Environ Sci Pollut Res DOI 10.1007/s11356-014-3580-6
RESEARCH ARTICLE
Perturbations in ROS-related processes of the fish Gambusia holbrooki after acute and chronic exposures to the metals copper and cadmium Bruno Nunes & Carina Caldeira & Joana Luísa Pereira & Fernando Gonçalves & Alberto Teodorico Correia
Received: 2 April 2014 / Accepted: 8 September 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Metallic contamination is a widespread phenomena, particularly in areas impacted by human activities, and has become a relevant environmental concern. However, the toxicity of metals on fish requires full characterization in terms of short- and long-term effects. Thus, the purpose of this study was to determine the acute and chronic oxidative stress response in liver and gills of Gambusia holbrooki exposed to copper and cadmium. To assess the effects of these two metals, we adopted a strategy of analyzing the pollution effects caused by salts of the two metallic elements, and we quantified the oxidative stress biomarkers catalase, glutathione reductase, glutathione-S-transferases, and lipid peroxidation after exposure (4 and 28 days) to ecologically relevant concentrations, thus simulating actual conditions of exposure in the wild. Our results showed that copper elicited strong effects in all tested biomarkers for both acute and chronic challenges. Cadmium Responsible editor: Philippe Garrigues B. Nunes (*) : C. Caldeira : J. L. Pereira : F. Gonçalves Departamento de Biologia, Centro de Estudos do Ambiente e do Mar (CESAM), Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal e-mail: [email protected] C. Caldeira e-mail: [email protected] J. L. Pereira e-mail: [email protected] F. Gonçalves e-mail: [email protected] A. T. Correia Faculdade de Ciências da Saúde, Universidade Fernando Pessoa, Rua Carlos da Maia 296, 4200-150 Porto, Portugal e-mail: [email protected] A. T. Correia Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR–CIMAR), Rua dos Bragas 289, 450-123 Porto, Portugal
caused a similar response and was shown to cause significant changes particularly in catalase and glutathione-S-transferases activities. These findings evidence that ecologically relevant concentrations of common anthropogenic contaminants are causative agents of serious imbalances (namely oxidative stress) that are likely to trigger life-threatening events. Keywords Freshwater fish . Metals . Oxidative stress . Enzymes . Ecologically relevant exposure . Biomarkers . Mosquito fish
Introduction The widespread presence of metals in the environment is causative of a series of deleterious imbalances in exposed organisms, which can result in toxic insult. Among the diversity of effects that are likely to occur as a consequence of chemical contamination by metals, oxidative modification/ damage is amongst the most studied. Despite the prolific use of metallic compounds by human activities, metals are not always regarded as important contaminants of aquatic ecosystems. In fact, some metals are essential to life, while others can cause important toxicological outcomes. It must be emphasized that exposure to metals, above a certain level, is always causative of deleterious effects. Among the most characterized effects that metals can exert, one can find oxidative stress. The occurrence of oxidative stress in aquatic organisms is a major area of research due to the vast number of pro-oxidative compounds that exist in the water compartment (Lushchak 2011a, b). The continuous release of several classes of chemical compounds, including metallic derivatives, can work in the genesis of multiple adverse effects, mostly on aquatic ecosystems (López-Galindo et al. 2010). Among aquatic organisms, fish are prone to the establishment of a large number of toxic effects
Environ Sci Pollut Res
consequent to pollution provided their bioaccumulation capability; consequently, fish may also be vectors of contamination to human consumers (Greco et al. 2010). In addition, xenobiotics can induce severe alterations, including oxidative stress response in various cell types, and a modification of the elimination profiles of foreign molecules from the body (Johnston et al. 2010). Thus, biomarkers (such as oxidative stress and others), interpreted as physiological tools that are directly linked to effects, damages, and adaptive responses towards pollution have become an important subject in aquatic toxicology (Jin et al. 2010; Johnston et al. 2010). Metallic species such as copper, nickel, chromium, iron, and zinc are fundamental for life, being part of macromolecules and enzymes (Serafim et al. 2012). However, metals (copper and iron; Saini et al. 2010; manganese, arsenic, and selenium; Rensing and Rosen 2009; cadmium, mercury, lead, and nickel; García-Fernández