Environ Sci Pollut Res DOI 10.1007/s11356-015-4329-6
RESEARCH ARTICLE
Biochemical effects of the pharmaceutical drug paracetamol on Anguilla anguilla Bruno Nunes 1 & Maria Francisca Verde 1 & Amadeu M. V. M. Soares 1
Received: 15 December 2014 / Accepted: 5 March 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract The ever-increasing presence of pharmaceutical drugs in the environment is a motif of concern, and humanuse drugs are of particular importance. This is the case of paracetamol, a widely employed drug in human therapeutics, as analgesic and antipyretic, whose toxicity on aquatic organisms is still not fully characterized. The present study aimed to assess the toxic deleterious effects of paracetamol on European eel, Anguila anguilla, by using a comprehensive battery of antioxidant biomarkers (activities of enzymes such as catalase (CAT) and glutathione S-transferases (GSTs)), and the quantification of oxidative damage (measurement of levels of lipid peroxidation (thiobarbituric acid reactive substances (TBARS) assay)). Other biochemical effects elicited by this substance were also quantified, in terms of anaerobic respiration (activity of lactate dehydrogenase, LDH) and neurotoxicity (acetylcholinesterase, AChE, activity). The obtained results showed the occurrence of an oxidative base response, and paracetamol also seemed to inhibit AChE, showing that this drug can also elicit neurotoxicity. The lack of response by both CAT and LDH show that, despite the occurrence of toxicity, eels have detoxification mechanisms that are effective to cope with paracetamol, preventing additional deleterious alterations, including in the main pathway by which they obtain energy.
Responsible editor: Cinta Porte * Bruno Nunes [email protected] 1
Department of Biology, Centro de Estudos do Ambiente e do Mar (CESAM), University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal
Keywords Drugs . Paracetamol . Enzymatic biomarkers . Oxidative stress . European eel . Detoxification
Introduction The presence of anthropogenic pollutants in the wild has systematically increased during the last decades and has been even more noticeable for the aquatic compartment (Altindag et al. 2008). The increase in the use of pharmaceuticals has had a decisive contribution for their dispersal of unaltered drugs and metabolites in the wild (Daughton and Ternes 1999; Jones et al. 2002; Miao et al. 2002). Consequently, during the last two decades, several studies have shown the occurrence of these substances in superficial waters (HallingSorensen et al. 1998; Hirsch et al. 1999; Kolpin et al. 2002), sewage (Marecos do Monte and Albuquerque 2010), groundwater (Newson 1994), seas, and oceans (Timbrell 1995). Pharmaceutical drugs and their residues/metabolites are continuously released in low concentrations into the aquatic ecosystem (Halling-Sorensen et al. 1998; Dorne et al. 2007), in amounts that roughly equal the environmental elimination rates. Thus, these substances can be considered pseudopersistent pollutants (Zenker et al. 2014), consequently constituting a risk for biota that may interact with them. In fact, pharmaceutical drugs share a common number of traits that favor their environmental relevance. Drugs are usually resistant to degradation, to increase their time in the patient’s organism, a factor that increases their environmental persistence (Gennaro 1995; Ansel et al. 1999; Vadas 2000). The therapeutic use of drugs may result in a large amount of unaltered, unmetabolized drug being released (50 to 90 %; Mulroy 2001) without being metabolized. Some drugs can be reverted in the wild to their initial forms, with not only pharmacological but also toxicological activity (Boxall et al. 2003, 2004).
Environ Sci Pollut Res
The metabolism of some pharmaceuticals can lead to the formation of very stable and non-biodegradable final products (Stuer-Lauridsen et al. 2000). Many pharmaceutical compounds may continue exerting their pharmacological activity, despite their extremely low concentrations in the wild, and may also resist to biodegradation (Fent et al. 2006). As a result, it is possible to observe that some of these substances have the potential to suffer bioaccumulation, a phenomenon that is also favored by a considerable lipophylicity that they have (Cleuvers 2003; Fent et al. 2006; Nunes et al. 2006). Due to the conservative nature of physiological processes, many aquatic species possess similar target molecules to those drugs that are intended to interact with in humans (Owen et al. 2007; Gunnarsson et al. 2008). It is possible to predict that from some of these interactions, effects at the subindividual level are possible, which may have consequences at the population and ecosystem levels (Livingstone 1993). Considering the intrinsic features of pharmaceuticals, and the possibility of exerting deleterious effects, some studies have been developed to understand the toxicity of drugs in exposed biota and natural communities (Chapman 2002). However, the assessment of effects is usually confronted with the necessity to prioritize substances, given the extremely large number of compounds of pharmaceutical use. The drug paracetamol is widely used in human therapeutics (Yang et al. 2008; Solé