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Chem. Res. Toxicol. 2001, 14, 1184-1192
A Putative Metabolite of Serotonin, Tryptamine-4,5-Dione, Is an Irreversible Inhibitor of Tryptophan Hydroxylase: Possible Relevance to the Serotonergic Neurotoxicity of Methamphetamine Monika Z. Wrona and Glenn Dryhurst* Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019 Received February 16, 2001
Tryptamine-4,5-dione (T-4,5-D) is formed as a result of oxidation of 5-hydroxytryptamine by superoxide (O2-‚), nitric oxide (NO‚), and peroxynitrite (ONOO-). T-4,5-D rapidly inactivates tryptophan hydroxylase (TPH), derived from rat brain, probably as a result of covalent modification of active site cysteine residues. The activity of TPH exposed to T-4,5-D cannot be restored by anaerobic reduction with dithiothreitol (DTT) and ferrous iron (Fe2+) indicating that the inactivation is irreversible. 7-S-Glutathionyl-tryptamine-4,5-dione, formed by the rapid reaction between T-4,5-D and glutathione, also inhibits TPH but in this case the activity is restored by anaerobic reduction with DTT/Fe2+. The results of this investigation may be relevant to the initial reversible and subsequent irreversible inactivation of TPH evoked by methamphetamine and 3,4-methylenedioxymethamphetamine. Methamphetamine (MA)1 can evoke neurotoxic effects on serotonergic and dopaminergic neurons in selected regions of the brain (1). This neurotoxicity is not mediated directly by MA or its known metabolites (2), and the underlying mechanisms are unknown. Nevertheless, a number of processes have been implicated as key steps in the serotonergic and dopaminergic neurotoxicity of MA. These include a rapid but transient MA-induced impairment of serotonergic and dopaminergic neuron ATP production (3-5) caused by inhibition of mitochondrial complex IV (5), hyperthermia (6, 7), activation of poly(ADP-ribose)polymerase (PARP) (8), and other metabolic perturbations (9). This MA-induced energy impairment and resultant neuronal depolarization (10), interference with the vesicular storage of 5-hydroxytryptamine (5-HT) and dopamine (DA) (11, 12) and reversal of the DA transporter (DAT) and 5-HT transporter (5-HTT) (13-15) together mediate a massive release of 5-HT and DA (14, 16). This release of 5-HT and DA is only transient. Thus, after reaching peak levels, extracellular concentrations of 5-HT and DA fall but without corresponding increases of their major metabolites 5-hydroxy* To whom correspondence should be addressed. Phone: (405) 3254811. Fax: (405) 325-6111. E-mail:
[email protected]. 1 Abbreviations: MeCN, acetonitrile; CySH, cysteine; COX, cytochrome c oxidase; 4,5-DHT, 4,5-dihydroxytryptamine; 6,7-di-S-GS-T4,5-D, 6,7-di-S-glutathionyl-tryptamine-4,5-dione; DTNB, 5,5’-dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol; DAQ, dopamine-o-quinone; Glu, L-glutamate; GSH, glutathione; 7-S-GS-4,5-DHT, 7-S-glutathionyl4,5-dihydroxytrypamine; 7-S-GS-T-4,5-D, 7-S-glutathionyl-tryptamine4,5-dione; HPLC, high-performance liquid chromatography; HPLCECox, HPLC with oxidative electrochemical detection; HPLC-ECRed, HPLC with reductive electrochemical detection; NSD-1015, 3-hydroxybenzylhydrazine; HO‚, hydroxyl radical; 5-HT, 5-hydroxytryptamine; 5-HTT, 5-hydroxytryptamine plasma transporter; 5-HTP, L-5-hydroxytryptophan; MA, methamphetamine; MDMA, 3,4-methylenedioxymethamphetamine; BH4, D,L-6-methyl-5,6,7,8-tetrahydrobiopterin; AMT, R-methyltyrosine; NMDA, N-methyl-D-aspartate; NO‚, nitric oxide; nNOS, neuronal nitric oxide synthase; ONOO-, peroxynitrite; PARP, poly(ADP-ribose)polymerase; RNS, reactive nitrogen species; ROS, reactive oxygen species; -SH, sulfhydryl group; O2-‚, superoxide radical anion; T-4,5-D, tryptamine-4,5-dione; TPH, tryptophan hydroxylase.
