Recent times have seen a growing concern about the effects of waterborne pharmaceuticals on human and ecosystem health as their detection in water has advanced to as low as nano-levels (1). Their effects on non-target organisms, however, have hardly been investigated so far (2, 3). Usually they contaminate drainage waters as the main compound, its metabolites, and/or conjugates at micromolar levels (4). Being relatively polar, they cannot be removed from waters easily. Psychotherapeutics such as antidepressants, cardiovascular drugs, and anti-infectives seem to stand out as particularly dangerous to aquatic life (5, 6).
In this study we focused on one particular antidepressant acting through inhibition of serotonin reuptake, that is, fluoxetine (Flx; IUPAC name:
Fluoxetine can bioaccumulate in
In this study we considered three scenarios to evaluate the toxicity of Flx, NorFlx, and their combinations: chronic exposure to a realistic environmental concentration; chronic exposure to a 10-fold environmental concentration, and subacute exposure to a 100-fold environmental concentration because of increased antidepressant use in the world. Besides morphological and brood production changes, we evaluated oxidative and anticholinesterase effects of Flx, NorFlx and their combinations in these three scenarios on cytochrome c and ERK1/2 protein levels and mitochondrial membrane polarisation (MMP).
Fluoxetine hydrochloride (Supelco PHR1394) and its human metabolite norfluoxetine hydrochloride (Supelco 40724) were obtained from Supelco, Inc. (Bellefonte, PA, USA). Serial dilution method was used to prepare the exposure media. The efficiency of our dilution method was confirmed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). 100 μL of water sample was injected into the LC-MS/MS device (LCMS-8040, Shimadzu, Kyoto, Japan) equipped with Inertsil® ODS-4 colon (3 μm, 2.1 x 50 mm, GL Sciences, Japan). Flow rate, colon temperature, and mobile phases A and B were 0.4 mL/min., 40 °C, and 0.2 mmol/L for ammonium formate + 0.004 % formic acid and methanol, respectively. Parent and daughter ions and retention time for Flx were 310.2, 148.1, and 8.033 min, respectively. Parent and daughter ions and retention time for NorFlx were 296.1, 134.1, and 8.011 min, respectively. As our device and method could not detect environmental concentrations, we spiked environmental solutions with 1 μg/L Flx or NorFlx and calculated the difference between 1 μg/L and spiked solutions. Method accuracy expressed in terms of recovery was ≥89.78 % for Flx and ≥93.00 % for NorFlx.
Confirmation of water concentrations of fluoxetine (Flx) and norfluoxetine (NorFlx) after serial dilution
Concentrations | 0.091 μg/L | 0.91 μg/L | 9.10 μg/L | 0.011 μg/L | 0.11 μg/L | 1.10 μg/L |
---|---|---|---|---|---|---|
0.090±0.007 | 1.18±0.06 | 9.39±0.05 | - | - | - | |
- | - | - | 0.010±0.000 | 0.15±0.01 | 1.14±0.01 |
Data are given as means ± standard deviations (
For the chronic toxicity experiments we used third-generation offspring younger than 24 h divided in seven groups and kept in beakers containing 800 mL of ASTM hard water: control group, Flx group (0.091 μg/L as environmentally realistic concentration), NorFlx group (0.011 μg/L as environmentally realistic concentration), Flx+NorFlx group (mixture of their environmentally realistic concentrations), 10 x Flx group (0.91 μg/L), 10 x NorFlx group (0.11 μg/L), and 10 x Flx+10 x NorFlx group (a mixture of their 10-fold environmental concentrations). Twenty-one animals were placed in each beaker, and four beakers for each group were used as replicates. The experiments lasted 21 days. Water was completely changed every third day. During the change, 1.5 mL/L of food was pipetted into beakers. Less than 24 h-old nestlings in each beaker were counted and collected into 50 mL Falcon tubes (containing 50 % ethyl alcohol) at 24 h intervals starting from the eighth day. Collected animals were stored at +4 °C before microscopy.
For subacute, four-day exposure experiments we used the fourth-generation offspring of 500 less than 24 h-old animals divided into two plastic 15 L containers filled with ASTM hard water. The animals were fed 1.5 mL/L trout chow/yeast/alfalfa mixture for 17 days. On day 17, 21 animals per group were placed into a beaker containing 800 mL of ASTM hard water. The groups included control, 100 x Flx (9.1 μg/L), 100 x NorFlx (1.1 μg/L), and 100 x Flx+100 x NorFlx (a mixture of their 100-fold environmental concentrations). Five beakers for each group were used as replicates. 1.5 mL/L of food was pipetted into each beaker. After 48 h, water was changed, and the animals stopped receiving food. At the end of 96 h exposure period, animals were 21 days old.
