Oxidative stress responses in different organs of Jenynsia multidentata exposed to endosulfan

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Ecotoxicology and Environmental Safety 72 (2009) 199–205 www.elsevier.com/locate/ecoenv

Oxidative stress responses in different organs of Jenynsia multidentata exposed to endosulfan M.L. Ballesterosa, D.A. Wunderlinb, M.A. Bistonia, a

Universidad Nacional de Co´rdoba, Facultad de Ciencias Exactas Fı´sicas y Naturales, Ca´tedra Diversidad Animal II, Avda. Ve´lez Sa´rsfield 299, 5000 Co´rdoba, Argentina b Universidad Nacional de Co´rdoba-CONICET, Facultad de Ciencias Quı´micas, Dto. Bioquı´mica Clı´nica-CIBICI, Haya de la Torre y Medina Allende, Ciudad Universitaria, 5000 Co´rdoba, Argentina Received 28 April 2007; received in revised form 2 January 2008; accepted 13 January 2008 Available online 4 March 2008

Abstract We evaluate antioxidant responses of Jenynsia multidentata experimentally exposed to sublethal concentrations of endosulfan (EDS). The main goal was to determine differences in the response between different organs to assess which one was more severely affected. Thus, we exposed females of J. multidentata to EDS during 24 h, measuring the activity of GST, GR, GPx, CAT and LPO in brain, gills, liver, intestine and muscle of both exposed fish and controls. GST activity was inhibited in gills, liver, intestine and muscle of exposed fish but was induced in brain. GR and GPx activities were increased in brain and gills at 0.014 and 0.288 mg L1, respectively. GPx activity was inhibited in liver and muscle at all studied concentrations whereas inhibition was observed in the intestine above 0.288 mg L1. Exposure to 1.4 mg L1 EDS caused CAT inhibition and increase of LPO levels in liver. LPO was also increased in brain at almost all concentrations tested. We find that the brain was the most sensitive organ to oxidative damage. Thus, J. multidentata could be used as a suitable bioindicator of exposure to EDS measuring activities of antioxidant enzymes in brain and liver as biomarkers. r 2008 Elsevier Inc. All rights reserved. Keywords: Antioxidant enzymes; Oxidative stress; Endosulfan; Fish; Lipid peroxidation

1. Introduction The widespread use of pesticides has resulted in the pollution of many aquatic habitats worldwide. Pesticides enter to the aquatic systems by different routes, including: direct application, urban and industrial discharges, surface runoff from non-point sources, including agricultural soil, aerosol, particulate deposition and rainfall, etc. (Sharma, 1990). Among different pollutants, organochlorine pesticides require special attention because of their high stability and toxicity to the aquatic organisms. Endosulfan (EDS) is an organochlorine insecticide belonging to the cyclodiene group. EDS is persistent in soils (60 days for alpha and 800 days for beta isomers) (Stewart and Cairns, 1974). EDS is partially soluble in water (60–100 mg L1) (ATDSR, Corresponding author. Fax: +54 351 4332099.

E-mail address: [email protected] (M.A. Bistoni). 0147-6513/$ - see front matter r 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2008.01.008

2000) where persists from 3 to 15 days (Eichelberger and Litchemberg, 1971). The low persistence of EDS in water and its relatively low toxicity to mammals and bees have justified its use in agriculture (Ghadiri et al., 1995). However, this compound is highly toxic for fish (Naqvi and Vaishnavi, 1993). It accumulates in fatty tissues of aquatic organisms that are continuously exposed to sublethal concentrations (Jonsson and Toledo, 1993). Most organochlorine insecticides are banned in many countries. However, EDS is still in use in Argentina (Miglioranza et al., 2003), where Baudino et al. (2003) found concentrations ranging from 0.97 to 2 mg L1 for alpha and beta isomers in surface and ground water. According to EPA, EDS concentrations above 0.22 mg L1 (acute) and 0.056 mg L1 (chronic) have an adverse impact on the health of aquatic organisms (Mersie et al., 2003). Negative effects of EDS on fish have been evaluated in several studies, including biochemical (Sharma, 1988,

