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Neurochemical effects of benzodiazepine and morphine on freshwater mussels
Comparative Biochemistry and Physiology, Part C 152 (2010) 207–214
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Comparative Biochemistry and Physiology, Part C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p c
Neurochemical effects of benzodiazepine and morphine on freshwater mussels F. Gagné ⁎, C. André, M. Gélinas Fluvial Ecosystem Research Section, Environment Canada, 105 McGill Street, Montréal, Quebec, Canada H2Y 2E7
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Article history: Received 17 March 2010 Received in revised form 8 April 2010 Accepted 8 April 2010 Available online 14 April 2010 Keywords: Benzodiazepines Morphine Dopamine Serotonin Glutamate GABA Acetylcholinesterase Oxidative stress Municipal effluents Mussels
a b s t r a c t The purpose of this study was to examine the neurochemical effects of morphine, diazepam, a common benzodiazepine, and an effluent concentrate on the endemic freshwater mussel Elliptio complanata. Mussels were exposed to the drugs and to the solid-phase concentrate of a municipal effluent and left to stand at 15 °C for 48 h. Neurochemical effects were determined by monitoring changes in dopamine, serotonin, glutamate and γ-aminobutyric acid (GABA) levels in the visceral mass (containing the nerve ganglia) of mussels. The activities of acetylcholinesterase (AChE), dopamine and serotonin-dependent adenylyl cyclase (ADC) were also determined in the mussels. Oxidative stress was determined by tracking changes in lipid peroxidation (LPO) in the mitochondrial and post-mitochondrial fractions. The results revealed that the drugs and the effluent extract were biologically active in mussels. Morphine reduced serotonin and increased dopamine in mussel tissues while reducing AChE activity and increasing GABA levels. This suggests the induction of a relaxation state in mussels. Diazepam also reduced serotonin levels but produced no change in dopamine levels. However, dopamine-sensitive ADC activity was readily activated, indicating the potential effect on opiate signaling. Diazepam increased glutamate levels slightly, but AChE remained stable. The increase in both dopamine ADC activity and glutamate concentrations was also associated with greater oxidative stress on the mitochondrial and post-mitochondrial fractions in cells. A comparison of the global response pattern of these drugs with those of the effluent extract revealed only a relative proximity to morphine. In conclusion, the data warrant more studies on the analysis of opiates and benzodiazepines in municipal effluents to better address the potential environmental hazard of these neuroactive drug classes to aquatic organisms. Crown Copyright © 2010 Published by Elsevier Inc. All rights reserved.
1. Introduction Municipal effluents are recognized as important sources of pollution to the aquatic environment. They have been reported to release a variety of biological and chemical contaminants including endocrine disruptors (mostly estrogenic), oxidizing, genotoxic and serotonergic compounds (Sumpter and Jobling, 1995; Gagné and Blaise, 2003; Vethaak et al., 2005). In addition, pharmaceutical and personal care products (PPCPs) have been identified in various municipal effluents (Kummerer, 2001). Numerous studies have also identified compounds like pain relievers, anti-inflammatory drugs (ibuprofen and acetaminophen), antibiotics (sulfonamines and tetracyclines), cholesterol regulators (clofibrates and statins), and neuroactive drugs (caffeine, carbamazepine, and selective serotonin reuptake inhibitors). Reports on benzodiazepines and morphine in urban effluents are scarce, at best. However, diazepam was sold in the United State reaching sales of 2.3 billion tablets i.e., circa 11,500–
⁎ Corresponding author. Fluvial Ecosystem Research, Aquatic Ecosystem Protection Research Division, Water Science and Technology, Environment Canada, 105 Mc Gill, Montréal, Québec, Canada H2Y 2E7. Tel.: + 1 514 496 7105. E-mail address: [email protected] (F. Gagné).
23,000 kg (Sample, 2005). Morphine was detected in urban effluents at the 0.1 µg/L (representing 220 g/day) using a competitive immunoassay method (Gagné et al., 2004), while reports on benzodiazepines in urban effluents are, to the best of our knowledge, lacking at the present time. Benzodiazepine acts through specific receptors located in the plasma and mitochondrial membranes of invertebrates (Giannaccini et al., 2004). Diazepam is a commonly prescribed benozodiazepine for the treatment of illnesses such as anxiety, convulsions, insomnia and muscle spasms. The mode of action of this class of compounds consists of binding to γ-aminobutyric acidA (GABAA) receptors, which involves relaxation by slowing synaptic depolarization. Morphine is a drug in the opiate receptor family that is used to treat various pain-related conditions. Derivatives of morphine are also used as cough suppressants and for the treatment of diarrhea. Morphine binds to the opiate receptors, which act as pain suppressants and reduce smooth-muscle contraction. Morphine signaling by the opiate receptor also involves the release of dopamine to synapses and turnover. In mussels, dopamine and morphine production are coupled (Zhu et al., 2005a). The evolutionary conservation of tyrosine as the common precursor of catecholamines and opiate signaling suggests that morphine and dopamine metabolisms are physiologically linked. However, dopamine signaling is also involved in oogenesis when vitellogenesis takes
1532-0456/$ – see front matter. Crown Copyright © 2010 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2010.04.007
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place in bivalves (Khotimchenko, 1991). Serotonin signaling is involved at the end of gametogenesis to complete the maturation of gametes and initiate spawning (Matsutani and Nomura, 1987). Studies also revealed that pharmaceutical products are likely to affect bivalve physiology. For example, increased levels of dopamine and norepinephrine were observed when exposed to monoamine oxidase (MAO) inhibitors such as Pargyline (type A inhibitor) and Deprenyl (type B inhibitor) in scallops (Pani and Croll, 1998). This suggests that bivalves possess mammalian-like metabolic pathways for catecholamines and indolamines. Indeed, catecholamines (dopamine, noradrenaline) and indolamines (serotonin, octopamine) are eliminated by a mitochondrial MAO enzyme complex whose expression could be influenced by environmental contaminants such as polyaromatic hydrocarbons (Boutet et al., 2004). Acetylcholine and glutamate are considered neuroexcitatory neurotransmitters and they are involved in neuromuscular stimulation, locomotion and stimulation of neural activity (García-Lavandeira et al., 2005). Acetylcholine is regulated by acetylcholinesterase (AChE), which is rendered inactive by hydrolysis into choline and acetate. Increased glutamate signaling is involved in excitotoxicity syndromes that lead to hyperactivity in nerve cells and their breakdown by oxidative stress and inflammation (Reiss et al., 1977; Gagné et al., 2007). Mussels and other bivalves are particularly at risk to compounds released by municipal effluents. They are sessile, long-lived and filter high volumes of water and suspended matter during respiration and feeding. The steady decrease in mussel recruitment has been attributed to numerous factors, including water quality (pollution), loss of habitat, fluctuations in water level and climatic changes (Lydeard et al., 2004). In a previous study, mussels injected with caffeine, carbamazepine and an effluent extract had elevated levels of cytochrome P4503A-like activity and lipid peroxidation for caffeine and carbamazepine (Martin-Diaz et al., 2009a). In another study, exposure to environmentally realistic concentrations of carbamazepine in water led to increased xenobiotic conjugation and catalase activity, with