Introduction

Abstract Introduction Materials & Methods Results Discussion References

Methcathinone (also known as ephedrone, Jeff or Mulka) is a Schedule I controlled substance that is chemically related to cathinone, a naturally occurring central nervous system stimulant found in the Khat plant (Catha edulis). Heavy patterns of methcathinone abuse have been reported in the former Soviet Union since the 1980s (Rosen, 1993; Zhingel et al., 1991). In the United States, clandestine laboratories manufacturing this illicit stimulant were first discovered in the early 1990s in the upper peninsula of Michigan (Rosen, 1993). Since then, manufacturing, distribution and abuse of methcathinone have increased. As of April 1995, clandestine laboratories had been seized in 13 states (Calkins et al., 1995). Like other amphetamine analogs, methcathinone exerts profound psychomotor stimulant effects (Glennon et al., 1995, 1987). Its short-term intoxication and severe addiction patterns have been compared with those of crack cocaine or methamphetamine (Calkins et al., 1995).

Relatively little is known regarding the neurotoxic potential of methcathinone. We demonstrated that a single administration of (±)-methcathinone (30 mg/kg s.c.) rapidly (within 30 min) decreases the activity of tryptophan hydroxylase, the rate-limiting enzyme in the synthesis of 5-HT (Gygi et al., 1996). In contrast, multiple administrations (4 injections, 30 mg/kg s.c. at 4-hr intervals) cause decreases in striatal concentrations of dopamine, 5-HT and their respective metabolites 18 hr after the last (±)-methcathinone injection (Gygi et al., 1996). This high-dose regimen also decreases activities of the monoamine-synthesizing enzymes tyrosine hydroxylase and tryptophan hydroxylase 18 and 72 hr after the last methcathinone administration. Recently, Sparago et al. (1996) demonstrated decreases in striatal dopamine concentrations 14 days after the administration of the (S)-()- or (R)-(+)-isomer of methcathinone (50 mg/kg s.c. twice daily for 4 days). Long-term decreases in hippocampal and neocortical 5-HT and its metabolite after multiple injections of (S)-()-methcathinone were likewise found (Sparago et al., 1996). These data suggest that methcathinone use has the potential to damage dopamine and 5-HT neurons.

The purpose of this study was to investigate further the long-term effects of methcathinone on monoaminergic systems. Because numerous studies have implicated dopamine as an important mediator of neurochemical deficits induced by amphetamine analogs (Nash et al., 1990; Schmidt et al., 1985), a second objective of this study was to investigate the role of dopamine in the effects of methcathinone. The results reveal that multiple administrations of methcathinone cause long-term neurochemical deficits in both dopaminergic and 5-hydroxytryptaminergic neuronal systems and that endogenous dopamine likely contributes to these effects.

	Materials and Methods

Abstract Introduction Materials & Methods Results Discussion References

Animals and treatments. Male Sprague-Dawley rats (180-275 g; Simonsen Laboratories, Gilroy, CA) were housed at 24°C in hanging wire cages with a 12-hr alternating light/dark cycle with food and water available ad libitum. On the day of the experiments, rats were killed by decapitation at 12:00 noon. All procedures were conducted in accordance with approved National Institutes of Health guidelines.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Dose-response effects of methcathinone on striatal extracellular dopamine concentrations in freely moving rats. Three base-line samples were collected at 20-min intervals, after which rats received a single injection of methcathinone (1-30 mg/kg s.c.) as indicated by the arrow. Values represent mean ± 1 S.E.M. of determinations in five rats.

