response to request for data and information on zebrafish … · 2020-05-04 · kanungo,...

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From: Kanungo, Jyotshnabala (FDA/NCTR) To: NIEHS NICEATM Subject: FW: NTP News: Request for Data and Information; NTP Update November 2016 now available Date: Friday, November 04, 2016 12:23:28 PM Attachments: Guo_et_al-2016-Journal_of_Applied_Toxicology.pdf

JAT 2013.pdf

Dear Sir/Madam:

In response to the email below, I am attaching two manuscripts. Please let me know if I need to provide any additional documents.

Thank you.

Sincerely,

Josna

Jyotshna Kanungo, Ph.D. Senior Investigator Lead Scientist, Zebrafish HTS Laboratory Division of Neurotoxicology National Center for Toxicological Research, FDA 3900 NCTR Road Bldg 53D, Rm 203O Jefferson, AR 72079

Tel: 870-543-7591 Fax: 870-543-7745 Email: [email protected]

"The contents of this message are mine and do not reflect any position of the U.S. government or Food & Drug Administration."

From: National Toxicology Program [mailto:[email protected]] On Behalf Of NTP News Sent: Friday, November 04, 2016 10:55 AM To: [email protected] Subject: NTP News: Request for Data and Information; NTP Update November 2016 now available

NTP News - November 4, 2016 In this email:

Request for Data and Information on Zebrafish Embryo Chemical Screening

NTP Update November 2016 now available
mailto:/O=NIH/OU=NIHEXCHANGE/CN=RECIPIENTS/CN=JKANUNGOmailto:[email protected]:[email protected]

Research article

Received: 22 February 2016, Revised: 31 March 2016, Accepted: 31 March 2016 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jat.3340

Acetyl L-carnitine targets adenosinetriphosphate synthase in protecting zebrafishembryos from toxicities induced by verapamiland ketamine: An in vivo assessment

Xiaoqing Guoa,b, Melanie Dumasa, Bonnie L. Robinsona, Syed F. Alia,Merle G. Paulea, Qiang Gua and Jyotshna Kanungoa*

ABSTRACT: Verapamil is a Ca2+ channel blocker and is highly prescribed as an anti-anginal, antiarrhythmic and antihyper-tensive drug. Ketamine, an antagonist of the Ca2+-permeable N-methyl-D-aspartate-type glutamate receptors, is a pediatricanesthetic. Previously we have shown that acetyl L-carnitine (ALCAR) reverses ketamine-induced attenuation of heart rateand neurotoxicity in zebrafish embryos. Here, we used 48 h post-fertilization zebrafish embryos that were exposed to rele-vant drugs for 2 or 4 h. Heart beat and overall development were monitored in vivo. In 48 h post-fertilization embryos, 2mMketamine reduced heart rate in a 2 or 4 h exposure and 0.5mM ALCAR neutralized this effect. ALCAR could reverseketamine’s effect, possibly through a compensatory mechanism involving extracellular Ca2+ entry through L-type Ca2+

channels that ALCAR is known to activate. Hence, we used verapamil to block the L-type Ca2+ channels. Verapamil was morepotent in attenuating heart rate and inducing morphological defects in the embryos compared to ketamine at specific timesof exposure. ALCAR reversed cardiotoxicity and developmental toxicity in the embryos exposed to verapamil or verapamilplus ketamine, even in the presence of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester, an inhibitor of intracellularCa2+ release suggesting that ALCAR acts via effectors downstream of Ca2+. In fact, ALCAR’s protective effect was blunted byoligomycin A, an inhibitor of adenosine triphosphate synthase that acts downstream of Ca2+ during adenosine triphosphategeneration. We have identified, for the first time, using in vivo studies, a downstream effector of ALCAR that is critical in ab-rogating ketamine- and verapamil-induced developmental toxicities. Published 2016. This article is a U.S. Government workand is in the public domain in the USA.

Keywords: verapamil; heart rate; ketamine; zebrafish; acetyl L-carnitine; developmental toxicity

*Correspondence to: Jyotshna Kanungo, Division of Neurotoxicology, NationalCenter for Toxicological Research, US Food and Drug Administration, 3900 NCTRRoad, Jefferson, AR 72079, USA.E-mail: [email protected]

aDivision of Neurotoxicology, National Center for Toxicological, Research, U.S. Foodand Drug Administration, 3900 NCTR Road, Jefferson, AR 72079, USA

bDivision of Genetic and Molecular Toxicology, National Center for ToxicologicalResearch, U.S. Food and Drug Administration, 3900 NCTR Road, Jefferson, AR72079, USA

IntroductionVerapamil belongs to the phenylalkylamine class of L-type Ca2+

channel blockers and is used to treat cardiac arrhythmias,hypertension and angina (Prisant, 2001). Overdoses of Ca2+

channel blockers cause rapid development of hypotension,bradydysrhythmia and cardiac arrest (Derlet and Horowitz,1995). Ketamine, a pediatric anesthetic, is an antagonist of N-methyl-D-aspartate (NMDA)-type glutamate receptors (Kohrsand Durieux, 1998). Ketamine is listed as a schedule III con-trolled substance in the USA, due to its potential for abuse (Mor-gan et al., 2010; Rowland, 2005). A number of studies on rodentsand non-human primates have shown that treatment with highdoses of ketamine or exposure for long durations during sus-ceptible periods of development (the brain growth spurt) caninduce abnormally high levels of neuroapoptosis (Habernyet al., 2002; Ikonomidou et al., 1999; Lahti et al., 2001; Larsenet al., 1998; Malhotra et al., 1997; Maxwell et al., 2006; Slikkeret al., 2007; Wang et al., 2006). Studies in humans and animalshave shown that ketamine has a direct negative (Aya et al.,1997; Graf et al., 1995; Hara et al., 1994; Yeh et al., 1994) or an in-direct positive (Cook et al., 1991; Endou et al., 1992; Riou et al.,1990) inotropic effect on myocardial contractility. Ketamine re-duces heart rate in rats (Zausig et al., 2009) and guinea pigs(Kim et al., 2006) and acts as a cardiac depressant in dogs after

J. Appl. Toxicol. 2016 Published 2016. This article is a U.S. Government

pharmacological blockade of the autonomic nervous system(Pagel et al., 1992). In rhesus monkeys, extended intravenousinfusions of ketamine result in decreased heart rates in preg-nant females and in postnatal day 5 and 35 infants (Hotchkisset al., 2007). However, the mechanism of how ketamine impactscardiac function is not clear.Several drugs that either directly or indirectly inhibit mito-

chondrial fatty acid oxidation (e.g., etomoxir, oxfenicine,dichloroacetate, trimetazidine, ranolazine, pyruvate, malonyl-coenzyme A decarboxylase inhibitors), improve cardiac functionin experimental models and humans with cardiac ischemia andfailure (Lopaschuk, 2002; Stanley, 2005). Acetyl L-carnitine (ALCAR)is an L-lysine derivative and its main role lies in the transport of

work and is in the public domain in the USA.

X. Guo et al.

long chain fatty acids into mitochondria to enter the β-oxidationcycle (Bohles et al., 1994). ALCAR preserves the mechanical func-tion of ischemic swine hearts perfused in the presence of free fattyacids (Liedtke and Nellis, 1979). Moreover, ALCAR has been shownto ameliorate ketamine-induced behavioral alterations and bodyweight deficits in rats (Boctor et al., 2008), in addition to havingneuroprotective effects in mammals (Zou et al., 2008), and bothneuroprotective and cardioprotective effects in zebrafish (Cuevaset al., 2013; Kanungo et al., 2012).

The zebrafish (Danio rerio) embryo has become an importantexperimental model for studying various diseases, protein func-tions, drug toxicity and various physiological responses(Kanungo et al., 2014; Vogel, 2000). Accordingly, the therapeuticand toxic responses for both cardiac and non-cardiac drugs canbe assessed by simply immersing the embryos in the water withthe test drug (Kanungo et al., 2012; Langheinrich et al., 2003;Milan et al., 2003). We have previously shown that consistentwith its effects on mammals (Hotchkiss et al., 2007; Irifuneet al., 1991, 1997; Kim et al., 2006; Zausig et al., 2009), inzebrafish embryos, ketamine modulates the serotonergic anddopamine systems (Robinson et al., 2015, 2016) and heart rate(Kanungo et al., 2012). Based on reported observations that ve-rapamil affects cardiac function in mammals including humans(Derlet and Horowitz, 1995; Prisant, 2001), the goal of this studywas to test whether verapamil has similar effects and ketamineaccentuates verapamil’s effects in zebrafish, an emerging alter-nate vertebrate animal model for drug screening and drugsafety assessments. Here, we have undertaken in vivomechanis-tic studies to demonstrate how ALCAR reverses ketamine- andverapamil-induced cardiac and developmental toxicities.

Materials and methods

Animals (zebrafish)

Adult wild-type zebrafish (D. rerio, AB strain) were obtained fromthe Zebrafish International Resource Center (www.zirc.org)(Eugene, OR, USA). The fish were kept in fish tanks (AquaticHabitats, FL, USA) at the NCTR/FDA zebrafish facility containingbuffered water (pH 7.5) at 28 °C, and were fed daily live brineshrimp and Zeigler dried flake food (Zeiglers, Gardeners, PA,USA). Each 3 liter tank housed eight adult males or eight females.Handling and maintenance of zebrafish complied with the NIHGuide for the Care and Use of Laboratory Animals and were ap-proved by the NCTR/FDA IACUC. The day–night cycle was main-tained at 14: 10 h. For in-system breeding, crosses of males andfemales were set up the previous day with partitions that weretaken off the following morning at the time of light onset at07.30h to stimulate spawning and fertilization. Fertilized zebrafisheggs were collected from the bottom of the tank as soon as theywere laid. The eggs were placed in Petri dishes and washedthoroughly with buffered egg water (reverse osmosis water con-taining 60 mg sea salt [Crystal Sea®; Aquatic Eco-systems, Inc.,Apopka, FL, USA] per liter of water [pH 7.5]) and then allowed todevelop in an incubator at 28.5 °C for later use.

Reagents

Ketamine hydrochloride was purchased from Vedco, Inc. (St.Joseph, MO, USA). ALCAR, oligomycin A (oligomycin) andTMB-8 (3,4,5-trimethoxybenzoic acid 8-(diethylamino)octylester) were purchased from Sigma (St. Louis, MO, USA).

Published 2016. This aand is in the

wileyonlinelibrary.com/journal/jat

Verapamil hydrochloride was purchased from TOCRIS Biosci-ences (Ellisville, MO, USA). All other reagents including dinitro-phenol used in this study were purchased from Sigma unlessmentioned otherwise. Verapamil (0.05 M) and ALCAR stock(1 M) solutions were made fresh with buffered egg water.Stocks of oligomycin A (10mM), TMB-8 (0.5 M) and dinitrophe-nol (1 M) were prepared using dimethyl sulfoxide as the solvent.