indole-3-acetic acid and 3,4-dihydroxyphenylacetic acid, respectively (17). Such observations suggest that as the transient MA-induced energy impairment begins to subside increasing ATP production initiates repolarization of neuronal membranes which returns the reversed 5-HTT and DAT to their normal function with resultant reuptake of released 5-HT and DA, respectively (18). Under conditions of profoundly impaired neuronal energy metabolism, the voltage-dependent Mg2+ block of N-methyl-D-aspartate (NMDA) receptor ion channels is relieved and they can be activated by endogenous extracellular L-glutamate (Glu). This, in turn, mediates an influx of Ca2+ through the receptor ion channel (19) with resultant intraneuronal superoxide (O2-‚) production (20), neuronal nitric oxide synthase (nNOS) activation and consequent nitric oxide (NO‚), and thence peroxynitrite (ONOO-) generation (21, 22). Indeed, NMDA receptor activation (23, 24), elevated intraneuronal O2-‚ production (25), activation of nNOS (26) and NO‚ and ONOOgeneration (27, 28) are all implicated in the serotonergic and dopaminergic neurotoxic mechanisms triggered by MA. Furthermore, ONOO--mediated DNA damage with resultant activation of the repair enzyme PARP contributes significantly to the depletion of intraneuronal ATP evoked by MA (8). Many of the factors implicated with MA neurotoxicity have also been associated with the neurotoxic effects of 3,4-methylenedioxymethamphetamine (MDMA). However, in most species, MDMA is a selective serotonergic neurotoxin (9, 29, 30). A single dose of MA or MDMA evokes a rapid decrease of tryptophan hydroxylase (TPH) activity that subsequently recovers in vivo or can be restored in vitro by anaerobic reduction with dithiothreitol (DTT) and ferrous iron (Fe2+) (29-32). However, these and other neurotoxic amphetamines have no direct effect on TPH activity in vitro (33). One interpretation of these observations is that MA and MDMA mediate oxidation of active site cysteine (CySH) residues of TPH by reactive oxygen species (ROS)
10.1021/tx010037c CCC: $20.00 © 2001 American Chemical Society Published on Web 08/04/2001
Tryptophan Hydroxylase Inhibition by Tryptamine-4,5-dione
and/or reactive nitrogen species (RNS) forming protein disulfide linkages that can be reduced both in vivo and in vitro thus restoring activity (30-33). However, at longer times, particularly following multiple or large acute doses of MA or MDMA, the activity of TPH cannot be restored in vivo or in vitro implying the development of irreversible enzyme damage (30-33). Both NO‚ (34) and ONOO- (35) can irreversibly inactivate TPH in vitro, suggesting that these reactive RNS may be directly responsible for the delayed irreversible inhibition of this enzyme evoked by MA and MDMA in vivo. However, other factors implicated with MA- and MDMA-induced irreversible inhibition of TPH and serotonergic neurotoxicity are difficult to reconcile with this suggestion. For example, both the DA synthesis inhibitor R-methyltyrosine (AMT) (36, 37) and 5-HT uptake inhibitors such as fluoxetine (38, 39) block MA- and MDMA-induced reversible and irreversible decreases of TPH activity and serotonergic neurotoxicity. Indeed, fluoxetine reverses the decline of TPH activity even when administered a few hours after MDMA administration (33). These findings have led to the suggestion that DA, released by MA and MDMA (40), may compete with released 5-HT for the 5-HTT for entry into serotonergic neurons and, once inside, contributes to damage to these neurons (36, 41). Indeed, the ability of MA and MDMA to mediate generation of intraneuronal O2-‚ (25, 42) and ONOO- (27, 28, 43) that both oxidize DA to dopamine-o-quinone (DAQ) (44, 45) which, in vitro, irreversibly inactivates TPH by covalent attachment to active site CySH residues (46) provides