J. Pineal Res. 2006; 40:204–213
2005 The Authors Journal compilation
2005 Blackwell Munksgaard Doi:10.1111/j.1600-079X.2005.00299.x Journal of Pineal Research Endogenous melatonin protects L-DOPA from autoxidation in the striatal extracellular compartment of the freely moving rat: potential implication for long-term L-DOPA therapy in Parkinson’s disease Abstract: We previously showed, using microdialysis, that autoxidation of exogenous L-dihydroxyphenylalanine (l-DOPA) occurs in vivo in the extracellular compartment of the freely moving rat, with a consequent formation of l-DOPA semiquinone (l-DOPA-SQ). In the present study, intrastriatal infusion of l-DOPA (1.0 lm for 200 min) increased dialysate l-DOPA concentrations (maximum increases up to 116-fold baseline values); moreover, l-DOPA-SQ was detected in dialysates. Individual dialysate concentrations of l-DOPA were negatively correlated with those of l-DOPA-SQ. Co-infusion of N-acetylcysteine (100 lm) or melatonin (50 lm) increased l-DOPA (up to 151- and 246-fold, respectively) and decreased l-DOPA-SQ (by about 53% and 87%, respectively) dialysate concentrations. Systemic l-DOPA [25 mg/kg intraperitoneally (i.p.) twice in a 12-h interval] significantly increased striatal baseline dialysate concentrations of l-DOPA and decreased dopamine (DA) and ascorbic acid (AsAc) concentrations, when compared with controls. Following systemic l-DOPA, l-DOPA-SQ was detected in dialysates. Endogenous melatonin was depleted in rats maintained on a 24-h light cycle for 1 wk. In melatonin-depleted rats, systemic l-DOPA induced a smaller increase in dialysate l-DOPA, a greater increase in l-DOPA-SQ formation, and a greater reduction in DA and AsAc dialysate concentrations. Co-administration of melatonin (5.0 mg/kg, i.p., twice in a 12-h interval) with l-DOPA, in control as well as in light-exposed rats, significantly increased dialysate l-DOPA concentrations, greatly inhibited l-DOPA-SQ formation, and restored up to the control values dialysate DA and AsAc concentrations. These findings demonstrate that endogenous melatonin protects exogenous l-DOPA from autoxidation in the extracellular compartment of the striatum of freely moving rats; moreover, systemic co-administration of melatonin with l-DOPA markedly increases striatal l-DOPA bioavailability in control as well as in melatonin-depleted rats. These results may be of relevance to the long-term l-DOPA therapy of Parkinson’s disease. Introduction Parkinson’s disease (PD) is characterized by a selective loss of dopaminergic neurons in the substantia nigra (SN), with a consequent decrease in neostriatal dopamine (DA) content and impairment of the functioning of the nigrostriatal dopaminergic system. A major problem for researchers and clinicians is that, by the time patientsÕ symptoms become apparent, about 70–80% of their dopaminergic neurons may have already died [1]. Although cellular and molecular pathways leading to neuronal death in PD are still unknown, major biochemical processes such as oxidative stress and impaired energy metabolism may be involved. Current concepts also suggest a genetic predis204 Gaia Rocchitta1,2, Rossana Migheli1, Giovanni Esposito1, Bianca Marchetti1,2, Maria S. Desole1, Egidio Miele1 and Pier Andrea Serra1 1 Department of Pharmacology, University of Sassari, Sassari; 2OASI Institute for Research and Care on Mental Retardation and Brain Aging (IRCCS), Neuropharmacology Section, Troina, Italy Key words: antioxidant melatonin, ascorbic acid, autoxidation, L-DOPA, Parkinson’s disease, striatum Address reprint requests to Pier Andrea Serra, MD, Department of Pharmacology, University of Sassari, viale S.Pietro 43B, 07100 Sassari, Italy. E-mail: pharmaco@uniss.it Received August 16, 2005; accepted October 25, 2005. position to a toxic process involving oxidative stress and mitochondrial dysfunction [2]. In addition, evidence is accumulating for the involvement of microglial activation [3]. In PD, activated microglia are present in proximity to damaged nigral cells, suggesting their possible role in triggering or amplifying neuronal injury as well as in removing the debris of injured cells [4]. In the past two decades a key role for DA has been emphasized in the PD pathogenesis [5]. DA neurotoxicity may result both from its autoxidation and monoamine oxidase-mediated oxidation. DA autoxidation generates free radicals, melanin, and catechol-quinones. Quinonic compounds are toxic intermediates capable of reacting with various nucleophilic groups in the cell. DA-derived Melatonin and in vivo striatal l-DOPA autoxidation quinones can act as oxidants, producing toxic hydroxy radicals, and can act as electrophiles, covalently binding to and inhibiting cellular sulphydryl-containing compounds [6]. The most suggestive data showing an involvement of DA oxidation products in PD is the presence of the specific dopaminergic toxin 6-hydroxydopamine (6-OHDA) in the urine of parkinsonian patients treated with l-DOPA [7]. l-DOPA is the drug of choice in PD therapy. Parkinsonian symptoms are relieved by administration