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1990), histological (Cengiz and U¨nlu¨, 2003; Ballesteros et al., 2007), and behavioural studies (Rehman, 2006). The effects on brain acetylcholinesterase and endocrine-disrupting potential of EDS were demonstrated in vivo experiments using animals (Dutta and Arends, 2003) as well as in vitro assays using isolated adrenocortical cells of rainbow trout (Bisson and Hontela, 2002). Fish living in polluted areas are continuously exposed to toxic compounds, many of them exerting cytotoxic effects by the production of reactive oxygen species (ROS) (Di Giulio et al., 1989). ROS induce damage on most biomolecules, namely lipids, proteins and DNA (Winston and Di Giulio, 1991; Kelly et al., 1998). Endogenous enzymatic and non-enzymatic antioxidants are essential for the conversion of ROS to harmless metabolites as well as to protect and restore normal cellular metabolism and functions (Bebe and Panemangalore, 2003). The key enzymes for the detoxication of ROS in all organisms are superoxide dismutase (SOD; 1.15.1.1), catalase (CAT; EC 1.11.1.6), glutathione reductase (GR; EC 1.8.1.7) and glutathione peroxidase (GPx; EC 1.11.1.9). Glutathione-S-transferase (GST; 2.5.1.18) catalyzes the conjugation of GSH with a variety of electrophilic metabolites. This enzyme participates in the defence against oxidative stress as these enzymes are able to detoxify endogenous harmful compounds like hydroxyalkenals and base propenals or DNA hydroperoxides and electrophilic xenobiotics (Cnubben et al., 2001). Apart from their essential functions in the cell, a critical role for GSTs is obviously defence against oxidative damage. Thus, the induction of GST is considered beneficial to handle environmental stress (van der Oost et al., 2003). Estimation of lipid peroxidation has been found to have predictive importance from a number of studies as a biomarker for oxidative stress (Lackner, 1998). Lipid peroxidation can also occur as a consequence of imbalance between antioxidant system and pro-oxidant state generated by pesticide toxicity (Winston and Di Giulio, 1991). Induced oxidative stress has been reported in fish exposed to EDS, namely, cultured adrenocortical cells of rainbow trout, Oncorhynchus mykiss (Dorval and Hontela, 2003; Dorval et al., 2003). Pandey et al. (2001) found induction in the activities of GPx and GST as well as elevated levels of LPO and inhibition of CAT activity in gills, liver and kidney of freshwater fish Channa punctatus. Also, EDS-induced toxicity was found in tissues of rat, which has been associated with oxidative damage generating ROS as well as depletion of GSH (Bebe and Panemangalore, 2003). As a model species to evaluate the negative effects of EDS we used the widely distributed fish Jenynsia multidentata (Anablepidae, Cyprinodontiformes). This is a viviparous species, presenting external sexual dimorphism between males and females. It inhabits both polluted and non-polluted areas in relative high number of individuals in the Neotropical region of South America (Haro and Bistoni, 1996; Malabarba et al., 1998; Hued and Bistoni,

2005). Several studies have been performed using J. multidentata as a bioindicator of pollution using both field and laboratory conditions. Guzman et al. (2004) investigated the recovery of Escherichia coli in the muscle and digestive tract of this species in the Suquı´ a River. Cazenave et al. (2005) evaluated the accumulation of microcystin-RR (MC-RR) on different organs of J. multidentata. Furthermore, Ballesteros et al. (2007) observed differences in the toxicity of EDS between males and females as well as histological alterations in gills and liver of this species. Cazenave et al. (2007) found alterations in the swimming behaviour in individuals of this species exposed to MC-RR. Our main goal was to assess if J. multidentata could be also used as bioindicator in areas polluted with EDS and similar compounds. Additionally, we looked to evaluate which organ is the most affected upon exposure of fish to this xenobiotic. 2. Materials and methods 2.1. Fish Female adults of the native widespread species J. multidentata were selected for their experimental properties such as small size, easy collection and maintenance in the laboratory (APHA et al., 1995). Fish were captured by a backpack electrofisher equipment from an unpolluted area on San Antonio River; San Antonio de Arredondo locality, 641320 W, 311280 S-Co´rdoba, Argentina (Hued and Bistoni, 2005) and transported to the laboratory within water tanks (20 L). Time interval from capturing to laboratory arrival was less than 4 h. Fish were acclimatized to laboratory conditions for 2 weeks prior to the experiments. They were maintained in 15 L aerated glass aquarium containing dechlorinated tap water: temperature 20–22 1C, pH 7.6–7.8, alkalinity 118 mg L1, hardness 75 mg L1, conductivity 325 mS cm1, dissolved oxygen 100% saturation (aerated). Acclimatization was performed in a temperature controlled room at 2171 1C, with a light:dark cycle of 12:12 h. Fish were fed ad libitum twice a day with commercial fish pellets and starved 24 h prior to experiment to avoid prandial effects and to prevent deposition of feces in the course of the assay. The remainder food was removed after feeding. Standard length and weight (mean7SD) were 35.3476.00 mm and 9207530 mg, respectively.