At the end of subacute and chronic experiments, one animal per beaker was placed into a microplate well and the microplate stored at -80 °C until 5,6-dichloro-2-[(E)-3-(5,6-dichloro-1,3-diethylbenzimidazol-3-ium-2-yl)prop-2-enylidene]-1,3-diethylbenzimidazole;iodide (JC-1; CAS No: 3520-43-2) staining for MMP analysis. The remaining animals from each beaker were carefully collected on a filter, washed with clean ASTM hard water, blotted dry, placed into one micro tube, weighed (all these steps were done on ice as much as possible), and stored at -80 °C until biochemical analysis. The procedure was repeated with four beakers as replicates until we acquired an adequate sample for Western blotting of cytochrome c and ERK1/2.
Before biochemical analysis, a sample of 20 animals from each microtube were homogenised with chilled 100 mmol/L KCl and 1 mmol/L EDTA (containing pH 7.4 100 mmol/L potassium phosphate buffer on ice at a ratio of 1:4 w/v in a homogeniser equipped with stainless steel shaft (HS30T, WiseStir, Daihan, Korea). The homogenate was then centrifuged at 10000
Glutathione peroxidase (GPx) protects the cell against oxidative stress by reducing hydrogen peroxide (H2O2) and organic peroxides via glutathione consumption (20). It was analysed according to the method described by Beutler (21). Briefly, 25 μL of supernatant was incubated with 10 μL of 1 mol/L pH 8.0 Tris buffer, 25 μL of 0.1 mol/L glutathione (GSH), 100 μL of 10 U/mL glutathione reductase (GR), and 100 μL of 2 mmol/L reduced β-nicotinamide adenine dinucleotide phosphate (NADPH) at +37 °C for 10 min. The reaction was started with the addition of 7 mmol/L
Cholinesterases (ChE) are proposed as biomarkers for apoptosis (and neurotoxicity) because of increased expression during apoptotic conditions (22). Cholinesterase activity was analysed according to the method of Ellman et al. (23). Briefly, 100 μL of supernatant were added to a 125 μL mixture of 0.1 mol/L potassium phosphate buffer (pH 7.2), 10 mmol/L 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB), and 10 mmol/L sodium bicarbonate and incubated at room temperature for 10 min. The reaction was started with the addition of 100 μL of acetylthiocholine iodide. Absorbance change was measured at 405 nm for 5 min using the 13.6 L/mmol/cm extinction coefficient.
TBARS is a universal measure of reactive oxygen species (ROS) damage on unsaturated fatty acids (24). Its levels were measured according to the method described by Wills (25). 25 μL of supernatant was mixed with 25 μL of 10 % trichloroacetic acid (TCA). After centrifugation at 500
Total protein levels were measured with a modified Lowry method (26). 10 μL of supernatant were mixed with 45 μL of Lowry reagent and incubated at room temperature for 10 min. Then, 45 μL of Folin & Ciocâlteu’s phenol reagent were added, and the mixture incubated at room temperature for another 30 min. The absorbance was measured at 750 nm and converted to concentration values using a standard calibration curve prepared with bovine serum albumin (BSA).
