Illicit drug ketamine induces adverse effects from behavioral alterations and oxidative stress to p53-regulated apoptosis in medaka fish under environmentally relevant exposures

Environmental Pollution 237 (2018) 1062e1071

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Illicit drug ketamine induces adverse effects from behavioral alterations and oxidative stress to p53-regulated apoptosis in medaka fish under environmentally relevant exposures* Pei-Han Liao, Wen-Kai Yang, Ching-Hsin Yang, Chun-Hon Lin, Chin-Chu Hwang, Pei-Jen Chen* Department of Agricultural Chemistry, College of Bio-Resources and Agriculture, National Taiwan University, Taipei, Taiwan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 June 2017 Received in revised form 27 October 2017 Accepted 6 November 2017 Available online 14 November 2017

With increasing problems of drug abuse worldwide, aquatic ecosystems are contaminated by human pharmaceuticals from the discharge of hospital or municipal effluent. However, ecotoxicity data and related toxic mechanism for neuroactive controlled or illicit drugs are still lacking, so assessing the associated hazardous risk is difficult. This study aims to investigate the behavioral changes, oxidative stress, gene expression and neurotoxic or apoptosis effect(s) in larvae of medaka fish (Oryzias latipes) with environmentally relevant exposures of ketamine (KET) solutions for 1e14 days. KET exposure at an environmentally relevant concentration (0.004 mM) to 40 mM conferred specific patterns in larval swimming behavior during 24 h. At 14 days, such exposure induced dose- and/or time-dependent alteration on reactive oxygen species induction, the activity of antioxidants catalase and superoxide dismutase, glutathione S-transferase and malondialdehyde contents in fish bodies. KET-induced oxidative stress disrupted the expression of acetylcholinesterase and p53-regulated apoptosis pathways and increased caspase expression in medaka larvae. The toxic responses of medaka larvae, in terms of chemical effects, were qualitatively analogous to those of zebrafish and mammals. Our results implicate a toxicological impact of waterborne KET on fish development and human health, for potential ecological risks of directly releasing neuroactive drugs-containing wastewater into the aquatic environment. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Ketamine Medaka (Oryzias latipes) Fish locomotion Oxidative stress Neurotoxicity Apoptosis

1. Introduction With fast civilization and serious problems of drug abuse worldwide, our aquatic ecosystems are ubiquitously contaminated by a variety of human pharmaceuticals from frequent discharge of municipal or hospital effluents (Jones et al., 2001; Kolpin, 2002; Writer et al., 2013; Pal et al., 2013; Rosi-Marshall et al., 2015). Many pharmaceuticals can escape from chemical and biological processes of wastewater treatment plants and/or natural purification processes and remain active or toxic to the aquatic life (Lin et al., 2014a, 2014b; Wang and Lin, 2014; McCall et al., 2016). For instances, wastewater pharmaceuticals are prevailingly detected

* This paper has been recommended for acceptance by Dr. Harmon Sarah Michele. * Corresponding author. Department of Agricultural Chemistry, National Taiwan University, R319, No. 1, Sec. 4, Roosevelt Road, Taipei 106, Taiwan. E-mail address: [email protected] (P.-J. Chen).

https://doi.org/10.1016/j.envpol.2017.11.026 0269-7491/© 2017 Elsevier Ltd. All rights reserved.

worldwide and at environmentally relevant concentrations can induce adverse effects in endocrine disruption in wildlife and mammalians including humans for decades (Corcoran et al., 2010; € ffker and Tyler, 2012). Jones et al., 2001; So Recently, water pollution by a variety of neuroactive pharmaceuticals poses a substantial threat to the aquatic ecosystem and human safety (Corcoran et al., 2010; Jones et al., 2002; Writer et al., 2013). Neuroactive drugs are medically designed to act with the central nervous system, thus altering behavior or physiology (Jones et al., 2002; Larsson et al., 2007; Lin et al., 2014a). These neuroactive drugs such as anaesthetics, psychotropics, narcotics and amphetamines have high potential for abuse, so accessing and using these substances are carefully controlled (Lin et al., 2010; Zuccato et al., 2008). However, the abuse of these controlled drugs is widespread among the general population because they can provide pain relief or a pleasurable or hallucinogenic consciousness when improperly used (Morgan and Curran, 2012). As compared to weekdays or normal days, many studies reported high