decreased cAMP-mediated cell signaling and mRNA encoding for the multixenobiotic resistance gene in the blue mussel Mytilus galloprovincialis (Martín-Díaz et al., 2009b). These authors concluded that carbamazepine, at the persistent environmental concentrations in respect to municipal effluents, affects specific biochemical pathways that are seemingly evolutionarily conserved in these invertebrates. The purpose of the present study was to document the neurochemical effects of morphine and benzodiazepine on freshwater mussels in response to the current lack of toxicological information and given the likelihood of their being found in municipal effluents. The neurotoxicity of these compounds in freshwater mussels was tracked by measuring changes in serotonin, dopamine, dopamine- or serotonin-dependent adenylyl cyclase activities, AChE and glutamate in the visceral mass containing ganglia. In addition, oxidative stress was measured by tracking lipid peroxidation (LPO) in the homogenate, the mitochondrial (cellular respiration), and post-mitochondrial fractions (metabolism) to determine the intracellular location of oxidative stress in cells. The response of mussels exposed to morphine and diazepam were compared to those exposed to a domestic municipal wastewater extract, with the aim of identifying some common features of the observed neurochemical responses. 2. Materials and methods 2.1. Mussel handling and exposure Mussels (Elliptio complanata) were collected by hand in the Richelieu River (Quebec, Canada) in late May (2007), during their period of late gametogenesis. They were placed in aerated tanks for two weeks at 15 °C and fed with Pseudokirchneriella subcapitata algal preparations (10–30 million cells/L) bi-weekly. The mussels
were divided up (eight each) among the four treatment groups, i.e. the control, the diazepam, the morphine and the municipal effluent extract group. Mussels were chosen to minimize size distribution (30–44 g whole wet weight) and variations in soft tissue size or mass in these experiments. The mode of exposure by injection was chosen to ensure that the drugs were readily delivered in mussel tissues and eliminated the chance for drug adsorption to the exposure vessel walls, aggregation in the water phase and adsorption to the shells. Mussels were injected (50 µL) via the adductor muscle using a 1-mL hypodermic syringe with increasing concentrations of morphine sulfate (0.03, 0.15 and 0.75 μg/g wet weight) and diazepam (0.07, 0.35 and 1.75 μg/g). These concentrations corresponded to the same molar concentrations (4, 20 and 100 nmol per mussel). Morphine is a controlled substance and obtained by permission under the Food and Drug Act of Health Canada and purchased from Sigma Chemical Company, USA. The drugs were prepared in 100% dimethyl sulfoxide (DMSO); the control group received an equal amount of DMSO only. Mussels were also treated with a municipal effluent extract as described below. The extract was prepared by filtering 200 mL of municipal effluent on a C8 reverse-phase cartridge (200 mg), washing with 10 mL of bi-distilled water, and eluting with 1 mL of 100% ethanol (200× concentrate). The ethanol fraction was then evaporated under nitrogen stream and resuspended in 100% DMSO. Mussels were then injected with the extract as described above. The mussels were then placed in glass aquaria (40-L) containing drinking tapwater (UV-treated and passed through an activated charcoal column) at 15 °C for 48 h. The visceral mass containing the nerve ganglia without the digestive gland was dissected on ice and immediately homogenized in 50 mM Hepes–NaOH buffer, pH 7.4, containing 100 mM NaCl, 1 mM EDTA and 1 mM dithiothreitol, at 4 °C at a 1:5 weight/volume ratio. A Teflon pestle tissue grinder apparatus was used (five passes at 4 °C). A portion of the homogenate was centrifuged at 1500 g for 15 min at 2 °C to remove cell debris and nuclei. The supernatant was centrifuged at 9000 g for 20 min at 2 °C to obtain the crude mitochondrial fraction (pellet) and the remaining supernatant was centrifuged at 15 000 g for 20 min at 2 °C to obtain the post-mitochondrial supernatant. The mitochondrial pellet was resuspended in two volumes of 100 mM NaCl containing 2 mM KH2PO4, 0.1 mM EDTA and 10 mM Hepes– NaOH, pH 7.4. Total proteins were determined in each fraction using serum bovine albumin for calibration (Bradford, 1976). The homogenate, mitochondrial and post-mitochondrial fractions were stored at − 85 °C until biochemical analyses. 2.2. Neurochemical assessments The levels of glutamate and GABA were measured using the GABA transaminase and succinate semialdehyde dehydrogenase procedure (Gagné et al., 2007; Woff and Klemish, 1991). The postmitochondrial fraction was heat-treated at 100 °C for 5 min in 0.2 M HCl to remove NADPH and centrifuged at 10,000 g to remove denatured proteins. The resulting supernatant was pre-incubated with one unit of GABA decarboxylase (Sigma Chemical Company) in 0.1 mM sodium phosphate, pH 7, 1 mM 2-aminoethylisothironium bromide, 0.1% Triton X-100, 0.2 mM pyridoxal 5-phosphate and 1 mM dithiothreitol. After incubating for 60 min at 30 °C, the reaction was stopped with one volume of HCl 0.2 M. The formation of GABA from the decarboxylation of glutamate was measured using the GABA transaminase/succinate semialdehyde system as described. Standard solutions of glutamate and GABA were used for calibration. The data were expressed as µg equivalents of glutamate or GABA/mg proteins. The levels of serotonin and dopamine were determined in the homogenates using a fluorescence-based method (Orsinger et al., 1980; Szabo et al., 1983). A portion of the homogenate was mixed with one volume of 0.1 N HCl and 0.2 mM of
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reduced glutathione followed by the addition of one volume of nbutanol. The mixture was mixed for 2 min and centrifuged at 2000 g for 15 min to remove precipitated material. The “supernatant” was mixed with one volume of hexane. For serotonin, one volume of aqueous phase was mixed with one volume o-phthalaldehyde in 2 N HCl and heated at 70 °C for 15 min in capped Eppendorf tubes. Fluorescence was measured at 360 nm excitation and 480 nm emission. For dopamine, the aqueous phase was mixed with 0.1 volume of alkaline potassium iodide solution (I2/KI, Lugol, Sigma Chemical Company) and heated at 75 °C for 5 min. Fluorescence was measured at 325 nm excitation and 385 nm emission. Standards of dopamine and serotonin were used for calibration. Data were expressed as ng of monoamine/mg proteins. The activity of acetylcholinesterase (AChE) in the post-mitochondrial fraction was determined using acetylthiocholine as the substrate and detection with Ellman's reagent (Bonacci et al., 2004). Oxidative-mediated damage was tracked using the lipid peroxidation (LPO) assay (Wills, 1987). The assay procedure was performed in the homogenate and the mitochondrial and post-mitochondrial fractions. A standard solution of malonaldehyde (tetramethoxypropane) was used for calibration. The data were expressed as µg thiobarbituric acid reactants/mg proteins. 2.3. Serotonin and dopamine adenylyl cyclase activity Serotonin and dopamine adenylyl cyclase (ADC) activity was determined in synaptosome preparations isolated from the 1500 g supernatant of the homogenate using a sucrose density gradient (Sherman, 1989). Serotonin- and dopamine-stimulated ADC was determined in synaptosomes in the presence of ATP using the inorganic phosphate assay (Kakko et al., 2003; Gagné et al., 2007). The reaction contained 100 µM ATP, 10 µM of either dopamine or serotonin and synaptosome (50–100 µg/mL) in 100 mM NaCl, 10 mM Hepes–NaOH, pH 7.4, 1 mM MgCl2 and 1 mM KCl. After a 30 and 60 min incubation at 25 °C, the levels of phosphates from the hydrolysis of ATP were determined by the phosphomolybdate spectrophotometric methodology (Stanton, 1968). The basal activity of (Na/K−) ADC was also determined and remained constant across the treatment groups (ANOVA p N 0.05).
