Ecotoxicology and Environmental Safety 99 (2014) 21–27
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Effects of in vivo chronic exposure to pendimethalin on EROD activity and antioxidant defenses in rainbow trout (Oncorhynchus mykiss) Morgane Danion a,b,n, Stéphane Le Floch c, François Lamour a,b, Claire Quentel a,b a
ANSES, Ploufragan-Plouzané Laboratory, Unit of Viral Pathology in Fish, Technopôle Brest-Iroise, 29280 Plouzané, France Université Européenne de Bretagne, France c CEDRE, Center of Documentation, Research and Experimentation on Accidental Water Pollution, 715 Rue Alain Colas, 29200 Brest, France b
art ic l e i nf o
a b s t r a c t
Article history: Received 20 February 2013 Received in revised form 13 September 2013 Accepted 14 September 2013 Available online 29 October 2013
Pendimethalin, an herbicide active substance frequently used in terrestrial systems, has detected in European aquatic ecosystems. Reliable indicators still need to be found in order to properly assess the impact of pesticides in fish. After an in vivo chronic exposure to pendimethalin, the detoxification process and the antioxidant defense system were assessed in 120 adult rainbow trout, Oncorhynchus mykiss. Four nominal exposure conditions were tested: control (C), 500 ng L 1 (P500), 800 ng L 1 (P800) and the commercial formulation Prowls at 500 ng L 1 (Pw500). Fish samples were made after a 28 day exposure period (D28) and after a fifteen day recovery period in clean fresh water (D43). At D28, ethoxyresorufinO-deethylase (EROD) activity was not activated in liver in spite of the pendimethalin uptake in fish. At D43, EROD activity in fish exposed to the commercial product was lower than in control fish, which may be explained by the high presence of herbicide in fish (613 7163 ng g bile 1). Furthermore, antioxidant defense responses were set up by trout in gills and liver following chronic exposure to 800 ng L 1 of pendimethalin concentration. While the glutathione content (GSH) decreased in gills, it increased in liver associated with higher activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD). These disturbances could lead to reactive oxygen species production and oxidative stress in the vital organs in fish. After fifteen days in clean water, while the SOD activity was restored, the GSH content and GPx activity were still significantly disturbed in fish exposed to pendimethalin in comparison with control. These significant differences between treatments in antioxidant defenses parameters measured, attesting to the irreversibility of the effects. Crown Copyright & 2013 Published by Elsevier Inc. All rights reserved.
Keywords: Pendimethalin Oncorhynchus mykiss EROD GSH SOD GPx
1. Introduction Pendimethalin (N-(1-ethylpropyl)-2,6-dinitro-3,4-xylidine) is a dinitroaniline herbicide active substance, frequently used in terrestrial systems. Due to the common usage of various formulations composed of pendimethalin, this chemical compound has been detected at high concentrations in European aquatic ecosystem i.e. 352 ng L 1 in Denk (Asman et al., 2005), 370 ng L 1 in Spain (Barba-Brioso et al., 2010) and 840 ng L 1 in France (CORPEP, 2010). Taken up by fish due to its high bioconcentration factor (5100, Agritox database), pendimethalin is accumulated in specific organs and could disturb physiological parameters such as immune system components (Danion et al., 2012a,b). Despite these facts, reliable indicators still need to be found in order to measure and properly assess the impact of low and prolonged n Corresponding author at: Unit of Viral Pathology in Fish, Ploufragan-Plouzané Laboratory, Technopôle Brest-Iroise, 29280 Plouzané, France. Fax: þ33 298 055 165. E-mail address: [email protected] (M. Danion).
exposure to pesticides in fish. Danion et al. (2012a) have already demonstrated that chronic exposure to 200 ng L 1 of pendimethalin involves a high bioconcentration in flesh, attesting to the uptake of the pesticide. Indeed, following exposure to pollutants, organisms usually attempt to metabolize and depurate them, minimizing some of the cellular damage they cause and hence acquire fast elimination rates of compounds through bile and urine (Oliveira-Ribeiro et al., 2005). Phase I and phase II biotransformation parameters such as ethoxyresorufin-O-deethylase (EROD) and glutathione-S-transferase activities take part to a set of biochemical parameters which were applied to monitor biological effects in fish in the context of the Joint Assessment and Monitoring Program (JAMP) developed in the European framework (Sanchez and Porcher, 2009). The first step is usually catalyzed by cytochrome P450-dependent monooxygenases (phase I) and their products are subsequently coupled to endogenous metabolites (phase II) (Buhler and Williams, 1988; James and Whitlock, 1999). Moreover, depends on their chemical structure and their biotransformation, pesticides are known to cause the generation of reactive oxygen species (ROS), leading to oxidative stress.
0147-6513/$ - see front matter Crown Copyright & 2013 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.09.024
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M. Danion et al. / Ecotoxicology and Environmental Safety 99 (2014) 21–27
Pesticide-induced oxidative stress has been a focus of toxicological research for the last decade as a possible mechanism of toxicity (Banerjee et al., 2001; Abdollahi et al., 2004; Dorval et al., 2003; Moraes et al., 2009; Jin et al., 2011). In response to these potential harmful impacts, every living organism has a variety of antioxidant system defenses to protect themselves against the production of oxygen radicals uncoupling at various electron transfer sites or via autoxidation reactions. In this context, thiols play an important role against the pernicious effects of pro-oxidant challenges, and glutathione in particular provides a first line of defense against ROS (Pastore et al., 2003). This non-enzymatic antioxidant participates in many cellular reactions taking away ROS directly (Fang et al., 2002). Antioxidant enzymes are also commonly used to understand the associated toxic mechanisms of xenobiotics (Jensen et al., 1991; Sanchez et al., 2005). Superoxide dismutase (SOD) catalyzes the transformation of superoxide radicals to H2O2 and O2 and is the first enzyme to deal with oxyradicals. Then, H2O2 can be reduced to water and oxygen by glutathione peroxidase (GPx) and catalase (CAT) (Kappus, 1985; Oliveira et al., 2008). So, variations in antioxidant defenses can be very sensitive in revealing a pro-oxidant condition and have been used as oxidative stress markers in fish exposed to pesticides (Anguiano et al., 2001; Dorval and Hontela, 2003; Kavitha and Rao, 2008). Previous studies focusing on xenobiotic effects in fish have found that antioxidant enzyme activities may differ considerably between organs (Ahmad et al., 2003; Jee and Kang, 2005), highlighting the need to assess antioxidant responses in the main target organs when a comprehensive evaluation of the oxidative stress risk at individual level is intended. So, antioxidant defense parameters should be assessed in gills and liver, selected on the basis of functional criteria which made them preferential targets, i.e. xenobiotic uptake (gills) and xenobiotic metabolism (liver). The aim of the present study was to evaluate the effects of chronic exposure to 200 ng L 1 pendimethalin, active substance alone or the commercial product (Prowls) on the detoxification process and the antioxidant defense system were tested in vivo in rainbow trout (Oncorhynchus mykiss). To this end, the pendimethalin concentration in bile, the EROD activity and antioxidant defense parameters were monitored in gills and liver.
