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Cocaethylene synthesis in Drosophila

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Cocaethylene synthesis in Drosophila
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  Cocaethylene synthesis in  Drosophila  German Torres a, *, Judith M. Horowitz b a  Behavioral Neuroscience Program, Department of Psychology, State University of New York at Buffalo, Buffalo, NY 14260, USA b  Social Sciences, Department of Psychology, Medaille College, Buffalo, NY 14214, USA Received 8 January 1999; received in revised form 5 February 1999; accepted 5 February 1999 Abstract Cocaethylene is an active cocaine metabolite that targets mammalian neural reward pathways and thus contributes to thereinforcingand addictivepropertiesof ethanolandcocaine.Using gas chromatography-massspectrometry, we find that fruit flies( Drosophila melanogaster  ) possess a cellular mechanism through which cocaine can be converted to cocaethylene, presumablyvia ethanol-sensitive enzymes. These findings illustrate the striking similarity of gene products in humans and flies, which mightreflect a homologous role in the metabolic inactivation of cocaine. Further, this conservation of metabolic steps suggests that Drosophila  can be used to study cellular, molecular and biochemicalprocesses leadingto polydrug abuse and addiction.  ©  1999Elsevier Science Ireland Ltd. All rights reserved. Keywords:   Ethanol; Cocaine; Esterases; Evolution; Addiction; Metabolism One of the most prevalent patterns of drug abuse inhumans is the simultaneous consumption of ethanol andcocaine. This particular drug combination appears toenhance the perception of euphoria (reward) and may there-fore explain, in part, the pathogenesis of ethanol-cocaineabuse [11]. The simultaneous consumption of these twodrugs results in the enzymatic synthesis of an active cocainemetabolite, cocaethylene, that when administered alone pro-duces euphsrcenic effects similar to those of cocaine [12,15]. Cocaethylene blocks dopamine (DA) transporterswithin specific brain structures thereby leading to increasedsynaptic activity related to states of reward and motor beha-viors [8,10]. Although studies on cocaethylene to date haveall been in mammals, behavioral experiments suggest thatthis active metabolite evokes striking patterns of motoractivity in fruit flies as well [17].  Drosophila  is a particu-larly useful model for studying the effects of ethanol andcocaine because of its well defined behaviors, genetic tract-ability and simple nervous system. Further,  Drosophila  con-tains many core genes that are equally important in thedevelopment of a drug-dependent phenotype in humans.These latter findings suggest therefore a profound sharingnot only of DNA sequence, but also of detailed biologicalfunction in these two animal lineages (see below). Againstthese outstanding experimental features, we tested whetherthe simultaneous exposure of ethanol and cocaine to adult  Drosophila  could lead to the in vivo synthesis of cocaethy-lene.Wild-type male and female  Drosophila  (Canton-S strain)were raised on standard medium with dry yeast added. Allexperiments were performed on 4–8 day-old flies during thelight phase of a 12:12 h light-dark cycle. A solution of 0.2ml (100%) ethanol and 3.8 ml (0.4 mg/20 ml) cocaine HCLwas added to 2 g of food medium. The dose of ethanol wasselected because it represents natural concentrations of thetwo-carbon molecule in  Drosophila  habitats [1]. Further, a5% ethanol volume (0.2 ml/4 ml) is a sublethal dose therebyposing little or no detrimental effects on cellular structuresand physiological processes [9]. The concentration of cocaine was selected on the basis of pilot experimentswhich showed that it produced no untoward effects on flysurvival.Ethanol concentration was measured in fly homogenatesby gas chromatography. For ethanol analysis, 0.1 ml ali-quots of each sample were obtained and transferred intoseparate 12  ×  75 mm culture tubes. A 0.1 ml volume of 0.20 g/100ml n-propanol internal standard solution was Neuroscience Letters 263 (1999) 201–2040304-3940/99/$ - see front matter  ©  1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3940(99)00156-1 * Corresponding author. Tel.: +1-716-6453650 ext. 678; fax: +1-716-6453801; e-mail: gtorres@acsu.buffalo.edu  added to each tube. A 1.5  m l volume of the sample prepara-tion was injected directly