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Keywords:

  • Corbicula fluminea;
  • paracetamol;
  • biomarkers;
  • oxidative stress;
  • bivalve species

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. REFERENCES

The Asian clam Corbicula fluminea is an invasive bivalve that has recently spread in Europe and currently represents a large portion of the aquatic biomass in specific areas. Because of the impacts that the species may have in invaded ecosystems, increased knowledge on the physiologic features of the species life-cycle under different environmental scenarios (e.g., contamination events) is critical to understand the dynamics of the invasion and resulting ecosystem imbalance. The presence of pharmaceutical residues in the aquatic environment has recently received great attention since high levels of contamination have been found, not only in sewage treatment plant effluents, but also in open waters. The present article reports toxicological biochemical effects of paracetamol to Corbicula fluminea following short- and long-term exposures. Oxidative stress parameters were specially focused namely catalase (CAT), glutathione S-transferases (GSTs), and glutathione reductase (GRed). The effect of tested substances on lipid peroxidation was also investigated. Paracetamol did not induce alterations on CAT activity, caused a significant decrease of GSTs activity following short- and long-term exposure (LOEC values of 532.78 mg L−1 and 30.98 μg L−1, respectively), and was responsible for a significant and dose-dependent decrease of GRed activity in short- and long-term exposures. These results indicate that exposure to paracetamol can provoke significant alterations on the cellular redox status of C. fluminea. 2011 Wiley Periodicals, Inc. Environ Toxicol 29: 74–83, 2014.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. REFERENCES

During the last decades, many pharmaceutical residues (PhaR) have been detected in the aquatic environment (Winker et al., 2008); sewage effluents and wastewater in the proximity of sewage treatment plants (STPs), surface and ground water, and drinking water (Ganiyat, 2008; Yang et al., 2008) have been reported to be contaminated. Pharmaceuticals commonly found in aquatic environments include antibiotics, anticonvulsants, antipyretics, cytostatic drugs, and hormones. These compounds reach water bodies through various pathways, such as direct disposal of domestic surplus drugs, excretion in faeces/urine after therapeutic use, and inadequate treatment of effluents from the manufacturing industry (Ganiyat, 2008; Yang et al., 2008). Additional sources include treatment and prophylaxis through the water in fish farms (aquaculture) (Brillas et al., 2005; Thomas et al., 2007), and livestock treatment and pet care (Thomas et al., 2007).

Pharmaceutical drugs, and personal care products, are somewhat particular when considering anthropogenic chemicals that can be found in the aquatic compartment. These substances have a widespread environmental distribution, as a consequence of their continuous use by human populations. Pharmaceuticals have biological activity, which is the most important parameter to be considered when evaluating their toxicological impact in the wild (Halling-Sörensen et al., 1998; Daughton and Ternes, 1999; Jones et al., 2002; Miao et al., 2002). Moreover, these compounds are specifically designed to resist metabolic degradation, and high lipophylicity is generally a basic requirement to maximize absorption by target organisms. A variety of metabolites may be formed and toxicological interactions are very likely to occur among PhaR in the wild (Cleuvers, 2003). Therefore, a considerable part of these xenobiotics may be considered as active, effective, and persistent environmental contaminants.

One the most extensively used drugs worldwide is paracetamol (N-acetyl-p-aminophenol, also known as acetaminophen) (Yang et al., 2008; Lourenção et al., 2009; Solé et al., 2010). Paracetamol is an acylated aromatic amide that has been considered safe (Xu et al., 2008) for human therapeutics and effective when used as an antipyretic/analgesic drug. Paracetamol has been found in concentrations of up to 6 μg L−1 in European STP effluents (Ternes, 1998), up to 10 μg L−1 in natural waters in USA (Kolpin et al., 2002), and above 65 μg L−1 in the Tyne River, UK (Robert and Tomas, 2006).