Tyrosine hydroxylase. Frozen tissues were weighed and homogenized in 125 µl of 50 mM HEPES, pH 7.4, containing 0.2% Triton X-100 and 6 mM dithiothreitol and centrifuged for 15 min at 22,000 × g. Duplicate 10-µl aliquots of the supernatant were added to 40 µl of double-distilled water and assayed for tyrosine hydroxylase activity according to a modification of the tritium-release method of Nagatsu (1983). In this procedure, L-[3,5-³H]tyrosine is converted to either L-[5-³H]dihydroxyphenylalanine or L-[3-³H]dihydroxyphenylalanine, and the displaced tritium that equilibrates with water is counted by liquid scintillation spectroscopy. Fifty µl of the reaction medium containing 0.4 µCi L -[3,5-³H]tyrosine (Amersham, Arlington Heights, IL) dried under nitrogen gas, 0.2 mM L -tyrosine, 4 mM ferrous ammonium sulfate, 6.6 mM 6-methyltetrahydropterin (6 MPH₄), 96 µM 2-mercaptoethanol, 0.66 M sodium acetate were added to each sample. Blanks contained 0.02% Triton X-100. The final pH of the reaction medium at 37°C was 6.0. The reaction medium was incubated for 15 min in a metabolic shaker at 37°C, after which the enzyme reaction was stopped by the addition of 1 ml of a 7.5% (w/v) charcoal suspension in 1 M HCl. The mixture was centrifuged for 30 min at 1850 × g, and aliquots of the supernatants were counted on a liquid scintillation detector (2000 CAA Tri-Carb; Packard, Meriden, CT).

Tryptophan hydroxylase. Tryptophan hydroxylase activity was determined in tissue homogenates using HPLC by measuring 5-HTP formation resulting from the hydroxylation in vitro of tryptophan according to a modification of the method described by Johnson et al. (1992). Briefly, frozen tissue samples were sonicated in 150 to 300 µl of ice-cold 50 mM HEPES buffer, pH 7.4, containing 0.2% Triton X-100 and 6 mM dithiothreitol. The resulting suspension was centrifuged (16,000 × g for 15 min at 4°C). Duplicate aliquots (15 µl) of supernatant were then incubated for 10 min (37°C) with 10-µl reaction mixture [52.8 mM HEPES, 50 mM -mercaptoethanol, 8 mM tryptophan, 1.25 mM m-hydroxybenzylhydrazine (NSD 1015) and 3.38 mM DL-6-methyl-5,6,7,8-tetrahydrobiopterin dihydrochloride (6 MPH₄)]. Boiled supernatant was used for blanks. The reaction was terminated by placing the tubes on ice and adding HPLC mobile phase (see below). The resulting mixture was centrifuged (2500 × g for 10 min at 4°C), and the supernatant was retained for 5-HTP quantification using HPLC coupled with electrochemical detection (C-18 Microsorb column; Rainin, Woburn, MA; glassy carbon electrode set at +0.73 V relative to a Ag/AgCl reference electrode). The HPLC mobile phase (pH 2.7) consisted of 0.05 M sodium phosphate dibasic, 0.03 M citric acid, 0.1 mM EDTA, 0.6 mM sodium octyl sulfate and 15% methanol.

Monoamines and metabolites. Concentrations of dopamine, 5-HT and their respective metabolites were assessed using HPLC with electrochemical detection according to a modification of methods described previously (Nielson and Moore, 1982). Tissues were homogenized in buffer (0.15 M monochloroacetic acid, 0.2 mM EDTA, 108 µM octyl sulfate sodium and 12.5% methanol; pH 3.1) and centrifuged at 3300 × g for 30 min. The supernatant was filtered though 0.2-µm regenerated cellulose membranes, and 50 µl was injected onto a Whatman PartiSphere C18 column (5 µm); the mobile phase used was the same buffer as used for homogenizing brain tissue. The eluent was monitored using a glassy carbon electrode with a potential of +0.73V (vs. Ag/AgCl reference electrode). Where indicated, protein content was determined as described previously (Lowry et al., 1951).