into the injector port of the gaschromatograph instrument. The analysis was performed ona Varian 3700 gas chromatograph with a flame ionizationdetector. A 0.2% Carbowax 1500 on 80/100 ml Carbopack C column was used for the separation. The carrier gas wasnitrogen. The peak integration and quantification was mea-sured using a Varian Vista 402 data system. A single pointcalibration at 0.158 g/100 ml was used. The linearity of theanalysis was validated from 0.01 to 0.237 g/100 ml by ana-lyzing aqueous standards within that range. Samples withethanol concentrations higher than the standard curve werediluted with water and reanalyzed. The limit of quantifica-tion was 0.01 g/100 ml.Cocaine and cocaethylene samples were extracted fromfruit fly homogenates using a solid phase extraction proce-dure (SPE) that employed a vacuum manifold device andZSDAU020 Solid Phase Extraction columns (WorldwideMonitoring-United Chemical Technologies; Bristol, PA).For the samples, 0.5 ml aliquots of each sample wereobtained and 50 ng of cocaine-D 3  and 50 ng of cocaethy-lene-D 3  were added as internal standards. Samples wereprepared by adding 3 ml of H 2 O and 1 ml of 100 mMsodium acetate. After being centrifuged for 5 min at1000  ×  g , the supernatants were collected into clean culturetubes. Sample supernatants were loaded onto SPE columnsconditioned with one 3 ml volume of methanol, followed byone 3 ml volume of H 2 O, and one 2 ml volume of 100 mMsodium phosphate buffer (pH 6.0). After the sample loading,SPE columns were washed with a 3 ml volume of H 2 O, a 1ml volume of 100 mM HCL followed by a 3 ml volume of methanol. After the methanol wash, the SPE columns werevacuum dried for 5 min and eluted with 3 ml of methylenechloride: isopropanol: concentrated ammonium hydroxide(80:20:2). Extracts were collected into glass culture tubesand evaporated to dryness. The residue was reconstitutedwith 50  m l of chloroform and transferred to conical autosampler vials. The analysis was performed on a Finnigan4500 gas chromatograph-mass spectrometer using a 15meter DB-5, 1  m m film capillary column. The injectorport temperature and interface oven temperature were 260and 255 ° C, respectively. The carrier gas was helium, thechemical ionization reagent gases were methane and ammo-nia and the ionization temperature was 130 ° C. The extractresidues were analyzed using positive chemical ionizationunder selected ion monitoring mode: ion currents forcocaine and cocaine-D 3  were m/z 304 and 307, whereasion currents monitored for cocaethylene and cocaethylene-D 3  were m/z 318 and 321. The concentrations of cocaineand cocaethylene in the sample were determined by extra-polation from a standard curve generated from plasma stan-dards that were spiked with known amounts of cocaine andcocaethylene ranging from 1 to 1000 ng/ml. The limit of quantification for cocaine was 1 ng/ml and for cocaethylene2.5 ng/ml. Data were analyzed by non-directional  t  -testswith statistically significant differences defined as P    0.05. Relative ethanol, cocaine and cocaethylenelevels are expressed as means  ±  SEM.Approximately 50 flies were placed for 5 consecutivedays in vials containing food medium laced with 5% ethanoland 0.02% cocaine HCl. To control for evaporation of etha-nol, flies were transferred twice daily (~8 h apart) to freshvials containing fixed ethanol-cocaine doses. We found thatthe aforementioned drug combination was tolerated well bythe flies; mortality was relatively low (  5%). Further, noapparent ataxia was observed in flies randomly selected forbehavioral assays (data not shown).  Drosophila  exposed tothe above two drugs contained appreciable levels of ethanoland cocaine. Remarkably, the same flies also containedmeasurable levels of cocaethylene (Table 1). As in humansand rats, levels of cocaethylene in  Drosophila  were signifi-cantly lower than those of its parent compound, cocaine(non-directional  t  -tests,  P    0.05). Frequency of exposureto fresh medium appeared to be a critical variable for thesynthesis of cocaethylene, as flies ( n  =  50) exposed to etha-nol once every 2 days did not show detectable levels of either ethanol or the ethyl metabolite. Cocaine levels,36.8  ±  10.6 ng/ml (mean  ±  SEM of five vials) however,were measurable and comparable with those reported inTable 1. It should be noted that synthesis of cocaethyleneoccurred specifically in the presence of