et al. 2002) are also prone to establish redox cycles if in the presence of molecular oxygen, giving rise to the production of short-lived, highly unstable and chemically reactive intermediates, designated reactive oxygen species (ROS). Consequently, metals are dual in their effects: albeit vital metals are eminently toxic (Liu and Thiele 1997). ROS production can occur and/or be enhanced through several mechanisms, such as interference in electron transport in the mitochondrial membrane (and subsequent accumulation of reactive intermediates), inactivation of antioxidant enzymes, depletion of non-enzymatic antioxidants, and membrane lipid peroxidation (Modesto and Martinez 2010). Being highly deleterious in nature, as a consequence of their high reactivity, ROS must be promptly removed from the cell by antioxidant defenses. It is noteworthy that a continuous balance between these molecules and the antioxidant defense efficacy must be achieved fundamentally to protect organisms from oxidative stress (Modesto and Martinez 2010). Additionally, ROS posses other biological activities, being highly important for cells as essential cellular messengers, and redox regulators (Wang et al. 2013); the balance between its synthesis and degradation/control is thus of high significance. Nevertheless, the exposure to toxic chemical pollutants (including metals) may alter this equilibrium and can, consequently, induce a decrease in the antioxidant defenses efficiency. Moreover, and considering the partial or total failure of the antioxidant defense system to cope with ROS overproduction (as occurring in the case of chemical contamination), the oxidative stress can cause irreversible oxidative damages, such as oxidation of DNA and other macromolecules, or even cellular death (Jin et al. 2010; Modesto and Martinez 2010). In general, organisms deal with oxidative stress effects through the onset and development of adaptive responses that may include expression of key enzymes involved in biotransformation and metabolism of a wide range of environmental contaminants (Ognjanovic et al. 2008; Modesto and Martinez
2010). Among the adaptive responses, one can elicit enzymes of the antioxidant defense system, such as glutathione-S-transferase (GST), catalase (CAT), and glutathione reductase (GR), which are the most frequently studied biomarkers in fish (Jin et al. 2010; Modesto and Martinez 2010). Lipoperoxidation (LPO) is not a biological response, being a direct estimate of oxidative damage. Quantification of LPO consists of a biomarker of toxic effects since this parameter reflects the oxidation of cell membrane lipids (Ognjanovic et al. 2008). Gambusia holbrooki was selected to serve as test organism for the present study. It is commonly known as mosquito fish and is a euryhaline organism widely distributed in both freshwater systems and estuaries of temperate regions. G. holbrooki possesses high fecundity and has been considered as a representative secondary consumer in aquatic ecosystems. Furthermore, this species is abundant, easy to capture, and easy to maintain in the laboratory. These characteristics make this organism a suitable animal model in ecotoxicology (Nunes et al. 2004, 2008). The aim of the present study was to evaluate the acute and chronic effects induced by exposure to environmentally realistic concentrations of the metals cadmium and copper (under the forms of their respective salts) on oxidative stress biomarkers of G. holbrooki. Catalase (CAT), glutathione-S-transferases (GSTs), glutathione reductase (GR), and thiobarbituric acid-reactive substances (TBARS) were measured in hepatic and gill tissues. These parameters were selected for use as putative biomarkers of effect in this study since they are involved in key biological processes, determinant for the survival of the individuals, such as detoxification (by phase II metabolism, namely conjugation with GSH), oxidative stress, and lipoperoxidation. As target organs, we chose liver and gills because the liver is the main organ of xenobiotic metabolism in fish, and the gills are the primary barrier against the entrance of xenobiotics into the body and probably are also the first line of detoxification and elimination of deleterious compounds. Copper was selected as model toxicant due to its wide presence in the environment and to its well-known oxidative effects that involve the cycling between two oxidation states (Cu(I) and Cu(II) in a Fenton-like chemical reaction favored by hydrogen peroxide) thoroughly described by Ninh Pham et al. (2013). The second selected metal, cadmium, despite being a redox inert metal (Jomova and Valko 2011) is also capable of exerting oxidative stress, but by distinct, not totally clear, complex mechanisms (Shaikh et al. 1999).