of l-DOPA, which is converted by neuronal aromatic l-amino acid decarboxylase (EC 4.1.1.28) into DA, hence restoring DA levels in surviving neurons, which, however, continue to die despite the l-DOPA treatment [8]. l-DOPA therapy dramatically improves parkinsonian symptoms. However, after years of therapy, disabling motor complications develop greatly and limit the effectiveness of the drug [9]. l-DOPA, as a catechol-containing compound, can undergo autoxidation [8, 10] to generate an o-semiquinone (l-DOPA-SQ) which, after disproportionation, gives rise to the corresponding o-quinone and reactive oxygen species (ROS), which might further load the pre-existing condition of oxidative stress at nigro-striatal sites [8]. The quinones generated by l-DOPA oxidation may react with cysteine to form 5-S-cysteinyl-DOPA. Indeed, Spencer et al. [11] have shown that cysteinyl-conjugates of DA and l-DOPA in PD are higher than in normal SN. In a previous study [12], we showed that systemic l-DOPA underwent autoxidation in the striatal extracellular compartment of freely moving rats, with a consequent formation of l-DOPA semiquinone (l-DOPA-SQ). Moreover, systemic l-DOPA decreased baseline levels of DA and ascorbic acid (AsAc). Following systemic l-DOPA, intrastriatal infusion of the antioxidant N-acetylcysteine (NAC) decreased l-DOPA-SQ formation, increased dialysate recovery of l-DOPA, restored baseline levels of DA, but failed to restore baseline AsAc concentrations. Melatonin is a serotonin derivative which is synthesized in the pineal gland during the night [13]. Its lipophilicity ensures that melatonin rapidly crosses cellular membranes [14]. Many in vitro and in vivo studies have shown that melatonin is a powerful and broad-spectrum free radical scavenger [15]. For instance, melatonin proved to be more active than vitamin E or AsAc in inhibiting iron-catalyzed DA oxidation [16] or copper-catalyzed DA oxidation [17]. Moreover, melatonin protected the nigro-striatal system against oxidative stress caused by the neurotoxins 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) in the mouse [18] and 6-OHDA in the rat [19]. In a previous study, performed using microdialysis, we demonstrated that endogenous melatonin actively co-operates with endogenous AsAc in maintaining the oxidative homeostasis of the extracellular striatal compartment of the freely moving rat [20]. Moreover, endogenous melatonin protected extracellular endogenous DA and l-DOPA from oxidation. In light of these findings, we deemed it of interest to assess whether endogenous melatonin would protect exogenous l-DOPA from autoxidation in the extracellular striatal compartment of the freely moving rats. In addition, we looked into the effects of melatonin co-administration with systemic l-DOPA on both l-DOPA autoxidation and l-DOPA-induced changes in baseline levels of DA and AsAc. Materials and methods Sources of compounds l-DOPA, melatonin and NAC were purchased from SigmaAldrich (Milano, Italy). Animals Male Wistar rats (Morini, R. Emilia, Italy), weighing between 280 and 330 g were used in all experiments. The rats were maintained under standard animal care conditions (12:12 hr light/dark cycle, lights coming on at 07:00 hr; room temperature 21C), with food and water ad libitum. Prior to the start of any experiment, the health of each rat was assessed according to published guidelines [21]. All procedures were specifically licensed under the European Community directive 86/609 included in Decreto No. 116/1992 of the Italian Ministry of Public Health. Drug administration Systemic l-DOPA treatment schedule [25 mg/8.0 mL/kg intraperitoneally (i.p.) twice in a 12-h interval] was chosen according to the previous study [12]. Melatonin was dissolved in 5% ethanol in saline and administered i.p. at 5.0 mg/3 mL/kg twice in a 12-h interval. Melatonin was injected 5 min before each l-DOPA administration. Intrastriatal NAC and melatonin concentrations were chosen according to previous studies [12, 20]. Striatal microdialysis probe The striatal probe, which combines two independent microdialysis probes of concentric design with two separate inlets and two corresponding outlets, was previously described in detail [20, 22]. The two inlets with two corresponding separate outlets permit separate co-infusion of drugs and separate dialysate sample collection from the same intrastriatal site. In the present study, the determination of both l-DOPA and l-DOPA-SQ in dialysates from the outlet contralateral to the inlet of l-DOPA infusion allow us to affirm that detected concentrations reflect those in the extracellular compartments. On the contrary, the determination in dialysates from the ipsilateral outlet would include also the nondialyzed quota of infused l-DOPA. Moreover, separate sample collection is useful when one or more drugs which may have either pro-oxidant or antioxidant properties are infused. Briefly, the probe was constructed using two sections of plastic-coated silica tubing (diameter 0.15 mm; Scientific Glass Engineering, Milton Keynes, UK) each