some support for a possible role of DA in the serotonergic neurotoxicity of these drugs. However, other lines of evidence argue against a role for DA in MA- and MDMA-induced irreversible inhibition of TPH and serotonergic neurotoxicity. For example, when coadministered with MA or MDMA, AMT blocks hyperthermia normally evoked by these drugs which is known to be a key factor associated with their neurotoxicity (47-49). Furthermore, MA-induced decreases of TPH activity and damage to serotonergic neurons occurs not only in brain regions innervated with DA terminals but also in structures having little or no dopaminergic input (1). Finally, a recent study indicates that the 5-HTT is not able to mediate DA uptake (38). Nevertheless, fluoxetine blocks MA- and MDMA-induced decreases of TPH activity and damage to serotonergic terminals without affecting hyperthermia evoked by these drugs (38, 39). This raises the possibility that the 5-HTT-mediated reuptake of released 5-HT and its intraneuronal oxidation by ROS/ RNS as a MA- or MDMA-induced serotonergic energy impairment subsides may result in an endogenously formed intraneuronal toxin that contributes to TPH inhibition and neuronal damage. A major product of the in vitro oxidation of 5-HT by O2-‚ (50), NO‚, and ONOO(unpublished results) is tryptamine-4,5-dione (T-4,5-D, Scheme 1). Furthermore, T-4,5-D reacts rapidly with the cysteinyl -SH residue of GSH forming 7-S-glutathionyl4,5-dihydroxytryptamine (7-S-GS-4,5-DHT) which is readily autoxidized to 7-S-glutathionyl-T-4,5-D (7-S-GST-4,5-D) (Scheme 1) (51). Thus, the ROS/RNS-mediated oxidation of 5-HT as it returns via the 5-HTT into the cytoplasm of serotonergic terminals as a MA- or MDMAinduced energy impairment begins to subside might form T-4,5-D that may inactivate TPH by covalent modification of active site CySH residues. In this communication we report that T-4,5-D irreversibly inactivates TPH. 7-S-
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GS-T-4,5-D also inhibits TPH although this inactivation can be largely reversed by anaerobic reduction with DTT/Fe2+.
Materials and Methods Chemicals. D- and L-Tryptophan, L-5-hydroxytryptophan (5HTP), 5-HT, d,L-6-methyl-5,6,7,8-tetrahydrobiopterin (BH4), D,LDTT, catalase (EC.1.11.1.6 from bovine liver), Sephadex G-25 and 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB) were obtained from Sigma (St. Louis, MO), 3-hydroxybenzylhydrazine (NSD1015) from RBI (Natick, MA), and ethylene glycol-bis(β-aminoethyl ether)-N,N,N1,N1-tetraacetic acid (EGTA) from Aldrich (Milwaukee, WI). The synthesis and spectroscopic evidence for the structures of T-4,5-D and 7-S-GS-T-4,5-D have been described in detail elsewhere (50, 52, 53). HPLC with Electrochemical Detection (HPLC-EC). Two HPLC systems equipped with glassy carbon detector electrodes were employed. One, with the detector electrode set in the oxidative mode (HPLC-ECox), was employed to quantitate 5-HTP in the method used to assay for TPH activity. The other, equipped with a glassy carbon detector electrode set in the reductive mode (HPLC-ECRed), was used to determine T-4,5-D and 7-S-GS-T-4,5-D. HPLC-ECox. This system consisted of a Gilson (Middleton, WI) model 307 pump, a Rheodyne (Cotati, CA) 7125 injector with a 5.0 µL sample loop, a BAS (Bioanalytical Systems, West Lafeyette, IN) LC-4C amperometric detector equipped with a glassy carbon detector electrode set at + 800 mV vs a Ag/AgCl reference electrode, a reversed-phase column (BAS Phase II-ODS, 3 µm, 100 × 3.2 mm) and guard column (BAS Phase II-ODS, 7 µm, 15 × 3.2 mm). The mobile phase was 0.85% (v/v) diethylamine, 0.63 mM Na2EDTA, 0.26 mM sodium octyl sulfate, 0.1 M citric acid dissolved in deionized water containing 5% (v/v) HPLC-grade acetonitrile (MeCN) having a pH of 2.20. The flow rate was 0.6 mL min-1. The