Material and methods Chemicals The metals (cadmium and copper) to which fish were exposed were dosed as stable salts, cadmium chloride (CdCl2; Acros
Environ Sci Pollut Res
Organics, Geel, Belgium), and copper sulfate (CuSO4·5H2O; EMSURE®, Merck, Darmstadt, Germany) in degrees of purity of 99.9 % and 99.0–101.0 %, respectively. Stock solutions of the tested compounds were prepared in ultrapure water immediately before dilution into test solutions. Fish exposure The experiments were carried out with G. holbrooki specimens (adult males and immature females, 2.5–3 cm; weight of 0.153±0.035 g), which were collected in the natural lagoon of Pateira de Fermentelos located in the central region of Portugal (40° 34′ 48″ N, 8° 31′ 12″ W). Pateira de Fermentelos is characterized by low levels of anthropogenic pollution (Ferreira et al. 2003). Fish were captured using hand nets and immediately transported to the laboratory in a plastic container with air supply. Prior to the toxicity tests, specimens were kept in plastic tanks during 15 days for acclimation and quarantine and fed ad libitum. The tanks were supplied with dechlorinated tap water, continuous aeration, and constant temperature (20±1 °C). Inspections were conducted twice a day in order to discard diseased and dead specimens. After this period of quarantine, fish were maintained in tanks under laboratory-controlled conditions before exposure. The specimens were fed on a daily basis with commercially available fish food (Sera Vipan® flakes). The water was renewed once a week. The experimental design was performed according to the OECD guidelines, and acute exposures were run for 96 h according to OECD 203 (OECD 1992), while chronic exposures lasted for 28 days according to OECD 215 (OECD 2000). Specimens were individually exposed in plastic bottles previously rinsed with distilled water. The bottles were filled with 200 ml of dechlorinated tap water. Three replicates were used per concentration; each replicate was composed by ten individually exposed fish (1 fish/200 ml of test media) to obtain enough biological material to perform all determinations. Fish were randomly distributed into the experimental containers and submitted to the following treatments: 3.125, 6.25, 12.5, 25, and 50 μg L−1 (copper, acute exposure); 0.1875, 0.375, 0.75, 1.5, and 3 ng L−1 (copper, chronic exposure); 0.468, 0.9375, 1.875, 3.75, and 7.5 mg L−1 (cadmium, acute exposure); and 31.25, 62.5, and 125 μg L−1 (cadmium, chronic exposure). Metal content was measured by inductively coupled plasma mass spectrometry (ICP-MS). ICPMS-SB analyses were made using a double-focusing magnetic sector field instrument ICP-SF-MS (Thermo ICP-MS x series, Thermo Electron Corporation). This instrument is equipped with a compact double-focusing magnetic sector mass spectrometer of reversed Nier-Johnson geometry. All measurements were made at a medium resolution setting (m/Δm= 4000) to avoid false readings from spectral interferences. The instrument was equipped with a microflow nebulizer
(PFAAR35-1-C1E, Glass Expansion) operated in the selfaspirating mode (sample uptake rate ∼0.93 L min −1 ). Concentrations were calculated by linear interpolation (sum of least squares) based on normalization with the internal standard and on calibration curves made from single element standards (Merck KGaA) covering the individual expected concentration ranges. A calibration was made at the beginning of each session. Since measured concentrations were within 90 and 110 % of the nominal concentrations, we used the latter to express toxicity following the OECD guidelines (OECD 2002). All assays included one control with only dechlorinated tap water. Concentrations of metal salts tested were based on a combination of previous studies (LC50 values) and quantified levels already reported in Portuguese estuaries (Mucha et al. 2003; Fernandes et al. 2008) to simulate a low-concentration exposure scenario for both acute and chronic