Characterization Of Psychoactive, Addictive, And .
ResearchCharacterization of Psychoactive, Addictive, and NeurotoxicEffects of New Synthetic Amphetamine-like 2C-B, 2C-I andPMMA in MiceChen Chang-Mu1,†, Wu Cheng-Tien2, Lin Jen-Kun4, Liu Shing-Hw1,2, Lin-Shiau Shoei-Yn3,†AbstractRationale:The traditional illicit drugs such as cocaine, marijuana, and methamphetamine (MA) havehad the attention of general public. Designer drugs were created in the 1960s by preparinganalogs or derivatives of currently available drugs to avoid legal restrictions. MA is apsychostimulating drug with significant abuse potential and neurotoxic effects. 4-bromo-2,5dimethoxyphenethylamine (2C-B), 4-iodo-2,5-dimethoxyphenethylamine (2C-I), and paramethoxy-methamphetamine (PMMA) are all phenylethylamine derivatives that are popularnew synthetic psychoactive phenylethylamines. There is a substantial lack of evidence abouttheir psychostimulating, addictive, and neurotoxic effects in the scientific literature.Objectives:In this study, we performed behavioral tests to explore the psychoactive, addictive, andneurotoxic effects induced in mice after drug treatments.Methods:Mice were treated with intraperitoneal (i.p.) injections of saline, MA, 2C-B, 2C-I, and PMMA for 4consecutive days. After no injections for one week, another 4 days of injections were repeated.Results:The behavioral tests showed impaired locomotion performance, rotarod balance performance,and water maze tests, increased conditioned preference place (CPP), and decreased timespent in the open field in the plus maze after injection. Neurochemical studies showedabnormal NOx, LPO, and Na ,K -ATPase activities in brain tissues after injection. Body weightdecrease and gross appearances like untidy fur, back hump, and awkward stance were alsonoted in mice receiving injections. Our results demonstrated that injections of MA, 2C-I, 2C-Band PMMA in mice can impair motor activities, balance and memory performance, induceaddiction and depression, and cause neurochemical abnormalities in brain tissues.Conclusion:In studies of the psychoactive, addictive, and neurotoxic effects of MA, 2C-I, 2C-B and PMMA inDepartment of Surgery, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan1Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan2Institute of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan3Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Taiwan4Author for correspondence: Chang-Mu Chen, Department of Surgery, National Taiwan University Hospital and National TaiwanUniversity College of Medicine, Taipei, Taiwan, #7, Section 1, Chung-Shan S. Rd., Taipei, Taiwan. Tel: 886 (2) 2312-3456, ext: 65324, Fax: 886 (2) 2312-3456, ext: 63110; email: cmchen10@ms27.hinet.net†Lin-Shiau Shoei-Yn, Institute of Pharmacology, College of Medicine, National Taiwan University, #1, Section 1, Jen-Ai Road, Taipei,Taiwan. Tel: 886 (2) 2312-3456, ext: 88313, Fax: 886 (2) 2391-5297; email: syshiau@ntu.edu.tw10.4172/Neuropsychiatry.1000293 2017Neuropsychiatry (London) (2017) 7(6), 880–891p- ISSN 1758-2008e- ISSN 1758-2016880
