Aquatic Toxicology 151 (2014) 68–76

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Growth inhibition and coordinated physiological regulation of zebrafish (Danio rerio) embryos upon sublethal exposure to antidepressant amitriptyline Ming Yang a,∗,1 , Wenhui Qiu a,1 , Jingsi Chen a , Jing Zhan a , Chenyuan Pan a , Xiangjie Lei a , Minghong Wu b,∗ a b

School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, China Shanghai Applied Radiation Institute, Shanghai University, Shanghai 200444, China

a r t i c l e

i n f o

Article history: Received 24 August 2013 Received in revised form 21 December 2013 Accepted 27 December 2013 Available online 8 January 2014 Keywords: Pharmaceuticals Tricyclic antidepressant Zebrafish embryo Adrenocorticotropic hormone Oxidative stress Nitric oxide

a b s t r a c t Amitriptyline is a tricyclic antidepressant used for decades. It is present at low detectable concentrations in the aquatic environment, but relative few studies have focused on its ecotoxicological effects on non-target aquatic animals. The present study conducted an acute toxicity test of waterborne amitriptyline exposure using zebrafish (Danio rerio) embryos 4 to 124 h-post-fertilization. Time-dependent lethal concentrations were firstly determined and at mg/L levels. Effects of amitriptyline on zebrafish embryos were then evaluated under amitriptyline exposure at sublethal concentrations of 1, 10, 100 ng/L, 1, 10, 100 ␮g/L and 1 mg/L. Our results showed that amitriptyline significantly reduced the hatching time and body length of embryos after exposure in a concentration-dependent manner. Our study also revealed that the exposure evoked a coordinated modulation of physiological and biochemical parameters in exposed zebrafish embryos, including alterations of adrenocorticotropic hormone (ACTH) level, oxidative stress and antioxidant parameters, as well as nitric oxide (NO) production and total nitric oxide synthase (TNOS) activity. A U-shaped concentration-dependent response curve was observed in ACTH level in response to amitriptyline exposure. However, both U-shaped and inversed U-shaped curves were indicated in the responses of antioxidant parameters, including total antioxidant capacity, antioxidant enzyme activities (catalase, superoxide dismutase and peroxidase), glutathione content and glutathione reductase activity. Correspondingly, hydroxyl radical formation and lipid peroxidation indices changed in similar U-shaped concentration-dependent patterns, which together the results of antioxidant parameters suggested induction of oxidative stress in embryos exposed to amitriptyline at high concentrations. Moreover, NO production and TNOS activity were both significantly affected by amitriptyline exposure. Notably, significant correlations between these measured parameters were revealed, which suggested a dynamic adaptation process and coordinated regulation of multiple physiological systems in fish embryos to amitriptyline treatment. Furthermore, our study demonstrated that the effective concentrations of amitriptyline for measured parameters in zebrafish embryos were as low as 10 ng/L, and thus revealed the potential risk of amitriptyline and other antidepressants to aquatic life. © 2014 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding authors at: School of Environmental and Chemical Engineering/Shanghai Applied Radiation Institute, Shanghai University, Shangda Road 99, Shanghai 200444, China. Tel.: +86 21 66137801/+86 21 66137507; fax: +86 21 66137801. E-mail addresses: [email protected] (M. Yang), [email protected] (W. Qiu), [email protected] (J. Chen), [email protected] (J. Zhan), [email protected] (C. Pan), [email protected] (X. Lei), [email protected] (M. Wu). 1 These authors contributed equally. 0166-445X/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquatox.2013.12.029

Pharmaceuticals are considered as emerging contaminants during the last few years due to their ubiquity in the environment at trace levels (Togola and Budzinski, 2008). Pharmaceuticals are designed for combating diseases and promoting human health, but the active pharmaceutical ingredients and their metabolites can be release into the environment through human excretion or through direct disposal of unused and expired medicines (Cunningham et al., 2006). Antidepressants are one of the most commonly prescribed pharmaceuticals in the United States (Schultz et al., 2010). Increasing prevalence of psychiatric disorders and awareness of