2. Materials and methods This experiment was conducted in accordance with the European Commission recommendation 2007/526/EC on revised guidelines for the accommodation and care of animals used for experimental and other scientific purposes. The center of documentation, research and experimentation on accidental water pollution (CEDRE) is authorized to conduct experimentation on animals in its facilities as a certified establishment according to the administrative order no. 2006-0429. Furthermore, the experimentation carried out as part of this study was conducted under the responsibility and supervision of Dr. Claire Quentel, who holds a certificate to direct scientific experimentation on animals (certificate no. 29-008). 2.1. Fish The 120 rainbow trout, 79.8 7 1.6 g, used for this experiment were sourced from virus-free fish rearing facilities at ANSES Laboratory (French Agency for Food, Environmental and Occupational Health and Safety, Plouzané, France). At CEDRE (Brest, France), the fishes were acclimated for one week in eight tanks (300 L) with a flow of 8 L h 1 at 157 1 1C. The fishes were fed with commercial dry pellets at 1.5 percent body weight (Neo Prima 4, Le Gouessant Aquaculture) once a day. 2.2. Tested substances The chemical and physical characteristics of pendimethalin have previously been described by Strandberg and Scott-Fordsmand (2004). The active substance and the commercial herbicide formulation were obtained from Sigma– Aldrich (98.8 percent purity; Germany) and BASF (400 g L 1; France), respectively. The stock solutions were prepared and stored as previously described by Danion et al. (2012a).
2.3. Experimental design 2.3.1. Experimental system The experimental system has already been described by Danion et al. (2012a). Briefly, the experimental system consisted of four similar independent units, each composed of three tanks. One stock tank containing a concentrated solution of the pollutant supplied two exposure tanks to expose fish in duplicate using a peristaltic pump with a two-channel pump head. All the exposure tanks were also provided with clean fresh water to dilute the concentrated solution in order to obtain the required pendimethalin concentration. The four units were placed in a thermoregulated greenhouse, in which the air was totally renewed every 6 h and with a natural light/dark cycle (12/12 h approximately). 2.3.2. Exposure conditions and recovery period Four nominal exposure conditions were tested, one per unit: (i) fresh water as control (C), (ii) 500 ng L 1 of active substance (P500), (iii) 800 ng L 1 of active substance (P800) and (iv) 500 ngCL 1 of pendimethalin with the commercial herbicide formulation of Prowls (Pw500). P500 represents the nominal concentration just below the predicted no-effect concentration (PNEC) estimated at 550 ng L 1 by Council Directive 2003/31/EC, while P800 represents the maximal concentration measured in rivers in Brittany in 2007. For the exposure period, the concentrated solution was flowed at 1 L h 1 from the stock tanks to the exposure tanks. In addition, a clean fresh water flow at a rate of 5 L h 1 was supplied to the exposure tanks to dilute the exposure concentration. Then, 120 trout were randomly distributed among the four units (or fifteen fishes/exposure tank). During the 28 days of the exposure period, the trout were exposed to a diluted pendimethalin concentration. At the end of the exposure period, the stock tanks were disconnected and clean fresh water was flowed into the exposure tanks at a rate of 8 L h 1 during the two week recovery period. Throughout the experiment, the trout were maintained in the same tanks and were fed once a day. 2.3.3. Samples and sampling date The oxygen level and temperature were monitored daily in each tank (Oxymeter WTW-OXI315I), while nitrate and nitrite levels were measured weekly (Colorimetric test JBLs). Fresh water parameters were stable during the acclimation period and throughout the experiment: dissolved oxygen 93 7 3 percent, pH 87 0.1, temperature 157 1 1C, free of nitrate and nitrite. Ten fishes were sampled from each unit (or five fishes/exposure tank) on the last day of exposure and recovery periods. The fishes were killed with an overdose of anesthetic phenoxy-2ethanol and weighed. From each fish, the bile, liver and two branchial arcs were carefully removed and immediately frozen at 80 1C to measure the EROD activity and antioxidant defense parameters. 2.4. Analytical methods 2.4.1. Pendimethalin concentration in bile The extraction technique used was stir bar sorptive extraction (SBSE) and the quantification was performed using gas chromatography equipment coupled with a mass spectrometer (GC–MS). From each fish, 300 mL of bile was diluted in 100 mL of distilled water with 100 mL of a solution of quintozene Pestanals (internal standard; Sigma–Aldrich). The SBSE and GC–MS analysis conditions and the quantification of pendimethalin were as described by Danion et al. (2012a). 2.4.2. Liver EROD activity The livers were homogenized in an ice-cold HEPES buffer using the tissue homogenizer Precellys 24 (Bertin Technologies, France). EROD activity in the liver S9 fraction was measured as described by Couillard et al. (2004) by a microplate spectrofluorometric assay using a Spectrofluorometers plate reader (excitation 530 nm, emission 585 nm). Reaction mixture contained S9 in HEPES buffer (0.1 M, pH 7.8), 7-ethoxyresorufin (0.024 mg mL 1 in DMSO) and NADPH (20 mg mL 1). The activity was quantified by measuring the fluorescence of resorufin at 60 s intervals over a 13 min total scan time. Liver EROD activity was calculated as nmol min 1 mg 1 protein with resorufin used as standard.All samples were assayed in duplicate. 2.4.3. Antioxidant defenses The gills and livers were homogenized in an ice-cold phosphate buffer (0.1 M, pH 7.8) with 20 percent glycerol and 0.2 mM phenylmethylsulfonyl fluoride as a serine protease inhibitor with the tissue homogenizer Precellys 24 (Bertin Technologies, France). The homogenates were centrifuged at 9000 g at 4 1C, for 15 min and the supernatant was splitted into five aliquots and stored at 80 1C for biochemical assays. The total protein concentrations in gill and liver samples were spectrophotometrically estimated using the method of Bradford (1976) with bovine serum albumin (Sigma–Aldrich Chemicals, France) used as a standard. Biomarker assays including GSH content and activities of SOD, GPx and CAT in the gills and liver of rainbow trout were adapted for use in microplates after preliminary tests using several dilutions. All samples were assayed in duplicate.