ResearchLin-Shiau Shoei-Ynmice, we found that these designer drugs had fewer psychostimulating effects than MA, butcomparable addictive and neurotoxic effects. Based on our results, proper legal restriction ofand information campaigns against these designer drugs are warranted to control drug abuseand counter the adverse effects of these drugs.KeywordsMA, 2C-I, 2C-B, PMMA, Psychostimulation, Addiction, NeurotoxicityIntroductionThe traditional illicit drugs such as cocaine,marijuana, and methamphetamine (MA) havehad the attention of the general public andthe authorities. However, designer drugs werecreated in the 1960s by preparing analogs orderivatives of currently available drugs to avoidlegal restrictions [1]. In order to stay ahead ofthe drug laws, manufacturers of designer drugspurposely changed the structure of these drugs,making it difficult for clinicians to discern.Structural manipulation to produce newcompounds, advertisement of these compoundsas “legal” highs, and ease of access to designerdrugs on the internet, at raves and night clubs,and on sale in head shops have rejuvenated andpromoted the current designer drug resurgence[2].MA is a psychostimulating drug with significantabuse potential and neurotoxic effects. Itsprincipal mechanism of action is to cause therelease of central and peripheral monoaminesand their subsequent effects [3]. In 1971, MAwas restricted by US law, but oral MA continuesto be used today clinically as a second-linetreatment for a number of medical conditions,including attention deficit hyperactivity disorder(ADHD) and refractory obesity [4]. The 2008United Nations Office on Drugs and Crime(UNODC) world drug report estimated thatthere were approximately 25 million abusers ofMA worldwide, exceeding that for cocaine (14million) and heroin (11 million) [5]. The 2011UNODC report describes the MA problem as aglobal epidemic, citing an unprecedented rise inuse compared to other illicit substances [6].Manynewsyntheticpsychoactivephenylethylamines have spread through theillicit market around the world during the last2 decades. Among these drugs, 4-bromo-2,5dimethoxyphenethylamine (2C-B) , 4-iodo-2,5dimethoxyphenethylamine (2C-I), and paramethoxy-methamphetamine (PMMA) are allphenylethylamine derivatives that are popularin their use (Dean et al. 2013). In spite of their881Neuropsychiatry (London) (2017) 7(6)popularity among recreational drug users, thereis a substantial lack of evidence about theirpsychostimulating, addictive, and neurotoxiceffects in the scientific literature. There have beenreports recently of drug intoxication after usingthese designer drugs, and side effects includingseizure, psychosis, and even death [7-9].Therefore, a detailed animal study to characterizethe neurobehavioral and neurotoxic effects ofthese drugs is needed for guidance and regulationof their use. In this study, we performedbehavioural tests to explore the psychoactive,addictive, and neurotoxic effects of these drugsin mice. The neurochemical profiles in differentparts of the brain of mice were also examined todetermine the underlying pathophysiology afterdrug injection.Materials and Methods MiceThe experiment protocols were approved bythe Hospital Animal Research Committee ofNational Taiwan University Hospital. Adultmale ICR mice, aged 4-5 weeks and weighing18-25 g, were used in this study. Mice werehoused under air-conditioned and illuminatedconditions to maintain the temperature at 22 1.5 , humidity at 40-60%, and a 12-12-hourdark-light cycle. Treatment of the miceIn order to elicit behavioural sensitization, micereceived intraperitoneal injections of drugs(saline, 2C-I, 2C-B, PMMA) for 4 consecutivedays (10 mg/kg/day), and after one week withoutan injection, the mice were given another 4 daysof injections (20 mg/kg/day). There are 5 groupsthat receiving different kinds of drug injection.Each group consisted of 6 mice. Locomotor activity testLocomotor activities of the mice were assessedwith TruScan photobeam tracking as previouslydescribed [10]. In brief, 10 minutes afterinjection, the mice were put in a tracking device.