2.2. Chemicals A commercial formulation of the organochlorine insecticide EDS (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3-benzodioxathiepine-3-oxide, CAS number 115-29-7)—Galgofan 35 ECs-was used. Other chemicals and reagents were purchased from Sigma-Aldrich Chemical Corporation (USA).

2.3. Experimental design Fish were exposed by immersion to four sublethal concentrations of EDS (0.014, 0.072, 0.288 and 1.4 mg L1) and a control group for 24 h. These sublethal concentrations were chosen according to the EDS 24-h LC50 value previously determined for J. multidentata (2.8 mg L1) (Ballesteros et al., 2007). Enzyme measurements and lipid peroxidation assays were carried out by separate experiments using three individuals at each (n ¼ 3). Each experiment was carried out in a 15 L aerated glass aquarium containing three fish and supplemented with EDS to reach the reported concentrations. EDS concentrations in water was verified at the beginning of each experiment by GC-ECD (Gitahi et al., 2002), showing

ARTICLE IN PRESS M.L. Ballesteros et al. / Ecotoxicology and Environmental Safety 72 (2009) 199–205 recoveries 495% of the nominal value. Controls were also performed using a 15 L glass aquarium without EDS. After 24 h exposure, fish were decapitated by transecting the spinal cord and dissected. Brain (971 mg), gills (1975 mg), intestine (1573 mg), liver (671 mg) and muscle (2574 mg) were carefully removed, washed with physiological saline (0.9% NaCl) and immediately frozen into liquid nitrogen and stored at 80 1C until analysis (within 1 month after extraction).

2.4. Enzyme extraction and measurement Enzyme extracts from each organ were prepared from individual fish (not pooled) according to Cazenave et al. (2006). Briefly, organs were homogenized in 0.1 M potassium phosphate buffer, pH 6.5 containing 20% (v/v) glycerol, 1 mM EDTA and 1.4 mM dithioerythritol (DTE) using a glass homogenizer (Potter Elvehjem), affording a tissue weight of ca. 10% per volume. The samples were centrifuged at 13 000g for 10 min to separate cell debris and the supernatant was further centrifuged at 105 000g for 60 min using an ultracentrifuge (Sorvalls Ultraspeed). The resultant supernatant was used to assess all the enzyme activities. Enzyme activities were determined by triplicate using a spectrophotometer Shimadzu Corporation MultiSpec-1501, equipped with a multiple cell holder and temperature control. The activity of GST was determined using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, according to Habig et al. (1974). Glutathione reductase (GR) activity was assayed according to Tanaka et al. (1994). The activity of glutathione peroxidase (GPx) was determined according to Drotar et al. (1985), using H2O2 as substrate. Catalase activity (CAT) was determined according to Chang and Kao (1998). The enzymatic activity was calculated in terms of the protein content of the sample (Bradford, 1976), and is reported in nanokatals per milligram of protein (nkat (mg prot)1), where 1 kat is the conversion of 1 mol of substrate per second.

2.5. Lipid peroxidation The thiobarbaturic acid (TBA) method was used to evaluate the peroxidation of lipids (LPO) (Fatima et al., 2000) in brain, gills, intestine, liver and muscle of exposed and control fish. The rate of LPO was expressed as nanomoles of thiobarbituric acid reactive substances (TBARS) formed per hour, per milligram of proteins (nmol TBARS/ (mg prot)). Each extract was measured by triplicate.