bioassays that reflect realistic environmental conditions. During exposures, abiotic conditions (temperature and aeration) were similar to those described above for fish maintenance. Fish were fed once every 2 days ad libitum along with renewal of test medium. Parameters such as mortality, pH, temperature, oxygen concentration, and conductivity were measured daily during the experimental period. Enzymatic assays After each exposure period, fish were sacrificed by decapitation on ice-cold phosphate buffer, and gills and liver were separated. Gills and livers were homogenized in ice-cold phosphate buffer (50 mM, pH 7.0, with 0.1 % Triton X-100) and then the homogenized tissues were centrifuged at 15,000g for 10 min at 4 °C. Supernatants were divided into aliquots, which were used for different enzymatic determinations. Each homogenate sample was composed by ten livers or ten gills. Samples were stored at −80 °C until further enzymatic determinations. Enzymatic activity was determined for catalase (CAT), glutathione-S-transferases (GSTs), and glutathione reductase (GR). Lipoperoxidation extent was assessed by the quantification of thiobarbituric acid-reactive substances (TBARS). Catalase activity was assayed by the procedure described by Aebi (1984); CAT activity was quantified based on the degradation rate of the substrate H2O2 monitored at 240 nm in samples previously homogenized in 50 mM, pH 7.0 phosphate buffer. The results were expressed by considering that one unit of activity was equal to the number of moles of H2O2 degraded per minute per milligram of protein. GSTs catalyze the conjugation of the substrate 1-chloro-2,4-dinitrobenzene (CDNB) with glutathione forming a thioether that can be followed by the increment of absorbance at 340 nm. Thereby, GST activity was determined by spectrophotometry (spectrophotometer with microplate reader LABSYSTEMS, model MULTISKAN EX), according to Habig et al. (1974),
Environ Sci Pollut Res
and the results were expressed as nanomoles of thioether produced per minute per milligram of protein. Samples for GST determination were prepared in 0.1 M, pH=6.5 phosphate buffer. The activity of GR was determined by spectrophotometry (spectrophotometer with microplate reader LABSYSTEMS, model MULTISKAN EX), according to the protocol of Carlberg and Mannervik (1985). In this assay, the GR activity can be monitored by the NADPH oxidation, with decrease in absorbance values at 340 nm. Samples for GR activity determination were prepared with 200 mM, pH 7.0, and EDTA 2 mM phosphate buffer. Enzymatic activity was expressed as micromoles of NADPH oxidized per minute and per milligram of protein. The extent of lipid peroxidation was measured by the quantification of thiobarbituric acid-reactive substances (TBARS), according to the protocol described by Buege and Aust (1978). Samples, after homogenisation, were treated with 10 % trichloroacetic acid to precipitate soluble proteins and then boiled with 1 % thiobarbituric acid. Absorbance readings of each sample were performed in triplicate at 535 nm. This methodology is based on the reaction of compounds such as malondialdehyde (MDA) formed by degradation of initial products of free radical attack with 2thiobarbituric acid (TBA). TBARS content was expressed as MDA equivalents. Protein quantification of samples was determined according to the Bradford method (Bradford 1976) adapted to microplate in order to express protein content of the analyzed tissues. Statistical analysis Enzymatic activities were analyzed in order to compare the results between metal exposure of each treatment group (Cu or Cd) and control values. Data were checked for normality and homoscedasticity prior to statistical analysis. One-way ANOVA, followed by the Dunnet multicomparison test, was used to discriminate significant differences among treatments. The significance level adopted for all analyses (α) was 0.05. Data in the figures were expressed as mean±standard error (SE).
