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TOXICOLOGY
Division of Pharmacology and Toxicology, College of Pharmacy, University of Texas at Austin, Austin, Texas
Received March 31, 2004; accepted May 27, 2004.
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Abstract |
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-methyldopamine [5-(GSyl)--MeDA] and 2,5-bis(glutathion-S-yl)--methyldopamine [2,5-bis(GSyl)--MeDA], metabolites of MDA and MDMA, are also selective serotonergic neurotoxicants and produce behavioral and neurochemical changes similar to those seen with MDA and MDMA. We now show that 5-(GSyl)--MeDA and 2,5-bis(GSyl)--MeDA are more potent than MDA and MDMA (Ki = 69, 50, 107, and 102 µM, respectively) at inhibiting 5-hy-droxytryptamine (serotonin) transport into SK-N-MC cells transiently transfected with the human serotonin transporter (hSERT). Moreover, 5-(GSyl)--MeDA and 2,5-bis(GSyl)--MeDA simultaneously stimulated dopamine (DA) transport into the hSERT-expressing cells, an effect attenuated by fluoxetine, indicating that stimulated DA transport was hSERT-dependent. Finally, 5-(GSyl)--MeDA and 2,5-bis(GSyl)--MeDA, and to a lesser extent MDA and MDMA, induced a concentration and time-dependent increase in reactive oxygen species (ROS) in both hSERT and human dopamine transporter-transfected cells. Fluoxetine attenuated the increase in ROS generation in hSERT-expressing cells. The results are consistent with the view that the serotonergic neurotoxicity of MDA and MDMA may be mediated by the metabolism-dependent stimulation of DA transport into hSERT-expressing cells and ROS generation by redox active catechol-thioether metabolites and DA.
; Commins et al., 1987). The recreational use of these amphetamines, especially MDMA, is prevalent (Johnston et al., 2000) despite warnings of damage to the central nervous system, (McCann et al., 1999; Parrott, 2000). MDA and MDMA are serotonergic neurotoxicants (Ricaurte et al., 2000), the long-term consequences of which are manifest as depletions in 5-HT and 5-hydroxyindoleacetic acid concentrations (Commins et al., 1987). In addition, inhibition of function (Fleckenstein et al., 1999) and loss of 5-HT transporters (SERT) (Battaglia et al., 1987; Aguirre et al., 1998) likely contributes to long-term impairments of the serotonergic system, including the degeneration of 5-HT axonal projections and nerve terminals (O'Hearn et al., 1988).
Interestingly, direct injection of MDA and MDMA into the brain fails to reproduce the acute or long-term neurotoxic effects evident after peripheral administration (Molliver et al., 1986; Paris and Cunningham, 1992; Esteban et al., 2001). Moreover, inhibition of cytochrome P450-mediated MDMA metabolism attenuates MDMA-induced neurotoxicity (Gollamudi et al., 1989). Therefore, systemic metabolism of the parent drugs likely plays an important role in the development of neurotoxicity (Esteban et al., 2001; for review, see Monks and Jones, 2002; O'Shea et al., 2002). Esteban et al. (2001) provide persuasive evidence that peripheral metabolism of MDMA is required for neurotoxicity. Sufficient amounts of MDMA were perfused into the hippocampus to achieve the range of concentrations observed after peripheral administration of neurotoxic doses of MDMA. After perfusion, acute monoamine release was observed in the absence of long-term depletions in 5-HT levels. These data are consistent with the hypothesis that peripheral generation of neurotoxic metabolites contributes to MDMA-induced serotonergic neurotoxicity. However, several MDMA metabolites, including -MeDA, fail to elicit 5-HT neurotoxicity (Zhao et al., 1992; Miller et al., 1997). -MeDA undergoes rapid oxidation to o-quinones, which are readily scavenged by glutathione (GSH) (Hiramatsu et al., 1990; Patel et al., 1991) to yield 5-(glutathion-S-yl)--MeDA [5-(GSyl)--MeDA] and 2,5-bis(glutathion-S-yl)--MeDA [2,5-bis(GSyl)--MeDA] (Miller et al., 1995; Fig. 1). These catechol-thioethers retain the electrophilic and redox properties of the parent catechols and produce neurotoxic responses similar to MDMA and MDA (Miller et al., 1995, 1997; Bai et al., 1999), suggesting that such metabolites contribute to the neurotoxicity of MDA and MDMA. Furthermore, 
-glutamyl transpeptidase is enriched in blood brain-barrier endothelial cells and catalyzes the first step in GSH and S-conjugate metabolism. The finding that the inhibition of -glutamyl transpeptidase potentiates both the brain uptake (Miller et al., 1996) and the neurotoxicity of systemically administered MDA and MDMA (Bai et al., 2001), suggests that the neurotoxicity is mediated in part by metabolites that are substrates for this enzyme.