et al. 2010), as an analgesic and antipyretic drug (Xu et al. 2008), especially for pediatric purposes (Pandolfini and Bonati 2005). Despite its undisputable virtues, paracetamol when released into the wild can cause environmental impacts. Paracetamol is, according to Voogt et al. (2009), a drug whose environmental fate and ecotoxicological assessment should be prioritized, since it is highly dispersed, and some studies have shown its involvement in toxic deleterious effects (Heberer 2002). In vertebrates, paracetamol is metabolized by conjugation with both glucuronic acid and sulfate, being these nontoxic conjugates promptly excreted (Sebben et al. 2010). A small amount of paracetamol is oxidized via cytochrome p450 (Jaeschke and Bajt 2006; Xu et al. 2008). This last pathway is responsible for the formation of N-acetyl-p-benzoquinone imine (NAPQI) (He et al. 2011), a highly oxidant and reactive intermediate, which can be conjugated with glutathione. If the amount of ingested paracetamol is high, all cofactors are exhausted, and only NAPQI is formed, leading to the oxidation of thiol groups of cellular proteins and causing the fragmentation of DNA and RNA and finally the oxidation of membrane lipids (Jaeschke and Bajt 2006; Xu et al. 2008; Parolini et al. 2010). Anguilla anguilla, also known as European eel, is a species with a complex life cycle, since it spawns at the Sargasso Sea, where eggs are fertilized. After the eclosion of eggs, juveniles (glass eels) migrate across the Atlantic Ocean and enter freshwater system (rivers and lakes) of Europe and of the Mediterranean area (Capoccioni et al. 2010). Individuals of European
eel are subjected to extreme modifications in their physiology during the migratory period; considering that the length of the migration comprises a distance of approximately 6000 km, at depths varying from 200 to 1000 m (with changes from high to low hydrostatic pressure and extreme temperature variations), and that individuals are not fed (Kopecka-Pilarczyk and Coimbra 2010), it is possible to conclude that this phase of the life cycle is extremely demanding and challenging. In spite of this extremely difficult process, the European eel is considered a highly important organism, in ecological, commercial, and cultural terms (Domingos 2003; Caruso et al. 2008). Nevertheless, European eel populations have been strongly declining in the past two decades, a trend that is possibly related with habitat change, namely by the introduction of anthropogenic contaminants. Pollutants seem to conditionate the reproductive success of European eels (Robinet and Feunteun 2002; Belpaire and Goemans 2007). In addition, evidence for changes in lipid metabolism due to chemical exposure exists (Belpaire and Goemans 2007), raising concern because supplementary amounts of energy required for detoxification deplete energetic reserves that should otherwise be used to reach spawning areas, jeopardizing migration efforts (Maes et al. 2005). It is of the uttermost importance to study the effects of environmental pollutants, including pharmaceutical drugs, on the metabolic features of A. anguilla such as its antioxidant defense mechanism and detoxification capabilities. Furthermore, taking into consideration that some toxicants may affect the biochemical pathways involved in energy production, this is an important insight into the toxic phenomena. To attain this objective, we quantified levels of lactate dehydrogenase (LDH) activity in muscle tissue of A. anguilla. The impairment of this enzyme’s activity can inhibit energy production and response to stimuli, including capture of prey and escape from predators (Diamantino et al. 2001); on the other hand, its increase indicates that the organism preferentially uses the anaerobic pathway to comply with its energy demands (Li et al. 2009). The importance of this aspect is highlighted by the increase of anaerobic metabolism under low oxygen content, such as deep-sea conditions. LDH quantification allows estimating the metabolic status of organisms, under low levels of oxygen (Moreira et al. 2006). Neurotransmission is a feature that may also be affected by exposure to anthropogenic compounds and can have extreme consequences in organisms subjected to extremely long migratory periods, such as eels. Specific enzymes involved in neurotransmission (e.g., acetylcholinesterase) have already been involved in swimming disturbances reported in fish species, as a consequence of exposure to widespread contaminants, such as pesticides (Pereira et al. 2012). Consequently, the present study aimed to determine the toxicological effects caused by acute paracetamol exposure (at ecological relevant levels) on adult individuals of A. anguilla. The assessed toxicological
Environ Sci Pollut Res
parameters included the quantification of the activities of antioxidant enzymes (glutathione S-transferases, GSTs; catalase, CAT) and lipid peroxidation (thiobarbituric acid reactive substance (TBARS) assay), in both gills and liver tissues. Additionally, we also evaluated the neurotoxicity and respiratory effects by this drug by measuring the activities of acetylcholinesterase (AChE) and lactate dehydrogenase (LDH) in eyes and muscle tissue, respectively.