Results and discussion Copper exposure No mortality was recorded for copper exposure. The results indicate that the acute exposure to copper caused a significant increase in catalase activity in liver tissue of G. holbrooki (p
RESEARCH ARTICLE
Perturbations in ROS-related processes of the fish Gambusia holbrooki after acute and chronic exposures to the metals copper and cadmium Bruno Nunes & Carina Caldeira & Joana Luísa Pereira & Fernando Gonçalves & Alberto Teodorico Correia
Received: 2 April 2014 / Accepted: 8 September 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Metallic contamination is a widespread phenomena, particularly in areas impacted by human activities, and has become a relevant environmental concern. However, the toxicity of metals on fish requires full characterization in terms of short- and long-term effects. Thus, the purpose of this study was to determine the acute and chronic oxidative stress response in liver and gills of Gambusia holbrooki exposed to copper and cadmium. To assess the effects of these two metals, we adopted a strategy of analyzing the pollution effects caused by salts of the two metallic elements, and we quantified the oxidative stress biomarkers catalase, glutathione reductase, glutathione-S-transferases, and lipid peroxidation after exposure (4 and 28 days) to ecologically relevant concentrations, thus simulating actual conditions of exposure in the wild. Our results showed that copper elicited strong effects in all tested biomarkers for both acute and chronic challenges. Cadmium Responsible editor: Philippe Garrigues B. Nunes (*) : C. Caldeira : J. L. Pereira : F. Gonçalves Departamento de Biologia, Centro de Estudos do Ambiente e do Mar (CESAM), Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal e-mail: [email protected] C. Caldeira e-mail: [email protected] J. L. Pereira e-mail: [email protected] F. Gonçalves e-mail: [email protected] A. T. Correia Faculdade de Ciências da Saúde, Universidade Fernando Pessoa, Rua Carlos da Maia 296, 4200-150 Porto, Portugal e-mail: [email protected] A. T. Correia Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR–CIMAR), Rua dos Bragas 289, 450-123 Porto, Portugal
caused a similar response and was shown to cause significant changes particularly in catalase and glutathione-S-transferases activities. These findings evidence that ecologically relevant concentrations of common anthropogenic contaminants are causative agents of serious imbalances (namely oxidative stress) that are likely to trigger life-threatening events. Keywords Freshwater fish . Metals . Oxidative stress . Enzymes . Ecologically relevant exposure . Biomarkers . Mosquito fish
Introduction The widespread presence of metals in the environment is causative of a series of deleterious imbalances in exposed organisms, which can result in toxic insult. Among the diversity of effects that are likely to occur as a consequence of chemical contamination by metals, oxidative modification/ damage is amongst the most studied. Despite the prolific use of metallic compounds by human activities, metals are not always regarded as important contaminants of aquatic ecosystems. In fact, some metals are essential to life, while others can cause important toxicological outcomes. It must be emphasized that exposure to metals, above a certain level, is always causative of deleterious effects. Among the most characterized effects that metals can exert, one can find oxidative stress. The occurrence of oxidative stress in aquatic organisms is a major area of research due to the vast number of pro-oxidative compounds that exist in the water compartment (Lushchak 2011a, b). The continuous release of several classes of chemical compounds, including metallic derivatives, can work in the genesis of multiple adverse effects, mostly on aquatic ecosystems (López-Galindo et al. 2010). Among aquatic organisms, fish are prone to the establishment of a large number of toxic effects
Environ Sci Pollut Res
consequent to pollution provided their bioaccumulation capability; consequently, fish may also be vectors of contamination to human consumers (Greco et al. 2010). In addition, xenobiotics can induce severe alterations, including oxidative stress response in various cell types, and a modification of the elimination profiles of foreign molecules from the body (Johnston et al. 2010). Thus, biomarkers (such as oxidative stress and others), interpreted as physiological tools that are directly linked to effects, damages, and adaptive responses towards pollution have become an important subject in aquatic toxicology (Jin et al. 2010; Johnston et al. 2010). Metallic species such as copper, nickel, chromium, iron, and zinc are fundamental for life, being part of macromolecules and enzymes (Serafim et al. 2012). However, metals (copper and iron; Saini et al. 2010; manganese, arsenic, and