Hepatotoxic effects of paracetamol may result from overdose, and were documented in experimental animals and humans (Prescott, 1980; Jaeschke et al., 2003; Hinson et al., 2004; Jaeschke and Bajt, 2006; Brind, 2007; Xu et al., 2008). Normal therapeutic doses of paracetamol may undergo conjugation with co-factors, forming the non-toxic conjugated metabolites acetaminophen glucuronide and acetaminophen sulphate (Patel et al., 1993; Klaassen, 2001; Jaeschke and Bajt, 2006; Xu et al., 2008). These detoxification pathways account for over 90% of the paracetamol metabolism by the liver. Under normal conditions, less than 10% of paracetamol is metabolized by the cytochrome P450 enzymes (primarily CYP 2E1, 1A2, and 3A4) producing the toxic metabolic intermediate N-acetyl-p-benzoquinoemine (NAPQI) (Xu et al., 2008). The small amount of NAPQI formed conjugates with intracellular glutathione for detoxification (Prescott, 1980; Patel et al., 1993; Klaassen, 2001; Xu et al., 2008). At high drug load and/or low intracellular glutathione reserve, the highly reactive and electrophilic NAPQI can exert multiple toxic effects, such as covalent modifications of thiol groups on cellular proteins (Xu et al., 2008), DNA and RNA damage, and oxidation of membrane lipids, resulting in necrosis and cellular death (Prescott, 1980; Jaeschke et al., 2003; Hinson et al., 2004; Jaeschke and Bajt, 2006).

As shown, oxidative stress should have a role on the mechanism of toxicity of paracetamol. Following oxidative stress, adaptive responses of the protective (antioxidant) systems, modification of cellular macromolecules, and ultimately tissue damage, may occur. Changes in oxidative systems and modified macromolecules can hence serve as biomarkers of exposure to paracetamol. Measures of oxidative stress in different non-target species (vertebrates and invertebrates) have been used as indicators of adverse effects in contaminated aquatic habitats (Livingstone, 2001; Conners, 2004; Nunes et al., 2006, 2008). In this way, oxidative stress in bivalve species following exposure to different xenobiotics has been fairly studied (Vidal et al., 2001; Conners, 2004; Solé et al., 2010). However, little is known about the overall impact of pharmaceutical drugs in these invertebrates. Thus, the purpose of the present study was to evaluate the effects of short- and long-term exposures to paracetamol on biomarkers of oxidative stress in Corbicula fluminea. Three key enzymes were selected as indicative parameters of toxic stress, namely (1) glutathione reductase (GRed), (2) glutathione S-transferases (GSTs), (3) catalase (CAT). Lipid peroxidation (thiobarbituric acid reactive substances, TBARS) was also addressed as a measure of peroxidative damage resulting from oxidative stress.

The Asian clam Corbicula fluminea is a freshwater infaunal bivalve originating from Southeast Asia that has massively spread worldwide over the last century (Araujo et al., 1993), being nowadays considered one of the 100 most concerning non-indigenous invasive species (DAISIE, 2008). Life-history traits such as rapid growth rates, early sexual maturity, short life span, high fecundity, as well as high filtration rates, feature the Asian clam invasive potential (Stryer, 1999; Sousa et al., 2008a), hence this species ability to rapidly become a major component of benthic communities in invaded ecosystems (Sousa et al., 2008b). Any alteration in its population dynamics caused by pollution may translate into changes at the ecosystem level being thus a matter of concern. C. fluminea has high adaptation capabilities towards environmental variations and it is known to accumulate various pollutants such as metals or policyclic aromatic hydrocarbons (Doherty, 1990 ; Inza et al., 1997; Cataldo et al., 2001; Vidal et al., 2001; Diniz et al., 2007). Given its bioaccumulation abilities, C. fluminea has been considered a promising sentinel species for the biomonitoring of freshwater ecosystems. Detailed assessment of biomarker responses after exposure to different environmental contaminants (Vidal et al., 2001) applies to the comprehensive study of this ability, regardless the contamination scenario. It should be noticed in addition that C. fluminea is currently being used as a test species responding to water contamination, for instance, in the national French program ECODYN (Diniz et al., 2007). Furthermore, C. fluminea is a convenient model in toxicological studies provided the mentioned features as well as considering that the species is of easy maintenance and testing under laboratory conditions, and can be used as an adequate representative of freshwater benthic bivalves. In this way, this studyaimed specifically: (i) to characterize the response of C. fluminea to anthropogenic contaminants using an oxidative stress biomarker approach; (ii) to assess the potential toxicological effects of paracetamol on C. fluminea.