P.-H. Liao et al. / Environmental Pollution 237 (2018) 1062e1071

concentrations of illicit drugs or controlled substances in wastewater flows or surface waters on weekends and during holidays, sport seasons or youth festivals, implying the serious problems of drug abuse and addiction (Gerrity et al., 2011; Huerta-Fontela et al., 2008; Jiang et al., 2015). As a result of continuous input of abused or illicit drugs into the surface water from hospital or municipal wastewaters and other special routes, it is urgent to understand the consequent impact of this class of pharmaceutical pollution on environmental safety and human health. Ketamine (KET) is a controlled anesthetic frequently used in human and veterinary for maintaining sedation and anaesthesia (Kohrs and Durieux, 1998; Morgan and Curran, 2012). It has been increasingly used for complex pain management for decades and recently considered for treating major depressive disorder (Hocking and Cousins, 2003; Mathew et al., 2012). The environmental occurrence of KET can be attributed to two major sources: use in veterinary, clinical and paediatric practice or abuse by individuals (Lin et al., 2014a). In cities with large populations, the maximum environmental concentration for KET ranges from 51 to 420 ng L
1 in river water and up to 10,000 ng L
1 in hospital wastewater effluent (Tables S1 and SI). The KET concentrations can reach to 9533 ng L
1 in river water and 138,000 ng L
1 in wastewater during a week of the youth festival in Taiwan (Jiang et al., 2015). Thus, the environmental presence of this drug poses an unknown risk to aquatic ecosystems and to humans if surface water is a major source of drinking water. Studies have reported several ecotoxicological outcomes (e.g., physiological, biochemical or behavioral alterations) for a wide range of human pharmaceuticals in several aquatic organisms such as fish, daphnia or algae with acute to subchronic exposure at broad concentration ranges (Brodin et al., 2013; Cleuvers, 2004; Kim et al., 2007). However, evidence is still lacking for the chronic adverse effects and underlying toxic mechanisms of illicit drugs such as KET for aquatic life, especially in fish. For mammalians including humans, the long-term illicit use of controlled drugs including KET can lead to abnormal messaging by the central nervous system, thereby causing behavioral changes or drug dependence and addiction (Davidson et al., 2001; Cubells et al., 1994). The KET at clinical doses induced toxicity is involved in induction of oxidative stress or neurotoxicity and/or physical or psychological alteration in mammals (Morgan and Curran, 2012). Microtox results show greater toxicity in photobyproduct mixtures of KET (including metabolite norketamine) than the parent compound (Lin et al., 2014a). For the higher tropic level of aquatic organisms such as fish, acute exposure to KET over clinical doses (e.g., high mg L
1 to mg L
1 levels) causes developmental toxic and/or neurotoxic effects in zebrafish embryos (Felix et al., 2014; Kanungo et al., 2013; Zakhary et al., 2011). We recently demonstrated that the 7 day-developmental exposure to KET (from ng L
1 - mg L
1 levels) can alter physiology of embryos and larval locomotion in medaka fish (Oryzias latipes) (Liao et al., 2015). Similarly, 24 h of ketamine embryonic exposure (mg/L levels) induced developmental abnormalities and behavioral changes in zebrafish (Felix et al., 2017a, 2017b). We especially need a reliable method for environmentally relevant chronic exposure to understand ecotoxicological impact and related biological toxic mechanisms of KET on higher tropic levels of aquatic vertebrate species. Medaka fish (Oryzias latipes) possess several unique characteristics Medaka fish (Oryzias latipes) possess several unique characteristics including small size, short generation time, asynchronous spawning, ease of breeding and relatively economic husbandry and longer developmental window, all of which make them superior for use in basic biology, medical and pharmaceutical research and for toxicity assessments of chemicals or drugs (Wittbrodt et al., 2002; Ishikawa, 2000). To understand whether the waterborne controlled