selenium; Rensing and Rosen 2009; cadmium, mercury, lead, and nickel; García-Fernández et al. 2002) are also prone to establish redox cycles if in the presence of molecular oxygen, giving rise to the production of short-lived, highly unstable and chemically reactive intermediates, designated reactive oxygen species (ROS). Consequently, metals are dual in their effects: albeit vital metals are eminently toxic (Liu and Thiele 1997). ROS production can occur and/or be enhanced through several mechanisms, such as interference in electron transport in the mitochondrial membrane (and subsequent accumulation of reactive intermediates), inactivation of antioxidant enzymes, depletion of non-enzymatic antioxidants, and membrane lipid peroxidation (Modesto and Martinez 2010). Being highly deleterious in nature, as a consequence of their high reactivity, ROS must be promptly removed from the cell by antioxidant defenses. It is noteworthy that a continuous balance between these molecules and the antioxidant defense efficacy must be achieved fundamentally to protect organisms from oxidative stress (Modesto and Martinez 2010). Additionally, ROS posses other biological activities, being highly important for cells as essential cellular messengers, and redox regulators (Wang et al. 2013); the balance between its synthesis and degradation/control is thus of high significance. Nevertheless, the exposure to toxic chemical pollutants (including metals) may alter this equilibrium and can, consequently, induce a decrease in the antioxidant defenses efficiency. Moreover, and considering the partial or total failure of the antioxidant defense system to cope with ROS overproduction (as occurring in the case of chemical contamination), the oxidative stress can cause irreversible oxidative damages, such as oxidation of DNA and other macromolecules, or even cellular death (Jin et al. 2010; Modesto and Martinez 2010). In general, organisms deal with oxidative stress effects through the onset and development of adaptive responses that may include expression of key enzymes involved in biotransformation and metabolism of a wide range of environmental contaminants (Ognjanovic et al. 2008; Modesto and Martinez
2010). Among the adaptive responses, one can elicit enzymes of the antioxidant defense system, such as glutathione-S-transferase (GST), catalase (CAT), and glutathione reductase (GR), which are the most frequently studied biomarkers in fish (Jin et al. 2010; Modesto and Martinez 2010). Lipoperoxidation (LPO) is not a biological response, being a direct estimate of oxidative damage. Quantification of LPO consists of a biomarker of toxic effects since this parameter reflects the oxidation of cell membrane lipids (Ognjanovic et al. 2008). Gambusia holbrooki was selected to serve as test organism for the present study. It is commonly known as mosquito fish and is a euryhaline organism widely distributed in both freshwater systems and estuaries of temperate regions. G. holbrooki possesses high fecundity and has been considered as a representative secondary consumer in aquatic ecosystems. Furthermore, this species is abundant, easy to capture, and easy to maintain in the laboratory. These characteristics make this organism a suitable animal model in ecotoxicology (Nunes et al. 2004, 2008). The aim of the present study was to evaluate the acute and chronic effects induced by exposure to environmentally realistic concentrations of the metals cadmium and copper (under the forms of their respective salts) on oxidative stress biomarkers of G. holbrooki. Catalase (CAT), glutathione-S-transferases (GSTs), glutathione reductase (GR), and thiobarbituric acid-reactive substances (TBARS) were measured in hepatic and gill tissues. These parameters were selected for use as putative biomarkers of effect in this study since they are involved in key biological processes, determinant for the survival of the individuals, such as detoxification (by phase II metabolism, namely conjugation with GSH), oxidative stress, and lipoperoxidation. As target organs, we chose liver and gills because the liver is the main organ of xenobiotic metabolism in fish, and the gills are the primary barrier against the entrance of xenobiotics into the body and probably are also the first line of detoxification and elimination of deleterious compounds. Copper was selected as model toxicant due to its wide presence in the environment and to its well-known oxidative effects that involve the cycling between two oxidation states (Cu(I) and Cu(II) in a Fenton-like chemical reaction favored by hydrogen peroxide) thoroughly described by Ninh Pham et al. (2013). The second selected metal, cadmium, despite being a redox inert metal (Jomova and Valko 2011) is also capable of exerting oxidative stress, but by distinct, not totally clear, complex mechanisms (Shaikh et al. 1999).