JC-1 probe was used as qualitative measure of change in mitochondrial membrane potential (27). Membrane depolarisation is a common biomarker of apoptotic cell death (28, 29). At low potential, the membrane turns fluorescent green, but at higher potential it turns fluorescent red. Each
In healthy cells cytochrome c is found between mitochondrial membranes and is mainly responsible for oxidative phosphorylation. However, its release into the cytosol triggers the intrinsic pathway of apoptosis through the formation of mitochondrial permeability transition pore (MPTP) (30). The other two apoptosis-related proteins we studied – extracellular signal-regulated kinases 1 and 2 (ERK1/2) – act together to increase the activity of anti-apoptotic proteins or their transcription factors to prevent apoptosis. However, they also behave as an apoptotic factor through yet unknown mechanisms (31). For Western blotting of cytochrome c and ERK1/2, 20
For morphometric analysis nestlings were placed in a concave microscope slide, which contained lactic acid solution. They were photographed under a binocular microscope (BX53, Olympus, Tokyo, Japan) equipped with a Canon EOS1200D camera (Canon Inc., Tokyo, Japan) with 40x magnification. Carapace length was measured between the anterior head and caudal spine basement (32) using the Micam v. 2.0 imaging software (
Biochemical and morphometric data were analysed with the Statistical Package for Social Sciences for Windows, version 17 (SPSS, SPSS Inc., Chicago, IL, USA). Normality of data distribution was tested with the Kolmogorov-Smirnov test. For data that were not distributed normally we used the Kruskal-Wallis non-parametric test. If the Kruskal-Wallis test revealed significant difference, we further tested it with the Mann-Whitney
Table 2 shows neurotoxic and oxidative effects of chronic and subacute exposure to Flx, NorFlx, and their combinations. Cholinesterase activity was particularly high at 100-fold environmental concentrations, but NorFlx at environmental concentration and at ten times that also increased ChE activity, while the increase with Flx and Flx+NorFlx was not statistically significant. Li and Tan (33) also reported a rise in ChE in
Effects of fluoxetine (Flx), norfluoxetine (NorFlx), and their combinations on GPx, ChE, TBARS, and total protein levels in
GPx (μmol/min/mg protein) | ChE (μmol/min/mg protein) | TBARS (nmol/mg protein) | Total proteins (mg/mL) | |
---|---|---|---|---|
Chronic | ||||
Control | 0.047±0.010 |
0.010±0.003 |
8.169±2.892 |
1.143±0.271 |
Flx | 0.048±0.002 |
0.012±0.001 |
8.413±0.999 |
0.972±0.038 |
NorFlx | 0.051±0.004 |
0.014±0.001 |
11.670±1.347 |
0.996±0.032 |
Flx+NorFlx | 0.056±0.010 |
0.012±0.002 |
8.750±1.106 |
0.948±0.037 |
10 x Flx | 0.086±0.003 |
0.014±0.001 |
16.354±3.315 |
0.909±0.028 |
10 x NorFlx | 0.062±0.010 |
0.010±0.001 |
21.840±6.003 |
0.966±0.125 |
10 x Flx+10 x NorFlx | 0.050±0.005 |
0.007±0.002 |
15.600±3.283 |
1.029±0.098 |
Subacute* | ||||
Control | 0.063±0.005 |
0.016±0.003 |
10.635±3.731 |
0.681±0.107 |
100 x Flx | 0.175±0.071 |
0.058±0.020 |
67.963±32.735 |
0.365±0.048 |
100 x NorFlx | 0.273±0.221 |
0.053±0.009 |
24.030±7.179 |
0.278±0.071 |
100 x Flx+100 x NorFlx | 0.129±0.042 |
0.054±0.012 |
35.388±15.764 |
0.297±0.069 |
Data are presented as means ± standard deviations. Data that do not share the same letters are significantly different (
Considering that ChE activity is increased by apoptosis inducers
At 100-fold environmental concentrations Flx, NorFlx, and their combinations decreased total protein levels, but this effect was not observed in chronic toxicity experiments involving environmental and 10-fold environmental concentrations (Table 2). Concentration- and duration-dependent decrease in total protein levels was also observed in
In contrast to our findings, Campos et al. (47) reported no significant changes in total protein and lipid content in adult
Glutathione peroxidase as first-line defence against oxidative stress also increased at both 10 and 100 times Flx and NorFlx environmental concentrations. At environmental levels, however, its basal activity seems to have been sufficient to counter ROS and prevent oxidative damage (Table 2). A case in point could by the anticholinesterase insecticide chlorpyrifos study (49), which increased cytosolic ChE and GPx activities and H2O2 levels but not lipid peroxidation. In our study, the concentration-dependent increase in GPx activity and lipid peroxidation levels should be viewed through redox balance, which tipped toward oxidation at higher Flx and NorFlx concentrations. Similar concentration-dependent changes in