placed in the center of semipermeable polyacrylonitrile dialysis fibers (molecular cutoff weight of 12 kDa; Filtral 16 Hospal Industrie, Meyzieu Cedex, France). Each probe had a final diameter of 0.22 mm. The tips of the dialysis fibers were sealed and joined using quick-drying epoxy glue. The two sections of silica tubing served as inlets; the outlets were also made with a section of plastic-coated silica tubing, positioned in the center of the polythene tubing. The semi-permeable 205 Rocchitta et al. membrane was coated with epoxy leaving an active length of 4 mm. The diameter of the final probe was approximately 0.50 mm. Stereotaxic surgery Stereotaxic surgery was performed under chloral hydrate (400 mg/kg, i.p.) anesthesia. The microdialysis probes were implanted in the right striatum using the following coordinates from the atlas of Paxinos and Watson [23]: A/P + 0.5 mm from bregma, )2.5 mm M/L, and )6.0 mm D/V from dura. Body temperature during anesthesia was maintained at 37C by means of an isothermal-heating pad (Harvard Apparatus, Kent, UK). Following surgery the animals were placed in large plastic bowls (50 · 55 cm), and maintained in a temperature- and light-controlled environment, with free access to food and water. Experiments were carried out 24 hr after probe implantation with the animal in its home bowl. This arrangement allowed the rats free movement. 0.1 nm), l-DOPA (detection limit 0.2 nm), and AsAc (detection limit 0.05 lm) were quantified by HPLC-EC as previously described [20, 22], using an Alltech 426 HPLC pump equipped with a Rheodyne injector (mod. 7725), column 15 cm · 4.6 mm i.d. (Toso Haas ODS80TM C18), electrochemical detector BAS mod. LC4B and a PC-based analog-to-digital converter system (Varian Star Chromatographic Workstation, Valnut Creek, CA, USA). The mobile phase was citric acid 0.1 m, sodium acetate 0.1 m, ethylenediaminetetraacetic acid 1.0 mm, MeOH 9% and sodium octylsulphate 50 mg/L (pH ¼ 2.9); the flow rate was 1.3 mL/min. The first sample was collected after 60 min of stabilization (time 0), then dialysates were collected, at 20-min intervals, for 40 min prior to the start of experiments. In rats given systemic l-DOPA or l-DOPA + melatonin, the second i.p. dose was given 15 min before the start of stabilization. As shown previously [12], following l-DOPA 1.0 lm intrastriatal infusion, HPLC-EC chromatograms of the striatal dialysate revealed a peak (retention time 5.9 min) which was not present in the striatal dialysate of untreated rats. Light-exposed rats Rats were maintained under constant light for 6 days [24]. Early in the morning of the sixth day, animals underwent surgery, which lasted no more than 1 hr. Following surgery, the animals were placed in large plastic bowls (50 · 55 cm), and maintained in a temperature- and lightcontrolled environment, with free access to food and water. Light was kept on overnight. Experiments were carried out 24 hr after probe implantation with the animal in its home bowl. Experiments were carried out as above. Baseline dialysates were collected after 60 min of stabilization. Microdialysis procedure The composition of the Ringer solution used was as follows (in mm): NaCl 147.0, KCl 4.0, CaCl2 1.2, MgCl2 1.0 (pH 6.0). A microinfusion pump (CMA/100; Microdialysis, Solna, Sweden) pumped Ringer solution at a flow rate of 1.5 lL/min using two separate syringes connected to the inlets by a length of polythene tubing; every 20 min, two 30 lL dialysate samples were collected manually in 250 lL micro-centrifuge tubes (Alpha Laboratories, Eastleigh, UK) attached to the outlets. Subsequently, a 20-lL aliquot of each collected dialysate was injected into each of two parallel analytical systems. Drugs were added to the Ringer solution and infused via the striatal probe implanted in the striatum. Chromatographic analysis l-DOPA-SQ was quantified by high-performance liquid chromatography with electrochemical detection (HPLCEC) according to the procedure previously described in detail [12]. The peak, identified as l-DOPA-SQ, appeared within 20 min from the start of l-DOPA infusion. We were not able to follow the fate of l-DOPA-SQ (further oxidation to l-DOPA-3,4-o-quinone) as our HPLC apparatus was not suitable for the detection of the latter l-DOPA oxidation product. Analogously, DA (detection limit 206 Histology Following the experiments, rats were killed with an overdose of chloral hydrate (800 mg/kg, i.p.). The location of each microdialysis probe was confirmed by postmortem histology. Brains were fixed in formal saline and 50 lm coronal sections were made with a cryostat. The slices were stained with cresyl violet and examined under a microscope. Statistical analysis Concentrations of neurochemicals in dialysates were expressed in nm (DA l-DOPA, l-DOPA-SQ) or lm (AsAc) and given as mean ± S.E.M. Drug effects on neurochemicals were statistically evaluated in terms of changes in absolute dialysate concentrations. Statistical significance was assessed using ANOVA for difference between groups and over time. Difference within or between groups