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2.4. Statistical analysis The data (from N = 8 mussels per treatment group) were subjected to the Brown–Forsythe test to ensure homogeneity of variances. The data were log-transformed as necessary to minimize the effect of the heterogeneity of variances. An analysis of variance was performed in which the critical difference between the controls and the three treatment groups (i.e. diazepam, morphine and effluent extract) was determined using the Dunnett t test. Pearson-moment correlations, factorial and discriminant function analyses were also determined. Significance was set at the p b 0.05 level (Statistica software version 8, France). 3. Results Serotonin significantly decreased at a threshold concentration of 0.35 µg diazepam/g mussel (Fig. 1). Morphine was more potent than diazepam in reducing serotonin tissue levels, with a threshold concentration b0.07 µg/g mussel. The effluent extract did not produce any significant changes in serotonin content in visceral tissues. Dopamine content was not affected by diazepam while there was a transient 2.6-fold rise at the lowest morphine exposure dose (i.e., 0.07 µg/g mussel). The levels of dopamine returned to control values with increasing concentrations of morphine. However, dopamine content was increased 7-fold by the effluent extract. No significant correlations were observed between serotonin and dopamine contents in mussels. Serotonin and dopamine ADC activity were also determined in mussels exposed to diazepam, morphine and a solidphase (C8) municipal effluent extract (Fig. 2). Serotonin ADC activity was significantly increased 2.3-fold at the highest dose of diazepam, with an estimated threshold concentration of 0.35 µg of diazepam/g mussel. Neither morphine nor the effluent extract produced significant changes in serotonin ADC activity, but dopamine ADC activity was significantly affected by these treatments. Diazepam readily stimulated dopamine ADC activity in the mussels at a threshold concentration of b0.03 µg/g mussel, reaching 5.5-fold activation at diazepam dosage of 0.15 μg/g. Morphine had no significant effect on dopamine ADC activity. Conversely, the effluent extract increased such activity 2.8-fold relative to control mussels (i.e. those injected
Fig. 1. Change in monoamine levels in mussels treated with morphine, diazepam and an effluent extract. Mussels were injected with the compound into the adductor muscle and left to stand for 48 h at 15 °C. Tissue monoamines (serotonin and dopamine) were determined in the visceral mass. * indicates significant difference from the controls (pb0.05).
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with DMSO only). Based on this biological response, the final effluent extract would contain roughly 100 µg/L of diazepam-like substances. However this only shows the scale of the observed response (i.e. not a real estimate of diazepam or other benzodiazepines) and should be validated with proper chemical tissue analysis. A correlation analysis revealed that serotonin ADC activity was correlated with dopamine ADC activity (r = 0.68; p b 0.001). Dopamine ADC activity was correlated with serotonin content (r = 0.41; p b 0.01). The activity of AChE, glutamate and GABA were also determined in mussels (Fig. 3A and B). AchE was significantly affected by morphine and by the effluent extract. Treatment with morphine reduced AChE activity 1.5-fold at the lowest concentration tested (0.07 ng/g mussel). AChE activity was significantly increased (1.2-fold) by the effluent extract. A correlation analysis revealed that AChE was significantly correlated with dopamine ADC activity (r = 0.4; p b 0.01) and serotonin levels (r = 0.55; p b 0.001). The levels of the excitatory neurotransmitter glutamate were also determined in tissues (Fig. 3B). Glutamate levels were significantly increased by diazepam and reduced by morphine at a threshold concentration of 0.07 µg/g mussel and 0.8 µg/g mussel, respectively. A correlation analysis revealed that glutamate levels were correlated with dopamine content (r = 0.44; p b 0.01). The relaxant neurotransmitter GABA was also significantly affected by the treatments (Fig. 3B). GABA levels were higher in mussels exposed to morphine and diazepam at the lowest concentration tested (0.07 and 0.03 µg/g mussel for morphine and diazepam, respectively). However, only a transitorily significant increase was observed with diazepam. The effluent extract did not produce any significant changes. A correlation analysis revealed that GABA tissue levels were significantly correlated with dopamine ADC activity (r = − 0.42; p b 0.01), serotonin (r = − 0.49; p = 0.001) and AChE (r = − 0.60; p b 0.001). Oxidative stress was determined by tracking changes in LPO in mussel tissues (Fig. 4). LPO was measured in the homogenate, mitochondrial and post-mitochondrial fractions to determine the general location of the oxidative stress. While LPO in the mitochondrial fraction is the results of oxidative stress from cellular respiration, LPO in the post-mitochondrial fraction represents a measure of oxidative stress resulting from phase 1 and 2 biotransformation
activities which occurs in microsomes and the soluble fraction of cells. LPO in the homogenate was significantly increased by the treatments. LPO was significantly affected by diazepam at a threshold concentration of 0.07 µg/g mussel, while morphine did not increase LPO in the homogenate. The effluent extract significantly reduced (by 3-fold) LPO in tissues. LPO was significantly correlated with serotonin ADC activity (r = 0.52; p b 0.001), dopamine ADC activity (r = 0.43; p b 0.01) and dopamine (r = −0.48; p b 0.01). LPO was also measured in mitochondria (Fig. 4). LPO in mitochondria was significantly induced by diazepam at a threshold concentration of 0.07 µg/g mussel. Morphine did not influence mitochondrial LPO. However, the effluent extract increased LPO in mitochondria. LPO in the postmitochondrial fraction containing light membrane vesicles from plasma, lysosomes and microsomes was significantly induced by diazepam at a threshold concentration of 0.03 µg/g mussel. Morphine also elevated LPO in the post-mitochondrial fraction at a threshold concentration of 0.8 µg morphine/g mussel. LPO in the postmitochondrial fraction was significantly correlated with dopamine (r = −0.37; p b 0.05), glutamate (r = −0.28; p b 0.05) and LPO in the homogenate (r = 0.61; p b 0.001). Mitochondrial LPO was significantly correlated with dopamine ADC activity (r = 0.38; p b 0.01), glutamate (r = 0.49; p b 0.001) and LPO in the homogenate (r = 0.43; p = 0.01). The biomarker data obtained for the four treatment groups (i.e. controls, diazepam, morphine and effluent extract) were analyzed using discriminant function and factorial analyses in an attempt to determine the similarities of these pharmaceutical agents with the municipal effluent extract, and the interrelationships between the various neurochemical responses (Fig. 5A). The analyses revealed that exposure to both drugs and to the effluent extract were well classified, with the exception of the control group (60%), which was misclassified in the morphine treatment group in some cases. The centre of gravity of the diazepam cluster was farther from the effluent extract cluster than from the morphine cluster. The biomarkers with the highest factorial weights were dopamine, GABA, AChE activity and LPO in the homogenate. Based on this analysis, the global effects of effluent extract appear closer to the general responses of opiate-like effects than those of benzodiazepine-like effects. A factorial analysis revealed that dopamine ADC activity was closely related with
Fig. 2. Monoamine adenylate cyclase (ADC) activity in mussels treated with morphine and diazepam and an effluent extract. Mussels were injected to the drugs and effluent extract into the adductor muscle and left to stand for 48 h at 15 °C. Serotonin or dopamine Na/K-ATPase activity was determined in the synaptosome fraction. * indicates significant difference from the the controls (pb0.05).