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drug could induce adverse outcomes in fish, we exposed medaka larvae to KET solutions from environmentally relevant concentrations (ng L
1) through mg L
1 levels for 1e14 days, followed by a 7day recovery in drug-free solutions. We used different organizational biomarkers to assess toxic responses regarding behavioral changes, oxidative stress and neurotoxic-related effect(s) of KET in fish. We discuss the toxic mechanism of KET across species in the views of ecologically relevant conditions and their association with ecological risk assessment. 2. Materials and methods 2.1. Dosing solutions The hydrochloride salt of KET was obtained from Sigma-Aldrich (USA) with permission. The KET salt was dissolved in deionized milli-Q water to make a stock solution. A series of dosing solutions was diluted from the stock with embryo-rearing media (ERM, containing 1 g NaCl, 0.03 g KCl, 0.04 g CaCl2$2H2O, and 0.163 g MgSO4$7H2O in 1 L DI water, adjusted to pH 7.2, Iwamatsu, 1994) for treating fish. We choose test concentrations of 0.004e40 mM KET, as these ranges showed no acute mortality to medaka larvae based on (Liao et al., 2015), which agree with the literature that KET over mg L
1 to g L
1 levels causes fish mortality (Felix et al., 2014). The lowest tested concentrations used (0.004 mM ¼ 951 ng L
1) referred to the literature data for KET occurrence in the aquatic compartment (e.g., the maximum environmental concentration for KET is 53.7e420 ng L
1 in river water and 278e10000 ng L
1 in wastewater effluent, Tables S1 and SI). The concentration of KET in dosing solutions was confirmed by HPLCeMS/MS as described (Lin et al., 2010). Actual concentrations of each dosing solution were about 95%e108% of their nominal values, as previously described (Liao et al., 2015). 2.2. The larval locomotion test Medaka fish (orange red strain) were bred and used based on the animal research protocols approved by the Institutional Animal Care and Use Committee, National Taiwan University. Briefly, fish were reared in dechlorinated tap water with stable water quality under rigorously environmental conditions at 27 ± 1  C, with a 14hr light/10-hr dark photoperiod. Egg clutches spawned from 1-day spawn of the same stock of females were separated and embryos of an early blastula stage were selected for experiments. Free swimming larvae were randomly assigned to 24-well microplates containing ERM (one per well) one day before chemical exposure for acclimation. Then fish (3 days post-hatching, dph) were treated with 2-ml KET at 0.004, 0.1, 0.4, 10 and 40 mM (equal to 951 ng L
1, 24 mg L
1, 95 mg L
1, 2.4 mg L
1 and 9.5 mg L
1) or a blank control for 24 h. A total of 32 larvae (4 larvae in one plate per concentration for 8 plates) were used for each concentration. At 1 and 24 h, larvae were assessed for behavior with the animal movement tracking system and software (EthoVison XT, Noldus Information Technology, The Netherlands) as described (Chou et al., 2010) with minor modification (Liao et al., 2015). Briefly, after 180 s of acclimation, the locomotor parameters were recorded for each fish in dark with infrared illumination for 180 s with a tracking rate of 25 frames/s without any disturbances. The velocity (cm/s) was the distance traveled by the center column of larvae per unit time. The percentage time active (%) was the movement duration to total time interval measured. The absolute or relative turn angle (degree) was the change in movement direction (clockwise or anti-clockwise). During the following 180 s, the holding stage was slightly knocked with a hammer every 30 s with consistent intensity and

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the movement of the fish was recorded to calculate the maximum velocity due to stimulation (the stimulate startle response). 2.3. Subchronic exposure After 1-day acclimation, larvae (7 dph) were treated with KET (0.004e10 mM) or blank control continuously for 14 days (40 fish in 200 mL solution). At the end of KET exposure, the larvae were transferred to clean ERM for a 7 day-recovery period. Each concentration was replicated with four beakers. The details in exposure procedure were described in the supporting information. At days 1, 7, 14 and 21, larvae from each treatment (3 larvae per beaker) were collected by polyethylene dropper with wide opening and homogenized in phosphate buffered saline and then centrifuged (12,000  g; 30 min at 4  C) with the high-speed centrifuge (Avanti J-26S Beckman Coulter, IN, USA) as described (Tu et al., 2016). The supernatant was immediately used for total protein concentrations measured with the BCA Protein Assay Kit (Thermo Fisher Scientific, USA) and the reactive oxygen species (ROS) level by the 20 70 dichlorofluorescin diacetate assay (LeBel et al., 1992). Additional fish were collected from certain time points for gene expression assays. 2.4. Measurements of oxidative stress responses and acetylcholinesterase (AChE) activity

0.4 and 4 mM) or blank control (four replicates per concentration) continuously for 14 days based on transcriptional expression results of caspase-3a. At the endpoint, larvae (6 fish per beaker were collected and 3 fish were pooled as a subsample for n ¼ 8) were anesthetized in ice-cold water, harvested and homogenized in lysis buffer (with 200 mM TRIS, 2 M NaCl, 20 mM EDTA, 0.2% TRITON X100; pH 7.5). The homogenate was centrifuged (12,000  g) with the high-speed centrifuge (Avanti J-26S Beckman Coulter, IN, USA) at 4  C for 30 min, and the supernatant was immediately used for Caspase-3 activity with EnzChek Caspase-3 Assay Kit #2 (Molecular probes, Leiden, The Netherlands) based on the manufacture's instruction. 2.7. Statistical analysis Data are shown as mean ± SD. The ShapiroeWilk's test was used to test normality for all data. One-way ANOVA analysis was used for data with a normal distribution, and then followed by post-hoc analysis with the least significant difference (LSD) or Dunnett's test with SAS Learning Edition 4.1 (SAS Inst., Cary, NC). Statistically significant differences were considered at p < 0.05. For data which violated the assumption of normality, a nonparametric KruskaleWallis test was used with a significant a level being Bonferroni-adjusted to 0.01 to avoid type I error. 3. Results