M. Danion et al. / Ecotoxicology and Environmental Safety 99 (2014) 21–27 GSH concentration was measured according to Vandeputte et al. (1994). Briefly, 10 mL of TCA deproteinized sample was mixed with phosphate buffer containing 0.3 mM NADPH and 1 mM Ellman reagent. The enzymatic reaction was monitored spectrophotometrically at 405 nm. The results were expressed in mmol mg 1 protein. SOD activity was measured according to Marklund and Marklund (1974). Briefly, samples were assayed in a solution of 8.7 mL of 50 mM phosphate buffer pH 8 and 0.3 mL of 3 mM pyrogallol. The rate of pyrogallol autoxidation was measured with a UV-220 spectrophotometer at 325 nm. SOD activity was calculated in terms of U mg 1 protein, whereby one unit of SOD activity was defined as the amount of the enzyme which gave 50 percent inhibition of the oxidation rate of 0.1 nM pyogallol in the 1 mL of solution. GPx activity was determined using 15 mL of a 4.5 g protein/L diluted sample according to the Paglia and Valentine method (1967). Cumene hydroperoxide was used as the substrate and enzymatic activity was assessed at 340 nm. GPx activity was calculated in terms of nmol min 1 mg 1 protein. CAT activity was assayed using the method of Babo and Vasseur (1992). Briefly, the assay mixture consisted of phosphate buffer (100 mM, pH 7) and H2O2 (28 mM). The variation of H2O2 absorbance in 60 s was measured with a UV-220 spectrophotometer at 225 nm. CAT activity was calculated in terms of mmol min 1 mg 1 protein with bovine erythrocyte catalase used as standard. 2.5. Statistical analysis All statistics were performed using XLstat Statistical Software 2007.0 with a significance level of 5 percent. There were significant variations between control groups during the exposure and recovery periods. So, verification of normality and of homogeneity of covariance matrices was conducted using the Anderson–Darling test and the Bartlett test, respectively, on exposure and recovery data separately, before using a one way analysis of variance (ANOVA). Newman–Keuls significant difference tests were used a posteriori to detect differences between means. All data were expressed as the mean7 standard deviation (SD).
3. Results 3.1. Pendimethalin concentration in bile No pendimethalin concentration above the quantification limit of the method used (1 ng g 1) was detected throughout the experimental period in all control fishes (Table 1). After the exposure period (D28), fish exposed to P500, P800 and Pw500 showed pendimethalin concentrations of 264 7256, 456 7362 and 263 7177 ng g bile 1, respectively, without any significant differences between them. After fifteen days in clean fresh water (D43), fish exposed to P800 and Pw500 showed significantly more pendimethalin in bile, with 4517 148 and 613 7163 ng g bile 1, than fish contaminated by P500 (176 7163 ng g bile 1) (Table 1).
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commercial formulation (4.17 70.34 nmol min 1 mg 1 of protein) (Fig. 1). 3.3. Antioxidant defenses In gills, throughout the experiment, the antioxidant defense parameters were not disturbed by the lowest exposure concentration (P500). After the 28 days exposure period, a significant decrease in the GSH content was only noted in fish exposed to the highest pendimethalin concentration (P800; 28.673.4 mmol mg 1 of protein) compared to control fish (34.272.7 mmol mg 1 of protein) (Fig. 2A). The other antioxidant defense parameters (SOD, CAT and GPx) were not disturbed after chronic exposure to pendimethalin (Fig. 2B–D). After the recovery period (D43), the GSH content significantly decreased only in fish exposed to the commercial formulation (Pw500) compared to control fish. The GPx activity was lower in all fish contaminated by pendimethalin than in control fish, but this difference was significant only for the fish exposed to Pw500, with 48.675.4 and 34.672.9 nmol min 1 mg 1 of protein, respectively (Fig. 2A and C). Moreover, CAT activity in fishes which were exposed to P800 was significantly higher than in control fish, with 11.471.1 and 9.071.3 mmol min1 mg 1 of protein (Fig. 2D). In liver, as in gills, the antioxidant defense parameters were not disturbed by the lowest exposure concentration (P500) throughout the experiment. Furthermore, after the 28 days exposure period, the SOD and GPx activities had significantly increased in fish exposed to the highest exposure concentration (P800) (Fig. 2B and C). Moreover, CAT activity in all fishes contaminated by pendimethalin had decreased compared to control fish (2.870.3 mmol min 1 mg 1 of protein), but the difference was significant only for fish exposed to Prowls (4.470.4 mmol min 1 mg 1 of protein) (Fig. 2D). At D43, while SOD activity was no longer disturbed, the GSH content and GPx activity were significantly higher in fishes which were exposed to P800 than in the control fish (Fig. 2A and C).
4. Discussion The aim of this study was to evaluate the impacts of chronic exposure to pendimethalin active substance alone or the
3.2. Liver EROD activity After the 28 days exposure period, no significant difference was observed in liver EROD activity in fish whatever the experimental exposure conditions. After the recovery period, at D43, a decrease was noted in all contaminated fish by pendimethalin but this decrease was only significant compared to the control (5.86 70.65 nmol min 1 mg 1 of protein) for fish exposed to the Table 1 Concentration of pendimethalin in bile of rainbow trout after the exposure (D28) and recovery (D43) periods in control and contaminated fish by 500 ng L 1 (P500), 800 ng L 1 (P800) and Prowls (Pw500). The results are expressed in ng g 1 of bile. N ¼10, blq¼ below limit of quantification ( o 1 ng bile 1). Mean values 7standard deviation (SD) having similar letters are not significantly different (ANOVA, Newman–Keuls, p 40.05). Pendimethalin concentration in fish bile (ng g 1 7SD) Sampling date
D28 D43
Exposure conditions Control
P500
P800
Pw500
Blq Blq
264 7256a 176 7163a
456 7 362a 4517 148b
263 7 177a 6137 163b
Fig. 1. Liver ethoxyresorufin-O-deethylase (EROD) activity after the exposure (D28) and recovery (D43) periods in control and contaminated rainbow trout by 500 ng L 1 (P500), 800 ng L 1 (P800) and Prowls (Pw500). N ¼ 10. Mean values ( 7SD) having similar letters are not significantly different (ANOVA; Newman– Keuls, p4 0.05).
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Fig. 2. (A) Glutathione content (GSH); (B) superoxide dismutase (SOD) activity; (C) glutathione peroxidase (GPx) activity; (D) catalase activity; in gills and liver after the exposure (D28) and recovery (D43) periods in control and contaminated fish by 500 ng L 1 (P500), 800 ng L 1 (P800) and Prowls (Pw500). N ¼ 10. Mean values ( 7SD) having similar letters are not significantly different (ANOVA; Newman–Keuls, p 40.05).