2.6. Statistical analysis All data were expressed as mean7SD (n ¼ 3, considering nine data arising from three individuals at each concentration, measuring each parameter by triplicate). Statistical analyses were carried out using Infostat Software Package (Grupo Infostat, 2002). Shapiro–Wilks test was applied to evaluate normality while Levene test was used to test the homogeneity of variance. One way analysis of variance (ANOVA) followed by a Multiple Comparison Test (Tukey) were performed. Pvalues below 0.05 were regarded as significant. Kruskal–Wallis test was applied to those variables with non-normal distribution or variance heterogeneity (Sokal and Rohlf, 1999).

3. Results 3.1. Enzymatic activity 3.1.1. Glutathione S-transferase The activity of GST in studied organs is shown in Table 1, where we can observe that exposure to EDS caused a significant inhibition in gills, muscle and liver of exposed fish. On the contrary, GST was significantly increased in brain of fish exposed at 0.014, 0.072 and

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Table 1 Activity of glutathione-S-transferase (nkat (mg prot)–1) in brain, gills, intestine, liver and muscle of Jenynsia multidentata after exposure to sublethal concentrations of endosulfan for 24 h Brain Control 0.014 0.072 0.288 1.4

Gills

4.571.7a 16.172.0c 42.778.2c 2.772.0a ab 14.872.8 7.473.7b 22.473.5bc 6.071.9ab a 14.571.7 7.072.7b

Intestine

Liver

7.673.0bc 16.975.0c 4.972.3ab 11.272.3bc 8.373.2c 8.974.9ab 4.770.9a 5.272.4a abc 5.671.5 8.572.0ab

Muscle 3.271.4c 0.470.1a 1.370.8bc 0.870.5ab 0.970.5ab

Mean7SD; n ¼ 3 (means and SD are calculated from nine measurements, considering three individuals at each concentration, measuring each parameter by triplicate). Means not sharing the same superscript (a, b, or c) in each column are significantly different at Po0.05. Table 2 Activity of glutathione reductase (nkat (mg prot)–1) in brain, gills, intestine, liver and muscle of Jenynsia multidentata after exposure to sublethal concentrations of endosulfan for 24 h

Control 0.014 0.072 0.288 1.4

Brain

Gills

Intestine

Liver

Muscle

2.871.4a 7.273.0b 3.172.0a 4.572.3ab 2.472.0a

3.572.4a 4.37 2.9a 3.170.6a 10.374.2b 2.271.4a

2.971.1a 2.670.7a 3.071.4a 2.070.6a 2.270.9a

5.773.4ab 9.374.3b 6.871.6b 4.672.4ab 2.171.0a

5.472.5c 3.071.9bc 4.872.3c 1.070.7ab 0.870.6a

Mean7SD; n ¼ 3 (means and SD are calculated from nine measurements, considering three individuals at each concentration, measuring each parameter by triplicate). Means not sharing the same superscript (a, b, or c) in each column are significantly different at Po0.05.

0.288 mg L1. In intestine, there were no significant changes in enzymatic activity except of 0.288 mg L1 where the activity was inhibited. 3.1.2. Glutathione reductase Table 2 shows that GR did not exhibit significant changes in fish exposed to EDS, with a few exemptions, namely: activation in brain and gills at 0.014 and 0.288 mg L1, respectively, as well as with inhibition in muscle of fish exposed to 0.288 and 1.4 mg L1. 3.1.3. Glutatione peroxidase Table 3 shows results obtained with GPx activities. Exposure to EDS induced a significant inhibition in liver and muscle of exposed fish. Gills exposed to 0.288 mg L1 show significant activity GPx activation, followed by inhibition at 1.4 mg L1. In brain, the exposure to 0.014 and 0.288 mg L1 caused a significant activation of GPx. In intestine there was significant reduction in the enzyme activity above 0.288 mg L1. 3.1.4. Catalase CAT activity was only detected in liver of fish, where its activity was inhibited at the highest concentration (1.4 mg L1), while at lower concentrations there were not significant changes (Fig. 1).