were determined by paired or unpaired t-tests with either Bonferroni multiple comparison adjustment or Student– Newman–Keuls t-test post hoc analysis. Pearson’s correlation coefficient between individual concentrations of l-DOPA and l-DOPA-SQ was calculated in some instances The null hypothesis was rejected when P < 0.05. Results l-DOPA 1.0 lm (n ¼ 4) was infused through the ipsilateral inlet for 200 min. l-DOPA concentrations in dialysates from the contralateral outlet increased up to 116-fold baseline levels 20 min after the start of infusion; thereafter, l-DOPA concentrations showed a trend to decrease, despite continuous l-DOPA infusion (Fig. 1). l-DOPASQ detection in dialysates from the contralateral outlet occurred about 20 min after the start of l-DOPA infusion. l-DOPA-SQ concentrations increased over-times (Fig. 1). Individual dialysate concentrations of l-DOPA were negatively correlated with those of l-DOPA SQ [r values range between )0.786 (P < 0.005) and )0.842 (P < 0.0001), Melatonin and in vivo striatal l-DOPA autoxidation Fig. 1. Detection of l-DOPA-SQ in dialysates from the striatum of freely moving rats following intrastriatal infusion of l-DOPA (n ¼ 4) and effects of N-acetylcysteine (NAC, n ¼ 4) or melatonin (n ¼ 4) co-infusion on dialysate l-DOPA (A) and l-DOPA-SQ (B) concentrations. Dialysates were collected, at 20-min intervals, during continuous intrastriatal infusion of l-DOPA through the ipsilateral inlet. NAC or melatonin co-infusion through the contralateral inlet started 5 min before l-DOPA infusion. Values are given as mean ± S.E.M. and refer to the concentrations in dialysates from the contralateral outlet. §P < 0.05 compared with l-DOPA group (A); +(thin horizontal bar) P < 0.05 compared with both l-DOPA and l-DOPA + NAC groups (A); +(thin horizontal bar) P < 0.05 compared with both l-DOPA + NAC and l-DOPA + melatonin groups (B); §(thin horizontal bar) P < 0.05 compared with l-DOPA + NAC group (B). Bonferroni multiple comparison adjustment test. d.f. ¼ 8]. l-DOPA infusion induced a short-lasting increase in DA concentrations in dialysates from the contralateral outlet (maximum increase 107% of baseline after 40 min) and did not affect AsAc dialysate concentrations (Fig. 2). A concentration of 100 lm NAC (n ¼ 4) was infused through the contralateral inlet. The infusion started 5 min before l-DOPA 1.0 lm infusion through the ipsilateral inlet for 200 min. NAC co-infusion induced the following: (i) significant increases in l-DOPA concentrations (up to 151-fold baseline levels) in dialysates from the contralateral outlet, when compared with l-DOPA group (Fig. 1); (ii) significant decreases (by about 53%) of l-DOPA-SQ concentrations at the end of drug infusion (Fig. 1); (iii) further and more sustained increases in dialysate DA (up to 153% of baseline after 40 min) (Fig. 2); (iv) slight decrease Fig. 2. Effects of intrastriatal infusion of l-DOPA on dopamine (DA, A) and ascorbic acid (AsAc, B) concentrations in dialysates from the striatum of freely moving and effects of N-acetylcysteine (NAC) or melatonin co-infusion on l-DOPA-induced changes. Same groups as in Fig. 1. Dialysates were collected, at 20-min intervals, during continuous intrastriatal infusion of l-DOPA through the ipsilateral inlet. NAC or melatonin co-infusion through the contralateral inlet started 5 min before l-DOPA infusion. Values are given as mean ± S.E.M. and refer to the concentrations in dialysates from the contralateral outlet. *P < 0.05 compared with pertinent baseline values (A); +(thin horizontal bar) P < 0.05 compared with both l-DOPA and l-DOPA + NAC groups (A); §P < 0.05 compared with l-DOPA group (A); §P < 0.05 compared with l-DOPA group (B); +(thin horizontal bar) P < 0.05 compared with l-DOPA + melatonin group (B). Bonferroni multiple comparison adjustment test. in AsAc dialysate concentrations, when compared with baseline values (Fig. 2). A concentration of 50 lm melatonin (n ¼ 4) was infused through the contralateral inlet. The infusion started 5 min before l-DOPA 1.0 lm infusion through the ipsilateral inlet for 200 min. Melatonin co-infusion induced the following: (i) greater and significant increases in l-DOPA concentrations (up to 246-fold baseline levels) in dialysates from the contralateral outlet, when compare with both l-DOPA and l-DOPA + melatonin groups (Fig. 1); (ii) greater and significant decreases (by about 87%) of l-DOPA-SQ concentrations at the end of drug infusion (Fig. 1); (iii) greater and more sustained increases in dialysate DA (up to 232% of baseline after 40 min) (Fig. 2); (iv) slight increases in AsAc dialysate concentrations, when com207 Rocchitta et al. pared with baseline levels. Dialysate AsAc concentrations, however, were significantly higher than concentrations in both l-DOPA and l-DOPA + NAC groups during last 80 and 160 min, respectively, of drug infusion (Fig. 2). As shown previously [12], l-DOPA given systemically significantly increased striatal dialysate baseline levels of l-DOPA and decreased those of DA and AsAc. Moreover, l-DOPA-SQ was detected in