The supernatant of fish homogenates from each time point was used for measuring the antioxidant activity including catalase (CAT, by measuring the change of substrate hydrogen peroxide in absorbance of 240 nm; Beers and Sizer, 1952), superoxide dismutase (SOD, by measuring the change of nitroblue tetrazolium to formazan in absorbance at 560 nm; Beaucham and Fridovic, 1971), glutathione reductase (GR, by following the decrease in absorbance at 340 nm due to NADPH oxidation; Foster and Hess, 1980) and glutathione S-transferase (GST, by using 1-chloro-2,4dinitrobenzene as a substrate and absorbance at 340 nm was measured; Grundy and Storey, 1998). The lipid peroxidation product malondialdehyde (MDA) and acetylcholinesterase (AChE) activity were measured by the TBARS Assay Kit (Cayman Chemical, Ann Arbor, MI, USA) and Amplite Colorimetric AChE Assay Kit (AAT Bioquest, Inc., Sunnyvale, CA, USA), respectively. 2.5. Quantitative real time PCR (qPCR) Based on the results of oxidative stress responses, total RNA from frozen larvae collected at day 14 was extracted with TRI REAGENT (Molecular Research Center, OH, USA) and 1-bromo-3chloropropane according to the manufacturer's instruction. Total RNA was treated with the Turbo DNA-free kit (Ambion Inc., TX, USA) to remove DNA contamination. cDNA synthesis from the same quantity of purified RNA involved the high-capacity cDNA reversetranscriptase kit (Applied Biosystems, ABI, CA, USA) with random hexamers used as primers, as described (Chen et al., 2011). The qPCR assays were performed with the StepOne Real-Time PCR System (Applied Biosystems, ABI, Foster City, USA), the Power SYBR Green PCR Master Mix (ABI) and custom-designed primers (MDbio Inc., Taipei) (Tables S2 and SI). The expression profile of the target gene was normalized to that of ribosomal protein L7 (rpl7, a housekeeping gene; Zhang and Hu, 2007) and expressed as a fold change by the relative quantification method (DDCt method) (Livak and Schmittgen, 2001). 2.6. Measurement of Caspase-3 activity In an additional study, fish (7 dph) were dosed with KET (0.004,

3.1. Larval locomotor activity altered by acute KET exposures from environmentally relevant concentrations to 40 mM We assessed larval locomotor activity at 1 and 24 h with 24-hr exposure to KET solutions (Fig. 1). With 1-hr exposure, KET (0.004e40 mM) conferred an inverted U-shaped curve in doseresponse of average velocity: lower doses (0.004e0.4 mM) evoked hyperactivity, which was mitigated at higher doses (10e40 mM) (p < 0.05 at 0.004e10 mM) (Fig. 1a). With 24-hr exposure, KET did not significantly change the mean velocity (Fig. 1a) or percent time active (Fig. S1a-b, SI) as compared with controls. Furthermore, medaka larvae with KET exposure showed a significant decrease in the relative turn angle at lower dosages with 1-hr exposure (Fig. 1b), indicating a less tortuous swimming path. This alteration was returned to the control level with higher concentrations or longer time (24 h) (Fig. 1b). However, the absolute turn angle was not significantly changed within 24 h (Fig. S1i and Fig. S1j). The maximum velocity was dose-dependently increased with KET treatment at both times but significantly only at 24 h (p < 0.05 at 0.004e40 mM) (Fig. 1c). 3.2. ROS-mediated oxidative stress responses from subchronic KET exposure ROS production and activity of the antioxidants SOD, CAT and GR and lipid oxidized product MDA were determined in larvae after 1to 14-day exposure to KET at 0.004e10 mM, followed by a 7 dayrecovery (day 21) (Fig. 2). KET induced a dose- and timedependent oxidative stress response with 14-day exposure by inducing ROS levels and altering the balance of antioxidant activities in larvae. With 1-day exposure, ROS level did not differ from the control level (Fig. 2a). However, exposure to KET for 7e14 days dose-dependently increased ROS level (p < 0.05 at 10 mM on day 7 and 0.004e10 mM on day 14). KET conferred an inverted U-shaped curve in SOD activity at assessed time points, which was dosedependently enhanced at lower doses (p < 0.05 at certain doses < 10 mM), and then mitigated at higher doses (Fig. 2b). After a 7-day recovery, the induced ROS or SOD activity was not

