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Effects of the antidepressant fluoxetine on the immune parameters and acetylcholinesterase activity of the clam Venerupis philippinarum
Marine Environmental Research 94 (2014) 32e37
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Effects of the antidepressant fluoxetine on the immune parameters and acetylcholinesterase activity of the clam Venerupis philippinarum Marco Munari, Maria Gabriella Marin, Valerio Matozzo* Department of Biology, University of Padova, Via Ugo Bassi 58/B, 35131 Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 October 2013 Received in revised form 15 November 2013 Accepted 20 November 2013
Fluoxetine is an antidepressant used worldwide for the treatment of depression and other psychological disorders. The occurrence of fluoxetine in aquatic environments has been demonstrated. However, there is a lack of information about the effects of fluoxetine on non-target species, such as bivalve molluscs. In the present study, the effects of fluoxetine on the immune parameters of the clam Venerupis philippinarum were evaluated for the first time. Clams were exposed to various sublethal concentrations of fluoxetine (0, 1, 5, 25, 125, 625 mg l1) for 7 days, and the effects on the total haemocyte count (THC), the diameter and volume of haemocytes, haemocyte proliferation, Neutral Red uptake (NRU), and lysozyme activity in cell-free haemolymph (CFH) were evaluated. In addition, acetylcholinesterase (AChE) activity was measured in clam gills as a biomarker of neurotoxicity. A significant increase in THC values was observed in clams exposed to 25 mg l1 compared with controls, whereas no significant variations were recorded in either the diameter or the volume of haemocytes. Haemocyte proliferation increased significantly in animals exposed to 25, 125 and 625 mg l1 compared with controls. NRU decreased significantly in the haemocytes of clams exposed to 1 or 5 mg l1, whereas NRU returned to control values in clams exposed to the highest fluoxetine concentrations tested (25e625 mg l1). No significant alterations were observed in CFH lysozyme activity, whereas gill AChE activity decreased significantly in clams exposed to 1 or 5 mg l1. Overall, the obtained results demonstrated that fluoxetine markedly affected immune parameters and AChE activity in clams. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Antidepressant Clams Fluoxetine Pharmaceuticals Haemocytes Gills Immunomarkers Acetylcholinesterase
1. Introduction Pharmaceuticals and personal care products (PCCPs) are a class of emerging environmental contaminants that can be detected in various aquatic ecosystems, such as seawater, surface waters, groundwater and effluents from wastewater treatment plants (Daughton and Ternes, 1999; Kolpin et al., 2002; Metcalfe et al., 2003). Although PCCPs are generally considered not to be persistent, their continuous release into aquatic ecosystems is a matter of concern. Indeed, PCCPs can be found in the environment either as unmetabolised substances or as metabolites (Daughton and Ternes, 1999; Kolpin et al., 2002; Metcalfe et al., 2003; Bringolf et al., 2010). Among pharmaceuticals, fluoxetine, also known by the brand name of ProzacÒ, is a selective serotonin reuptake inhibitor (SSRI) that is prescribed as an antidepressant in large amounts worldwide to treat depression and other psychological disorders (Brooks et al., 2003; Nentwig, 2007). A relatively high percentage of fluoxetine
* Corresponding author. Tel.: þ39 049 8276201; fax: þ39 049 8276199. E-mail addresses: [email protected], [email protected] (V. Matozzo). 0141-1136/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marenvres.2013.11.007
(approximately 20e30%) is excreted, unchanged, by humans, whereas the remainder is excreted as the active metabolites fluoxetine glucuronide and norfluoxetine (Hartke and Mutschler, 1993). This drug has been detected in aquatic environments in the range of ng l1emg l1 (Kolpin et al., 2002; Chen et al., 2006; Calisto and Esteves, 2009; Bringolf et al., 2010; Metcalfe et al., 2010). In Ontario (Canada), fluoxetine concentrations ranging from 0.054 to 0.141 mg l1 were measured in surface waters, whereas a maximum concentration of 0.191 mg l1 was detected in untreated wastewaters (Metcalfe et al., 2010). A maximum concentration of 0.509 mg l1 was reported for treated wastewaters in Alberta (Canada) (Chen et al., 2006). Regarding seawater, Vasskog et al. (2008) reported quite low concentrations of SSRIs (not exceeding total concentrations of 3 ng l1) in Tromsø and Longyearbyen, where effluents from sewage treatment plants are discharged into the sea. In addition, it has been demonstrated that fluoxetine can accumulate in aquatic organisms, such as molluscs (Bringolf et al., 2010). Due to the drug’s serotonergic action, most studies have mainly addressed the evaluation of fluoxetine’s effects on the reproduction of non-target species (Nentwig, 2007). For example, it has been demonstrated that fluoxetine can significantly
M. Munari et al. / Marine Environmental Research 94 (2014) 32e37
reduce reproduction in the freshwater mudsnail Potamopyrgus antipodarum (Nentwig, 2007; Gust et al., 2009). In the zebra mussel Dreissena polymorpha, exposure to fluoxetine (200 ng l1) markedly decreases both the number of oocytes per follicle and the spermatozoa density in the tubules and significantly increases the endogenous levels of esterified estradiol (Lazzara et al., 2012). Significant increases in fecundity were observed in Daphnia magna after exposure to 36 mg l1 fluoxetine for 30 days (Flaherty and Dodson, 2005). In the mussel Elliptio complanata, exposure to 3000 mg l1 fluoxetine induces a significant release of spermatozeugmata by males and of nonviable larvae by females (Bringolf et al., 2010). Regarding other effects, fluoxetine was shown to alter mantle flap lure display behaviour in the freshwater mussels Lampsilis cardium and Lampsilis fasciola (Bringolf et al., 2010). Fluoxetine and other antidepressants cause foot detachment from a substrate in five marine snail species (Fong and Molnar, 2013). In the crab Carcinus maenas, it was shown that exposure to fluoxetine for 7 days increased locomotion at concentrations equal to or above 120 mg l1; fluoxetine-treated animals spent more time moving and walked for longer distances than controls (Mesquita et al., 2011). In addition, significant increases in the activity of cholinesterases, glutathione S-transferases and glutathione reductase were recorded in fluoxetine-exposed crabs (Mesquita et al., 2011). Despite this evidence, there is a lack of information concerning the effects of fluoxetine on immune parameters in aquatic organisms, such as bivalves. Consequently, in the present study, the effects of fluoxetine on the immune parameters of the clam Venerupis philippinarum were evaluated for the first time. Bivalves were exposed to sublethal fluoxetine concentrations for 7 days, and the effects on the total haemocyte count (THC), haemocyte diameter and volume, haemocyte proliferation, uptake of the vital dye Neutral Red (NRU) (an indicator of pinocytosis), cell-free haemolymph (CFH) lysozyme activity and were evaluated. In addition, gill acetylcholinesterase (AChE) activity was measured as a biomarker of neurotoxicity (Cajaraville et al., 2000).
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Eppendorf tubes and stored at 4 C. Eight pools of haemolymph (from three bivalves each) for each experimental condition were prepared (final volume of at least 2.5 ml). Pooling was necessary to obtain a sufficient volume of haemolymph for the analyses. Aliquots of pooled haemolymph from control and fluoxetine-exposed clams were added to an equal volume of 0.38% sodium citrate (Sigma) in 0.45 mm-filtered seawater (FSW) (pH 7.5) to prevent clotting. To measure AChE activity, gills from 10 clams per concentration were excised, frozen in liquid nitrogen and stored at 80 C until processing. 2.3. THC and haemocyte diameter and volume determination The THC and haemocyte diameter and volume were determined using a Model Z2 Coulter Counter electronic particle counter/size analyser (Coulter Corporation, FL, USA). Haemolymph (100 ml) was added to 19.9 ml of FSW. Five counts for each haemolymph sample were performed. The THC was expressed as the number of haemocytes (106) ml1 of haemolymph, whereas haemocyte diameter and volume were expressed in mm and femtolitres (fl), respectively. 2.4. Haemocyte proliferation
2. Materials and methods
Haemocyte proliferation was evaluated using a colourimetric method with a commercial kit (Cell Proliferation Kit II, Roche). The assay is based on the cleavage of the yellow tetrazolium salt XTT to form an orange formazan dye in metabolically active (viable) cells. This assay has previously been validated in clams (Matozzo et al., 2012a). Briefly, the XTT labelling reagent and electron-coupling reagent were thawed at 37 C and mixed immediately before use to obtain the XTT labelling mixture. A total of 200 ml of the mixture was added to 400 ml of pooled haemolymph and incubated for 4 h in a dark, humidified chamber. The absorbance at 450 nm was subsequently recorded using a Beckman 730 spectrophotometer. The data were normalised to the THC values that were recorded for clams from each experimental condition and expressed as optical density (OD) values at 450 nm.