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Fig. 3. Change in acetylcholinesterase activity and glutamate metabolism in mussels treated to morphine, diazepam and an effluent extract. Mussels were injected with the compounds into the adductor muscle and left to stand for 48 h at 15 °C. AChE (A), glutamate/GABA (B) were determined in the visceral mass. * indicates significant difference from the controls (pb0.05).
oxidative stress in the mitochondrial fraction (Fig. 5B). Serotonin was also closely related with AChE activities and GABA was negatively related with serotonin, dopamine ADC and AChE activities. 4. Discussion Municipal effluents are well known to contain a plethora of pharmaceutical product residues such as caffeine, carbamazepine, analgesics, selective serotonin reuptake inhibitors and antibiotics. Benzodiazepines (e.g. diazepam or valium) and morphine-based substances (analgesics, cough suppressant and anti-diarrheal drugs) are also likely to be found in these wastewaters. Morphine was reported in a physically and chemically treated municipal (City of Montréal, QC, Canada) effluent at the 0.1 µg/L level (Gagné et al.,
2004). Diazepam was found in turbot liver at concentrations ranging from 23 to 110 ng/g (wet weight), pointing up their occurrence in polluted aquatic environments (Kwon et al., 2009). Mussels possess benzodiazepine and opiate receptors in their nervous systems (Stefano and Scharrer, 1996; Betti et al., 2003). Mussels collected at a heavy-metal-polluted site showed a significantly higher number of binding sites for benzodiazepines than mussels collected at a less contaminated site (Betti et al., 2003). An increase in the number of available binding sites might be the consequence of decreased GABA production in tissues. A similar response was observed in the clam Mya arenaria contaminated by tributyltin, which showed less estradiol-17β and a high number of estradiol-binding sites (Gagné et al., 2003). In the present study, levels of glutamate and GABA were not significantly affected by the effluent extract. However, morphine
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Fig. 4. Induction of LPO in mussels treated with diazepam, morphine and an effluent extract. Mussels were injected with the compounds into the adductor muscle and left to stand for 48 h at 15 °C. LPO was determined in the visceral mass. * indicates significant difference from the controls (pb0.05).
was able to increase GABA (and reduce glutamate) levels in mussels while lowering AChE activity, suggesting a state of relaxation in mussels. Studies examining the interaction of GABA and morphinerelated effects in aquatic animals are scarce. In rats, the activation of GABAA receptors (but not GABAB) appeared to accentuate the effects of morphine (Yoon et al., 2009). This suggests that GABA signaling could potentiate the effects of morphine. In mussels, morphine stimulates dopamine turnover and urban effluents increase dopamine levels and dopamine ADC activity (Gagné et al., 2007). However, the primary-treated effluent decreased GABA levels, while the effluent extract produced no changes. The effluent extract was from a biologically-treated sewage station which treats domestic wastewaters with an input of hospital complex of 300 beds. Morphine and dopamine metabolisms are coupled in mussels, where morphine synthesis is produced by dopamine in nerve ganglia (Zhu et al., 2005b). Moreover, morphine reduces the immune response in blue mussels (Ottaviani et al., 1995) and in the freshwater mussel E. complanata (Gagné et al., 2006). Hence, the release of dopamine by diazepam could stimulate morphine signaling, which in turn suppresses the immune system. Immunosuppression is a common syndrome in mussels exposed to municipal effluents (Gagné et al., 2006). Diazepam and morphine are biotransformed by cytochrome P450 3A4 and 2C19, which involve oxidative N-dealkylation, hydroxylation and conjugation to glucuronide (Charney et al., 2001). Xenobiotic biotransformation is an oxidative process in which the production of reactive oxygen species and the formation of more polar (reactive) intermediates occur. In the present study, both diazepam and morphine increased LPO in the post-mitochondrial fraction where xenobiotic biotransformation processes take place. Diazepam was more potent than morphine in inducing LPO in mussels with a threshold concentration of 0.06 µg/g mussel. Interestingly, only diazepam and the effluent extract were able to elicit LPO in the mitochondria. GABA receptors have been identified in the mitochondria of mussels (Giannaccini et al., 2004) and can be blocked by reactive oxygen species like H2O2 in cardiomyocytes (Zhang et al., 2002). Lead ions (Pb2+) inhibited benzodiazepine (PK 1195) binding in mussel mitochondria in a non-competitive manner and basal ADC
activity in plasma membranes. Dopamine is also eliminated by monoamine oxidases located in mitochondria (Gilloteaux, 1979) and dopamine-dependent ADC activity was significantly correlated with LPO in mitochondria in the present study. This is in agreement with a previous study in which the large proportion of dopaminesensitive ADC activity was located in the crude mitochondrial fraction in rat cerebral cortex homogenates (von Hungen and Roberts, 1973). Dopamine was able to activate mitochondrial ADC receptors and was seemingly independent of β-blockers such as propanolol but was very sensitive (blocked) to the anti-psychotic drug Haloperidol. This suggests that increased synaptosomal dopamine-sensitive ADC activity was associated with increased LPO in mitochondria, perhaps through metabolism by monoamine oxidase activity in mitochondria, which involves the release of H2O2. The release of H2O2 would block GABA receptors and maintain high levels of GABA, as shown in the morphine-treated group. Multivariate analyses of the neurochemical data were performed in an attempt to identify similarities between the observed responses with diazepam or morphine and those found with the effluent extract. They revealed that the drug responses did not overlap with those of the effluent extract, suggesting a different response pattern (Fig. 5A). However, the response pattern of morphine was closer to the effluent extract, which suggests some morphine-like properties in this effluent. In a previous study, an opiate-like effect was observed in mussels exposed to a primary-treated effluent (Gagné et al., 2007), as evidenced by increased dopamine turnover and adenylyl cyclase activity in nerve tissues. Interestingly, diazepam was also able to increase dopamine levels and dopamine ADC in mussels, which suggest that benzodiazepines might have contributed to increased dopamine metabolism. In conclusion, morphine and diazepam (a benzodiazepine) were biologically active in freshwater mussels. Morphine reduced serotonin and increased dopamine in the visceral tissues. This was accompanied by reduced AChE activity and increased GABA levels, suggesting the induction of a relaxation state in mussels. Diazepam also lowered serotonin levels, but produced no change in dopamine levels. However, dopamine-sensitive ADC activity was readily activated, perhaps indicating facilitated opiate signaling. Diazepam slightly increased glutamate levels, indicating a mild
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Fig. 5. Multivariate analysis of the biomarker response data. Biomarkers were analyzed using discriminant function (A) and factorial (B) analyses to determine the ability of the biomarkers to discriminate between the treatment groups and to represent the interrelationships of the biomarkers. The letters (C, D, M and E) are placed at the centres of gravity of the corresponding treatments. The dotted circle corresponds to a highly correlated cluster of biomarkers. The biomarkers in parentheses represent the biomarkers with the highest factorial weight.
stimulatory activity in mussels. The increase in both dopamine ADC activity and glutamate concentration was also associated with increased oxidative stress at the mitochondrial and post-mitochondrial fractions in cells. A comparison of the global response pattern of these drugs with those of the effluent extract revealed only a relative proximity with morphine. However, further experiments will be needed to confirm whether these effluents contain benzodiazepine at sufficient quantities to elicit effects in mussels exposed to them. Acknowledgements This work was funded by the municipal effluent research program of Environment Canada. The authors thank Sophie Trépanier for the biomarker assessments and Patricia Potvin for editing the manuscript.
