Does selective serotonin reuptake inhibitor (SSRI) fluoxetine affects mussel Mytilus galloprovincialis?
Highlights
► Short-time exposure of Mytilus galloprovincialis to antidepressant fluoxetine. ► Tissue-specific transient antioxidant enzymes activities alteration. ► Lipid peroxidation (LPO) induction in exposed-tissues. ► Acetylcholinesterase (AChE) activity upregulation in exposed gills. ► ALP levels downregulation in exposed sex-differentiated mussels.
Introduction
Ecotoxicological risks associated to the ubiquitous occurrence of active pharmaceutical ingredients (APIs) in aquatic ecosystems are far from known. As the detection technology improves, a larger variety of APIs are being detected in the aquatic environment (see reviews: Calisto and Esteves, 2009; Kümmerer, 2009; Li and Randak, 2009; Alonso et al., 2010; Pal et al., 2010; Kümmerer, 2010; Santos et al., 2010; Brausch and Rand, 2011). Waste water treatment plants (WWTPs) are still ill-equipped for the constant APIs discharge load removal, consequently these bioactive compounds end up entering surface waters (rivers, estuaries and lakes) particularly via household and hospital treated effluents, and from there often recycled to drinking water (Ternes, 2001; Ternes et al., 2002; Stackelberg et al., 2004; Jones et al., 2005; Gibs et al., 2007; Kim et al., 2007; Daughton, 2010) posing potential risks to non-target aquatic life (Fong, 2001; Brooks et al., 2005; Fent et al., 2006).
Fluoxetine (FLX) present in antidepressant Prozac® the most widely prescribed psychoactive drug in the market and like others (e.g. citalopram, fluvoxamine, paroxetine, and sertraline) act as selective serotonin reuptake inhibitor (SSRI) in the treatment of depression, and other mood disorders by increasing the serotonin (5-hydroxytrypamine – 5-HT) levels in neuron synaptic space (Brosen, 1993; De Vane, 1999; Hiemke and Härtter, 2000; Fent et al., 2006). Even though, FLX is excreted via urine approximately 10–30% as unchanged parent compound or metabolized to norfluoxetine (De Vane, 1999; Hiemke and Härtter, 2000; Fong and Molnar, 2008) is resilient to hydrolysis, photolysis and microbial degradation processes (Kwon and Armbrust, 2006) occurring in the aquatic environment at ng L−1 (Table 1). Since SSRIs alter neurotransmitter 5-HT regulation, which has been associated to the modulation of important functions in hormonal and neuronal mechanisms in both vertebrates and invertebrates (Fong, 2001; Fent et al., 2006; Stanley et al., 2007; Painter et al., 2009; Styrishave et al., 2011) most peer reviews on FLX exposure ecotoxicological effects focus on acute FLX toxicity and/or physiological, behavioral (mobility, feeding habits and aggression) and reproductive fitness alterations (Table 2). Nevertheless, FLX has also been related to affect antioxidant system in mice (Djordjevic et al., 2011). Oxidative stress is characterized by the imbalance when xenobiotic-mediated enhancement of reactive oxygen species (ROS) (e.g. superoxide anion (OH−), hydrogen peroxide (H2O2) and hydroxyl radicals) exceed exposed aerobic organism's antioxidant defense mechanisms. One of these mechanisms involves the counteracting response of antioxidant enzymes activities such as, superoxide dismutase (SOD) and catalase (CAT) and glutathiones (peroxidase – GPx and reductase – GR) (Livingstone, 2001; Regoli et al., 2002a,b; Valavanidis et al., 2006). Additionally, Phase II glutathione S-transferase enzyme also enables detoxification, acting as a catalyst in conjugation reactions between glutathione with xenobiotic compounds electrophilic centers (Regoli and Principato, 1995). When the antioxidant system response is compromised by an ROS excess, lipid peroxidation (LPO) occurs, resulting in the damage of phospholipids membrane (Valavanidis et al., 2006). The fluctuation of antioxidant enzymes parallels to lipid peroxidation generation due to contaminant exposure have been used successfully as oxidative stress and damage biomarkers in mussel species (Regoli and Principato, 1995; Regoli et al., 2002a; Santovito et al., 2005; Bebianno et al., 2005).
