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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 10, 2006 as doi:10.1096/fj.05-4996fje. |
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* Department of Brain Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan; and
Department of Medical Technology, Nagoya University Graduate School of Medicine, Nagoya, Japan
1Correspondence: Department of Brain Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikatacho, Okayama 700-8558, Japan. E-mail: asachan{at}cc.okayama-u.ac.jp
SPECIFIC AIMS
Methamphetamine (METH) is a drug of abuse that causes damage to striatal dopaminergic nerve terminals. Several studies have demonstrated that endogenous dopamine (DA) plays an important role in mediating METH-induced neuronal damage. Recently, the neurotoxicity of DA quinone formation by auto-oxidation of DA has focused on dopaminergic neuron-specific oxidative stress. The aim of this study was to clarify the involvement of DA quinone formation in METH-induced dopaminergic neurotoxicity and possible modulating or protective effects of quinone formation-related molecules, quinone reductase inducer and tyrosinase, using METH-treated dopaminergic CATH.a cells and METH-injected mouse brain.
PRINCIPAL FINDINGS
1. DA quinone formation is involved in the METH-induced dopaminergic neurotoxicity
METH exposure (1–4 mM) for 24 h dose-dependently induced cell death in dopaminergic CATH.a cells with increases in LDH release (IC50: 
2 mM). Levels of quinoprotein formation, which represents generation of DA quinones, also increased in a dose-dependent manner with METH treatment, coinciding with cell toxicity.
Repeated METH injections have been reported to cause dopaminergic terminal loss shown as reduction of DA transporter (DAT) -positive signals in the striatum of animals. In this study, we confirmed the reduction of DAT-positive signals in the striatum of BALB/c mice 1, 3, and 14 days after repeated METH injections (4 mg/kg x 4, i.p. with 2 h intervals). Marked reduction of DAT signals was observed in the striatum 3 days after METH injections, in agreement with many reports, starting at 1 day through to 14 days. Quinoprotein levels in the striatum were significantly increased 3 and 14 days after the repeated METH injections, coinciding with reduction of DAT signals.
2. Quinone reductase inducer and DA depletion provide protective effects against METH neurotoxicity
To confirm the possible involvement of quinone species formed by DA auto-oxidation in METH-induced cell death, we examined whether induction of intracellular quinone reductase [NAD(P)H: quinone oxidereductase-1 (NQO-1)], known to protect against the toxic effects of quinone by catalyzing two electron reduction of quinone to the redox-stable hydroquinone, might attenuate METH toxicity. Up-regulation of NQO-1 was achieved by treating CATH.a cells with quinone reductase inducer, butylated hydroxyanisol (BHA), and confirmed by Western blot analysis (167% of control). Pretreatment with BHA (25-100 µM) for 6 h on CATH.a cells significantly and dose dependently reduced METH (2 mM) -induced neurotoxicity (Fig. 1
A). BHA pretreatment also dramatically blocked METH-induced elevation of quinoprotein levels in a dose-dependent manner (Fig. 1B
), in parallel with the cell toxicity results (Fig. 1A
).
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To examine the effects of DA depletion on METH-induced quinoprotein formation, CATH.a cells were pretreated with 1 µM reserpine or 100 µM 
-methyl-p-tyrosine (-MT) for 24 h and then cotreated with 2 mM METH. We confirmed DA depletion caused by treatment with reserpine or -MT by HPLC analysis; the DA content in CATH.a cells was reduced to almost 30% of control cells. Pretreatment with reserpine and -MT significantly reduced the METH-induced elevation of quinoprotein.
