Chemosphere 65 (2006) 952–962 www.elsevier.com/locate/chemosphere
Anguilla anguilla L. oxidative stress biomarkers: An in situ study of freshwater wetland ecosystem (Pateira de Fermentelos, Portugal) Iqbal Ahmad, Ma´rio Pacheco, Maria Ana Santos
*
Animal Physiology/Ecotoxicology Sector, Department of Biology, University of Aveiro, 3810-193 Aveiro, Portugal Received 24 November 2005; received in revised form 17 February 2006; accepted 18 March 2006 Available online 24 May 2006
Abstract Pateira de Fermentelos (PF) is a natural freshwater wetland in the central region of Portugal. In the last decade, the introduction of agricultural chemicals, heavy metals, domestic wastes, as well as eutrophication and incorrect utility of resources resulted in an increased water pollution. The present research work was carried out to check the various oxidative stress biomarker responses in European eel (Anguilla anguilla L.) gill, kidney and liver due to this complex water pollution. Eels were caged and plunged at five different PF sites (A– E) for 48 h. A reference site (R) was also selected at the river spring where no industrial contamination should be detected. Lipid peroxidation (LPO), catalase (CAT), glutathione peroxidase (GPX), glutathione S-transferase (GST) and reduced glutathione (GSH) were the oxidative stress biomarkers studied. In gill, site A exposure induced a significant GST activity increase and site B exposure induced CAT activity increase when compared to R. Site C exposure showed a significant CAT and GPX activity increase. Data concerning site D exposure were not determined due to cage disappearance. Site E exposure displayed a significant CAT and GST activity increase. In kidney, site A exposure induced a significant CAT and GPX decrease as well as a GST increase. Site B exposure showed a significant decrease in GPX activity and GSH content. However, site C exposure demonstrated a significant increase in CAT and a decrease in GPX. Site E exposure showed a significant decrease in GPX and increase in GST. In liver, site A exposure showed a significant GST activity decrease as well as GSH content increase. Site B exposure showed a significant CAT, GST and LPO decrease. Site C exposure showed only GST activity decrease, while site E exposure induced a significant increase in GPX. These investigation findings provide a rational use of oxidative stress biomarkers in freshwater ecosystem pollution biomonitoring using caged fish, and the first attempt reported in Portugal as a study of this particular watercourse under the previous conditions. The presence of pollutants in the PF water was denunciated even without a clear relation to the main pollution source distance. The organ specificity was evident for each parameter but without a clear pattern. 2006 Elsevier Ltd. All rights reserved. Keywords: Freshwater ecosystem; Pateira de Fermentelos; Anguilla anguilla L.; Oxidative stress; Antioxidants; Organ specificity
1. Introduction Lakes and reservoirs are more prone to receive and accumulate contaminants discharging from sewages, domestic wastes and agriculture runoff due to their specific water dynamic and configuration differing from other aquatic ecosystems. Pateira de Fermentelos (PF), a natural freshwater wetland, is an important fishing and recreation *
Corresponding author. Tel.: +351 234370965; fax: +351 234426408. E-mail addresses:
[email protected] (I. Ahmad), mpacheco@bio. ua.pt (M. Pacheco),
[email protected] (M.A. Santos). 0045-6535/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2006.03.042
place in the central region of Portugal, receiving water mainly from the Ce´rtima River and discharging into the ´ gueda River. In the last decade, eutrophication caused A by sewage and agricultural fields’ nutrient runoff, pesticides leachates, electroplating industry discharges as well as incorrect utility of resources with the introduction of new organisms have aggravated the pollution problems, leading to an increased organisms’ health threat where fish are included (Calado et al., 1991; Calado and Craveiro, 1995). PF heavy metal analysis demonstrated the presence of nickel, zinc, aluminium and manganese, as a clear demonstration of water quality deterioration (Almeida, 1998).
