Bath Salt Combinations

, , ,

*Research & Development Service, John D. Dingell VA Medical Center, Detroit, Michigan, USA

†Department of Psychiatry & Behavioral Neurosciences, Wayne State University School of Medicine,

Detroit, Michigan, USA

AbstractMethylone, 3,4-methylenedioxypyrovalerone (MDPV), andmephedrone are psychoactive ingredients of ‘bath salts’ andtheir abuse represents a growing public health care concern.These drugs are cathinone derivatives and are classifiedchemically as b-ketoamphetamines. Because of their closestructural similarity to the amphetamines, methylone, MDPV,and mephedrone share most of their pharmacological, neuro-chemical, and behavioral properties. One point of divergencein their actions is the ability to cause damage to the CNS.Unlike methamphetamine, the b-ketoamphetamines do notdamage dopamine (DA) nerve endings. However, mephed-rone has been shown to significantly accentuate metham-phetamine neurotoxicity. Bath salt formulations containnumerous different psychoactive ingredients, and individualswho abuse bath salts also coabuse other illicit drugs. There-fore, we have evaluated the effects of methylone, MDPV,mephedrone, and methamphetamine on DA nerve endings.

The b-ketoamphetamines alone or in all possible two-drugcombinations do not result in damage to DA nerve endings butdo cause hyperthermia. MDPV completely protects against theneurotoxic effects of methamphetamine while methyloneaccentuates it. Neither MDPV nor methylone attenuates thehyperthermic effects of methamphetamine. The potent neuro-protective effects of MDPV extend to amphetamine-, 3,4-methylenedioxymethamphetamine-, and MPTP-induced neu-rotoxicity. These results indicate that b-ketoamphetaminedrugs that are non-substrate blockers of the DA transporter(i.e., MDPV) protect against methamphetamine neurotoxicity,whereas those that are substrates for uptake by the DAtransporter and which cause DA release (i.e., methylone,mephedrone) accentuate neurotoxicity.Keywords: dopamine nerve ending, dopaminetransporter, neurotoxic amphetamines, neurotoxicity,b-ketoamphetamines.J. Neurochem. (2015) 133, 211–222.

Methylone, 3,4-methylenedioxypyrovalerone (MDPV), andmephedrone are synthetic psychoactive drugs that havereceived worldwide notoriety as components of ‘bath salts’.These agents are abused singly or with other psychoactivedrugs like alcohol, cannabis, and 3,4-methylenedioxymeth-amphetamine (Winstock et al. 2011; Miller and Stogner2014). The high abuse potential of these illicit compoundsand the clinically significant dangers associated with theirintake has resulted in their classification as Schedule Icompounds by the US Drug Enforcement Administration.These same drugs are also now banned by all member statesof the European Monitoring Centre for Drugs and DrugAddiction. Despite regulatory efforts to restrict their distri-

bution and sale, growing abuse of methylone, MDPV andmephedrone continues to be a significant public health, lawenforcement, and societal concern (Miotto et al. 2013).

Received December 12, 2014; revised manuscript received January 13,2015; accepted January 21, 2015.Address correspondence and reprint requests to Donald M. Kuhn,

Research & Development Service, John D. Dingell VA Medical Center,R&D Service (11R), 4646 John R, Detroit, MI 48201, USA. E-mail:[email protected] used: 5-HT, serotonin; DA, dopamine; DAT, dopamine

transporter; GAPDH, glyceraldehyde 3-phosphate dehydrogenase;GFAP, glial fibrillary acidic protein; MDMA, 3,4-methylenedioxymeth-amphetamine; MDPV, 3,4-methylenedioxypyrovalerone; TH, tyrosinehydroxylase.

© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 133, 211--222 211

JOURNAL OF NEUROCHEMISTRY | 2015 | 133 | 211–222 doi: 10.1111/jnc.13048

Bath salt constituents are cathinone derivatives and arealso referred to as b-ketoamphetamines as they bear veryclose structural similarity to the amphetamines. In fact, theirpossession of a beta-keto moiety is the only feature thatdifferentiates them from their respective amphetamine cong-eners [e.g., 3,4-methylenedioxymethamphetamine (MDMA)is the deketo form of methylone]. Not surprisingly, methy-lone, MDPV, and mephedrone share many of the pharma-cological and behavioral characteristics commonlyassociated with the amphetamine psychostimulants toinclude increased locomotor activity (Huang et al. 2012;Lisek et al. 2012; Lopez-Arnau et al. 2012; Marusich et al.2012, 2014; Motbey et al. 2012a; Wright et al. 2012; Aardeet al. 2013a,b; Fantegrossi et al. 2013; Gatch et al. 2013;Miller et al. 2013; Shortall et al. 2013b; Varner et al. 2013),altered learning and memory (Motbey et al. 2012b; denHollander et al. 2014), disruptions in thermoregulation(Baumann et al. 2012; Aarde et al. 2013a; Fantegrossi et al.2013; Shortall et al. 2013a; Lopez-Arnau et al. 2014),induction of behavioral sensitization (Gregg et al. 2013a,b), and the ability to serve as discriminative drug stimuli(Fantegrossi et al. 2013; Gatch et al. 2013; Varner et al.2013). The abuse potential of these drugs has been affirmedin pre-clinical studies that document their ability to supportthe formation of a conditioned place preference (Lisek et al.2012; Karlsson et al. 2014), sustain self-administration(Hadlock et al. 2011; Watterson et al. 2012; Aarde et al.2013a,b), and enhance intracranial self-stimulation (Robin-son et al. 2012; Watterson et al. 2012; Bonano et al. 2014).These drugs also elicit the release of dopamine (DA),serotonin (5-HT), and norepinephrine and block the uptakeof these monoamines by their respective transporters (Had-lock et al. 2011; Baumann et al. 2012; Eshleman et al.2013; Marusich et al. 2014).Based on the close chemical and pharmacological similar-