both ethanol andcocaine, as flies ( n  =  50) exposed to one or the other drugfailed to show traces of the active cocaine metabolite (seeTable 1). This finding adds further credence to the observa-tion that neither ethanol nor cocaine alone possess the abil-ity to generate cocaethylene [5]. Table 1Whole body homogenate extracts from drug-exposed  Drosophila  Vial # Ethanol*(g/100 ml)Cocaine(ng/ml)Cocaethylene(ng/ml)1 0.01 54 122 0.01 55 103 0.01 19 54 0.01 18 5*Ethanol levels (i.e. 0.01 g/100 ml) in SI units are equivalent to 2.17mmol/l.Data (in duplicate) were obtained from drug-treated flies 2 h after thelast transfer to vials. Ethanol and cocaine levels from residual foodmedium were: 0.26 g/100 ml and 1887 ng/ml, respectively. Druglevels from fresh food medium were: 0.47 g/100 ml for ethanol and1628 ng/ml for cocaine. As expected, cocaethylene was not found inthese samples.  Drosophila   homogenates from ethanol- or cocaine-only vials also failed to show traces of cocaethylene. At a higherethanol dose (i.e. 10% in volume), there was an increased mortalityrate. Body homogenate extracts from surviving flies ( n   =  20/vial)contained ethanol levels of 0.01  ±  0.0 g/100 ml (mean  ±  SEM oftwo vials) and cocaine levels of 30.5  ±  1.5 ng/ml (mean  ±  SEM oftwo vials). Cocaethylene levels, however, were not detected in thesesamples, perhaps due to (1) the small number of surviving flies or (2)toxic and detrimental effects of the two-carbon molecule on cellularstructures and physiological processes. Differences in cocaine andcocaethylene levels between samples could reflect differences indistribution of cocaine in the  Drosophila   food medium. 202  G. Torres, J.M. Horowitz / Neuroscience Letters 263 (1999) 201–204   Cocaethylene is synthesized in vivo through the transes-terification of cocaine in the presence of ethanol by a non-specific mammalian carboxylesterase localized to liver andkidney cells [5]. The fact that this active metabolite issynthesized not only in humans but also in rodents indicatesa profound conservation of detailed enzymatic function inregulating the metabolism of cocaine. In this context, amajor advancement in our understanding of evolution isthe appreciation that many genes in vertebrate and phylo-genetically distant invertebrate lineages encode similarprotein products. For instance, fruit flies contain dehydro-genases, specific enzymes with the capacity to oxidize etha-nol [9]. Further, brain neurons and thoracic ganglia of fliesharbor DA molecules, biogenic transmembrane receptorsand cocaine-sensitive serotonin transporter proteins thatmodulate a variety of physiological and behavioral phenom-ena [3,4,6,7,16]. Our data extend this evolutionary conceptby demonstrating the striking conservation of an endogen-ous enzymatic mechanism in  Drosophila  that catalyzescocaine into cocaethylene in the presence of ethanol (seeFig. 1). Previously, we provided evidence that cocaethylene,indirectly acting through DA and  N  -methyl- d -aspartate(NMDA) receptor systems, can induce significant patternsof behavioral activity in  Drosophila  [17]. Therefore,cocaethylene is not only synthesized in flies but also exertsmarked behavioral effects in this animal lineage. Theseproperties are advantageous for further studies, particularlyin view of the fact that flies develop behavioral sensitizationto repeated cocaine exposure [13] and become inebriated tovarious doses of ethanol [14]. This opens the possibility of using the fruit fly to study basic processes that are altered inthe addictive brain.In conclusion, the present study indicates that the fruit fly  Drosophila  synthesizes cocaethylene. This neuroactivemetabolite is detected in body homogenates five days afterdietary ethanol and cocaine consumption. This findingpoints to a conserved mechanism underlying the metabo-lism of cocaine in both humans and drosophilids.This research was supported in part by a research grant toGerman Torres from the Johns Hopkins Center forAlternatives to Animal Testing. [1] Ashburner, M., Speculations on the subject of alcohol dehydro-genase and its properties in  Drosophila   and other flies,BioEssays, 20 (1998) 949–954.[2] Boyer, C.S. and Petersen, D.R., Enzymatic basis for the trans-esterification of cocainein the presenceof ethanol:evidenceforthe participation of microsomal carboxylesterases, J. Pharma-col. Exp. Ther., 260 (1992) 939–946.