I. Ahmad et al. / Chemosphere 65 (2006) 952–962
In these environments, organisms are exposed to mixtures of pollutants, whose synergistic/antagonistic effects are hardly interpreted and predicted exclusively from the chemical analyses; some contaminants strongly accumulate in tissues without inducing toxic effects, while others are characterised by elevated toxicity at low levels of exposure. Moreover, the impracticality to analyze all the individual chemicals pooled in a mixture of contaminants also increases the problem of aquatic pollutant characterization. Thus, during the past two decades, the use of biological responses (biomarkers) on particular test species has become relevant in toxicological assessments since it allows the early detection of overall effects of contaminants, providing information, even at the sub-lethal level, which reflects eventual chemical interactions (Passino, 1984; Goksøyr and Fo¨rlin, 1992; Livingstone, 1993; Peakall and Shugart, 1993). In this direction, the study of oxidative stress seems to be particularly promising (Lackner, 1998; Livingstone, 2001). Many environmental pollutants are capable of inducing oxidative stress in aquatic animals including fish. An important role in toxicity of several pollutants is assumed by the enhancement of intracellular reactive oxygen species (ROS) and perturbation of antioxidant efficiency which often prelude the onset of alterations like DNA damage, lipid peroxidation (LPO) and enzyme inhibition (Winston and Di Giulio, 1991). The overall enhancement or reduction of pollutant-induced toxicity greatly depends on the imbalance between pro-oxidant and antioxidant status as a result of xenobiotic interferences. Hence, enhanced pollutant oxygenation rates may increase toxicity via oxidative stress, rendering fish antioxidants less effective. In this perspective, a number of studies confirmed the successful employment of antioxidant enzymes and non-enzymatic antioxidant modulation in identifying environmental stress (Regoli et al., 1998; Ahmad et al., 2000, 2004, 2005; Livingstone, 2001; Santos et al., 2004). In addition, LPO estimation in particular has also been found to have a high predictive importance as revealed from a credible number of research papers describing its suitability as a biomarker of effect (Lackner, 1998; Ahmad et al., 2000, 2004, 2005; Santos et al., 2004). Previous field studies using European eel (Anguilla anguilla L.) as a model, on harbour water and paper mill effluent contaminated river exposure under natural environmental condition as well as laboratory studies on sub-lethal naphthalene, b-naphthoflavone/copper or chromium exposures showed that antioxidants are useful biomarkers of pollution. Moreover, a correlation between exposure and biomarker response, especially the induction of antioxidants was also observed (Santos et al., 2004, 2006; Ahmad et al., 2005). As per the best of our knowledge, none of the previously published papers includes PF water-body assessment in this context. Keeping in view the applicability and suitability of A. anguilla L. oxidative stress responses for different classes of compounds, the same approach was applied in the present study as a rational use for PF monitoring which is
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likely to present a complex mixture of pollutants. The duration of exposure (48 h) is based on our previous field investigation (Ahmad et al., 2004; Santos et al., 2004, 2006) showing measurable oxidative stress responses at 48 h exposure. Thus, A. anguilla were caged and plunged in situ at five study sites located at increasing distances from the entrance point of the Ce´rtima River, as a main source of contamination, following the same exposure strategy as adapted by Fenet et al. (1996, 1998), Doyotte et al. (2001), Livingstone et al. (2000) and Pacheco and Santos (1998) for different aquatic ecosystems biomonitoring using A. anguilla and other fish species. The study was focussed on the role of antioxidant enzymes such as catalase (CAT), glutathione peroxidase (GPX), glutathione S-transferase (GST) and non-enzymatic antioxidants such as reduced glutathione (GSH) against peroxidative damage (measured as LPO increase). It is intended to evaluate the relationships between different PF site exposure and the A. anguilla oxidative stress response modulation in the gill, kidney and the liver with a key question of various antioxidant status against peroxidative damage. Moreover, the assessment A. anguilla organ specificity was also intended. In addition, the suitability and sensitivity of A. anguilla oxidative stress biomarkers in early detection for the freshwater ecosystem health was evaluated. 