M. Danion et al. / Ecotoxicology and Environmental Safety 99 (2014) 21–27
commercial formulation Prowls, in rainbow trout. To this end, the active substance concentration in bile, the EROD detoxification parameter and antioxidant defenses were monitored. Numerous studies have evaluated these biomarkers in fish exposed to pollution such as pesticides (Elia et al., 2002; Dorval and Hontela, 2003; Sayeed et al., 2003; Kavitha and Rao, 2008), polycyclic aromatic hydrocarbons (PAHs) (Wessel et al., 2010), polychlorobiphenyls (Huuskonen et al., 1996; Saera-Vila et al., 2009) and metals (Pandey et al., 2008; Kerambrun et al., 2011). Concerning pesticide pollution, few studies have evaluated in vivo chronic exposure and to our knowledge, no data is available on the effect of pendimethalin on these parameters in fish. The experimental system and protocol used in this study were elaborated to realistically reconstitute the state of river water. According to a number of predefined criteria, pendimethalin appeared to be the most relevant pesticide to perform this study. In fact, the selected substance was required to (i) be listed in Annex I of Council Directive 91/414/EEC, (ii) be detected in the aquatic environment at high concentrations in rivers in Brittany, (iii) have a bioconcentration factor (BCF) 43000 and (iv) have an ecotoxicity that was already known in aquatic fauna. In a previous study, some data concerning the experimental design and the characteristics of the exposure have been discussed (Danion et al., 2012a). In this study, the presence of pendimethalin in the bile (o 456 ng g 1) attested to the uptake of herbicide by fish as well as Danion et al. (2012a) has been already demonstrated it by the bioconcentration in flesh. However, only free chemical compounds were measured because we did not know the structure of the conjugated metabolites released into the bile and the potential elimination process was not sought yet in fish. Organic xenobiotics can be detoxified in aquatic organisms’ liver which is the main tissue of metabolism (Livingstone, 1991). 7-Ethoxyresorufin O-deethylase (EROD) is one biomarker that has been used extensively as it is induced by exposure to a wide variety of environmental contaminants (Lemaire and Livingstone, 1995; Van der Oost et al., 2003; Ferreira et al., 2006). Indeed, most of the studies conducted in fish have reported significant induction of EROD activity following exposure to CYP1A inducers such as polycyclic aromatic hydrocarbons (PAH), polychlorated biphenyl (PCB) or polychlorodibenzo-p-dioxine (PCDD) (Peters et al., 1994; Arnic et al., 2000). In this study, although exposure to pendimethalin was attested to the uptake in fish, liver EROD activity was not affected in trout following the 28 days exposure period, whatever the exposure conditions. This result agreed with those of Jensen et al. (1991) and Savelli et al. (1997) who have previously demonstrated that fish exposure to pesticide, contrary to PAHs and PCB, could lead to only mild induction in EROD, questioning (i) the role of the aryl hydrocarbon receptor (AhR) in the activation of CYP1A1/EROD by several environmental pollutants and (ii) the role of the structural–functional relationships of ligands in the mechanism of the EROD induction. Navas et al. (2004) have classified molecules according to the structural– functional relationships of ligands during biotransformation. According to Fatima and Ahmad (2006), pendimethalin could be included in a molecule group with low affinity AhR ligands like malathion and DDT due to its unexpected classic chemical structure for binding to the AhR. On the other hand, after a fifteen day recovery period, the liver EROD activity had decreased in all contaminated fishes, but a significant difference was only noted between fish exposed to Prowls and control fish. In fact, at D43, while the active substance was no longer quantified in fish muscles (Danion et al., 2012a), a high concentration of pendimethalin was detected in the bile of fish exposed to the commercial product (613 7163 ng g 1). This result questioned the real biotranformation process of this compound in liver before being excreted through the bile. Indeed, no pendimethalin metabolites
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have been found in fish yet and it would be interesting to monitor one phase II biotransformation parameter such as glutathione transferase activity. Furthermore, the decreased inductive response of EROD in the present study in fish exposed to commercial product could be explained by an impairment of cellular protein synthesis due to a severe exposure to contaminants (Livingstone et al., 1993; Klumpp et al., 2002). Antioxidant defense has been also a focus of toxicological research for the last decade as a possible mechanism of pesticide toxicity (Dautremepuits et al., 2009; Kavitha and Rao, 2008; Moraes et al., 2009). ROS, when they are produced in excess by pesticide, cause oxidative stress and damage macromolecules, such as DNA, proteins, and membranes. To fight these harmful impacts, an antioxidant defense system is set up in fish cells. The protective and adaptive roles of glutathione content (GSH) against oxidative stress-induced toxicity are well established in aquatic animals (Oliveira et al., 2008; Saera-Vila et al., 2009) and provide a first line of defense against ROS (Ahmad et al., 2000; Pastore et al., 2003; Li et al., 2007). Previous studies have shown that GSH content inhibits free radical formation through its ability to stabilize components in its oxidative state, preventing redox cycling and free radical generation (Pandey et al., 2008). In gills, no changes were observed in fish exposed to 500 ng L 1 of pendimethalin. On the other hand, the GSH level decreased in fish exposed to P800. Our results concurred with those obtained by Parvez and Raisuddin (2006) who have reported a decrease in GSH level leading to an adaptive response of fish either due to the induction of xenobiotics like oxyradical scavengers or due to overutilization to challenge the prevailing oxidative stress. In parallel, the three most important specialized antioxidant enzymes (SOD, CAT and GPx) were not disturbed by the pollution, whatever the experimental conditions. An over-utilization of GSH to protect organisms against ROS could also explain the non-activation of the enzymatic antioxidant defense in this study. On the other hand, the non-activation could express an impaired antioxidant defense mechanism due to excessive generation of free radicals generated by pendimethalin exposure and could highlight a potential oxidative stress. To attest this hypothesis, oxidative stress parameters like lipoperoxidation, protein oxidation or DNA damage could be measured in the future. After fifteen days in clean fresh water, fishes which were exposed to the commercial product seemed to be the most disturbed with a GSH content and GPx activity lower than in control fish. The correlation between the evolution of GSH content and GPx activity has already been demonstrated as this enzyme is GSH-dependant, utilizing GSH as an electron donor (Ahmad, 1995). Furthermore, the induction of CAT activity in fish exposed to 800 ng L 1 in response to the decrease in GPx has already been noted by Oliveira et al. (2008) who have shown that CAT activity was induced in fish exposed to xenobiotic as an alternative to GPx and as a second line of enzymatic defense against H2O2 generation. Concerning the antioxidant defense system in the liver, the GSH content was not disturbed following chronic exposure to pendimethalin, whatever the exposure concentration tested. This is in discrepancy with Ahmad et al. (2000) who found an increase in the liver glutathione level in fish exposed to paper mill effluent, suggesting a pollutant-induced adaptive response. Our result could indicate (i) that GSH content in liver is not sensitive to pendimethalin and probably not suitable as an indicator of chronic pesticide exposure, (ii) an inhibition of GSH synthesis or (iii) hyper activity of the redox cycle with GSSG and GR associated with overconsumption of GSH for many cellular reactions. In addition, the increased GPx activity in fish exposed to the highest concentration would support the last hypothesis. Furthermore, SOD activity increased while CAT activity decreased, indicating a possible disturbance of antioxidant defense following chronic
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exposure to herbicide. After the recovery period, the GSH content had increased in all fishes contaminated by pendimethalin and could suggest an adaptation synthesis de novo as previously demonstrated in fish (Thomas and Wofford, 1984; Gallagher et al., 1992) in response to an oxidative stress following the exposure period. In the same way, GPx activity in fish contaminated by 800 ng L 1 was still higher than in control fish, in correlation with the GSH content.
5. Conclusion After a 28 day exposure period to pendimethalin, liver EROD activity was not activated in fish. Antioxidant defense responses were set up by trout in the gills and liver following chronic exposure to herbicide. While the GSH content decreased in gills, it increased in liver. The mechanism of disturbance of glutathione status is not clear. It would be interesting to monitor the GSH/ GSSG ratio as a useful index of the precarious state of the cell (Oliveira et al., 2008). These effects on antioxidant defense responses could lead to oxidative stress in the vital organs of fish. After fifteen days in clean water, while SOD activity was restored, significant differences remained between treatments in other antioxidant defense parameters measured, attesting to the irreversibility of the effects. As we have already shown (Danion et al., 2012a), fishes which were exposed to the commercial product Prowls seemed to be the most disturbed.