2.1. Clams and exposure to fluoxetine
2.5. NR uptake (NRU) assay
Specimens of V. philippinarum (3.4e3.7 cm shell length) were collected from a reference site that was located inside a licensed area for clam culture in the southern basin of the Lagoon of Venice (Italy) and were acclimatised in the laboratory for 5 days before exposure to fluoxetine. The clams were maintained in large aquaria that contained a sandy bottom and aerated seawater (salinity of 35 1 psu, temperature of 17 0.5 C) and were fed with microalgae (Isochrysis galbana) every two days. A stock solution (1 g l1) of fluoxetine (SigmaeAldrich, Milano, Italy) was prepared in distilled water, whereas working solutions were prepared daily by diluting the stock solution in seawater. Clams (25 per concentration) were exposed for 7 days to 0, 1, 5, 25, 125 and 625 mg l1 fluoxetine. The nominal concentrations were chosen based on data on fluoxetine toxicity in aquatic invertebrates (Calisto and Esteves, 2009; Bringolf et al., 2010; Mesquita et al., 2011). Clams were maintained in 35 l capacity glass aquaria (without sediment) containing aerated seawater (1 l per animal) under the same thermohaline conditions that were used during the acclimatisation period. Every day, the water was changed, and fluoxetine and microalgae were added.
The cationic probe NR was used to evaluate the ability of haemocytes to perform pinocytosis (Cajaraville et al., 1996; Matozzo et al., 2002). Pooled haemolymph (500 ml) from fluoxetinetreated and untreated (control) clams was centrifuged at 780 g for 10 min. The haemocytes (at a final concentration of 106 cells ml1) were resuspended in an equal volume of 8 mg l1 NR dye (Merck) solution in FSW and incubated at 20 C for 30 min. The haemocytes were centrifuged at 780 g for 10 min, resuspended in distilled water, sonicated at 0 C for 30 s using a Braun Labsonic U sonifier at 50% duty cycles, and centrifuged at 12,000 g for 15 min at 4 C. The supernatant, corresponding to the haemocyte lysate (HL), was collected for the NRU assay. The absorbance at 550 nm was recorded using a Beckman 730 spectrophotometer. The data were expressed as the OD at 550 nm.
2.2. Haemolymph and gills collection After fluoxetine exposure, haemolymph was collected from the anterior adductor muscle using a 1-ml plastic syringe, placed in
2.6. Lysozyme activity in cell-free haemolymph (CFH) Pooled haemolymph was centrifuged at 780 g for 10 min at room temperature, and the supernatant, corresponding to CFH, was collected. In total, 50 ml of CFH was added to 950 ml of a 0.15% suspension of Micrococcus lysodeikticus (Sigma) in 66 mM phosphate buffer (pH 6.2), and the decrease in absorbance (DA min1) was continuously recorded at 450 nm for 3 min at room temperature. The results were expressed as mg lysozyme mg1 of protein.
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Protein concentrations in the CFH were quantified according to Bradford (1976). 2.7. Gill homogenate preparation and AChE activity assay Gills were thawed on ice and individually homogenised in four volumes of 0.1 M TriseHCl buffer, pH 7.5, containing 0.15 M KCl, 0.5 M sucrose, 1 mM EDTA, 1 mM dithiothreitol (DTT; Sigma) and 40 mg ml1 aprotinin (Sigma). The gills were then sonicated for 1 min at 0 C with a Braun Labsonic U sonifier at 50% duty cycles and centrifuged at 10,000 g for 30 min at 4 C. Supernatants (SN) were collected for an enzyme assay. The method of Ellman et al. (1961), adapted to a microplate reader, was adopted as follows. Enzyme activity was followed in 96-well microplates by measuring the colourimetric reaction between acetylthiocholine (ATC) and the reagent dithiobisnitrobenzoate (DTNB). SN and buffer blanks (50 ml) were incubated for 5 min in microplates at room temperature with 200 ml of 0.75 mM DTNB (Sigma) in 0.1 M TriseHCl buffer, pH 7.5. The reaction was started by the addition of 50 ml of 3 mM ATC (Sigma). Samples were incubated for 10 min at room temperature. Changes in absorbance at 405 nm were then recorded for 5 min on a microplate reader at room temperature. The results were expressed as nmol min1 mg1 of protein. The protein concentration was quantified according to Bradford (1976). 2.8. Statistical analysis For all immunomarkers, the normal distribution (Shapiroe Wilk’s test) and the homogeneity of the variance (Bartlett’s test) were assessed. The data were compared using a one-way ANOVA followed by a post-hoc test (Duncan’s test) and expressed as the means standard deviation (SD). The STATISTICA 10 software package (StatSoft, Tulsa, OK) was used for statistical analyses. 3. Results No clam mortality was recorded during the experiments. A significant increasing trend (ANOVA: p < 0.05; df: 5; F: 2.188) in the THC was recorded, with significantly (Duncan’s test: p < 0.05) higher values (5.6 106 cells ml1 of haemolymph) in clams exposed to 25 mg l1 fluoxetine than in control clams (3.9 106 cells ml1 of haemolymph) (Fig. 1). Conversely, the THC decreased, although not significantly, in clams exposed to the two highest concentrations tested. No significant alterations in either
Fig. 1. Total haemocyte count (THC), expressed as the number of haemocytes (106) ml1 of haemolymph, in V. philippinarum after exposure to fluoxetine. The values are mean þ SD (n ¼ 8). The asterisk indicates significant differences in comparison with controls: *p < 0.05.
the diameter (ANOVA: p ¼ 0.391; df: 5; F: 1.069) or the volume (ANOVA: p ¼ 0.521; df: 5; F: 0.852) of haemocytes was observed compared with controls (Figs. 2 and 3). Haemocyte proliferation was shown to increase significantly (ANOVA: p < 0.000; df: 5; F: 25.32) in animals exposed to 25 (Duncan’s test: p < 0.05), 125 (Duncan’s test: p < 0.01) and 625 (Duncan’s test: p < 0.001) mg l1 fluoxetine compared with controls (Fig. 4). NRU decreased significantly (ANOVA: p < 0.01; df: 5; F: 4.473) in the haemocytes of clams exposed to 1 and 5 mg l1 fluoxetine (Duncan’s test: p < 0.01) compared with controls, whereas NRU returned to control values in clams exposed to the highest fluoxetine concentrations tested (Fig. 5). No significant alterations were observed in CFH lysozyme activity compared with controls (ANOVA: p ¼ 0.375; df: 5; F: 1.099) (Fig. 6). After 7 days of exposure, AChE activity was shown to decrease significantly (ANOVA: p < 0.05; df: 5; F: 4.922) in the gills of clams exposed to 1 and 5 mg l1 (p < 0.05) compared with controls (Fig. 7). 4. Discussion Consolidated knowledge concerning the involvement of haemocytes in the immune responses of bivalves is available in the literature (Renwrantz, 1990; Hine, 1999; Cima et al., 2000; Matozzo et al., 2007; Donaghy et al., 2009). At the same time, numerous studies have demonstrated that contaminants can markedly alter bivalve haemocyte functionality, potentially increasing the susceptibility of the animals to diseases (Pipe and Coles, 1995; Parry and Pipe, 2004). In the present study, alterations in the immune parameters of V. philippinarum were recorded after a 7-day exposure to fluoxetine. The THC is one of the immunomarkers most commonly used to evaluate the negative effects of stressors (including pollutants) on bivalves (Oliver and Fisher, 1999). Generally, an increased THC may be due to either the proliferation or the movement of cells away from tissues and into haemolymph, whereas a decreased THC is caused by cell lysis or increased cell movement away from haemolymph and into tissues (Pipe and Coles, 1995; Parry and Pipe, 2004). In the present study, a nonlinear trend of variation in the THC was observed in fluoxetine-exposed clams. Indeed, THC values increased significantly in clams exposed to 25 mg l1 and decreased in animals exposed to the two highest concentrations tested. The obtained results suggest a biphasic response of clams to fluoxetine exposure. This peculiar dose response is known as hormesis, a phenomenon characterised by low-dose stimulation and high-dose inhibition. Regarding the effects of other pharmaceuticals on mollusc THCs, it has been demonstrated that ibuprofen (IBU), a propanoic acid derivative widely used as an analgesic,
Fig. 2. Effects of fluoxetine on haemocyte diameter, expressed in mm, in V. philippinarum. The values are mean þ SD (n ¼ 8).