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Although the importance of the SERT in MDA- and MDMA-induced neurotoxicity is well established, the precise function of the transporter remains elusive. Fluoxetine and citalopram, SERT inhibitors, protect against MDMA-induced neurotoxicity (Aguirre et al., 1998; Liechti et al., 2000; Sanchez et al., 2001), and MDMA-induced depletions of 5-HT are absent in SERT-deficient mice (Bengel et al., 1998), supporting the involvement of a functional SERT in MDMA-induced neurotoxicity. MDMA-induced serotonergic neurotoxicity also seems to be coupled to increases in dopamine (DA) release (Bankson and Cunningham, 2001). MDMA stimulates the release of DA (Gudelsky and Nash, 1996; Koch and Galloway, 1997), and functional SERTs are capable of transporting DA into 5-HT cells (Schmidt and Lovenberg 1985; Faraj et al., 1994). The subsequent monoamine oxidase (MAO)-B-mediated oxidation of DA within the 5-HT nerve terminal may contribute to MDMA-induced generation of reactive oxygen species (ROS; Sprague and Nichols, 1995; Sprague et al., 1998). Indeed, the generation of ROS plays an important role in MDMA-induced neurotoxicity (Colado et al., 1997). Consequently, antioxidants attenuate MDMA-induced neurotoxicity (Gudelsky, 1996; Shankaran et al., 2001), and overexpression of superoxide dismutase protects against the effects of MDMA (Cadet et al., 1995). Lipid peroxidation and protein nitration, morphological markers of ROS-induced damage, are also apparent after MDMA administration (Sprague and Nichols, 1995). The source of ROS generation induced by MDMA remains debatable, although two pathways seem possible: 1) metabolism of MDMA to redox active metabolites (Miller et al., 1995, 1996; Bai et al., 2001; Esteban et al., 2001), and 2) oxidation of DA (Sprague et al., 1998; Shankaran et al., 1999).
We herein demonstrate that thioether metabolites of -MeDA inhibit 5-HT transport into hSERT-transfected SK-N-MC cells more effectively than either MDA or MDMA and that they concomitantly stimulate the uptake of DA into hSERT-expressing cells. Moreover, thioether metabolites of -MeDA stimulate ROS generation in hSERT-transfected SK-N-MC cells in a hSERT-dependent manner. The combination of the oxidation of DA and of the thioether metabolites of -MeDA likely contributes to the generation of ROS within 5-HT neurons and to MDA- and MDMA-induced neurotoxicity.
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Materials and Methods |
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-MeDA and 2,5-bis(GSyl)--MeDA were synthesized and purified as described previously (Miller et al., 1995). Briefly, 5-(GSyl)--MeDA was prepared by reacting -MeDA (Merck Research Labs, Rahway, NJ) (2 mM), GSH (10 mM), and mushroom tyrosinase (100 U/ml) in 100 ml of sodium phosphate buffer (50 mM, pH 7.4). The product was purified by HPLC (LC-6A; Shimadzu, Kyoto, Japan) using an Ultrasphere ODS-5 reverse-phase semipreparative column (Beckman Coulter, Fullerton, CA). Fractions were collected at 
= 280 nm/retention time of 12 min. Collected fractions were combined, concentrated by rotary evaporation, frozen over dry ice/acetone, and lyophilized to dryness. The resulting powder was reanalyzed by HPLC and coulometric electrode array detection. 2,5-bis(GSyl)--MeDA was synthesized by dissolving 100 mg of 5-(GSyl)--MeDA in 100 ml of 10% formic acid. Sodium periodate (50 mg) was added to the solution to promote quinone formation before saturating the reaction with GSH. The resulting mixture was concentrated by rotary evaporation, frozen over dry ice/acetone, and lyophilized to dryness. The product was purified by HPLC, and the major UV-absorbing product was eluted with water. Fractions were collected at = 280/retention time of 11 min, rotary evaporated, and lyophilized to dryness. Reanalysis of the product by HPLC with UV and coulometric electrode array detection produced a single peak. All other reagents were purchased from commercial sources.