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Comparative Biochemistry and Physiology, Part C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p c
Neurochemical effects of benzodiazepine and morphine on freshwater mussels F. Gagné ⁎, C. André, M. Gélinas Fluvial Ecosystem Research Section, Environment Canada, 105 McGill Street, Montréal, Quebec, Canada H2Y 2E7
a r t i c l e
i n f o
Article history: Received 17 March 2010 Received in revised form 8 April 2010 Accepted 8 April 2010 Available online 14 April 2010 Keywords: Benzodiazepines Morphine Dopamine Serotonin Glutamate GABA Acetylcholinesterase Oxidative stress Municipal effluents Mussels
a b s t r a c t The purpose of this study was to examine the neurochemical effects of morphine, diazepam, a common benzodiazepine, and an effluent concentrate on the endemic freshwater mussel Elliptio complanata. Mussels were exposed to the drugs and to the solid-phase concentrate of a municipal effluent and left to stand at 15 °C for 48 h. Neurochemical effects were determined by monitoring changes in dopamine, serotonin, glutamate and γ-aminobutyric acid (GABA) levels in the visceral mass (containing the nerve ganglia) of mussels. The activities of acetylcholinesterase (AChE), dopamine and serotonin-dependent adenylyl cyclase (ADC) were also determined in the mussels. Oxidative stress was determined by tracking changes in lipid peroxidation (LPO) in the mitochondrial and post-mitochondrial fractions. The results revealed that the drugs and the effluent extract were biologically active in mussels. Morphine reduced serotonin and increased dopamine in mussel tissues while reducing AChE activity and increasing GABA levels. This suggests the induction of a relaxation state in mussels. Diazepam also reduced serotonin levels but produced no change in dopamine levels. However, dopamine-sensitive ADC activity was readily activated, indicating the potential effect on opiate signaling. Diazepam increased glutamate levels slightly, but AChE remained stable. The increase in both dopamine ADC activity and glutamate concentrations was also associated with greater oxidative stress on the mitochondrial and post-mitochondrial fractions in cells. A comparison of the global response pattern of these drugs with those of the effluent extract revealed only a relative proximity to morphine. In conclusion, the data warrant more studies on the analysis of opiates and benzodiazepines in municipal effluents to better address the potential environmental hazard of these neuroactive drug classes to aquatic organisms. Crown Copyright © 2010 Published by Elsevier Inc. All rights reserved.
1. Introduction Municipal effluents are recognized as important sources of pollution to the aquatic environment. They have been reported to release a variety of biological and chemical contaminants including endocrine disruptors (mostly estrogenic), oxidizing, genotoxic and serotonergic compounds (Sumpter and Jobling, 1995; Gagné and Blaise, 2003; Vethaak et al., 2005). In addition, pharmaceutical and personal care products (PPCPs) have been identified in various municipal effluents (Kummerer, 2001). Numerous studies have also identified compounds like pain relievers, anti-inflammatory drugs (ibuprofen and acetaminophen), antibiotics (sulfonamines and tetracyclines), cholesterol regulators (clofibrates and statins), and neuroactive drugs (caffeine, carbamazepine, and selective serotonin reuptake inhibitors). Reports on benzodiazepines and morphine in urban effluents are scarce, at best. However, diazepam was sold in the United State reaching sales of 2.3 billion tablets i.e., circa 11,500–
⁎ Corresponding author. Fluvial Ecosystem Research, Aquatic Ecosystem Protection Research Division, Water Science and Technology, Environment Canada, 105 Mc Gill, Montréal, Québec, Canada H2Y 2E7. Tel.: + 1 514 496 7105. E-mail address: [email protected] (F. Gagné).
23,000 kg (Sample, 2005). Morphine was detected in urban effluents at the 0.1 µg/L (representing 220 g/day) using a competitive immunoassay method (Gagné et al., 2004), while reports on benzodiazepines in urban effluents are, to the best of our knowledge, lacking at the present time. Benzodiazepine acts through specific receptors located in the plasma and mitochondrial membranes of invertebrates (Giannaccini et al., 2004). Diazepam is a commonly prescribed benozodiazepine for the treatment of illnesses such as anxiety, convulsions, insomnia and muscle spasms. The mode of action of this class of compounds consists of binding to γ-aminobutyric acidA (GABAA) receptors, which involves relaxation by slowing synaptic depolarization. Morphine is a drug in the opiate receptor family that is used to treat various pain-related conditions. Derivatives of morphine are also used as cough suppressants and for the treatment of diarrhea. Morphine binds to the opiate receptors, which act as pain suppressants and reduce smooth-muscle contraction. Morphine signaling by the opiate receptor also involves the release of dopamine to synapses and turnover. In mussels, dopamine and morphine production are coupled (Zhu et al., 2005a). The evolutionary conservation of tyrosine as the common precursor of catecholamines and opiate signaling suggests that morphine and dopamine metabolisms are physiologically linked. However, dopamine signaling is also involved in oogenesis when vitellogenesis takes
1532-0456/$ – see front matter. Crown Copyright © 2010 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2010.04.007
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place in bivalves (Khotimchenko, 1991). Serotonin signaling is involved at the end of gametogenesis to complete the maturation of gametes and initiate spawning (Matsutani and Nomura, 1987). Studies also revealed that pharmaceutical products are likely to affect bivalve physiology. For example, increased levels of dopamine and norepinephrine were observed when exposed to monoamine oxidase (MAO) inhibitors such as Pargyline (type A inhibitor) and Deprenyl (type B inhibitor) in scallops (Pani and Croll, 1998). This suggests that bivalves possess mammalian-like metabolic pathways for catecholamines and indolamines. Indeed, catecholamines (dopamine, noradrenaline) and indolamines (serotonin, octopamine) are eliminated by a mitochondrial MAO enzyme complex whose expression could be influenced by environmental contaminants such as polyaromatic hydrocarbons (Boutet et al., 2004). Acetylcholine and glutamate are considered neuroexcitatory neurotransmitters and they are involved in neuromuscular stimulation, locomotion and stimulation of neural activity (García-Lavandeira et al., 2005). Acetylcholine is regulated by acetylcholinesterase (AChE), which is rendered inactive by hydrolysis into choline and acetate. Increased glutamate signaling is involved in excitotoxicity syndromes that lead to hyperactivity in nerve cells and their breakdown by oxidative stress and inflammation (Reiss et al., 1977; Gagné et al., 2007). Mussels and other bivalves are particularly at risk to compounds released by municipal effluents. They are sessile, long-lived and filter high volumes of water and suspended matter during respiration and feeding. The steady decrease in mussel recruitment has been attributed to numerous factors, including water quality (pollution), loss of habitat, fluctuations in water level and climatic changes (Lydeard et al., 2004). In a previous study, mussels injected with caffeine, carbamazepine and an effluent extract had elevated levels of cytochrome P4503A-like activity and lipid peroxidation for caffeine and carbamazepine (Martin-Diaz et al., 2009a). In another study, exposure to environmentally realistic concentrations of carbamazepine in water led to increased xenobiotic conjugation and catalase activity, with decreased cAMP-mediated cell signaling and mRNA encoding for the multixenobiotic resistance gene in the blue