Material and methods Quarantine, acclimation, depuration, and exposure Organisms used in this study were purchased from a commercial aquaculture facility (Viveiros da Boca Torta, Aveiro). These animals were previously captured at Ria de Aveiro, which is an estuarine area, located in the northwest of the Portuguese shore, circa 42 km long and 10 km wide (Lopes and Silva 2006). This estuary is highly irregular and complex in topography as it is comprised of several canals in contact with the Atlantic Ocean (Leandro et al. 2007); thus, tidal influence is the main driving factor determining water circulation (Morgado et al. 2003). Ria de Aveiro is an estuary of great ecological value. While it is subjected to a considerable degree of deterioration caused by human pressure, especially by industrial and urban sources (Kennish 2002; Lopes 2003; Silva et al. 2002), it is considered a reference site because it is protected as a Special Protection Zone (ZPE, Zona de Proteção Especial; Directive 79/409/CEE 1979) and integrated in the Rede Natura 2000 (Diretive n. 92/43/ CEE 1992). Sixty animals were purchased and were immediately transported to the laboratory facilities in plastic thermal boxes. Upon arrival, animals were subjected to a quarantine, depuration, and acclimation period, for approximately 20 days. Animals were kept in opened plastic boxes, with total capacity of 60 L. Animals were kept in dechlorinated and filtered (5 μm) tap water, at a temperature of 20±1 °C, and a photoperiod of 12hL:12hD. Water was continuously aerated and filtered by mechanical filters. After 1 week of acclimation, animals were fed ad libitum with portions of squid for human consumption. Immediately after feeding, the remaining not ingested portions of squid were removed. Animals during this initial period were apparently healthy, without any apparent sign of stress (e.g., malformation, depigmentation), fulfilling all prerequisites to be used in standard ecotoxicological assays (OECD 1992). After the acclimation period, during which no deaths were registered, 30 animals were randomly selected. These were divided into 6 groups of 5 organisms each, to be exposed to 5 treatments of paracetamol + a control (non-exposed) treatment. The selected paracetamol concentrations were 5, 25,
125, 625, and 3125 μg/L. Paracetamol concentrations were based on ecological relevance criteria; the two first concentrations were close to those already reported for freshwater systems (6–65 μg/L; Ternes 1998; Roberts and Thomas 2006). The two intermediate concentrations were 1 order of magnitude above the observed levels in the wild; the highest concentration was used to test the responsiveness of eels to the oxidant effect of acetaminophen (Antunes et al. 2013). Test solutions were prepared by preparing a stock solution of 500, 000 μg/L in water; this paracetamol concentration was confirmed through colorimetric quantification following an adapted version of the procedure no. 430 by Sigma Diagnostics (Brandão et al. 2014). Test concentrations nominated hereinafter are actual concentrations estimated on the basis of the paracetamol quantification made on the stock solution. Exposure was conducted in agreement with OECD guideline no. 203, for acute testing of fish (OECD 1992), and lasted for 96 h (with media renewal at 48 h); all abiotic conditions were the same as described above. No food was provided during exposure. Immediately after exposure, animals were euthanized by immersion in an ice-cold (4 °C) water bath (Wilson et al. 2009) and immediately sacrificed by decapitation. Animals were then dissected on ice-cold phosphate buffer, and the necessary tissues/organs were collected and frozen at −80 °C. Tissues were dissected for biomarker assays as follows: liver and gills for determination of catalase activity (CAT), gills for determination of glutathione S-transferase activity (GST) and lipid peroxidation (TBARS assay), muscle for lactate dehydrogenase (LDH) activity determination, and eyes for the quantification of acetylcholinesterase activity (AChE). Liver was selected due to its primary detoxification function (Heath 1995) while gills due to their continuous close contact with the external media (Au 2004). The determination of the ezymatic activity of LDH was performed in muscle because it contains especially high amounts of this enzyme (Nunes et al. 2004). Finally, AChE activity was determined in the eyes due to their high enervation and particularly elevated content of this enzyme in the selected species (Nunes et al. 2014a). All tissues (except eyes) were homogenized in 50 mM, pH=7 phosphate buffer, with 0.1 % Triton X-100, and centrifuged at 15,000×g for 10 min at 4 °C. Eyes were homogenized with 0.1 M, pH = 7.2 phosphate buffer, and centrifuged at 6000×g for 3 min at 4 °C. Biomarker assays CAT activity was determined according to the method described by Aebi 1984. CAT is involved in the decomposition of hydrogen peroxide (H2O2), preventing its accumulation inside the cell (Atli et al. 2006) and the onset of oxidative stress. Its quantification was performed by spectrophotometrically monitoring the decomposition of H2O2 at a wavelength
Environ Sci Pollut Res
of 240 nm. CAT activity was expressed as the quantity of the enzyme that catalyzes the conversion of 1 μmol of substrate per min (U/mg of protein). GSTs are a group of isoenzymes of the phase II metabolic pathway that catalyzes the conjugation of the substrate 1chloro-2,4-dinitrobenzene (CDNB) with glutathione, forming a thioether, whose formation can be spectrophotometrically followed by the increment of absorbance at a wavelength of 340 nm, according to Habig et al. (1974). GST activity was expressed by the quantity of the enzyme that catalyzes the conversion of 1 μmol of substrate per min (U/mg of protein). According to the protocol described by Buege and Aust (1978), the extent of lipid peroxidation was measured by the quantification of TBARSs. This methodology is based on the reaction of LPO by-products (Gutteridge 1995; Scoccia et al. 2001), such as malondialdehyde (MDA), with 2-thiobarbituric acid. The amount of TBARS was spectrophotometrically measured at a wavelength of 535 nm (ε=156 mM−1 cm− 1), and results were expressed as nanomoles of MDA equivalents per milligram of protein. AChE is a carboxylesterase of the B type that promotes the hydrolysis of acetylcholine, a neurotransmitter fundamental for the neuromuscular transmission. AChE acts by cleaving acetylcholine into thiocholine and acetic acid (Ellman et al. 1961). The predominant form of cholinesterase found in tissues of A. anguilla is acetylcholinesterase, as shown by Valbonesi et al. (2011). AChE activity was spectrophotometrically monitored according to the method described by Ellman et al. (1961), at a wavelength of 412 nm. Lactate dehydrogenase (LDH) is a cytosolic enzyme involved in the glicolytic metabolism in the majority of tissues. Determination of its activity was performed following the technique described by Vassault (1983), by spectrophotometrically measuring the reduction of absorbance caused by the oxidation of the reduced form of nicotinamide adenine dinucleotide (NADH) in the presence of pyruvate, at a wavelength of 340 nm. Quantification of total soluble protein Protein concentration was determined according to the spectrophotometric (wavelength of 595 nm) method of Bradford (1976), based on the reaction that occurs between Coomassie Brilliant Blue G-250 with soluble proteins, adapted to microplate, using bovine serum albumin (BSA) as standard. Statistical analyses Biochemical parameters were statistically analyzed with oneway ANOVA, followed by the Dunnett’s multicomparison test to discriminate significant differences between toxicant concentrations and the control treatment (Dunnett 1955). The adopted level of significance was of 0.05.