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
Effects of in vivo chronic exposure to pendimethalin on EROD activity and antioxidant defenses in rainbow trout (Oncorhynchus mykiss) Morgane Danion a,b,n, Stéphane Le Floch c, François Lamour a,b, Claire Quentel a,b a
ANSES, Ploufragan-Plouzané Laboratory, Unit of Viral Pathology in Fish, Technopôle Brest-Iroise, 29280 Plouzané, France Université Européenne de Bretagne, France c CEDRE, Center of Documentation, Research and Experimentation on Accidental Water Pollution, 715 Rue Alain Colas, 29200 Brest, France b
art ic l e i nf o
a b s t r a c t
Article history: Received 20 February 2013 Received in revised form 13 September 2013 Accepted 14 September 2013 Available online 29 October 2013
Pendimethalin, an herbicide active substance frequently used in terrestrial systems, has detected in European aquatic ecosystems. Reliable indicators still need to be found in order to properly assess the impact of pesticides in fish. After an in vivo chronic exposure to pendimethalin, the detoxification process and the antioxidant defense system were assessed in 120 adult rainbow trout, Oncorhynchus mykiss. Four nominal exposure conditions were tested: control (C), 500 ng L 1 (P500), 800 ng L 1 (P800) and the commercial formulation Prowls at 500 ng L 1 (Pw500). Fish samples were made after a 28 day exposure period (D28) and after a fifteen day recovery period in clean fresh water (D43). At D28, ethoxyresorufinO-deethylase (EROD) activity was not activated in liver in spite of the pendimethalin uptake in fish. At D43, EROD activity in fish exposed to the commercial product was lower than in control fish, which may be explained by the high presence of herbicide in fish (613 7163 ng g bile 1). Furthermore, antioxidant defense responses were set up by trout in gills and liver following chronic exposure to 800 ng L 1 of pendimethalin concentration. While the glutathione content (GSH) decreased in gills, it increased in liver associated with higher activities of glutathione peroxidase (GPx) and superoxide dismutase (SOD). These disturbances could lead to reactive oxygen species production and oxidative stress in the vital organs in fish. After fifteen days in clean water, while the SOD activity was restored, the GSH content and GPx activity were still significantly disturbed in fish exposed to pendimethalin in comparison with control. These significant differences between treatments in antioxidant defenses parameters measured, attesting to the irreversibility of the effects. Crown Copyright & 2013 Published by Elsevier Inc. All rights reserved.
Keywords: Pendimethalin Oncorhynchus mykiss EROD GSH SOD GPx
1. Introduction Pendimethalin (N-(1-ethylpropyl)-2,6-dinitro-3,4-xylidine) is a dinitroaniline herbicide active substance, frequently used in terrestrial systems. Due to the common usage of various formulations composed of pendimethalin, this chemical compound has been detected at high concentrations in European aquatic ecosystem i.e. 352 ng L 1 in Denk (Asman et al., 2005), 370 ng L 1 in Spain (Barba-Brioso et al., 2010) and 840 ng L 1 in France (CORPEP, 2010). Taken up by fish due to its high bioconcentration factor (5100, Agritox database), pendimethalin is accumulated in specific organs and could disturb physiological parameters such as immune system components (Danion et al., 2012a,b). Despite these facts, reliable indicators still need to be found in order to measure and properly assess the impact of low and prolonged n Corresponding author at: Unit of Viral Pathology in Fish, Ploufragan-Plouzané Laboratory, Technopôle Brest-Iroise, 29280 Plouzané, France. Fax: þ33 298 055 165. E-mail address: [email protected] (M. Danion).
exposure to pesticides in fish. Danion et al. (2012a) have already demonstrated that chronic exposure to 200 ng L 1 of pendimethalin involves a high bioconcentration in flesh, attesting to the uptake of the pesticide. Indeed, following exposure to pollutants, organisms usually attempt to metabolize and depurate them, minimizing some of the cellular damage they cause and hence acquire fast elimination rates of compounds through bile and urine (Oliveira-Ribeiro et al., 2005). Phase I and phase II biotransformation parameters such as ethoxyresorufin-O-deethylase (EROD) and glutathione-S-transferase activities take part to a set of biochemical parameters which were applied to monitor biological effects in fish in the context of the Joint Assessment and Monitoring Program (JAMP) developed in the European framework (Sanchez and Porcher, 2009). The first step is usually catalyzed by cytochrome P450-dependent monooxygenases (phase I) and their products are subsequently coupled to endogenous metabolites (phase II) (Buhler and Williams, 1988; James and Whitlock, 1999). Moreover, depends on their chemical structure and their biotransformation, pesticides are known to cause the generation of reactive oxygen species (ROS), leading to oxidative stress.
0147-6513/$ - see front matter Crown Copyright & 2013 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ecoenv.2013.09.024
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M. Danion et al. / Ecotoxicology and Environmental Safety 99 (2014) 21–27
Pesticide-induced oxidative stress has been a focus of toxicological research for the last decade as a possible mechanism of toxicity (Banerjee et al., 2001; Abdollahi et al., 2004; Dorval et al., 2003; Moraes et al., 2009; Jin et al., 2011). In response to these potential harmful impacts, every living organism has a variety of antioxidant system defenses to protect themselves against the production of oxygen radicals uncoupling at various electron transfer sites or via autoxidation reactions. In this context, thiols play an important role against the pernicious effects of pro-oxidant challenges, and glutathione in particular provides a first line of defense against ROS (Pastore et al., 2003). This non-enzymatic antioxidant participates in many cellular reactions taking away ROS directly (Fang et al., 2002). Antioxidant enzymes are also commonly used to understand the associated toxic mechanisms of xenobiotics (Jensen et al., 1991; Sanchez et al., 2005). Superoxide dismutase (SOD) catalyzes the transformation of superoxide radicals to H2O2 and O2 and is the first enzyme to deal with oxyradicals. Then, H2O2 can be reduced to water and oxygen by glutathione peroxidase (GPx) and catalase (CAT) (Kappus, 1985; Oliveira et al., 2008). So, variations in antioxidant defenses can be very sensitive in revealing a pro-oxidant condition and have been used as oxidative stress markers in fish exposed to pesticides (Anguiano et al., 2001; Dorval and Hontela, 2003; Kavitha and Rao, 2008). Previous studies focusing on xenobiotic effects in fish have found that antioxidant enzyme activities may differ considerably between organs (Ahmad et al., 2003; Jee and Kang, 2005), highlighting the need to assess antioxidant responses in the main target organs when a comprehensive evaluation of the oxidative stress risk at individual level is intended. So, antioxidant defense parameters should be assessed in gills and liver, selected on the basis of functional criteria which made them preferential targets, i.e. xenobiotic uptake (gills) and xenobiotic metabolism (liver). The aim of the present study was to evaluate the effects of chronic exposure to 200 ng L 1 pendimethalin, active substance alone or the commercial product (Prowls) on the detoxification process and the antioxidant defense system were tested in vivo in rainbow trout (Oncorhynchus mykiss). To this end, the pendimethalin concentration in bile, the EROD activity and antioxidant defense parameters were monitored in gills and liver.