Transient Transfection of hSERT and hDAT in SK-N-MC Cells. SK-N-MC cells were purchased from the American Type Culture Collection (Manassas, VA). Cells were cultured in Eagle's minimal essential medium (American Type Culture Collection) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The parental cDNAs, hSERT, and hDAT, inserted into pCDNA 3.1 (Invitrogen, Carlsbad, CA), were used for transient expression of the transporter proteins. Transfections were accomplished using the Lipofectamine reagent (Invitrogen) following the protocol supplied by the manufacturer. Briefly, 24 h before transfection, cells were seeded in 24-well plates (
5 x 105 cells/well). Opti-MEM I reduced serum medium (Invitrogen) containing the cDNA was combined with the Lipofectamine reagent and incubated at room temperature for 30 min. Cells were washed with serum-free medium (Opti-MEM), and the cDNA-liposomal solution was added to each well. Cells were incubated with the cDNA complexes at 37°C, 5% CO2 for 5 h. Growth medium supplemented with 10% fetal bovine serum was added to each well, and the cells were incubated for 24 h before being replaced with Eagle's minimal essential medium. Expression of the transporter proteins was confirmed by Western analysis using polyclonal antibodies against the hDAT and hSERT (Research Diagnostics, Flanders, NJ) (Fig. 2) and cellular uptake assays. Transfected cells were used for experiments 48 to 72 h post-transfection.
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Cellular Neurotransmitter Uptake Experiments. Cellular uptake of [3H]DA and [3H]5-HT into hDAT- and hSERT-transfected SK-N-MC cells was measured 48 h post-transfection, as described previously (Mortensen et al., 2001). Several uptake experiments were conducted, and details for each experiment can be found in the figure legends. Briefly, cells were washed with Krebs-Ringer (KR) buffer (125 mM NaCl, 5 mM KCl, 25 mM HEPES, 6 mM glucose, 5 mM NaHCO3, 1.2 mM MgSO4·7H2O, 1.2 mM KH2PO4, 2.4 mM CaCl2·2H2O, pH, 7.4). Cells were then incubated in Krebs-Ringer buffer containing 20 nM [3H]5-HT, 100 µM pargyline, and 100 µM ascorbate plus MDA, MDMA, 5-(GSyl)--MeDA, or 2,5-bis(GSyl)--MeDA (100 µM). Cellular uptake was terminated by washing the cells with KR buffer and uptake of [3H]5-HT into mock-, hDAT-, and hSERT-transfected cells was determined by liquid scintillation spectroscopy (LS 5000TD; Beckman Coulter) and cpm normalized per cell number. Specific [3H]5-HT uptake is defined as that fraction of tritium uptake inhibitable by fluoxetine. The cellular uptake of [3H]DA into the hDAT-transfected cells was examined using a procedure similar to that used for [3H]5-HT, and specific uptake defined as that fraction of tritium uptake inhibitable by nomifensine.
The kinetics of 5-(GSyl)--MeDA- and 2,5-bis(GSyl)--MeDA-induced inhibition of 5-HT cellular uptake was examined with saturation and inhibition experiments. Saturation transport analysis was conducted by incubating the cells with increasing concentrations of substrate ([3H]5-HT) for 30 min in KR buffer containing 100 µM pargyline and 100 µM ascorbate. Nonspecific uptake was determined in parallel using fluoxetine (10 µM). Assays were terminated by washing the cells with KR buffer, and intracellular accumulation of [3H]5-HT was determined by liquid scintillation spectroscopy. For inhibition experiments, cells were incubated in KR buffer containing 20 nM [3H]5-HT and increasing concentrations of MDA, MDMA, 5-(GSyl)--MeDA, or 2,5-bis(GSyl)--MeDA for 30 min, and intracellular [3H]5-HT accumulation was determined. Assuming MichaelisMenten kinetics, substrate Km and inhibitor Ki values were determined by nonlinear least-squares curve fit (GraphPad Prism; GraphPad Software Inc., San Diego, CA; Adkins et al., 2001). Experiments were carried out in triplicate, and Ki values are presented as the mean (n = 4) ± the standard error of four independent transfections.