Results Animals exposed to paracetamol had an average weight of 35.63±0.05 g and an average length of 27.69±0.05 cm. During exposure, a single death was registered, among organisms exposed to the concentration of 5 μg/L, at the final day of the experiment. No significant effects were observed in terms of catalase activity. However, a slight albeit non-significant increase was observed for the lowest tested concentrations, and for both tissues, liver (F=1.33, df=5.23, p=0.287; Fig. 1a), and gills (F=0.92, df=5.23, p=0.490, Fig. 1b). GST significantly increased in liver between the control and the two highest concentrations of paracetamol (625 and 3125 mg/L; F=8.92, df=5.23, p=8.07E−5; Fig. 2a). However, GST activity significantly decreased in the gills, especially for the highest tested concentration (F = 12.59, df= 5.23, p = 5.86E −6 ; Fig. 2b; no observed effect concentration (NOEC) =25 μg/L; lowest observed effect concentration (LOEC)=125 μg/L). TBARS levels remained unaltered in liver tissue following paracetamol exposure, as shown in Fig. 3a (F=1.53, df=5.23, p=0.220). In gills, it was possible to observe an increase in lipid peroxidation that was significant for the highest tested concentration of paracetamol (F=4.58, df=5.23, p=0.005; Fig. 3b; NOEC=625 μg/L; LOEC=3125 μg/L). Data analysis concerning LDH activity did not show any statistical significant difference in comparison with control animals LDH (F=2.19, df=5.22, p=0.090; Fig. 4). The biomarker AChE was not significantly changed after exposure to paracetamol (F=2.04, df=5.22, p=0.112), despite a significant decrease in its activity for an intermediate concentration (625 μg/L) which is of uncertain biological meaning (Fig. 5).
Discussion The main role attributed to catalase is the degradation of hydrogen peroxide, consequently protecting organisms from its oxidant deleterious effects. Whenever molecular oxygen is used in aerobic processes, and in a more prominent way during the occurrence of oxidative stress, catalase may be overexpressed and its activity may rise, in response to the increased levels of oxygen peroxide (Oruç et al. 2004). Thus, it is possible to assume that a catalase activity increase may signal a compensatory physiological response to oxidative alterations (van der Oost et al. 2003). In the present study, such a response was not registered, since no significant modifications were reported concerning CAT activity, indicating that exposure to this drug, under the selected conditions, was not capable of inducing a response both in liver and in gill tissues. This set of responses contradicts previous findings by
Environ Sci Pollut Res Fig. 1 Effects of paracetamol on catalase (CAT) activity of A. anguilla. Results are expressed as mean±standard deviation, corresponding to the average of five replicates. a Liver. b Gills. Data were analyzed with a one-way ANOVA, followed by the Dunnett test
other authors, which obtained significant pro-oxidative responses after exposing other fish species, such as Oreochromis mossambicus, to paracetamol (Kavitha et al. 2011). A somewhat similar response, with significant effects in terms of catalase enhancement, was also reported by Shivashri et al. (2013); this study described the oxidative effects of paracetamol on the freshwater fish species Pangasius sutchi. Additional evidence pointing to an involvement of the oxidative pathway in aquatic organisms exposed to this drug were given by Ramos et al. (2014) and Parolini et al. (2010) with the mollusk species Corbicula fluminea and Dreissena polymorpha, respectively. Despite the lack of clear indicators suggesting the occurrence of oxidative-related effects in our study (namely by alteration of GST or CAT activities), it is possible to conclude that paracetamol may exert toxicity via this specific pathway, but only in sensitive species or under favorable conditions that enhance uptake or long durations of exposure. Similarly to what was postulated for the enzyme catalase, the isoenzymes glutathione S-transferases may also exert an antioxidant effect. By facilitating the conjugation with the biologically active reduced form of glutathione, this group of enzymes is of the uttermost importance in the detoxification
of xenobiotics, since they augment the biotransformation and excretion rates of numerous compounds, reducing their overall toxicity (Habig et al. 1974). Furthermore, these enzymes are part of an antioxidant system capable of converting reactive oxygen species (ROS) into less damaging substances, but at a high cost: by doing so, these enzymes deplete the cofactor reduced glutathione (GSH); when this cofactor is no longer present at the intracellular level, oxidative damage is likely to occur (Vlahogianni et al. 2007). At higher dosages, however, a paradoxical effect may occur, with impairment of the catalytic activity of GSTs; low levels of exposure did not cause any significant effects (Manimaran et al. 2010; Brandão et al. 2014). The here-obtained results, on the contrary, showed a significant increase of these enzymes’ activity and for the highest tested concentrations of paracetamol. This effect was particularly evident in liver tissue, implicating this specific organ in the biologic response to paracetamol. Gill tissue evidenced a contrary pattern, but only for concentrations of 125 and 3125 μg/L of paracetamol. According to Kavitha et al. (2011), fish gills are characterized by lower GST activity when compared to liver, only responding when faced with higher levels of contamination. The most likely effect may be connected to an increase of the antioxidant capacity of liver,
Environ Sci Pollut Res Fig. 2 Effects of paracetamol on glutathione S-transferases (GSTs) activity of A. anguilla. Results are expressed as mean±standard deviation, corresponding to the average of five replicates. a Liver. b Gills. Data were analyzed with a one-way ANOVA, followed by the Dunnett test; *p
RESEARCH ARTICLE
Biochemical effects of the pharmaceutical drug paracetamol on Anguilla anguilla Bruno Nunes 1 & Maria Francisca Verde 1 & Amadeu M. V. M. Soares 1
Received: 15 December 2014 / Accepted: 5 March 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract The ever-increasing presence of pharmaceutical drugs in the environment is a motif of concern, and humanuse drugs are of particular importance. This is the case of paracetamol, a widely employed drug in human therapeutics, as analgesic and antipyretic, whose toxicity on aquatic organisms is still not fully characterized. The present study aimed to assess the toxic deleterious effects of paracetamol on European eel, Anguila anguilla, by using a comprehensive battery of antioxidant biomarkers (activities of enzymes such as catalase (CAT) and glutathione S-transferases (GSTs)), and the quantification of oxidative damage (measurement of levels of lipid peroxidation (thiobarbituric acid reactive substances (TBARS) assay)). Other biochemical effects elicited by this substance were also quantified, in terms of anaerobic respiration (activity of lactate dehydrogenase, LDH) and neurotoxicity (acetylcholinesterase, AChE, activity). The obtained results showed the occurrence of an oxidative base response, and paracetamol also seemed to inhibit AChE, showing that this drug can also elicit neurotoxicity. The lack of response by both CAT and LDH show that, despite the occurrence of toxicity, eels have detoxification mechanisms that are effective to cope with paracetamol, preventing additional deleterious alterations, including in the main pathway by which they obtain energy.