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mental health issues correlate with the mounting prescription and consumption of antidepressants (Silva et al., 2012). Several studies have demonstrated common methods of removal of antidepressants in most waste water treatment plants (WWTPs) are ineffective, thus lead to continuous contamination of water with accumulating concentration of antidepressants (Gros et al., 2007; Lajeunesse et al., 2008). As a result, the concentration of antidepressants in water, sludge and biological tissues of aquatic organisms have ranged from ng to ␮g/L (Calisto and Esteves, 2009). Amitriptyline is the most widely prescribed tricyclic antidepressant. It is commonly prescribed for depression and several neuropathic and inflammatory illnesses in humans and animals (Bautista-Ferrufino et al., 2011; Calisto and Esteves, 2009). Amitriptyline is listed as the fourth most consumed antipsychotic and antidepressant drugs in the Province of Quebec, Canada (Lajeunesse et al., 2008). Meanwhile, the presence of amitriptyline and its metabolites in aqueous environment has been reported in several studies. For examples, 0.5–21 ng/L of amitriptyline has been reported in surface water in UK (Kasprzyk-Hordern et al., 2008), and 1.4 ng/L of this drug has been reported in the drinking water in France (Togola and Budzinski, 2008). Amitriptyline and its metabolite nortriptyline have been detected in the rivers in Canada, and are fairly stable, lasting for 7 days, in effluent under defined storage conditions, while being eliminated at extremely low efficiency by common sewage treatment (Lajeunesse et al., 2008). Since active ingredients of pharmaceuticals possess their intrinsic properties to cause biological effects, usually at very low doses, the continuous infusion of those chemicals into the aquatic environment generates great ecotoxicological concern on aquatic non-target species (van der Ven et al., 2006). The bioaccumulative potential of amitriptyline has been found to be increased by up to 78 fold in brook trout livers after exposure to diluted effluents from that WWTP, suggesting a potential risk fish species living in the receiving river (Lajeunesse et al., 2011). To our knowledge, previous studies regarding the potential adverse effects of antidepressants on aquatic organisms have mainly focused on selective serotonin reuptake inhibitors (SSRIs). It has been reported that SSRIs have a potential to disrupt important physiological processes in fish and affect their hormone levels, leading to alteration of reproductive behaviors, a reduction of reproductive capability, abnormalities in embryo development, and a delay in physiological development and sexual maturation (Mennigen et al., 2008; Calisto and Esteves, 2009; Park et al., 2012). Most studies on amitriptyline thus far have been performed in human and rodent models, either in vivo or in vitro, however, the adverse effects of tricycle antidepressants such as amitriptyline on aquatic organisms has not been thoroughly studied. The efficacy of amitriptyline in the treatment of depression is owing to its inhibitory effect on the serotonin and norepinephrine uptake in the presynaptic nerve endings, thereby reducing the hyperactivity of the hypothalamo–pituitary–adrenocortical (HPA) axis present in major depression (Moreno-Fernández et al., 2008). Accordingly, the neurotoxic side-effects of amitriptyline have been reported (Mannerström and Tähti, 2004; Mannerström et al., 2006). The potential mechanisms for amitriptyline neurotoxicity have been proposed to be associated with caspase-mediated apoptosis (Lirk et al., 2006) or its chemical nature as a detergent (Kitagawa et al., 2006). However, more recent reports demonstrated that toxicity of amitriptyline is a result of increase in oxidative stress, indicated by an increasing level of reactive oxygen species (ROS) and intracellular lipid peroxidation (Moreno-Fernández et al., 2008). In addition, the anti-inflammatory activity of amitriptyline has been observed and suggested to be associated with the inhibition of release of proinflammatory cytokines by the immune cells and a decrease in nitric oxide production (Vismari et al., 2012).

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The zebrafish (D. rerio) embryotoxicity test is considered as a fast and simple method for investigating the chemical toxicity during embryogenesis (Hermsen et al., 2011). Zebrafish embryos depend on the yolk sac as a nutrient source and can survive without food intake until 7 days post fertilization. This dependence confers an amenability of the chemical toxicity studies on these organisms owing to the reliable experimental conditions without maternal and external interference. The present study evaluated the adverse effects of amitriptyline on fish species in vivo. The zebrafish embryos were used to test amitriptyline toxicity after waterborne exposure for 120 h during embryogenesis and their early subsequent larval developmental stages (4 h post-fertilization (hpf) to 124 hpf). The lethal concentrations were firstly determined and then sublethal exposure to amitriptyline was performed. Various physiological and biochemical parameters were determined after exposure including hormone levels, antioxidant capacity and oxidative stress, and nitrite oxide production which all have been reported to be associated with amitriptyline toxicity in human and rodent studies. Our results, hereby, present preliminary data on amitriptyline effects on non-target aquatic species and accordingly, help to better understand how those organisms respond this chemical treatment at physiological and biochemical levels.

2. Materials and methods 2.1. Chemicals Amitriptyline hydrochloride (CAS number 549-18-8; ≥98%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The compound was dissolved in ultrapure water to obtain stock solutions of 10 g/L and stored at 4 ◦ C. Methanol for chemical analysis was obtained from Merck (Darmstadt, Germany). All other reagents used were of analytical grade and obtained from Sangon (Shanghai, China).

2.2. Zebrafish embryos Wild type zebrafish adults were purchased from a local commercial fish market in Shanghai, China. The fish were acclimated to laboratory conditions in dechlorinated tap water at 28 ± 0.5 ◦ C under a 14:10-h light:dark cycle and were then allowed for breeding weekly. The fish were fed with brine shrimp (Artemia nauplii) twice daily. Fish embryos were collected in the morning after adult spawning and were examined under a stereomicroscope to remove the unfertilized embryos and select those that had reached the blastula stage, which are considered to have developed normally, for exposure experiments at 4 hpf. The embryos were kept in a constant temperature and light incubator at 28 ± 0.5 ◦ C under a 14:10-h light:dark cycle. Fresh egg water (0.1 × E3 medium, 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.3 mM MgSO4, methylene blue free) or exposure solutions (composition described below) were replaced completely every 24 h.