Evaluation of ROS Generation. 2',7'-Dichlorofluorescein diacetate (DCF-DA) is oxidized to the fluorescent 2',7'-dichlorofluorescein by cellular oxidants and is used as a marker for intracellular generation of ROS, in particular hydrogen peroxide and the hydroxyl radical. Intracellular ROS generation was monitored as described previously (Jones et al., 2000) with modifications. Transfected SK-N-MC cells were loaded with DCF-DA (final concentration of 10 µM) in KR buffer for 15 min in the dark. Cells were washed with KR buffer and exposed to MDA, MDMA, 5-(GSyl)--MeDA, or 2,5-bis(G-Syl)--MeDA in the presence or absence of the transporter blockers, nomifensine (DAT), and fluoxetine (SERT). Assays were terminated by washing cells with KR buffer and ROS generation was monitored at 475 nm (excitation) and 525 nm (emission) using a FL600 microplate fluorescence reader (Bio-Tek Instruments, Winooski, VT). Changes in fluorescence are expressed as percentage of control, and represent DCF fluorescence in treated samples minus the "background" fluorescence of the sample (cells treated with the Krebs-Ringer vehicle alone, minus drugs and metabolites, and without DCF-DA). Vehicle controls represent DCF fluorescence in cells to which only DCF-DA was added. This "vehicle control" fluorescence presumably reflects the inherent endogenous level of ROS production in these cells.
Statistics. Data are expressed as the mean ± S.E.M. A one-way analysis of variance was performed on the data and multiple pairwise comparisons were made using Student-Newman-Keuls tests. Differences within and between treatment groups were considered significant at p < 0.05.
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Results |
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-MeDA, and 2,5-bis (GSyl)--MeDA on the cellular uptake of 5-HT. Confirmation of protein expression was demonstrated by Western analysis and by quantifying the cellular uptake of [3H]5-HT and [3H]DA into hDAT- and hSERT-transfected cells (Fig. 2). Fluoxetine and nomifensine significantly inhibited [3H]5-HT and [3H]DA uptake into hSERT- and hDAT-transfected cells, respectively. The cellular uptake of 5-HT into untreated hSERT-expressing cells was rapid and accumulation continued for 8 h, before slowly returning to baseline levels by 48 h (Fig. 3). MDA, MDMA, 5-GSyl--MeDA, and 2,5-bis(GSyl)--MeDA maximally inhibited 5-HT uptake between 2 and 4 h after drug treatment (Fig. 3). After 4 h of exposure, MDA and MDMA inhibited 5-HT uptake by approximately 30% (Fig. 4). 5-GSyl--MeDA and 2,5-bis(GSyl)--MeDA were more potent inhibitors of 5-HT transport than either MDA or MDMA, inhibiting uptake by approximately 60 and 70%, respectively. Nomifensine, a selective DAT inhibitor, had no effect on 5-HT uptake into hSERT-transfected cells (Fig. 4) and did not influence drug or metabolite mediated inhibition of 5-HT uptake. In contrast, 100 µM fluoxetine inhibited 5-HT uptake into hSERT-transfected cells to a similar extent as 5-GSyl--MeDA and 2,5-bis(GSyl)--MeDA. None of the compounds tested had any effect on mock- (pcDNA) or hDAT-transfected cells (data not shown).
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Kinetic analysis of the inhibition of 5-HT uptake was performed on hSERT-transfected cells. Km and Vmax values for specific [3H]5-HT uptake were determined by saturation transport analysis using increasing concentrations of [3H]5-HT (Fig. 5A). Ki values for each of the compounds were determined by measuring uptake of a single concentration of [3H]5-HT (20 nM) and various concentrations of MDA, MDMA, 5-(GSyl)--MeDA, and 2,5-bis(GSyl)--MeDA. Consistent with the single concentration and time course results, 5-(GSyl)--MeDA, and 2,5-bis(GSyl)--MeDA were more potent at inhibiting [3H]5-HT uptake (Ki = 69 and 49 µM, respectively) than either MDA or MDMA (Ki = 107 and 102 µM, respectively) (Fig. 5B).