Responsible editor: Cinta Porte * Bruno Nunes [email protected] 1
Department of Biology, Centro de Estudos do Ambiente e do Mar (CESAM), University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal
Keywords Drugs . Paracetamol . Enzymatic biomarkers . Oxidative stress . European eel . Detoxification
Introduction The presence of anthropogenic pollutants in the wild has systematically increased during the last decades and has been even more noticeable for the aquatic compartment (Altindag et al. 2008). The increase in the use of pharmaceuticals has had a decisive contribution for their dispersal of unaltered drugs and metabolites in the wild (Daughton and Ternes 1999; Jones et al. 2002; Miao et al. 2002). Consequently, during the last two decades, several studies have shown the occurrence of these substances in superficial waters (HallingSorensen et al. 1998; Hirsch et al. 1999; Kolpin et al. 2002), sewage (Marecos do Monte and Albuquerque 2010), groundwater (Newson 1994), seas, and oceans (Timbrell 1995). Pharmaceutical drugs and their residues/metabolites are continuously released in low concentrations into the aquatic ecosystem (Halling-Sorensen et al. 1998; Dorne et al. 2007), in amounts that roughly equal the environmental elimination rates. Thus, these substances can be considered pseudopersistent pollutants (Zenker et al. 2014), consequently constituting a risk for biota that may interact with them. In fact, pharmaceutical drugs share a common number of traits that favor their environmental relevance. Drugs are usually resistant to degradation, to increase their time in the patient’s organism, a factor that increases their environmental persistence (Gennaro 1995; Ansel et al. 1999; Vadas 2000). The therapeutic use of drugs may result in a large amount of unaltered, unmetabolized drug being released (50 to 90 %; Mulroy 2001) without being metabolized. Some drugs can be reverted in the wild to their initial forms, with not only pharmacological but also toxicological activity (Boxall et al. 2003, 2004).
Environ Sci Pollut Res
The metabolism of some pharmaceuticals can lead to the formation of very stable and non-biodegradable final products (Stuer-Lauridsen et al. 2000). Many pharmaceutical compounds may continue exerting their pharmacological activity, despite their extremely low concentrations in the wild, and may also resist to biodegradation (Fent et al. 2006). As a result, it is possible to observe that some of these substances have the potential to suffer bioaccumulation, a phenomenon that is also favored by a considerable lipophylicity that they have (Cleuvers 2003; Fent et al. 2006; Nunes et al. 2006). Due to the conservative nature of physiological processes, many aquatic species possess similar target molecules to those drugs that are intended to interact with in humans (Owen et al. 2007; Gunnarsson et al. 2008). It is possible to predict that from some of these interactions, effects at the subindividual level are possible, which may have consequences at the population and ecosystem levels (Livingstone 1993). Considering the intrinsic features of pharmaceuticals, and the possibility of exerting deleterious effects, some studies have been developed to understand the toxicity of drugs in exposed biota and natural communities (Chapman 2002). However, the assessment of effects is usually confronted with the necessity to prioritize substances, given the extremely large number of compounds of pharmaceutical use. The drug paracetamol is widely used in human therapeutics (Yang et al. 2008; Solé et al. 2010), as an analgesic and antipyretic drug (Xu et al. 2008), especially for pediatric purposes (Pandolfini and Bonati 2005). Despite its undisputable virtues, paracetamol when released into the wild can cause environmental impacts. Paracetamol is, according to Voogt et al. (2009), a drug whose environmental fate and ecotoxicological assessment should be prioritized, since it is highly dispersed, and some studies have shown its involvement in toxic deleterious effects (Heberer 2002). In vertebrates, paracetamol is metabolized by conjugation with both glucuronic acid and sulfate, being these nontoxic conjugates promptly excreted (Sebben et al. 2010). A small amount of paracetamol is oxidized via cytochrome p450 (Jaeschke and Bajt 2006; Xu et al. 2008). This last pathway is responsible for the formation of N-acetyl-p-benzoquinone