M. Munari et al. / Marine Environmental Research 94 (2014) 32e37
Fig. 3. Effects of fluoxetine on haemocyte volume, expressed in fl, in V. philippinarum. The values are mean þ SD (n ¼ 8).
Fig. 4. Effects of fluoxetine on haemocyte proliferation, expressed as OD450, in V. philippinarum. The values are mean þ SD (n ¼ 8). The asterisks indicate significant differences in comparison with controls: *p < 0.05, **p < 0.01, ***p < 0.001.
antirheumatic and antipyretic in nonsteroidal anti-inflammatory drugs, significantly decreased the number of circulating haemocytes in clams (Matozzo et al., 2012b). Haemocyte count was significantly increased (3.1-fold) in the snails (Lymnaea stagnalis) exposed to a global mixture of different pharmaceutical classes (Gust et al., 2013). However, information about the effects of contaminants on the THC in molluscs is conflicting. For example,
Fig. 5. Effects of fluoxetine on NRU, expressed as OD550, of haemocytes from V. philippinarum. The values are mean þ SD (n ¼ 8). The asterisks indicate significant differences in comparison with controls: **p < 0.01.
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exposure to cadmium and fluoranthene induces a significant increase in the THC in Mytilus edulis (Coles et al., 1994, 1995), whereas exposure to phenanthrene does not induce a significant change in THC values in M. edulis, Cerastoderma edule or Ensis siliqua (Wootton et al., 2003). Exposure to 4-nonylphenol (NP) for 7 days significantly increases the THC values in Ruditapes philippinarum (Matozzo and Marin, 2005), whereas no significant alterations in the THC are observed in the clam Chamelea gallina after 12 days of exposure to benzo(a)pyrene (Matozzo et al., 2009). We hypothesised that the increased THC in clams exposed to low levels of fluoxetine was due to the increased mobilisation of haemocytes away from peripheral tissues and into the haemolymph or to an increase in haemocyte proliferation. Regarding the second hypothesis, it is important to note that circulating haemocytes from V. philippinarum can divide in the haemolymph (Matozzo et al., 2008a) and that contaminants (Mayrand et al., 2005), including pharmaceuticals (Matozzo et al., 2012b), can stimulate the mitotic activity of bivalve haemocytes. Consequently, the increase in haemocyte proliferation observed in fluoxetine-exposed clams may partially account for the increased THC values, at least at 25 mg l1. Surprisingly, the significant increase in haemocyte proliferation that was observed in clams exposed to the two highest fluoxetine concentrations tested did not correspond to a significant increase in the number of circulating haemocytes. We formulated two hypotheses to explain the inconsistency of these results. First, it is possible that clams that were exposed to the two highest fluoxetine concentrations underwent a reduction in the number of circulating haemocytes to such an extent that new haemocytes were not enough to compensate for cell number reduction. Alternatively, it is possible that new haemocytes moved towards peripheral tissues to increase immunosurveillance in these tissues. In any case, further studies are needed to better elucidate these aspects. Fluoxetine did not induce significant alterations in either the diameter or the volume of haemocytes in V. philippinarum. The reduction in haemocyte diameter is assumed to be a consequence of alterations in cytoskeletal organisation, whereas the reduction in cell volume may be due to the release of fluids from the cells. Based on the results obtained in this study, we can exclude the idea that fluoxetine induces haemocyte shrinkage in clams, at least under the tested experimental conditions. Similarly, no significant alterations in the diameter or volume of haemocytes are observed in IBUexposed clams (Matozzo et al., 2012b). Conversely, the exposure of clams to triclosan, an antibacterial agent, significantly decreases
Fig. 6. Effects of fluoxetine on lysozyme activity, expressed as mg lysozyme mg1 of protein, in cell-free haemolymph (CFH) of V. philippinarum. The values are mean þ SD (n ¼ 8).
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M. Munari et al. / Marine Environmental Research 94 (2014) 32e37
Fig. 7. AChE activity, expressed as nmol min1 mg1 of protein, in gills from V. philippinarum exposed to fluoxetine. The values are mean þ SD (n ¼ 10). The asterisks indicate significant differences in comparison with controls: *p < 0.05.
the diameter and volume of haemocytes, suggesting cell shrinkage (Matozzo et al., 2012a). The cationic probe NR is commonly used to evaluate the effects of stressors on lysosomal membrane stability in bivalve haemocytes (Hauton et al., 1998; Matozzo et al., 2001; Canesi et al., 2007a; Binelli et al., 2009; Aguirre-Martínez et al., 2013). The uptake of NR by haemocytes occurs either by pinocytosis or by passive diffusion across cell membranes (Coles et al., 1995). Consequently, alterations in dye uptake reflect damage to cell membranes (including lysosomal membranes) and/or weakening of haemocyte pinocytotic capabilities. In this study, we demonstrated that fluoxetine (1 and 5 mg l1) significantly reduced NRU by haemocytes, suggesting a reduction in pinocytotic activity. In our previous studies, we observed increases in NRU by haemocytes from clams exposed to the highest tested NP concentrations (Matozzo and Marin, 2005) and significant reductions in the haemocytes of clams exposed to triclosan (Matozzo et al., 2012a). A biphasic response of clams to fluoxetine exposure was also recorded in this study: NRU decreased in clams exposed to the lowest fluoxetine concentrations and returned to control values in bivalves exposed to the highest concentrations tested. Lysozyme is one of the most important bivalve bacteriolytic agents against several species of gram-positive and gram-negative bacteria (Cheng and Rodrick, 1974). In the present study, the ability of fluoxetine to induce lysozyme release from haemocytes into the haemolymph was evaluated to ascertain whether the compound alters haemocyte membrane stability. Based on results obtained, we can exclude this hypothesis, at least for the species studied and under the tested experimental conditions. An induction of haemocyte degranulation, followed by an increase in extracellular lysozyme activity, has been observed in haemocytes from Mytilus galloprovincialis exposed in vitro to estradiol (Canesi et al., 2004). Significant increases in acid phosphatase and lysozyme activities were also observed in the CFH of cockles (Cerastoderma glaucum) exposed to NP, suggesting that the contaminant caused the destabilisation of lysosomal membranes and the consequent release of hydrolytic enzymes into the haemolymph (Matozzo et al., 2008b). NP has also been shown to induce a significant increase in lysozyme release in M. galloprovincialis haemocytes treated in vitro (Canesi et al., 2007b). It is well known that AChE plays an important role in the functioning of the neuromuscular system by preventing continuous muscular contraction. AChE activity has been proposed as a biomarker of exposure to neurotoxic compounds in aquatic organisms (Cajaraville et al., 2000). Numerous studies have
demonstrated that AChE is mainly inhibited by organophosphorus compounds and carbamates, which are pesticides that are widely used in agriculture (Cajaraville et al., 2000). However, other environmental pollutants, such as heavy metals (Hamza-Chaffai et al., 1998) and pharmaceuticals (ibuprofen) (Milan et al., 2013), can inhibit AChE activity in bivalves. In this study, we tested the hypothesis that fluoxetine is neurotoxic to V. philippinarum. The results of our study partially support this hypothesis. Indeed, gill AChE activity decreased significantly in clams exposed to the lowest concentrations of fluoxetine, whereas enzyme activity returned to control values in clams exposed to the highest fluoxetine concentrations. Although the results obtained in the present study are surprising, it is important to note that controversial results were also recorded in a recent study on mussels (M. galloprovincialis) that were exposed to fluoxetine (75 ng l1) for 15 days (Gonzalez-Rey and Bebianno, 2013). In that study, gill AChE activity increased significantly after 3 days of exposure, followed by progressive inhibition, reaching a significantly lower activity than controls after 15 days of exposure. Conversely, AChE activity was shown to increase significantly in the muscle of crabs (C. maenas) after 7 days of exposure to fluoxetine (120 and 750 mg l1) (Mesquita et al., 2011). Based on both the results of this study and data from the literature, we can conclude that AChE activity may vary markedly in response to fluoxetine exposure, depending on the exposure concentration and duration and on the species and tissues analysed. Regarding the effects of other pharmaceuticals, Solé et al. (2010) demonstrated that acetaminophen significantly inhibits AChE activity in gills from the mussel M. galloprovincialis, while Gagné et al. (2010) observed a significant reduction in AChE activity in the visceral mass of mussels (E. complanata) exposed to morphine. In conclusion, the results of the present study demonstrate that fluoxetine affects haemocyte parameters in the clam V. philippinarum. In addition, the lowest concentrations of fluoxetine significantly reduce AChE activity in clams. Overall, the present study revealed a biphasic response of clams to fluoxetine exposure and suggested that fluoxetine can pose risks to aquatic invertebrates. However, the nonlinear responses of biomarkers that have been measured in V. philippinarum indicate that further investigations (e.g., long-term exposures, tissue collection at different time intervals) are required to completely assess the impact of this compound in bivalves.