MATERIAL AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. REFERENCES

Corbicula fluminea Rearing

A batch of C. fluminea (Müller, 1993) individuals was randomly collected in a Portuguese river (the River Minho) using a trawl net. The clams were transported to the laboratory and were kept under laboratory-controlled conditions for 2 weeks prior testing. Clams were reared in plastic trays over a sand substrate (quartz sand) under 2 cm of dechlorinated tap water. Rearing trays were kept at constant temperature (20 ± 1)°C, photoperiod of 16 hL:8 hD and continuous aeration. Two sets of rearing trays were established according to the size of the clams (maximum shell length: less than or equal to 2 ± 0.05 cm; larger than 2 ± 0.05 cm).

A preliminary experiment was carried out to test the nutritional needs of C. fluminea, i.e., the optimal feeding conditions, using the microalgae Pseudokirchneriella subcapitata. Forty-five individuals were selected (shell length: 2–2.5 ± 0.05 cm), and distributed by three replicated test containers filled with 200 mL dechlorinated tap water per treatment with five animals (n = 5) each. Treatments consisted in providing clams with different amounts of P. subcapitata cells (2.12 × 108, 3.18 × 108, and 4.24 × 108 cells mL−1, expressed as final number of algal cells per treatment) and were kept under continuous aeration. Algal suspensions were obtained from our batch culture, which has been kept continuously in the laboratory following the protocol by Stein (1973). In order to monitor the daily requirements of the clam culture in terms of P. subcapitata cells, daily consumption of algae was estimated. The test vessels were agitated to prevent measurement error related to algae sedimentation on the bottom of the vessel, and a 400-μL sample volume was withdrawn and preserved with a Lugol solution for further cell counting (1 sample per replicate). Sample collection was performed daily during a period of 5 days. Cell counting was performed in a haemotocytometer (Neubauer improved; Marienfeld, Germany). We concluded that a C. fluminea individual requires about 2.15 × 105 cells mL−1 every 2 days, since half of this ration was in average consumed by the clams on a daily basis.

Exposure Conditions

Stock Solutions

Paracetamol (CAS number 103-90-2) was acquired from Sigma-Aldrich (Germany) in a purity degree of 98.0–101.0%. Test solutions used in both the acute and the chronic exposures were prepared by dilution of paracetamol stock solutions, which were freshly prepared prior testing in dechlorinated tap water. Nominal concentrations of the stock solutions were 1000, 500, and 100 mg L−1. These paracetamol concentrations were confirmed through colorimetric quantification following an adapted version of the procedure no. 430 by Sigma Diagnostics. Test concentrations nominated hereinafter are actual concentrations estimated on the basis of the paracetamol quantification made on stock solutions.

Clam 96 h Exposure

Tests were performed under laboratory-controlled conditions as stated for the rearing procedure. C. fluminea (2–2.5 cm shell length, which was the most abundant size class in the obtained sample and represents adult clams) were individually exposed for 96 h to paracetamol in plastic bottles, filled with 100 mL dechlorinated tap water continuously aerated. Tests were performed following an adapted version of the Ecological Effects Test Guidelines: OPPTS 850. 1710. Oyster BCF (EPA, b ). Ten replicates were used per treatment and treatments consisted of a blank control plus five concentrations of paracetamol. Test concentrations were: 0 (control), 0.05, 0.48, 4.82, and 532.78 mg L−1 paracetamol. Test solutions were renewed after a 48 h exposure-period. Each animal was used to quantify all the selected biomarkers. Measurements of pH, temperature, and dissolved oxygen were monitored every 24 h, for test validation purposes.