imine (NAPQI) (He et al. 2011), a highly oxidant and reactive intermediate, which can be conjugated with glutathione. If the amount of ingested paracetamol is high, all cofactors are exhausted, and only NAPQI is formed, leading to the oxidation of thiol groups of cellular proteins and causing the fragmentation of DNA and RNA and finally the oxidation of membrane lipids (Jaeschke and Bajt 2006; Xu et al. 2008; Parolini et al. 2010). Anguilla anguilla, also known as European eel, is a species with a complex life cycle, since it spawns at the Sargasso Sea, where eggs are fertilized. After the eclosion of eggs, juveniles (glass eels) migrate across the Atlantic Ocean and enter freshwater system (rivers and lakes) of Europe and of the Mediterranean area (Capoccioni et al. 2010). Individuals of European
eel are subjected to extreme modifications in their physiology during the migratory period; considering that the length of the migration comprises a distance of approximately 6000 km, at depths varying from 200 to 1000 m (with changes from high to low hydrostatic pressure and extreme temperature variations), and that individuals are not fed (Kopecka-Pilarczyk and Coimbra 2010), it is possible to conclude that this phase of the life cycle is extremely demanding and challenging. In spite of this extremely difficult process, the European eel is considered a highly important organism, in ecological, commercial, and cultural terms (Domingos 2003; Caruso et al. 2008). Nevertheless, European eel populations have been strongly declining in the past two decades, a trend that is possibly related with habitat change, namely by the introduction of anthropogenic contaminants. Pollutants seem to conditionate the reproductive success of European eels (Robinet and Feunteun 2002; Belpaire and Goemans 2007). In addition, evidence for changes in lipid metabolism due to chemical exposure exists (Belpaire and Goemans 2007), raising concern because supplementary amounts of energy required for detoxification deplete energetic reserves that should otherwise be used to reach spawning areas, jeopardizing migration efforts (Maes et al. 2005). It is of the uttermost importance to study the effects of environmental pollutants, including pharmaceutical drugs, on the metabolic features of A. anguilla such as its antioxidant defense mechanism and detoxification capabilities. Furthermore, taking into consideration that some toxicants may affect the biochemical pathways involved in energy production, this is an important insight into the toxic phenomena. To attain this objective, we quantified levels of lactate dehydrogenase (LDH) activity in muscle tissue of A. anguilla. The impairment of this enzyme’s activity can inhibit energy production and response to stimuli, including capture of prey and escape from predators (Diamantino et al. 2001); on the other hand, its increase indicates that the organism preferentially uses the anaerobic pathway to comply with its energy demands (Li et al. 2009). The importance of this aspect is highlighted by the increase of anaerobic metabolism under low oxygen content, such as deep-sea conditions. LDH quantification allows estimating the metabolic status of organisms, under low levels of oxygen (Moreira et al. 2006). Neurotransmission is a feature that may also be affected by exposure to anthropogenic compounds and can have extreme consequences in organisms subjected to extremely long migratory periods, such as eels. Specific enzymes involved in neurotransmission (e.g., acetylcholinesterase) have already been involved in swimming disturbances reported in fish species, as a consequence of exposure to widespread contaminants, such as pesticides (Pereira et al. 2012). Consequently, the present study aimed to determine the toxicological effects caused by acute paracetamol exposure (at ecological relevant levels) on adult individuals of A. anguilla. The assessed toxicological
Environ Sci Pollut Res
parameters included the quantification of the activities of antioxidant enzymes (glutathione S-transferases, GSTs; catalase, CAT) and lipid peroxidation (thiobarbituric acid reactive substance (TBARS) assay), in both gills and liver tissues. Additionally, we also evaluated the neurotoxicity and respiratory effects by this drug by measuring the activities of acetylcholinesterase (AChE) and lactate dehydrogenase (LDH) in eyes and muscle tissue, respectively.