Acknowledgements This study was funded by the University of Padova to Dr. V. Matozzo. University-funded research project titled “Immunotoxicity, neurotoxicity and estrogenicity of pharmaceuticals and personal care products in bivalve mollusks” (CPDA095545). The English text was revised by American Journal Experts.
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M. Munari et al. / Marine Environmental Research 94 (2014) 32e37 Brooks, B.W., Foran, C.M., Richards, S.M., Weston, J., Turner, P.K., Stanley, J.K., Solomon, K.R., Slattery, M., La Point, T.W., 2003. Aquatic ecotoxicology of fluoxetine. Toxicol. Lett. 142, 169e183. Cajaraville, M.P., Olabarrieta, I., Marigomez, I., 1996. In vitro activities in mussel hemocytes as biomarkers of environmental quality: a case study in the Abra (Biscay Bay). Ecotoxicol. Environ. Saf. 35, 253e260. Cajaraville, M.P., Bebianno, M.J., Blasco, J., Porte, C., Sarasquete, C., Viarengo, A., 2000. The use of biomarkers to assess the impact of pollution in coastal environments of the Iberian Peninsula: a practical approach. Sci. Total Environ. 247, 295e311. Calisto, V., Esteves, V.I., 2009. Psychiatric pharmaceuticals in the environment. Chemosphere 77, 1257e1274. Canesi, L., Ciacci, C., Betti, M., Lorusso, L.C., Marchi, B., Burattini, S., Falcieri, E., Gallo, G., 2004. Rapid effects of 17b-estradiol on cell signalling and function of Mytilus hemocytes. General Comp. Endocrinol. 136, 58e71. Canesi, L., Ciacci, C., Lorusso, L.C., Betti, M., Gallo, G., Poiana, G., Marcomini, A., 2007a. Effects of Triclosan on Mytilus galloprovincialis hemocyte function and digestive gland enzyme activities: possible modes of action on non target organisms. Comp. Biochem. Physiol. 145C, 464e472. Canesi, L., Lorusso, L.C., Ciacci, C., Betti, M., Rocchi, M., Poiana, G., Marcomini, A., 2007b. Immunomodulation of Mytilus hemocytes by individual estrogenic chemicals and environmentally relevant mixtures of estrogens: in vitro and in vivo studies. Aquat. Toxicol. 81, 36e44. Chen, M., Ohman, K., Metcalfe, C., Ikonomou, M.G., Amatya, P.L., Wilson, J., 2006. Pharmaceuticals and endocrine disruptors in wastewater treatment effluents and in the water supply system of Calgary, Alberta, Canada. Water Qual. Res. J. Can. 41, 351e364. Cheng, T.C., Rodrick, G.E., 1974. Identification and characterization of lysozyme from the hemolymph of the soft shelled clam, Mya arenaria. Biol. Bull. 147, 311e320. Cima, F., Matozzo, V., Marin, M.G., Ballarin, L., 2000. Haemocytes of the clam Tapes philippinarum (Adams & Reeve, 1850): morphofunctional characterisation. Fish Shellfish Immunol. 10, 677e693. Coles, J.A., Farley, S.R., Pipe, R.K., 1994. Effects of fluoranthene on the immunocompetence of the common marine mussel, Mytilus edulis. Aquat. Toxicol. 30, 367e379. Coles, J.A., Farley, S.R., Pipe, R.K., 1995. Alteration of the immune response of the common marine mussel Mytilus edulis resulting from exposure to cadmium. Dis. Aquat. Org. 22, 59e65. Daughton, C.G., Ternes, T.A., 1999. Pharmaceuticals and personal care products in the environment: agents of subtle change? Environ. Health Perspect. 107, 907e938. Donaghy, L., Lambert, C., Choi, K.S., Soudant, P., 2009. Hemocytes of the carpet shell clam (Ruditapes decussatus) and the Manila clam (Ruditapes philippinarum): current knowledge and future prospects. Aquaculture 297, 10e24. Ellman, G.L., Courtney, K.D., Andres, V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88e95. Flaherty, C.M., Dodson, S.I., 2005. Effects of pharmaceuticals on Daphnia survival, growth, and reproduction. Chemosphere 61, 200e207. Fong, P.P., Molnar, N., 2013. Antidepressants cause foot detachment from substrate in five species of marine snail. Mar. Environ. Res. 84, 24e30. Gagné, F., André, C., Gélinas, M., 2010. Neurochemical effects of benzodiazepine and morphine on freshwater mussels. Comp. Biochem. Physiol. 152C, 207e214. Gonzalez-Rey, M., Bebianno, M.J., 2013. Does selective serotonin reuptake inhibitor (SSRI) fluoxetine affects mussel Mytilus galloprovincialis? Environ. Pollut. 173, 200e209. Gust, M., Buronfosse, T., Giamberini, L., Ramil, M., Mons, R., Garrii, J., 2009. Effects of fluoxetine on the reproduction of two prosobranch mollusks: Potamopyrgus antipodarum and Valvata piscinalis. Environ. Pollut. 157, 423e429. Gust, M., Fortier, M., Garric, J., Fournier, M., Gagné, F., 2013. Effects of short-term exposure to environmentally relevant concentrations of different pharmaceutical mixtures on the immune response of the pond snail Lymnaea stagnalis. Sci. Total Environ. 445-446, 210e218. Hamza-Chaffai, A., Roméo, M., Gnassia-Barelli, M., El Abed, A., 1998. Effect of copper and lindane on some biomarkers measured in the clam Ruditapes decussatus. Bull. Environ. Contam. Toxicol. 61, 397e404. Hartke, K., Mutschler, E., 1993. In: . In: Hartke, K., Mutschler, E. (Eds.), Deutsches Arzneibuch DAB 10-Kommentar, tenth ed., vol. II and III. Deutscher ApothekerVerlag, Stuttgart. 3rd Suppl. Hauton, C., Hawkins, L.E., Hutchinson, S., 1998. The use of the neutral red retention assay to examine the effects of temperature and salinity on haemocytes of the European flat oyster Ostrea edulis (L). Comp. Biochem. Physiol. 119B, 619e623.