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the phase II metabolizing enzyme GST activity was significantly increased in larvae with 7- and 14-day exposure (p < 0.05 at 4e10 mM), but GST activity did not differ from the control level at 1 day; the induced activity was ameliorated to the control level after 7-day recovery (Fig. 3a). 3.4. Gene expression of neurotoxic related gene AChE activity was measured in larvae after 1- to 14-day exposure to KET at 0.004e10 mM, followed by 7-day recovery in drugfree ERM (Fig. 3b). With 14-day exposure to KET, the AChE activity was significantly elevated only at 0.004 mM but did not differ from the control level with all concentrations before and after 7day recovery in ERM (Fig. 3b). However, mRNA expression of ache in larvae was significantly suppressed with 14-day KET exposure (0.004e4 mM) as compared with the control (p < 0.05 at 0.4e4 mM; Fig. 4a). 3.5. Gene expression involved in p53-related apoptosis pathways We measured the mRNA levels of several key genes in p53regulated pathways (Figs. S5 and SI) in larvae after 14-day exposure to KET (0.004e4.0 mM) (Fig. 4a). As compared with the control, larvae with 14-day exposure to KET showed elevated mRNA level of p53 (p < 0.05 at 0.4e4 mM, Fig. 4). We further showed that transcriptional expression of apoptotic genes including baxa (an apoptotic inducer), bcl-2 (an anti-apoptotic gene) (p < 0.05 at 0.004e0.4 mM for both genes) and caspase-3a (an apoptotic effecter) (p < 0.05 at 0.004e4 mM) were significantly upregulated in 14 day-KET treated fish as compared with the control, especially with a >2.5-fold increase for caspsae-3a at 0.4e4 mM (Fig. 4a). We also showed that the Caspase-3 activity was marginally enhanced in KET-treated fish at 4 mM (with a >1.5-fold increase) with the 14 day-exposure (Fig. 4b). In contrast, the mRNA expression of p21 (a cyclin-dependent kinase inhibitor) was suppressed with KET (p < 0.05 at 0.4 and 4 mM) (Fig. 4a). 4. Discussion

Fig. 1. The locomotor activity of (a) average velocity, (b) relative turn angle and (c) maximum velocity for medaka larvae with 1- and 24-hr aqueous exposure to ketamine (KET). Data are mean responses from 32 fish * p < 0.05 vs the control. The original dataset is provided in Figs. S1 and SI.

completely recovered to the control level. With 1- or 7-day exposure, CAT activity was not significantly changed by KET treatments at lower concentrations (&4 mM), except 10 mM (p < 0.05), as compared with the control (Fig. 2c). However, with 14-day exposure, KET significantly decreased CAT activity at all concentrations (p < 0.05). As well, the production of MDA was significantly elevated with 7- and 14-day KET treatment at 10 mM (Fig. 2d). After a 7-day recovery in ERM, CAT activity and MDA production returned to the control level (Fig. 2ced). However, KET did not significantly alter GR activity with all treatments (Figs. S2 and SI) or change the transcriptional expression of sod, cat and glutathione peroxidase (gpx) at day 14 (Figs. S3 and SI). 3.3. Gene expression of metabolism enzymes The mRNA expression profiles of selected phase I cytochrome P450 (CYPs, cyp1a and cyp3a) and related receptors ahr and pxr (Figs. S4 and SI) were not changed in larvae with 14-day KET exposure (0.004e4 mM) as compared with the control. However,

With the frequent use of controlled or abused (illicit) drugs and continuous input from hospital and domestic wastewater treatment plants resulting in their persistence in the aquatic environment, an assessment of the toxicity of these neuroactive drugs is critical to achieve a full understanding of their impact on the aquatic ecosystem and human health. Currently most KET-related studies focus on its pharmacological or medical effects in treated animals under high clinical dosages of exposures to predict its biological responses in humans. Ecotoxicological studies about KET impact on aquatic organisms is still lacking under environmental relevant concentrations of chronic exposures. KET was reported as no significant ecological risk (e.g., risk quotient, RQ < 1), based on the calculation from human therapeutic plasma levels expected to cause a pharmacological effect in fish (as the only available ecotoxicity data for KET are 40 mg/L from the ECOSAR model), which are substantially greater than their surface water concentrations (Fick et al., 2010; Van der Aa et al., 2013). Because of low acute mortality and a lack of studies of the chronic or sublethal effects for this class of drugs (e.g., KET) on aquatic organisms, a final ecological risk assessment is difficult. As we observed in this study, the ecotoxicological risk of KET is substantial to early life stages of aquatic organisms such as medaka fish, although the environmental detected concentrations of KET in the aquatic environment is low (ng L
1 - low mg L
1 levels; Tables S1 and SI). We report adverse outcomes of KET regarding different levels of toxic responses observed in medaka fish. KET at

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Fig. 2. Dose-dependent oxidative stress responses of (a) reactive oxygen species (ROS) level and activity of antioxidants (b) superoxide dismutase (SOD) and (c) catalase (CAT) and (d) malondialdehyde (MDA) content in medaka larvae during 14-day aqueous exposure and 7-day recovery from 0.004e10 mM KET solutions. Data are mean ± SD (3 fish were pooled as a subsample; n ¼ 4). *p < 0.05 vs the control.