detector response for 5-HTP was linear between 0.02 and 1.7 ng/5 µL injection with a signal-to-noise ratio ) 2. HPLC-ECRed. A BAS 200B instrument was used equipped with a Rheodyne 9125 injection valve (5.0 µL sample loop) and dual parallel thin-layer glassy carbon detector electrodes. One electrode was set at -50 mV and the other at -550 mV vs Ag/AgCl. The entire system was purged with ultrahigh purity He for 2 h to remove molecular oxygen. The exhaust valve was then closed and the system pressurized at 4 psi with He. The mobile phase, detector oven, cell and column were maintained at 35 ( 0.1 °C. The analytical and guard columns were the same as used with the HPLC-ECox system. The mobile phase was the same as used for HPLC-ECox except it contained 7.5% (v/v) MeCN and the flow rate was 1.0 mL min-1. Quantitative determinations of T-4,5-D and 7-S-GS-T-4,5-D were based on linear calibration curves prepared as described previously (50). TPH Assay. Soluble protein containing TPH (EC 1.14.16.4) was obtained from the brains of male albino Sprague-Dawley rats (Harlan Sprague-Dawley, Madison, WI) weighing approximately 300 g. Frozen brain tissue was weighed and then homogenized with a Brinkman PT1200 Polytron in 2 parts (w/v) of ice-cold 50 mM sodium phosphate buffer (pH 7.4) containing 1 mM EGTA and 1 mM DTT. The homogenate was centrifuged (35000g, 4 °C, 40 min). The clear pink supernatant was then further purified on a column of Sephadex G-25 (30 × 1.5 cm), using 50 mM phosphate buffer (pH 7.4) as the mobile phase, to remove GSH and other low molecular weight compounds (54). The pink band which eluted in the void volume was collected and used as the source of soluble GSH-free TPH. Protein content was determined by the biuret assay method (55). Approximately, 130 µg of the GSH-free rat brain protein preparation was first incubated for a predetermined time at 37 °C in the presence of catalase (900 units; 340 µg) and different concentrations of T-4,5-D or 7-S-GS-T-4,5-D in 225 µL of phosphate buffer (pH 7.4). Positive controls contained no T-4,5-D or 7-S-GS-T-4,5-D. Then, in the following sequence, was added
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Chem. Res. Toxicol., Vol. 14, No. 9, 2001
Wrona and Dryhurst Scheme 1
25 µL each of NSD-1015 (3.0 mM; aromatic amino acid decarboxylase inhibitor), BH4 (1.2 mM), and L-tryptophan (6.0 mM) all dissolved in 50 mM phosphate buffer (pH 7.4) except BH4 which was dissolved in HCl/H2O (pH 3) and maintained icecold in order to minimize autoxidation. For negative controls, L-tryptophan was replaced by D-tryptophan. The resulting solution was vortexed and then incubated for 1 h at 37 °C. The final volume of the assay solution was 300 µL and final concentrations were NSD-1015 (0.25 mM), BH4 (0.1 mM) and L- (or D-)tryptophan (0.5 mM). The reaction was terminated by addition of 25 µL of 60% HClO4 and the mixture was centrifuged (17500g, 4 °C, 25 min). An aliquot (5 µL) of the supernatant was injected into the HPLC-ECox system to determine the
concentration of 5-HTP. The activity of TPH was calculated as pmol 5-HTP formed/mg protein/h. The assay was run under saturating concentration of tryptophan. The apparent Km for tryptophan in these experiments was 109.5 ( 31 µM (mean ( SD; n ) 4). All assays were run as triplicates except negative controls which were duplicates. Reactivation of Inhibited TPH. TPH samples inhibited by T-4,5-D, 7-S-GS-T-4,5-D, and/or air-deactivated were exposed to DTT (5 mM) and Fe2+ (50 µM) according to a procedure similar to that described by Stone et al. (30). Briefly, 50 µL of TPH samples (ca. 130 µg of protein) were placed in 20 vials containing 25 µL of catalase (900 units) in 50 mM phosphate buffer (pH 7.4). To 10 of these vials was added 110 µL of T-4,5-D
Tryptophan Hydroxylase Inhibition by Tryptamine-4,5-dione