NTLI. The NTLI content was determined by a modified solid-phase radioimmunoassay described by Maidment et al. (1991). Removable 96-well immunoplates (MaxSorb; Nunc, Naperville, CT) were incubated overnight at 4°C with a protein G solution (50 ng/100 µl in 0.1 M sodium bicarbonate; pH 9.0). This solution was aspirated, and the wells were washed three times with buffer (0.15 M K₂HPO₄, 0.02 M NaH₂PO₄, 0.2 mM ascorbic acid, 0.2% Tween-20, 0.1% sodium azide, pH 7.5). NT antiserum (Letter et al., 1986) was diluted 1:20,000 in assay buffer (i.e., wash buffer with 0.1% gelatin), and 50 µl was added to each well and incubated at room temperature for 4 hr to allow antibody attachment to the protein G-coated surface. After incubation, antiserum was washed from the wells. Samples (homogenized in 0.1 N HCl and then lyophilized) were resuspended in assay buffer, and 50 µl of samples or standards was added. Standards and samples were incubated at 4°C overnight. The next day, ¹²⁵I-NT (Dupont-New England Nuclear, Boston, MA) was diluted to 9000 dpm/100 µl in assay buffer, and 25 µl was added to each well and to four wells treated with only the protein G solution to assess nonspecific binding. Labeled NT was incubated in the wells for 2 hr. Wells were then washed, and radioactivity was counted on a four-channel -counter. This procedure allowed reliable detection of 250 fg of NT/sample.

[³H]Dopamine and [³H]5-HT uptake. Uptake of [³H]monoamines was determined according to a modification of a method described by Boja et al. (1992). Fresh tissue was homogenized in ice-cold 0.32 M sucrose and centrifuged (800 × g for 12 min at 4°C). The resulting supernatant was then centrifuged (22,000 × g for 10 min at 4°C), and the pellets were resuspended in ice-cold 0.32 M sucrose. Assays were conducted in modified Kreb's buffer (in mM: 126 NaCl, 4.8 KCl, 1.3 CaCl₂, 16 sodium phosphate, 1.4 MgSO₄, 11 dextrose, 1 ascorbic acid; pH 7.4). Each assay tube contained synaptosomal tissue obtained from 1.5 mg of striatal or 5 mg of hippocampal tissue for [³H]dopamine uptake and [³H]5HT uptake, respectively, and 1 mM pargyline. Nonspecific values were determined in the presence of 1 mM cocaine (for [³H]dopamine uptake) or 1 µM citalopram (for [³H]5-HT uptake). After preincubation of assay tubes for 10 min at 37°C, assays were initiated by the addition of [³H]dopamine or [³H]5-HT (0.5 nM or 5 nM final concentrations). Samples were incubated at 37°C for 3 min and then filtered through Whatman GF/B filters previously soaked in 0.05% polyethylenimine. Filters were washed rapidly three times with 5 ml of ice-cold 0.32 M sucrose using a Brandel filtering manifold. Radioactivity trapped in filters was counted using a liquid scintillation counter. Citalopram hydrobromide and pargyline hydrochloride were supplied kindly by H. Lundbeck and Co. and Abbott Laboratories (North Chicago, IL), respectively. [7,8-³H]Dopamine (43 Ci/mmol) and [³H]5-HT (30 Ci/mmol) were purchased from Amersham Life Sciences (Arlington Heights, IL) and New England Nuclear (Boston, MA), respectively.

Statistical analysis. Statistical analyses between two groups were conducted using a two-tailed Student's t test. Analyses among three or more groups were conducted using analysis of variance followed by Fisher's Least Significant Difference test. Differences among groups were considered significant if the probability of error was <5%.