environmentally relevant concentrations (0.004 mM ¼ 951 ng L
1) and subchronic exposure in medaka larvae induced an acute alteration in swimming behavior and oxidative stress responses. With 14-day exposure, KET dose- and time-dependent altered the oxidative stress profiles and GST activity in medaka, which were not completely recovered after 7-day exposure in drug-free solutions for ROS and SOD at certain concentrations. As well, the 14-day KET exposure altered AChE activity and disrupted p53-regulated apoptosis pathways that led to increase of caspase-3 expression (both mRNA expression and enzyme activity) in larvae. Therefore, the KET-induced oxidative stress is involved in AChE-associated neurotoxicity and p53-regulated apoptosis pathways with subchronic exposure. As compared with our previous study in medaka embryos with developmental exposure (Liao et al., 2015), KET conferred similar larval locomotor activities from environmentally relevant concentrations to 40 mM within 1 h, but showed distinct patterns after 24hr exposure (Fig. 1a). Similarly, acute exposure (10e70 min) to alcohol, abused drugs (including KET) or hallucinogens also conferred such an inverted U shape of doseeresponse in larval locomotion of zebrafish (Irons et al., 2010; Lockwood et al., 2004; Stewart et al., 2011). Acute doses of amphetamines or other hallucinogens in young (Campbell et al., 1969; Niculescu et al., 2005) and adult rodents (Porrino et al., 1984; Antoniou et al., 1998) and humans (Caligiuri and Buitenhuys, 2005) often conferred a

rewarding activity (e.g., accelerated locomotor activity at low doses and reduced locomotion at higher doses). However, chronic anesthetic KET treatments induced antidepressant effects or lower anxiety (Krystal et al., 1994), whereas higher doses or long-term of 3,4-methylenedioxymethamphetamine treatments produced hyperactivity or anxiety-like behaviors in mammalian models (Baumann et al., 2007). Zebrafish treated with abused drugs also showed a robust drug-evoked neurobehavioral phenotype (Neelkantan et al., 2013; Stewart et al., 2011). Early embryonic exposure to KET (200e800 mg L
1, 20 min) induced a significant increment of the anxiety-like behavior and a decrease in avoidance behavior (Felix et al., 2017a). The behavioral responses and sensitivity of medeka larvae to KET in general compare well with those observed in zebrafish (Riehl et al., 2011; Zakhary et al., 2011), rodents (Porrino et al., 1984; Antoniou et al., 1998; Baumann et al., 2007) and humans (Caligiuri and Buitenhuys, 2005; Krystal et al., 1994). We also found larvae with an acute KET exposure exhibited increased locomotion and more straight swimming paths in the previous (Liao et al., 2015) and current studies (Fig. 1b). This adaption may help fish maintain their position and balance in water (Kane et al., 2004). However, different abused drugs (e.g., MET)treated larvae tended to move in a clockwise direction under a longer exposure (Liao et al., 2015). Fish with acute exposure to KET and other hallucinogens often show similar larval locomotor

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Fig. 3. The activity of (a) glutathione S-transferase (GST) and (b) acetylcholinesterase (AChE) in medaka larvae during 14-day aqueous exposure and 7-day recovery from 0.004e10 mM KET solution. Data are mean ± SD (3 fish were pooled as a subsample; n ¼ 4). *p < 0.05 vs the control.

activities, but distinct swimming patterns on long-term exposure (Irons et al., 2010; Liao et al., 2015; Lockwood et al., 2004; Stewart et al., 2011). From ecological perspectives, the fright (startle) response is a crucial capability for neonatal fish to escape from predators and danger in the hostile environment (Swearer et al., 1999; Wolter and Arlinghaus, 2003). Many toxic chemicals disrupt the fright response of aquatic organisms and then change their swimming activity and/ or the ability to escape from a predator (Carlson et al., 1998). An environmentally relevant concentration of KET exposure significantly disrupted the fright response (e.g., enhanced maximum velocity was due to stimuli, as compared to the control fish without stimulation, Fig. 1a) in medaka larvae with acute exposure and embryos with 10-day developmental exposures (Liao et al., 2015). However, over sensitivity to external stimulation may affect the timing and decision of the escape response for neonatal fish (Fuiman and Magurran, 1994). In general, the KET induced alteration in locomotor activity is more sensitive in medaka larvae with acute KET exposure (within 24 h, as shown in Fig. 1) than those observed from our previous study with 10 day-development exposures in medaka embryos (Liao et al., 2015). In rodents, KET at clinical doses may cause neurotoxic effects on the developing brain by altering neurogenesis and then cause