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Table 3 Activity of glutathione peroxidase (nkat (mg prot)–1)in brain, gills, intestine, liver and muscle of Jenynsia multidentata after exposure to sublethal concentrations of endosulfan for 24 h

Control 0.014 0.072 0.288 1.4

Brain

Gills

Intestine

Liver

Muscle

6.473.7ab 34.2710.0c 7.071.9ab 12.576.2bc 4.272.0a

13.274.4b 9.472.5b 9.072.8b 25.375.8c 4.771 .8a

14.472.3cd 22.378.4d 12.774.0bc 9.073.5ab 3.971.7a

23.375.9b 17.073.4a 17.672.8a 15.472.0a 12.972.7a

8.573.4d 2.871.2bc 4.571.3cd 2.070.6ab 1.370.5a

Mean7SD; n ¼ 3 (means and SD are calculated from nine measurements, considering three individuals at each concentration, measuring each parameter by triplicate). Means not sharing the same superscript (a, b, c, or d) in each column are significantly different at Po0.05.

Fig. 1. Activity of catalase in liver of Jenynsia multidentata after exposure to sublethal concentrations of endosulfan for 24 h. Mean7SD; n ¼ 3 (means and SD are calculated from nine measurements, considering three individuals at each concentration, measuring each parameter by triplicate). *Po0.05 respect to the control group.

Table 4 Levels of lipid peroxidation (nmol TBARS/(mg prot)) in brain, gills, intestine, liver and muscle of Jenynsia multidentata after exposure to sublethal concentrations of endosulfan for 24 h Brain Control 0.014 0.072 0.288 1.4

Gills a

0.1570.03 0.5570.25b 0.3270.08ab 0.7870.07b 0.4370.04b

Intestine a

0.1170.05 0.5570.40a 0.3370.24a 0.6070.75a 0.2770.20a

Liver a

0.2470.07 0.5470.26a 0.2870.20a 0.7770.14a 0.2770.05a

Muscle a

0.5670.31 1.0070.23ab 0.4470.13a 0.5670.12a 1.7770.51b

0.0670.05a 0.5470.21a 0.5170.41a 0.1370.10a 0.0670.05a

Mean7SD; n ¼ 3 (means and SD are calculated from nine measurements, considering three individuals at each concentration, measuring each parameter by triplicate). Means not sharing the same superscript (a or b) in each column are significantly different at Po0.05.

3.2. Lipid peroxidation Exposures to 1.4 mg L1 caused an elevation in LPO in the liver of exposed fish. Additionally, LPO levels were

significantly increased in brain of exposed fish at almost all concentrations tested (Table 4). Conversely, in gills, intestine and muscle there were not changes in LPO levels. 4. Discussion 4.1. Enzyme activity The antioxidant defence system is being increasingly studied because of its potential utility to provide biochemical biomarkers that could be used in environmental monitoring systems (Winston and Di Giulio, 1991; Orucet al., 2004). Fish respond to exposure to pollutants by altering or adapting their metabolic functions (Bebe and Panemangalore, 2003). Alterations found in the activity of antioxidants enzymes upon exposure to sublethal concentrations of EDS suggest that changes observed could be an adaptative response to ROS. The activity of antioxidant enzymes may be increased or inhibited under chemical stress depending on the intensity and the duration of the stress applied as well as the susceptibility of the exposed species. It is not a general rule that an increase in xenobiotic concentrations induces antioxidant activity (Cheung et al., 2001). In the present work, the studied enzymes responded in a different way in different organs. So, our resent results confirm the need of evaluating biomarkers at several organs at a give species to fully assess its usefulness as bioindicator. In the present study, the GST activity was inhibited in gills, liver and muscle but activated in brain. In intestine the changes were not significant except at 0.288 mg L1 where inhibition was observed. This could be due to the route of exposure to EDS. Gill is the first organ exposed to dissolved pollutants (Heath, 1987) and, therefore, exposed to higher concentrations of EDS than other organs tested. Other enzymes showed differential response under different route of exposure. For instance, van Veld et al. (1997) demonstrated different patterns of cellular CYP1A expression resulting from different benzo[a]pyrene exposure routes. Aqueous exposures resulted in high levels of CYP1A induction in gill pillar cells, heart endothelium, and general vascularization. In contrast, dietary exposures resulted in high levels of CYP1A only in the gut mucosal epithelium with only mild to moderate expression elsewhere. In the present study, fish was exposed to EDS by immersion, thus gills are exposed continuously to EDS at the highest possible concentrations. The liver also might be exposed to a high concentration of this pesticide due to its important role in the detoxication of xenobiotics; thus, showing GST inhibition similarly to those observed in gills. 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 (Monteiro et al., 2006). The inhibition of GST could be explained by different hypothesis. First biotransformation steps by cytochrome P450 enzymes may