2.3. Experimental design 2.3.1. Acute toxicity test Zebrafish embryos (4 hpf) were randomly distributed in 6-well plates, 20 embryos per well and four replicates per exposure, for 120 h-exposure to a series of diluted amitriptyline solution in egg water (6 mL per well). The exposure concentrations were set at 0, 0.1, 1, 2, 3, 5, 10, 25, 50, 75 and 100 mg/L of amitriptyline, based on the reported 24 and 48 h toxicity data for cultured rat pheochromocytoma cells (Kolla et al., 2005). Mortality was measured every 12 h to determine time-dependent lethal concentrations.

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2.3.2. Sublethal exposure Zebrafish embryos (4 hpf) were randomly distributed in 6-well plates, 60 embryos per well (10 mL per well) and four replicate per exposure, for 120 h exposure to sublethal concentrations of 1, 10, 100 ng/L, 1, 10, 100 ␮g/L and 1 mg/L amitriptyline in egg water. A control group was cultured in egg water. Mortality was measured every 24 h. Hatchability, defined as the percentage of hatched embryos to all survived ones, was recorded every 2 h starting from when the first embryo hatched until 60 hpf. After 120 h exposure (124 hpf), body length of 20 larvae randomly selected from each exposure group was measured under a stereomicroscope. And then, larvae from each exposure well were pooled as one sample, and were separately snap-frozen in liquid nitrogen and stored at −80 ◦ C for subsequent bioassays. 2.4. Chemical analysis method The exposure solutions were collected after 24 h-exposure with zebrafish embryos and were centrifuged at 1000g for 5 min. Then, the actual exposure concentrations of amitriptyline in these supernatants were determined by liquid chromatography-tandem mass spectrometry (LC–MS/MS). The samples were analyzed by Agilent Technologies 1260 infinity installed with a Poroshell 120 EC-C 18 column (3 × 100 mm, 2.7 ␮m particles), coupled to an Agilent Technologies 6460 Triple Quad LC mass spectrometer through an electro-spray interface (ESI). Amitriptyline detection was conducted following the method as described by Ho et al. (2007), by a 0–80% methanol gradient elution, with a 0.4 mL/min flow rate. The injection volume was 5 ␮L. The conditions for MS analysis of each HPLC peak included a capillary voltage of 4000 V, a nebulizing pressure of 30.0 psi, and the sheath gas temperature at 350 ◦ C, with 3 L/min nitrogen (99.99%, Shanghai, China) as the cone gas. The mass spectrometer was operated with the following ion transition: amitriptyline (278 → 233). 2.5. Sample preparation Each sample was homogenized on ice, in 1 mL of cold 0.15 M NaCl solution, pH 7.2–7.4, using a Dounce homogenizer. The homogenate was centrifuged at 10,000g for 20 min at 4 ◦ C. The supernatant was aliquoted for subsequent biochemical assays. The protein concentration of each sample was determined by the Bradford assay, using bovine serum albumin as standards. The protein concentration of sample input for subsequent biochemical assays was calculated accordingly and used to calibrate the detected value of each measured parameter. 2.6. Adrenocorticotropic hormone (ACTH) level Levels of ACTH were determined using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) according to the protocol provided by Nanjing Jiancheng Bioengineering Institute. 2.7. Oxidative stress indices Hydroxyl radical and lipid peroxidation were determined using commercially available kits (Nanjing Jiancheng Bioengineering Institute). Hydroxyl radical was assayed by monitoring the decrease in hydrogen peroxide level according to Halliwell’s method (1978). Lipid peroxidation was determined by monitoring the reaction of malondialdehye (MDA) with thiobarbituric acid according to Sinnhuber et al. (1958). Antioxidant parameters, including the activity of catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), and total antioxidant capacity (TAC), were determined according protocols provided