Characterization of Psychoactive, Addictive, and Neurotoxic Effects of New Synthetic Amphetamine-like2C-B, 2C-I and PMMA in MiceThe first 5 minutes were used for adaptation, andthen continuous tracking for 1 hour. The resultswere recorded every 10 minutes. Motor equilibrium performance on therotarodThe mice were tested for their ability to balanceon a slowly rotating rod (60 revolutions per min)as described previously [11]. One day beforethe experiment, the ability of the mice to stayon the rod and reach the cut-off time of 180 sfor 10 consecutive sessions was tested. One dayafter injection, the mice were tested again. Theretention time, defined as the total time (sec)remaining on the rod, was recorded for eachsession. Conditioned preference place (CPP)The CPP tests were performed as described byItzhak and Martin with some modifications[12]. In the preconditioning test, the mice wereallowed to move freely between 2 chamberswith the sliding door opened for 15 minutesper day. The tests were continued for 3 days.In the conditioning test, the mice were injectedwith 2C-I, 2C-B or PMMA, and put in a darkchamber with the sliding door closed for 40minutes. The next day, mice were injected withsaline and put in a stripe chamber with thesliding door closed for 40 minutes. The sameprocess was repeated 3 times for a total of 6 days.In the post-conditioning test, the mice were putin chambers with the sliding door opened, andthe time the mice spent in the dark or stripechambers was recorded. Elevated plus mazeThe apparatus consisted of a wooden, plusshaped maze, elevated 60 cm above ground andcomprised of two dark, enclosed arms with 24cm walls, and two lit, open arms with a 1 cmlip. The size of each arm was 30 cm 5 cm witha 5 cm 5 cm center area. Each mouse wasindividually placed in the center of the maze andits behavior digitally recorded from above for a 5min session. Recordings served to manually scorethe time spent in the open and the closed armsand the number of entries to each arm. Time inthe open arms, in the closed arms, number ofentries to the open arms or the closed arms andthe ratio of open arms/closed arms time servedfor statistical analysis. Morris water mazeThe water maze consisted of a featureless circularpool (diameter: 2.0 m, 60 cm high) that wasResearchfilled with water and 2 L milk to a depth of 30cm. A hidden black escape platform (diameter:21 cm) was placed in one of its quadrants suchthat its top lay 1 cm beneath the water surface.Each trial was initiated by placing the mice inone of the 3 other quadrants near the wall of thepool. The mice were allowed to search for theescape platform for a maximum of 90 s. Thosethat failed to locate the platform within thisperiod were guided to it by the experimenter.The mice remained on the platform for 15 s priorto removal from the maze. They were trained 4times per day for 9 days. The average time neededto find the escape platform could be shortenedthrough learning and memory and representedmemory consolidation. After 9 days, the escapeplatform was removed, and the mice were put inthe pool for 90 s. The time that target quadrantmice stayed represented memory retention. Lipid peroxidation (LPO) of brain tissueThe measurement of LPO in brain tissue wasconducted using the method described byKim, et al. with some modifications [13]. Themice were anesthetized with pentobarbital (50mg/kg), and then sacrificed by decapitation.The brain tissue was dissected and stored at-70 . Then, 100 mg of brain tissue (cerebralcortex, cerebellum, brainstem, and striatum)was harvested and homogenized in 20 mMTris-HCl buffer, pH 7.4, containing 0.5 mMbutylated hydroxytoluene to prevent sampleoxidation. After centrifugation, 3.25 volumesof diluted R1 reagent (10.3 mM N-methyl-2phenylindole in acetonitrile) were added to thesupernatant, followed by gentle vortex mixing.After addition of 0.75 volumes of 37% HCl, themixtures were incubated at 45 C for 60 min.After cooling and centrifugation, the absorbanceof the clear supernatant was read at 590 nm.The protein concentration was determined usingthe bicinchoninic acid protein assay kit (Pierce,Rockford, IL). Nitric oxide (NO) in brain tissueNext, 100 mg of brain tissue (cerebral cortex,cerebellum, brainstem, and striatum) washarvested and homogenized in 10% (w:v)homogenate buffer (10% sucrose buffer), andthen centrifuged at 0 C for 20 min at 10 000 g. To avoid incomplete protein denaturation,we added 70% ethanol to the tissue pellet, andallowed the mix to stand overnight. On thefollowing day, all samples were centrifuged at 4 Cfor 20 min at 12 000 g, and the supernatantsfrom the brain tissue were collected and assayed882