2. Materials and methods 2.1. Chemicals 1-Chloro-2,4-dinitrobenzene (CDNB), 2,5-dithiobis-tetranitrobenzoic acid (DTNB), 2-thiobarbituric acid (TBA), trichloroacetic acid (TCA), NADPH were purchased from Sigma (Spain) and other routine chemicals and reagents (analytical grade) were purchased from local sources. 2.2. Test animals European eels (A. anguilla L.) with an average weight 49.50 ± 0.6 g were captured from a non-polluted Aveiro lagoon area – Murtosa, Portugal. Eels were acclimated for 7 days in 160-l aquaria under standard laboratory conditions as described by Santos and Pacheco (1996). Briefly, fish were kept under a natural photoperiod at room temperature in aerated, filtered, dechlorinated and recirculating tap water with the following physico-chemical conditions: salinity 0&, pH 7.4 ± 0.2, dissolved oxygen 8.67 ± 0.5 mg/l. Fish were fed neither during laboratory adaptation nor during field exposure. 2.3. Experimental design In order to evaluate the A. anguilla oxidative stress responses to PF water contamination, five groups of eel were caged and plunged into the five selected sites of PF water-body (sites A–E), differing in their distances to the main known pollution source (Ce´rtima River), for 48 h
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cal characteristics such as temperature, pH, conductivity, dissolved oxygen (DO), biological oxygen demand (BOD), total solid (TS), total dissolved solid (TDS), total suspended solids (TSS) and depth using procedures described in APHA (1998). Other parameters such as ammonium, nitrate, nitrite, phosphate, carbonate hardness (acidbinding capacity). Total hardness tests were performed using Compact laboratory for water testing (Merck, Germany).
(Fig. 1). The study sites (A–E) are affected primarily by the pollutants input from the Ce´rtima River, since point-specific sources around the study sites are not known, excluding agricultural leachates and domestic discharges. One more site as a reference (site R) was selected at the Ce´rtima River spring, without any industrial and domestic sewage contamination. The geographical location of study sites has been described in Table 1. The experiment was carried out in December 2004. Fish were placed into an 80-l net cage, transported and plunged into PF pre-selected sites according to Santos et al.’s (2006) previous technique, adopted for this fish species. Fish cages were maintained 15 cm from the bottom to avoid a direct contact with the sediment. After 48 h exposure, A. anguilla liver, kidney and gill were sampled from each fish and individually assayed for LPO, CAT, GPX, GST and GSH.
2.5. Estimation of lipid peroxidation (LPO) in fish tissues LPO was estimated in liver, kidney and gill of all exposed fish. The gills were excised out of the sacrificed fish, gill rackers removed and gill lamellae were used for homogenization. All the tissues were homogenized in chilled phosphate buffer (0.1 M, pH 7.4) containing KCl (1.17%), using a Potter–Elvehjem homogenizer. The LPO was determined in the homogenate as per the method of Utley et al. (1967), modified and adapted by Fatima et al. (2000). The absorbance of each aliquot was measured at 535 nm. The rate of lipid peroxidation was expressed as
2.4. Water physico-chemical parameters Water samples collected from the bottom of each PF study site were analyzed (Table 2) for some physico-chemi-
Fig. 1. Representation of A. anguilla L. caging sites (A–E) on Pateira de Fermentelos water-body in the central region of Portugal.