Chronic effects of fluoxetine (Fix), norfluoxetine (NorFlx), and their combinations at environmental or 10-fold environmental concentrations on carapace length, maximum carapace width, and caudal spine length in less than 24 h-old
Carapace length (pm) | Max. carapace width (pm) | Caudal spine length (pm) | Carapace length (pm) | Max. carapace width (pm) | Caudal spine length (pm) | Carapace length (pm) | Max. carapace width (pm) | Caudal spine length (pm) | |
---|---|---|---|---|---|---|---|---|---|
1st generation offspring | 2nd generation offspring | 3rd generation offspring | |||||||
777.6±50.4 | 430.0±41.0 | 403.2±30.4 | 880.9±85.9a | 473.7±86.9acd | 415.3±31.5 | 847.0±46.1a | 490.6±52.8ad | 411.5±42.8a | |
773.0±37.3 | 414.5±43.1 | 385.2±26.4 | 827.8±76.1b | 437.5±62.9c | 400.6±33.1 | 806.9±45.7b | 475.6±53.6ac | 371.2±42.0b | |
748.5±30.4 | 408.5±38.9 | 354.0±34.6 | 842.3±72.5b | 448.5±66.1ac | 403.5±28.6 | 838.7±41.8a | 475.0±38.9ac | 378.1±44.4bc | |
774.2±33.9 | 420.9±47.8 | 367.0±29.3 | 857.3±56.1a | 466.0±55.1ad | 397.4±32.3 | 812.4±45.2b | 469.2±45.4bc | 369.7±39.9b | |
742.6±32.7 | 400.9±45.8 | 345.1±25.9 | 874.1±107.5ab | 496.4±97.7de | 406.1±43.0 | 906.2±80.9c | 524.4±70.2e | 397.3±40.2ac | |
764.2±34.6 | 407.6±36.9 | 358.8±26.2 | 893.8±101.5ac | 521.6±78.7be | 401.4±41.7 | 857.9±69.4a | 492.8±68.0abf | 379.9±41.5bc | |
733.7±34.1 | 388.5±43.2 | 349.4±41.9 | 925.6±102.1c | 544.5±81.9b | 408.0±53.6 | 915.7±105.3c | 524.0±90.6def | 380.3±56.7bc | |
4th generation offspring | 5th generation offspring | Average | |||||||
887.6±22.5a | 483.0±31.2a | 392.0±31.3ae | 899.7±64.5a | 497.7±49.2a | 385.8±30.3ac | 865.0±70.3a | 478.3±59.6a | 401.0±35.4a | |
842.9±27.4bd | 456.7±33.7bc | 414.1±32.4b | 783.7±58.2b | 446.4±35.1b | 363.0±51.7a | 805.9±58.5b | 444.5±50.0b | 386.8±42.6b | |
852.2±55.1bc | 449.7±52.4b | 372.0±28.7cd | 890.9±100.1c | 503.9±71.5af | 389.8±49.7cb | 831.1±78.7c | 455.8±62.4bd | 378.8±41.5c | |
878.8±56.8a | 473.4±60.4ac | 367.7±31.6d | 891.2±94.0ac | 532.3±63.7c | 395.5±35.5cb | 836.7±74.8c | 468.3±65.8cef | 378.7±36.0c | |
845.3±47.1bd | 454.4±37.5b | 395.9±29.0a | 836.9±35.9d | 462.1±30.5ed | 394.0±25.7b | 841.0±86.0c | 467.6±73.6deg | 387.9±39.6bd | |
855.9±22.2c | 480.4±24.8a | 394.0±41.5a | 838.4±98.5e | 476.5±69.7def | 386.7±44.6ab | 837.8±84.4c | 472.2±71.4afg | 382.3±41.5cd | |
833.0±42.3d | 465.9±25.9c | 381.0±23.8ce | 833.0±88.7de | 470.2±61.0be | 381.0±45.3ab | 849.9±105.7c | 480.2±84.1afg | 381.0±48.9cd |
Data were presented as means ± standard deviations. There is a statistical difference between the data that does not share the same letters (
Mitochondrial membrane depolarisation is a common marker of cell death and physiological condition of cells and tissues (28, 53). In our study, mitochondrial membrane depolarisation was observed in all the Flx, NorFlx, and combination groups in both chronic and subacute exposure (Figures 1 and 2) and especially with NorFlx. This suggests that Flx and NorFlx exert their toxicity via mitochondrial mechanisms directly or indirectly. Similar mechanisms have been observed in other studies in different models, including
Chronic effects of fluoxetine, norfluoxetine, and their combinations at environmental or 10-fold environmental concentrations on mitochondrial membrane potential in
Effects of fluoxetine, norfluoxetine, and their combinations at subacute, 100-fold environmental concentrations on mitochondrial membrane potential in
Mitochondrial membrane depolarisation is an important mechanism for the release of cytochrome c from the mitochondria (28), and cytochrome c release induces mitochondrial apoptosis in mammalian cells (56). In our study, cytochrome c protein levels were generally increased, except for the Flx and 10 x Flx+10 x NorFlx combination groups (Figures 3 and 4). However, increased cytochrome c protein content alone does not provide strong evidence of the mitochondrial release and apoptosis in our study, but in combination with increased lipid peroxidation and mitochondrial membrane depolarisation it does give grounds for such an assumption. We believe that increased cytochrome c levels in our study are related to intensive stress conditions and higher mitochondrial damage induced by Flx and NorFlx. Such a result was also found in the liver of mercury chloride-exposed rats (57).