Material and methods Quarantine, acclimation, depuration, and exposure Organisms used in this study were purchased from a commercial aquaculture facility (Viveiros da Boca Torta, Aveiro). These animals were previously captured at Ria de Aveiro, which is an estuarine area, located in the northwest of the Portuguese shore, circa 42 km long and 10 km wide (Lopes and Silva 2006). This estuary is highly irregular and complex in topography as it is comprised of several canals in contact with the Atlantic Ocean (Leandro et al. 2007); thus, tidal influence is the main driving factor determining water circulation (Morgado et al. 2003). Ria de Aveiro is an estuary of great ecological value. While it is subjected to a considerable degree of deterioration caused by human pressure, especially by industrial and urban sources (Kennish 2002; Lopes 2003; Silva et al. 2002), it is considered a reference site because it is protected as a Special Protection Zone (ZPE, Zona de Proteção Especial; Directive 79/409/CEE 1979) and integrated in the Rede Natura 2000 (Diretive n. 92/43/ CEE 1992). Sixty animals were purchased and were immediately transported to the laboratory facilities in plastic thermal boxes. Upon arrival, animals were subjected to a quarantine, depuration, and acclimation period, for approximately 20 days. Animals were kept in opened plastic boxes, with total capacity of 60 L. Animals were kept in dechlorinated and filtered (5 μm) tap water, at a temperature of 20±1 °C, and a photoperiod of 12hL:12hD. Water was continuously aerated and filtered by mechanical filters. After 1 week of acclimation, animals were fed ad libitum with portions of squid for human consumption. Immediately after feeding, the remaining not ingested portions of squid were removed. Animals during this initial period were apparently healthy, without any apparent sign of stress (e.g., malformation, depigmentation), fulfilling all prerequisites to be used in standard ecotoxicological assays (OECD 1992). After the acclimation period, during which no deaths were registered, 30 animals were randomly selected. These were divided into 6 groups of 5 organisms each, to be exposed to 5 treatments of paracetamol + a control (non-exposed) treatment. The selected paracetamol concentrations were 5, 25,
125, 625, and 3125 μg/L. Paracetamol concentrations were based on ecological relevance criteria; the two first concentrations were close to those already reported for freshwater systems (6–65 μg/L; Ternes 1998; Roberts and Thomas 2006). The two intermediate concentrations were 1 order of magnitude above the observed levels in the wild; the highest concentration was used to test the responsiveness of eels to the oxidant effect of acetaminophen (Antunes et al. 2013). Test solutions were prepared by preparing a stock solution of 500, 000 μg/L in water; this paracetamol concentration was confirmed through colorimetric quantification following an adapted version of the procedure no. 430 by Sigma Diagnostics (Brandão et al. 2014). Test concentrations nominated hereinafter are actual concentrations estimated on the basis of the paracetamol quantification made on the stock solution. Exposure was conducted in agreement with OECD guideline no. 203, for acute testing of fish (OECD 1992), and lasted for 96 h (with media renewal at 48 h); all abiotic conditions were the same as described above. No food was provided during exposure. Immediately after exposure, animals were euthanized by immersion in an ice-cold (4 °C) water bath (Wilson et al. 2009) and immediately sacrificed by decapitation. Animals were then dissected on ice-cold phosphate buffer, and the necessary tissues/organs were collected and frozen at −80 °C. Tissues were dissected for biomarker assays as follows: liver and gills for determination of catalase activity (CAT), gills for determination of glutathione S-transferase activity (GST) and lipid peroxidation (TBARS assay), muscle for lactate dehydrogenase (LDH) activity determination, and eyes for the quantification of acetylcholinesterase activity (AChE). Liver was selected due to its primary detoxification function (Heath 1995) while gills due to their continuous close contact with the external media (Au 2004). The determination of the ezymatic activity of LDH was performed in muscle because it contains especially high amounts of this enzyme (Nunes et al. 2004). Finally, AChE activity was determined in the eyes due to their high enervation and particularly elevated content of this enzyme in the selected species (Nunes et al. 2014a). All tissues (except eyes) were homogenized in 50 mM, pH=7 phosphate buffer, with 0.1 % Triton X-100, and centrifuged at 15,000×g for 10 min at 4 °C. Eyes were homogenized with 0.1 M, pH = 7.2 phosphate buffer, and centrifuged at 6000×g for 3 min at 4 °C. Biomarker assays CAT activity was determined according to the method described by Aebi 1984. CAT is involved in the decomposition of hydrogen peroxide (H2O2), preventing its accumulation inside the cell (Atli et al. 2006) and the onset of oxidative stress. Its quantification was performed by spectrophotometrically monitoring the decomposition of H2O2 at a wavelength
Environ Sci Pollut Res
of 240 nm. CAT activity was expressed as the quantity of the enzyme that catalyzes the conversion of 1 μmol of substrate per min (U/mg of protein). GSTs are a group of isoenzymes of the phase II metabolic pathway that catalyzes the conjugation of the substrate 1chloro-2,4-dinitrobenzene (CDNB) with glutathione, forming a thioether, whose formation can be spectrophotometrically followed by the increment of absorbance at a wavelength of 340 nm, according to Habig et al. (1974). GST activity was expressed by the quantity of the enzyme that catalyzes the conversion of 1 μmol of substrate per min (U/mg of protein). According to the protocol described by Buege and Aust (1978), the extent of lipid peroxidation was measured by the quantification of TBARSs. This methodology is based on the reaction of LPO by-products (Gutteridge 1995; Scoccia et al. 2001), such as malondialdehyde (MDA), with 2-thiobarbituric acid. The amount of TBARS was spectrophotometrically measured at a wavelength of 535 nm (ε=156 mM−1 cm− 1), and results were expressed as nanomoles of MDA equivalents per milligram of protein. AChE is a carboxylesterase of the B type that promotes the hydrolysis of acetylcholine, a neurotransmitter fundamental for the neuromuscular transmission. AChE acts by cleaving acetylcholine into thiocholine and acetic acid (Ellman et al. 1961). The predominant form of cholinesterase found in tissues of A. anguilla is acetylcholinesterase, as shown by Valbonesi et al. (2011). AChE activity was spectrophotometrically monitored according to the method described by Ellman et al. (1961), at a wavelength of 412 nm. Lactate dehydrogenase (LDH) is a cytosolic enzyme involved in the glicolytic metabolism in the majority of tissues. Determination of its activity was performed following the technique described by Vassault (1983), by spectrophotometrically measuring the reduction of absorbance caused by the oxidation of the reduced form of nicotinamide adenine dinucleotide (NADH) in the presence of pyruvate, at a wavelength of 340 nm. Quantification of total soluble protein Protein concentration was determined according to the spectrophotometric (wavelength of 595 nm) method of Bradford (1976), based on the reaction that occurs between Coomassie Brilliant Blue G-250 with soluble proteins, adapted to microplate, using bovine serum albumin (BSA) as standard. Statistical analyses Biochemical parameters were statistically analyzed with oneway ANOVA, followed by the Dunnett’s multicomparison test to discriminate significant differences between toxicant concentrations and the control treatment (Dunnett 1955). The adopted level of significance was of 0.05.