Table 1 Geographical positions of different Pateira de Fermentelos study sites Geographical positions
Exposure sites Site R
Longitude Lattitude
0
Site A 00
4020 50 N 00824 0 0800 W
0
Site B 00
4033 34 N 00830 0 3700 W
0
Site C 00
4034 24 N 00830 0 4800 W
0
Site D 00
4034 34 N 00831 0 0600 W
0
Site E 00
4034 44.60 N 00831 0 38.0600 W
4035 0 19.5500 N 00831 0 44.1900 W
I. Ahmad et al. / Chemosphere 65 (2006) 952–962 Table 2 Physico-chemical analysis of water at different Pateira de Fermentelos study sites Physico-chemical parameters
Exposure sites Site R
Site A
Site B
Site C
Site E
Temperature (C) pH Conductivity (ls/cm) DO (mg/1) BOD (mg/1) Ammonium (mg/1) Nitrate (mg/1) Nitrite (mg/1) Phosphate (mg/1) Carbonate hardness (acid-binding capacity) (mmol/l) Total hardness (mmol/l) Total solids (mg/l) Total dissolved solids (mg/l) Total suspended solids (mg/l) Depth (m)
10.9 7.937 289 10.34 2.33 0.5 0 0.075 0 0.9
9.3 7.999 492 8.86 1.38 0.5 0 0.5 0.125 2.3
9.1 9.640 151 8.45 1.03 0.3 0 0.05) using the Shapiro–Wilk and Levene test respectively. Analysis of variance was followed by the LSD test to compare results between fish groups (Zar, 1996) and the significance of the results was ascertained at p < 0.05. 3. Results 3.1. Physico-chemical parameter of water (Table 2) The water temperature showed no important variations neither to reference site nor PF study sites. All the sites
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*
* 10
5
Site R
(GST)
(GPX)
150
15
300
3.2.1. Gill responses (Fig. 2) In general, no clear relation could be established between gill oxidative stress responses and the distance to the main source of pollution (river Ce´rtima). However, an overall induction of antioxidant enzymes is perceptible. Specifying, site A exposure induced a significant GST
(CAT) *
0
Biochemical findings have been described in an organspecific manner, keeping in view only significant changes at PF exposure sites in comparison to reference site (site R). Site D exposed fish could not be processed for various biochemical analyses because of the cage disappearance after 48 h exposure.
nmol NADPH oxidized/min/mg protein
20
3.2. Oxidative stress profile
Site A Site B Site C
*
120 90
60 30 0
Site E
Site R Site A Site B Site C Site E
(GSH)
*
0.015
* nmol/g tissue
nmol CDNB formed/min/mg protein
nmol H2O 2 consumed/min/ mg protein
were alkaline in nature with an incremental pH trend from site A (minimum) to site C (maximum), followed by a decrease at site E. Conductivity measurement showed no major differences among all the study sites. Concerning the DO level, the observed variation ranged from 6.62 (site E) to 10.34 mg/l (site R). BOD also showed a slight variation along all the sites, being highest at site E. Among the ammonium, nitrates, nitrites, phosphate, carbonates hardness and total hardness parameters, only nitrite concentration showed its considerable presence at sites B and E. In terms of TS and TSS, major differences were measured since a higher level was detected at the reference site. The recorded depth was minimum at the reference site, whereas PF study sites showed an increase from sites A to C, followed by a decrease at site E.
200
100
0.01
0.005
0
0
nmol TBARS formed/hr/mg protein
Site R Site A Site B Site C
2
Site R Site A Site B Site C Site E
Site E
(LPO)
1.5 1 0.5 0 Site R
Site A
Site B
Site C
Site E
Fig. 2. Represents CAT, as expressed in nanomol H2O2 consumed/min/mg protein; GPX, as expressed in nanomol NADPH oxidized/min/mg protein; GST, as expressed in nanomol CDNB formed/min/mg protein; GSH, as expressed in nanomole/g tissue and LPO, as expressed in nanomole TBARS released during 1 h incubation in gill PMS of A. anguilla L. exposed to Pateira de Fermentelos water-body for 48 h. The significance level observed is *p < 0.05 when compared with the values of reference (site R).
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25
(CAT)
*
20 15 *
10 5 0
Site R Site A Site B Site C Site E
100
(GPX)
100
*
75
*
*
50
* *
25 0 Site R Site A Site B Site C Site E
(GSH)
(GST)
*
*
75 50 25 0
3.2.3. Liver responses (Fig. 4) Site A exposed fish showed a significant GST activity decrease and GSH content increase. Site B exposed fish showed a significant decrease in CAT and GST activities as well as in LPO. Site C exposure showed only GST activity decrease, while site E exposure induced a significant increase in GPX activity. Therefore, a clear relation between liver oxidative stress responses and the distance to the main pollution input could not be established.