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5-(GSyl)--MeDA and 2,5-Bis(GSyl)--MeDA-Induced ROS Generation. MDA, MDMA, 5-(GSyl)--MeDA, and 2,5-bis(GSyl)--MeDA induce rapid ROS generation in hSERT- and hDAT-transfected SK-N-MC cells (Fig. 6). The rate of ROS generation declines rapidly after the initial burst and quickly returns to baseline levels, presumably due to either metabolism of the drugs and/or exhaustion of reducing equivalents required to support redox cycling. 5-(GSyl)--MeDA and 2,5-bis(GSyl)--MeDA were more efficient inducers of ROS generation, in comparison with MDA and MDMA, in both hSERT- and hDAT-transfected cells (Fig. 7). Although ROS generation was slightly greater in hSERT-transfected cells, the kinetics of ROS generation were similar in both cell types. None of the compounds tested had any effect on ROS generation in mock-transfected cells (data not shown), indicating the requirement for hSERT or hDAT for ROS generation. Interestingly, pretreatment with nomifensine had no effect on ROS generation in hDAT-transfected cells (Fig. 8B), despite the fact that nomifensine inhibited DA uptake into hDAT-transfected cells (Fig. 2), indicating that ROS generation, although hDAT-dependent, is insensitive to nomifensine. In contrast, fluoxetine significantly inhibited ROS generation in hSERT-transfected cells (Fig. 8A), indicating that MDA, MDMA, 5-(GSyl)--MeDA, and 2,5-bis(GSyl)--MeDA-induced ROS generation in hSERT-expressing cells requires a functional SERT.
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5-(GSyl)--MeDA- and 2,5-Bis(GSyl)--MeDA-Induced Stimulation of DA Uptake into hSERT-Transfected Cells. DA may contribute to the serotonergic neurotoxicity of MDMA via its ability to generate ROS and reactive quinones (Sprague et al., 1998; Shankaran et al., 1999). MDA, MDMA, 5-(GSyl)--MeDA, and 2,5-bis(GSyl)--MeDA all stimulated the cellular uptake of [3H]DA into hSERT-transfected cells (Fig. 9). 5-(GSyl)--MeDA and 2,5-bis(GSyl)--MeDA increased DA uptake 6- to 7-fold over control levels, whereas MDA and MDMA increased DA uptake by only 3.5-fold. The maximum stimulation of DA uptake occurred between 4 and 8 h (Fig. 9). Thus, the interaction of the thioether metabolites with the hSERT 1) inhibits the uptake of 5-HT and 2) stimulates the simultaneous uptake of DA into hSERT-expressing cells. Moreover, fluoxetine inhibited the uptake of DA-(Fig. 10), supporting the contention that DA is transported into hSERT-transfected cells via the hSERT. Subsequent MAO-mediated DA oxidation and metabolism may then contribute to the generation of ROS and to the serotonergic neurotoxicity of MDA and MDMA.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-MeDA and 2,5-bis(GSyl)--MeDA stimulate the transport of DA into hSERT-expressing SK-N-MC cells (Fig. 9), while simultaneously inhibiting 5-HT uptake (Figs. 3, 4, 5). The stimulation of DA uptake into "serotonergic" cells is a significant finding, because although a relationship between DA- and MDMA-induced serotonergic neurotoxicity has long been appreciated, the mechanisms underlying this relationship are unknown. Consistent with the hypothesis that systemic metabolism is required for MDA and MDMA neurotoxicity, 5-(GSyl)--MeDA- and 2,5-bis(GSyl)--MeDA-stimulated uptake of DA into hSERT-expressing SK-N-MC cells was greater than that caused by the parent amphetamines. Fluoxetine attenuated the stimulation of DA uptake (Fig. 10), indicating that DA entered the cell via the hSERT. Indeed, functional SERT proteins are capable of transporting DA into 5-HT cells (Schmidt and Lovenberg, 1985; Faraj et al., 1994). Although the mechanism by which MDMA/MDA and the metabolites stimulate DA uptake by the hSERT is not known, Saldana and Barker (2004) have recently reported that elevated temperature alters SERT-mediated 5-HT and DA transport, such that the relative selectivity of the SERT for DA increases significantly at the higher temperature. Thus, drug and/or metabolite-induced alterations in hSERT structure may similarly alter the relative preference of the transporter for DA and 5-HT.