ities shared by the amphetamines and b-ketoamphetamines,we hypothesized that the b-ketoamphetamines would causeneurotoxicity to monoamine nerve terminals in the striatum,hippocampus, and cortex like methamphetamine, amphet-amine, and MDMA. Initial studies revealed the surprisingfinding that at least mephedrone did not cause damage to DAnerve endings, even when administered in a high-dose bingeregimen (Angoa-Perez et al. 2012). Some studies havedocumented mild damage to 5-HT nerve endings bymephedrone (Hadlock et al. 2011) and the toxicity of thisdrug can be unmasked to a small degree when given in highdoses over a 2-day span at elevated ambient temperature(Lopez-Arnau et al. 2014; Martinez-Clemente et al. 2014).In balance, a large number of emerging studies indicate thatmethylone, MDPV, and mephedrone do not appear to causechronic depletions of DA, 5-HT, or norepinephrine thatwould be indicative of neurotoxicity (Kehr et al. 2011;Angoa-Perez et al. 2012, 2014; Baumann et al. 2012;Motbey et al. 2012b; Shortall et al. 2013b). Failure to

document a neurotoxic profile for mephedrone promptedthe alternative hypothesis that its ability to block the uptake ofDA by the DA transporter (DAT) would provide protectionagainst methamphetamine neurotoxicity as is seen with more‘classical’ DAT blockers (Schmidt and Gibb 1985; Pu et al.1994). This prediction also proved incorrect when we foundthat mephedrone caused a significant enhancement of theneurotoxicity caused by methamphetamine, amphetamine,and MDMA (Angoa-Perez et al. 2013).Illicit bath salt formulations are not pure and forensic

analyses reveal that they contain mixtures of variouspsychoactive ingredients (Spiller et al. 2011). In light ofthis fact and considering that individuals who abuse bathsalts coabuse numerous other substances (Winstock et al.2011; Miller and Stogner 2014), we expanded the study of b-ketoamphetamine effects on the DA nerve ending by treatingmice with combinations of methylone, MDPV, mephedrone,and methamphetamine. The results revealed that any com-bination of bath salt agents did not lead to neurotoxicity. Onthe other hand, MDPV potently and completely protectedagainst methamphetamine neurotoxicity, whereas methylone,like mephedrone (Angoa-Perez et al. 2013), significantlyenhanced the toxic effects of methamphetamine. Theseresults indicate that the non-neurotoxic b-ketoamphetaminescan be differentiated as protectants or enhancers of neuro-toxicity by virtue of their interaction with the DAT asnon-substrate blockers or substrates, respectively.

Materials and methods

Drugs and reagents

Methylone hydrochloride, MDPV hydrochloride, (�) mephedronehydrochloride, and (�) MDMA hydrochloride were obtained fromthe NIDA Research Resources Drug Supply Program. (+) metham-phetamine hydrochloride, D-amphetamine sulfate, MPTP hydro-chloride, DA, polyclonal antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and all buffers and HPLCreagents were purchased from Sigma-Aldrich (St. Louis, MO,USA). Bicinchoninic acid protein assay kits were obtained fromPierce (Rockford, IL, USA). Polyclonal antibodies against rattyrosine hydroxylase (TH) were produced as described previously(Kuhn and Billingsley 1987). Monoclonal antibodies against ratDAT were generously provided by Dr Roxanne A. Vaughan(University of North Dakota, Grand Forks, ND, USA). Polyclonalantibodies against glial fibrillary acidic protein (GFAP) wereobtained from Thermo Scientific (Rockford, IL, USA). IRDyesecondary antibodies for Odyssey Imaging Systems were purchasedfrom LiCor Biosciences (Lincoln, NE, USA).

Animals

Female C57BL/6 mice (Harlan, Indianapolis, IN, USA) weighing20–25 g at the time of experimentation were housed five per cage inlarge shoe-box cages in a light (12-h light/dark) and temperature-controlled room. Female mice were used because they are known tobe very sensitive to neuronal damage by the neurotoxic ampheta-mines and to maintain consistency with our previous studies of

© 2015 International Society for Neurochemistry, J. Neurochem. (2015) 133, 211--222

212 J. H. Anneken et al.

methamphetamine and b-ketoamphetamine interactions (Angoa-Perez et al. 2012, 2013, 2014). Mice had free access to food andwater. The Institutional Care and Use Committee of Wayne StateUniversity approved the animal care and experimental procedures.All procedures were also in compliance with the NIH Guide for theCare and Use of Laboratory Animals and were conducted incompliance with ARRIVE guidelines.