Material and methods Chemicals The metals (cadmium and copper) to which fish were exposed were dosed as stable salts, cadmium chloride (CdCl2; Acros
Environ Sci Pollut Res
Organics, Geel, Belgium), and copper sulfate (CuSO4·5H2O; EMSURE®, Merck, Darmstadt, Germany) in degrees of purity of 99.9 % and 99.0–101.0 %, respectively. Stock solutions of the tested compounds were prepared in ultrapure water immediately before dilution into test solutions. Fish exposure The experiments were carried out with G. holbrooki specimens (adult males and immature females, 2.5–3 cm; weight of 0.153±0.035 g), which were collected in the natural lagoon of Pateira de Fermentelos located in the central region of Portugal (40° 34′ 48″ N, 8° 31′ 12″ W). Pateira de Fermentelos is characterized by low levels of anthropogenic pollution (Ferreira et al. 2003). Fish were captured using hand nets and immediately transported to the laboratory in a plastic container with air supply. Prior to the toxicity tests, specimens were kept in plastic tanks during 15 days for acclimation and quarantine and fed ad libitum. The tanks were supplied with dechlorinated tap water, continuous aeration, and constant temperature (20±1 °C). Inspections were conducted twice a day in order to discard diseased and dead specimens. After this period of quarantine, fish were maintained in tanks under laboratory-controlled conditions before exposure. The specimens were fed on a daily basis with commercially available fish food (Sera Vipan® flakes). The water was renewed once a week. The experimental design was performed according to the OECD guidelines, and acute exposures were run for 96 h according to OECD 203 (OECD 1992), while chronic exposures lasted for 28 days according to OECD 215 (OECD 2000). Specimens were individually exposed in plastic bottles previously rinsed with distilled water. The bottles were filled with 200 ml of dechlorinated tap water. Three replicates were used per concentration; each replicate was composed by ten individually exposed fish (1 fish/200 ml of test media) to obtain enough biological material to perform all determinations. Fish were randomly distributed into the experimental containers and submitted to the following treatments: 3.125, 6.25, 12.5, 25, and 50 μg L−1 (copper, acute exposure); 0.1875, 0.375, 0.75, 1.5, and 3 ng L−1 (copper, chronic exposure); 0.468, 0.9375, 1.875, 3.75, and 7.5 mg L−1 (cadmium, acute exposure); and 31.25, 62.5, and 125 μg L−1 (cadmium, chronic exposure). Metal content was measured by inductively coupled plasma mass spectrometry (ICP-MS). ICPMS-SB analyses were made using a double-focusing magnetic sector field instrument ICP-SF-MS (Thermo ICP-MS x series, Thermo Electron Corporation). This instrument is equipped with a compact double-focusing magnetic sector mass spectrometer of reversed Nier-Johnson geometry. All measurements were made at a medium resolution setting (m/Δm= 4000) to avoid false readings from spectral interferences. The instrument was equipped with a microflow nebulizer
(PFAAR35-1-C1E, Glass Expansion) operated in the selfaspirating mode (sample uptake rate ∼0.93 L min −1 ). Concentrations were calculated by linear interpolation (sum of least squares) based on normalization with the internal standard and on calibration curves made from single element standards (Merck KGaA) covering the individual expected concentration ranges. A calibration was made at the beginning of each session. Since measured concentrations were within 90 and 110 % of the nominal concentrations, we used the latter to express toxicity following the OECD guidelines (OECD 2002). All assays included one control with only dechlorinated tap water. Concentrations of metal salts tested were based on a combination of previous studies (LC50 values) and quantified levels already reported in Portuguese estuaries (Mucha et al. 2003; Fernandes et al. 2008) to simulate a low-concentration exposure scenario for both acute and chronic bioassays that reflect realistic environmental conditions. During exposures, abiotic conditions (temperature and