3. Involvement of tyrosinase in METH-induced neurotoxicity and quinoprotein formation
Because tyrosinase in the brain enzymatically and rapidly oxidizes excessive amounts of cytosolic DA to form melanin, we examined the effect of tyrosinase inhibitor PTU on METH-induced neurotoxicity by LDH assay. PTU (50–250 µM) significantly enhanced METH neurotoxicity in CATH.a cells in a dose-dependent manner. We further examined the possible regulatory effect of tyrosinase in protecting DA neurons from METH injection-induced neurotoxicity using albino tyrosinase null C57BL/6J-Tyrc-2J/Tyrc-2J and wild-type C57BL/6J mice. Repeated METH injections (4 mg/kg x 4, i.p. with 2 h intervals) showed moderate reduction of the DAT signal in the striatum of wild-type C57BL/6J mice 3 days after the injections (30% reduction of control) (Fig. 2
A, B), which was less than the reduction in BALB/c mice (70% reduction of control). In contrast, severe reduction of DAT signals was observed in the striatum of tyrosinase null Tyrc-2J/Tyrc-2J mice 3 days after the METH injection (almost 90% reduction of control) (Fig. 2A, B
). Basal quinoprotein levels in the striatum of Tyrc-2J/Tyrc-2J mice were higher than those of C57BL/6J mice. METH injections significantly increased quinoprotein levels in the striatum of both wild-type and tyrosinase null mice. On day 3 after the METH injections, levels of striatal quinoprotein in Tyrc-2J/Tyrc-2J mice were much higher than those in C57BL/6J mice (Fig. 2C
). There were no differences in plasma and brain METH concentrations and the brain distributions between C57BL/6J and Tyrc-2J/Tyrc-2J mice 2 h after a single METH (4 mg/kg, i.p.) injection.
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CONCLUSIONS AND SIGNIFICANCE
In the present study, we confirmed that DA quinone formation is involved in the METH-induced dopaminergic neurotoxicity in vitro and in vivo as a dopaminergic neuron-specific neurotoxic factor. We also demonstrated that quinone formation-related molecules such as quinone reductase and tyrosinase protect against METH neurotoxicity to reduce intracellular free DA and DA quinone (Fig. 3
).
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Auto-oxidation of cytosolic free DA and consequent generation of reactive oxygen species have been reported to be involved in METH-induced neurotoxicity in dopaminergic neurons. Recently, the neurotoxicity of DA quinone formation by auto-oxidation of DA has focused on dopaminergic neuron-specific oxidative stress. DA quinones exert cytotoxicity by interacting with the sulfhydryl group of the amino acid cysteine on various bioactive molecules, resulting predominantly in the formation of 5-cysteinyl-DA. Since the sulfhydryl group on cysteine is often found at the active site of functional proteins, covalent modification of cysteine residues by quinones to form 5-cysteinyl-catechols irreversibly alters or inhibits protein function. Indeed, DA quinone covalently binds to key molecules of DA neurons, tyrosine hydroxylase, and DAT, to consequently inactivate those molecules.
The induction of quinone reductase by BHA treatment almost completely prevented METH-induced quinone generation, in parallel with the cell toxicity. These findings confirm that DA quinone formation is involved in METH-induced dopaminergic neurotoxicity. Intracellular DA depletion with reserpine or -MT treatment significantly prevented the elevation of quinoprotein formation induced by METH exposure, suggesting that the reduction of endogenous DA could attenuate quinone toxicity. Several reports show that -MT prevents the toxic effects of METH. Our present study has provided in vitro and in vivo evidence indicating that DA quinone formation by auto-oxidation of endogenous DA may play an important role in METH-induced neurotoxicity. We also demonstrated possible protection by quinone formation-related molecules such as quinone reductase and tyrosinase against METH toxicity. The reduction of striatal DAT induced by the METH injection was dramatically aggravated in the tyrosinase null mice, which showed higher quinoprotein levels than those in wild-type mice. As shown in Fig. 3
, the melanin-synthetic enzyme tyrosinase in the brain enzymatically oxidizes excess amounts of cytosolic DA and DA quinone to form melanin, thereby preventing slowly progressive cell damage by auto-oxidation of DA.
These experimental findings would open a new chapter of quinone involvement as a dopaminergic neuron-specific toxic factor in the field of METH-induced neurotoxicity. Enhancing activities of quinone formation-related molecules such as quinone reductase would be a novel approach to prevent METH-induced neurotoxicity.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4996fje;
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