Pharmacological and physiological procedures

Mice were treated with methylone (30 mg/kg), MDPV (30 mg/kg),or mephedrone (40 mg/kg) using a binge-like regimen comprisedfour injections with a 2-h interval between each injection. This bingetreatment regimen, when used to inject substituted amphetaminesresults in extensive DA nerve ending damage. Doses of all drugsused fall well within the range used in similar published studies(Angoa-Perez et al. 2012, 2013; Marusich et al. 2012; Fantegrossiet al. 2013; den Hollander et al. 2013; Karlsson et al. 2014).Independent groups of mice were treated with each drug separatelyor with all possible combinations of two of the drugs. Forcombination treatment of mice with methylone or MDPV withmethamphetamine, mice were treated with varying doses of either b-ketoamphetamine (49 – 10, 20, or 30 mg/kg) concurrent eachinjection of varying doses of methamphetamine (49 – 2.5, 5, or10 mg/kg). The combination treatment of mephedrone + metham-phetamine was carried out in a previous study (Angoa-Perez et al.2013) and was not repeated presently. Controls received injectionsof physiological saline on the same schedule used for the b-ketoamphetamines and methamphetamine (alone or in combination).The effect of MDPV (49 – 30 mg/kg) on amphetamine (49 –5 mg/kg)- and MDMA (49 – 20 mg/kg)-induced damage to DAnerve endings was also tested. The latter drugs were administeredusing the same binge regimen described above for the b-ketoam-phetamines and methamphetamine. To determine if MDPV neuro-protection would extend to non-amphetamine neurotoxins, micewere treated with MDPV (29 – 10 mg/kg) prior to each of twoinjections of MPTP (20 mg/kg). All injections were given via thei.p. route. Mice were killed 2 days after the last drug treatment whenamphetamine-associated neurotoxicity has reached maximum. Bodytemperature was monitored by telemetry using IPTT-300 implant-able temperature transponders from Bio Medic Data Systems, Inc.(Seaford, DE, USA). Temperatures were recorded non-invasivelyevery 20 min starting 60 min before the first drug injection andcontinuing for 9 h thereafter using the DAS-5001 console systemfrom Bio Medic Data Systems, Inc.

Determination of striatal DA content

Striatal tissue was dissected from the brain after treatment and storedat �80°C. Frozen tissues were weighed and sonicated in 10 volumesof 0.16 N perchloric acid at 4°C. Insoluble protein was removed bycentrifugation and DA was determined by HPLC with electrochem-ical detection as described previously (Angoa-Perez et al. 2012,2013).

Determination of DAT, TH, and GFAP protein levels by

immunoblotting

The effects of drug treatments on striatal DAT and TH levels, highlyspecific markers for striatal DA nerve endings, were determined byimmunoblotting as an index of toxicity. GFAP levels were used as

an index of nerve ending damage as described previously (Angoa-Perez et al. 2012). Mice were killed by decapitation after treatmentand striatum was dissected bilaterally. Tissue was stored at �80°C.Frozen tissue was disrupted by sonication in 1% sodium dodecylsulfate at 95°C and insoluble material was sedimented by centri-fugation. Protein was determined by the bicinchoninic acid methodand equal amounts of protein (70 lg/lane) were resolved by sodiumdodecyl sulfate–polyacrylamide gel electrophoresis and then elec-troblotted to nitrocellulose. Blots were blocked in Odyssey blockingbuffer (phosphate-buffered saline) for 1 h at 23°C. Primaryantibodies against DAT (1 : 1000), TH (1 : 1000), GFAP(1 : 2000) or GAPDH (1 : 10 000) were added to blots andallowed to incubate for 16 h at 4°C. Blots were washed three timesin Tris-buffered saline to remove unreacted antibodies and thenincubated with IRDye secondary antibodies (1 : 4000) for 1 h at23°C. Immunoreactive bands were visualized by enhanced fluores-cence and the relative densities of TH-, DAT-, GFAP-, andGAPDH-reactive bands were determined by imaging with anOdyssey CLx Infrared Image System (LiCor Biosciences) andquantified using ImageJ software (NIH, Bethesda, MD, USA). TH,DAT and GFAP relative densities were normalized to GAPDH.

Data analysis

The effects of drug treatments on core body temperature over timewere analyzed using two-way ANOVAs and post hoc comparisonswere carried out using Bonferroni’s test. Two-way ANOVAs wereperformed to analyze the dose effects of methylone, MDPV, andmethamphetamine, and their combinations, on striatal levels of DA,DAT, TH, and GFAP. The effects of individual drug treatments onDA, DAT, TH, and GFAP content were also tested for significanceby one-way ANOVA and post hoc comparisons were carried out withthe Holm–Sidak test for multiple comparisons. The effects ofMDPV on amphetamine, MDMA, and MPTP toxicity wereanalyzed with a one-way ANOVA and post hoc comparisons werecarried out with the Holm–Sidak test for multiple comparisons.Differences were considered significant if p < 0.05. Because manyof the treatments using multiple doses of methylone or MDPV incombination with methamphetamine resulted in such large numbersof planned comparisons, the outcomes of the statistical tests aredescribed minimally in Results (i.e., only p values presented) anddetailed descriptions of all statistical outcomes are presented inTables S1–S6. Likewise, the details of the statistical analyses ofmethylone, MDPV, and mephedrone (single and combined treat-ments) effects on body temperature are presented in Supportinginformation. All statistical analyses were carried out using GraphPadPrism version 6.01 for Windows (GraphPad Software, San Diego,CA, USA, www.graphpad.com).