by available kits (Nanjing Jiancheng Bioengineering Institute). Catalase activity was estimated by monitoring the disappearance of H2 O2 at 240 nm, as described by Luck (1963). Superoxide dismutase activity was determined by measuring its antibody-dependent ability to inhibit the auto-oxidation of pyrogallol (Marklund and Marklund, 1974). Peroxidase activity was measured using a modified procedure described by Chérif et al. (1994). Total antioxidant capacity was measured by monitoring the decrease content of Fe3+ , which is directly related to hydroxyl radical scavenging capability (Stephanson et al., 2003). Glutathione reductase (GR) activity and reduced glutathione (GSH) were determined using commercially available kits (Nanjing Jiancheng Bioengineering Institute). Contents of GSH were determined using a reaction of dithiobisnitrobenzoicacid with sulfhydryl compound (Baker et al., 1990). The GR activity was measured by determined by monitoring the decrease of nicotinamide adenine dinucleotide phosphate levels (Cribb et al., 1989). 2.8. Nitric oxide (NO) and total nitric oxide synthase (TNOS) Nitric oxide can be oxidized and ultimately converted to nitrate or nitrite (Tarpey and Fridovich, 2001). The NO concentration was indirectly reflected by the nitrate chromogenic agent. The activity of total nitric oxide synthase (TNOS) was assayed by monitoring the formation of [3 H]-citrulline from l-arginine (Palmer and Moncada, 1989). 2.9. Statistical analysis Data are presented as means ± standard deviation (SD). They were normalized using the Kolmogorov–Smirnov one-sample test and Levene’s test. Statistical analyses were performed using SPSS Statistics 18.0 (SPSS Inc., Chicago, IL, USA). The intergroup differences were assessed using one-way analysis of variance (ANOVA) followed by Fisher’s least significant difference (LSD’s) test or Dunnett’s test. In all comparisons, p < 0.05 was considered to be statistically significant. 3. Results 3.1. Time-dependent lethal concentrations in acute toxicity test Mortality was determined every 12 h of zebrafish embryos exposed to various concentrations of amitriptyline during the 120 h-exposure period of acute toxicity test, as shown in Fig. 1. Concentrations at 100% mortality (LC) was observed; and concentrations at 50% (LC50 s) and 5% mortality (LC5 s) occurred among the tested embryos were determined by the trimmed Spearman–Karber method and Probit analysis, respectively (Table 1). The no-observed effect concentration (NOEC) for 120 h-exposure was 100 ␮g/L (3.19 ␮M). These results were confirmed by three independent experiments of acute toxicity test. The LC50 of zebrafish embryos to amitriptyline was estimated at 1.4 mg/L by Spearman–Karber method, and hence the sublethal exposure experiments were subsequently performed with this concentration of amitriptyline. 3.2. Amitriptyline concentrations for sublethal exposure To confirm the constant concentrations of amitriptyline during the sublethal exposure, the concentrations of amitriptyline levels in exposure solutions were measured daily during the first 3 days of the exposure experiment using LC–MS/MS (Table 2). Compared to nominal concentrations, the detected exposure concentrations were of slight deviation but within the range of allowable error. For the control and nominal amitriptyline concentrations lower than

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Fig. 1. Acute toxicity of amitriptyline to zebrafish embryos/larvae. Mortality was observed every 12 h during a 120 h-exposure period (from 4 to 124 hpf). Data are shown as means (n = 4, 20 larvae/well × 4 wells). Table 1 Acute toxicity values for zebrafish embryos/larvae upon exposure to amitriptyline for 4 to 124 hpf. Exposure time (h)

LCa (mg/L)

LC50 b (mg/L)

LC5 c (mg/L)

12 24 36 48 60 72 84 96 108 120

>100 75–100 50–75 25–50 10–25 10–25 5–10 5–10 3–5 2–3

59.3 (53.8–65.2) 27.2 (22.8–32.5) 15.5 (13.2–18.3) 6.6 (5.5–7.8) 3.9 (3.2–4.6) 2.8 (2.3–3.3) 2.4 (2.0–2.9) 1.9 (1.6–2.3) 1.5 (1.2–1.8) 1.4 (1.1–1.7)

19.15 1.63 1.44 0.84 0.50 0.41 0.32 0.25 0.17 0.17

a Lethal concentration observed. The maximum concentration tested was 100 mg/L. b Median lethal concentration calculated by the trimmed Spearman–Karber (TS–K) method. Values in parentheses represent 95% confidence levels. c The concentration that would cause 5% mortality of tested populations calculated by Probit analysis method.

Table 2 Measured concentrations of amitriptyline in the sublethal exposure experiment by liquid chromatography-tandem mass spectrometry (LC–MS/MS). Nominal concentrations Control 1 (␮g/L) 10 (␮g/L) 100 (␮g/L) 1 (mg/L) a

Measured concentrations after exposure 1st day (n = 4)

2nd day (n = 4)

3rd day (n = 4)

NDa 0.873 ± 0.296 8.64 ± 1.08 92.6 ± 10.4 0.902 ± 0.107

ND 1.29 ± 0.121 11.8 ± 0.721 108 ± 3.73 1.05 ± 0.103

ND 1.28 ± 0.476 9.32 ± 0.321 95.8 ± 12.0 0.964 ± 0.0551

The concentration was not detectable.

exposure, as shown in Table 3. This observation was consistent with the results from the acute toxicity test. The normal hatching timing of zebrafish embryos occurs on day 2 to 3 post fertilization. We measured the hatchability of each group at different time points, and observed an apparent early hatching time in fish embryos upon exposure to sublethal concentrations of amitriptyline. At 60 hpf, all the embryos in the control group had completely hatched, there was no significant hatching rate difference between the exposed groups and the control. At 36 and 48 hpf however, the hatching rates of exposed embryos were significantly higher than the control (p < 0.05) (Fig. 2A). The results suggest that exposure to amitriptyline did not affect the hatchability of fish embryos but significantly shortened their hatching time. Exposure to amitriptyline also reduced the body length of zebrafish embryos in a concentrationdependent manner, as shown in Fig. 2B and C. In three independent sublethal exposure experiments, the body length of fish larvae among groups exposed to amitriptyline equal to or higher than 100 ng/L (3.19 nM) was significantly reduced after 120 h of exposure (p < 0.05). 3.4. ACTH levels The ACTH levels were determined in zebrafish embryos after 120 h sublethal amitriptyline exposure by ELISA assay. The ACTH levels significantly decreased upon exposure to 10 and 100 ng/L of amitriptyline compared to control (p < 0.05), but elevated upon increasing exposure concentrations, particularly at the highest exposure concentration of 1 mg/L (p < 0.05) (Fig. 3). 3.5. Antioxidant parameters