To our knowledge, this is the first study focused on the potential antioxidant alteration status of an environmental realistic concentration of FLX (75 ng L−1) exposure in mussels M. galloprovincialis through the assessment of antioxidant enzyme activities: SOD, CAT; phase II GST activity, and LPO in mussels' gills and digestive gland. In parallel, SSRI FLX potential to cause neurotoxic effects response was tested by assessing the activity of an essential neurotransmission modulator, enzyme acetylcholinesterase (AChE) in mussel gills. AChE activity has been reported to be inhibited in the presence of several organic contaminants (such as pesticides, detergents and pharmaceuticals) (Almeida et al., 2010; Solé et al., 2010). Finally, alkali-labile phosphate (ALP) method was applied on sex-differentiated mussel gonads to assess FLX as an endocrine disruption inducer, since ALP levels are positively correlated with those from vitellogenin-like proteins which are naturally synthesized in females and inactive in males (Blaise et al., 1999; Gagné et al., 2002; Matozzo et al., 2008).
Section snippets
Chemicals
R-(-) fluoxetine hydrochloride (F1678, >98%, CAS: 114247-09-5); 1.1.3.3. tetramethoxypropane (MDA) (108383, CAS: 102-52-3); 1-methyl-2-phenylindole (99%, CAS: 3558-24-5); 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (D8130, ≥98% TLC, CAS: 69-78-3); acetyl thiocholine iodide (ATC) (A5751, ≥98% TLC, CAS: 1866-15-5); bovine albumin serum (BSA) (A9418, >98%, CAS: 9048-46-8); butylated hydroxytoluene (BHT) (B1378, ≥99.0% GC, CAS: 128-37-0); cytochrome c from equine heart (C7752, >95%, CAS: 9007-43-6);
CI
A significant decrease was observed in control mussels from the beginning (26.0 ± 2.6%) to the 3rd day (22.2 ± 3.3%), remaining unchanged until the end of the experiment (min. 19.5 ± 4.4%). Additionally, FLX exposed mussels' condition index was not affected throughout the exposure (day 3: 21.9 ± 4.0; day 7: 22.0 ± 3.3; day 15: 20.0 ± 3.8%) being not significantly different from respective controls.
Antioxidant enzymes
SOD activity is significantly higher in gills than in digestive glands for both non- and FLX
Oxidative stress
To our knowledge this is the first study concerning the effect of FLX as a potential oxidative stress inducer applying antioxidant enzymes response activities in mussels. The results reveal a transient antioxidant status alteration in both mussels' tissues. As the first organ in direct contact with the contaminant, the significant SOD activity downregulation was inversely related only in the gills, the concomitant inhibition tendency of SOD activity in digestive glands resulted in a significant
Conclusions
FLX exposure to mussels for two weeks induced a transient antioxidant enzyme activities alteration particularly in gills. However these alterations were not as severe as overall ALP sex-differentiated downregulation in gonadal tissues, highlighting a higher endocrine disruption effect of FLX rather than an oxidative stress inducer. Furthermore, resulting from the influence of FLX as a SSRI, on 5-HT levels increase as mentioned before, AChE activity was clearly altered throughout the experiment
Conflict of interest statement
The authors declare the inexistence of any conflicts of interest.
Acknowledgments
Maria Gonzalez-Rey Ph.D. fellowship (SFRH/BD/41606/2007) from the Portuguese Science and Technology Foundation (FCT) of the Portuguese Ministry of Science and Technology. The authors would like to thank the support of subproject EMECORISK: “Effect of emerging contaminants in aquatic ecosystems” of 0432-I2TEP-5-E (I2TEP) project and FP7-People-2009-IRSES GENERA project. Deborah Legibre, Mathilde Muller and Catarina Pereira for technical assistance in ALP method protocol.
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