Clam 28 Days Exposure

Long-term exposures were carried out similarly to what was previously described for short-term testing. Animals were fed with a suspension of P. subcapitata at the previously optimized rate of 2.15 × 105 cells mL−1 every 2 days. Test concentrations were: 0 (control), 3.88, 7.74, 15.49, 30.98, 61.95 μg L−1 paracetamol. The test period was 28 days and test solutions were renewed every other day. Mortality was checked on a daily basis.

Biochemical Assays

After the exposure period, living animals were sacrificed and homogenized in 2 mL of ice-cold phosphate buffer with rotary homogenizer, for 3–6 s at medium intensity. 50 mM, pH = 7.0 with 0.1% TRITON X-100 phosphate buffer was used in the preparation of homogenates for the determination of CAT, GRed, GSTs, and TBARS. Homogenized tissues were centrifuged at 15,000 G for 10 min and supernatants were divided into aliquots, to be further used for the different enzymatic determinations. Homogenized tissues were stored at −80°C until the performance of enzymatic determinations.

CAT activity was measured by the decomposition of H2O2 to water and O2 that can be followed by the decrease of absorbance at 240 nm as described by Aebi (1984). To maximize the effectiveness of this test with C. fluminea, the supernatant resulting from the homogenization of the animal was diluted 1:20. Changes in sample absorbance were spectrophotometrically monitored at 240 nm for 30 s, and activities were expressed as μmol H2O2 consumed per min per mg protein.

GSTs activity was determined by spectrophotometry, according to Habig et al. (1974). To maximize the effectiveness of this test with C. fluminea, the supernatant from the homogenization of the animal was diluted 1:40. GSTs catalyse the conjugation of the substrate 1-chloro-2,4-dinitrobenzene (CDNB) with glutathione, forming a thioeter that can be followed by the increment of absorbance at 340 nm. Results were expressed as nanomoles of thioeter produced per minute, per mg protein.

GRed activity was determined by spectrophotometry, according to the protocol by Carlberg and Mannervik (1985). The appropriate dilution for the determination of this enzyme in C. fluminea was 1:1. In this assay, the GRed mediated oxidation of NADPH was monitored at 340 nm. Enzymatic activity was expressed as nanomoles of NADPH oxidized per minute and per mg protein.

The extent of lipid peroxidation was measured by the quantification of TBARS, according to the protocol described by Buege and Aust (1978). Samples were diluted 1:1. TBARS were expressed as malondialdehyde (MDA) equivalents, calculated using an extinction coefficient of 1.56 × 105 M−1 cm−1. This methodology is based on the reaction of compounds such as MDA (formed by degradation of initial products from lipid membranes by free radical attack), with 2-thiobarbituric acid (TBA).

Protein concentration of samples was determined for every sample dilutions according to the protocol by Bradford (1976), in order to express enzymatic activities taking into account the protein content of the analysed tissues.

Statistical Analysis

A one-way ANOVA was run for each endpoint, followed by the Dunnet multicomparison test to discriminate significantly different chemical treatments relatively to control and determine the lowest observed concentration (LOEC) values whenever applicable. A significance level of 0.05 applied to all analyses.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. REFERENCES

No mortality was observed either in the short- or in the long-term exposures of C. fluminea to paracetamol. In the short-term exposure, we tested an additional paracetamol concentration of 5000 mg L−1; however, this concentration was eliminated from data analysis since a mortality rate of 100% was observed following exposure, thus corresponding to a lethal concentration.