2. Materials and methods This experiment was conducted in accordance with the European Commission recommendation 2007/526/EC on revised guidelines for the accommodation and care of animals used for experimental and other scientific purposes. The center of documentation, research and experimentation on accidental water pollution (CEDRE) is authorized to conduct experimentation on animals in its facilities as a certified establishment according to the administrative order no. 2006-0429. Furthermore, the experimentation carried out as part of this study was conducted under the responsibility and supervision of Dr. Claire Quentel, who holds a certificate to direct scientific experimentation on animals (certificate no. 29-008). 2.1. Fish The 120 rainbow trout, 79.8 7 1.6 g, used for this experiment were sourced from virus-free fish rearing facilities at ANSES Laboratory (French Agency for Food, Environmental and Occupational Health and Safety, Plouzané, France). At CEDRE (Brest, France), the fishes were acclimated for one week in eight tanks (300 L) with a flow of 8 L h 1 at 157 1 1C. The fishes were fed with commercial dry pellets at 1.5 percent body weight (Neo Prima 4, Le Gouessant Aquaculture) once a day. 2.2. Tested substances The chemical and physical characteristics of pendimethalin have previously been described by Strandberg and Scott-Fordsmand (2004). The active substance and the commercial herbicide formulation were obtained from Sigma– Aldrich (98.8 percent purity; Germany) and BASF (400 g L 1; France), respectively. The stock solutions were prepared and stored as previously described by Danion et al. (2012a).
2.3. Experimental design 2.3.1. Experimental system The experimental system has already been described by Danion et al. (2012a). Briefly, the experimental system consisted of four similar independent units, each composed of three tanks. One stock tank containing a concentrated solution of the pollutant supplied two exposure tanks to expose fish in duplicate using a peristaltic pump with a two-channel pump head. All the exposure tanks were also provided with clean fresh water to dilute the concentrated solution in order to obtain the required pendimethalin concentration. The four units were placed in a thermoregulated greenhouse, in which the air was totally renewed every 6 h and with a natural light/dark cycle (12/12 h approximately). 2.3.2. Exposure conditions and recovery period Four nominal exposure conditions were tested, one per unit: (i) fresh water as control (C), (ii) 500 ng L 1 of active substance (P500), (iii) 800 ng L 1 of active substance (P800) and (iv) 500 ngCL 1 of pendimethalin with the commercial herbicide formulation of Prowls (Pw500). P500 represents the nominal concentration just below the predicted no-effect concentration (PNEC) estimated at 550 ng L 1 by Council Directive 2003/31/EC, while P800 represents the maximal concentration measured in rivers in Brittany in 2007. For the exposure period, the concentrated solution was flowed at 1 L h 1 from the stock tanks to the exposure tanks. In addition, a clean fresh water flow at a rate of 5 L h 1 was supplied to the exposure tanks to dilute the exposure concentration. Then, 120 trout were randomly distributed among the four units (or fifteen fishes/exposure tank). During the 28 days of the exposure period, the trout were exposed to a diluted pendimethalin concentration. At the end of the exposure period, the stock tanks were disconnected and clean fresh water was flowed into the exposure tanks at a rate of 8 L h 1 during the two week recovery period. Throughout the experiment, the trout were maintained in the same tanks and were fed once a day. 2.3.3. Samples and sampling date The oxygen level and temperature were monitored daily in each tank (Oxymeter WTW-OXI315I), while nitrate and nitrite levels were measured weekly (Colorimetric test JBLs). Fresh water parameters were stable during the acclimation period and throughout the experiment: dissolved oxygen 93 7 3 percent, pH 87 0.1, temperature 157 1 1C, free of nitrate and nitrite. Ten fishes were sampled from each unit (or five fishes/exposure tank) on the last day of exposure and recovery periods. The fishes were killed with an overdose of anesthetic phenoxy-2ethanol and weighed. From each fish, the bile, liver and two branchial arcs were carefully removed and immediately frozen at 80 1C to measure the EROD activity and antioxidant defense parameters. 2.4. Analytical methods 2.4.1. Pendimethalin concentration in bile The extraction technique used was stir bar sorptive extraction (SBSE) and the quantification was performed using gas chromatography equipment coupled with a mass spectrometer (GC–MS). From each fish, 300 mL of bile was diluted in 100 mL of distilled water with 100 mL of a solution of quintozene Pestanals (internal standard; Sigma–Aldrich). The SBSE and GC–MS analysis conditions and the quantification of pendimethalin were as described by Danion et al. (2012a). 2.4.2. Liver EROD activity The livers were homogenized in an ice-cold HEPES buffer using the tissue homogenizer Precellys 24 (Bertin Technologies, France). EROD activity in the liver S9 fraction was measured as described by Couillard et al. (2004) by a microplate spectrofluorometric assay using a Spectrofluorometers plate reader (excitation 530 nm, emission 585 nm). Reaction mixture contained S9 in HEPES buffer (0.1 M, pH 7.8), 7-ethoxyresorufin (0.024 mg mL 1 in DMSO) and NADPH (20 mg mL 1). The activity was quantified by measuring the fluorescence of resorufin at 60 s intervals over a 13 min total scan time. Liver EROD activity was calculated as nmol min 1 mg 1 protein with resorufin used as standard.All samples were assayed in duplicate. 2.4.3. Antioxidant defenses The gills and livers were homogenized in an ice-cold phosphate buffer (0.1 M, pH 7.8) with 20 percent glycerol and 0.2 mM phenylmethylsulfonyl fluoride as a serine protease inhibitor with the tissue homogenizer Precellys 24 (Bertin Technologies, France). The homogenates were centrifuged at 9000 g at 4 1C, for 15 min and the supernatant was splitted into five aliquots and stored at 80 1C for biochemical assays. The total protein concentrations in gill and liver samples were spectrophotometrically estimated using the method of Bradford (1976) with bovine serum albumin (Sigma–Aldrich Chemicals, France) used as a standard. Biomarker assays including GSH content and activities of SOD, GPx and CAT in the gills and liver of rainbow trout were adapted for use in microplates after preliminary tests using several dilutions. All samples were assayed in duplicate.