dialysates. l-DOPA 25 mg/8.0 mL/kg was given i.p. twice in a 12-hr interval to three groups of four rats. Baseline concentrations of DA, l-DOPA, l-DOPA-SQ and AsAc were determined 1 hr after last l-DOPA administration in dialysates from both outlets, as l-DOPA was given systemically. The data were pooled in order to calculate baseline values for each rat. The results are given in Table 1. Systemic l-DOPA significantly decreased baseline DA (by about 39%) and AsAc (by about 43%) and increased baseline l-DOPA (by about 11-fold). In untreated rats, l-DOPA-SQ was not detectable in dialysates; however, following systemic l-DOPA, l-DOPA-SQ was detected in concentrations even greater (by about 40%) than l-DOPA ones. In the first group of systemic l-DOPA-treated rats, dialysate concentrations of DA, l-DOPA, l-DOPA-SQ and AsAc were monitored for 200 min after baseline samples collection. Neurochemicals were determined in the dialysate from the outlet conventionally indicated as contralateral. Dialysate l-DOPA concentrations progressively declined (maximum decreases by about 51% at the end of the monitoring period), while those of l-DOPA-SQ progressively increased (by about 77% at the end of the monitoring period) (Fig. 3). Dialysate concentrations of both DA and AsAc did not show significant changes, when compared with baseline values (Fig. 4). In the second group, 100 lm NAC was infused intrastriatally through the ipsilateral inlet for 200 min. Dialysates were collected from the contralateral outlet. NAC infusion increased dialysate l-DOPA (maximum increase by about 101% after 40 min) and decreased dialysate l-DOPA-SQ concentrations by about 57% at the end of the drug infusion. NAC infusion restored dialysate DA concentrations (maximum increase 101% after 40 min), but failed to restore dialysate AsAc concentrations (Fig. 4). In the third group, 50 lm melatonin was infused intrastriatally through the ipsilateral inlet for 200 min. Dialysates were collected from the contralateral outlet. Melatonin infusion induced a long-lasting increase in dialysate l-DOPA (maximum increase by about 207% after 40 min) and a great decrease in dialysate l-DOPA-SQ concentrations (by about 96% at the end of the infusion) (Fig. 3). Melatonin infusion induced a long-lasting increase in dialysate DA concentrations (maximum increase by about 269% after 80 min) and fully restored dialysate AsAc concentrations (Fig. 4). To evaluate the effects of systemic co-administration of melatonin on systemic l-DOPA-induced changes in dialysate concentrations of striatal neurochemicals, melatonin 5.0 mg/3.0 mL/kg i.p. was co-administered with l-DOPA 25 mg/8.0 mL/kg i.p. twice, at 12-hr interval, in a group of four rats. We could not evaluate the effects of systemic co-administration of NAC, as NAC, although readily crosses the blood–brain barrier when given into the carotid artery, it does not reach the brain when given systemically, owing to the its rapid body clearance [25]. Melatonin was injected 5 min before each l-DOPA administration. Baseline concentrations of DA, l-DOPA, l-DOPA-SQ and AsAc were determined 1 hr after last l-DOPA administration in dialysates from both outlets. The data were pooled in order to calculate baseline values for each rat and are given in Table 1. Melatonin co-administration significantly increased dialysate DA concentrations, when compared with both untreated and systemic l-DOPAtreated rats (by about 29% and 111%, respectively). Moreover, melatonin co-administration significantly increased baseline l-DOPA by about 75% and decreased baseline l-DOPA-SQ concentration by about 81%, when compared with systemic l-DOPA-treated rats. Finally, melatonin coadministration fully restored baseline AsAc concentrations. After baseline samples collection, dialysate concentrations of neurochemicals were monitored for 200 min. Dialysate l-DOPA progressively increased, while l-DOPA-SQ progressively decreased; l-DOPA increases reached statistical significance during the last 60 min of monitoring, when compared with baseline levels (Fig. 5). Dialysate DA concentrations showed a progressive significant increase that reached statistical significance during the last 140 min of Table 1. Effects of systemic l-DOPA on baseline striatal dialysate concentration of neurochemicals in control, light-exposed and systemic melatonin-treated freely moving rats. See text for detail Treatment Neurochemical DA (nm) l-DOPA (nm) l-DOPA-SQ (nm) AsAc (lm) None (n ¼ 12) l-DOPA (n ¼ 12) 4.24 ± 0.32 2.59 ± 2.81 ± 0.36 30.84 ± ND 43.06 ± 9.45 ± 0.48 5.03 ± l-DOPA + melatonin (n ¼ 4) l-DOPA + light exposure (n ¼ 4) l-DOPA + melatonin + light exposure (n ¼ 4) 0.22a ()38.9) 5.47 ± 0.81ab (+29.0) 1.36 ± 0.37ab ()67.9) 4.84 ± 0.95bd (+14.1) 1.28a (+1098) 53.96 ± 5.76ab (+1920) 18.15 ± 2.28ab (+646) 50.88 ± 5.21abd (+1811) 3.17 8.11 ± 1.61b ()81.1) 63.45 ± 9.57b (+47.4) 9.96 ± 2.48bd ()76.9.0) 8.49 ± 1.08b ()8.2) 2.38 ± 0.26ab ()74.3) 7.97 ± 0.63bd ()13.8) 0.26a ()43.4) ND, not detectable; DA, dopamine; AsAc, ascorbic acid. Data are given as mean ± S.E.M. Values in parentheses are