	Results

Abstract Introduction Materials & Methods Results Discussion References

Results presented in figures 1 and 2 demonstrate that multiple administrations of methcathinone (30 mg/kg s.c., four administrations at 4-hr intervals) cause persistent impairment of monoaminergic systems in rat brain. For example, 30 days after treatment, tyrosine hydroxylase activity was reduced, with concurrent decreases in tissue concentrations of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid. Striatal tryptophan hydroxylase activity was likewise reduced, with concurrent decreases in striatal content of 5-HT and 5-HIAA (fig. 1). Striatal [³H]dopamine and hippocampal [³H]5-HT synaptosomal uptake were diminished by 31% and 52%, respectively (fig. 2). In separate experiments, similar effects on enzyme activity, monoamine and metabolite concentrations and synaptosomal uptake were observed 7 days after the last methcathinone administration (data not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Striatal dopaminergic and 5-hydroxytryptaminergic neurochemical parameters 30 days after multiple administrations of methcathinone. Rats received four injections of methcathinone (30 mg/kg s.c.; 4-hr intervals) or saline vehicle (1 ml/kg s.c., 4-hr intervals) and were decapitated 30 days after the last injection. Saline control values for tyrosine hydroxylase and tryptophan hydroxylase activity were 1793 ± 211 and 94.4 ± 15.5 nmol/hr/g of tissue, respectively. Striatal concentrations of dopamine, DOPAC, HVA, 5-HT and 5-HIAA were 22.7 ± 0.4, 0.64 ± 0.05, 0.35 ± 0.02, 0.51 ± 0.07 and 0.18 ± 0.02 µg/g of tissue, respectively. Columns represent mean ± 1 S.E.M. of determinations in five or six rats. *P < .05 vs. corresponding saline control.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Synaptosomal striatal [3H]dopamine and hippocampal [3H]5-HT uptake 30 days after multiple administrations of methcathinone. Rats received four injections of methcathinone (30 mg/kg s.c.; 4-hr intervals) or saline vehicle (1 ml/kg s.c., 4-hr intervals) and were decapitated 30 days after the last injection. Saline control values for [3H]dopamine uptake and [3H]5-HT uptake were 35.1 and 12.8 fmol/mg of tissue, respectively. Columns represent mean ± 1 S.E.M. of determinations in five or six rats. *P < .05 vs. corresponding saline control.

The role of dopamine in the methcathinone-induced impairment of aminergic neurons described above was first investigated by using microdialysis to assess the acute effect of a single methcathinone administration on extracellular dopamine concentrations (fig. 3). The administration of 1 mg/kg methcathinone increased extracellular dopamine levels to 419% of control values. The administration of 10 to 30 mg/kg comparably increased extracellular dopamine levels by 1100%. Because tissue NT concentrations have been demonstrated to reflect changes in extracellular dopamine concentrations (for review, see Kitabgi, 1989; Nemeroff, 1986; Nemeroff et al., 1983), the effects of methcathinone on striatal NTLI content were also assessed. A single administration of 10, 20 or 30 mg/kg methcathinone increased striatal tissue NTLI 75% relative to saline-treated controls (fig. 4).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Dose-response effects of methcathinone on striatal NTLI. Rats received a single injection of methcathinone (10-30 mg/kg s.c.) or saline vehicle (1 ml/kg s.c.) and were decapitated 18 hr later. The mean control NTLI level was 205 pg/mg of protein. Values represent mean ± 1 S.E.M. of determinations in six rats. *P < .05 vs. control.

The role of D₁ and D₂ dopamine receptors in methcathinone-induced monoaminergic neuronal impairment was determined by treatment with the D₁ receptor antagonist SCH23390 on the D₂ antagonist eticlopride before multiple methcathinone administrations and measuring changes in tyrosine hydroxylase and tryptophan hydroxylase activity. Pretreatment with either SCH23390 (0.5 mg/kg) or eticlopride (0.5 mg/kg) completely prevented the methcathinone-induced decrease in tyrosine hydroxylase activity (fig. 5). However, pretreatment with SCH23390 or eticlopride did not prevent the methcathinone-induced decrease in tryptophan hydroxylase activity (fig 6).

View larger version (41K): [in this window] [in a new window]   Fig. 5.   Effects of D1 and D2 dopamine receptor antagonists on methcathinone-induced changes in striatal tyrosine hydroxylase activity. Rats received four injections of methcathinone (30 mg/kg s.c.; 4-hr intervals) or saline vehicle (1 ml/kg s.c., 4-hr intervals) and were decapitated 18 hr after the last injection. SCH23390 (0.5 mg/kg i.p.) or eticlopride (0.5 mg/kg i.p.) was administered 5 min before each methcathinone injection. Saline control values were 1363 and 2050 nmol/hr/g of tissue in the SCH23390 and eticlopride experiments, respectively. Columns represent mean ± 1 S.E.M. of determinations in six rats. *P < .05 vs. corresponding saline control.