Figure 1. Changes in T-4,5-D concentration during incubation with the GSH-free rat brain protein preparation used as a source of TPH. Concentrations at each time point are expressed as percentage of the initial T-4,5-D concentration (14 µM). (2) T-4,5-D in homogenization buffer (pH 7.4) containing no protein; (9) T-4,5-D in the presence of 0.64 mg mL-1 protein. The concentration of T-4,5-D was determined by HPLC-ECRed with the glassy carbon detector electrode set at -50 mV vs Ag/AgCl. or 7-S-GS-T-4,5-D (30 µM) in 50 mM phosphate buffer (pH 7.4). A total of 110 µL of phosphate buffer alone was added to the remaining 10 vials. All vials were vortexed and incubated for 20 min at room temperature. The solutions in five experimental and five control vials were assayed for TPH activity as described above (triplicates with L-tryptophan, duplicates with D-tryptophan). To the remaining 10 vials were added 20 µL each of DTT and Fe2+ to bring the final concentrations to 5 mM and 50 µM, respectively. These vials were placed in a Nalgene plastic desiccator at room temperature. The covered desiccator was partially evacuated for 10 min and then purged with nitrogen (ca. 5 psi) for 22-24 h. The vials were then removed and immediately assayed for TPH activity as described previously. Measurement of Free Sulfhydryl (-SH) Groups of Proteins. Free -SH groups in the GSH-free soluble rat brain protein preparation employed as a source of TPH was determined with Ellman’s reagent (56). Briefly, a 50 µL aliquot of the protein preparation, containing approximately 130 µg of protein, was added to 980 µL of 80 mM phosphate buffer (pH 8.0). Then, 50 µL of a DTNB solution (400 mg of DTNB in 100 mL of 80 mM phosphate buffer, pH 8.0) was added. After 15 min at room temperature, the absorbance at 410 nm was measured. In experiments with TPH inhibitors, T-4,5-D or 7-SGS-T-4,5-D was preincubated with the protein preparation in 980 µL of phosphate buffer for a predetermined time after which 50 µL of the DTNB reagent solution was added. Controls contained no T-4,5-D or 7-S-GS-T-4,5-D. All samples were analyzed as triplicates. Statistics. TPH inhibition data were analyzed by nonlinear regression (57) using GraphPad Prism 2.0 software (GraphPad, San Diego, CA). IC50 values were determined from the best fit of experimental data to a general sigmoidal dose-response equation. Data are presented as mean ( standard deviation (SD). Differences between groups were analyzed by the twotailed t-test and were considered statistically significant for p < 0.05.
Results Interaction of T-4,5-D and 7-S-GS-T-4,5-D with TPH. Previous studies have established that T-4,5-D reacts rapidly with GSH forming 7-S-GS-T-4,5-D (Scheme 1). Accordingly, to investigate the interaction between T-4,5-D and TPH, the protein preparation used in this study as the source of TPH was filtered through Sephadex G-25 to remove GSH and other low molecular weight species. Figure 1 shows representative time-dependent
Chem. Res. Toxicol., Vol. 14, No. 9, 2001 1187
Figure 2. Effects of (O) T-4,5-D and (2) 7-S-GS-T-4,5-D on the activity of TPH using the assay conditions described in Materials and Methods. T-4,5-D and 7-S-GS-T-4,5-D were incubated with the GSH-free rat brain protein preparation used as the source of TPH for 8 and 20 min, respectively, prior to assaying for TPH activity. Each data point is the mean ( SD of three experiments run in triplicate.
changes in the concentration of T-4,5-D when incubated with the GSH-free protein preparation and in the homogenization medium (pH 7.4) containing no protein. These results indicate that at pH 7.4 in the absence of protein low concentrations of T-4,5-D (14 µM) are relatively stable. However, in the presence of the GSH-free protein preparation the concentration of T-4,5-D decreased to
