilhelm-Filho et al., 2001; Oakes and Van der Kraak, 2003; Oakes et al., 2004). Given that LPO is considered a valuable indicator of oxidative damage of cellular components, our results suggest that the exposure to MP enhanced ROS synthesis in the white muscle and gill of B. cephalus and that antioxidant defenses were not totally able to effectively scavenge them, thus leading to lipid

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peroxidation. According to Zhang et al. (2004), GPx plays an important role against the LPO, since it is mainly involved in the removal of organic and, in a small extent, hydrogen peroxides. Thus, GPx is considered to play an especially important role in protecting membranes from damage due to LPO (Oost et al., 2003). This observation suggests that the major detoxification function of GPx is the termination of the radical chain propagation (Oost et al., 2003). In this context, the GPx inhibition observed in the present study might reflect a possible antioxidant defense failure responsible for the observed increase in LPO levels. Although the detoxification functions occur primarily in the liver, skeletal muscle is also involved in these processes. Skeletal muscle cells have been shown to express different types of xenobiotic-metabolizing enzymes, including cytochrome P450 (Otto and Moon, 1995; Bainy et al., 1999; Smith et al., 2000) and glutathione S-transferases (Hussey et al., 1991; Van der Weiden et al., 1992; Nam et al., 2005). Although the relative amount of enzymes in skeletal muscle is lower than in the liver, its importance in the detoxification processes may be considered since it represents a high body mass percentage. Gills, due to their large surface area and permeability, are the primary sites for absorption of xenobiotics and may be the first sites for the MP effects (Sancho et al., 1997). Therefore, it seems obvious that there is a high level of LPO such as the decreases in the antioxidant enzyme GPx and GSH content in the gills and white muscle, as observed in the present study after exposure to MP. Studies on the effects of endosulfan and paper mill effluent in fish have shown that gills are the most sensitive organs to the LPO induced by xenobiotic and their antioxidant potential is also weak compared to that of other organs (Fatima et al., 2000; Pandey et al., 2001; Sayeed et al., 2003). Organophosphates may enhance lipid peroxidation by direct interaction with the cell membrane (Hazarika et al., 2003). Conversely, Yang et al. (1996) and Yang and Dettbarn (1996) studied the effects of the OPC diisopropylphosphofluoridate and suggested that cholinergic hyperactivity induced by the inhibition of the AChE initiates the accumulation of ROS, leading to lipid peroxidation, which may cause cell injuries. These effects could contribute to the high LPO levels in the gills and white muscle of matrinxã exposed to MPc in the present study. Hepatic LPO content did not vary significantly after MP treatment, indicating that this organ resisted to the oxidative stress by means of antioxidant mechanisms and prevented LPO increases. Differing from the majority of the Amazon basin fish species, high vitamin E content was found in the liver of B. cephalus (Wilhelm-Filho and Marcon, 1996). These high vitamin E levels in the liver of matrinxã probably provided an additional protecting effect against the LPO, and, consequently, prevented increases in the hepatic LPO levels. In this study, tissue-specific responses related to antioxidant defenses and oxidative damage were observed after 96 h exposure to MP. The results indicate that gills and white muscle are the most sensitive organs to oxidative stress in comparison to liver. The main cause for these differences could be the different rates of free radical

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generation and different antioxidant potentials in the tissues. The antioxidant system of these tissues is not as efficient as that of liver, which increases their vulnerability towards ROS (Winston, 1991; Fatima et al., 2000). The present work demonstrated that MPc induces oxidative stress in B. cephalus. It is evident that, from an eco-physiological point of view, the use of methyl parathion in agriculture and aquaculture must be carefully evaluated. Our results suggest that the parameters analyzed could be good biomarkers of exposure to oxidative stress caused by methyl parathion. However, more experiments at lower MPc concentrations are needed to validate these parameters as biomarkers of oxidative stress in large-scale environmental monitoring programs. Acknowledgments The authors are thankful to CAPES (D.A. Monteiro fellowship) and Águas Claras fish farm, which provided the fish. They are also grateful to Prof. F.R.M. Laurindo, Dr. C.X. Santos and Mrs. M.A. Bertolini (Laboratory of Vascular Biology, the Heart Institute of the University of São Paulo Medical School, São Paulo, Brazil) for the HPLC technical assistance. This study was supported by FAPESP (São Paulo State Research Foundation — Proc. 03/06105-7). References Adams, S.M., Greeley, M.S., 2000. Ecotoxicological indicators of water quality: using multi-response indicators to assess the health of aquatic ecosystems. Water Air Soil Pollut. 123, 103–115. Aebi, H., 1974. Catalase. In: Bergmayer, H.U. (Ed.), Methods of Enzymatic Analysis. Academic Press, London, New York, pp. 671–684. Aguiar, L.H., Moraes, G., Avilez, I.M., Altran, A.E., Correa, C.F., 2004. Metabolical effects of Folidol 600 on the neotropical freshwater fish matrinxã, Brycon cephalus. Environ. Res. 95, 224–230. Ahmad, I., Hamid, T., Fatima, M., Chand, H.S., Jain, S.K., Athar, M., Raisuddin, S., 2000. Induction of hepatic antioxidants in freshwater catfish (Channa punctatus Bloch) is a biomarker of paper mill effluent exposure. Biochim. Biophys. Acta 1519, 37–48. Almeida, J.A., Diniz, Y.S., Marques, S.F.G., Faine, L.A., Ribas, B.O., Burneiko, R.C., Novelli, E.L.B., 2002. The use of oxidative stress responses as biomarkers in Nile tilapia (Oreochromis niloticus) exposed to in vivo cadmium contamination. Environ. Int. 27, 673–679. Almeida, J.S., Meletti, P.C., Martinez, C.B.R., 2005. Acute effects of sediments taken from an urban stream on physiological and biochemical parameters of the neotropical fish Prochilodus lineatus. Comp. Biochem. Physiol. C 140, 356–363. Alves, S.R.C., Severino, P.C., Ibbotson, D.P., 2002. Effects of furadan in the brown mussel Perna perna and in the mangrove oyster Crassostrea rhizophorae. Mar. Environ. Res. 54, 1–5. Avilez, I.M., Aguiar, L.H., Altran, A.E., Moraes, G., 2004. Acute toxicity of nitrite to matrinxã, Brycon cephalus (Günther, 1869), (Teleostei–Characidae). Cienc. Rural 34, 1753–1756. Bagnasco, M., Camoirano, A., De Flora, S., Melodia, F., Arillo, A., 1991. Enhanced liver metabolism of mutagens and carcinogens in fish living in polluted seawater. Mutat. Res. 262, 129–137. Bainy, A.C.D., 1996. Oxidative stress as biomarker of polluted aquatic sites. In: Val, A.L., Almeida-Val, V.M.F., Randall, D.J. (Eds.), Physiology and Biochemistry of the Fishes of the Amazon. INPA, Manaus, Brazil, pp. 101–110. Bainy, A.C.D., Arisi, A.C.M., Azzalis, L.A., Simizu, K., Barios, S.B.M., Videla, L.A., Jungueira, V.B.C., 1993. Differential effects of short-term lindane administration on parameters related to oxidative stress in rat liver and erythrocytes. J. Biochem. Toxicol. 8, 187–194.

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