[3] Budnik, V. and White, K., Catecholamine-containing neurons in Drosophila melanogaster  : distribution and development, J.Comp. Neurol., 268 (1988) 400–413.[4] Corey, J.L., Quick, M.W., Davidson, N., Lester, H.A. andGuastella, J., A cocaine-sensitive  Drosophila   serotonin trans-porter: cloning, expression, and electrophysiologicalcharacterization, Proc. Natl. Acad. Sci. USA, 91 (1994)1188–1192.[5] Dean, R.A., Christian, C.D., Sample, R.H.B. and Bosron, W.F.,Effects of ethanol on cocaine metabolism: formation ofcocaethylene and norcocaethylene, Toxicol. Appl. Pharmacol.,117 (1991) 1–8.[6] Demchyshyn, L.L., Pristupa, Z.B., Sugamori, K.S., Barker, E.L.,Blakely, R.D., Wolfgang, W.J., Forte, M.A. and Niznik., Cloning,expression, and localization of a chloride-facilitated, cocaine-sensitive serotonin transporter from  Drosophila melanogaster  ,Proc. Natl. Acad. Sci. USA, 91 (1994) 5158–5162.[7] Feng, G., Hannan, F., Reale, V., Hon, Y.Y., Kousky, C.T.,Evans, P.D. and Hall, L.M., Cloning and functional character-ization of a novel dopamine receptor from  Drosophila melanogaster  , J. Neurosci., 16 (1996) 3925–3933.[8] Hearn, W.L., Flynn, D.D., Hime, G.W., Rose, S., Cofino, J.C.,Mantero-Atienza, E., Wetli, C.V. and Mash, D.C., Cocaethy-lene: a unique cocaine metabolite displays high affinity for thedopamine transporter, J. Neurochem., 56 (1991) 698–701.[9] Heinstra, P.W.H., Evolutionary genetics of the  Drosophila   alco-hol dehydrogenase gene-enzyme system, Genetica, 92 (1993)1–22.[10] Katz, J.L., Terry, P. and Witkin, J.M., Comparative behavioralpharmacology and toxicologyof cocaineand its ethanol-derivedmetabolite, cocaine ethyl-ester (cocaethylene), Life Sci., 50(1992) 1351–1361.[11] McCance-Katz, E., Kosten, T.R. and Jatlow, P., Concurrent useof cocaine and alcoholis more potent and potentiallymore toxicthan use of either alone: a multiple-dose study, Biol. Psychiatry,44 (1998) 250–259.[12] McCance, E., Price, L.H., Kosten, T.R. and Jatlow, P.I.,Cocaethylene: pharmacology, physiology and behavioraleffects in humans, J. Pharmacol. Exp. Ther., 274 (1995)215–223.[13] McClung, C. and Hirsh, J., Stereotypic behavioral responses tofree-base cocaine and the developmentof behavioralsensitiza-tion in  Drosophila  , Curr. Biol., 8 (1998) 109–112.[14] Moore, M.S., DeZazzo, J., Luk, A.Y., Tully, T., Singh, C.M. andHeberlein, U., Ethanol intoxication in  Drosophila  : genetic andpharmacological evidence for regulation by the cAMP signalingpathway, Cell, 93 (1998) 997–1007.[15] Rafla, F.K. and Epstein, R.L., Identification of cocaine and itsmetabolites in human urine in the presence of ethyl alcohol,Anal. Toxicol., 3 (1979) 59–63.Fig. 1. Schematic diagram depicting the primary metabolic pathwayof cocaethylene in mammals (modified from Torres and Horowitz[18]). The general mechanism(s) of carboxylesterase-mediated cat-alysis of cocaine in the presence of ethanol is the transesterificationof the 2-carbomethoxy group of cocaine into its 2-carboxyethyl deri-vative, cocaethylene. The findings that cocaethylene is detected in Drosophila   suggest that an ethanol-dependent synthetic pathwayalso exists in invertebrates. Carboxylesterases are a class of  b -esterases which have a broad substrate specificity [2]. Of interest,fruit flies also contain esterase-coding DNA fragments [9], presum-ably localized to the fat body and alimentary tract. Activity of this(homologue) enzyme could be responsible for the biotransformationof cocaine into cocaethylene. It cannot be excluded, however, thatanother esterase-like enzyme (or pathway) for cocaethylene synth-esis exists in  Drosophila  . 203 G. Torres, J.M. Horowitz / Neuroscience Letters 263 (1999) 201–204   [16] Sugamori, K.S., Demchyshyn, L.L., McConkey, F., Forte, M.A.and Niznik, H.B., A primordial dopamine D 1 -like adenylylcyclase-linked receptor from  Drosophila melanogaster   display-ing poor affinity for benzazepines, FEBS Lett., 362 (1995)131–138.[17] Torres, G. and Horowitz, J.M., Activating properties of cocaineand cocaethylene in a behavioral preparation of  Drosophila melanogaster  , Synapse, 29 (1998) 148–161.[18] Torres, G. and Horowitz, J.M., Individual and combined effectsof ethanol and cocaine on intracellular signals and geneexpression, Prog. Neuropsychopharmacol. Biol. Psychiatry,20 (1996) 561–596. 204  G. Torres, J.M. Horowitz / Neuroscience Letters 263 (1999) 201–204 
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