Results

Effects of treatment with individual or combinations of

b-ketoamphetamines on DA nerve endings

Mice were treated with methylone (30 mg/kg), MDPV,(30 mg/kg) or mephedrone (40 mg/kg) singly or in allpossible combinations of two of these drugs and the effectson striatal DA, DAT, and TH levels are presented in Fig. 1. Itcan be seen that none of the drugs changed the levels fromcontrol of any marker for DA nerve endings to include DA


b-ketoamphetamines and methamphetamine neurotoxicity 213

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(Fig. 1a), DAT (Fig. 1b) or TH (Fig. 1c). Likewise, com-bined treatment with MDPV + mephedrone, methylone +mephedrone, or methylone + MDPV did not alter striatallevels of DA, DAT, or TH. The effects of these drugs singlyor in combination on striatal GFAP are presented in Fig. 1(d)and show that this marker for astrogliosis was not changedfrom control values by any treatment. In agreement withpublished accounts, each b-ketoamphetamine alone and in alltwo-drug combinations resulted in significant hyperthermiaand these results are described in more detail in Figures S1–S3.

Effects of MDPV on methamphetamine-, amphetamine-,

and MPTP-induced toxicity to DA nerve endings

Mice were treated with MDPV and methamphetamine invarying doses of each drug alone or in combination andFig. 2 shows the results of these treatments on DA (Fig. 2a),DAT (Fig. 2b), TH (Fig. 2c), and GFAP (Fig. 2d). The maineffects of both drugs and their interactions on DA, DAT, TH,and GFAP were all highly statistically significant (p < 0.01–0.0001). MDPV alone at any dose did not change the levelsof any of the dependent variables, whereas with only oneexception, all doses of methamphetamine alone significantlyreduced DA, DAT, and TH and significantly increased

GFAP (p < 0.05–0.0001). The 2.5 mg/kg dose of metham-phetamine alone did not lower TH levels significantly. Alldoses of MDPV provided complete protection against theeffects of each dose of methamphetmine on all DA nerveending markers and GFAP as evidenced by two differentstatistical comparisons. First, none of the MDPV + meth-amphetamine treatments differed from control at any dosecombination for both drug. Second, with only a fewexceptions, all MDPV + methamphetamine treatments weresignificantly different from methamphetamine alone for eachrespective dose of this drug (p < 0.05–0.0001). The excep-tions involved the lowest dose of methamphetamine. Thespecific outcomes for the statistical tests of all main effectsand interactions as well as all post hoc comparisons for thedata in Fig. 2 are presented in detail in Tables S1 and S2.The effects on body temperature of the highest doses of

MDPV (30 mg/kg) given in combination with methamphet-amine (10 mg/kg) were measured and the results arepresented in Fig. 3. The main effects of time (F(30,480) =7.861, p < 0.0001) and treatment (F(3,16) = 157.1, p <0.0001) as well as their interaction (F(90,480) = 6.789,p < 0.0001) were statistically significant. When given alone,methamphetamine (p < 0.0001) and MDPV (p < 0.0001)caused significant increases in body temperature by

(a) (b)

(c) (d)

Fig. 1 Effects of mephedrone (49 – 40 mg/kg), methylone (49 –

30 mg/kg), and MDPV (49 – 30 mg/kg) alone or in combination on DAnerve endings of the striatum. Mice were treated with mephedrone(MEPH; 49 – 40 mg/kg), MDPV (49 – 30 mg/kg), or methylone

(MTHY; 49 – 30 mg/kg) singly or in the indicated two-drug combina-tions and the levels of DA (a), dopamine transporter (DAT) (b), tyrosinehydroxylase (TH), (c) and glial fibrillary acidic protein (GFAP) (d) were

determined 2 days after treatment. Controls were injected with

physiological saline on the same binge schedule used for the b-

ketoamphetamines. DA levels were determined by HPLC and arereported as % control. Relative pixel densities for immunoblots of DAT,TH, and GFAP were quantified using ImageJ, normalized to glycer-

aldehyde 3-phosphate dehydrogenase (GAPDH) and expressed asrelative band density by comparison to the respective control. Data areexpressed as mean � SEM for n = 4–5 mice per group.



comparison to controls. Combined treatment with metham-phetamine +MDPV significantly increased body temperatureby comparison to control (p < 0.0001) and Fig. 3 also showsthat MDPV did not reduce the hyperthermic effect ofmethamphetamine (p > 0.05). The effects of MDPV on bodytemperature differed significantly from both methamphet-amine alone (p < 0.0001) and from methamphetamine +MDPV (p < 0.0001). Taken together, these results indicatethat MDPV protects against methamphetamine neurotoxicitywithout interfering with its hyperthermic effects.The generality of the neuroprotective effects of MDPV

was examined by testing it for protection against amphet-amine-, MDMA-, and MPTP-induced damage to DA nerveendings and the results are presented in Fig. 4. BecauseMDPV does not change DA nerve ending markers (seeFigs 1 and 2), data from groups treated with this drug aloneare omitted from Fig. 4. The main effect of drug treatment onstriatal levels of DA (Fig. 4a), DAT (Fig. 4b) and TH(Fig. 4c) was significant for amphetamine (p < 0.009–0.0001), MDMA (p < 0.0002–0.0001), and MPTP(p < 0.0025–0.0001). Individually, all test drugs signifi-cantly decreased DA, DAT, and TH, and increases in GFAPby comparison to controls (p < 0.05–0.0001) were observedafter all drugs except MPTP. MDPV provided protection