mussel Mytilus galloprovincialis (Martín-Díaz et al., 2009b). These authors concluded that carbamazepine, at the persistent environmental concentrations in respect to municipal effluents, affects specific biochemical pathways that are seemingly evolutionarily conserved in these invertebrates. The purpose of the present study was to document the neurochemical effects of morphine and benzodiazepine on freshwater mussels in response to the current lack of toxicological information and given the likelihood of their being found in municipal effluents. The neurotoxicity of these compounds in freshwater mussels was tracked by measuring changes in serotonin, dopamine, dopamine- or serotonin-dependent adenylyl cyclase activities, AChE and glutamate in the visceral mass containing ganglia. In addition, oxidative stress was measured by tracking lipid peroxidation (LPO) in the homogenate, the mitochondrial (cellular respiration), and post-mitochondrial fractions (metabolism) to determine the intracellular location of oxidative stress in cells. The response of mussels exposed to morphine and diazepam were compared to those exposed to a domestic municipal wastewater extract, with the aim of identifying some common features of the observed neurochemical responses. 2. Materials and methods 2.1. Mussel handling and exposure Mussels (Elliptio complanata) were collected by hand in the Richelieu River (Quebec, Canada) in late May (2007), during their period of late gametogenesis. They were placed in aerated tanks for two weeks at 15 °C and fed with Pseudokirchneriella subcapitata algal preparations (10–30 million cells/L) bi-weekly. The mussels
were divided up (eight each) among the four treatment groups, i.e. the control, the diazepam, the morphine and the municipal effluent extract group. Mussels were chosen to minimize size distribution (30–44 g whole wet weight) and variations in soft tissue size or mass in these experiments. The mode of exposure by injection was chosen to ensure that the drugs were readily delivered in mussel tissues and eliminated the chance for drug adsorption to the exposure vessel walls, aggregation in the water phase and adsorption to the shells. Mussels were injected (50 µL) via the adductor muscle using a 1-mL hypodermic syringe with increasing concentrations of morphine sulfate (0.03, 0.15 and 0.75 μg/g wet weight) and diazepam (0.07, 0.35 and 1.75 μg/g). These concentrations corresponded to the same molar concentrations (4, 20 and 100 nmol per mussel). Morphine is a controlled substance and obtained by permission under the Food and Drug Act of Health Canada and purchased from Sigma Chemical Company, USA. The drugs were prepared in 100% dimethyl sulfoxide (DMSO); the control group received an equal amount of DMSO only. Mussels were also treated with a municipal effluent extract as described below. The extract was prepared by filtering 200 mL of municipal effluent on a C8 reverse-phase cartridge (200 mg), washing with 10 mL of bi-distilled water, and eluting with 1 mL of 100% ethanol (200× concentrate). The ethanol fraction was then evaporated under nitrogen stream and resuspended in 100% DMSO. Mussels were then injected with the extract as described above. The mussels were then placed in glass aquaria (40-L) containing drinking tapwater (UV-treated and passed through an activated charcoal column) at 15 °C for 48 h. The visceral mass containing the nerve ganglia without the digestive gland was dissected on ice and immediately homogenized in 50 mM Hepes–NaOH buffer, pH 7.4, containing 100 mM NaCl, 1 mM EDTA and 1 mM dithiothreitol, at 4 °C at a 1:5 weight/volume ratio. A Teflon pestle tissue grinder apparatus was used (five passes at 4 °C). A portion of the homogenate was centrifuged at 1500 g for 15 min at 2 °C to remove cell debris and nuclei. The supernatant was centrifuged at 9000 g for 20 min at 2 °C to obtain the crude mitochondrial fraction (pellet) and the remaining supernatant was centrifuged at 15 000 g for 20 min at 2 °C to obtain the post-mitochondrial supernatant. The mitochondrial pellet was resuspended in two volumes of 100 mM NaCl containing 2 mM KH2PO4, 0.1 mM EDTA and 10 mM Hepes– NaOH, pH 7.4. Total proteins were determined in each fraction using serum bovine albumin for calibration (Bradford, 1976). The homogenate, mitochondrial and post-mitochondrial fractions were stored at − 85 °C until biochemical analyses. 2.2. Neurochemical assessments The levels of glutamate and GABA were measured using the GABA transaminase and succinate semialdehyde dehydrogenase procedure (Gagné et al., 2007; Woff and Klemish, 1991). The postmitochondrial fraction was heat-treated at 100 °C for 5 min in 0.2 M HCl to remove NADPH and centrifuged at 10,000 g to remove denatured proteins. The resulting supernatant was pre-incubated with one unit of GABA decarboxylase (Sigma Chemical Company) in 0.1 mM sodium phosphate, pH 7, 1 mM 2-aminoethylisothironium bromide, 0.1% Triton X-100, 0.2 mM pyridoxal 5-phosphate and 1 mM dithiothreitol. After incubating for 60 min at 30 °C, the reaction was stopped with one volume of HCl 0.2 M. The formation of GABA from the decarboxylation of glutamate was measured using the GABA transaminase/succinate semialdehyde system as described. Standard solutions of glutamate and GABA were used for calibration. The data were expressed as µg equivalents of glutamate or GABA/mg proteins. The levels of serotonin and dopamine were determined in the homogenates using a fluorescence-based method (Orsinger et al., 1980; Szabo et al., 1983). A portion of the homogenate was mixed with one volume of 0.1 N HCl and 0.2 mM of
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reduced glutathione followed by the addition of one volume of nbutanol. The mixture was mixed for 2 min and centrifuged at 2000 g for 15 min to remove precipitated material. The “supernatant” was mixed with one volume of hexane. For serotonin, one volume of aqueous phase was mixed with one volume o-phthalaldehyde in 2 N HCl and heated at 70 °C for 15 min in capped Eppendorf tubes. Fluorescence was measured at 360 nm excitation and 480 nm emission. For dopamine, the aqueous phase was mixed with 0.1 volume of alkaline potassium iodide solution (I2/KI, Lugol, Sigma Chemical Company) and heated at 75 °C for 5 min. Fluorescence was measured at 325 nm excitation and 385 nm emission. Standards of dopamine and serotonin were used for calibration. Data were expressed as ng of monoamine/mg proteins. The activity of acetylcholinesterase (AChE) in the post-mitochondrial fraction was determined using acetylthiocholine as the substrate and detection with Ellman's reagent (Bonacci et al., 2004). Oxidative-mediated damage was tracked using the lipid peroxidation (LPO) assay (Wills, 1987). The assay procedure was performed in the homogenate and the mitochondrial and post-mitochondrial fractions. A standard solution of malonaldehyde (tetramethoxypropane) was used for calibration. The data were expressed as µg thiobarbituric acid reactants/mg proteins. 2.3. Serotonin and dopamine adenylyl cyclase activity Serotonin and dopamine adenylyl cyclase (ADC) activity was determined in synaptosome preparations isolated from the 1500 g supernatant of the homogenate using a sucrose density gradient (Sherman, 1989). Serotonin- and dopamine-stimulated ADC was determined in synaptosomes in the presence of ATP using the inorganic phosphate assay (Kakko et al., 2003; Gagné et al., 2007). The reaction contained 100 µM ATP, 10 µM of either dopamine or serotonin and synaptosome (50–100 µg/mL) in 100 mM NaCl, 10 mM Hepes–NaOH, pH 7.4, 1 mM MgCl2 and 1 mM KCl. After a 30 and 60 min incubation at 25 °C, the levels of phosphates from the hydrolysis of ATP were determined by the phosphomolybdate spectrophotometric methodology (Stanton, 1968). The basal activity of (Na/K−) ADC was also determined and remained constant across the treatment groups (ANOVA p N 0.05).