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neurofunctional impairment and/or neuronal cell death during susceptible or sensitive developmental stages (Dong and Anand, 2013; Yan and Jiang, 2014). Few recent studies reported that high doses of KET dose-dependently altered behavior and motor neuron toxicity in wild type or transgenic zebrafish, possibly by affecting differentiating neurons (Zakhary et al., 2011; Kanungo et al., 2013). None of studies report the environmentally relevant concentrations of KET exposure on neurotoxicity in fish population and association with behavioral alteration. KET-altered AChE expression or neurotoxicity may be associated with disrupted larval locomotion, as the inhibited brain AChE activity by a neurotoxic pesticide endosulfan was associated with impaired swimming performance in adult zebrafish (Pereira et al., 2012). In this study, we found that low concentrations of KET significantly suppressed the mRNA expression of ache in larvae with 14-day exposure, but the AChE activity was elevated only at 0.004 mM with 14-day exposure to KET. Differential expression of AChE activity and mRNA expression after xenobiotic exposure was also reported in rat (Hammond et al., 1994), carp (Cyprinus carpio) (Xing et al., 2013) and zebrafish (Chen et al., 2012; Pereira et al., 2012; Richetti et al., 2011; Rico et al., 2007). These results may be ascribed to complex mechanisms such as differential responses from distinct organs/tissues or cell signaling pathways, different types of cholinesterases (AChE and butyrylcholinesterase) to hydrolyze acetylcholine, and/or posttranscriptional or posttranslational modulation of AChE (Ballesteros et al., 2009; Pezzementi et al., 2011; Rico et al., 2007; Xing et al., 2013). Xenobiotics cause oxidative stress and damage by producing ROS via CYP-mediated metabolism (Morita et al., 2013). Our previous study showed that KET (0.9e9000 mg/L) can induce SOD activity (converting internal O
2 to H2O2) and CAT activity (a H2O2 scavenger) in medaka larvae with 10 day-developmental exposures of medaka embryos, which indicates a turnover transformation of ROS species inside the treated fish (Liao et al., 2015). Our current study on KET-induced oxidative stress responses agreed well with our previous study (Liao et al., 2015), but with more sensitive and higher extent of responses. Similarly, KET treatments (200e800 mg/L, 20 min) induced an increase in SOD and CAT enzymatic activities along with an increase in GSSG levels during lix et al., 2016). developmental exposures to zebrafish embryos (Fe These changes in antioxidant activities indicated the status of an imbalance between pro- and antioxidant defense action (e.g., protective mechanisms) against chemical induced oxidative stress in lix the fish body with KET exposure as shown in the literature (Fe et al., 2016) and current study. Overload of exogenous chemicals can further result in excessive ROS accumulation due to depletion of antioxidants, which then cause persistently damage in lipids, protein and DNA and/or stimulate the tumor-promoting effect such as abnormal cell apoptosis (Kohen and Nyska, 2002). As well, the oxidative stress and misregulation of N-methyl-D-aspartate glutamate receptors are associated with KET-induced neuronal apoptosis (Bai et al., 2013; Zou et al., 2009), but the exact mechanism of KET-induced neurotoxicity or cell apoptosis remains unknown. Potential bioaccumulation of KET was reported in humans and other organisms due to its quick-absorbed and slow-excretion rates and ineffective metabolism (Cohen et al., 1973; Cuevas et al., 2013; Meyer and Maurer, 2011). We did not observe significal induction in CYPs-related gene expression with the KET exposure (Figs. S4 and SI), whereas the phase II metabolizing enzyme GST was induced in embryos (Fig. 3a) or hatchlings with 10-day KET or MET exposure (Liao et al., 2015). Similarly, adult rats treated with 10e80 mg/kg KET produced 18e25% increases in hepatic cytosolic GST activity (Chan et al., 2008). Several xenobiotics often induced the GST activity by an adaptive mechanism to slight oxidative stress (Hayes

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Fig. 4. (a) Transcriptional expression of ache, p53 and related genes (bcl-2, baxa, baxb, caspase-3a and p21) and (b) caspase-3 activity in medaka larvae with 14-day aqueous exposure from 0.004e4.0 mM KET solutions. Data are mean ± SD (3 fish were pooled as a subsample; n ¼ 4 for gene expression and n ¼ 8 for caspase activity). *p < 0.05 vs the control by the LSD test. #p < 0.05 vs the control by the t-test.

and McLellan, 1999). GST induction may be associated with glutathione-modulated antioxidant system and chemical induced carcinogenesis (Tew, 1994; Dong et al., 2010; Egaas et al., 1999; Li et al., 2010), as the GST family may regulate intracellular concentrations of the lipid peroxidation products (e.g., MDA) and are involved in the signaling mechanisms of apoptosis (Yang et al., 2001). Like KET in this study; for instance, subchronic exposure to carcinogenic propiconazole also induces ROS-related oxidative stress, GST induction and lipid peroxidation in fish (Tu et al., 2016). Furthermore, we demonstrated medaka larvae showed elevated mRNA level of p53 with 14-day exposure to KET (Fig. 4a). Tumor suppressor p53 plays an important role in inhibiting carcinogenesis and is a regulator of metabolic stress (Attardi and DePinho, 2004; Vousden and Lane, 2007; Vousden and Ryan, 2009). Activation of p53 induces a series of cellular pathways induced by different stresses, such as apoptosis, cell-cycle arrest, stress resistance, and senescence (Attardi and DePinho, 2004; Vousden and Lane, 2007). Mice lacking p53 showing increased ROS levels and deficit in antioxidant defense systems is related with increased susceptibility to cancer (Donehower et al., 1992; Sablina et al., 2005). Like mammals, p53 may also play a similar role in chemical induced tumorigenesis in fish (Chen et al., 2001). For instance, we