1 ␮g/L, amitriptyline was not detectable due to such concentrations being below the minimum detection limit of the MS/MS analyses. 3.3. Mortality, hatching time and body length The exposure to amitriptyline at 1 mg/L significantly increased the mortality of zebrafish embryos (p < 0.05) after 120 h sublethal

The total antioxidant capacity in zebrafish embryos was significantly promoted in the 100 ng/L and 1 ␮g/L amitriptyline treated groups (p < 0.05), follow by a significant decrease in the 1 mg/L group (p < 0.05) (Fig. 4A). The trends of antioxidant enzyme activities including SOD, CAT and POD also displayed this inverse U-shaped concentration-dependent patterns (Fig. 4B–D). The highest reduced antioxidant enzyme activities were observed among

Table 3 Mortality (%) of zebrafish embryos/larvae after subleathal exposure to various concentrations of amitriptyline for 4 to 124 hpf. Exposure time (h)

Contorl

1 ng/L

10 ng/L

100 ng/L

1 ␮g/L

10 ␮g/L

100 ␮g/L

1 mg/L

24 48 72 96 120

0.417 0.417 1.67 1.67 1.67

1.25 1.25 1.67 1.67 1.67

0.833 0.833 1.67 1.67 1.67

1.25 1.25 1.67 2.08 2.08

2.08 2.08 2.50 2.50 2.50

2.50 2.50 2.92 2.92 2.92

2.50 2.50 4.17* 4.58 4.58

2.92* 5.00* 6.67* 12.1* 15.0*

*

Significant differences versus control are indicated, p < 0.05 (ANOVA, Dunnett’s test). Values are means (n = 4).

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were altered in a concentration-dependent manner in zebrafish embryos responded to amitriptyline exposure as shown in Fig. 5. The results suggest that hydroxyl radical formation and lipid peroxidation were significantly suppressed in 100 ng/L amitriptyline treated group. As the concentrations increased however, hydroxyl radical formation and lipid peroxidation were elevated, indicating occurrence of oxidative stress in embryos exposed to concentrations of amitriptyline higher than 100 ng/L. As a result, a significant induction of hydroxyl radical formation and lipid peroxidation was observed in the 1 mg/L group (p < 0.05). 3.7. NO content and TNOS activity The NO content and TNOS activity decreased in a concentrationdependent manner in embryos upon amitriptyline exposure at concentrations less than 1 mg/L (Fig. 6). However, there was a sudden increase of NO content and NOS activity upon amitriptyline exposure at the concentration from 100 ␮g/L to 1 mg/L, as oppose to the concentration-dependent inhibition of NO content and NOS activity correlated with exposure to amitriptyline concentrations that were lower than 1 mg/L. 3.8. Correlation analysis

Fig. 2. Effects of amitriptyline on hatching time (A) and body length (B and C) of zebrafish embryos/larvae after exposure to amitriptyline for 4 to 124 h postfertilization (hpf). Hatchability was measured at 36 and 48 hpf. Body length was measured under a stereomicroscope after exposure to amitriptyline at 124 hpf and shown as means ± standard deviation (n = 20, 5 larvae/well × 4 cells, random picking). Significant difference is indicated as * p < 0.05 versus control (ANOVA, Dunnett’s test). Visual comparisons of body length were conducted between fish larvae exposed to 1 mg/L amitriptyline and the control ones.

the embryos exposed to 100 ng/L of amitriptyline, while the lowest inhibited enzyme activities were observed among the embryos exposed to 1 mg/L of the compound. The GSH content was significantly lowered at concentrations of 100 ng/L and 1 ␮g/L compared to the control (p < 0.05), however, it was significantly induced at 1 mg/L (Fig. 4E). Similarly, GR activity was inhibited at concentrations of 100 ng/L, 1 and 10 ␮g/L (p < 0.05), but it significantly enhanced at 1 mg/L compared to the control (p < 0.05) (Fig. 4F). 3.6. Oxidative stress The levels of two oxidative stress indicators, hydroxyl radical formation and lipid peroxidation (monitored by MDA level),

Fig. 3. Adrenocorticotropic hormonea (ACTH) levels in zebrafish embryos after exposure to amitriptyline for 4 to 124 hpf. Values are means ± standard deviation (n = 4). Significant differences versus control are indicated as ∗ p < 0.05 (ANOVA, LSD’s test). The ACTH content in the control group was 176.8 ± 19.6 ng/g protein.