Treatment of zebrafish embryos with verapamil, ketamine,acetyl L-carnitine and 3,4,5-trimethoxybenzoic acid8-(diethylamino)octyl ester

For treatment with verapamil, ketamine, ALCAR, TMB-8 andoligomycin A, 48 h post-fertilization (hpf ) manually dechorionatedembryos were used. For every experiment, in each treatment, 10embryos were placed in the six-well plates (Nunc non-treatedplates) with 5ml buffered eggwater. Ketamine (2.0mM), verapamil(10 μM–1 mM), ALCAR (0.5mM), TMB-8 (0.25–0.5mM) andoligomycin A (0.25–5.0 μM) treatments, alone or in combinationcontinued for 2 or 4 h (static exposures). Control groups treatedwith equivalent amount of vehicle (dimethyl sulfoxide) were exam-ined in parallel. As we previously reported, ALCAR at 0.5mM didnot have any effect on heart rate at 2 and 4h of exposure. In theearlier report we also showed that 0.1mM ALCAR did not affectthe ketamine-induced decrease in heart rate, doses of 0.5 and1.0mM ALCAR were equally effective in completely inhibiting thateffect (Kanungo et al., 2012). Based on these data we chose to use0.5mM ALCAR for all our experiments in the present study. For theketamine dose, we have earlier determined that 2mM ketamineexposure results in an internal exposure of the anesthetic level ofplasma concentrations in humans (Robinson et al., 2015; Trickleret al., 2014). For co-exposure to various drug combinations, thedrugs were added together at the same time. For acute toxicityassessments, drug exposure continued for 2 or 4 h.

Embryo imaging, scoring heart rate and measurement of thepericardial sac area

Live images of the embryos in the six-well plates were acquiredusing an Olympus SZX 16 binocular microscope and DP72 cameraand heart rate of each embryo was measured by visually countingventricular beats per minute on the live video using a timer (stopwatch). The data are presented by converting heart beats perminute to percentage of control (100%). Data for each group(n=10) were used to calculate the mean and standard deviation(SD). The extent of pericardial edema in the embryos was deter-mined by quantitation of the pericardial sac area (Carney et al.,2004). The pericardial sac area was outlined in lateral view imagesof the embryos and the area was quantitated using the DP2 BSWmicroscope digital camera software (Olympus, Tokyo, Japan). Thestatistical significance of the effects of the various treatments onheart rate and pericardial edema was determined by one-wayANOVA (Sigma Stat) using Holm–Sidak pairwise multiple compar-ison post-hoc analysis.

ResultsTo assess the effects of verapamil on heart rate, we usedzebrafish embryos at 48 hpf (Fig. 1). In these embryos, treat-ment with various doses of verapamil altered heart rate dose-dependently in 2 and 4 h post-treatment (Fig. 1). Doses up to20 μM did not have any significant effect, whereas doses 50 μM

J. Appl. Toxicol. 2016rticle is a U.S. Government workpublic domain in the USA.

http://www.zirc.org

Figure 1. Effect of verapamil on heart rate in zebrafish embryos. Embryos(48 hpf ) were exposed to various doses of verapamil for 2 or 4 h. Eachgroup consisted of 10 embryos and the experiment was repeated at leastthree times. Heart rate was measured in beats per minute and the datafrom all the embryos in a group were averaged and shown as mean ± SD.One-way ANOVAwas used to compare the heart rates between the controland the treated groups. Significance (*) was set at P< 0.05.

Figure 2. Effects of verapamil and ALCAR on heart rate in zebrafish em-bryos. Embryos (48 hpf ) were exposed to various doses of verapamil aloneor with 0.5mM ALCAR. Each exposure (static exposure) group consisted of10 embryos and the experiment was repeated at least three times. Heartrate was measured in beats per minute and the data from all the embryosin a group were averaged and shown as mean ± SD. One-way ANOVA wasused to compare the heart rates between the control and the treatedgroups. Significance (*) was set at P< 0.05. Heart rates are shown for 2and 4 h exposures. ALCAR, acetyl L-carnitine.

Figure 3. Effects of verapamil and ketamine in the presence or absenceof ALCAR on heart rate in zebrafish embryos. Embryos (48 hpf ) were ex-posed to various doses of verapamil alone or with 2mM ketamine and inthe presence of 0.5mM ALCAR. Each exposure group consisted of 10 em-bryos and the experiment was repeated at least three times. Heart rate, at2 and 4 h post-exposure, was measured in beats per minute and the datafrom all the embryos in a group were averaged and shown as mean ± SD.One-way ANOVA was used to compare the heart rates between the controland the treated groups. Significance (*) was set at P< 0.05. ALCAR, acetyl L-carnitine.

ALCAR prevents toxicities induced by ketamine and verapamil

and above significantly reduced heart rate. Ketamine decreasesCa2+ release from intracellular stores of rat ventricular myocytes(Kanaya et al., 1998) and its cardiac depressive effects could becaused by interference with Ca2+ influx (Endou et al., 1992;Kunst et al., 1999). L-carnitine, a naturally occurring amino acidthat induces fatty acid oxidation, neutralizes ketamine-inducedtoxicities, such as neuronal apoptosis in the developing ratfrontal cortex (Zou et al., 2008). In our previous studies, wehad shown that ALCAR counteracted ketamine-induced attenu-ation of heart rate, implying that ALCAR potentially acted torestore the intracellular Ca2+ levels that could have beenreduced due to NMDA receptor blockade by ketamine. Here, wehypothesized that were it a plausible scenario, ALCAR would alsocounteract verapamil (an L-type Ca2+ channel inhibitor)-inducedcardiotoxicity. We chose to use a verapamil dose, 150 μM, whichcould cause ~50% reduction in heart rate in 2 h of exposure. At thisdose, as expected, verapamil reduced heart rate at 2 and 4h(Fig. 2). ALCAR reversed the effects of not only 150 μM verapamilbut also of a higher dose of verapamil (500 μM) (Fig. 2).

It has been reported that pretreatment of surgical patients withverapamil may reduce the dose of ketamine required for anesthe-sia due to increased duration of sleeping time and can potentiatethe effects of some central nervous system drugs such as ketaminein mice. The latter effect has been attributed to blocked neuronalCa2+ channels although ketamine does not interact with verapamilbinding site on the L-type Ca2+ channels. We tested whetherzebrafish embryos also reproduce similar effects observed inmammals. In our experiments, we used an anesthetic dose of keta-mine (2mM in water) and co-treated 48 hpf embryos with increas-ing doses of verapamil (50 μM–1.0mM). The results showed thatverapamil significantly potentiated ketamine’s effects on theattenuating heart rate (Fig. 3). These results indicated the effectsof ketamine and verapamil were additive on the heart rate dueto blockade of Ca2+ entry through NMDA receptors and the L-typeCa2+ channels by the drugs, respectively.

To examine whether in the presence of ALCAR (0.5mM), theeffects of ketamine plus verapamil on the heart rate could becompletely reversed, as ALCAR could reverse the effects of

J. Appl. Toxicol. 2016 Published 2016. This article is aand is in the public dom

ketamine and verapamil alone, we treated the embryos with allthree drugs. The results showed that ALCAR indeed was effectivein restoring the attenuated heart rate induced by ketamine andverapamil together to the control level (Fig. 3). These results fur-ther suggested that ALCAR could potentially induce intracellularCa2+ release to compensate for the blockade of Ca2+ entry throughthe NMDA receptors and the L-type Ca2+ channels by ketamineand verapamil, respectively and thus could circumvent the dropin heart rate due to Ca2+ deprivation.

U.S. Government workain in the USA.


X. Guo et al.

In a previous study, we showed that ketamine (2mM with20 h exposure) acted as a developmental toxicant in zebrafish.However, when treated for 2–4h, ketamine’s developmentaltoxicity at the 2mM dose was not apparent compared to control(Fig. 4A,B), although contrary to controls (Fig. 5A), the pericardial

Figure 4. Morphology of 48 hpf zebrafish embryos treatedwith verapamil or kof embryos show control (A), and groups treated with 2mM ketamine (B), 1 mM v200 μM verapamil (E), 2mM ketamine with 1 mM verapamil (F), 1 mM verapamil wALCAR (H). Curved tails are indicated (*). ALCAR, acetyl L-carnitine; Ket, ketamin



edema was evident in the ketamine-treated embryos (Fig. 4B).With 1 mM verapamil, a number of embryos showed specificdeformities, particularly curved tails (41.2 ± 3.3%) (Fig. 4C)and severe pericardial edema (Fig. 5C) and within minutes,their hearts would stop beating, subsequently leading to

etamine or both, in the presence or absence of ALCAR for 2 h. Group pictureserapamil (C), 2mM ketaminewith 100 μM verapamil (D), 2mM ketamine withith 0.5mM ALCAR (G) and 2mM ketamine with 1 mM verapamil and 0.5mMe; Ver, verapamil.


Figure 5. Morphology showing pericardial area of 48 hpf zebrafish embryos treated with verapamil or ketamine or both in the presence or absence ofALCAR for 2 h. Control (A) and embryos treated with 2mM ketamine (B), 1 mM verapamil (C), 2mM ketamine with 1 mM verapamil (D) and 2mM ketaminewith 1 mM verapamil and 0.5mM ALCAR (E). Arrows indicate the pericardium. Pericardial area is indicated by dotted lines and the pericardial area measure-ments are shown (F). Values are averaged and shown as mean ± SD. One-way ANOVA was used to compare the pericardial areas between the control andthe treated groups. Significance (*) was set at P< 0.05. Bar =125 μM. ALCAR, acetyl L-carnitine.


death (data not shown). However, embryos treated with ve-rapamil (0.1 or 0.2mM) and 2mM ketamine did not exhibitthe curved tail phenotype (Fig. 4D,E) indicating that the effectwas specific to the 1 mM verapamil dose. Embryos treatedwith 2mM ketamine and 1 mM verapamil were more adverselyaffected (Figs. 4F and 5D) than those treated with 1 mMverapamil alone (Figs. 4C and 5C) and would eventually diefaster than the latter (data not shown). A number of theseembryos also had curved tails (41.5 ± 3.4%) (Fig. 4F) similarto the group treated with 1 mM verapamil alone (Fig. 4C).Embryos treated with 2mM ketamine do not die even whenthe static exposure continues for a few days (data not shown).However, in the presence of 0.5mM ALCAR, none of the ef-fects of verapamil alone (Fig. 4G) or with ketamine (Figs. 4Hand 5E) were observed indicating that ALCAR reversed thetoxic effects of ketamine and verapamil as it did for the heartrate. The extent of the edematous pericardia is quantitativelypresented showing the adverse effects of ketamine andverapamil alone and in combination (Fig. 5F).