Results Animals exposed to paracetamol had an average weight of 35.63±0.05 g and an average length of 27.69±0.05 cm. During exposure, a single death was registered, among organisms exposed to the concentration of 5 μg/L, at the final day of the experiment. No significant effects were observed in terms of catalase activity. However, a slight albeit non-significant increase was observed for the lowest tested concentrations, and for both tissues, liver (F=1.33, df=5.23, p=0.287; Fig. 1a), and gills (F=0.92, df=5.23, p=0.490, Fig. 1b). GST significantly increased in liver between the control and the two highest concentrations of paracetamol (625 and 3125 mg/L; F=8.92, df=5.23, p=8.07E−5; Fig. 2a). However, GST activity significantly decreased in the gills, especially for the highest tested concentration (F = 12.59, df= 5.23, p = 5.86E −6 ; Fig. 2b; no observed effect concentration (NOEC) =25 μg/L; lowest observed effect concentration (LOEC)=125 μg/L). TBARS levels remained unaltered in liver tissue following paracetamol exposure, as shown in Fig. 3a (F=1.53, df=5.23, p=0.220). In gills, it was possible to observe an increase in lipid peroxidation that was significant for the highest tested concentration of paracetamol (F=4.58, df=5.23, p=0.005; Fig. 3b; NOEC=625 μg/L; LOEC=3125 μg/L). Data analysis concerning LDH activity did not show any statistical significant difference in comparison with control animals LDH (F=2.19, df=5.22, p=0.090; Fig. 4). The biomarker AChE was not significantly changed after exposure to paracetamol (F=2.04, df=5.22, p=0.112), despite a significant decrease in its activity for an intermediate concentration (625 μg/L) which is of uncertain biological meaning (Fig. 5).
Discussion The main role attributed to catalase is the degradation of hydrogen peroxide, consequently protecting organisms from its oxidant deleterious effects. Whenever molecular oxygen is used in aerobic processes, and in a more prominent way during the occurrence of oxidative stress, catalase may be overexpressed and its activity may rise, in response to the increased levels of oxygen peroxide (Oruç et al. 2004). Thus, it is possible to assume that a catalase activity increase may signal a compensatory physiological response to oxidative alterations (van der Oost et al. 2003). In the present study, such a response was not registered, since no significant modifications were reported concerning CAT activity, indicating that exposure to this drug, under the selected conditions, was not capable of inducing a response both in liver and in gill tissues. This set of responses contradicts previous findings by
Environ Sci Pollut Res Fig. 1 Effects of paracetamol on catalase (CAT) activity of A. anguilla. Results are expressed as mean±standard deviation, corresponding to the average of five replicates. a Liver. b Gills. Data were analyzed with a one-way ANOVA, followed by the Dunnett test
other authors, which obtained significant pro-oxidative responses after exposing other fish species, such as Oreochromis mossambicus, to paracetamol (Kavitha et al. 2011). A somewhat similar response, with significant effects in terms of catalase enhancement, was also reported by Shivashri et al. (2013); this study described the oxidative effects of paracetamol on the freshwater fish species Pangasius sutchi. Additional evidence pointing to an involvement of the oxidative pathway in aquatic organisms exposed to this drug were given by Ramos et al. (2014) and Parolini et al. (2010) with the mollusk species Corbicula fluminea and Dreissena polymorpha, respectively. Despite the lack of clear indicators suggesting the occurrence of oxidative-related effects in our study (namely by alteration of GST or CAT activities), it is possible to conclude that paracetamol may exert toxicity via this specific pathway, but only in sensitive species or under favorable conditions that enhance uptake or long durations of exposure. Similarly to what was postulated for the enzyme catalase, the isoenzymes glutathione S-transferases may also exert an antioxidant effect. By facilitating the conjugation with the biologically active reduced form of glutathione, this group of enzymes is of the uttermost importance in the detoxification
of xenobiotics, since they augment the biotransformation and excretion rates of numerous compounds, reducing their overall toxicity (Habig et al. 1974). Furthermore, these enzymes are part of an antioxidant system capable of converting reactive oxygen species (ROS) into less damaging substances, but at a high cost: by doing so, these enzymes deplete the cofactor reduced glutathione (GSH); when this cofactor is no longer present at the intracellular level, oxidative damage is likely to occur (Vlahogianni et al. 2007). At higher dosages, however, a paradoxical effect may occur, with impairment of the catalytic activity of GSTs; low levels of exposure did not cause any significant effects (Manimaran et al. 2010; Brandão et al. 2014). The here-obtained results, on the contrary, showed a significant increase of these enzymes’ activity and for the highest tested concentrations of paracetamol. This effect was particularly evident in liver tissue, implicating this specific organ in the biologic response to paracetamol. Gill tissue evidenced a contrary pattern, but only for concentrations of 125 and 3125 μg/L of paracetamol. According to Kavitha et al. (2011), fish gills are characterized by lower GST activity when compared to liver, only responding when faced with higher levels of contamination. The most likely effect may be connected to an increase of the antioxidant capacity of liver,
Environ Sci Pollut Res Fig. 2 Effects of paracetamol on glutathione S-transferases (GSTs) activity of A. anguilla. Results are expressed as mean±standard deviation, corresponding to the average of five replicates. a Liver. b Gills. Data were analyzed with a one-way ANOVA, followed by the Dunnett test; *p