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2.4. Statistical analysis The data (from N = 8 mussels per treatment group) were subjected to the Brown–Forsythe test to ensure homogeneity of variances. The data were log-transformed as necessary to minimize the effect of the heterogeneity of variances. An analysis of variance was performed in which the critical difference between the controls and the three treatment groups (i.e. diazepam, morphine and effluent extract) was determined using the Dunnett t test. Pearson-moment correlations, factorial and discriminant function analyses were also determined. Significance was set at the p b 0.05 level (Statistica software version 8, France). 3. Results Serotonin significantly decreased at a threshold concentration of 0.35 µg diazepam/g mussel (Fig. 1). Morphine was more potent than diazepam in reducing serotonin tissue levels, with a threshold concentration b0.07 µg/g mussel. The effluent extract did not produce any significant changes in serotonin content in visceral tissues. Dopamine content was not affected by diazepam while there was a transient 2.6-fold rise at the lowest morphine exposure dose (i.e., 0.07 µg/g mussel). The levels of dopamine returned to control values with increasing concentrations of morphine. However, dopamine content was increased 7-fold by the effluent extract. No significant correlations were observed between serotonin and dopamine contents in mussels. Serotonin and dopamine ADC activity were also determined in mussels exposed to diazepam, morphine and a solidphase (C8) municipal effluent extract (Fig. 2). Serotonin ADC activity was significantly increased 2.3-fold at the highest dose of diazepam, with an estimated threshold concentration of 0.35 µg of diazepam/g mussel. Neither morphine nor the effluent extract produced significant changes in serotonin ADC activity, but dopamine ADC activity was significantly affected by these treatments. Diazepam readily stimulated dopamine ADC activity in the mussels at a threshold concentration of b0.03 µg/g mussel, reaching 5.5-fold activation at diazepam dosage of 0.15 μg/g. Morphine had no significant effect on dopamine ADC activity. Conversely, the effluent extract increased such activity 2.8-fold relative to control mussels (i.e. those injected
Fig. 1. Change in monoamine levels in mussels treated with morphine, diazepam and an effluent extract. Mussels were injected with the compound into the adductor muscle and left to stand for 48 h at 15 °C. Tissue monoamines (serotonin and dopamine) were determined in the visceral mass. * indicates significant difference from the controls (pb0.05).
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with DMSO only). Based on this biological response, the final effluent extract would contain roughly 100 µg/L of diazepam-like substances. However this only shows the scale of the observed response (i.e. not a real estimate of diazepam or other benzodiazepines) and should be validated with proper chemical tissue analysis. A correlation analysis revealed that serotonin ADC activity was correlated with dopamine ADC activity (r = 0.68; p b 0.001). Dopamine ADC activity was correlated with serotonin content (r = 0.41; p b 0.01). The activity of AChE, glutamate and GABA were also determined in mussels (Fig. 3A and B). AchE was significantly affected by morphine and by the effluent extract. Treatment with morphine reduced AChE activity 1.5-fold at the lowest concentration tested (0.07 ng/g mussel). AChE activity was significantly increased (1.2-fold) by the effluent extract. A correlation analysis revealed that AChE was significantly correlated with dopamine ADC activity (r = 0.4; p b 0.01) and serotonin levels (r = 0.55; p b 0.001). The levels of the excitatory neurotransmitter glutamate were also determined in tissues (Fig. 3B). Glutamate levels were significantly increased by diazepam and reduced by morphine at a threshold concentration of 0.07 µg/g mussel and 0.8 µg/g mussel, respectively. A correlation analysis revealed that glutamate levels were correlated with dopamine content (r = 0.44; p b 0.01). The relaxant neurotransmitter GABA was also significantly affected by the treatments (Fig. 3B). GABA levels were higher in mussels exposed to morphine and diazepam at the lowest concentration tested (0.07 and 0.03 µg/g mussel for morphine and diazepam, respectively). However, only a transitorily significant increase was observed with diazepam. The effluent extract did not produce any significant changes. A correlation analysis revealed that GABA tissue levels were significantly correlated with dopamine ADC activity (r = − 0.42; p b 0.01), serotonin (r = − 0.49; p = 0.001) and AChE (r = − 0.60; p b 0.001). Oxidative stress was determined by tracking changes in LPO in mussel tissues (Fig. 4). LPO was measured in the homogenate, mitochondrial and post-mitochondrial fractions to determine the general location of the oxidative stress. While LPO in the mitochondrial fraction is the results of oxidative stress from cellular respiration, LPO in the post-mitochondrial fraction represents a measure of oxidative stress resulting from phase 1 and 2 biotransformation
activities which occurs in microsomes and the soluble fraction of cells. LPO in the homogenate was significantly increased by the treatments. LPO was significantly affected by diazepam at a threshold concentration of 0.07 µg/g mussel, while morphine did not increase LPO in the homogenate. The effluent extract significantly reduced (by 3-fold) LPO in tissues. LPO was significantly correlated with serotonin ADC activity (r = 0.52; p b 0.001), dopamine ADC activity (r = 0.43; p b 0.01) and dopamine (r = −0.48; p b 0.01). LPO was also measured in mitochondria (Fig. 4). LPO in mitochondria was significantly induced by diazepam at a threshold concentration of 0.07 µg/g mussel. Morphine did not influence mitochondrial LPO. However, the effluent extract increased LPO in mitochondria. LPO in the postmitochondrial fraction containing light membrane vesicles from plasma, lysosomes and microsomes was significantly induced by diazepam at a threshold concentration of 0.03 µg/g mussel. Morphine also elevated LPO in the post-mitochondrial fraction at a threshold concentration of 0.8 µg morphine/g mussel. LPO in the postmitochondrial fraction was significantly correlated with dopamine (r = −0.37; p b 0.05), glutamate (r = −0.28; p b 0.05) and LPO in the homogenate (r = 0.61; p b 0.001). Mitochondrial LPO was significantly correlated with dopamine ADC activity (r = 0.38; p b 0.01), glutamate (r = 0.49; p b 0.001) and LPO in the homogenate (r = 0.43; p = 0.01). The biomarker data obtained for the four treatment groups (i.e. controls, diazepam, morphine and effluent extract) were analyzed using discriminant function and factorial analyses in an attempt to determine the similarities of these pharmaceutical agents with the municipal effluent extract, and the interrelationships between the various neurochemical responses (Fig. 5A). The analyses revealed that exposure to both drugs and to the effluent extract were well classified, with the exception of the control group (60%), which was misclassified in the morphine treatment group in some cases. The centre of gravity of the diazepam cluster was farther from the effluent extract cluster than from the morphine cluster. The biomarkers with the highest factorial weights were dopamine, GABA, AChE activity and LPO in the homogenate. Based on this analysis, the global effects of effluent extract appear closer to the general responses of opiate-like effects than those of benzodiazepine-like effects. A factorial analysis revealed that dopamine ADC activity was closely related with
Fig. 2. Monoamine adenylate cyclase (ADC) activity in mussels treated with morphine and diazepam and an effluent extract. Mussels were injected to the drugs and effluent extract into the adductor muscle and left to stand for 48 h at 15 °C. Serotonin or dopamine Na/K-ATPase activity was determined in the synaptosome fraction. * indicates significant difference from the the controls (pb0.05).