As ALCAR prevented ketamine- and verapamil-inducedcardiotoxicity, we hypothesized that intracellular Ca2+ releasecould compensate for the lack of extracellular Ca2+ via NMDA


receptors (by ketamine) and L-type Ca2+ channels (by verapamil).Therefore, we used the intracellular Ca2+ release inhibitor TMB-8to block such a compensatory potential. TMB-8 modulates cellularCa2+ metabolism by preventing intracellular Ca2+ mobilization viainhibition of the inositol 1,4,5-trisphosphate-induced Ca2+ release(Nishizuka, 1988). In zebrafish embryos, TMB-8 dose-dependentlyreduced heart rate (data not shown) and a higher dose (0.5mM)completely caused the heart to stop beating in 2 h of co-exposurewith either ketamine or verapamil alone or in combination (Fig. 6).However, in the presence of ALCAR, the adverse effect of TMB-8was completely obliterated. These data suggested that ALCARfunction is potentially carried out by an effector moleculedownstream of Ca2+.With the reversal of heart rate to control levels by ALCAR even in

the presence of ketamine, verapamil and TMB-8, the latter threeinhibiting intracellular Ca2+, we redirected our focus to selectivetargets downstream of Ca2+. One of the major downstream effec-tors of Ca2+ is adenosine triphosphate (ATP) synthase. The additionof oligomycin A (an inhibitor of the mitochondrial F-ATP synthase)to embryos alongwith ALCAR, ketamine and verapamil obliteratedthe protective effects of ALCAR on ketamine- and verapamil-induced cardiotoxicity (Fig. 7). Doses of oligomycin less than



Figure 6. TMB-8, an inhibitor of intracellular Ca2+ release does not inter-rupt ALCAR’s protection from ketamine- and verapamil-inducedcardiotoxicity. Embryos (48 hpf ) were exposed for 2 h with 1 mM verapamilalone or with 2mM ketamine and in the presence of 0.5mM ALCAR andTMB-8 (either 0.25 or 0.5mM). Each exposure group consisted of 10 em-bryos and the experiment was repeated three times. Post-exposure, heartrate was measured in beats per minute and the data from all the embryosin a group were averaged and shown as mean ± SD. One-way ANOVA wasused to compare the heart rates between the control and the treatedgroups. Significance (*) was set at P< 0.05. ALCAR, acetyl L-carnitine;TMB-8, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester.

Figure 7. Oligomycin disrupts ALCAR’s protective effects on ketamine-and verapamil- induced cardiotoxicity. Embryos (48 hpf ) were exposedfor 2 h with 1 mM verapamil alone or with 2mM ketamine and in the pres-ence of 0.5mM ALCAR, and oligomycin (5 μM). Each exposure groupconsisted of 10 embryos and the experiment was repeated three times.Post-exposure, heart rate was measured in beats per minute and the datafrom all the embryos in a group were averaged and shown as mean ± SD.One-way ANOVAwas used to compare the heart rates between the controland the treated groups. Significance (*) was set at P< 0.05. ALCAR, acetylL-carnitine.

X. Guo et al.

0.5mM were ineffective in interfering with ALCAR’s beneficialeffects (data not shown). These results indicated that ALCAR’sfunction is not dependent on Ca2+ but is mediated by the latter’sdownstream target, ATP synthase.

DiscussionOur previous study has shown that ALCAR reverses ketamine-induced attenuated heart rate in zebrafish embryos and we



hypothesized that ALCAR, due to its ability to upregulate L-typeCa2+ channels (Tewari et al., 1995), could compensate forketamine-induced blockade of Ca2+-permeable NMDA recep-tors (Kanungo et al., 2012) (Fig. 8). It has been shown that keta-mine decreases Ca2+ release from intracellular stores of ratventricular myocytes (Kanaya et al., 1998). There are also reportsthat the cardiac depressive effects of ketamine could be causedby interference with Ca2+ influx (Endou et al., 1992; Kunst et al.,1999). It has also been suggested that pretreatment of surgicalpatients with verapamil may reduce the dose of ketamine re-quired for anesthesia due to increased duration of sleeping time(Saha et al., 1990) and can potentiate the effects of some centralnervous system drugs such as ketamine in mice (Shen andSangiah, 1995). The latter effect has been attributed to blockedneuronal Ca2+ channels although ketamine does not interactwith the verapamil binding site on the L-type Ca2+ channels(Hirota and Lambert, 1996). In the current study, we provide ev-idence that ALCAR can reverse verapamil-inducedcardiotoxicity caused by verapamil treatment alone or with ke-tamine. Verapamil is an L-type Ca2+ channel blocker (Prisant,2001), and we show that verapamil dose-dependently attenu-ated the heart rate in zebrafish embryos. At high dose, it in-duced severe developmental anomalies and cardiotoxicity.With these results, what remained unanswered however, iswhether in the presence of all three compounds, verapamil, ke-tamine and ALCAR, normalization of overall development andheart rate occurs or not. To answer this question, we followeda completely non-invasive in vivo approach. Instead of directlymonitoring Ca2+ in the heart by imaging, which would need mi-croinjection of fluorescent Ca2+ trackers, and therefore, may in-terfere with survival rate and normal physiology in addition tobeing exposed to the drugs of interest, we chose to use variousinhibitors of Ca2+ and monitored the downstream physiologicaleffect on the heart rate. The zebrafish embryo is an intact organ-ism in which the heart beat is observed in its native environ-ment (water) rather than in isolation in a culture medium (Kimet al., 2006), an advantage that can be utilized to test for safetyand efficacy of drugs in vivo.

The effect of ketamine, an NMDA receptor antagonist, onheart rate has been reported for a variety of mammals, suchas rats (Zausig et al., 2009), rabbits (Yeh et al., 1994) and guineapigs (Kim et al., 2006). The zebrafish embryo response to keta-mine with regard to heart rate is also concordant with that ofhuman infants (Saarenmaa et al., 2001) and non-human primateinfants at postnatal days 5 and 35 (Hotchkiss et al., 2007).Zebrafish embryos can accurately model drug effects onmammals and humans (Kanungo et al., 2014), and our earlierstudies have shown that in 48 hpf embryos, a 2 h exposure toketamine was effective in significantly lowering the heart rate(Kanungo et al., 2012).

In humans, neonatal myocardium appears more dependent onthe entry of extracellular Ca2+ rather than the release of Ca2+ fromintracellular sarcoplasmic reticulum (Kudoh et al., 2003). Ketaminedecreases Ca2+ release from intracellular stores of rat ventricularmyocytes (Kanaya et al., 1998). There are also reports that thecardiac depressive effects of ketamine could be caused by interfer-ence with Ca2+ influx (Endou et al., 1992; Kunst et al., 1999). Amongthe compounds that appear to have a neutralizing effect onketamine-induced toxicities, such as neuronal apoptosis in thedeveloping rat frontal cortex, is L-carnitine (Zou et al., 2008), anaturally occurring amino acid that induces fatty acid oxidation.The mechanism underlying the effect of L-carnitine to inhibit such


Figure 8. Schematic presentation of a potential mechanism of ALCAR’s reversal of ketamine- and verapamil-induced cardiotoxicity. NMDA receptors areCa2+ permeable. Ketamine as an antagonist of NMDA receptors can inhibit Ca2+ entry into the cells. In the absence of intracellular Ca2+, the mitochondrialATP synthase activity is inhibited that could result in blunted ATP generation. ALCAR potentially bypasses the need for Ca2+ to induce ATP synthase activityand instead directly induces ATP synthase activity. ALCAR, acetyl L-carnitine; ATP, adenosine triphosphate; NMDAR, N-methyl-D-aspartate receptor.


neurotoxicity is not clear. On the other hand, ketamine is known toinduce blockade of lipid oxidation in rat heart (Ovchinnikov et al.,1989). Here, we show that ALCAR not only neutralized theketamine-induced decrease in heart rate as we reported in ourearlier study (Kanungo et al., 2012), but also reversed verapamil’sadverse effects on the heart rate in the presence or absence of ke-tamine, suggesting that ALCAR may have altered cellular Ca2+

transport or intracellular Ca2+ release or both. Carnitine is knownto be an endogenous promoter of Ca2+ efflux from rat heart mito-chondria (Baydoun et al., 1988) and acyl-carnitines increase Ca2+

concentrations in the cytoplasm of rat ventricular cardiomyocytes(Berezhnov et al., 2008). In patients withmild diastolic heart failure,ALCAR improves their symptoms (Serati et al., 2010). Differentialeffects of ketamine and ALCAR have been reported, which showthat ketamine induces a blockade of lipid oxidation and a decreasein ATP content in rat heart, whereas ALCAR facilitates fatty acid ox-idation to support ATP production (Hagen et al., 2002). Althoughour study shows the differential effects of ketamine (with or with-out verapamil) and ALCAR on the zebrafish heart rate, directevidence on whether such counteracting effects are exclusivelymediated by ATP, remains to be uncovered. Our studies showedthat ALCAR completely reverses the adverse effects on heart rate(even lethality) induced by 0.5mM dinitrophenol alone or withketamine co-treatment (data not shown). These findings suggestthat ALCAR counteracts acute cardiotoxicity in the zebrafishembryos by replenishing ATP in a way that is not dinitrophenol-sensitive. This would mean that ATP production might besustained via beta-oxidation of fatty acids instead of the oxidativephosphorylation. ALCAR can induce fatty acid metabolism viabeta-oxidation (Hagen et al., 2002).

In the current study, ALCAR also reversed the reduced heartrate induced by verapamil, an L-type Ca2+ channel blocker, inthe zebrafish embryos even in the presence of ketamine, indi-cating that ALCAR may be regulating heart rate by modulatingintracellular Ca2+ levels. In our in vivo approach to modulatethe availability of cellular Ca2+ to determine how ketamine, ve-rapamil and ALCAR modulate heart rate, we used the intracel-lular Ca2+ inhibitor, TMB-8. TMB-8 acts as an intracellular Ca2+

antagonist. In mammalian skeletal or smooth muscle, TMB-8,an amphiphilic ester, acts to inhibit intracellular Ca2+ availabil-ity (Chiou and Malagodi, 1975; Malagodi and Chiou, 1974a,b).TMB-8 enhanced ketamine inhibition of inositol 1,4,5-trisphosphate production in rat heart myocytes (Kudoh et al.,2002). Our study showed that just like verapamil, TMB-8 alone


could drastically reduce heart rate followed by lethality (datanot shown) and its effects were exacerbated in the presenceof ketamine; however, it was ineffective in altering heart rateor overall development in the presence of ALCAR alone or incombination with ketamine and verapamil, suggesting thatreversal of this acute toxicity depended on targets actingdownstream of Ca2+.It has been reported that verapamil has the potential to change

myocardial Ca2+ sensitivity in zebrafish and the mechanism bywhich this occurs may involve myofilament Ca2+ sensitivity ofthe heart muscle (Haustein et al., 2015). A potential mechanismfor the effects of ketamine and ALCAR on mitogen-activatedprotein kinase (MAPK)/extracellular signal-regulated kinase (ERK)linked to Ca2+ in zebrafish was discussed in our previous study(Kanungo et al., 2012). Involvement of MAPK/ERK pathway inketamine-induced neurotoxicity in mice has been reported(Straiko et al., 2009). However, a distinct effector molecule down-stream of the MAPK/ERK signaling pathway, has not beenidentified that can explain ketamine-induced toxicity, in whichcase intervention at the MAPK/ERK level might prove deleteriousas this signaling pathway is ubiquitous and critical for overallcellular physiology.We were focused on identifying the effector(s) downstream

of Ca2+ signaling that could be activated by ALCAR. In muscle-derived cell cultures from patients with collagen type VImyopathies, addition of oligomycin results in mitochondrial de-polarization (Angelin et al., 2007). In the present investigation,we show that oligomycin blunted ALCAR’s beneficial effectson toxicities induced by either ketamine or verapamil or TMB-8 alone or all in combination. Oligomycin A, an inhibitor of themitochondrial F-ATP synthase, blocks ATP synthesis in the mito-chondria. Our results thus point to a direct role of ALCAR-mediated ATP synthase activity in protecting zebrafish embryosfrom toxicities induced by ketamine, verapamil and TMB-8,alone or in combination (Fig. 8). Our future studies would focuson how ALCAR affects ATP synthase activity, whether at thetranscriptional or post-translational levels or by upregulatingthe expression of any inhibitor(s) of ATPase. Nonetheless, forthe first time, we have identified a molecule that is responsiblefor imparting ALCAR’s protective effects on ketamine- andverapamil-induced toxicities. These results provide mechanisticdata with an insight on how these drugs might work in humansand how their reported toxicities could be ameliorated with po-tential intervention.