ResearchLin-Shiau Shoei-Ynby a NO:ozone chemiluminescence assaymethod (NO-Analyzer 280A, Sievers ResearchInc., Boulder, CO, USA) for quantitative NOassay, as described previously [10].an injection, the mice were given another 4 daysof injections (20 mg/kg/day). Under this dosage,mice showed decreased body weight, untidy fur,back hump, and awkward stance after injection. Measurements of Na , K -ATPase Locomotor activitiesactivityATPase activities were determined by measuringthe amount of inorganic phosphate (Pi)released from the substrate ATP, according to apreviously described colorimetric method [14].In brief, ATPase reactions were initiated in amixture containing NaCl (354 mM), KCl (14.4mM), MgCl2 (3.6 mM), NaHCO3 (37.5 mM),ethyleneglycol bis(amino-ethylether) tetraacetate(EGTA, 1.5 mM), glucose (33.3 mM) andATP (9 mM), and in the absence or presenceof ouabain (1 mM). Brain tissues from differentbrain areas were incubated at 37 0.5 C for 30min in the reaction mixture. Reactions wereterminated by the addition of 150 μl of a solutioncontaining ammonium molybdate (1.05%),malachite green hydrochloride (0.034%) andTriton-X (0.6%). To stabilize the color reaction,10 μl of a sodium citrate solution (34%) wasadded, and the assay solution was held at roomtemperature for 20 min. The optical density at630 nm was determined by an ELISA reader(Dynatech MR-7000). The absorbance valuesobtained were converted to activity values bylinear regression using a standard curve forsodium monobasic phosphate included in theassay at various concentrations. Pi released (inmmol/l) was taken to represent the concentrationof inorganic phosphate released by the enzymatichydrolysis of ATP. Na , K -specific ATPaseactivity was determined by subtracting ouabaininsensitive Mg2 -ATPase activity from totalNa , K - and Mg2 -ATPase activities. Proteinconcentration was determined colorimetricallywith a commercial bicinchoninic acid kit (Pierce,Rockford, IL).Statistical analysisStatistical comparisons between study groupswere performed using the ANOVA test followedby Dunnett’s test. P values of less than 0.05 wereconsidered to be biologically significant.ResultsIn order to elicit behavioral sensitization, micereceived intraperitoneal injections of drugs(saline, 2C-I, 2C-B, PMMA) for 4 consecutivedays (10 mg/kg/day), and after one week without883Neuropsychiatry (London) (2017) 7(6)With injection of 2C-I, the mice showeddecreased ambulation distance in the first 4days (first phase), which persisted through thelast 4 days (second phase). Injection with 2C-Bshowed slightly decreased ambulation distancein the first phase, but significantly increasedambulation distance in the second phase. ForPMMA, the ambulation distance increased inthe first phase, but significantly decreased in thesecond phase (Figure 1A).In contrast, rest time increased after injectionof 2C-I and 2C-B, but decreased after PMMAinjection in the first phase. During the secondphase, rest time increased after 2C-I and PMMAinjection, but decreased after 2C-B injection(Figure 1B).Injection of 2C-I and 2C-B significantlydecreased jumping activities, but the decreasewas less significant after PMMA injection inthe first phase. In the second phase, injectionof 2C-I and PMMA significantly decreased, but2C-B injection significantly increased jumpingactivities ((Figure 1C).Injection of 2C-I, 2C-B and PMMA significantlydecreased stereotype episodes in both the firstand second phase. The decrease in stereotypeepisodes was most evident after 2C-B injection(Figure 1D).Injection of 2C-I and 2C-B significantlydecreased vertical plane entries, but the decreasewas less significant after PMMA injection in thefirst phase. In the second phase, injection with2C-I and PMMA significantly decreased, but2C-B injection significantly increased verticalplane entries (Figure 1E).In brief, the psychostimulating effects of PMMAwere evident in the first phase, but decreased in thesecond phase. With 2C-I, no psychostimulating,and even negative psychostimulating effects werenoted during the first and second phase. With2C-B, initial negative psychostimulating effectswere noted in the first phase, but pronouncedpsychostimulating effects were noted in thesecond phase.In addition, the mice received methamphetamine(MA, 0.75 mg/kg) intra-peritoneal injection, andthe locomotor activities were comparable to those