against each treatment by comparison to drug alone(p < 0.05–0.0001), with a few exceptions. In most cases,the protective effect of MDPV was complete (i.e., drug +MDPV did not differ from control values). Exceptions wereobserved for amphetamine + MDPV, where DA levels wereactually elevated above those of controls, for MDMA +MDPV effects on DAT and for MDMA + MDPV effects onTH, where protection was significant but partial. AlthoughMDPV provided some protection against the MDMA-induced reduction in TH, this effect did not reach statisticalsignificance. MPTP alone did not elevate GFAP levelspresently so it was not possible to observe a protective effectof MDPV on this dependent variable. The specific outcomesfor the statistical tests of all main effects and interactions aswell as all post hoc comparisons for the data in Fig. 4 arepresented in detail in Tables S3 and S4.

Effects of methylone on methamphetamine-induced

toxicity to DA nerve endings

Mice were treated with varying doses of methylone (49 – 10,20 or 30 mg/kg) alone or in combination with 2.5 mg/kgmethamphetamine and Fig. 5 shows the results of the effectsof these treatments on DA (Fig. 5a), DAT (Fig. 5b), TH(Fig. 5c), and GFAP (Fig. 5d). The main effect of treatment

(a) (b)

(c) (d)

Fig. 2 Effects of MDPV (49 – 10, 20, or 30 mg/kg) on methamphet-amine (2.5, 5, or 10 mg/kg)-induced neurotoxicity to DA nerve endings.Mice were treated with MDPV (49 – 10, 20 or 30 mg/kg), metham-

phetamine (Meth; 2.5, 5 or 10 mg/kg), or their combination in theindicated doses and the levels of DA (a), dopamine transporter (DAT)(b), tyrosine hydroxylase (TH) (c), and glial fibrillary acidic protein(GFAP) (d) were determined 2 days after treatment. Controls were

injected with physiological saline on the same binge schedule used forMDPV and methamphetamine. DA levels were determined by HPLC

and are reported as % control. Relative pixel densities for immunoblotsof DAT, TH, and GFAP were quantified using ImageJ, normalized toglyceraldehyde 3-phosphate dehydrogenase (GAPDH), and

expressed as relative band density by comparison with the respectivecontrol. Data are means for n = 4–8 mice per group. SEM bars (< 5%of means) and symbols indicating p values are omitted from the figurefor the sake of clarity. Specific details of all statistical comparisons for

the data in this figure are included in Tables S1 and S2.



on DA, DAT, TH, and GFAP was highly significant(p < 0.01–0.0001). Methylone alone at any dose did notchange the levels of any dependent variable. Methamphet-amine (2.5 mg/kg) significantly changed the levels of DA(p < 0.0001) and GFAP (p < 0.01) and while DAT and THwere reduced by this low dose of methamphetamine, thechanges were not significant. All doses of methylonesignificantly accentuated the effects of methamphetamineon DA, DAT, TH, and GFAP by comparison with controls(p < 0.05–0.0001) with the exception of the 10 mg/kg dose,which did not significantly increase the methamphetamineeffect on TH. Figure 5 also shows that methylone signifi-cantly enhanced methamphetamine effects on DA and DATat all doses by comparison with methamphetamine alone.The enhancing effect of methylone was observed only at the30 mg/kg dose in the case of TH but not for GFAP. Doses ofmethamphetamine higher than 2.5 mg/kg were not tested toavoid a floor effect on depletion of DA nerve endingmarkers. The specific outcomes for the statistical tests of allmain effects and interactions as well as all post hoc

Fig. 3 Effects of MDPV (49 – 30 mg/kg), methamphetamine (49 –

10 mg/kg), and their combination on core body temperature. Micewere treated with MDPV (49 – 30 mg/kg), methamphetamine (49 –

10 mg/kg), or their combination and body temperature was mea-

sured via telemetry at 20-min intervals starting 40 min before thefirst drug injection and continuing for 560 min during drug treat-ments. Controls were injected with physiological saline on the same

binge schedule used for MDPV and methamphetamine. Data aremeans for n = 5–6 mice per group. SEM bars (< 5% of means) andsymbols indicating p values are omitted from the figure for the sake

of clarity.

(a) (b)

(c) (d)

Fig. 4 Effects of MDPV (49 – 30 mg/kg) on amphetamine (49 – 5 mg/kg)-, 3,4-methylenedioxymethamphetamine (MDMA) (49 – 20 mg/kg)-, and MPTP (29 – 20 mg/kg)-induced neurotoxicity to DA nerve

endings. Mice were treated with MDPV (MV; 49 – 30 mg/kg) incombination with amphetamine (AM; 49 – 5 mg/kg), MDMA (MD; 49 –