X. Guo et al.

Conflict of interestThe authors did not report any conflict of interest.

ReferencesAngelin A, Tiepolo T, Sabatelli P, Grumati P, Bergamin N, Golfieri C,

Mattioli E, Gualandi F, Ferlini A, Merlini L, Maraldi NM, Bonaldo P,Bernardi P. 2007. Mitochondrial dysfunction in the pathogenesis ofUllrich congenital muscular dystrophy and prospective therapy withcyclosporins. Proc. Natl. Acad. Sci. U. S. A. 104: 991–996.

Aya AG, Robert E, Bruelle P, Lefrant JY, Juan JM, Peray P, Eledjam JJ,de La Coussaye JE. 1997. Effects of ketamine on ventricular con-duction, refractoriness, and wavelength: potential antiarrhythmiceffects: a high-resolution epicardial mapping in rabbit hearts. An-esthesiology 87: 1417–1427.

Baydoun AR, Markham A, Morgan RM, Sweetman AJ. 1988. Palmitoylcarnitine: an endogenous promotor of calcium efflux from rat heartmitochondria. Biochem. Pharmacol. 37: 3103–3107.

Berezhnov AV, Fedotova EI, Nenov MN, Kokoz Iu M, Zinchenko VP,Dynnik VV. 2008. Destabilization of the cytosolic calcium level andcardiomyocyte death in the presence of long-chain fatty acid deriv-atives. Biofizika 53: 1025–1032.

Boctor SY, Wang C, Ferguson SA. 2008. Neonatal PCP is more potent thanketamine at modifying preweaning behaviors of Sprague–Dawley rats.Toxicol. Sci. 106: 172–179.

Bohles H, Evangeliou A, Bervoets K, Eckert I, Sewell A. 1994. Carnitine estersin metabolic disease. Eur. J. Pediatr. 153: S57–S61.

Carney SA, Peterson RE, Heideman W. 2004. 2,3,7,8-Tetrachlorodibenzo-p-dioxin activation of the aryl hydrocarbon receptor/aryl hydrocar-bon receptor nuclear translocator pathway causes developmentaltoxicity through a CYP1A-independent mechanism in zebrafish.Mol. Pharmacol. 66: 512–521.

Chiou CY, Malagodi MH. 1975. Studies on themechanism of action of a newCa2+ antagonist, 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoatehydrochloride in smooth and skeletal muscles. Br. J. Pharmacol. 53:279–285.

Cook DJ, Carton EG, Housmans PR. 1991. Mechanism of the positive inotro-pic effect of ketamine in isolated ferret ventricular papillary muscle.Anesthesiology 74: 880–888.

Cuevas E, Trickler WJ, Guo X, Ali SF, Paule MG, Kanungo J. 2013. AcetylL-carnitine protects motor neurons and Rohon-Beard sensory neu-rons against ketamine-induced neurotoxicity in zebrafish embryos.Neurotoxicol. Teratol. 39: 69–76.

Derlet RW, Horowitz BZ. 1995. Cardiotoxic drugs. Emerg. Med. Clin. NorthAm. 13: 771–791.

Endou M, Hattori Y, Nakaya H, Gotoh Y, Kanno M. 1992. Electrophysiologicmechanisms responsible for inotropic responses to ketamine in guineapig and rat myocardium. Anesthesiology 76: 409–418.

Graf BM, Vicenzi MN, Martin E, Bosnjak ZJ, Stowe DF. 1995. Ketamine hasstereospecific effects in the isolated perfused guinea pig heart. Anesthe-siology 82: 1426–1437 .discussion 1425A

Haberny KA, Paule MG, Scallet AC, Sistare FD, Lester DS, Hanig JP,Slikker W, Jr. 2002. Ontogeny of the N-methyl-D-aspartate (NMDA)receptor system and susceptibility to neurotoxicity. Toxicol. Sci.68: 9–17.

Hagen TM, Moreau R, Suh JH, Visioli F. 2002. Mitochondrial decay in theaging rat heart: evidence for improvement by dietary supplementa-tion with acetyl-L-carnitine and/or lipoic acid. Ann. N. Y. Acad. Sci.959: 491–507.

Hara Y, Tamagawa M, Nakaya H. 1994. The effects of ketamine on conduc-tion velocity and maximum rate of rise of action potential upstroke inguinea pig papillary muscles: comparison with quinidine. Anesth. Analg.79: 687–693.

Haustein M, Hannes T, Trieschmann J, Verhaegh R, Koster A, Hescheler J,Brockmeier K, Adelmann R, Khalil M. 2015. Excitation-contraction cou-pling in zebrafish ventricular myocardium is regulated by trans-sarcolemmal Ca2+ influx and sarcoplasmic reticulum Ca2+ release. PLoSOne 10 .e0125654

Hirota K, Lambert DG. 1996. I.v. anaesthetic agents do not interact with theverapamil binding site on L-type voltage-sensitive Ca2+ channels. Br. J.Anaesth. 77: 385–386.

Hotchkiss CE, Wang C, Slikker W, Jr. 2007. Effect of prolonged ketamine ex-posure on cardiovascular physiology in pregnant and infant rhesusmonkeys (Macaca mulatta). J. Am. Assoc. Lab. Anim. Sci. 46: 21–28.



Ikonomidou C, Bosch F, Miksa M, Bittigau P, Vockler J, Dikranian K,Tenkova TI, Stefovska V, Turski L, Olney JW. 1999. Blockade of NMDAreceptors and apoptotic neurodegeneration in the developingbrain. Science 283: 70–74.

Irifune M, Shimizu T, Nomoto M. 1991. Ketamine-induced hyperlocomotionassociated with alteration of presynaptic components of dopamineneurons in the nucleus accumbens of mice. Pharmacol. Biochem. Behav.40: 399–407.

Irifune M, Fukuda T, Nomoto M, Sato T, Kamata Y, Nishikawa T, Mietani W,Yokoyama K, Sugiyama K, Kawahara M. 1997. Effects of ketamine ondopamine metabolism during anesthesia in discrete brain regions inmice: comparison with the effects during the recovery andsubanesthetic phases. Brain Res. 763: 281–284.

Kanaya N, Murray PA, Damron DS. 1998. Propofol and ketamine only inhibitintracellular Ca2+ transients and contraction in rat ventricular myocytesat supraclinical concentrations. Anesthesiology 88: 781–791.

Kanungo J, Cuevas E, Ali SF, Paule MG. 2012. L-Carnitine rescues ketamine-induced attenuated heart rate and MAPK (ERK) activity in zebrafishembryos. Reprod. Toxicol. 33: 205–212.

Kanungo J, Cuevas E, Ali SF, Paule MG. 2014. Zebrafish model in drug safetyassessment. Curr. Pharm. Des. 20: 5416–5429.

Kim SJ, Kang HS, Lee MY, Lee SJ, Seol JW, Park SY, Kim IS, Kim NS, Kim SZ,Kwak YG, Kim JS. 2006. Ketamine-induced cardiac depression is associ-ated with increase in [Mg2+]i and activation of p38 MAP kinase and ERK1/2 in guinea pig. Biochem. Biophys. Res. Commun. 349: 716–722.

Kohrs R, Durieux ME. 1998. Ketamine: teaching an old drug new tricks.Anesth. Analg. 87: 1186–1193.

Kudoh A, Kudoh E, Katagai H, Takazawa T. 2002. Ketamine suppressesnorepinephrine-induced inositol 1,4,5-trisphosphate formation viapathways involving protein kinase C. Anesth. Analg. 94: 552–557. tableof contents

Kudoh A, Kudoh E, Katagai H, Takazawa T. 2003. Norepinephrine-inducedinositol 1,4,5-trisphosphate formation in atrial myocytes is regulatedby extracellular calcium, protein kinase C, and calmodulin. Jpn. Heart J.44: 547–556.

Kunst G, Martin E, Graf BM, Hagl S, Vahl CF. 1999. Actions of ketamine and itsisomers on contractility and calcium transients in human myocardium.Anesthesiology 90: 1363–1371.

Lahti AC, Weiler MA, Tamara Michaelidis BA, Parwani A, Tamminga CA.2001. Effects of ketamine in normal and schizophrenic volunteers.Neuropsychopharmacology 25: 455–467.

Langheinrich U, Vacun G, Wagner T. 2003. Zebrafish embryos express anorthologue of HERG and are sensitive toward a range of QT-prolonging drugs inducing severe arrhythmia. Toxicol. Appl. Pharmacol.193: 370–382.

Larsen B, Hoff G, WilhelmW, Buchinger H, Wanner GA, Bauer M. 1998. Effectof intravenous anesthetics on spontaneous and endotoxin-stimulatedcytokine response in cultured human whole blood. Anesthesiology 89:1218–1227.

Liedtke AJ, Nellis SH. 1979. Effects of carnitine in ischemic and fatty acidsupplemented swine hearts. J. Clin. Invest. 64: 440–447.

Lopaschuk GD. 2002. Metabolic abnormalities in the diabetic heart. HeartFail. Rev. 7: 149–159.

Malagodi MH, Chiou CY. 1974a. Pharmacological evaluation of a newCa2+ antagonist, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoatehydrochloride (TMB-8): studies in smooth muscles. Eur. J. Pharmacol.27: 25–33.

Malagodi MH, Chiou CY. 1974b. Pharmacological evaluation of a new Ca++antagonist, 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydro-chloride (TMB-8): studies in skeletal muscles. Pharmacology 12: 20–31.

Malhotra AK, Pinals DA, Adler CM, Elman I, Clifton A, Pickar D, BreierA. 1997. Ketamine-induced exacerbation of psychotic symptomsand cognitive impairment in neuroleptic-free schizophrenics.Neuropsychopharmacology 17: 141–150.

Maxwell CR, Ehrlichman RS, Liang Y, Trief D, Kanes SJ, Karp J, Siegel SJ. 2006.Ketamine produces lasting disruptions in encoding of sensory stimuli.J. Pharmacol. Exp. Ther. 316: 315–324.

Milan DJ, Peterson TA, Ruskin JN, Peterson RT, MacRae CA. 2003. Drugs thatinduce repolarization abnormalities cause bradycardia in zebrafish.Circulation 107: 1355–1358.

Morgan CJ, Muetzelfeldt L, Curran HV. 2010. Consequences of chronic keta-mine self-administration upon neurocognitive function and psycholog-ical wellbeing: a 1-year longitudinal study. Addiction 105: 121–133.

Nishizuka Y. 1988. The molecular heterogeneity of protein kinase C and itsimplications for cellular regulation. Nature 334: 661–665.



Ovchinnikov IV, Gimmel’farb GN, Abidova SS. 1989. The action of anaprilinand ketamine on fatty acid metabolism in the myocardium. Farmakol.Toksikol. 52: 37–40.