The importance of the SERT in contributing to MDA- and MDMA-induced neurotoxicity is firmly established (Shankaran et al., 1999; Liechti et al., 2000), although the precise role of the SERT remains unclear. MDMA-induced depletion of 5-HT and hyperactivity are both absent in SERT-deficient mice (Bengel et al., 1998), suggesting that a functional SERT is essential for MDMA-induced neurotoxicity. The SERT inhibitor fluoxetine protects against MDMA-induced ROS generation (Shankaran et al., 1999, 2001) and neurotoxicity (Aguirre et al., 1998; Sanchez et al., 2001) and citalopram attenuates the effects of MDMA in humans (Liechti et al., 2000), suggesting that the SERT participates in MDMA-mediated cellular oxidant generation and neurotoxicity. Our data are also consistent with an important role for the SERT in MDA and MDMA induced neurotoxicity. For instance, 5-(GSyl)--MeDA and 2,5-bis(GSyl)--MeDA significantly inhibited [3H]5-HT transport into hSERT-transfected cells (Fig. 3) and to a greater extent than that produced by either MDA or MDMA. Indeed, the Ki for the thioether metabolites were substantially less than that for either MDA or MDMA (Fig. 5). The interaction between MDMA/MDA and the SERT in vivo may therefore involve both direct (MDMA) and indirect (metabolite) effects. Fluoxetine potentiated MDA, MDMA, 5-(GSyl)--MeDA and 2,5-bis(GSyl)--MeDA inhibition of 5-HT uptake, suggesting the compounds act in synergy with fluoxetine, although the precise location and nature of this interaction with hSERT is unknown. However, the electrophilic nature of 5-(GSyl)--MeDA and 2,5-bis(GSyl)--MeDA provides a means by which the metabolites may interact with nucleophilic sites in the SERT, such as the multiple cysteine residues found in the extracellular domain (Chen et al., 1998), to modify SERT function.
Inhibition of SERT function has been attributed to MDMA-induced ROS generation (Sprague and Nichols, 1995; Falk et al., 2002), whereas, conversely, an increase in ROS generation may be a consequence of MDMA-induced SERT inhibition (Shankaran et al., 1999, 2001). In accord with previous reports (Shankaran et al., 1999, 2001), our data demonstrate that MDA and MDMA induced significant increases in ROS generation in hSERT-expressing cells (Fig. 6). However, 5-(GSyl)--MeDA and 2,5-bis(GSyl)--MeDA were more effective ROS generators than either MDA or MDMA, supporting the view that systemic metabolism contributes to the serotonergic neurotoxicity of the parent drugs. Moreover, fluoxetine attenuated the increase in ROS generation in hSERT-expressing cells (Fig. 8), indicating that MDA, MDMA, 5-(GSyl)--MeDA, and 2,5-bis(GSyl)--MeDA induced ROS generation in serotonergic cells is SERT-dependent.
DA oxidation and the subsequent generation of ROS have been implicated in MDMA-induced serotonergic neurotoxicity (Sprague and Nichols, 1995; Aguirre et al., 1998; Sprague et al., 1998; Shankaran et al., 1999; Bankson and Cunningham, 2001), although the mechanism by which MDMA produces these effects is not known. Both DA (Simantov and Tauber, 1997) and L-dopa (Schmidt et al., 1991; Aguirre et al., 1998) potentiate the neurotoxicity of MDMA. Further more, inhibition of MAO-B, which metabolizes DA within the serotonin cell, protects against serotonergic cell damage (Sprague and Nichols, 1995; Falk et al., 2002) presumably by preventing the oxidation of DA and the subsequent ROS generation. We have now shown that not only can the hSERT modestly transport dopamine into hSERT-expressing cells but also that such hSERT-mediated DA transport is greatly stimulated by 5-(GSyl)--MeDA and 2,5-bis(GSyl)--MeDA, and to a lesser extent by MDMA and MDA (Figs. 9 and 10). These data may provide the crucial link between MDMA- and MDA-mediated serotonergic neurotoxicity and the long recognized requirement for DA for these effects. However, we note that the maximum stimulation of DA uptake by 5-(GSyl)--MeDA and 2,5-bis(GSyl)--MeDA occurred subsequent to maximum ROS generation. This is likely a consequence of the rapid depletion of reducing equivalents, required to support redox cycling, which typically occurs in cultured cells. Nonetheless, the data do reveal a second wave of ROS generation in hDAT-transfected cells (Fig. 6) concomitant with maximum drug-induced DA uptake (Fig. 9). DA uptake and oxidation within serotonergic neurons may thus contribute to sustained ROS generation subsequent to that initially catalyzed by MDA, MDMA, and their metabolites. MDMA-induced ROS generation therefore probably involves multiple mechanisms, including the oxidation and redox cycling of 5-(GSyl)--MeDA and 2,5-bis(GSyl)--MeDA (Hiramatsu et al., 1990; Miller et al., 1996; Bai et al., 1999) and the oxidation of DA within serotonergic nerve terminals (Sprague and Nichols, 1995; Aguirre et al., 1998; Sprague et al., 1998) after drug and/or metabolite stimulated DA uptake.