20 mg/kg) or MPTP (MP; 29 – 20 mg/kg) and the levels DA (a),dopamine transporter (DAT) (b), tyrosine hydroxylase (TH) (c), and

glial fibrillary acidic protein (GFAP) (d) were determined 2 days aftertreatment. Controls were injected with physiological saline on the samebinge schedule used for all drugs. DA levels were determined by HPLC

and are reported as % control. Relative pixel densities for immunoblotsof DAT, TH and GFAP were quantified using ImageJ, normalized toglyceraldehyde 3-phosphate dehydrogenase (GAPDH) and expressed

as relative band density by comparison with the respective control.Data are mean � SEM for n = 5–6 mice per group. *p < 0.05,**p < 0.01, ***p < 0.001 and ****p < 0.0001 by comparison withuntreated controls. #p < 0.05, ##p < 0.01, ###p < 0.001 and####p < 0.0001 by comparison with AMPH, MDMA or MPTP alone.Specific details of all statistical comparisons for the data in this figureare included in Tables S3 and S4.



comparisons for the data in Fig. 5 are presented in detail inTables S5 and S6.The effect on body temperature of the highest doses of

methylone (30 mg/kg) and methamphetamine (2.5 mg/kg)given in combination were measured and the resultspresented in Fig. 6 indicate that the main effects of time (F(30,480) = 7.16, p < 0.0001) and treatment (F(3,16) =480.7, p < 0.0001) as well as their interaction (F(90,480)= 6.77, p < 0.0001) were statistically significant. Comparedto controls, methamphetamine (p < 0.0001), methylone(p < 0.0001), and their combination (p < 0.0001) signifi-cantly increased body temperature. The effects of metham-phetamine on body temperature also differed significantlyfrom those of methylone (p < 0.0001) and the combinationof methamphetamine + methylone (p < 0.0001). Finally,methylone effects on body temperature were significantlydifferent from those caused by methamphetamine + methy-lone (p < 0.01).

Discussion

The goal of the present work was to expand our previousstudies of how mephedrone influences DA nerve ending

damage caused by methamphetamine. Most pre-clinicalinvestigations of the neuropharmacological actions of theb-ketoamphetamines have studied the effects of single drugson the CNS. However, illicit bath salt formulations generallycontain mixtures of psychoactive ingredients (Spiller et al.2011) and individuals who abuse these drugs often coabuseother substances along with bath salts (Winstock et al. 2011;Miller and Stogner 2014). Therefore, a compelling rationaleexists for testing bath salts in combination as well as withother psychostimulants like methamphetamine, amphet-amine, and MDMA. Mephedrone, despite its close structuraland pharmacological similarities to the neurotoxic amphet-amines, does not cause neurotoxicity when administered tomice in a high-dose binge regimen (Angoa-Perez et al.2012). Instead, this drug exerts the surprising property ofenhancing the depletions in striatal DA, DAT, and THcaused by methamphetamine, amphetamine, and MDMA(Angoa-Perez et al. 2013). These results raise at least twovery interesting questions about the mechanisms of action ofthe b-ketoamphetamines and their amphetamine congenersthat cause damage to the CNS. First, if mephedrone shareswith methamphetamine the ability to release DA, inhibit itsuptake, and cause hyperthermia, factors that are thought to be

(a) (b)

(c) (d)

Fig. 5 Effects of methylone (49 – 10, 20 or 30 mg/kg) on metham-phetamine (49 – 2.5 mg/kg)-induced neurotoxicity to DA nerve

endings. Mice were treated with methylone (49 – 10, 20, or 30 mg/kg) alone or in combination with methamphetamine (49 – 2.5 mg/kg)and the levels DA (a), dopamine transporter (DAT) (b), tyrosinehydroxylase (TH) (c), and glial fibrillary acidic protein (GFAP) (d) were

determined 2 days after treatment. Controls were injected withphysiological saline on the same binge schedule used for all drugs.DA levels were determined by HPLC and are reported as % control.

Relative pixel densities for immunoblots of DAT, TH and GFAP werequantified using ImageJ, normalized to glyceraldehyde 3-phosphate

dehydrogenase (GAPDH), and expressed as relative band density bycomparison with the respective control. Data are mean � SEM forn = 4–6 mice per group. **p < 0.01, ***p < 0.001 and ****p < 0.0001by comparison with untreated controls. #p < 0.05, ##p < 0.01,###p < 0.001 and ####p < 0.0001 by comparison with methamphet-amine alone. Specific details of all statistical comparisons for the datain this figure are included in Tables S5 and S6.



essential for methamphetamine neurotoxicity, why is it notneurotoxic? Second, if mephedrone can block DA uptake viaits interaction with the DAT, why does it not preventmethamphetamine-induced neurotoxicity like other DATblockers (Schmidt and Gibb 1985; Pu et al. 1994; Angoa-Perez et al. 2013)? The answer to these questions may lie inthe mechanisms by which mephedrone and other bath saltconstituents interact with the DAT.When given alone or in two-drug combinations, mephed-

rone, MDPV, and methylone cause significant increases inbody temperature. The most remarkable effect is associatedwith mephedrone which causes a profound hypothermiaimmediately after injection that reverts to hyperthermia after30–40 min, in agreement with our previous studies with thisdrug (Angoa-Perez et al. 2012, 2013). When methylone orMDPV are given with mephedrone, this hypothermic effectof mephedrone is retained and slightly exaggerated. Com-bined treatment with MDPV and methylone results in a 1–2°C steady increase in core body temperature that becomesevident within 15 min of treatment and persists for at least 8–9 h. Each of these drugs increases the synaptic levels of DAby stimulating release and blocking uptake (i.e., mephedrone,methylone) or by acting as a pure DAT antagonist (i.e.,MDPV). Despite exerting significant hyperthermic effectsoverall, none of the b-ketoamphetamines alone or incombination result in changes in striatal DA, DAT, TH, orGFAP that would be indicative of neurotoxicity. Takentogether, these results leave open the question of why the b-ketoamphetamines do not cause DA nerve ending damagedespite exerting most of the effects that are consideredimportant for methamphetamine-induced neurotoxicity.We found presently that methylone shares with mephed-