Pagel PS, Kampine JP, Schmeling WT, Warltier DC. 1992. Ketamine de-presses myocardial contractility as evaluated by the preload recruitablestroke work relationship in chronically instrumented dogs with auto-nomic nervous system blockade. Anesthesiology 76: 564–572.

Prisant LM. 2001. Verapamil revisited: a transition in novel drug deliverysystems and outcomes. Heart Dis. 3: 55–62.

Riou B, Viars P, Lecarpentier Y. 1990. Effects of ketamine on the cardiacpapillary muscle of normal hamsters and those with cardiomyopathy.Anesthesiology 73: 910–918.

Robinson BL, DumasM, PauleMG, Ali SF, Kanungo J. 2015. Opposing effectsof ketamine and acetyl L-carnitine on the serotonergic system ofzebrafish. Neurosci. Lett. 607: 17–22.

Robinson BL, Dumas M, Cuevas E, Gu Q, Paule MG, Ali SF, Kanungo J. 2016.Distinct effects of ketamine and acetyl l-carnitine on the dopaminesystem in zebrafish. Neurotoxicol. Teratol. 54: 52–60.

Rowland LM. 2005. Subanesthetic ketamine: how it alters physiology andbehavior in humans. Aviat. Space Environ. Med. 76: C52–C58.

Saarenmaa E, Neuvonen PJ, Huttunen P, Fellman V. 2001. Ketamine forprocedural pain relief in newborn infants. Arch. Dis. Child. Fetal NeonatalEd. 85: F53–F56.

Saha N, Chugh Y, Sankaranarayanan A, Datta H. 1990. Interactions of verap-amil and diltiazemwith ketamine: effects onmemory and sleeping timein mice. Methods Find. Exp. Clin. Pharmacol. 12: 507–511.

Serati AR, Motamedi MR, Emami S, Varedi P, MovahedMR. 2010. L-carnitinetreatment in patients with mild diastolic heart failure is associated withimprovement in diastolic function and symptoms. Cardiology 116:178–182.


Shen Y, Sangiah S. 1995. Effects of cadmium and verapamil on ketamine-induced anesthesia in mice. Vet. Hum. Toxicol. 37: 201–203.

Slikker W, Jr, Paule MG, Wright LK, Patterson TA, Wang C. 2007. Systemsbiology approaches for toxicology. J. Appl. Toxicol. 27: 201–217.

Stanley WC. 2005. Rationale for a metabolic approach in diabetic coronarypatients. Coron. Artery Dis. 16(Suppl. 1): S11–S15.

StraikoMM, Young C, Cattano D, Creeley CE,Wang H, Smith DJ, Johnson SA,Li ES, Olney JW. 2009. Lithium protects against anesthesia-induceddevelopmental neuroapoptosis. Anesthesiology 110: 862–868.

Tewari K, Simard JM, Peng YB, Werrbach-Perez K, Perez-Polo JR. 1995.Acetyl-L-carnitine arginyl amide (ST857) increases calcium channel den-sity in rat pheochromocytoma (PC12) cells. J. Neurosci. Res. 40: 371–378.

Trickler WJ, Guo X, Cuevas E, Ali SF, Paule MG, Kanungo J. 2014. Ketamineattenuates cytochrome p450 aromatase gene expression andestradiol-17beta levels in zebrafish early life stages. J. Appl. Toxicol. 34:480–488.

Vogel G. 2000. Zebrafish earns its stripes in genetic screens. Science 288:1160–1161.

Wang JH, Fu Y, Wilson FA, Ma YY. 2006. Ketamine affects memory consoli-dation: differential effects in T-maze and passive avoidance paradigmsin mice. Neuroscience 140: 993–1002.

Yeh FC, Tzeng CC, Chang CL, Chen HI. 1994. The direct cardiac effects ofketamine studied on the intact isolated rabbit heart. Acta Anaesthesiol.Sin. 32: 209–212.

Zausig YA, Busse H, Lunz D, Sinner B, Zink W, Graf BM. 2009. Cardiac effectsof induction agents in the septic rat heart. Crit. Care 13: R144.

Zou X, Sadovova N, Patterson TA, Divine RL, Hotchkiss CE, Ali SF, Hanig JP,Paule MG, Slikker W, Jr, Wang C. 2008. The effects of L-carnitine onthe combination of, inhalation anesthetic-induced developmental, neu-ronal apoptosis in the rat frontal cortex. Neuroscience 151: 1053–1065.



Research Article

Received: 13 November 2012, Revised: 19 January 2013, Accepted: 7 February 2013 Published online in Wiley Online Library: 20 May 2013

(wileyonlinelibrary.com) DOI 10.1002/jat.2888

480

Ketamine attenuates cytochrome p450aromatase gene expression and estradiol-17blevels in zebrafish early life stagesWilliam J. Tricklera,b, Xiaoqing Guoa, Elvis Cuevasa, Syed F. Alia,Merle G. Paulea and Jyotshna Kanungoa*

ABSTRACT: Ketamine, a dissociative anesthetic, is a noncompetitive antagonist of N-methyl-D-aspartate-type glutamatereceptors. In rodents and non-human primates as well as in zebrafish embryos, ketamine has been shown to be neurotoxic.In cyclic female rats, ketamine has been shown to decrease serum estradiol-17b (E2) levels. E2 plays critical roles inneurodevelopment and neuroprotection. Cytochrome p450 (CYP) aromatase catalyzes E2 synthesis from androgens.Although ketamine down-regulates a number of CYP enzymes in rodents, its effect on the CYP aromatase (CYP19) is not known.Zebrafish have been used as a model system for examining mechanisms underlying drug effects. Here, using wild-type (WT)zebrafish (Danio rerio) embryos, we demonstrate that ketamine significantly reduced E2 levels compared with the control.However, the testosterone level was elevated in ketamine-treated embryos. These results are concordant with data frommammalian studies. Ketamine also attenuated the expression of the ovary form of CYP aromatase (cyp19a1a) at the transcrip-tional level but not the brain form of aromatase, cyp19a1b. Exogenous E2 potently induced the expression of cyp19a1b andvtg 1, both validated biomarkers of estrogenicity and endocrine disruption, but not cyp19a1a expression. Attenuation ofactivated ERK/MAPK levels, reportedly responsible for reduced human cyp19 transcription, was also observed in ketamine-treatedembryos. These results suggest that reduced E2 levels in ketamine-treated embryos may have resulted from the suppression ofcyp19a1a transcription. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

Keywords: ketamine; zebrafish; estradiol-17b; CYP aromatase; gene expression; testosterone

*Correspondence to: Jyotshnabala Kanungo, Division of Neurotoxicology, NationalCenter for Toxicological Research, U.S. Food and Drug Administration, 3900 NCTRRoad, Jefferson, AR 72079, USA.Email: [email protected]

aDivision of Neurotoxicology, National Center for Toxicological Research, USFood and Drug Administration, 3900 NCTR road, Jefferson, AR, 72079, USA

bToxicologic Pathology Associates, National Center for Toxicological Research,US Food and Drug Administration, 3900 NCTR road, Jefferson, AR, 72079, USA

IntroductionKetamine [2-(2-chlorophenyl)-2-(methylamino)-cyclohexanone],a pediatric anesthetic, is thought to act primarily through block-ade of N-methyl-D-aspartate (NMDA)-type glutamate receptorsto provide analgesia/anesthesia to children for painful proce-dures (Kohrs and Durieux, 1998). Several previous reports haveillustrated that ketamine can induce neuronal apoptosis whenadministered in high doses and/or for prolonged durations dur-ing susceptible periods of development in rodents (Lahti et al.,2001; Larsen et al., 1998; Malhotra et al., 1997; Maxwell et al.,2006) and primates (Haberny et al., 2002; Slikker et al., 2007;Wang et al., 2006) and these effects can manifest as later disrup-tions in cognitive function (Paule et al., 2011). We have previ-ously reported that ketamine induces motor neuron toxicity inzebrafish embryos (Kanungo et al., 2013).

Estrogens are known to play key roles in a wide range of phys-iological functions, including immune responses, the central ner-vous system and normal somatic cell growth (Filby and Tyler2005; Gustafsson 2003). Among the anesthetics, ketamine, butnot pentobarbital, has been shown to reduce serum concentra-tions of estradiol-17b (E2) in adult cyclic female rats (Lee et al.,2000) although any direct action of anesthetics on the ovary isnot known. Ketamine does not affect ovulation and serum levelsof FSH (follicle-stimulating hormone) and LH (luteinizing hor-mone) in cyclic rats (Clarke and Doughton, 1983). E2 synthesisfrom androgens in gonadal and extragonadal tissues is carriedout by cytochrome p450 (CYP) aromatase encoded by the gene

J. Appl. Toxicol. 2014; 34: 480–488 Published 2013. This article is a U.S. G

cyp19 (Meinhardt and Mullis, 2002). CYPs are a diverse superfamilyof the heme-containing monooxygenase enzymes (Nebert andDalton 2006; Nebert et al., 1991) involved in the metabolism andbioactivation of both endogenous and exogenous chemicals(Nebert et al., 1991; Zhang and Yang, 2009).

Zebrafish have a total of 94 CYP genes, distributed among 18gene families found also in mammals (Goldstone et al., 2010).While expressions of CYPs are transcriptionally regulated by avariety of xenobiotics (Gotoh, 2012), it has been shown thatketamine suppresses CYP3A4 gene expression in mammals(Chang et al., 2009; Chen and Chen, 2010) that could result inendoplasmic reticulum stress, heat shock and apoptotic response(Acharya et al., 2009). However, ketamine’s effect on CYP19 aro-matase is not known.

With its small size, prolific reproductive capacity and easymaintenance, zebrafish maintain the typical complexity of verte-brate systems and accumulating evidence advocates its use inseveral areas of research with the prospect of extrapolating

overnment work and is in the public domain in the USA.

Ketamine attenutaes estrogen levels and aromatase gene expression

findings to other vertebrates and humans (Briggs 2002;Parng et al., 2002; Powers 1989; Vascotto et al., 1997). Exposureto environmental estrogens in oviparous organisms, such aszebrafish, induces a molecular response that is mediated bythe estrogen receptor (ER) where binding leads to induction ofmore ER, increased production of estrogen and subsequent syn-thesis of the egg yolk precursor protein vitellogenin (vtg) in theliver (Jobling et al., 2006). Induction of vtg is a widely acceptedbiomarker of exposure to estrogenic compounds (Sumpter andJobling, 1995). Cyp19a1b is the brain form of aromatase inzebrafish (Chiang et al., 2001; Tchoudakova et al., 2001) and ishighly responsive to estrogens (Menuet et al., 2005). Both theER and aromatases expressed early in development in zebrafishand xenobiotic response elements have been shown to existin the promoter region of cyp19a1b, thus, providing anothermechanistic link between these systems (Kazeto et al., 2004).Data obtained from studies using a transgenic cyp19a1b-GFPzebrafish line indicate that cyp19a1b expression increasesbetween 24 and 48 h post fertilization (hpf) whereas the expres-sion is blocked in embryos exposed to anti-estrogens (Mouriecet al., 2009). As the aromatase is highly expressed in radial glialcells that are progenitors for generating neurons throughoutthe zebrafish life cycle (Adolf et al., 2006; Pellegrini et al., 2005),estrogens are now strongly implicated in the process of embry-onic, adult or reparative neurogenesis (Barha et al., 2009;De Nicola et al., 2009; Galea 2008; Garcia-Segura et al., 2001;Martinez-Cerdeno et al., 2006).