The correlations between measured antioxidant parameters, oxidative stress indices, NO and TNOS, as well as ACTH level, were analyzed according to Spearman’s test (Table 4). Our analyses indicate a statistically significant relationship between oxidative stress and antioxidant defense parameters (p < 0.05). The oxidative stress indices, hydroxyl radical formation and lipid peroxidation, were inversely related to TAC level and antioxidant enzyme activities, but were positively related to GSH content and GR activity. In addition, nitric oxide and TNOS activity were positively related to oxidative stress and GSH system, but negatively related to TAC and some antioxidant enzyme activities. Notably, we observed that ACTH level significantly correlated with all antioxidant and oxidative stress parameters, as well as NO and TNOS (p < 0.05). 4. Discussion In the present study, we evaluated the effects of amitriptyline on non-target aquatic animals using zebrafish embryos. Our results demonstrated that the adverse effects of amitriptyline on fish embryos could be measured at concentrations as low as ng/L levels. The early hatching and significant growth inhibition of fish larvae, judged from body length reduction, were observed among embryos exposed to sublethal concentration of amitriptyline. Fish larvae exposed to amitriptyline also displayed dynamic physiological response including alterations of ATCH level, antioxidant and oxidative stress parameters, as well as NO content and NOs activity. While we observed U-shaped and inverse U-shaped dose response curves for most of the measured parameters as amitriptyline concentrations increased, there were significant correlations among these parameters. These results suggested that sublethal amitriptyline exposure might evoke a dynamic coordinated regulation of physiological responses in zebrafish embryos, which could be crucial for the survival of fish embryos while they were adapting to increased stress of amitriptyline exposure. There are relatively few studies reported on the amitriptyline toxicity to aquatic organisms. The acute toxicity test showed that the LCs and LC50 s at different time points during 120 h exposure (from 4 to 124 hpf) are at mg/L levels. Compared to results from previous studies, our study showed that fish embryos are more tolerant to amitriptyline exposure than aquatic invertebrates. The 24-h LC50 s of two freshwater crustaceans (Daphnia magna and Streptocephalus proboscideus) to amitriptyline are 20 and 2.8 ␮M,

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Fig. 4. Total antioxidant capacity (TAC, (A)), activities of catalase (CAT, (B)), superoxide (SOD, (C)) and peroxidase (POD, (D)), as well as reduced glutathione content (GSH, (E)) and glutathione reductase antivity (GR, (F)) in zebrafish embryos after exposure to amitriptyline for 4 to 124 hpf. Values are means ± standard deviation (n = 4). The scales of axes for all graphs are the same. Significant differences versus control are indicated as ∗ p < 0.05 (ANOVA, LSD’s test). The TAC content in the control group was 24.7 ± 2.3 U/mg protein. The activities of CAT, SOD and POD in the control group were 0.43 ± 0.023 U/mg protein, 34.7 ± 3.67 U/mg protein, and 2.78 ± 0.29 U/mg protein, respectively. The GSH content and GR activity in the control group were 7.36 ± 0.63 mg/g protein and 13.4 ± 1.88 U/g protein, respectively.

respectively. The 24-h LC50 of marine crustacean (Artemia salina) to amitriptyline is 133 ␮M (Calleja et al., 1994). The 24-h LC50 of zebrafish embryos (from 4 to 28 hpf) to amitriptyline in our study was 75 mg/L (239 ␮M). Furthermore, our observation of the reduction of body length of fish larvae upon exposure to amitriptyline exposure is consistent with the previous study that reported the change of body length of fish larvae in response to exposure to antidepressants fluoxetine, sertraline, venlafaxine, and bupropion (singularly and in mixture) (Painter et al., 2009). The hyperactivity of HPA axis contributes to the development of depression in humans, accompanied with stimulation of ACTH release and the subsequent peripheral release of cortisol from the adrenal grand (Zhu et al., 2012). The mechanism of clinically effective antidepressant usually involves the regulation of HPA homeostasis to increase the ability of organisms to cope with stressful conditions. Our results showed that low concentrations of amitriptyline exposure (10 and 100 ng/L) possibly exerted its “therapeutic action” by reducing the ACTH levels in zebrafish embryos. However, as exposure concentrations increased, the excessive amitriptyline treatment seemed to have triggered the adaptive response of organisms to lower the effect of amtriptyline, and eventually a high concentration of amitriptyline treatment at 1 mg/L completely resulted in a significant induction of ACTH level as observed at 1 mg/L. The antioxidant defense system is comprised of antioxidant enzymes and non-enzymatic antioxidant molecules; together they