aeration) were similar to those described above for fish maintenance. Fish were fed once every 2 days ad libitum along with renewal of test medium. Parameters such as mortality, pH, temperature, oxygen concentration, and conductivity were measured daily during the experimental period. Enzymatic assays After each exposure period, fish were sacrificed by decapitation on ice-cold phosphate buffer, and gills and liver were separated. Gills and livers were homogenized in ice-cold phosphate buffer (50 mM, pH 7.0, with 0.1 % Triton X-100) and then the homogenized tissues were centrifuged at 15,000g for 10 min at 4 °C. Supernatants were divided into aliquots, which were used for different enzymatic determinations. Each homogenate sample was composed by ten livers or ten gills. Samples were stored at −80 °C until further enzymatic determinations. Enzymatic activity was determined for catalase (CAT), glutathione-S-transferases (GSTs), and glutathione reductase (GR). Lipoperoxidation extent was assessed by the quantification of thiobarbituric acid-reactive substances (TBARS). Catalase activity was assayed by the procedure described by Aebi (1984); CAT activity was quantified based on the degradation rate of the substrate H2O2 monitored at 240 nm in samples previously homogenized in 50 mM, pH 7.0 phosphate buffer. The results were expressed by considering that one unit of activity was equal to the number of moles of H2O2 degraded per minute per milligram of protein. GSTs catalyze the conjugation of the substrate 1-chloro-2,4-dinitrobenzene (CDNB) with glutathione forming a thioether that can be followed by the increment of absorbance at 340 nm. Thereby, GST activity was determined by spectrophotometry (spectrophotometer with microplate reader LABSYSTEMS, model MULTISKAN EX), according to Habig et al. (1974),
Environ Sci Pollut Res
and the results were expressed as nanomoles of thioether produced per minute per milligram of protein. Samples for GST determination were prepared in 0.1 M, pH=6.5 phosphate buffer. The activity of GR was determined by spectrophotometry (spectrophotometer with microplate reader LABSYSTEMS, model MULTISKAN EX), according to the protocol of Carlberg and Mannervik (1985). In this assay, the GR activity can be monitored by the NADPH oxidation, with decrease in absorbance values at 340 nm. Samples for GR activity determination were prepared with 200 mM, pH 7.0, and EDTA 2 mM phosphate buffer. Enzymatic activity was expressed as micromoles of NADPH oxidized per minute and per milligram of protein. The extent of lipid peroxidation was measured by the quantification of thiobarbituric acid-reactive substances (TBARS), according to the protocol described by Buege and Aust (1978). Samples, after homogenisation, were treated with 10 % trichloroacetic acid to precipitate soluble proteins and then boiled with 1 % thiobarbituric acid. Absorbance readings of each sample were performed in triplicate at 535 nm. This methodology is based on the reaction of compounds such as malondialdehyde (MDA) formed by degradation of initial products of free radical attack with 2thiobarbituric acid (TBA). TBARS content was expressed as MDA equivalents. Protein quantification of samples was determined according to the Bradford method (Bradford 1976) adapted to microplate in order to express protein content of the analyzed tissues. Statistical analysis Enzymatic activities were analyzed in order to compare the results between metal exposure of each treatment group (Cu or Cd) and control values. Data were checked for normality and homoscedasticity prior to statistical analysis. One-way ANOVA, followed by the Dunnet multicomparison test, was used to discriminate significant differences among treatments. The significance level adopted for all analyses (α) was 0.05. Data in the figures were expressed as mean±standard error (SE).
Results and discussion Copper exposure No mortality was recorded for copper exposure. The results indicate that the acute exposure to copper caused a significant increase in catalase activity in liver tissue of G. holbrooki (p