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Hine, P.M., 1999. The inter-relationships of bivalve haemocytes. Fish Shellfish Immunol. 9, 367e385. Kolpin, D.W., Furlong, E.T., Meyer, M.T., Thurman, E.M., Zaugg, S.D., Barber, L.B., Buxton, H.T., 2002. Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999e2000: a national reconnaissance. Environ. Sci. Technol. 36, 1202e1211. Lazzara, R., Blázquez, M., Porte, C., Barata, C., 2012. Low environmental levels of fluoxetine induce spawning and changes in endogenous estradiol levels in the zebra mussel Dreissena polymorpha. Aquat. Toxicol. 106-107, 123e130. Matozzo, V., Ballarin, L., Pampanin, D.M., Marin, M.G., 2001. Effects of copper and cadmium exposure on functional responses of hemocytes in the clam, Tapes philippinarum. Arch. Environ. Contam. Toxicol. 41, 163e170. Matozzo, V., Ballarin, L., Marin, M.G., 2002. In vitro effects of tributyltin on functional responses of haemocytes in the clam Tapes philippinarum. Appl. Organomet. Chem. 16, 169e174. Matozzo, V., Marin, M.G., 2005. 4-Nonylphenol induces immunomodulation and apoptotic events in the clam Tapes philippinarum. Mar. Ecol. Prog. Ser. 285, 97e 106. Matozzo, V., Rova, G., Marin, M.G., 2007. Haemocytes of the cockle Cerastoderma glaucum: morphological characterisation and involvement in immune responses. Fish Shellfish Immunol. 23, 732e746. Matozzo, V., Marin, M.G., Cima, F., Ballarin, L., 2008a. First evidence of cell division in circulating haemocytes from the Manila clam Tapes philippinarum. Cell Biol. Int. 32, 865e868. Matozzo, V., Rova, G., Ricciardi, F., Marin, M.G., 2008b. Immunotoxicity of the xenoestrogen 4-nonylphenol to the cockle Cerastoderma glaucum. Mar. Pollut. Bull. 57, 453e459. Matozzo, V., Monari, M., Foschi, J., Cattani, O., Serrazanetti, G.P., Marin, M.G., 2009. First evidence of altered immune responses and resistance to air exposure in the clam Chamelea gallina exposed to benzo(a)pyrene. Arch. Environ. Contam. Toxicol. 56, 479e488. Matozzo, V., Costa Devoti, A., Marin, M.G., 2012a. Immunotoxic effects of Triclosan in the clam Ruditapes philippinarum. Ecotoxicology 21, 66e74. Matozzo, V., Rova, S., Marin, M.G., 2012b. The nonsteroidal anti-inflammatory drug, ibuprofen, affects the immune parameters in the clam Ruditapes philippinarum. Mar. Environ. Res. 79, 116e121. Mayrand, E., St-Jean, S.D., Courtenay, S.C., 2005. Haemocyte responses of blue mussels (Mytilus edulis L.) transferred from a contaminated site to a reference site: can the immune system recuperate? Aquac. Res. 36, 962e971. Mesquita, S.R., Guilhermino, L., Guimarães, L., 2011. Biochemical and locomotor responses of Carcinus maenas exposed to the serotonin reuptake inhibitor fluoxetine. Chemosphere 85, 967e976. Metcalfe, C.D., Miao, X.S., Koenig, B.G., Struger, J., 2003. Distribution of acidic and neutral drugs in surface waters near sewage treatment plants in the Lower Great Lakes, Canada. Environ. Toxicol. Chem. 22, 2881e2889. Metcalfe, C.D., Chu, S., Judt, C., Li, H., Oakes, K.D., Servos, M.R., Andrews, D.M., 2010. Antidepressants and their metabolites in municipal wastewater, and downstream exposure in an urban watershed. Environ. Toxicol. Chem. 29, 79e89. Milan, M., Pauletto, M., Patarnello, T., Bargelloni, L., Marin, M.G., Matozzo, V., 2013. Gene transcription and biomarker responses in the clam Ruditapes philippinarum after exposure to ibuprofen. Aquat. Toxicol. 126, 17e29. Nentwig, G., 2007. Effects of pharmaceuticals on aquatic invertebrates. Part II: the antidepressant drug fluoxetine. Arch. Environ. Contam. Toxicol. 52, 163e170. Oliver, L.M., Fisher, W.S., 1999. Appraisal of prospective bivalve immunomarkers. Biomarkers 4, 510e530. Parry, H.E., Pipe, R.K., 2004. Interactive effects of temperature and copper on immunocompetence and disease susceptibility in mussels (Mytilus edulis). Aquat. Toxicol. 69, 311e325. Pipe, R.K., Coles, J.A., 1995. Environmental contaminants influencing immune function in marine bivalve molluscs. Fish Shellfish Immunol. 5, 581e595. Renwrantz, L., 1990. Internal defence system of Mytilus edulis. In: Stefano, G.B. (Ed.), Studies in Neuroscience: Neurobiology of Mytilus Edulis. Manchester University Press, Manchester, pp. 256e275. Solé, M., Shaw, J.P., Frickers, P.E., Readman, J.W., Hutchinson, T.H., 2010. Effects on feeding rate and biomarker responses of marine mussels experimentally exposed to propranolol and acetaminophen. Anal. Bioanal. Chem. 96, 649e656. Vasskog, T., Anderssen, T., Pedersen-Bjergaard, S., Kallenborn, R., Jensen, E., 2008. Occurrence of selective serotonin reuptake inhibitors in sewage and receiving waters at Spitsbergen and in Norway. J. Chromatogr. 1185A, 194e205. Wootton, E.C., Dyrynda, E.A., Pipe, R.K., Ratcliffe, N.A., 2003. Comparisons of PAHinduced immunomodulation in three bivalve molluscs. Aquat. Toxicol. 65, 13e25.