Previously, we have shown that ketamine induces develop-mental neurotoxicity in zebrafish embryos (Kanungo et al.,2013). The goal of the present study was to evaluate whetherin these embryos, ketamine modulated steroid hormone levelsand expression of aromatase genes, cyp19a1a and cyp19a1b.

Materials and Methods

Materials

Ketamine (ketamine HCL) and E2 were purchased from Phoenix(St. Joseph, MO, USA) and Sigma (St. Louis, MO, USA), respec-tively. All high-performance liquid chromatography (HPLC)reagents, unless otherwise indicated, were obtained from Sigma(St. Louis, MO, USA).

48

Animals

Adult wild-type (WT) zebrafish (Danio rerio, AB strain) wereobtained from the Zebrafish International Resource Center atthe University of Oregon (Eugene, OR, USA). The fish were keptin fish tanks (Aquatic Habitats) at the NCTR/FDA zebrafish facilitycontaining buffered water (pH 7.5) at 28 �C, and were fed dailylive brine shrimp and Zeigler dried flake food (Zeiglers,Gardeners, PA, USA). Each 3-l tank housed 8 adult males or8 females. Handling and maintenance of zebrafish were in com-pliance with the NIH Guide for the Care and Use of LaboratoryAnimals and were approved by the NCTR/FDA IACUC. The day–night cycle was maintained at 14:10 h, and spawning and fertili-zation were stimulated by the onset of light at 08.30 hours. Forin-system breeding, crosses of males and females were set upthe previous day with partitions that were taken off the follow-ing morning at the time of light onset at 08.30 hours. Fertilizedzebrafish embryos were collected from the bottom of the tankas soon as they were laid. The eggs/embryos were placed in

J. Appl. Toxicol. 2014; 34: 480–488 Published 2013. This article isand is in the public d

Petri dishes and washed thoroughly with buffered egg water[reverse osmosis water containing 60mg of sea salt (CrystalSeaW, Aquatic Eco-systems, Inc., Apopka, FL, USA) per liter ofwater (pH 7.5)] and then allowed to develop in an incubator at28.5 �C for further experiments.

Treatment of Zebrafish Embryos with Ketamine and E2

Embryos were developmentally staged according to methodsdescribed previously (Kimmel et al., 1995). For ketamine expo-sure, ketamine (2.0mM) was added directly to the embryomedium. E2 was used at a 1-nM concentration. Exposure wasfor 24 h in 6-well cell culture plates (20 embryos per well).

Determination of Ketamine Levels in Zebrafish EmbryosUsing Reverse Phase HPLC

The embryos were collected in microcentrifuge tubes (1.5ml),centrifuged in a microcentrifuge (1700 X g) and the aqueouslayer was discarded and replaced with fresh deionized water(0.5ml) three additional times. After the final centrifugation,the embryo samples were alkalinized with 0.35ml of 0.2mol l–1

borate buffer and sonicated at 30% intensity for 15 s and thenextracted with dichloromethane: ethyl acetate (80:20 v/v) bymixing (75 x g, 10min). The samples were centrifuged (1500 g,3min) and the organic layer was transferred to a borate test,and the samples were extracted again using dichloromethane:ethyl acetate (80:20 v/v). The combined organic layer was back-extracted with perchloric acid (2N), and the organic layer wasdiscarded. The acidic aqueous layer was evaporated to drynessat 45 �C. The dried residue was reconstituted in 0.1ml of mobilephase and 50ml was injected into the column, and the amountof ketaminewas determined by an HPLCmethod described below.The amount of ketamine was determined by a rapid HPLC

method on a Waters 2695 separations module coupled to aWaters 2996 PDA (Milford, MA, USA). Briefly, the isocratic HPLCseparation of ketamine was achieved on a SB-C18 Zorbax col-umn (150� 4.6mm, 5mm) (Agilent, Santa Clara, CA, USA) witha simple mobile phase consisting of acetonitrile: 0.03mol l–1

phosphate buffer (23:77% v/v) adjusted to pH 7.2 at a flow rateof 1.5mlmin–1. The effluents were monitored at 210 nm andquantified using the area under the peak from standard solu-tions dissolved in deionized water (0.75–200mmol l–1). The accu-mulated amount of ketamine per embryo was empiricallyderived by determining the fraction of the dose absorbed inthe embryo from triplicate samples. The data are expressed asmeans (amount per embryo)� SD.

Determination of E2 Levels in Zebrafish Embryos by ReversePhase HPLC

The embryos were collected in microcentrifuge tubes (1.5ml),centrifuged in a microcentrifuge (2000 rpm) and the aqueouslayer was discarded and replaced with fresh deionized water(0.5ml) three additional times. After the final centrifugation,the embryo samples were alkalinized with 0.35ml of perchloricacid (0.2 N) and sonicated at 30% intensity for 15 s and thencentrifuged (14000 g, 20min) and the aqueous layer was trans-ferred to a borate test tube and evaporated to dryness at45 �C. The dried residue was reconstituted in 0.1ml of mobilephase and 50 ml was injected into the column, and the amountof E2 was determined by an HPLC method described below.

a U.S. Government workomain in the USA.


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The amount of E2 was determined using a rapid HPLC methodon a Waters 2695 separations module coupled to a Waters 2996PDA (Milford, MA, USA). Briefly, the isocratic HPLC separation ofE2 was achieved on a SB-C18 Zorbax column (150� 4.6mm,5 um) (Agilent) with a simple mobile phase consisting of ace-tonitrile: water (35:65% v/v) at a flow rate of 1.5mlmin–1. Theeffluents were monitored at 215 nm and quantified using thearea under the peak from standard solutions dissolved inmobile phase (0.15 to 50 mmol l–1). The data are expressedas mean (amount/embryo)� SD from triplicate samples.

RNA Extraction and cDNA Synthesis

Total RNA (from 50 pooled embryos/treatment group) wasextracted from whole embryos using the Trizol reagent(Invitrogen, Carlsbad, CA, USA). An aliquot of each RNA sam-ple was used to spectrophotometrically (using a NanoDropND-1000; NanoDrop Technology, Wilmington, DE, USA) determineRNA quality (A260/A280> 2.0) and concentration. First-strandcDNA was synthesized from total RNA (1 mg; 20 ml final reactionvolume) with oligo(dT) priming using SuperScript II reversetranscriptase (Invitrogen) according to the manufacturer’sinstructions.

Primers

Zebrafish gene-specific primers (Table 1) were used for the quan-titative real-time polymerase chain reaction (RT-qPCR) assays toquantify GAPDH, cyp19a1a (GenBank entry no. AF226620),cyp19a1b (GenBank entry no. AF226619) and vtg1 (GenBank entryno. AF406784.1).

RT-qPCR

Real-time qPCR was performed using a CFX96 C1000 (Bio-Rad,Hercules, CA, USA) detection system with SYBR green fluores-cent label (Bio-Rad). Samples (25 ml final vol) contained the fol-lowing: 1� SYBR green master mix (Bio-Rad), 5 pmol of eachprimer and 0.25 ml of the reverse transcriptase (RT) reaction mix-ture. Samples were run in triplicate in optically clear 96-wellplates. Cycling parameters were as follows: 50 C� 2min, 95 � C10min, then 40 cycles of 95 �C� 15 s, 60 � C� 1min. A meltingtemperature-determining dissociation step was performed at95 C� 15 s, 60 � C� 15 s and 95 �C� 15 s at the end of theamplification phase. The 2�ΔΔCt method was used to deter-mine the relative gene expression (Livak and Schmittgen,2001). The GAPDH gene was the internal control for all qPCRexperiments. Data from each group (n= 3) were averaged andshown as normalized gene expression� SD. One-way ANOVA andHolm–Sidak pair-wise multiple comparison post-hocs (Sigma Stat

Table 1. List of primers used in quantitative real-time polymerase

Gene Forward primer

cyp19a1a 50-TCTGCTTCAGAAGATTCATAAATACTcyp19a1b 50-AAAGAGTTACTAATAAAGATCCACCvtg 1 50-CTGCGTGAAGTTGTCATGCT-30

GAPDH 50-GATACACGGAGCACCAGGTT-30

Published 2013. This article isand is in the public do


3.1 for analysis) were used to determine statistical significancewith P< 0.05.

Sample Preparation for ELISA to Determine Testosteroneand E2 Levels in Embryos

The embryos (125 per sample in triplicate) at 48 hpf were treatedeither with or without ketamine (2mM) for 24 h. Post-treatment,embryos were collected in microcentrifuge tubes (1.5ml). Theembryos were suspended in deionized water (0.5ml), sonicated(30% intensity for 30 s) and then centrifuged (14000 g, 20min).The aqueous layer was transferred to a glass vial anddichloromethane (2ml) was added followed by thoroughmixing. The mixture was incubated on a shaker for 2 h. Afterincubation, the samples were centrifuged (1000 g, 30 s.), andthe bottom organic layers was transferred to a clean test tubeand evaporated to dryness at 45 �C overnight. The dried residuewas reconstituted in 0.4ml of EIA buffer supplied with the ELISAkits, and the amount of E2 or testosterone was determined byELISA methods as described in the manufacturer’s protocol(Cayman Chemical Company, Ann Arbor, MI, USA). The dataare presented as the amount of compound of interest per mgprotein as determined by the Pierce BCA protein assay.

Electrophoresis and Western Blot Analysis

Embryos were prepared for immunoblotting (Western blot)through sonication (one embryo per sample) in Eppendorf tubesin loading buffer [0.125M Tris–HCl, pH 6.8; 4% sodium dodecylsulfate (SDS); 20% glycerol; 0.2M dithiothreitol (DTT); 0.02%bromophenol blue in distilled water) along with an additional0.1mM DTT and 1mM phenylmethylsulfonyl fluoride (proteaseinhibitor)]. Protein samples (one embryo equivalent/lane) wererun in 4–20% gradient SDS-polyacrylamide gels purchased fromBio-Rad (Hercules, CA, USA) at 30–32mA for protein separation.Proteins were then transferred onto a nitrocellulose membranein transfer buffer (20% methanol, 25mM Tris, 192mM glycine,0.1% SDS in distilled water). Transfer membranes were blockedwith 2% bovine serum albumin (BSA) in Tris-borate saline with0.5% Tween 20 (blocking buffer) and probed with phospho-MAPK specific antimouse IgG diluted in blocking buffer(1:2500; Sigma-Aldrich, St. Louis, MO, USA). After developmentfor chemiluminescent signals, the same membrane was strippedfrom the bound antibodies using the stripping buffer (ThermoScientific, Rockford, IL, USA) and reprobed with the total ERK1anti-rabbit IgG (1:500; Santa Cruz Biotech., Santa Cruz, CA,USA). Advanced ECL Western Blotting Substrate (GE Healthcare,Piscataway, NJ, USA) was used to detect HRP-conjugate boundsecondary antibodies. For quantification, densitometry of theimmunoblot signals was performed using ImageJ (National Insti-tutes of Health, Bethesda, MD).