percentage. a P < 0.05 compared with untreated rats; bP < 0.05 compared with l-DOPA-treated rats; cP < 0.05 compared with l-DOPA + melatonin-treated group; dP < 0.05 compared with l-DOPA + light-exposed group. Student–Newman–Keuls test. 208 Melatonin and in vivo striatal l-DOPA autoxidation Fig. 3. Effects of systemic l-DOPA on l-DOPA (A) and l-DOPASQ (B) concentrations in dialysates from the striatum of freely moving and effects of N-acetylcysteine (NAC) or melatonin intrastriatal infusion on systemic l-DOPA-induced changes. NAC (100 lm) or melatonin (50 lm) was infused for 200 min (solid horizontal bar a) through the ipsilateral inlet. N ¼ 4 for each group. Dialysates were collected at 20-min intervals. Values are given as mean ± S.E.M. and refer to concentrations in dialysates from the contralateral outlet. *P < 0.05 compared with pertinent baseline values (A, B); +(thin horizontal bar) P < 0.05 compared with systemic l-DOPA group (A), systemic l-DOPA group (B) and systemic l-DOPA + melatonin group (B); §(thin horizontal bar) P < 0.05 compared with systemic l-DOPA + NAC group (A); # P < 0.05 compared with systemic l-DOPA group (B). Bonferroni multiple comparison adjustment test. monitoring (maximum increase by about 96%); AsAc concentrations showed only a slight increase (Fig. 6). Two groups of four rats were exposed to light for 7 days (see Materials and methods for details). The first group was given l-DOPA 25 mg/8.0 mL/kg i.p. twice in a 12-hr interval. Baseline concentrations of DA, l-DOPA, l-DOPA-SQ and AsAc were determined 1 hr after last l-DOPA administration in dialysates from both outlets. The data were pooled in order to calculate baseline values for each rat. The results are given in Table 1. Baseline levels of l-DOPA were significantly lower (by about 41%) than rats given systemic l-DOPA, while baseline levels of lDOPA-SQ were significantly higher (by about 47%). Baseline DA and AsAc concentrations were significantly lower than rats given systemic l-DOPA, by about 47% and 53%, respectively. After baseline samples collection, dialysate concentrations of neurochemicals were monitored Fig. 4. Effects of systemic l-DOPA on dopamine (DA) and ascorbic acid (AsAc) concentrations in dialysates from the striatum of freely moving rats, and effects of N-acetylcysteine (NAC) or melatonin intrastriatal infusion on systemic l-DOPA-induced changes. NAC (100 lm) or melatonin (50 lm) was infused for 200 min (solid horizontal bar a) through the ipsilateral inlet. Same groups as in Fig. 3. Dialysates were collected at 20-min intervals. Values are given as mean ± S.E.M. and refer to concentrations in dialysates from the contralateral outlet. *P < 0.05 compared with pertinent baseline values (A, B): +(thin horizontal bar) P < 0.05 compared with both systemic l-DOPA and systemic l-DOPA + NAC groups (A, B); §(thin horizontal bar) P < 0.05 compared with systemic l-DOPA + NAC groups (A). Bonferroni multiple comparison adjustment test. for 200 min. Dialysate l-DOPA progressively decreased, while l-DOPA-SQ progressively increased; l-DOPA-SQ increases reached statistical significance during the last 60 min of monitoring, when compared with baseline levels (Fig. 5). Dialysate DA and AsAc concentrations did not show significant changes (Fig. 6). In the second group of light-exposed rats, melatonin (5.0 mg/3.0 mL/kg, i.p.) was co-administered with l-DOPA 25 mg/8.0 mL/kg i.p. twice at 12-hr interval. Melatonin was injected 5 min before each l-DOPA administration. Baseline concentrations of DA, l-DOPA, l-DOPA-SQ, and AsAc were determined 1 hr after last l-DOPA administration in dialysates from both outlets. The data were pooled in order to calculate baseline values for each rat. The results are given in Table 1. Baseline levels of l-DOPA were significantly higher (by about 379%) than baseline values in light-exposed group, while l-DOPA-SQ were significantly lower (by about 84%). Both baseline 209 Rocchitta et al. Fig. 5. Effects of systemic l-DOPA on l-DOPA and l-DOPA-SQ concentrations in dialysates from the striatum of freely moving control or light-exposed rats and effects of systemic melatonin coadministration on systemic l-DOPA-induced changes. N ¼ 4 for each group. Dialysates were collected, at 20-min intervals, 1 hr after last systemic administration. Values are given as mean ± S.E.M. and refer to concentrations in dialysates from both outlets. *P < 0.05 compared with pertinent baseline values (A, B); + (thin horizontal bar) P < 0.05 compared with pertinent systemic l-DOPA groups (A, B); +(thin horizontal bar) P < 0.05 compared with pertinent systemic l-DOPA and systemic l-DOPA + light exposure groups (A, B); §(thin horizontal bar) P < 0.05 compared with systemic l-DOPA group (A, B). Bonferroni multiple comparison adjustment test. Fig. 6. Effects of systemic l-DOPA on dopamine (DA) and ascorbic acid (AsAc) concentrations in dialysates from the striatum of freely moving control or light-exposed rats and effects of systemic melatonin co-administration on systemic l-DOPA-induced changes. Same groups as in Fig. 5. Dialysates