Catalase

Following both short- and long-term exposures to paracetamol, CAT did not respond according to a clear dose-response pattern, generally showing maximum activities at treatments other than the control [see e.g., 0.48 mg L−1 and 30.98 μg L−1 in Fig. 1(A,E) respectively]. The fluctuating pattern of response did not translate into significant differences between CAT activity of whole body homogenates of C. fluminea in the control and chemical treatments [short-term exposure: F = 1.26; df = 4, 39; p = 0.30; Fig. 1(A); long-term exposure: F = 0.76; df = 5, 46; p = 0.58); Fig. 1(E)].

image

Figure 1. Acute (left-hand panel) and chronic (right-hand panel) effects of acetominophen on the activity of different antioxidant parameters of C. fluminea homogenates: catalase activity (A,E), GSTs activity (B,F), GRed activity (C,G), and TBARS content. Values correspond to the mean of three replicated assays and error bars represent standard error. *Significant differences, p < 0.05.

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Glutathione S-Transferases

Short-term effects of paracetamol included a significant decrease of GSTs activity in the whole body homogenates of C. fluminea [F = 20.09; df = 4, 40; p < 0.001; Fig. 1(B)] with a LOEC of 0.48 mg L−1. Long-term exposure was responsible for a significant decrease of GSTs activity in the homogenates of C. fluminea [F = 3.51; df = 5, 54; p = 0.008; Fig. 1(F)] and a LOEC of 30.98 μg L−1 was found.

Glutathione Reductase

Short-term effects of paracetamol in homogenates of C. fluminea included a significant and dose-dependent decrease of GRed activity [F = 8.43; df = 4, 45; p < 0.001; Fig. 1(C)]. It should be noticed that at 532.78 mg L−1 this monotonic trend in GRed activity was reversed. Conversely, the GRed activity after chronic exposure fluctuated following increasing paracetamol concentrations, and did not differ significantly from control in any treatment [F =1.41; df = 5, 50; p = 0.237; Fig. 1(G)].

Lipid Peroxidation

Short-term exposure to paracetamol [Fig. 1(D)] caused an increase in lipid peroxidation in homogenates of C. fluminea, which was significant for the two highest concentrations [F = 15.97; df = 4, 45; p < 0.001; Fig. 1(D)]; a LOEC of 4.82 mg L−1 was found. Indeed, the biomarker in these treatments was responsive, and TBARS concentration increased more than three times relatively to control. Noxious effects of long-term exposure to paracetamol [Fig. 1(H)] were significant [F = 15.07; df = 5, 54; p < 0.001; Fig. 1(H)] and evidenced by: (i) a decrease in lipid peroxidation in homogenates of C. fluminea, significant for the two lowest concentrations with a LOEC lower than or equal to 3.88 μg L−1; and (ii) an increase in lipid peroxidation following exposure to 30.98 and 61.95 μg L−1 paracetamol, which was found statistically significant at 61.95 μg L−1.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. REFERENCES

CAT is a heme-containing enzyme that decomposes hydrogen peroxide into water and molecular oxygen. The formation of hydrogen peroxide is a common feature under oxidative stress scenarios, due to the degradative activity of an upstream enzyme, superoxide dismutase (SOD), in the oxidative defense cascade. SOD is responsible for the degradation of the superoxide radical, which is amongst the most unstable and reactive forms of oxygen that can result from oxidative insult. Thus, increased activity of CAT may result from higher, oxidative-derived levels of hydrogen peroxide. Our study showed that exposure of C. fluminea to paracetamol did not cause any significant change in CAT activity, following both short- and long-term exposures. This suggests that the catalytic degradation of hydrogen peroxide, one of the common steps of oxidative stress, was not accompanied by a rise in this enzyme's activity and/or the formation of hydrogen peroxide was not favored by exposure to paracetamol. In spite of this apparent paradox, similar results were already described in the literature. The study by Orhan and Sahin (2001) showed that exposure of human erythrocytes to paracetamol did not cause any measurable modification in CAT activity. Other authors, such as Olaleye and Rocha (2008) and Manimaran et al. (2010) reported significant decreases of CAT activity in paracetamol-exposed mice and rats. However, the likely establishment of an oxidative stress scenario should have been accompanied by a rise in antioxidant enzymes, including CAT, as described by Parolini et al. (2010) for the zebra mussel (Dreissena polymorpha). This particular enzyme (CAT) has been used as effective criteria in previous studies using C. fluminea as test organism, but not focusing specifically on pharmaceutical compounds: the study conducted by Oliveira et al. (2007) showed that CAT was responsive to the presence of lead, being significantly increased after exposure. In fact, the assessment of this toxicological parameter in C. fluminea was shown to be more responsive than in a freshwater crustacean species, Dilocarcinus pagei, as shown in the same study.