M. Danion et al. / Ecotoxicology and Environmental Safety 99 (2014) 21–27 GSH concentration was measured according to Vandeputte et al. (1994). Briefly, 10 mL of TCA deproteinized sample was mixed with phosphate buffer containing 0.3 mM NADPH and 1 mM Ellman reagent. The enzymatic reaction was monitored spectrophotometrically at 405 nm. The results were expressed in mmol mg 1 protein. SOD activity was measured according to Marklund and Marklund (1974). Briefly, samples were assayed in a solution of 8.7 mL of 50 mM phosphate buffer pH 8 and 0.3 mL of 3 mM pyrogallol. The rate of pyrogallol autoxidation was measured with a UV-220 spectrophotometer at 325 nm. SOD activity was calculated in terms of U mg 1 protein, whereby one unit of SOD activity was defined as the amount of the enzyme which gave 50 percent inhibition of the oxidation rate of 0.1 nM pyogallol in the 1 mL of solution. GPx activity was determined using 15 mL of a 4.5 g protein/L diluted sample according to the Paglia and Valentine method (1967). Cumene hydroperoxide was used as the substrate and enzymatic activity was assessed at 340 nm. GPx activity was calculated in terms of nmol min 1 mg 1 protein. CAT activity was assayed using the method of Babo and Vasseur (1992). Briefly, the assay mixture consisted of phosphate buffer (100 mM, pH 7) and H2O2 (28 mM). The variation of H2O2 absorbance in 60 s was measured with a UV-220 spectrophotometer at 225 nm. CAT activity was calculated in terms of mmol min 1 mg 1 protein with bovine erythrocyte catalase used as standard. 2.5. Statistical analysis All statistics were performed using XLstat Statistical Software 2007.0 with a significance level of 5 percent. There were significant variations between control groups during the exposure and recovery periods. So, verification of normality and of homogeneity of covariance matrices was conducted using the Anderson–Darling test and the Bartlett test, respectively, on exposure and recovery data separately, before using a one way analysis of variance (ANOVA). Newman–Keuls significant difference tests were used a posteriori to detect differences between means. All data were expressed as the mean7 standard deviation (SD).
3. Results 3.1. Pendimethalin concentration in bile No pendimethalin concentration above the quantification limit of the method used (1 ng g 1) was detected throughout the experimental period in all control fishes (Table 1). After the exposure period (D28), fish exposed to P500, P800 and Pw500 showed pendimethalin concentrations of 264 7256, 456 7362 and 263 7177 ng g bile 1, respectively, without any significant differences between them. After fifteen days in clean fresh water (D43), fish exposed to P800 and Pw500 showed significantly more pendimethalin in bile, with 4517 148 and 613 7163 ng g bile 1, than fish contaminated by P500 (176 7163 ng g bile 1) (Table 1).
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commercial formulation (4.17 70.34 nmol min 1 mg 1 of protein) (Fig. 1). 3.3. Antioxidant defenses In gills, throughout the experiment, the antioxidant defense parameters were not disturbed by the lowest exposure concentration (P500). After the 28 days exposure period, a significant decrease in the GSH content was only noted in fish exposed to the highest pendimethalin concentration (P800; 28.673.4 mmol mg 1 of protein) compared to control fish (34.272.7 mmol mg 1 of protein) (Fig. 2A). The other antioxidant defense parameters (SOD, CAT and GPx) were not disturbed after chronic exposure to pendimethalin (Fig. 2B–D). After the recovery period (D43), the GSH content significantly decreased only in fish exposed to the commercial formulation (Pw500) compared to control fish. The GPx activity was lower in all fish contaminated by pendimethalin than in control fish, but this difference was significant only for the fish exposed to Pw500, with 48.675.4 and 34.672.9 nmol min 1 mg 1 of protein, respectively (Fig. 2A and C). Moreover, CAT activity in fishes which were exposed to P800 was significantly higher than in control fish, with 11.471.1 and 9.071.3 mmol min1 mg 1 of protein (Fig. 2D). In liver, as in gills, the antioxidant defense parameters were not disturbed by the lowest exposure concentration (P500) throughout the experiment. Furthermore, after the 28 days exposure period, the SOD and GPx activities had significantly increased in fish exposed to the highest exposure concentration (P800) (Fig. 2B and C). Moreover, CAT activity in all fishes contaminated by pendimethalin had decreased compared to control fish (2.870.3 mmol min 1 mg 1 of protein), but the difference was significant only for fish exposed to Prowls (4.470.4 mmol min 1 mg 1 of protein) (Fig. 2D). At D43, while SOD activity was no longer disturbed, the GSH content and GPx activity were significantly higher in fishes which were exposed to P800 than in the control fish (Fig. 2A and C).
4. Discussion The aim of this study was to evaluate the impacts of chronic exposure to pendimethalin active substance alone or the
3.2. Liver EROD activity After the 28 days exposure period, no significant difference was observed in liver EROD activity in fish whatever the experimental exposure conditions. After the recovery period, at D43, a decrease was noted in all contaminated fish by pendimethalin but this decrease was only significant compared to the control (5.86 70.65 nmol min 1 mg 1 of protein) for fish exposed to the Table 1 Concentration of pendimethalin in bile of rainbow trout after the exposure (D28) and recovery (D43) periods in control and contaminated fish by 500 ng L 1 (P500), 800 ng L 1 (P800) and Prowls (Pw500). The results are expressed in ng g 1 of bile. N ¼10, blq¼ below limit of quantification ( o 1 ng bile 1). Mean values 7standard deviation (SD) having similar letters are not significantly different (ANOVA, Newman–Keuls, p 40.05). Pendimethalin concentration in fish bile (ng g 1 7SD) Sampling date
D28 D43
Exposure conditions Control
P500
P800
Pw500
Blq Blq
264 7256a 176 7163a
456 7 362a 4517 148b
263 7 177a 6137 163b
Fig. 1. Liver ethoxyresorufin-O-deethylase (EROD) activity after the exposure (D28) and recovery (D43) periods in control and contaminated rainbow trout by 500 ng L 1 (P500), 800 ng L 1 (P800) and Prowls (Pw500). N ¼ 10. Mean values ( 7SD) having similar letters are not significantly different (ANOVA; Newman– Keuls, p4 0.05).
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Fig. 2. (A) Glutathione content (GSH); (B) superoxide dismutase (SOD) activity; (C) glutathione peroxidase (GPx) activity; (D) catalase activity; in gills and liver after the exposure (D28) and recovery (D43) periods in control and contaminated fish by 500 ng L 1 (P500), 800 ng L 1 (P800) and Prowls (Pw500). N ¼ 10. Mean values ( 7SD) having similar letters are not significantly different (ANOVA; Newman–Keuls, p 40.05).