protect organisms from oxidative damage and maintain cellular redox homeostasis. Antioxidant enzymes such as SOD and CAT are vital first-line of defense against oxidative toxicity. Inhibition of these enzymes may lead to accumulation of hydrogen peroxide or its decomposition products. Glutathione is a key antioxidant that scavenges free radicals generated from oxidative metabolism and those not decomposed by antioxidant enzymes. The GSH and GSHrelated enzymes are considered as a second line of defense against oxidative damage (Wu et al., 2011). From our results, in the presence of low concentrations of amitriptyline, antioxidant enzyme activities positively responded to amitriptyline treatment. Upon exposure to concentrations that were higher than 100 ng/L however, antioxidant enzyme activities were significantly inhibited. While the GSH system was negatively responded to amitriptyline exposure at low exposure concentrations, it was significantly enhanced at the high concentration of 1 mg/L when antioxidant enzymes had been dramatically inhibited. From the results of TAC levels, we observed that the regulation of antioxidant enzymes mainly occurred when the fish embryos was exposed to low concentrations of amitriptyline; but when their positive regulation was disrupted, the GSH system subsequently functioned and might play as a complementary role to contribute to the total antioxidant capacity in organisms. Oxidative stress is defined as an imbalance between the production and the removal of ROS. Oxidative stress occurs when the production of ROS overwhelms the antioxidant capacity in

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Fig. 5. The levels of hydroxyl radical formation (A) and malondialdehyde content (MDA, (B)) in zebrafish embryos after exposure to amitriptyline for 4 to 124 hpf. Values are means ± standard deviation (n = 4). The scales of axes for all graphs are the same. Significant differences versus control are indicated as ∗ p < 0.05 (ANOVA, LSD’s test). The MDA content in the control group was 10.9 ± 1.24 nmol/mg protein.

cells. Lipid peroxidation is one of the prominent consequences of oxidative stress. Lipid peroxidation is the oxidative degradation of lipid in cell membrane that results in damage of the cells. Several in vivo and in vitro studies have implicated excess ROS production in the toxicity of amitriptyline and supposed mitochondrial dysfunction and increased mitochondrial ROS production to be involved

Fig. 6. The nitric oxide content (NO, (A)) and total nitric oxide synthase activity (TNOS, (B)) in zebrafish embryos after exposure to amitriptyline for 4 to 124 hpf. Values are means ± standard deviation (n = 4). The scales of axes for all graphs are the same. Significant differences versus control are indicated as ∗ p < 0.05 (ANOVA, LSD’s test). The NO content and NOS activity in the control group were 7.36 ± 0.63 mg/g protein and 13.4 ± 1.88 U/g protein, respectively.

in that toxicity (Slamon and Pentreath, 2000; Bartholomä et al., 2002; Cordero et al., 2009; Bautista-Ferrufino et al., 2011; MorenoFernandez et al., 2012). Consistent with our observation on the high concentrations of amitriptyline-exposed zebrafish embryos, a clear increase of lipid peroxidation and oxidative stress has also been found in human and mouse fibroblast cell cultures (Viola et al., 2000; Cordero et al., 2009), various tissues and serum of mice (Bautista-Ferrufino et al., 2011), and in peripheral blood cells from psychiatric patients (Moreno-Fernandez et al., 2012), after amitriptyline treatment. Furthermore, our results revealed a dynamic adaptive process of antioxidant system in fish embryos responded to oxidative stress induced by exposure to increasing concentrations of amitriptyline. These results showed that the positive regulation of antioxidant capacity in embryos parallels to the lowered levels of oxidative stress indices in embryos exposed to low concentrations of amitriptyline, while the occurrence of oxidative stress parallels to the decrease in total antioxidant capacity due to exposure to high concentrations of amitriptyline. Especially at 1 mg/L, significant intensive oxidative stress might occur in fish embryos where antioxidant capacity had been dramatically inhibited upon amitriptyline exposure. Therefore, the effect of amitriptyline on redox status in exposed fish embryos was more likely to be concentration-dependent, which consequently determines the occurrence of oxidative stress. Nitric oxide is the by-product of oxidative metabolism and is a proinflammatory mediator important for modulating immune system response (Xu et al., 2013). The levels of nitric oxide in organisms must be carefully regulated to maintain homeostasis (Karpuzoglu and Ahmed, 2006), since the appropriate levels of nitric oxide assist in mounting an effective defense against invading microbes. Conversely, an inability to generate nitric oxide results in serious, even fatal, susceptibility to infections. A prior study implicated dysregulation of NO in the pathogenesis of psychiatric disorders (Vismari et al., 2012). Our results showed that amitriptyline exposure significantly reduced NO levels and NOS activities in fish embryos at low concentrations, which was consistent with previous studies on the inhibitory capability of tricyclic or other antidepressants on NO production and NOS activity after antidepressant treatment (Vismari et al., 2012; Hwang et al., 2008; Yaron et al., 1999). However, there was a jump of NO content and NOS activity upon exposure to amitriptyline at the concentration from 100 ␮g/L to 1 mg/L. A sudden increase of NO and NOS was observed in embryos treated with 1 mg/L amitriptyline, which was completely opposite to the concentration-dependent inhibition of NO content and NOS activity cause by amitriptyline exposure at concentrations lower than 1 mg/L. This sharp increase might indicate a sudden change of physiological status in embryos upon exposure to 100 ␮g/L to 1 mg/L of amitriptyline. We showed that fish embryos exposed to 1 mg/L of amitriptyline displayed a significantly increased ACTH level, decreased the TAC level, and severe oxidative stress. However, the changes of these parameters embryos exposed to 100 ␮g/L amitriptyline were all significantly different from those in embryos exposed to 1 mg/L of amitriptyline. Therefore, the sudden promotion of NO content and NOS activity might be associated with elevated ACTH level and oxidative stress in exposed embryos. In addition, the sudden increased levels of NO and NOS occurred at the concentration that was exactly within the interval between the observed 120 h-NOEC and the LC50 of amitriptyline, i.e. from 100 ␮g/L to 1.4 mg/L, for mortality of zebrafish embryos in the acute toxicity test. This implies that upon exposure to 1 mg/L of amitriptyline, zebrafish embryos might have undergone an extremely intensive stress for survival. Accordingly, this stress resulted in a dramatic change of physiological status in fish embryos. Antidepressant drugs, including tricyclic antidepressants, exert potential immune-modulating effects that are related to