GSTs are a major group of xenobiotic detoxifying enzymes, and belong to a family of multifunctional enzymes involved in catalyzing nucleophilic attack of the sulfur atom of glutathione (γ-glutamyl-cysteinylglycine) to an electrophilic group on metabolic products or xenobiotic compounds (Blanchette et al., 2007). GSTs activity was significantly impaired after short- and long-term exposure to paracetamol, albeit apparently not according to a coherent or expected pattern: significant decrease in GSTs activity was recorded following both exposures. This is a major finding, since the impairment of a phase II metabolic pathway (conjugation of a xenobiotic with GSH), accounted for the majority of paracetamol metabolism, can only result in increased toxicity. It is of high toxicological importance if one considers that the described metabolism of paracetamol for most animal models involves the conjugation of paracetamol metabolites (namely NAPQI) with GSH, through the involvement of the isoenzymes GSTs (Allameh and Alikhani, 2002); and that paracetamol derived toxicity usually occurs through GSH depletion (Reicks and Crankshaw, 1993). The most reactive species (NAPQI), which is formed after in vivo oxidative degradation of paracetamol, is conjugated with the glutathione molecule (GSH), due to the activity of the mentioned phase II metabolic enzyme. GSH acts thus as an important scavenger for the oxidant metabolite, preventing the onset of oxidative damage. Paracetamol (in doses ranging from 90 to 150 mg kg−1) increased the activity of serum GSTs and reduced the activities of liver GST in mice, as described by Wang and Peng (1993). These results suggested that the conjugation levels in liver tissue were decreased in mice paracetamol-induced hepatotoxicity. Manimaran et al. (2010) reported that paracetamol toxic effects were dose-dependent, i.e., lower doses did not alter GSTs activities, but at higher doses significantly decreased enzymatic activity was observed. The potential binding of paracetamol to GSTs proteins is also a possibility to justify the decreased activity of these enzymatic forms. As shown by Wendel and Cikryt (1981), a significant portion of metabolites formed after paracetamol degradation in mouse liver is bound to GSTs.