rone the ability to enhance methamphetamine-induced

damage to DA nerve terminals, whereas MDPV providescomplete protection against neurotoxicity. This neuroprotec-tive effect of MDPV extends to amphetamine and MDMA,and to MPP+, the neurotoxic metabolite of MPTP that isstructurally unrelated to the neurotoxic amphetamines butwhich depends on uptake by the DAT to exert its damagingeffects on DA neurons (Javitch et al. 1985). These differingeffects of mephedrone, methylone, and MDPV on metham-phetamine neurotoxicity cannot be explained by their effectson body temperature because neither interferes with theability of methamphetamine to cause hyperthermia. Recentstudies of the mechanisms by which b-ketoamphetaminesinteract with the DAT offer significant insight into why thesedrugs have such divergent effects on neurotoxicity. The bathsalts have been classified as substrates and non-substratesbased on whether or not they are transported by the DAT.Mephedrone and methylone, like methamphetamine, aresubstrates for DAT-mediated uptake and they cause releaseof DA via carrier-mediated exchange (Baumann et al. 2012;Cameron et al. 2013a,b; Eshleman et al. 2013). MDPV isnot a substrate for transport and interacts with the DATstrictly as a blocker, like cocaine (Baumann et al. 2013;Cameron et al. 2013a,b; Eshleman et al. 2013; Kolanoset al. 2013; Simmler et al. 2013a). This dichotomy ofinteraction with the DAT by mephedrone and methylone onone hand and by MDPV on the other can explain theiropposing effects on methamphetamine-induced neurotoxic-ity. Mephedrone and methylone enhance the effects ofmethamphetamine, most likely by increasing the release ofDA above that caused by either drug alone. This possibilityhas not yet been tested but is supported by prior resultsshowing that treatments resulting in an increase in thereleasable pool of DA significantly accentuate methamphet-amine-induced damage in DA nerve endings (Thomas et al.2008, 2009). MDPV has an effect that is similar to moreclassical DAT blockers and protects against methamphet-amine-induced neurotoxicity. Compared to amfonelic acid(Schmidt and Gibb 1985; Pu et al. 1994) and nomifensine(Angoa-Perez et al. 2013), which provide partial but signif-icant protection, MDPV completely prevents the damagingeffects of methamphetamine on DA nerve endings. Byblocking DAT-mediated transport (inward or outward),MDPV blocks methamphetamine-induced efflux of DA(Simmler et al. 2013b). This property alone represents animportant mechanism by which it protects against DA nerveending damage caused by drugs that depend on inward DATtransport to exert their toxicity to include the neurotoxicamphetamines and MPTP (i.e., MPP+). MDPV may be oneof the most powerful blockers of the DAT yet described(Baumann et al. 2013; Eshleman et al. 2013; Simmler et al.2013a). It is also more effective than bupropion or methyl-phenidate in blocking methamphetamine-induced DA release(Simmler et al. 2013b) and is the most potent drug identified

Fig. 6 Effects of methylone (49 30 mg/kg) and methamphetamine(49 – 2.5 mg/kg) and their combination on core body temperature.Mice were treated with methylone (49 – 30 mg/kg), methamphetamine(49 – 2.5 mg/kg), or their combination and body temperature was

measured via telemetry at 20-min intervals starting 40 min before thefirst drug injection and continuing for 560 min during drug treatments.Controls were injected with physiological saline on the same binge

schedule used for MDPV and methamphetamine. Data are means forn = 5–8 mice per group. SEM bars (< 5% of means) and symbolsindicating p values are omitted from the figure for the sake of clarity.



to date for protecting against methamphetamine-induceddamage to DA nerve endings.Methamphetamine is probably the prototypical neurotoxic

amphetamine. Its ability to flood the synapse with DA,especially after binge administration (O’Dell et al. 1993), isthought to be the first step in a cascade that leads rapidly tomitochondrial dysfunction, enhanced excitatory neurotrans-mission, increases in glial reactivity and oxidative stress,nerve ending damage, and apoptosis (Halpin et al. 2014).The numerous facets of methamphetamine-induced neuro-toxicity have been studied in great detail over the past threedecades, whereas the dangers associated with bath salts haveemerged only recently. However, the b-ketoamphetaminesshould offer new possibilities for achieving a better under-standing of the mechanisms by which methamphetamine,amphetamine, and MDMA cause damage to monoaminenerve endings. Mephedrone and methylone cause little or noneurotoxicity yet they cause significant efflux of DA andinhibit its re-uptake through their interactions with the DAT.The interactions of mephedrone and methylone with theDAT are very similar to those of methamphetamine and thisleaves unanswered the question posed above of why theyenhance the neurotoxicity of the amphetamines as opposed tooffering protection. It could be predicted that mephedroneand methylone would dilute the effects of methamphetamineon DA release by substituting less toxic DAT substrates for amore toxic one. This does not appear to be the case.Several other possibilities offer sources for speculation