Chronic effects of fluoxetine, norfluoxetine, and their combinations at environmental or 10-fold environmental concentrations on cytochrome c and ERK 1/2 protein levels in
Either compound or their combination did not affect ERK1/2 protein levels at environmental concentrations but did at higher concentrations, except for 100 times higher NorFlx alone (Figures 3 and 4). ERK1/2 is activated by a toxicant and regulates response to cellular damage (58). It promotes cell survival against apoptotic mechanisms, but in certain condition it also favours apoptosis (31). This may explain different responses to NorFlx in our study and calls for further investigation. We believe that increase in ERK1/2 protein levels may be an adaptive response to apoptotic conditions.
Subacute effects of fluoxetine, norfluoxetine, and their combinations at 100-fold environmental concentrations on cytochrome c and ERK1/2 protein levels in
Table 3 shows a significant increase in carapace length and width in the 2nd and 3rd generation offspring of the 10 x Flx+10 x NorFlx and 10 x Flx groups, respectively. Width also increased in the 10 x NorFlx group. Prey animals experience a variety of changes to avoid their predators. They may include toxin production (physiological changes), late breeding (life-cycle changes), and production of spicules and sheathing (morphological changes) (59). Flx alone and in combination with its metabolite may have triggered this avoidance behaviour at 10-fold environmental concentrations. Decreased carapace length, width, and spine length seen in other groups since the 2nd generation offspring may be related to lower energy production, as reported elsewhere (60, 61). In a study by Pery et al. (62) Flx also decreased carapace length of less than 24 h-old 3rd generation
NorFlx alone and in combination with Flx significantly increased brood production at their environmental concentrations until the 5th generation offspring (Table 4). A similar increase in the first four generations was also observed with Flx and its combination with NorFlx at 10-fold concentrations. Campos et al. (47) suggested that brood production of
Chronic effects of fluoxetine, norfluoxetine, and their combinations at environmental or 10-fold environmental concentrations on brood production of
1st generation offspring | 2nd generation offspring | 3rd generation offspring | 4th generation offspring | 5th generation offspring | Average | |
---|---|---|---|---|---|---|
Control | 76.8±13.6 |
50.1±16.5 |
44.1±16.4 |
73.9±15.3 |
84.8±32.3 |
65.9±25.1 |
Flx | 79.8±24.4 |
46.2±5.7 |
62.7±18.1 |
78.7±32.1 |
81.3±29.7 |
69.7±26.9 |
NorFlx | 143.3±44.7 |
69.5±25.4 |
140.7±51.8 |
135.6±64.2 |
62.2±22.8 |
110.3±56.6 |
Flx+NorFlx | 126.3±29.9 |
94.3±43.4 |
144.3±29.5 |
135.3±68.1 |
45.4±9.3 |
109.1±53.7 |
10 x Flx | 129.5±59.0 |
53.5±29.2 |
60.6±9.3 |
84.9±58.6 |
41.8±17.4 |
74.1±48.0 |
10 x NorFlx | 70.6±21.9 |
42.3±26.8 |
57.7±29.9 |
52.7±33.2 |
67.6±27.6 |
58.2±29.1 |
10 10 x x Flx NorFlx + | 98.6±31.8 |
64.4±20.0 |
56.8±8.9 |
60.6±48.5 |
42.3±15.5 |
64.5±33.4 |
Data were presented as means ± standard deviations. There is a statistical difference between the data that does not share the same letters (
However, in the 5th generation offspring brood production dropped in all groups (Table 4), which may be related to the bioaccumulation of Flx and NorFlx in
In conclusion,