The tripeptide glutathione (GSH) participates in many important processes including detoxification of hydrogen peroxide, protein maintenance, synthesis of DNA precursors, and detoxification of metals and organic xenobiotics (Conners, 2004). Numerous reactions involve GSH as a reducing agent, and in most cases, GSH is converted into its oxidized disulfide form, GSSH. The enzyme responsible for converting the oxidized glutathione (GSSH) back to the reduced form is GRed, an intracellular enzyme occurring as ubiquitously as GSH itself. Paracetamol was clearly involved in the inhibition of GRed activity in our study, with the establishment of a concentration-dependent pattern. This finding is also toxicologically noteworthy, since impairment of GRed activity may challenge the antioxidant status of the organisms: the amount of GSH formed by reduction of the intracellular pool of oxidized glutathione may not be sufficient to cope with the amount of reactive oxygen species formed during oxidative stress periods, with deleterious consequences for the organism's physiology. Our results are in agreement with results previously obtained for rats, where paracetamol caused significant decrease in GRed activity at 420 mg kg−1 (Manimaran et al., 2010). However, and contrarily to these findings, Adamson and Harman (1989) reported that paracetamol induced the activity of GSH-Px/GSSG-Red in hepatocytes extracted from 2-week-old mice. According to this latter study, paracetamol toxicity was dependent on a complex activating metabolic enzymatic system, composed by glutathione peroxidase and GRed. It is thus possible to suggest a metabolic activation of paracetamol at least partly derived from the activity of GRed. Similar results were obtained by Kumar et al. (2005) after exposing rats to paracetamol, with significant inhibition of several antioxidant enzymes, including GRed. GRed inhibition following overdosage administration of paracetamol was also reported by Kozer et al. (2003), when following the oxidative status of children orally exposed to different amounts of this drug. Authors observed also a reduction in the effectiveness of the overall antioxidant status, making exposed children more susceptible to oxidative hepatic injury if higher doses are further used in therapeutics. On the contrary, exposure of rats to paracetamol has already evidenced a strong increase in GRed, consistent with a strong pattern of activation of the oxidative defense, as shown by Aouacheri et al. (2009). The general absence of data relating oxidative-based effects in invertebrate species, following exposure to pharmaceutical drugs, hardens the establishment of causal relationships justifying specific effects, such those here-described. However, and despite differences between the mammals and C. fluminea, the toxic effects of paracetamol are mediated via interference with glutathione metabolism. It is thus not difficult to ascertain that GRed impairment constitutes a decisive drawback for the antioxidant mechanism of any test species, with obvious deleterious consequences to the organism.

In spite of the antioxidant physiological mechanisms devoted to cope with reactive oxygen species, oxidative damage is sometimes likely to occur, due to extensive production of reactive species or reduced protective capacity of the organism. When free radicals formed in conditions of oxidative stress attack biological structures some compounds (such as malondialdehyde, MDA) are produced. The extent of lipid peroxidation can be measured through assessment of TBARS levels, which are mostly comprised by MDA (Nunes et al., 2006). Oxidative insult was a major outcome of the short- and long-term exposure of C. fluminea to the selected chemical, as reflected by the significant increase in lipid peroxidation shown by higher TBAR levels with increasing paracetamol concentrations. This might have occurred as a direct consequence of significant modifications in terms of GSTs and GRed activities; the absence of effective antioxidant mechanisms may be, at least partly, responsible for oxidative damages. Fairhurst et al. (1982) already evidenced the toxic effects of paracetamol, with the involvement of peroxidative membrane damage, when treating rodents with high doses of this chemical. In our case, a similar situation occurred, pointing to a potential oxidative outcome. The here-exposed organisms were more prone to damage after impairment of scavenging protective mechanisms, such as GSTs and GRed activities. Our results are in agreement with previous studies where paracetamol caused significant increase of lipid peroxidation in rats and mice (Ozdemirler et al. 1994; Olaleye and Rocha, 2008; Manimaran et al., 2010). Our data are also in line with the conclusions by Solé et al. (2010), where a consistent relationship between exposure to paracetamol and effects over enzymatic biomarkers in marine mussels is reported. They showed that paracetamol exposure did not cause any significant changes in CAT activity in digestive gland, while LPO was significantly increased. The work developed by Parolini et al. (2009, 2010) also evidenced significant physiological oxidative alterations caused by paracetamol in Dreissenia polymorpha.