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Effects of the antidepressant fluoxetine on the immune parameters and acetylcholinesterase activity of the clam Venerupis philippinarum Marco Munari, Maria Gabriella Marin, Valerio Matozzo* Department of Biology, University of Padova, Via Ugo Bassi 58/B, 35131 Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 October 2013 Received in revised form 15 November 2013 Accepted 20 November 2013
Fluoxetine is an antidepressant used worldwide for the treatment of depression and other psychological disorders. The occurrence of fluoxetine in aquatic environments has been demonstrated. However, there is a lack of information about the effects of fluoxetine on non-target species, such as bivalve molluscs. In the present study, the effects of fluoxetine on the immune parameters of the clam Venerupis philippinarum were evaluated for the first time. Clams were exposed to various sublethal concentrations of fluoxetine (0, 1, 5, 25, 125, 625 mg l1) for 7 days, and the effects on the total haemocyte count (THC), the diameter and volume of haemocytes, haemocyte proliferation, Neutral Red uptake (NRU), and lysozyme activity in cell-free haemolymph (CFH) were evaluated. In addition, acetylcholinesterase (AChE) activity was measured in clam gills as a biomarker of neurotoxicity. A significant increase in THC values was observed in clams exposed to 25 mg l1 compared with controls, whereas no significant variations were recorded in either the diameter or the volume of haemocytes. Haemocyte proliferation increased significantly in animals exposed to 25, 125 and 625 mg l1 compared with controls. NRU decreased significantly in the haemocytes of clams exposed to 1 or 5 mg l1, whereas NRU returned to control values in clams exposed to the highest fluoxetine concentrations tested (25e625 mg l1). No significant alterations were observed in CFH lysozyme activity, whereas gill AChE activity decreased significantly in clams exposed to 1 or 5 mg l1. Overall, the obtained results demonstrated that fluoxetine markedly affected immune parameters and AChE activity in clams. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Antidepressant Clams Fluoxetine Pharmaceuticals Haemocytes Gills Immunomarkers Acetylcholinesterase
1. Introduction Pharmaceuticals and personal care products (PCCPs) are a class of emerging environmental contaminants that can be detected in various aquatic ecosystems, such as seawater, surface waters, groundwater and effluents from wastewater treatment plants (Daughton and Ternes, 1999; Kolpin et al., 2002; Metcalfe et al., 2003). Although PCCPs are generally considered not to be persistent, their continuous release into aquatic ecosystems is a matter of concern. Indeed, PCCPs can be found in the environment either as unmetabolised substances or as metabolites (Daughton and Ternes, 1999; Kolpin et al., 2002; Metcalfe et al., 2003; Bringolf et al., 2010). Among pharmaceuticals, fluoxetine, also known by the brand name of ProzacÒ, is a selective serotonin reuptake inhibitor (SSRI) that is prescribed as an antidepressant in large amounts worldwide to treat depression and other psychological disorders (Brooks et al., 2003; Nentwig, 2007). A relatively high percentage of fluoxetine
* Corresponding author. Tel.: þ39 049 8276201; fax: þ39 049 8276199. E-mail addresses: [email protected], [email protected] (V. Matozzo). 0141-1136/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marenvres.2013.11.007
(approximately 20e30%) is excreted, unchanged, by humans, whereas the remainder is excreted as the active metabolites fluoxetine glucuronide and norfluoxetine (Hartke and Mutschler, 1993). This drug has been detected in aquatic environments in the range of ng l1emg l1 (Kolpin et al., 2002; Chen et al., 2006; Calisto and Esteves, 2009; Bringolf et al., 2010; Metcalfe et al., 2010). In Ontario (Canada), fluoxetine concentrations ranging from 0.054 to 0.141 mg l1 were measured in surface waters, whereas a maximum concentration of 0.191 mg l1 was detected in untreated wastewaters (Metcalfe et al., 2010). A maximum concentration of 0.509 mg l1 was reported for treated wastewaters in Alberta (Canada) (Chen et al., 2006). Regarding seawater, Vasskog et al. (2008) reported quite low concentrations of SSRIs (not exceeding total concentrations of 3 ng l1) in Tromsø and Longyearbyen, where effluents from sewage treatment plants are discharged into the sea. In addition, it has been demonstrated that fluoxetine can accumulate in aquatic organisms, such as molluscs (Bringolf et al., 2010). Due to the drug’s serotonergic action, most studies have mainly addressed the evaluation of fluoxetine’s effects on the reproduction of non-target species (Nentwig, 2007). For example, it has been demonstrated that fluoxetine can significantly
M. Munari et al. / Marine Environmental Research 94 (2014) 32e37
reduce reproduction in the freshwater mudsnail Potamopyrgus antipodarum (Nentwig, 2007; Gust et al., 2009). In the zebra mussel Dreissena polymorpha, exposure to fluoxetine (200 ng l1) markedly decreases both the number of oocytes per follicle and the spermatozoa density in the tubules and significantly increases the endogenous levels of esterified estradiol (Lazzara et al., 2012). Significant increases in fecundity were observed in Daphnia magna after exposure to 36 mg l1 fluoxetine for 30 days (Flaherty and Dodson, 2005). In the mussel Elliptio complanata, exposure to 3000 mg l1 fluoxetine induces a significant release of spermatozeugmata by males and of nonviable larvae by females (Bringolf et al., 2010). Regarding other effects, fluoxetine was shown to alter mantle flap lure display behaviour in the freshwater mussels Lampsilis cardium and Lampsilis fasciola (Bringolf et al., 2010). Fluoxetine and other antidepressants cause foot detachment from a substrate in five marine snail species (Fong and Molnar, 2013). In the crab Carcinus maenas, it was shown that exposure to fluoxetine for 7 days increased locomotion at concentrations equal to or above 120 mg l1; fluoxetine-treated animals spent more time moving and walked for longer distances than controls (Mesquita et al., 2011). In addition, significant increases in the activity of cholinesterases, glutathione S-transferases and glutathione reductase were recorded in fluoxetine-exposed crabs (Mesquita et al., 2011). Despite this evidence, there is a lack of information concerning the effects of fluoxetine on immune parameters in aquatic organisms, such as bivalves. Consequently, in the present study, the effects of fluoxetine on the immune parameters of the clam Venerupis philippinarum were evaluated for the first time. Bivalves were exposed to sublethal fluoxetine concentrations for 7 days, and the effects on the total haemocyte count (THC), haemocyte diameter and volume, haemocyte proliferation, uptake of the vital dye Neutral Red (NRU) (an indicator of pinocytosis), cell-free haemolymph (CFH) lysozyme activity and were evaluated. In addition, gill acetylcholinesterase (AChE) activity was measured as a biomarker of neurotoxicity (Cajaraville et al., 2000).
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Eppendorf tubes and stored at 4 C. Eight pools of haemolymph (from three bivalves each) for each experimental condition were prepared (final volume of at least 2.5 ml). Pooling was necessary to obtain a sufficient volume of haemolymph for the analyses. Aliquots of pooled haemolymph from control and fluoxetine-exposed clams were added to an equal volume of 0.38% sodium citrate (Sigma) in 0.45 mm-filtered seawater (FSW) (pH 7.5) to prevent clotting. To measure AChE activity, gills from 10 clams per concentration were excised, frozen in liquid nitrogen and stored at 80 C until processing. 2.3. THC and haemocyte diameter and volume determination The THC and haemocyte diameter and volume were determined using a Model Z2 Coulter Counter electronic particle counter/size analyser (Coulter Corporation, FL, USA). Haemolymph (100 ml) was added to 19.9 ml of FSW. Five counts for each haemolymph sample were performed. The THC was expressed as the number of haemocytes (106) ml1 of haemolymph, whereas haemocyte diameter and volume were expressed in mm and femtolitres (fl), respectively. 2.4. Haemocyte proliferation
2. Materials and methods
Haemocyte proliferation was evaluated using a colourimetric method with a commercial kit (Cell Proliferation Kit II, Roche). The assay is based on the cleavage of the yellow tetrazolium salt XTT to form an orange formazan dye in metabolically active (viable) cells. This assay has previously been validated in clams (Matozzo et al., 2012a). Briefly, the XTT labelling reagent and electron-coupling reagent were thawed at 37 C and mixed immediately before use to obtain the XTT labelling mixture. A total of 200 ml of the mixture was added to 400 ml of pooled haemolymph and incubated for 4 h in a dark, humidified chamber. The absorbance at 450 nm was subsequently recorded using a Beckman 730 spectrophotometer. The data were normalised to the THC values that were recorded for clams from each experimental condition and expressed as optical density (OD) values at 450 nm.
2.1. Clams and exposure to fluoxetine
2.5. NR uptake (NRU) assay
Specimens of V. philippinarum (3.4e3.7 cm shell length) were collected from a reference site that was located inside a licensed area for clam culture in the southern basin of the Lagoon of Venice (Italy) and were acclimatised in the laboratory for 5 days before exposure to fluoxetine. The clams were maintained in large aquaria that contained a sandy bottom and aerated seawater (salinity of 35 1 psu, temperature of 17 0.5 C) and were fed with microalgae (Isochrysis galbana) every two days. A stock solution (1 g l1) of fluoxetine (SigmaeAldrich, Milano, Italy) was prepared in distilled water, whereas working solutions were prepared daily by diluting the stock solution in seawater. Clams (25 per concentration) were exposed for 7 days to 0, 1, 5, 25, 125 and 625 mg l1 fluoxetine. The nominal concentrations were chosen based on data on fluoxetine toxicity in aquatic invertebrates (Calisto and Esteves, 2009; Bringolf et al., 2010; Mesquita et al., 2011). Clams were maintained in 35 l capacity glass aquaria (without sediment) containing aerated seawater (1 l per animal) under the same thermohaline conditions that were used during the acclimatisation period. Every day, the water was changed, and fluoxetine and microalgae were added.