View larger version (35K): [in this window] [in a new window]   Fig. 6.   Effects of D1 and D2 receptor antagonists on methcathinone-induced changes in striatal tryptophan hydroxylase activity. Rats received four injections of methcathinone (30 mg/kg s.c.; 4-hr intervals) or saline vehicle (1 ml/kg s.c., 4-hr intervals) and were decapitated 18 hr after the last injection. SCH 23390 (0.5 mg/kg i.p.) or eticlopride (0.5 mg/kg i.p.) was administered 5 min before each methcathinone injection. Saline control values were 156 and 119 nmol/hr/g of tissue in the SCH23390 and eticlopride experiments, respectively. Columns represent mean ± 1 S.E.M. of determinations in six rats. *P < .05 vs. corresponding saline control.

The role of dopamine in the methcathinone-induced decrease in tryptophan hydroxylase activity was investigated further by assessing the effects of the stimulant in dopamine-depleted rats. In the ipsilateral striata of animals receiving unilateral 6-OHDA lesions of the medial forebrain bundle, multiple administrations of methcathinone (30 mg/kg s.c., four injections at 4-hr intervals) were without effect on tryptophan hydroxylase activity (fig. 7, right). In the contralateral striata, methcathinone decreased tryptophan hydroxylase activity (fig. 7, left).

View larger version (35K): [in this window] [in a new window]   Fig. 7.   Effects of dopamine depletion on methcathinone-induced changes in striatal tryptophan hydroxylase activity. Rats received four injections of methcathinone (30 mg/kg s.c.; 4-hr intervals) or saline vehicle (1 ml/kg s.c., 4-hr intervals) and were decapitated 18 hr after the last injection. 6-OHDA or ascorbate vehicle was administered unilaterally 14 days before methcathinone treatment as described in the text. Tryptophan hydroxylase activity was measured in both contralateral and ipsilateral striatal tissue relative to the 6-OHDA or vehicle injections. 6-OHDA lesion caused a >99% decrease in dopamine concentration in the ipsilateral striata. Saline control values were 60 and 90 nmol/hr/g of tissue in the contralateral and ipsilateral striata, respectively. Columns represent mean ± 1 S.E.M. of determinations in four to seven rats. *P < .05 vs. sham control.

	Discussion

Abstract Introduction Materials & Methods Results Discussion References

Because of its potential to become a widespread drug of abuse, the possibilities of irreversible or prolonged neurochemical effects due to methcathinone use are of great concern. We and others have demonstrated that multiple administrations of methcathinone profoundly decrease concentrations of monoamines, their metabolites and their synthetic enzymes (Gygi et al., 1996; Sparago et al., 1996). These effects persist up to 2 weeks after the last methcathinone injection. The present study extends these observations by demonstrating depletions of tissue concentrations of dopamine, 5-HT and their metabolites that persist as long as 30 days after drug administration (fig. 1). Striatal [³H]dopamine and hippocampal [³H]5-HT synaptosomal uptake are likewise decreased after multiple methcathinone injections (fig. 2), as are activities of tyrosine hydroxylase and tryptophan hydroxylase (fig. 1). The precise mechanism(s) responsible for these deficits are unknown; however, we hypothesize that dopamine is an important mediator because an intact dopamine system is necessary for toxicity effected by the related compounds methamphetamine and methylenedioxymethamphetamine (Nash et al., 1990; Schmidt et al., 1985).

Consistent with a role for dopamine in mediating methcathinone-induced monoaminergic impairment, results from the present study demonstrate that methcathinone administration increases extracellular dopamine concentrations in vivo (fig. 3). These data confirm in conscious animals previous in vitro findings in slice preparations that methcathinone causes dopamine release (Glennon et al., 1987). Increases in striatal extracellular dopamine concentrations were observed after the administration of 1, 10, 20 or 30 mg/kg s.c. Interestingly, the maximal increases in striatal dopamine at the three higher doses did not differ significantly from each other: a similar "plateau" effect has been observed previously when comparing extracellular dopamine concentrations after the administration of 6, 12 or 18 mg/kg s.c. methamphetamine (Kuczenski et al., 1995).