In spite of a general absence of data concerning the mechanisms by which antioxidant defences are regulated in aquatic organisms, Correia et al. (2003) state that invertebrates share common differences when compared to vertebrates. Several antioxidant enzymatic activities, such as GPx of invertebrates (e.g., crustaceans) are 1 to 2 orders of magnitude lower than GPx of vertebrates; on the contrary, SOD activities are usually similar or even higher, when compared to SOD of vertebrates. The oxidative metabolism, mediated in most organisms by the cytochrome P450 super family of enzymes, is not well developed in aquatic invertebrates (Snyder, 2000), contributing to significant differences in toxicity development. These intrinsic differences between vertebrates and invertebrates indicate the physiological role attributed to distinct antioxidant defences. The interpretation of biochemical data after laboratory exposures of aquatic animals to several classes of contaminants is, according to the same review, difficult, due to the existence of transient rises of enzymatic activities, varying from species to species. Biomarker activities can also vary according to several factors, such as intrinsic characteristics of the tested organism (e.g., tissue analyzed, age/developmental stage, and diet type) and also external/environmental aspects (e.g., water temperature, oxygen concentration, pH; geographic location, season) (Domingues et al., 2010). Paracetamol exposure of invertebrates is related to oxidative alterations mediated by free radicals in experimental animals, as shown by Solé et al. (2010). These authors demonstrated that paracetamol exposure was responsible for elevated lipoperoxidation in mussels' digestive gland, but did not enhance CAT activity. Administration of 250–300 mg kg−1 paracetamol to mice induces oxidative stress in the liver and kidney, as shown by several classic biomarkers including lipid peroxidation products and changes in antioxidant enzyme activities (i.e., SOD, CAT, GST, GSH-Px, cytochrome P450) (Ghosh and Sil, 2007; Olaleye and Rocha, 2008). In spite of the specificity of these tissues as to paracetamol-induced damage, our study focused primarily on the use of whole body homogenates for the quantification of toxic effects. Thus, the patterns of response obtained in the present study, albeit significant in some cases, are somewhat diffuse and do not indicate the target organs of toxicity. At the same time, the contribution of structures involved in the primary line of chemical defense, such as gills, was not considered; this may be important, since such structures are biologically active, and were shown to be determinant in terms of oxidative stress response. This may be considered a limitation of the study that may be surpassed in further studies by separately analyzing organs or tissues.

In spite of having found significant results in terms of oxidative stress parameters, our data must not be interpreted strictly in terms of ecological relevance. Our experimental animals were short- and long-term exposed to concentrations considerably above those actually found in the environment. In fact, the ranges that have been found in aquatic ecosystems are in the ng L−1 or μg L−1 ranges (Ternes, 1998; Kolpin et al., 2002; Robert and Thomas, 2006), therefore, well below the μg L−1 or mg L−1 concentration ranges are used in our experiments. However, this study adopted the objective of studying specific oxidative stress responses of C. fluminea subjected to sub-lethal concentrations of paracetamol. This objective was satisfactorily attained, since C. fluminea was somewhat responsive to oxidative alterations. Furthermore, in long-term exposures to reduced paracetamol concentrations C. fluminea kept its responsiveness. These considerations are in line with the major conclusions by Geret et al. (2010), when studying the combined effects of a complex mixture of pharmaceutical compounds in C. fluminea. This study showed that a combination of several drug compounds, including paracetamol, was capable of exerting significant effects on differential protein expression of the Asian clam, underlining the potential role of this mollusk in environmental assessment of PhaR exposure. C. fluminea was also considered a sensitive species in terms of genetic damage, as described by Black and Belin (1998), when studying the impact of anthropogenic contamination (urban and industrial stormwater runoff).

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. REFERENCES

Exposures of C. fluminea to paracetamol did not cause any significant change in CAT activity, suggesting that the catalytic degradation of hydrogen peroxide, one of the common steps of oxidative stress defense, was not accompanied by a rise in this enzyme's activity. The conjugation capacity was significantly impaired following acute and chronic exposure to paracetamol, albeit not according to a coherent pattern. This is an important finding if one considers that glutathione conjugation is the main described mechanism by which paracetamol is excreted from most animal models. Impairment of conjugation with glutathione may have direct consequences in terms of the antioxidant defense, reflected by a significant decrease in GRed activity, which followed a concentration dependence pattern. Oxidative insult was a major outcome of short- and long-term exposure to the selected chemical (significant increase in peroxidation, with higher TBAR levels), and this may have occurred since organisms were more prone to damage after impairment of scavenging protective mechanisms, such as GSTs and GRed activities.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. REFERENCES
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