The cationic probe NR was used to evaluate the ability of haemocytes to perform pinocytosis (Cajaraville et al., 1996; Matozzo et al., 2002). Pooled haemolymph (500 ml) from fluoxetinetreated and untreated (control) clams was centrifuged at 780 g for 10 min. The haemocytes (at a final concentration of 106 cells ml1) were resuspended in an equal volume of 8 mg l1 NR dye (Merck) solution in FSW and incubated at 20 C for 30 min. The haemocytes were centrifuged at 780 g for 10 min, resuspended in distilled water, sonicated at 0 C for 30 s using a Braun Labsonic U sonifier at 50% duty cycles, and centrifuged at 12,000 g for 15 min at 4 C. The supernatant, corresponding to the haemocyte lysate (HL), was collected for the NRU assay. The absorbance at 550 nm was recorded using a Beckman 730 spectrophotometer. The data were expressed as the OD at 550 nm.
2.2. Haemolymph and gills collection After fluoxetine exposure, haemolymph was collected from the anterior adductor muscle using a 1-ml plastic syringe, placed in
2.6. Lysozyme activity in cell-free haemolymph (CFH) Pooled haemolymph was centrifuged at 780 g for 10 min at room temperature, and the supernatant, corresponding to CFH, was collected. In total, 50 ml of CFH was added to 950 ml of a 0.15% suspension of Micrococcus lysodeikticus (Sigma) in 66 mM phosphate buffer (pH 6.2), and the decrease in absorbance (DA min1) was continuously recorded at 450 nm for 3 min at room temperature. The results were expressed as mg lysozyme mg1 of protein.
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Protein concentrations in the CFH were quantified according to Bradford (1976). 2.7. Gill homogenate preparation and AChE activity assay Gills were thawed on ice and individually homogenised in four volumes of 0.1 M TriseHCl buffer, pH 7.5, containing 0.15 M KCl, 0.5 M sucrose, 1 mM EDTA, 1 mM dithiothreitol (DTT; Sigma) and 40 mg ml1 aprotinin (Sigma). The gills were then sonicated for 1 min at 0 C with a Braun Labsonic U sonifier at 50% duty cycles and centrifuged at 10,000 g for 30 min at 4 C. Supernatants (SN) were collected for an enzyme assay. The method of Ellman et al. (1961), adapted to a microplate reader, was adopted as follows. Enzyme activity was followed in 96-well microplates by measuring the colourimetric reaction between acetylthiocholine (ATC) and the reagent dithiobisnitrobenzoate (DTNB). SN and buffer blanks (50 ml) were incubated for 5 min in microplates at room temperature with 200 ml of 0.75 mM DTNB (Sigma) in 0.1 M TriseHCl buffer, pH 7.5. The reaction was started by the addition of 50 ml of 3 mM ATC (Sigma). Samples were incubated for 10 min at room temperature. Changes in absorbance at 405 nm were then recorded for 5 min on a microplate reader at room temperature. The results were expressed as nmol min1 mg1 of protein. The protein concentration was quantified according to Bradford (1976). 2.8. Statistical analysis For all immunomarkers, the normal distribution (Shapiroe Wilk’s test) and the homogeneity of the variance (Bartlett’s test) were assessed. The data were compared using a one-way ANOVA followed by a post-hoc test (Duncan’s test) and expressed as the means standard deviation (SD). The STATISTICA 10 software package (StatSoft, Tulsa, OK) was used for statistical analyses. 3. Results No clam mortality was recorded during the experiments. A significant increasing trend (ANOVA: p < 0.05; df: 5; F: 2.188) in the THC was recorded, with significantly (Duncan’s test: p < 0.05) higher values (5.6 106 cells ml1 of haemolymph) in clams exposed to 25 mg l1 fluoxetine than in control clams (3.9 106 cells ml1 of haemolymph) (Fig. 1). Conversely, the THC decreased, although not significantly, in clams exposed to the two highest concentrations tested. No significant alterations in either
Fig. 1. Total haemocyte count (THC), expressed as the number of haemocytes (106) ml1 of haemolymph, in V. philippinarum after exposure to fluoxetine. The values are mean þ SD (n ¼ 8). The asterisk indicates significant differences in comparison with controls: *p < 0.05.
the diameter (ANOVA: p ¼ 0.391; df: 5; F: 1.069) or the volume (ANOVA: p ¼ 0.521; df: 5; F: 0.852) of haemocytes was observed compared with controls (Figs. 2 and 3). Haemocyte proliferation was shown to increase significantly (ANOVA: p < 0.000; df: 5; F: 25.32) in animals exposed to 25 (Duncan’s test: p < 0.05), 125 (Duncan’s test: p < 0.01) and 625 (Duncan’s test: p < 0.001) mg l1 fluoxetine compared with controls (Fig. 4). NRU decreased significantly (ANOVA: p < 0.01; df: 5; F: 4.473) in the haemocytes of clams exposed to 1 and 5 mg l1 fluoxetine (Duncan’s test: p < 0.01) compared with controls, whereas NRU returned to control values in clams exposed to the highest fluoxetine concentrations tested (Fig. 5). No significant alterations were observed in CFH lysozyme activity compared with controls (ANOVA: p ¼ 0.375; df: 5; F: 1.099) (Fig. 6). After 7 days of exposure, AChE activity was shown to decrease significantly (ANOVA: p < 0.05; df: 5; F: 4.922) in the gills of clams exposed to 1 and 5 mg l1 (p < 0.05) compared with controls (Fig. 7). 4. Discussion Consolidated knowledge concerning the involvement of haemocytes in the immune responses of bivalves is available in the literature (Renwrantz, 1990; Hine, 1999; Cima et al., 2000; Matozzo et al., 2007; Donaghy et al., 2009). At the same time, numerous studies have demonstrated that contaminants can markedly alter bivalve haemocyte functionality, potentially increasing the susceptibility of the animals to diseases (Pipe and Coles, 1995; Parry and Pipe, 2004). In the present study, alterations in the immune parameters of V. philippinarum were recorded after a 7-day exposure to fluoxetine. The THC is one of the immunomarkers most commonly used to evaluate the negative effects of stressors (including pollutants) on bivalves (Oliver and Fisher, 1999). Generally, an increased THC may be due to either the proliferation or the movement of cells away from tissues and into haemolymph, whereas a decreased THC is caused by cell lysis or increased cell movement away from haemolymph and into tissues (Pipe and Coles, 1995; Parry and Pipe, 2004). In the present study, a nonlinear trend of variation in the THC was observed in fluoxetine-exposed clams. Indeed, THC values increased significantly in clams exposed to 25 mg l1 and decreased in animals exposed to the two highest concentrations tested. The obtained results suggest a biphasic response of clams to fluoxetine exposure. This peculiar dose response is known as hormesis, a phenomenon characterised by low-dose stimulation and high-dose inhibition. Regarding the effects of other pharmaceuticals on mollusc THCs, it has been demonstrated that ibuprofen (IBU), a propanoic acid derivative widely used as an analgesic,
Fig. 2. Effects of fluoxetine on haemocyte diameter, expressed in mm, in V. philippinarum. The values are mean þ SD (n ¼ 8).
M. Munari et al. / Marine Environmental Research 94 (2014) 32e37
Fig. 3. Effects of fluoxetine on haemocyte volume, expressed in fl, in V. philippinarum. The values are mean þ SD (n ¼ 8).
Fig. 4. Effects of fluoxetine on haemocyte proliferation, expressed as OD450, in V. philippinarum. The values are mean þ SD (n ¼ 8). The asterisks indicate significant differences in comparison with controls: *p < 0.05, **p < 0.01, ***p < 0.001.
antirheumatic and antipyretic in nonsteroidal anti-inflammatory drugs, significantly decreased the number of circulating haemocytes in clams (Matozzo et al., 2012b). Haemocyte count was significantly increased (3.1-fold) in the snails (Lymnaea stagnalis) exposed to a global mixture of different pharmaceutical classes (Gust et al., 2013). However, information about the effects of contaminants on the THC in molluscs is conflicting. For example,
Fig. 5. Effects of fluoxetine on NRU, expressed as OD550, of haemocytes from V. philippinarum. The values are mean þ SD (n ¼ 8). The asterisks indicate significant differences in comparison with controls: **p < 0.01.