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produce a cocktail of different metabolites, competing with GST substrates for the active sites on the GST enzyme (Egaas et al., 1999). Second, Gallangher and Sheehy (2000) postulates that lower activities may be caused by a decrease in the synthesis of GST proteins at molecular levels. Increase of GST activity in brain could appear as a result of LPO induction in this tissue (Table 4) which is more susceptible to ROS than other organs with higher polyunsaturated fatty acid contents (Mates, 2000). Other authors have studied the effects of EDS on the activity of GST. Pandey et al. (2001) found the GST activity induced in liver, gills and kidney of C. punctatus exposed to 5 mg L1 of EDS for 24 h. Our present results shows that the gills, liver and muscle of J. multidentata present inhibition of GST upon exposure to low amounts of EDS compared with the above-mentioned report, even when J. multidentata has lower weight than C. punctatus. Additionally, Barata et al. (2005) reported changes of GST in Daphnia magna juveniles exposed to 400, 600, and 800 mg L1 of this pesticide for 48 h. Once more, EDS concentrations used in our study were significantly lower than those reported for D. magna. Accordingly, J. multidentata could be suggested as a more sensitive species for biomonitoring EDS in water. The role of GR is to maintain the cytosolic concentration of GSH into the cells at expense of NADPH. GSH is substrate for GST and cofactor for GPx (van der Oost et al., 2003; Winston and Di Giulio, 1991). GR activity in brain of J. multidentata was increased at the lowest tested concentration (0.014 mg L1), while in gills it was increased at 0.288 mg1. GR activity in muscle of J. multidentata was inhibited at the highest concentration tested (1.4 mg L1). According to Zhang et al. (2004) and Moreno et al. (2005), the inhibition of GR activity could be due to the change in the availability of NADPH in the cell. However, it is important to point out that the evidence provided on the literature about the effects of EDS on muscle detoxication enzymes is scarce. Thus, the results obtained in this work gives additional information to consider changes in the activity of GR in muscle induced upon exposure to EDS. GPx catalyses the reduction of H2O2 and lipid hydroperoxides at expense of GSH (Moreno et al., 2005). We observed, the inhibition of GPx activity in gills, intestine, liver and muscle of fish exposed at the highest concentration (1.4 mg L1). This test result is in good agreement with Dorval and Hontela (2003) and Dorval et al. (2003), who reported GPx inhibition in cultured adrenocortical cells of O. mykiss exposed to 106, 105 and 104 M EDS for 60 min. Monteiro et al. (2006), pointed out that the enzyme activity can decrease by negative feedback either from excess of substrate or damage induced by oxidative modification. A reduced GPx activity in a given tissue could indicate that its antioxidant capacity was exceeded by the amount of hydroperoxide products. Thus, inhibition of GPx activity might reflect a possible failure of the antioxidant system in liver and muscle of exposed fish. On the contrary, increased GPx activity in brain and gills