0.018
nmol/g tissue
125
2.5
*
0.0135 0.009 0.0045 0
Site R Site A Site B Site C Site E
nmol TBARS formed/hr/mg protein
nmol CDNB formed/min/mg protein
nmol H2O2 consumed/min/mg protein
3.2.2. Kidney responses (Fig. 3) Considering the relation to the distance of the pollution source, only GPX activity showed a consistent variation (decrease) from sites A to E. Taking into account the individual site observations, site A exposed fish exhibited a significant CAT and GPX decrease as well as a GST increase when compared to R. At site B, a significant decrease in GPX activity and GSH content were detected. However,
site C exposure demonstrated a significant increase in CAT and decrease in GPX. Site E exposure showed a significant decrease in GPX activity and increase in GST activity.
nmol NADPH oxidized/min/mg protein
activity increase and site B exposure induced CAT activity increase when compared to site R. Site C exposure showed a significant CAT and GPX activity increase. Site E exposed fish displayed a significant CAT and GST activity increase.
957
Site R Site A Site B Site C Site E
(LPO)
2 1.5
1 0.5
0 Site R Site A Site B Site C Site E
Fig. 3. Represents CAT, as expressed in nanomol H2O2 consumed/min/mg protein; GPX, as expressed in nanomol NADPH oxidized/min/mg protein; GST, as expressed in nanomol CDNB formed/min/mg protein; GSH, as expressed in nanomole/g tissue and LPO, as expressed in nanomole TBARS released during 1 h incubation in kidney PMS of Anguilla anguilla L. exposed to Pateira de Fermentelos water-body for 48 h. The significance level observed is *p < 0.05 when compared with the values of reference (site R).
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(CAT)
75 50
*
25 0
300
(GPX)
40
nmol NADPH Oxidized/min/mg protein
100
Site R Site A
Site B Site C
*
30 20 10
Site E
(GST)
0
Site R Site A Site B Site C Site E
0.03 (GSH)
*
*
*
nmol/g tissue
0.024 200
*
100
0.018 0.012 0.006
0
Site R Site A
nmol TBARS formed/hr/mg protein
nmol CDNB formed/min/mg protein
nmol H2O2 consumed/min/mg protein
958
0
Site B Site C Site E
1
Site R Site A Site B Site C Site E
(LPO)
0.75
*
0.5
0.25
0
Site R
Site A
Site B
Site C
Site E
Fig. 4. Represents CAT, as expressed in nanomol H2O2 consumed/min/mg protein; GPX, as expressed in nanomol NADPH oxidized/min/mg protein; GST, as expressed in nanomol CDNB formed/min/mg protein; GSH, as expressed in nanomole/g tissue and LPO, as expressed in nanomole TBARS released during 1 h incubation in liver PMS of Anguilla anguilla L. exposed to Pateira de Fermentelos water-body for 48 h. The significance level observed is *p < 0.05 when compared with the values of reference (site R).
4. Discussion The physico-chemical analysis has long been employed to assess the water quality. In the current study, water quality parameters are, in general, on acceptable levels considering criteria given in APHA (1998) and in Aquamerck (Germany) guide lines, as well as the A. anguilla requirements in particular. However, some exceptions as observed for pH, BOD and nitrite levels must be considered. Water pH showed a considerably high level at site C (9.640) which can be assumed as an indication of strongly polluted to extremely polluted condition. All the other sites presented pH levels in a range belonging either to an unpolluted or to slightly polluted state. In the direction of pollution marking, BOD levels revealed site E as the most oxygen demanding, suggesting the existence of a moderate pollution state. Nitrite levels evidenced a moderately polluted
state at sites B and E. In terms of depth, slight differences were found along PF study sites; however, those differences were inversely related to DO levels, constituting a possible explanation for DO variations. Considering the previous water quality characterization, the observed fish oxidative stress responses are mainly discussed according to the different distances to the main pollution source. Nevertheless, some site-specific responses are also discussed keeping in view the respective water quality variables. Fish were not fed during holding period mainly to avoid the water quality depletion. We cannot exclude the hypothesis that starvation may pre-dispose fish to oxidative stress. However, results have been compared with control groups kept in the same conditions, reducing the relevance of the previous factor. In addition, fish starvation and fish without food often occur in the aquatic environment, mainly in winter.