chain reaction (RT-qPCR) assays

Reverse primer

TT-30 50-CCTGCAACTCCTGAGCATCTC-30

GGTAT-30 50-TCCACAAGCTTTCCCATTTCA-30

50-GACCAGCATTGCCCATAACT-30

50- GCCATCAGGTCACATACACG-30

J. Appl. Toxicol. 2014; 34: 480–488a U.S. Government workmain in the USA.

National Institutes of Health

National Institutes of Health

Figure 2. Effect of ketamine on E2 levels in zebrafish embryos. Embryosat 48 h post fertilization (hpf) were exposed to 2mM ketamine for 24 h(static exposure). Post exposure, embryos were washed extensively withquick changes of embryo water three times. Reverse-phase high-performance liquid chromatography (HPLC) was employed to measureestradiol-17b (E2) levels in the embryos (n=50 per group). Data from


Results

Time Course of Changes in Ketamine Accumulation inZebrafish Embryos

Based on the reports that ketamine decreases plasma levels ofE2 in rodents, and our previous studies showing ketamine asan inducer of neurotoxicity in zebrafish embryos (Kanungoet al., 2013), we tested whether similar effects of ketamine inreducing E2 also manifest in zebrafish embryos, a suitable verte-brate animal model for drug screening and drug safetyassessments. To this end, WT zebrafish embryos at 48 hpf weretreated with 2mM ketamine for up to 24 h (static exposure),the dose and duration selected on the basis of our previousreport on ketamine-induced neurotoxicity (Kanungo et al., 2013).Internal ketamine concentrations in these embryos were deter-mined by reverse-phase HPLC and were well within the limitsof quantification at 2, 4 and 20 h of static exposures. At a keta-mine dose 2mM, the amount of ketamine absorbed by theembryos was determined to be ~7.27 mM per embryo (Fig. 1).Differences in the amount of ketamine absorbed by the embryosafter 2, 4 and 20h were minimal and insignificant, indicating thatthe internal concentrations of ketamine reached steady-stateconditions in the embryos within 2 h of exposure.

three separate experiments were averaged and are presented as mean� SD. Student’s t test was used to determine significance P< 0.05 (*).

Reduced E2 and Elevated Testosterone Levels in Ketamine-Treated Embryos

Ketamine-treated embryos were processed to determine theinternal levels of E2 using reverse-phase HPLC and the observedamounts fell well within the limits of quantification. The resultsshowed a significant reduction in E2 levels (approximatelyfour-fold) in ketamine-treated embryos compared with untreatedcontrols (Fig. 2). The four-fold decrease in E2 suggested thatketamine may have interfered with E2 synthesis that is mediatedby cyp19 aromatase, otherwise known as estrogen synthase. It isimportant to note that without sex chromosomes, zebrafish earlylife stages up to 21days post-fertilization (dpf) develop as femalesand sex differentiation begins at 21 dpf.

Figure 1. Ketamine accumulation (internal ketamine concentrations) inzebrafish embryos after static exposure to 2mM ketamine. Embryos at48 h post fertilization (hpf) were exposed for 2, 4 and 24 h. Post exposure,embryos were washed extensively with quick changes of embryo waterthree times. Reverse-phase high-performance liquid chromatography(HPLC) was employed to measure ketamine concentrations in theembryos (n=15 per group). Data from three separate estimations wereaveraged and are presented as the mean� SD.


Further analysis using ELISA demonstrated a significant reduc-tion in E2 levels but elevated testosterone levels in ketamine-treated embryos compared with the controls (Fig. 3).

Effects of E2 on the Expression of Estrogen-Responsive andNon-Responsive Genes

In order to test whether ketamine also altered the expression ofcyp19 aromatases, we monitored the expression of bothcyp19a1a and cyp19a1b aromatase genes along with theirresponsiveness to exogenous E2. First, as well-established con-trols, after RT-qPCR analyzes of cDNA derived from RNA isolatedfrom 48 hpf embryos treated with E2 for 24 h (actual age of theembryo being 72 hpf during analysis), expression of both thearomatases and vtg 1, the gene encoding the estrogen-responsive, liver-synthesized protein vitellogenin, were quanti-fied. The brain form of aromatase, cyp19a1b, and vtg 1 were sig-nificantly induced by 1 nM E2, the lowest dose that reportedlyevokes a response by estrogen-inducible genes. These genesserve as biomarkers of estrogenicity or endocrine disruption.However, E2 did not alter cyp19a1a expression (Fig. 4A) as it isknown to be recalcitrant to E2 induction (Cheshenko et al.,2007; McElroy et al., 2012).

48

Ketamine’s Effect on the Expression of CYP19Aromatase Genes

The next step was to ascertain whether ketamine has any effecton the aromatase and vtg 1 gene expression, embryos at 48 hpfwere treated with 2mM ketamine for 24 h (actual age at the timeof analysis being 72 hpf). RT-qPCR analyzes showed that com-pared with the control, there was no significant difference inthe expression of vtg 1 or cyp19a1b transcripts (Fig. 4B). How-ever, there was a significant reduction (~ 12-fold) in cyp19a1aexpression. These results demonstrated that ketamine does notpossess estrogenic activity. While the expression of brain



3

Figure 3. Determination of the effects of ketamine on E2 (estradiol-17b)and testosterone levels in zebrafish embryos. Embryos (n=125 per sample)at 48 h post fertilization (hpf) were treated either with or without ketamine(2mM) for 24 h. Estimations of E2 (A) and testosterone (B) levels wereperformed by ELISA as described in the Materials and Methods. Datafrom three separate experiments were averaged and are presented asthe mean� SD. Student’s t test was used to determine significanceP< 0.05 (*).

Figure 4. Effects of E2 (A) and ketamine (B) on the expression of vtg 1,cyp19a1a and cyp19a1b genes in zebrafish embryos. Embryos at 48 hpost fertilization (hpf) were treated with either 1 nM estradiol-17b (E2)or 2mM ketamine for 24 h (static exposure). Total RNA was isolated fromthe embryos. After first strand cDNA synthesis from the RNA, quantitativereal-time polymerase chain reaction (RT-qPCR) was performed. The2-ΔΔCT method was used to determine relative gene expression. TheGAPDH gene was the internal control for all RT-qPCR experiments. Datawere averaged and shown as normalized (fold change over control)gene expression� SD. One-way ANOVA and Holm Sidak pair-wise multiplecomparison post-hocs (Sigma Stat 3.1 for analysis) were used to determinestatistical significance with P< 0.05 (*).


484

aromatase, cyp19a1b, remained unaltered, suppression of theexpression of ovary aromatase, cyp19a1a, by ketamine suggeststhat the significant reduction in E2 levels in the embryos couldbe the consequence of reduced expression of the latter,although both the aromatases are capable of mediating E2 syn-thesis from androgens.

Involvement of ERK/MAPK signaling in inducing cyp19 tran-scription in a human cell line (MCF-7) have been reported(Catalano et al., 2003; Ye et al., 2009) and our previous studyhas shown that ketamine attenuates activated (phosphorylated)ERK/MAPK (p-ERK) levels in 28 hpf zebrafish embryos treated for20 h with 2mM ketamine (Kanungo et al., 2012). Here, we ana-lyzed the ERK/MAPK levels in the 48 hpf embryos treated for24 h with 2mM ketamine and the results showed a significantreduction in p-ERK levels although E2 did not induce any signif-icant change (Fig. 5). These results suggested that similar tohuman MCF-7 cells, ERK/MAPK signaling may be involved incyp19a1a transcription.



DiscussionAnesthetics have complex neuroendocrine effects and ketamineis known to block spontaneous GnRH (gonadotropin-releasinghoromone) release and to decrease peripheral LH levels in adultrats (Cohen et al., 1983; Emanuele et al., 1987; Matzen et al., 1987;Sherwood et al., 1980). However, in male Sprague–Dawley rats,ketamine treatment increased testosterone concentrations, butdecreased LHRH (luteinizing hormone-releasing hormone) after1 of administration and continued to significantly decrease after24 h (Gould, 2008). In the present study, an effort to investigatewhether ketamine influenced E2 concentration and CYP aroma-tase expression in zebrafish embryos was based on the reportsthat ketamine regulates a number of CYPs in mammals (Chenand Chen, 2010) and alters the serum concentrations of severalhormones, such as progesterone, testosterone and E2 in cyclicrats (Lee et al., 2000). Estrogens are fundamental in the growth

J. Appl. Toxicol. 2014; 34: 480–488a U.S. Government workmain in the USA.

Figure 5. Effect of ketamine and estradiol-17b (E2) on activated ERK(phosphorylated ERK or p-ERK) levels in zebrafish embryos. Ketamine(2mM) or E2 (1 nM) treatment of 48 h post fertilization (hpf) embryoswas for 24 h (static exposure). (A) Western blot analysis conducted onwhole-embryo lysates (one embryo/lane) shows activated ERK (p-ERK)and total ERK levels (on the same blot used after stripping). (B) Densito-metric analysis (presented as the ratio of the densities of p-ERK to totalERK) of the Western blots. Data from three separate experiments wereaveraged and shown as the mean� SD. Student’s t test was used tocompare the densitometric values of the P-ERK/Total ERK of the controland the respective treated groups. *Significance was set at P< 0.05.


48

and development of the ovary in females (Richards et al., 1976),and spermatogenesis in males (Mahato et al., 2001; O’Donnellet al., 2001). In addition, estrogens are known to play key rolesin the central nervous system, and normal somatic cell growth(Filby and Tyler, 2005; Gustafsson, 2003). It has been suggestedthat differences in estrogen levels in zebrafish embryos and lar-vae occur from an early developmental stage (Lee et al., 2012).Our previous studies illustrated that 2mM ketamine had anadverse effect on the central nervous system (Kanungo et al.,2013). The present study shows the ketamine concentration tobe ~ 7.27 mM per embryo after exposure to 2mM ketamine for24 h. The data are comparable with 10 mM ketamine inducingneuronal damage in primary neurons from rat forebrain in cul-ture (Liu et al., 2012). Additionally, our current data demonstratethat 48 hpf embryos with such low internal ketamine concentra-tions had significantly lower levels of E2 compared with the con-trol, an effect concordant with that in mammals in which a singledose of ketamine reduces E2 levels (Lee et al., 2000).