were collected, at 20-min intervals, 1 hr after last systemic administration. Values are given as mean ± S.E.M. and refer to concentrations in dialysates from both outlets. *P < 0.05 compared with pertinent baseline values (A, B); +(thin horizontal bar) P < 0.05 compared with pertinent systemic l-DOPA groups (A, B); +(thin horizontal bar) P < 0.05 compared with pertinent systemic l-DOPA and systemic l-DOPA in light-exposed groups (A, B); §P < 0.05 compared with systemic l-DOPA group (A, B). Bonferroni multiple comparison adjustment test. 113%); AsAc concentrations showed only a slight increase (Fig. 6). levels of l-DOPA and l-DOPA-SQ did not statistically differ from baseline levels in l-DOPA + melatonin-treated group (Table 1). Baseline levels of DA and AsAc were significantly higher than baseline values in light-exposed group, by about 356% and 335%, respectively. Both baseline levels of DA and AsAc did not statistically differ from baseline levels in l-DOPA + melatonin-treated group (Table 1). After baseline samples collection, dialysate concentrations of neurochemicals were monitored for 200 min. Dialysate l-DOPA progressively increased, while l-DOPA-SQ progressively decreased; l-DOPA increases reached statistical significance during the last 60 min of monitoring, when compared with baseline levels (Fig. 5). Dialysate DA concentrations showed a progressive significant increase, that reached statistical significance during the last 140 min of monitoring (maximum increase by about 210 Discussion The key findings in the present study are the following: (i) both intrastriatally and systemically administered l-DOPA undergo autoxidation in the striatal extracellular compartment of the freely moving rat, with a consequent formation and detection of l-DOPA-SQ; (ii) systemic administration of l-DOPA decreases striatal baseline dialysate concentrations of DA and AsAc; (iii) systemic administration of l-DOPA in melatonin-depleted rats induces formation of l-DOPA-SQ and decreases in baseline levels of DA and AsAc significantly greater than in control rats; (iv) systemic co-administration of melatonin with l-DOPA, in control as well as in light-exposed rats, significantly increased both baseline and over-times concentrations of dialysate Melatonin and in vivo striatal l-DOPA autoxidation l-DOPA, greatly inhibited l-DOPA autoxidation and the consequent formation of l-DOPA-SQ, and fully restored both DA and AsAc dialysate concentrations. Intrastrial infusion of l-DOPA induced a short-lasting increase in dialysate DA. Co-infusion of the antioxidant NAC with l-DOPA increased dialysate DA concentrations, which were further significantly increased when melatonin was co-infused. Moreover, l-DOPA autoxidation was inhibited by NAC co-infusion and, to a greater extent, by melatonin co-infusion, with a consequent increase in dialysate recovery of l-DOPA. The finding that antioxidant drugs increased DA dialysate recovery strongly suggest that DA released following l-DOPA infusion undergoes oxidation in the extracellular compartment. Systemically administered l-DOPA undergoes biotransformation to DA in the nigro-striatal system of the rat [26]. In a previous study [12], we showed that stimulation of striatal dopaminergic endings induced increases in dialysate DA in rats given systemic l-DOPA much greater than in untreated rats. However, newly synthesized and released DA, unless appropriately shielded, easily undergoes autoxidation or ROS and/or reactive nitrogen species (RNS)-mediated oxidation and/or nitration in the extracellular compartment, mainly when DA is released in excess [20, 27, 28]. Thus, autoxidation of l-DOPA in the striatal extracellular compartment, with a consequent formation of l-DOPA-SQ, most likely promotes DA nonenzymatic oxidation. Spencer et al. [11] have shown that an acceleration of l-DOPA/DA oxidation occurs in PD, probably related to therapy with l-DOPA. Autoxidation of l-DOPA and DA generates quinones and superoxide anion (O
 2 ), which is scavenged by AsAc [29] and melatonin [13]. Intrastriatal l-DOPA infusion did not induce significant changes in dialysate AsAc. Dialysate AsAc concentrations reflect those found in vivo (0.2–0.4 mm) in the extracellular striatal compartment of the rat [30]. Moreover, an efficient in vivo homeostatic mechanism keeps constant striatal extracellular AsAc concentrations [30]. However, following co-infusion of NAC, dialysate concentrations of AsAc were significantly lower than those detected following melatonin co-administration. This finding may be explained on the basis of the activity of the respective oxidation products of NAC and melatonin. When NAC undergoes oxidation, it generates, like glutathione, a thiyl active free radical [31, 32], which needs to be scavenged in order to prevent hydroxyl radical formation [31]. On the contrary, melatonin oxidation products are also known to have antioxidant properties [33]. Endogenous AsAc [34, 35] and endogenous melatonin [20] cooperate in scavenging endogenous antioxidant (vitamin E, glutathione, uric acid)-derived