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4.1. Gill responses An overall antioxidant enzyme inducing trend was observed in the present gill responses. However, this trend was not clearly related to the distance of the main pollution source. Thus, CAT induction provides an indication of the higher H2O2 amount production at sites B, C and E suggesting the presence of redox active components in the PF water-body; however, GPX activity only corroborates this explanation for site C. Hence, fish exposed to sites B and E seem to cope efficiently with H2O2 production only through CAT activity as no peroxidative damage was observed, though, GPX involvement cannot be ignored since a slight induction was observed at those sites, despite being statistically non-significant. The absence of CAT induction at site A (closest to the main pollution source) appears to be unexpected since current gill GST as well as some kidney and liver responses, as discussed in the respective sections, showed the presence of pollutants at site A. In the author’s opinion it can demonstrate a fish incapability to induce gill CAT activity due to a high concentration of pollutants specific to CAT inhibition. Detoxification enzymes, such as GST, helps in eliminating reactive compounds by forming conjugates with glutathione and subsequently eliminating them as mercapturic acid, thereby protecting cells against ROS induced damage (Rodriguez-Ariza et al., 1991). A high level of GST was observed at sites A and E suggesting an activation of gill detoxification processes probably due to the presence of organic contaminants. This statement is partially in agreement with BOD data, since the highest oxygen demand was measured at site E. The conjugation of GSH with a xenobiotic can occur either spontaneously, or can be catalysed by GST (Elia et al., 2003). Thus, current results suggest a GST catalysed conjugation for sites A and E exposure. Moreover, the unchanged GSH levels, as observed in the present study, indicates that this potent free radical scavenger was not a limiting factor for GST or GPX activities. As a contributory part for water pollutants induced stress, the occurrence of high nitrite levels can be an important source of pro-oxidants for fish (Das et al., 2004), leading to nitric radicals production (nitrosative stress) as demonstrated in mammals by Lijima et al. (2003). In this direction, antioxidant responses observed at site B (CAT induction) and at site E (GST induction) could be correlated with the nitrite levels detected at these particular sites. However, Tomosso and Grosell (2005) demonstrated that freshwater adapted A. anguilla exhibit a high degree of resistance to NO 2 due to an absent or minimal gill Cl uptake activity. Thus, the observed eel’s antioxidant responses should be attributable mainly to other classes of compounds subsiding the nitrite effects. Since the typical reaction during oxidative stress is peroxidative damage to unsaturated fatty acids, the oxidative stress response could be conveniently used as biomarkers of oxidative stress inducing chemical pollutants (Ahmad
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et al., 2004; Santos et al., 2006). Thus, no gill LPO induction may be explained by an effective antioxidant action against the peroxidative PF pollutant’s potential. 4.2. Kidney response An overall trend on kidney stress responses is not discernible, since each studied parameter displayed a particular pattern. Therefore, the CAT activity displayed a decrease at site A and an increase at site C, reflecting an incremental trend in relation to the distance from PF entrance (from sites A to C). The significant CAT inhibition (site A) could be attributed to a high production of superoxide anion radical, which has been reported to inhibit CAT activity in case of excess production (Kono and Fridovich, 1982). In addition, the heavy metal presence previously reported by Almeida (1998) and their role in CAT activity decrease should also be considered, since Radi and Matkovics (1988) and Ahmad et al. (2000) reported the CAT decrease due to a high concentration of copper. GPX activity is reported to be induced by environmental pollutants (Radi et al., 1985). However, its protection against ROS requires more attention (Aksnes and Njaa, 1981; Hasspielar et al., 1994). Accordingly, the present results showed a regular kidney GPX activity decrease for all exposure sites, displaying a GPX decreasing pattern in concomitance to the increasing distance from the main pollution source. Bainy et al. (1996) has also reported a GPX decrease in Nile tilapia from a polluted lake, but an appropriated explanation was not provided. On the other hand, Janssens et al. (2000) found that general metabolic activity depletion associated with the depth increase may cause a GPX activity decrease. This explanation can be applicable to current observations since all PF sites were higher in depth as compared to the adopted reference site. This condition, in conjugation with a low water transparency, provides a less stimulating environment rendering eels more unperturbed and consequently reducing their metabolic activity. According to Janssens et al. (2000) the CAT activity behaves in a different manner being affected neither by the metabolic activity nor by the depth, corroborating our current CAT findings. Kidney GST showed a significant increase at sites A and E as observed in gills; thus, the same explanation can be suggested as described above for gills. The decrease in GSH content observed for site B exposed fish may be due to an increased use of GSH in spontaneous conjugation processes and an inefficient GSH regeneration. This GSH reduction coincides with the highest LPO level, though statistically insignificant, revealing a potential risk increase due to this non-enzymatic antioxidant depletion. Surprisingly, kidney LPO measurement revealed that fish were able to cope with the pollution stress, despite the depletion on some antioxidants. This is in agreement with Cossu et al. (2000) who stated that LPO increase cannot be predicted only on the basis of antioxidant depletion.