Characterization of Psychoactive, Addictive, and Neurotoxic Effects of New Synthetic Amphetamine-like2C-B, 2C-I and PMMA in MiceResearchFigure 1: Locomotor activities of the mice after intra-peritoneal injection of saline, 2C-I, 2C-B and PMMA. (A) ambulation distance (B) rest time (C) jumps (D)stereotype-1 episodes, and (E) vertical plane entries. (*p 0.05).884
ResearchLin-Shiau Shoei-YnCPP. However, injection of 2C-I and PMMAdecreased CPP (Figure 2B). The CPP resultsshowed 2C-B is far more addictive than 2C-Iand PMMA, and the reward effects of 2C-Iand PMMA were not evident after repeatedinjections. Plus mazeIn the plus maze, the mice showed no significantdifference in time spent at the open field, closedfield, and center before injection (Figure 3A).After injection of 2C-I and PMMA, the miceshowed a significantly shorter time in the openfield than the control group. Mice injected with2C-B showed an insignificantly shorter time inthe open field (Figure 3B). Water mazeThe mice showed no significant differencein water maze performance before injection.However, injection with PMMA in the firstphase, and injection of 2C-B and PMMA in thesecond phase both significantly impaired theirperformance in the water maze (Figure 4). NOx of brain tissuesInjection of 2C-I did not affect the productionof NOx in brain tissues. Injection of 2C-Bdecreased the production of NOx in the cerebralcortex (Figure 5A & 5B), but significantlyincreased the production of NOx in the brainstem and striatum (Figure 5C & 5D). Injectionof PMMA significantly increased the productionof NOx in the brain stem and striatum (Figure5C & 5D).Figure 2: Rotarod performance (A) and conditioned preference place (CPP) (B) ofthe mice after intra-peritoneal injection of saline, 2C-I, 2C-B and PMMA. (*p 0.05).of mice injected with 2C-I, 2C-B, and PMMA(10 mg/kg). Therefore, the psychostimulatingpotency of MA was at least 10 times that of 2CI, 2C-B, and PMMA. RotarodInjection of 2C-I, 2C-B and PMMA allsignificantly impaired rotarod performance inboth the first and second phase. The impairmentwas most evident after 2C-B injection(Figure 2A). Conditioned preference place (CPP)We performed CPP to test the addictive activityof the mice. In the first phase, injection of2C-I slightly increased CPP. In the secondphase, injection of 2C-B significantly increased885Neuropsychiatry (London) (2017) 7(6) LPO in brain tissuesInjection of 2C-I significantly increasedproduction of LPO in the cerebellumstriatum. Injection of 2C-B increasedproduction of LPO in the cerebral cortexcerebellum, but significantly decreasedproduction in the striatum (Figure 6).theandtheandits Na ,K -ATPase activity in brain tissuesInjection of 2C-I, 2C-B and PMMA did notaffect the Na ,K -ATPase activity in the cerebralcortex of the mice (Figure 7A). Injection ofPMMA caused Na ,K -ATPase activity to increasesignificantly in the cerebellum (Figure 7B), andinjection of 2C-I significantly increased Na ,K ATPase activity in the brain stem (Figure 7C).Injection of 2C-I, 2C-B and PMMA causedNa ,K -ATPase activity to decrease significantlyin the striatum (Figure 7D).
Characterization of Psychoactive, Addictive, and Neurotoxic Effects of New Synthetic Amphetamine-like2C-B, 2C-I and PMMA in MiceResearchFigure 3: Plus maze test of the mice before (A), and after intra-peritoneal injection of saline, 2C-I, 2C-B and PMMA (B). (*p 0.05).Figure 4: Water maze test of the mice before (A), 10 days (B), and 18 days (C) after intra-peritoneal injection of saline, 2C-I, 2C-B and PMMA. (*p 0.05).886
ResearchLin-Shiau Shoei-YnFigur
Characterization of Psychoactive, Addictive, and Neurotoxic Effects of New Synthetic Amphetamine-like Research 2C-B, 2C-I and PMMA in Mice The first 5 minutes were used for adaptation, and then continuous tracking for 1 hour. The results were recorded every 10 minutes. Motor equilibrium performance on the rotarod
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