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Table 4 Correlation coefficients between various biochemical parameters measured in zebrafish embryos upon 120 h-exposure to amitriptyline. LPO OH CAT SOD POD TAC GSH GR NO TNOS ACTH

OH.

CAT

SOD

POD

TAC

GSH

GR

NO

TNOS

−0.659* −0.638* −0.565* −0.587* 0.839* 0.648* 0.538* 0.584* 0.782*

0.68* 0.414* 0.613* −0.575* −0.609* −0.511* −0.421* −0.536*

0.700* 0.553* −0.551* −0.487* −0.208 −0.339 −0.515*

0.681* −0.636* −0.455* −0.073 −0.308 −0.517*

−0.635* −0.735* −0.423* −0.409 −0.541*

0.639* 0.539* 0.608* 0.815*

0.707* 0.658* 0.558*

0.788* 0.598*

0.729*

*

0.868 −0.531* −0.614* −0.656* −0.604* 0.893* 0.566* 0.454* 0.590* 0.761*

LPO = lipid peroxidation; OH = hydroxyl radical formation; CAT = catalase; SOD = superoxide dismutase; POD = peroxidase; TAC = total antioxidant capacity; GSH = reduced glutathione; GR = glutathione; NO = nitric oxide; TNOS = total nitric oxide synthase; ACTH = adrenocorticotropic hormone. * p < 0.05 according to Spearman’s test.

cell-mediated immune response, including the inhibition of immune cell proliferation, a decrease in NO production, and a reduction of the release of various cytokines in human and animal studies (Xia et al., 1996, 1997; Kubera et al., 1996; Ying et al., 2002; Abdel-Salam et al., 2003; Obuchowicz et al., 2006; Hashioka et al., 2007). It has been reported that amitriptyline displays anti-inflammatory activity in the carrageenan model of paw inflammation, reduces the release of proinflammatory cytokines in lipopolysaccharide-activated rat mixed glial and microglial cell cultures (Abdel-Salam et al., 2003; Obuchowicz et al., 2006). These anti-inflammatory effects of amitriptyline may be associated with the decrease of NO production (Vismari et al., 2012). Similarly, our results revealed significant alterations of the NO content and NOS activity upon exposure to amitriptyline, which were consistent with previous studies and implied a possible adverse effect of amitriptyline on the immune system of fish embryos. However, since the NO signaling pathway might be involved in regulation of multiple aspects of physiological response, not exclusively in the immune-related modulation, further study is needed to evaluate the putative immodulating effects of amitriptyline on fish. 5. Conclusions Our study demonstrated that upon exposure to amitriptyline, zebrafish embryos exhibited systematic physiological response. Amitriptyline treatment altered HPA axis-regulated hormone level and biochemical parameters of antioxidant system, and affected a pro-inflammatory mediator which might play a crucial role in modulating immune system response. Moreover, the regulation of these parameters closely correlates with each other and displayed an adaptation process as the concentration of amitriptyline increases, suggesting a crosstalk among the endocrine system, cellular antioxidant system and immune system upon amitriptyline exposure, which might be essential for promoting the survival of fish embryos upon exposure to environmental stress. Our results showed that the LCs of amitriptyline for zebrafish embryos at different time points were at mg/L levels, and the NOEC of amitriptyline for zebrafish embryo mortality was at 100 ␮g/L. However, upon exposure to 100 ng/L of amitriptyline, most measured parameters in the fish embryos were significantly affected. These effects included a decrease of hatching time and body length, an alteration of ACTH level, antioxidant parameters, oxidative stress indices, and TNOS activity. Particularly, some parameters such as ACTH level could be affected by amitriptyline at concentrations as low as 10 ng/L, four orders of magnitude lower than the NOEC value for mortality. It should be noted that during the early stage of fish, fish embryos/larvae are extremely sensitive to chemical exposure, and the alteration of their physiological status at this stage would significantly affected their development. Therefore, although the presence of amitriptyline in aquatic environments has

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