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4.3. Liver response Antioxidant responses in liver from exposure sites A to E showed no defined pattern suggesting that all the study sites are not equally polluted. Thus, CAT activity showed a decrease at site B indicating the presence of CAT inhibitors as described above for kidney at site A. The significant GPX increase at site E and no observable effect at other sites’ exposure may be due to the enzyme inducing effect of H2O2 on that particular site. The presence of mixtures of chemicals on PF water seems to affect the crucial role of GPX protection and therefore, current GPX responses reflect changes on the GPX inhibitors/inducers balance along the PF area, favouring the later on site E. GST activity decrease displayed at sites A–C suggests a significant reduction of fish capacity to detoxify chemicals. Although GST induction has been widely demonstrated following exposure to some organic contaminants (Eggens et al., 1995; Goksøyr, 1995; Beyer et al., 1996; Peters et al., 1997; Grinwis et al., 2000; Schlezinger and Stegeman, 2000; Stephensen et al., 2000), its inhibition has also been reported as a non-specific response to chemical challenge (Regoli et al., 2003). GSH exhibited a significant increase at site A and unaltered levels at the other sites. Hence, a GST decrease was not related to the liver GSH content suggesting a spontaneous GSH conjugation to xenobiotics at site A. In addition to its role as a cofactor for GPX and GST, GSH is by itself an effective protectant capable of oxyradical quenching (Ross, 1988). Therefore, the high liver GSH content observed in A. anguilla at site A indicates a protective role for GSH against chemical contaminants induced oxidative stress. A similar observation of high GSH levels in fish exposed to polluted sediment and river water have been reported by Di Giulio et al. (1993) and Pandey et al. (2003). The current liver LPO decrease at site B may be the result of a decreased DO level at this site (though in an acceptable level) when compared to the reference site. This finding agrees with Janssens et al.’s (2000) observations on deep-sea fish which were less prone to oxidative danger because of their poorly oxygenated environment and revealing that, under certain conditions, hypoxic environments can be beneficial as it relieves fish from a major oxidative stress risk. 4.4. Organ specificity Current results demonstrated an organ-specific antioxidant modulation, varying at different exposure sites. Organ-specific toxic responses are related to their anatomic location determining the exposure route and distribution of pollutants, as well as their defensive capacity. This is particularly evident on CAT activity as an overall increase was observed in gill, whereas liver and kidney showed no defined pattern. Both GPX and GST data demonstrated the presence of chemical inducers in the PF water; however, clear organ specificity is evident on their responses. In this direc-
tion, GPX activity showed a decrease in kidney, whereas the gill and the liver displayed their ability to induce this enzyme. On the other hand, GST activity showed a decrease in liver, while gill and kidney exhibited their ability to induce this conjugase. Therefore, the kidney seems to be the most vulnerable organ to GPX inhibition whereas the liver is most sensitive to GST inhibition, probably due to its specific propensity to bioaccumulate either chemical inhibitors or substrates up to inhibitory levels. Moreover, neither kidney GPX nor liver GST depletion can be justified by GSH overuse and insufficient regeneration. The overall analysis of GPX and GST data suggests two different explanations occurring either simultaneously or alternatively: first, the chemicals responsible for GPX and GST inhibition are different in nature; second, an organ-specific detoxification skill determining the overall responses in each organ. The latter explanation is corroborated by the difference in baseline levels (recorded for the reference site) among organs found for GPX or GST. Thus, kidney showed a higher basal GPX level than did the liver (around 80 vs. 24 nmol NADPH oxidized/min/ mg protein), whereas the opposite was observed in GST (around 60 vs. 250 nmol CDNB formed/min/mg protein). GSH showed its protective capability (though point specific) in kidney and liver, signed either by its increase (liver at site A) or decrease (kidney at site B); being