35
exposure to cadmium and fluoranthene induces a significant increase in the THC in Mytilus edulis (Coles et al., 1994, 1995), whereas exposure to phenanthrene does not induce a significant change in THC values in M. edulis, Cerastoderma edule or Ensis siliqua (Wootton et al., 2003). Exposure to 4-nonylphenol (NP) for 7 days significantly increases the THC values in Ruditapes philippinarum (Matozzo and Marin, 2005), whereas no significant alterations in the THC are observed in the clam Chamelea gallina after 12 days of exposure to benzo(a)pyrene (Matozzo et al., 2009). We hypothesised that the increased THC in clams exposed to low levels of fluoxetine was due to the increased mobilisation of haemocytes away from peripheral tissues and into the haemolymph or to an increase in haemocyte proliferation. Regarding the second hypothesis, it is important to note that circulating haemocytes from V. philippinarum can divide in the haemolymph (Matozzo et al., 2008a) and that contaminants (Mayrand et al., 2005), including pharmaceuticals (Matozzo et al., 2012b), can stimulate the mitotic activity of bivalve haemocytes. Consequently, the increase in haemocyte proliferation observed in fluoxetine-exposed clams may partially account for the increased THC values, at least at 25 mg l1. Surprisingly, the significant increase in haemocyte proliferation that was observed in clams exposed to the two highest fluoxetine concentrations tested did not correspond to a significant increase in the number of circulating haemocytes. We formulated two hypotheses to explain the inconsistency of these results. First, it is possible that clams that were exposed to the two highest fluoxetine concentrations underwent a reduction in the number of circulating haemocytes to such an extent that new haemocytes were not enough to compensate for cell number reduction. Alternatively, it is possible that new haemocytes moved towards peripheral tissues to increase immunosurveillance in these tissues. In any case, further studies are needed to better elucidate these aspects. Fluoxetine did not induce significant alterations in either the diameter or the volume of haemocytes in V. philippinarum. The reduction in haemocyte diameter is assumed to be a consequence of alterations in cytoskeletal organisation, whereas the reduction in cell volume may be due to the release of fluids from the cells. Based on the results obtained in this study, we can exclude the idea that fluoxetine induces haemocyte shrinkage in clams, at least under the tested experimental conditions. Similarly, no significant alterations in the diameter or volume of haemocytes are observed in IBUexposed clams (Matozzo et al., 2012b). Conversely, the exposure of clams to triclosan, an antibacterial agent, significantly decreases
Fig. 6. Effects of fluoxetine on lysozyme activity, expressed as mg lysozyme mg1 of protein, in cell-free haemolymph (CFH) of V. philippinarum. The values are mean þ SD (n ¼ 8).
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M. Munari et al. / Marine Environmental Research 94 (2014) 32e37
Fig. 7. AChE activity, expressed as nmol min1 mg1 of protein, in gills from V. philippinarum exposed to fluoxetine. The values are mean þ SD (n ¼ 10). The asterisks indicate significant differences in comparison with controls: *p < 0.05.
the diameter and volume of haemocytes, suggesting cell shrinkage (Matozzo et al., 2012a). The cationic probe NR is commonly used to evaluate the effects of stressors on lysosomal membrane stability in bivalve haemocytes (Hauton et al., 1998; Matozzo et al., 2001; Canesi et al., 2007a; Binelli et al., 2009; Aguirre-Martínez et al., 2013). The uptake of NR by haemocytes occurs either by pinocytosis or by passive diffusion across cell membranes (Coles et al., 1995). Consequently, alterations in dye uptake reflect damage to cell membranes (including lysosomal membranes) and/or weakening of haemocyte pinocytotic capabilities. In this study, we demonstrated that fluoxetine (1 and 5 mg l1) significantly reduced NRU by haemocytes, suggesting a reduction in pinocytotic activity. In our previous studies, we observed increases in NRU by haemocytes from clams exposed to the highest tested NP concentrations (Matozzo and Marin, 2005) and significant reductions in the haemocytes of clams exposed to triclosan (Matozzo et al., 2012a). A biphasic response of clams to fluoxetine exposure was also recorded in this study: NRU decreased in clams exposed to the lowest fluoxetine concentrations and returned to control values in bivalves exposed to the highest concentrations tested. Lysozyme is one of the most important bivalve bacteriolytic agents against several species of gram-positive and gram-negative bacteria (Cheng and Rodrick, 1974). In the present study, the ability of fluoxetine to induce lysozyme release from haemocytes into the haemolymph was evaluated to ascertain whether the compound alters haemocyte membrane stability. Based on results obtained, we can exclude this hypothesis, at least for the species studied and under the tested experimental conditions. An induction of haemocyte degranulation, followed by an increase in extracellular lysozyme activity, has been observed in haemocytes from Mytilus galloprovincialis exposed in vitro to estradiol (Canesi et al., 2004). Significant increases in acid phosphatase and lysozyme activities were also observed in the CFH of cockles (Cerastoderma glaucum) exposed to NP, suggesting that the contaminant caused the destabilisation of lysosomal membranes and the consequent release of hydrolytic enzymes into the haemolymph (Matozzo et al., 2008b). NP has also been shown to induce a significant increase in lysozyme release in M. galloprovincialis haemocytes treated in vitro (Canesi et al., 2007b). It is well known that AChE plays an important role in the functioning of the neuromuscular system by preventing continuous muscular contraction. AChE activity has been proposed as a biomarker of exposure to neurotoxic compounds in aquatic organisms (Cajaraville et al., 2000). Numerous studies have
demonstrated that AChE is mainly inhibited by organophosphorus compounds and carbamates, which are pesticides that are widely used in agriculture (Cajaraville et al., 2000). However, other environmental pollutants, such as heavy metals (Hamza-Chaffai et al., 1998) and pharmaceuticals (ibuprofen) (Milan et al., 2013), can inhibit AChE activity in bivalves. In this study, we tested the hypothesis that fluoxetine is neurotoxic to V. philippinarum. The results of our study partially support this hypothesis. Indeed, gill AChE activity decreased significantly in clams exposed to the lowest concentrations of fluoxetine, whereas enzyme activity returned to control values in clams exposed to the highest fluoxetine concentrations. Although the results obtained in the present study are surprising, it is important to note that controversial results were also recorded in a recent study on mussels (M. galloprovincialis) that were exposed to fluoxetine (75 ng l1) for 15 days (Gonzalez-Rey and Bebianno, 2013). In that study, gill AChE activity increased significantly after 3 days of exposure, followed by progressive inhibition, reaching a significantly lower activity than controls after 15 days of exposure. Conversely, AChE activity was shown to increase significantly in the muscle of crabs (C. maenas) after 7 days of exposure to fluoxetine (120 and 750 mg l1) (Mesquita et al., 2011). Based on both the results of this study and data from the literature, we can conclude that AChE activity may vary markedly in response to fluoxetine exposure, depending on the exposure concentration and duration and on the species and tissues analysed. Regarding the effects of other pharmaceuticals, Solé et al. (2010) demonstrated that acetaminophen significantly inhibits AChE activity in gills from the mussel M. galloprovincialis, while Gagné et al. (2010) observed a significant reduction in AChE activity in the visceral mass of mussels (E. complanata) exposed to morphine. In conclusion, the results of the present study demonstrate that fluoxetine affects haemocyte parameters in the clam V. philippinarum. In addition, the lowest concentrations of fluoxetine significantly reduce AChE activity in clams. Overall, the present study revealed a biphasic response of clams to fluoxetine exposure and suggested that fluoxetine can pose risks to aquatic invertebrates. However, the nonlinear responses of biomarkers that have been measured in V. philippinarum indicate that further investigations (e.g., long-term exposures, tissue collection at different time intervals) are required to completely assess the impact of this compound in bivalves.
Acknowledgements This study was funded by the University of Padova to Dr. V. Matozzo. University-funded research project titled “Immunotoxicity, neurotoxicity and estrogenicity of pharmaceuticals and personal care products in bivalve mollusks” (CPDA095545). The English text was revised by American Journal Experts.
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