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Fig. 3. Change in acetylcholinesterase activity and glutamate metabolism in mussels treated to morphine, diazepam and an effluent extract. Mussels were injected with the compounds into the adductor muscle and left to stand for 48 h at 15 °C. AChE (A), glutamate/GABA (B) were determined in the visceral mass. * indicates significant difference from the controls (pb0.05).
oxidative stress in the mitochondrial fraction (Fig. 5B). Serotonin was also closely related with AChE activities and GABA was negatively related with serotonin, dopamine ADC and AChE activities. 4. Discussion Municipal effluents are well known to contain a plethora of pharmaceutical product residues such as caffeine, carbamazepine, analgesics, selective serotonin reuptake inhibitors and antibiotics. Benzodiazepines (e.g. diazepam or valium) and morphine-based substances (analgesics, cough suppressant and anti-diarrheal drugs) are also likely to be found in these wastewaters. Morphine was reported in a physically and chemically treated municipal (City of Montréal, QC, Canada) effluent at the 0.1 µg/L level (Gagné et al.,
2004). Diazepam was found in turbot liver at concentrations ranging from 23 to 110 ng/g (wet weight), pointing up their occurrence in polluted aquatic environments (Kwon et al., 2009). Mussels possess benzodiazepine and opiate receptors in their nervous systems (Stefano and Scharrer, 1996; Betti et al., 2003). Mussels collected at a heavy-metal-polluted site showed a significantly higher number of binding sites for benzodiazepines than mussels collected at a less contaminated site (Betti et al., 2003). An increase in the number of available binding sites might be the consequence of decreased GABA production in tissues. A similar response was observed in the clam Mya arenaria contaminated by tributyltin, which showed less estradiol-17β and a high number of estradiol-binding sites (Gagné et al., 2003). In the present study, levels of glutamate and GABA were not significantly affected by the effluent extract. However, morphine
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Fig. 4. Induction of LPO in mussels treated with diazepam, morphine and an effluent extract. Mussels were injected with the compounds into the adductor muscle and left to stand for 48 h at 15 °C. LPO was determined in the visceral mass. * indicates significant difference from the controls (pb0.05).
was able to increase GABA (and reduce glutamate) levels in mussels while lowering AChE activity, suggesting a state of relaxation in mussels. Studies examining the interaction of GABA and morphinerelated effects in aquatic animals are scarce. In rats, the activation of GABAA receptors (but not GABAB) appeared to accentuate the effects of morphine (Yoon et al., 2009). This suggests that GABA signaling could potentiate the effects of morphine. In mussels, morphine stimulates dopamine turnover and urban effluents increase dopamine levels and dopamine ADC activity (Gagné et al., 2007). However, the primary-treated effluent decreased GABA levels, while the effluent extract produced no changes. The effluent extract was from a biologically-treated sewage station which treats domestic wastewaters with an input of hospital complex of 300 beds. Morphine and dopamine metabolisms are coupled in mussels, where morphine synthesis is produced by dopamine in nerve ganglia (Zhu et al., 2005b). Moreover, morphine reduces the immune response in blue mussels (Ottaviani et al., 1995) and in the freshwater mussel E. complanata (Gagné et al., 2006). Hence, the release of dopamine by diazepam could stimulate morphine signaling, which in turn suppresses the immune system. Immunosuppression is a common syndrome in mussels exposed to municipal effluents (Gagné et al., 2006). Diazepam and morphine are biotransformed by cytochrome P450 3A4 and 2C19, which involve oxidative N-dealkylation, hydroxylation and conjugation to glucuronide (Charney et al., 2001). Xenobiotic biotransformation is an oxidative process in which the production of reactive oxygen species and the formation of more polar (reactive) intermediates occur. In the present study, both diazepam and morphine increased LPO in the post-mitochondrial fraction where xenobiotic biotransformation processes take place. Diazepam was more potent than morphine in inducing LPO in mussels with a threshold concentration of 0.06 µg/g mussel. Interestingly, only diazepam and the effluent extract were able to elicit LPO in the mitochondria. GABA receptors have been identified in the mitochondria of mussels (Giannaccini et al., 2004) and can be blocked by reactive oxygen species like H2O2 in cardiomyocytes (Zhang et al., 2002). Lead ions (Pb2+) inhibited benzodiazepine (PK 1195) binding in mussel mitochondria in a non-competitive manner and basal ADC
activity in plasma membranes. Dopamine is also eliminated by monoamine oxidases located in mitochondria (Gilloteaux, 1979) and dopamine-dependent ADC activity was significantly correlated with LPO in mitochondria in the present study. This is in agreement with a previous study in which the large proportion of dopaminesensitive ADC activity was located in the crude mitochondrial fraction in rat cerebral cortex homogenates (von Hungen and Roberts, 1973). Dopamine was able to activate mitochondrial ADC receptors and was seemingly independent of β-blockers such as propanolol but was very sensitive (blocked) to the anti-psychotic drug Haloperidol. This suggests that increased synaptosomal dopamine-sensitive ADC activity was associated with increased LPO in mitochondria, perhaps through metabolism by monoamine oxidase activity in mitochondria, which involves the release of H2O2. The release of H2O2 would block GABA receptors and maintain high levels of GABA, as shown in the morphine-treated group. Multivariate analyses of the neurochemical data were performed in an attempt to identify similarities between the observed responses with diazepam or morphine and those found with the effluent extract. They revealed that the drug responses did not overlap with those of the effluent extract, suggesting a different response pattern (Fig. 5A). However, the response pattern of morphine was closer to the effluent extract, which suggests some morphine-like properties in this effluent. In a previous study, an opiate-like effect was observed in mussels exposed to a primary-treated effluent (Gagné et al., 2007), as evidenced by increased dopamine turnover and adenylyl cyclase activity in nerve tissues. Interestingly, diazepam was also able to increase dopamine levels and dopamine ADC in mussels, which suggest that benzodiazepines might have contributed to increased dopamine metabolism. In conclusion, morphine and diazepam (a benzodiazepine) were biologically active in freshwater mussels. Morphine reduced serotonin and increased dopamine in the visceral tissues. This was accompanied by reduced AChE activity and increased GABA levels, suggesting the induction of a relaxation state in mussels. Diazepam also lowered serotonin levels, but produced no change in dopamine levels. However, dopamine-sensitive ADC activity was readily activated, perhaps indicating facilitated opiate signaling. Diazepam slightly increased glutamate levels, indicating a mild
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Fig. 5. Multivariate analysis of the biomarker response data. Biomarkers were analyzed using discriminant function (A) and factorial (B) analyses to determine the ability of the biomarkers to discriminate between the treatment groups and to represent the interrelationships of the biomarkers. The letters (C, D, M and E) are placed at the centres of gravity of the corresponding treatments. The dotted circle corresponds to a highly correlated cluster of biomarkers. The biomarkers in parentheses represent the biomarkers with the highest factorial weight.
stimulatory activity in mussels. The increase in both dopamine ADC activity and glutamate concentration was also associated with increased oxidative stress at the mitochondrial and post-mitochondrial fractions in cells. A comparison of the global response pattern of these drugs with those of the effluent extract revealed only a relative proximity with morphine. However, further experiments will be needed to confirm whether these effluents contain benzodiazepine at sufficient quantities to elicit effects in mussels exposed to them. Acknowledgements This work was funded by the municipal effluent research program of Environment Canada. The authors thank Sophie Trépanier for the biomarker assessments and Patricia Potvin for editing the manuscript.
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