M. Danion et al. / Ecotoxicology and Environmental Safety 99 (2014) 21–27
commercial formulation Prowls, in rainbow trout. To this end, the active substance concentration in bile, the EROD detoxification parameter and antioxidant defenses were monitored. Numerous studies have evaluated these biomarkers in fish exposed to pollution such as pesticides (Elia et al., 2002; Dorval and Hontela, 2003; Sayeed et al., 2003; Kavitha and Rao, 2008), polycyclic aromatic hydrocarbons (PAHs) (Wessel et al., 2010), polychlorobiphenyls (Huuskonen et al., 1996; Saera-Vila et al., 2009) and metals (Pandey et al., 2008; Kerambrun et al., 2011). Concerning pesticide pollution, few studies have evaluated in vivo chronic exposure and to our knowledge, no data is available on the effect of pendimethalin on these parameters in fish. The experimental system and protocol used in this study were elaborated to realistically reconstitute the state of river water. According to a number of predefined criteria, pendimethalin appeared to be the most relevant pesticide to perform this study. In fact, the selected substance was required to (i) be listed in Annex I of Council Directive 91/414/EEC, (ii) be detected in the aquatic environment at high concentrations in rivers in Brittany, (iii) have a bioconcentration factor (BCF) 43000 and (iv) have an ecotoxicity that was already known in aquatic fauna. In a previous study, some data concerning the experimental design and the characteristics of the exposure have been discussed (Danion et al., 2012a). In this study, the presence of pendimethalin in the bile (o 456 ng g 1) attested to the uptake of herbicide by fish as well as Danion et al. (2012a) has been already demonstrated it by the bioconcentration in flesh. However, only free chemical compounds were measured because we did not know the structure of the conjugated metabolites released into the bile and the potential elimination process was not sought yet in fish. Organic xenobiotics can be detoxified in aquatic organisms’ liver which is the main tissue of metabolism (Livingstone, 1991). 7-Ethoxyresorufin O-deethylase (EROD) is one biomarker that has been used extensively as it is induced by exposure to a wide variety of environmental contaminants (Lemaire and Livingstone, 1995; Van der Oost et al., 2003; Ferreira et al., 2006). Indeed, most of the studies conducted in fish have reported significant induction of EROD activity following exposure to CYP1A inducers such as polycyclic aromatic hydrocarbons (PAH), polychlorated biphenyl (PCB) or polychlorodibenzo-p-dioxine (PCDD) (Peters et al., 1994; Arnic et al., 2000). In this study, although exposure to pendimethalin was attested to the uptake in fish, liver EROD activity was not affected in trout following the 28 days exposure period, whatever the exposure conditions. This result agreed with those of Jensen et al. (1991) and Savelli et al. (1997) who have previously demonstrated that fish exposure to pesticide, contrary to PAHs and PCB, could lead to only mild induction in EROD, questioning (i) the role of the aryl hydrocarbon receptor (AhR) in the activation of CYP1A1/EROD by several environmental pollutants and (ii) the role of the structural–functional relationships of ligands in the mechanism of the EROD induction. Navas et al. (2004) have classified molecules according to the structural– functional relationships of ligands during biotransformation. According to Fatima and Ahmad (2006), pendimethalin could be included in a molecule group with low affinity AhR ligands like malathion and DDT due to its unexpected classic chemical structure for binding to the AhR. On the other hand, after a fifteen day recovery period, the liver EROD activity had decreased in all contaminated fishes, but a significant difference was only noted between fish exposed to Prowls and control fish. In fact, at D43, while the active substance was no longer quantified in fish muscles (Danion et al., 2012a), a high concentration of pendimethalin was detected in the bile of fish exposed to the commercial product (613 7163 ng g 1). This result questioned the real biotranformation process of this compound in liver before being excreted through the bile. Indeed, no pendimethalin metabolites
25
have been found in fish yet and it would be interesting to monitor one phase II biotransformation parameter such as glutathione transferase activity. Furthermore, the decreased inductive response of EROD in the present study in fish exposed to commercial product could be explained by an impairment of cellular protein synthesis due to a severe exposure to contaminants (Livingstone et al., 1993; Klumpp et al., 2002). Antioxidant defense has been also a focus of toxicological research for the last decade as a possible mechanism of pesticide toxicity (Dautremepuits et al., 2009; Kavitha and Rao, 2008; Moraes et al., 2009). ROS, when they are produced in excess by pesticide, cause oxidative stress and damage macromolecules, such as DNA, proteins, and membranes. To fight these harmful impacts, an antioxidant defense system is set up in fish cells. The protective and adaptive roles of glutathione content (GSH) against oxidative stress-induced toxicity are well established in aquatic animals (Oliveira et al., 2008; Saera-Vila et al., 2009) and provide a first line of defense against ROS (Ahmad et al., 2000; Pastore et al., 2003; Li et al., 2007). Previous studies have shown that GSH content inhibits free radical formation through its ability to stabilize components in its oxidative state, preventing redox cycling and free radical generation (Pandey et al., 2008). In gills, no changes were observed in fish exposed to 500 ng L 1 of pendimethalin. On the other hand, the GSH level decreased in fish exposed to P800. Our results concurred with those obtained by Parvez and Raisuddin (2006) who have reported a decrease in GSH level leading to an adaptive response of fish either due to the induction of xenobiotics like oxyradical scavengers or due to overutilization to challenge the prevailing oxidative stress. In parallel, the three most important specialized antioxidant enzymes (SOD, CAT and GPx) were not disturbed by the pollution, whatever the experimental conditions. An over-utilization of GSH to protect organisms against ROS could also explain the non-activation of the enzymatic antioxidant defense in this study. On the other hand, the non-activation could express an impaired antioxidant defense mechanism due to excessive generation of free radicals generated by pendimethalin exposure and could highlight a potential oxidative stress. To attest this hypothesis, oxidative stress parameters like lipoperoxidation, protein oxidation or DNA damage could be measured in the future. After fifteen days in clean fresh water, fishes which were exposed to the commercial product seemed to be the most disturbed with a GSH content and GPx activity lower than in control fish. The correlation between the evolution of GSH content and GPx activity has already been demonstrated as this enzyme is GSH-dependant, utilizing GSH as an electron donor (Ahmad, 1995). Furthermore, the induction of CAT activity in fish exposed to 800 ng L 1 in response to the decrease in GPx has already been noted by Oliveira et al. (2008) who have shown that CAT activity was induced in fish exposed to xenobiotic as an alternative to GPx and as a second line of enzymatic defense against H2O2 generation. Concerning the antioxidant defense system in the liver, the GSH content was not disturbed following chronic exposure to pendimethalin, whatever the exposure concentration tested. This is in discrepancy with Ahmad et al. (2000) who found an increase in the liver glutathione level in fish exposed to paper mill effluent, suggesting a pollutant-induced adaptive response. Our result could indicate (i) that GSH content in liver is not sensitive to pendimethalin and probably not suitable as an indicator of chronic pesticide exposure, (ii) an inhibition of GSH synthesis or (iii) hyper activity of the redox cycle with GSSG and GR associated with overconsumption of GSH for many cellular reactions. In addition, the increased GPx activity in fish exposed to the highest concentration would support the last hypothesis. Furthermore, SOD activity increased while CAT activity decreased, indicating a possible disturbance of antioxidant defense following chronic
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exposure to herbicide. After the recovery period, the GSH content had increased in all fishes contaminated by pendimethalin and could suggest an adaptation synthesis de novo as previously demonstrated in fish (Thomas and Wofford, 1984; Gallagher et al., 1992) in response to an oxidative stress following the exposure period. In the same way, GPx activity in fish contaminated by 800 ng L 1 was still higher than in control fish, in correlation with the GSH content.
5. Conclusion After a 28 day exposure period to pendimethalin, liver EROD activity was not activated in fish. Antioxidant defense responses were set up by trout in the gills and liver following chronic exposure to herbicide. While the GSH content decreased in gills, it increased in liver. The mechanism of disturbance of glutathione status is not clear. It would be interesting to monitor the GSH/ GSSG ratio as a useful index of the precarious state of the cell (Oliveira et al., 2008). These effects on antioxidant defense responses could lead to oxidative stress in the vital organs of fish. After fifteen days in clean water, while SOD activity was restored, significant differences remained between treatments in other antioxidant defense parameters measured, attesting to the irreversibility of the effects. As we have already shown (Danion et al., 2012a), fishes which were exposed to the commercial product Prowls seemed to be the most disturbed.
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