A single methcathinone administration increased striatal NTLI (fig. 4). More specifically, the administration of 10 to 30 mg/kg methcathinone effected increases in NTLI that did not differ significantly from each other. Because tissue NT concentrations purportedly reflect changes in extracellular dopamine concentrations (for review, see Kitabgi, 1989; Nemeroff, 1986; Nemeroff et al., 1983), the lack of difference in the NT response to administration of 10, 20 or 30 mg/kg s.c. is consistent with the similar increase in extracellular dopamine concentrations caused by these three doses of methcathinone (i.e., the "plateau" effect noted above) (fig. 3).

To investigate further the role of dopamine in mediating the effects of methcathinone, the ability of the selective D₁ antagonist SCH23390 and the selective D₂ antagonist eticlopride to prevent methcathinone-induced deficits in dopaminergic and 5-hydroxytryptaminergic neuronal function was assessed (figs. 5 and 6). The results reveal that both the D₁ and D₂ antagonists prevented the decreases in tyrosine hydroxylase activity observed 18 hr after multiple methcathinone administrations (fig. 5), thereby demonstrating a role for dopamine receptors in the dopaminergic neuronal deficits caused by methcathinone.

The administration of neither SCH23390 nor eticlopride prevented the decrease in tryptophan hydroxylase activity observed 18 hr after multiple injections of methcathinone (fig. 6). To investigate further the role of dopaminergic systems, we examined the effects of depletion of dopamine on the methcathinone-induced 5-hydroxytryptaminergic deficits. Unilateral lesioning of the medial forebrain bundle with 6-OHDA eliminated the dopaminergic input from the substantia nigra to the striatum on the side of the lesion as demonstrated by a >99% loss of striatal dopamine content relative to control rats (data not shown). As reported previously (Johnson et al., 1987; Stone et al., 1988), the 6-OHDA lesion per se tended to increase tryptophan hydroxylase activity: the mechanism responsible for this phenomenon is unknown, and this tendency necessitates caution when interpreting data. Nevertheless, 6-OHDA lesioning prevented the decrease in tryptophan hydroxylase activity caused by multiple methcathinone administrations as assessed in the striatum ipsilateral to the lesion (fig. 7, right). The finding that tryptophan hydroxylase activity in the striata contralateral to the lesion was reduced in all animals treated with methcathinone supports the conclusion that the 6-OHDA lesion selectively blocked the tryptophan hydoxylase decrease on the ipsilateral side (fig. 7, left). Taken together, these data suggest that dopaminergic neurons contribute to methcathinone-induced 5-hydroxytryptaminergic deficits but that blockade of D₁ or D₂ receptors separately is not sufficient to prevent the 5-hydroxytryptamin-ergic neuronal impairment caused by the stimulant. Further investigation into the mechanism by which dopamine mediates the methcathinone-induced effects on 5-hydroxytryptaminergic systems is required.

In summary, the present findings reveal that multiple administrations of methcathinone cause deficits in dopaminergic and 5-hydroxytryptaminergic systems that persist at least for 30 days. A single high-dose administration of methcathinone increases striatal dopamine release, as measured extracellularly in conscious animals and reflected by increases in striatal NTLI content. Dopaminergic deficits observed 18 hr after multiple administrations of methcathinone were prevented using selective D₁ or D₂ receptor antagonists, whereas 5-hydroxytryptaminergic deficits were unaffected. 5-Hydroxytryptaminergic changes were, however, prevented in animals depleted of striatal dopamine after a 6-OHDA lesion. These studies indicate that dopaminergic systems are profoundly affected by methcathinone administration and that dopamine likely contributes to the monoaminergic effects of this stimulant.