Ketamine-treated embryos showed an increase in testoster-one level, which is in agreement with data reported in maleboars (Estienne and Barb 2002), male collared peccary (Tayassutajacu) (Hellgren et al., 1985) and female Norway rats (Gould,2008). In contrast, ketamine has been shown to cause a reduc-tion in plasma testosterone levels in men (Oyama et al., 1977),


domestic tom cats (Johnstone and Bancroft, 1988) and cyclicfemale Sprague–Dawley rats (Lee et al., 2000). In humans,although there have been some reports of decreased testoster-one levels after general anesthesia, possibly owing to surgicaltrauma, more often an increase in the testosterone leveloccurred with ketamine anesthesia 1 h after surgery (Cartensenet al., 1973). A lack of any significant change in testosteronelevels in ketamine-treated male rhesus monkeys (Puri et al.,1981; Zaidi et al., 1982) and male cynomolgus (Malaivijitnondet al., 1998) has also been reported. These differences may beattributed to variations in the dose and duration of ketaminetreatment as well as sex and age of the subjects.CYP19 aromatase is a crucial steroidogenic enzyme that cata-

lyzes the final, rate-limiting step in the conversion of androgensinto estrogens (Simpson et al., 1994). In mammals, with theexception of pigs, there is a single CYP19 gene which isexpressed in a variety of tissues (Simpson et al., 2002). However,owing to genome duplication during evolution, teleosts includ-ing zebrafish contain a pair of aromatase genes: cyp19a1a andcyp19a1b (Cheshenko et al., 2008). Changes in estradiol levelsduring sexual differentiation are directly correlated with thechanges in aromatase transcript expression in the gonads ofJapanese flounders (Kitano et al., 1999). It has been reported thatin zebrafish larvae exposed to E2 between 17 and 20 dpf, a timejust prior to the onset of sex differentiation, there was no signif-icant effect on cyp19a1a expression (Callard et al., 2001). Incontrast, short-term exposure of zebrafish larvae to E2 stronglyup-regulated cyp19a1b expression (Cheshenko et al., 2007; McElroyet al., 2012) supporting similar reports on the response ofzebrafish embryos and larvae exposed to E2 (Kishida and Callard,2001). Therefore, cyp19a1b has proven to be a promising markerof estrogenic compounds in zebrafish early life stages (Menuetet al., 2005). In contrast to the dramatic alteration of cyp19a1bexpression, E2 has no effect on cyp19a1a expression in zebrafishlarvae (Kazeto et al., 2004; Trant et al., 2001). Consistent withthese reports, our data show 1 nM E2 significantly inducingcyp19a1b but not cyp19a1a expression in 48-hpf embryos.Decades ago, in rodents, ketamine pretreatment was first

reported to result in a two-fold increase in metabolism in vitroin hepatic microsomes suggesting a self-induction of ketaminemetabolism (Marietta et al., 1976) and implicating ketamine asan inducer of CYP enzymes. However, later studies in rodentsshowed that ketamine decreased CYP (CYP1A, CYP2A, CYP2B,CYP2C, CYP2D1 and CYP3A) activities (Loch et al., 1995; Luppet al., 2003; Meneguz et al., 1999). In human hepatoma HepG2cells, ketamine suppresses CYP3A4 mRNA synthesis (Changet al., 2009). Moreover, ketamine-induced mitochondrial dys-function and inhibition of calcium mobilization has been linkedto suppression of CYP3A4 (Chang et al., 2009). However, theexact molecular mechanism of ketamine-induced suppressionof CYP gene expressions remains obscure. In our previous report,we have shown that ketamine-induced ERK/MAPK down regula-tion and an attenuated cardiac rate (Kanungo et al., 2012). Sup-pression of cyp19 aromatase gene transcription in a humanbreast cancer cell line (MCF-7) has been shown to occur whenERK/MAPK activation is prevented (Catalano et al., 2003; Yeet al., 2009). Although our results are consistent in showing thatketamine caused attenuation of activated ERK/MAPK (p-ERK),whether ketamine’s suppressive effect on cyp19a1a expressionand concomitant reduction of E2 in zebrafish embryos are directconsequences of the modulated ERK/MAPK signaling pathwayremain to be elucidated.



5


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Aromatase knockout (ArKO) female mice are infertile as theirreproductive organs do not develop properly (Simpson, 2004).These mice show impaired functionality in the amygdala andhypothalamus (Pierman et al., 2008) and increased apoptosis inthe frontal cortex (Hill et al., 2008). Various studies on ArKO micehave implicated estrogen in neurodevelopment (Balthazartet al., 1991; Sasahara et al., 2007). Whether ketamine-induceddown-regulation of the ovary form of aromatase (cyp19a1a)adversely affects the nervous system in zebrafish and is linkedto ketamine-induced neurotoxicity (Kanungo et al., 2013) needto be examined.

In conclusion, our data show that ketamine reduces E2 levelswhile elevating testosterone levels in zebrafish embryos. The sin-gle static exposure, however, did not alter estrogen-responsivegene (vtg 1 and cyp19a1b) transcription. Down- regulation ofthe ovary form of aromatase cyp19a1a further suggested thatthe reduced E2 levels in ketamine-treated embryos could resultfrom the inhibition of its synthesis from androgens. Finally, ourdata showing ketamine’s effects on E2 and testosteronelevels in zebrafish embryos are concordant with data frommammals. These findings further emphasize the importanceof cross-species comparisons to delineate molecular modeof action of drugs.

Disclaimer

This document has been reviewed in accordance with UnitedStates Food and Drug Administration (FDA) policy and approvedfor publication. Approval does not signify that the contents nec-essarily reflect the position or opinions of the FDA nor doesmention of trade names or commercial products constituteendorsement or recommendation for use. The findings andconclusions in this report are those of the authors and do notnecessarily represent the views of the FDA.

Acknowledgements

This work was supported by the National Center for ToxicologicalResearch (NCTR)/U.S. Food and Drug Administration (FDA). Wethank Melanie Dumas for zebrafish breeding.

ReferencesAcharya P, Engel JC, Correia MA. 2009. Hepatic CYP3A suppression by high

concentrations of proteasomal inhibitors: a consequence of endoplas-mic reticulum (ER) stress induction, activation of RNA-dependentprotein kinase-like ER-bound eukaryotic initiation factor 2alpha(eIF2alpha)-kinase (PERK) and general control nonderepressible-2eIF2alpha kinase (GCN2), and global translational shutoff. Mol.Pharmacol. 76: 503–515.

Adolf B, Chapouton P, Lam CS, Topp S, Tannhauser B, Strahle U, Gotz M,Bally-Cuif L. 2006. Conserved and acquired features of adultneurogenesis in the zebrafish telencephalon. Dev. Biol. 295: 278–293.

Balthazart J, Foidart A, Surlemont C, Harada N. 1991. Neuroanatomicalspecificity in the co-localization of aromatase and estrogen receptors.J. Neurobiol. 22: 143–157.

Barha CK, Lieblich SE, Galea LA. 2009. Different forms of oestrogen rap-idly upregulate cell proliferation in the dentate gyrus of adult femalerats. J. Neuroendocrinol. 21: 155–166.

Briggs JP. 2002. The zebrafish: a new model organism for integrativephysiology. Am. J. Physiol. Regul. Integr. Comp. Physiol. 282: R3–9.

Callard GV, Tchoudakova AV, Kishida M, Wood E. 2001. Differential tissuedistribution, developmental programming, estrogen regulation andpromoter characteristics of cyp19 genes in teleost fish. J. SteroidBiochem. Mol. Biol. 79: 305–314.



Catalano S, Marsico S, Giordano C, Mauro L, Rizza P, Panno ML, Ando S.2003. Leptin enhances, via AP-1, expression of aromatase in theMCF-7 cell line. J. Biol. Chem. 278: 28668–28676.

Chang HC, Chen TL, Chen RM. 2009. Cytoskeleton interruption in humanhepatoma HepG2 cells induced by ketamine occurs possibly throughsuppression of calcium mobilization and mitochondrial function.Drug Metab. Dispos. 37: 24–31.

Chen JT, Chen RM. 2010. Mechanisms of ketamine-involved regulation ofcytochrome P450 gene expression. Expert Opin. Drug Metab. Toxicol.6: 273–281.

Cheshenko K, Pakdel F, Segner H, Kah O, Eggen RI. 2008. Interference ofendocrine disrupting chemicals with aromatase CYP19 expression oractivity, and consequences for reproduction of teleost fish. Gen.Comp. Endocrinol. 155: 31–62.

Cheshenko K, Brion F, Le Page Y, Hinfray N, Pakdel F, Kah O, Segner H,Eggen RI. 2007. Expression of zebra fish aromatase cyp19a andcyp19b genes in response to the ligands of estrogen receptor andaryl hydrocarbon receptor. Toxicol. Sci. 96: 255–267.

Chiang EF, Yan YL, Guiguen Y, Postlethwait J, Chung B. 2001. Two Cyp19(P450 aromatase) genes on duplicated zebrafish chromosomes areexpressed in ovary or brain. Mol. Biol. Evol. 18: 542–550.

Clarke IJ, Doughton BW. 1983. Effect of various anaesthetics on restingplasma concentrations of luteinizing hormone, follicle-stimulating hor-mone and prolactin in ovariectomized ewes. J. Endocrinol. 98: 79–89.

Cohen H, Guillaumot P, Sabbagh I, Bertrand J. 1983. A newhypoprolactinemic rat strain. Prolactin, luteinizing hormone, follicle-stimulating hormone, testosterone and corticosterone levels in malesand effects of two anesthetics. Biol. Reprod. 28: 122–127.

De Nicola AF, Pietranera L, Beauquis J, Ferrini MG, Saravia FE. 2009.Steroid protection in aging and age-associated diseases. Exp. Gerontol.44: 34–40.

Emanuele MA, Tentler J, Kirsteins L, Reda D, Emanuele NV, Lawrence AM.1987. Anaesthesia with alphaxalone plus alphadolone acetatedecreases serum concentrations of LH in castrated rats. J. Endocrinol.115: 221–223.

Estienne MJ, Barb CR. 2002. Modulation of growth hormone, luteinizinghormone, and testosterone secretion by excitatory amino acids inboars. Reprod. Biol. 2: 13–24.

Filby AL, Tyler CR. 2005. Molecular characterization of estrogen receptors1, 2a, and 2b and their tissue and ontogenic expression profiles infathead minnow (Pimephales promelas). Biol. Reprod. 73: 648–662.

Galea LA. 2008. Gonadal hormone modulation of neurogenesis in thedentate gyrus of adult male and female rodents. Brain Res. Rev.57: 332–341.

Garcia-Segura LM, Azcoitia I, DonCarlos LL. 2001. Neuroprotection byestradiol. Prog. Neurobiol. 63: 29–60.

Goldstone JV, McArthur AG, Kubota A, Zanette J, Parente T, Jonsson ME,Nelson DR, Stegeman JJ. 2010. Identification and developmentalexpression of the full complement of Cytochrome P450 genes inZebrafish. BMC Genomics 11: 643.

Gotoh O 2012. Evolution of cytochrome p450 genes from the viewpointof genome informatics. Biol. Pharm. Bull. 35: 812–817.

Gould EM. 2008. The effect of ketamine/xylazine and carbon dioxide onplasma luteinizing hormone releasing hormone and testosteroneconcentrations in the male Norway rat. Lab. Anim. 42: 483–488.

Gustafsson JA. 2003. What pharmacologists can learn from recent ad-vances in estrogen signalling. Trends Pharmacol. Sci. 24: 479–485.

Haberny KA, Paule MG, Scallet AC, Sistare FD, Lester DS, Hanig JP, SlikkerW Jr. 2002. Ontogeny of the N-methyl-D-aspartate (NMDA) receptorsystem and susceptibility to neurotoxicity. Toxicol. Sci. 68: 9–17.

Hellgren EC, Lochmiller RL, Amoss MS Jr, Grant WE. 1985. Endocrine andmetabolic responses of the collared peccary (Tayassu tajacu) to im-mobilization with ketamine hydrochloride. J. Wildl. Dis. 21: 417–425.

Hill RA, Simpson ER, Boon WC. 2008. Evidence for the existence ofan estrogen-responsive sexually dimorphic group of cells in themedial preoptic area of the 129SvEv mouse strain. Int. J. Impot. Res.20: 315–323.

Jobling S, Williams R, Johnson A, Taylor A, Gro

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