the latter case probably related with an insufficient glutathione regeneration. Fish gill is in close contact with environmental water contaminants, being the main absorption organ for those aquatic toxicants (Sayeed et al., 2000). In our earlier field investigation on the recovery of bleached kraft paper mill effluent affected areas, we observed gill highest susceptibility towards peroxidative damage compared to kidney and liver, rendering gill antioxidants less effective (Santos et al., 2004, 2006). Moreover, studies have shown that gill and kidney antioxidant enzymes and non-enzymatic antioxidant molecules are less efficient than liver, thus increasing their vulnerability towards reactive oxidative radicals (Di Giulio et al., 1989; Winston and Di Giulio, 1991; Ahmad et al., 2000; Santos et al., 2004). Despite the previous statements, the present study showed significant gill antioxidant induction coupled with no peroxidative damage, demonstrating the gill efficiency to cope with the rapid contact of pro-oxidants present in the PF water body. In addition, a long-term exposure study may be suggested to validate the role of antioxidants to this particular ecosystem exposure. The nonaccepting trend of antioxidant induction in kidney and liver could be explained through differences in exposure route and pollutant uptake which is corroborated by Pitchard and Bend (1984) who stated that kidney is the target organ for chemicals after being taken up through the gill. 5. Conclusions The present results demonstrate that, despite the complexity of A. anguilla antioxidant mechanisms aggravated
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by the presence of mixtures of contaminants, the assessed biomarkers were able to express site-specific responses, demonstrating their utility on freshwater health assessment. The presence of pollutants in the PF water was denunciated even without a clear relation to the main pollution source distance. In this direction, the occurrence of non-pointed pollution sources and their contribution for antioxidant modulation should also be considered. Fish antioxidant responses in each organ proved to be dependent either on the anatomic position determining the uptake and distribution of xenobiotics or organ-specific physiology. The organ specificity was evident for each parameter but without a clear pattern. However, all the organs revealed a similar resistance to peroxidative damage, suggesting that the antioxidants are more responsive biomarkers than LPO for short-term exposure. Besides the activation of antioxidant enzymes (as observed in gill), their inhibition (as observed in kidney and liver) should also be considered as a clear indication of pollution presence and environmental health degradation. The overall fish responses should be explained by the type and degree of contamination as well as their interference to each exposure site. Moreover, the presence of different classes of contaminants both at the water column and sediments can undergo diverse dilution and degradation along the watercourse resulting in site specific chemical interactions and consequent antagonistic or synergistic effects. Keeping in view all the assessed parameters and the organ’s responsiveness, the authors suggest, for future biomonitoring studies, that a multiple organ strategy should be adopted in order to avoid misinterpretations. A. anguilla in situ trial proves its high ability for freshwater monitoring, especially on the basis of oxidative stress assessment. Acknowledgements The financial supports from FCT (Government of Portugal) provided through contract S.F.R.H/B.P.D/3603/ 2000 and by the Aveiro University Research Institute/ CESAM are gratefully acknowledged. References Ahmad, I., Hamid, T., Fatima, M., Chand, H.S., Jain, S.K., Athar, M., Raisuddin, S., 2000. Induction of hepatic antioxidants in freshwater fish (Channa punctatus Bloch) is a biomarker of paper mill effluent exposure. Biochim. Biophys. Acta 1523, 37–48. 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. Ahmad, I., Oliveira, M., Pacheco, M., Santos, M.A., 2005. Anguilla anguilla L. oxidative stress biomarkers responses to copper exposure with or without b-naphthoflavone pre-exposure. Chemosphere 61, 267–275. Aksnes, A., Njaa, L.R., 1981. Catalase, glutathione peroxidase and superoxide dismutase in different fish species. Comp. Biochem. Physiol. 69, 893–896.
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