previously showed higher hepatic p53 level with exposure of medaka adults to the hepatotumorigenic triadimefon fungicide than non-tumorigenic myclobutanil (Lin et al., 2014c). Also, oxidative stress and hepatic tumorigenic effects induced by a carcinogenic propiconazole are associated with p53-mediated pathways in medaka fish (Tu et al., 2016). Previous studies of rodents and primates showed KET-induced neuroapoptosis in developing nervous systems such as brain and neuronal cells (Brambrink et al., 2012; Ikonomidou et al., 1999; Wang et al., 2006). KET induces apoptotic alterations via a baxmitochondria-caspase protease pathway in human HepG2 cells (Lee et al., 2009). Similarly, we further showed that transcriptional expression of apoptotic genes including baxa, bcl-2 and caspase-3a were significantly upregulated in 14 day-KET treated fish as compared with the control (Fig. 4a). The BAX can heterodimerize with BCL-2 or other BCL family members to determine cell survival or apoptosis (Reed, 1997). The Caspase family play important role on programmed cell death (apoptosis) and Caspase-3 is a frequently expressed and crucial mediator (Porter and J€ anicke, 1999). The up-regulation of both bcl-2 and bax genes accompanied by increased caspase-3a expression in medaka larvae after KET exposure (Fig. 4a) indicated ongoing cell apoptosis via the p53-

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related apoptotic pathway. We also showed a >1.5-fold increase of Caspase-3 activity in KET-treated fish at 4 mM with the 14 dayexposure (Fig. 4b). Similarly, up-regulation of apoptosis-related genes including caspase-8 and caspase-9 were reported in zebrafish embryos during early life stages of KET exposure, whereas caspase-like enzymatic activities of Caspase-3 and Caspase-9 were not affected by KET treatments (Felix et al., 2017c). As well, the p53-p21 pathway is involved in the cell cycle checkpoint control (Attardi and DePinho, 2004; Somasundaram and El-Deiry, 2000), and p21 plays a key role in mediating G1phase arrest. Down regulation of p21 implies a possible activation of the cell cycle (i.e., increasing cell proliferation) or compensation for the elevated apoptosis (Somasundaram and El-Deiry, 2000). Taken together, subchronic exposure to environmentally relevant concentrations (e.g., 0.004 mM) or greater levels of KET can induce high cell apoptosis but may simultaneously reduce cell-cycle arrest. 5. Conclusions We highlight ecologically important adverse effects of humanabused drugs (e.g., KET) that enter aquatic ecosystems and modify fish behavior and development at minute levels. We show potential ecological risk (e.g., RQ > 1) of directly releasing KETcontaining wastewater into the aquatic environment, based on the lowest observed effect dose of 950.8 ng L
1 from this study, which at concentrations encountered in effluent-influenced surface waters (e.g., highest MEC values of KET in river water and wastewater effluents, Tables S1 and SI). The biomarkers we applied with medaka fish may be suitable as new, rapid and cost-effective test protocols to examine the full environmental impact of neuroactive drugs in the aquatic ecosystem. Hospitals and patients around the world use similar controlled drugs and excrete them or their metabolites into river water that could be sources of drinking water for humans. This work reports the toxic mechanism of KET regarding behavior, oxidative stress, neurotoxicity and p53-regulated abnormal apoptosis in a lower vertebrate medaka fish via acute to subchronic exposures. For risk assessments of human health or ecological safety, data on comparing toxic effects of controlled or abused drugs between humans and non-targeted organism are important. Revealing the toxic action of abused drugs in lower vertebrates may help us to understand their toxic impact in humans. Acknowledgements This work was supported by the National Science Council, Taiwan (NSC 100-2621-M-002-025 and NSC 101-2621-M-002019). The authors thank Dr. Te-Hao Chen (an associate professor at the National Museum of Marine Biology and Aquarium, Taiwan) for assistance in the larval locomotion test. Appendix A. Supplementary data Details in experimental procedures including fish exposure, measurements of each toxicity endpoints and statistical analysis are provided in the supporting information. Table S1 shows the maximum environmental concentration of ketamine (KET) detected in river water and effluent of wastewater treatment plants. Table S2 lists primer sequences of assessed genes for real-time quantitative PCR. Fig. S1 shows original datasets for altered medaka larval locomotor activity (including parameters of percent time active, average and maximum velocity and absolute and relative turn angles) at 1-hr and 24-hr KET exposure. Fig. S2 shows glutathione reductase (GR) activity in medaka larvae during 14-day KET exposure. Fig. S3 and Fig. S4 show gene expression of

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