Interestingly, neither MDMA/MDA- nor 5-(GSyl)--MeDA- and 2,5-bis(GSyl)--MeDA-stimulated ROS generation in hDAT-expressing cells was inhibited by nomifensine (Fig. 8B). Moreover, nomifensine failed to inhibit MDMA/MDA- or 5-(GSyl)--MeDA- and 2,5-bis(GSyl)--MeDA-stimulated DA uptake into hDAT-expressing cells (data not shown). We speculate that exposure of the hDAT to MDA/MDMA/metabolite causes physical alterations in the transporter that render it insensitive to nomifensine. Consistent with this view, the effects of MDMA on DA uptake in striatal synaptosomes were insensitive to the DAT ligand 2-
-carbomethoxy-3--(4-fluorophenyl)tropane 1,5-naphthalenedisulfonate (WIN35428 (Hansen et al., 2002), further suggesting that MDMA interacts with the DAT in a novel manner insensitive to modulation by classical inhibitors of DAT function.
In summary, 5-(GSyl)--MeDA and 2,5-bis(GSyl)--MeDA stimulate the transport of DA into hSERT-expressing SK-N-MC cells, while simultaneously inhibiting 5-HT uptake. The findings provide a possible mechanism underlying the interaction between the dopaminergic and serotonergic neurotransmitter systems during MDA- and MDMA-mediated neurotoxicity. In essence, the systemic metabolism of the parent amphetamines likely contributes to the serotonergic neurotoxicity by compromising SERT function and stimulating the uptake of DA into 5-HT cells, where it subsequently undergoes oxidation. Finally, the demonstration that 5-(GSyl)--MeDA and 2,5-bis(GSyl)--MeDA can inhibit hSERT-mediated 5-HT uptake not only illustrates the pharmacological properties of these metabolites but is consistent with their in vivo properties.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: MDA, 3,4-methylenedioxyamphetamine; MDMA, 3,4-methylenedioxymethamphetamine (ecstasy); 5-HT, 5-hydroxytryptamine (serotonin); SERT, serotonin transporter; GSH, glutathione; 5-(GSyl)--MeDA, 5-(glutathion-S-yl)--methyldopamine; 2,5-bis(GSyl)--MeDA, 2,5-bis(glutathion-S-yl)--methyldopamine; DA, dopamine; MAO, monoamine oxidase; ROS, reactive oxygen species; HPLC, high-performance liquid chromatography; hSERT, human serotonin transporter; hDAT, human dopamine transporter; DAT, dopamine transporter; KR, Krebs-Ringer; DCF-DA, dichlorofluorescein diacetate; DCF, dichlorofluorescein.
1 Current address: Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona Health Sciences Center, 1703 E. Mabel St., Tucson, AZ 85721-0207. 
Address correspondence to: Dr. Terrence J. Monks, Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona Health Sciences Center, 1703 E. Mabel St., Tucson, AZ 85721-0207. E-mail: scouser{at}pharmacy.arizona.edu
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), MDA- (100 µM, 
), MDMA- (100 µM, 
), 5-(GSyl)-
), or 2,5-bis(GSyl)-
)-treated SK-N-MC cells at various times (0.5, 1, 2, 4, 8, 16, 24, and 48 h) by liquid scintillation spectroscopy. Data are plotted as the percentage of specific (fluoxetine inhibitable) 5-HT uptake and expressed as the mean (n = 4) ± S.E.M. Differences between 1) control and treatment groups (*) and 2) MDMA/MDA and metabolite groups (
) are considered significant at p < 0.05.
, significantly different from untreated and nomifensine treated groups at p < 0.05.
), 5-(GSyl)-