regarding enhanced neurotoxicity when mephedrone ormethylone are combined with methamphetamine and includethe following points. First, the b-ketoamphetamines couldalter the pharmacokinetics or metabolism of methamphet-amine such that blood and brain drug levels are increased inamount and/or for longer periods of time over those seenafter methamphetamine alone. The b-keto group increasesthe polarity of mephedrone and methylone and reduces theirrelative penetration into the brain (Hill and Thomas 2011). Itis therefore possible that methamphetamine-induced alter-ation in the integrity of the blood–brain barrier (Northrop andYamamoto 2012) could make it more permeable to the b-ketoamphetamines. The net result of these possible effectswould be similar to treatment with higher doses of bothdrugs. Second, at the level of the DA nerve ending, it ispossible that the bath salts enhance methamphetaminetoxicity because of increased release of DA over that seenafter either drug alone. Methamphetamine collapses the pHgradient across the synaptic vesicle membrane, allowing DAleakage into the cytoplasm and subsequent efflux via reversetransport (Sulzer et al. 2005). Methylone and mephedronerelease cytoplasmic DA via reverse transport through theDAT, but they differ significantly from methamphetamine inthat they have little if any affinity for the vesicle monoaminetransporter and thus their inhibition of uptake and stimulationof release from synaptic vesicles is far lower than that of

methamphetamine (Eshleman et al. 2013). Because methy-lone and mephedrone do not likely release DA from synapticvesicles, combined treatment with either mephedrone ormethylone + methamphetamine could recruit greater num-bers of DAT molecules to result in heightened DA efflux intothe synapse because amphetamine-induced release is greaterwhen originating from both synaptic vesicle and cytoplasmicstores versus cytoplasmic stores only (Pifl et al. 1995).Third, mephedrone and methylone could enhance metham-phetamine toxicity by inhibiting monoamine oxidase A. Wehave shown previously that the monoamine oxidase Ainhibitor clorgyline significantly increases methamphet-amine-induced depletion of DA (Thomas et al. 2008) but itis not known if the bath salts inhibit monoamine oxidase.Ongoing studies in our laboratory are directed at achieving abetter understanding of these possible mechanisms by whichthe b-ketoamphetamines accentuate methamphetamine-induced neurotoxicity.From a strictly pre-clinical, mechanistic perspective,

MDPV has the potential to be an effective neuroprotectantin drug-induced neurotoxicity and in neurodegenerativeconditions such as Parkinson’s disease, or for treatment ofstimulant dependence. MDPV is not neurotoxic and is farmore potent in preventing methamphetamine-induced DArelease than bupropion or methylphenidate as mentionedabove (Simmler et al. 2013b). The latter two drugs havebeen tested as treatments for stimulant dependence (Tiihonenet al. 2007; Elkashef et al. 2008; Shoptaw et al. 2008) withmixed outcomes so far. However, as a powerful DATantagonist, the abuse potential of MDPV is high in humans(Coppola and Mondola 2012) as well as in animal models ofaddiction (Watterson et al. 2012; Aarde et al. 2013b;Bonano et al. 2014; Karlsson et al. 2014) and this propertyalone would limit its use as a therapeutic agent. Despite itsabuse potential in the illicit market, MDPV or a structuralvariant with lower abuse potential could have clinicaleffectiveness in treating substance abuse disorders as domethadone and buprenorphine, drugs that also possess highabuse potential in humans.

Acknowledgments and conflict of interestdisclosure

This work was supported by grants from the Department of VeteransAffairs. We thank Dr Roxanne Vaughn for her generous gift of DATantibodies. We would also like to thank the NIDA Drug SupplyProgram for providing methylone, MDPV, mephedrone, andMDMA for use in these studies. The authors declare no conflictof interest.

Author contributions

JHA, MAP, and DMK designed the experiments; JHA andMAP performed the experiments and collected the data;



JHA, MAP, and DMK analyzed the data; DMK wrote thefirst draft of the manuscript and all authors contributedsignificantly to the writing of the final version of the article.

Supporting information

Additional supporting information may be found in the onlineversion of this article at the publisher's web-site:

Figure S1. Effects of methylone or MDPV singly or incombination on body temperature.

Figure S2. Effects of mephedrone or methylone singly or incombination on body temperature.

Figure S3. Effects of mephedrone or MDPV singly or incombination on body temperature.

Table S1. Two-way ANOVAs for treatment effects of MDPV,methamphetamine, or their combination on DA, DAT, TH, andGFAP.

Table S2. Post hoc statistical comparisons (Holm–Sidak test formultiple comparisons) for treatment of mice with MDPV, metham-phetamine, or MDPV + methamphetamine.

Table S3. One-way ANOVAs for treatment effects of amphetamine,MDMA, and MPTP given singly or in combination with MDPV onDA, DAT, TH, and GFAP.

Table S4. Post hoc statistical comparisons (Holm–Sidak test formultiple comparisons) for treatment of mice with amphetamine,MDMA, or MPTP singly or in combination with MDPV.

Table S5. One-way ANOVAs for treatment effects of methylone,methamphetamine, and their combination on DA, DAT, TH, andGFAP.

Table S6. Post hoc statistical comparisons (Holm–Sidak test formultiple comparisons) for treatment of mice with methylone,methamphetamine, or methylone + methamphetamine.

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