active radicals, such as a-tocopheroxyl, thiyl/sulfenyl and urate radical. When given systemically, l-DOPA decreased dialysate baseline levels of both DA and AsAc. In systemic l-DOPAtreated rats, intrastriatal infusion of NAC increased overtimes dialysate concentrations of DA and l-DOPA, decreased l-DOPA-SQ generation, restored DA concentrations, but failed to restore those of AsAc. On the contrary, intrastriatal infusion of melatonin restored both DA and AsAc dialysate concentrations, greatly increased over-times dialysate l-DOPA concentrations and almost fully inhibited l-DOPA-SQ generation. These findings further demonstrate that following exogenous l-DOPA striatal loading, the l-DOPA autoxidation in the striatal extracellular compartment, with a consequent formation of l-DOPA-SQ, promotes nonenzymatic oxidation of released DA. As a consequence, AsAc and melatonin, the main components of the striatal extracellular antioxidant system [20, 34], are most likely markedly involved in maintaining oxidative homeostasis. The antioxidant NAC did protect exogenous l-DOPA and released DA from autoxidation, but the protection needed the cooperation of endogenous AsAc, which was probably involved in scavenging thiyl radical, the NAC oxidation product. On the contrary, co-infusion of melatonin not only afforded a greater protection, but fully restored dialysate AsAc. In a previous study [20], we suggested that endogenous melatonin might play an active role in maintaining the oxidative homeostasis in the extracellular compartment of the striatum of freely moving rats. The results of the present study confirm this hypothesis. In fact, systemic l-DOPA induced decreases in baseline DA and AsAc levels in melatonin-depleted rats significantly greater than in control rats; moreover, baseline levels of l-DOPA were significantly lower, while those of l-DOPA-SQ were significantly higher. Thus, following exogenous l-DOPA striatal loading in melatonin-depleted rats, the rate of l-DOPA and DA autoxidation in the striatal extracellular compartment was significantly increased, with a consequent greater involvement of endogenous AsAc in maintaining the oxidative homeostasis. The question arises as to whether the result of the present study might be of relevance to the l-DOPA long-term therapy of PD. Inhibitors of enzymatic l-DOPA metabolism, which do not cross the blood–brain barrier, are successfully administered with l-DOPA with the aim of increasing l-DOPA bioavailability at nigro-striatal site [36]. However, despite the drug-induced increase in l-DOPA bioavailability, some years after the start of therapy l-DOPA loses its beneficial effects as evidenced by motor fluctuations. The clinical effectiveness of dopaminergic agonists in controlling l-DOPA-associated motor fluctuation [37] allow us to speculate that, despite the l-DOPA loading, the postsynaptic dopaminergic input is greatly diminished. In this regard, an increase in l-DOPA/DA nonenzymatic oxidation [11] in the extracellular compartment, facilitated by an impaired oxidative homeostasis [38], might assume great relevance. However, no clinical data are available on the effectiveness of antioxidant drugs in controlling l-DOPA/DA nonenzymatic oxidation, which most likely occurs at nigro-striatal site in PD [11]. In the present study, systemic co-administration of melatonin with l-DOPA, in control as well as in light-exposed rats, significantly increased both baseline and over-times concentrations of dialysate l-DOPA, greatly inhibited l-DOPA autoxidation and the consequent formation of l-DOPA-SQ, and restored both DA and AsAc dialysate concentrations. Therefore, melatonin appears to be the most suitable antioxidant drug to be used as adjunctive drug with the aim of protecting l-DOPA and DA from 211 Rocchitta et al. nonenzymatic oxidation in the striatal extracellular compartment. We showed previously [12] that transition metals (manganese and iron) greatly increased l-DOPA autoxidation in the extracellular striatal compartment of the freely moving rats. Dysregulation of iron metabolism and iron-induced oxidative stress are widely believed to be important pathogenetic mechanisms of neuronal death in PD [38, 39]. Indeed, O
 2 releases iron from storage proteins and enzymic [4Fe-4S] clusters [40]. Thus, the hypothesis that endogenous iron might increase extracellular l-DOPA/DA oxidation in PD seems to be logical and further support the rationale of melatonin use as adjunctive drug to the l-DOPA therapy of PD. Clinical studies have shown that long-term administration of melatonin at pharmacological dosage, in PD [41] as well as in other neurologic disorders [42], is devoid of side effects. It is still claimed that melatonin is not an antioxidant because it must be given in what is referred to as pharmacological doses to repair the breach of antioxidant defenses by ROS, RNS, or toxic reactants leading to damage of critical cellular structures (DNA, lipids, proteins) with a consequent disruption of the cellular physiology [43]. 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