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showed the same pattern observed for GR activity (see Tables 2 and 3). The increase of GPx activity could trigger the increase of GR activity at the same exposure concentration to keep the cytosolic concentration of GSH. CAT is an enzyme located in peroxisomes and facilitates the removal of H2O2, which is metabolized to molecular oxygen and water (van der Oost et al., 2003). In our case, the activity of CAT was significantly decreased in the liver of fish exposed to the highest concentration tested (1.4 mg L1). Our present results are in good agreement with those found by Pandey et al. (2001) and Atif et al. (2005), reporting that, the exposure of C. punctatus to EDS produced a significant decrease of CAT activity. These authors pointed out that drop observed in CAT activity could be explained by the flux of superoxide radicals due to the oxidative stress caused by pollutants exposure. 4.2. Lipid peroxidation Impairment in antioxidant enzymes will produce an imbalance between pro and antioxidant systems causing the formation of toxic hydroxyl radicals, with direct consequences on cell integrity and cell function itself (Dorval et al., 2003). Production of LPO induced by EDS has been reported previously by other researchers. Hincal et al. (1995) demonstrated that either a single dose of 30 mg kg1 or repeated doses of 5 and 15 mg kg1 d1 EDS induced LPO in brain and liver of rat. Similarly, Pandey et al. (2001) and Atif et al. (2005) observed elevated levels of LPO in gills, liver and kidney of C. punctatus exposed to 5 mg L1 EDS for 24 h. Also, Dorval et al. (2003) found elevation in LPO levels in cultured adrenocortical cells of O. mykiss exposed to 107, 106 and 104 M EDS for 60 min. In our present study, only the liver and brain were affected with high levels of LPO. Brain is particularly susceptible to oxidative damages. In spite of the high rate of ROS production, from the high rate of oxidative metabolism and abundance of polyunsaturated fatty acids in cell membrane, brain has a relatively low antioxidant defence system (Mates, 2000). Additionally, our results show that enzymatic activities are lower in brain than in liver and gills of fish (see for instance values for control in Tables 1 and 3). Ahmad et al. (2004) have found that exposure to polluted water induces tissue-specific oxidative damage in gills, kidney and liver of Anguilla anguilla, being gills the most affected organ. They found that LPO levels in gills were significantly increased upon exposure to harbour water for 24 and 48 h while both CAT and GPx were strongly inhibited. In contrast, in this study, gills did not present high LPO levels and both GPx and GR activities were significantly increased at 0.288 mg L1, showing good capacity to scavenge ROS. CAT and GPx have complementary roles in hydrogen peroxide detoxication, having both, different subcellular localizations (peroxisomal and cytosolic, respectively) and

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target molecules (reduction of H2O2 for CAT as well as H2O2 and toxic hydroperoxides for GPx) (Barata et al., 2005). The decreased activities of GPx and CAT in the liver of fish exposed to 1.4 mg L1 EDS indicates its reduced capacity to scavenge H2O2 and lipid hydroperoxides produced in this tissue. Similar results were obtained by Moreno et al. (2005) in rats exposed to, microcystin-LR. We did not detect CAT activity in brain, gills, intestine and muscle of J. multidentata. Similar results were obtained by Cazenave et al. (2006) in Corydoras paleatus exposed to MC-RR. Jos et al. (2005) observed that CAT activity in gills was much lower than liver and kidney of tilapia. So, it is possible that the activity of CAT in this organs is too low to be detected by the method used during our study. 5. General conclusions Our present results demonstrate that exposure to a sublethal concentration of EDS results in oxidative stress in several organs of J. multidentata. A period of 24 h of exposure by immersion was enough to induce alterations in biotransformation and antioxidant enzymes such as GST, GR, GPx and CAT. The antioxidant system in intestine was the least altered compared with the other organs. This effect could be related to the route of exposure of this organ to EDS. The brain was the most sensitive organ to oxidative damage, particularly LPO levels were high in brain upon the lowest EDS concentrations tested. Considering the low LC50 and antioxidant response of J. multidentata exposed to low environmental relevant concentrations of EDS, we suggest that this fish could be effectively used as bioindicator to evaluate the pollution with EDS, using biomarkers measured in brain and liver as the most sensitive organs. Acknowledgments This work was supported by grants from the Agencia Nacional de Promocio´n Cientı´ fica y Te´cnica (FONCyT), Secretarı´ a de Ciencia y Te´cnica (SECyT) and National Research Council (CONICET). This work is a part of PhD thesis of M.L. Ballesteros, who gratefully acknowledges a fellowship from CONICET. Authors wish to thank J. Cazenave, A.C. Hued, for their assistance in the field, support in the laboratory, and critical review of this manuscript. References Ahmad, I., Pacheco, M., Santos, M.A., 2004. Enzymatic and nonenzymatic antioxidants as an adaptation to phagocyte-induced damage in Anguilla anguilla L. following in situ harbor water exposure. Ecotoxicol. Environ. Saf. 57, 290–302. APHA, AWWA, WEF, 1995. In: Greenberg, A.H., Clesceri, L.S., Eaton, A.D. (Eds.), Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington, DC.

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