Molecular Brain Research 93 (2001) 1–7 www.elsevier.com / locate / bres
Research report
3,4-Dihydroxyphenylacetaldehyde and hydrogen peroxide generate a hydroxyl radical: possible role in Parkinson’s disease pathogenesis a b a c, Shu Wen Li , Tien-Sung Lin , Shelly Minteer , William J. Burke * a
Department of Chemistry, Veterans Affairs Medical Center and St. Louis University Medical School, St. Louis, MO, USA b Department of Chemistry, Washington University, St. Louis, MO, USA c Department of Neurology, Veterans Affairs Medical Center and St. Louis University Medical School, 3635 Vista at Grand, St. Louis, MO, 63110, USA Accepted 3 April 2001
Abstract 3,4-Dihydroxyphenylacetaldehyde (DOPAL) and 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL), the monoamine oxidase (MAO) metabolites of dopamine (DA) and norepinephrine (NE), respectively, are toxic to catecholamine (CA) neurons in vitro and in vivo. DOPEGAL generates a free radical and activates mitochondrial permeability transition, a mechanism implicated in neuron death. To determine if DOPAL and other DA metabolites generate the hydroxyl radical in the presence of H 2 O 2 , we used HPLC–EC to detect salicylate hydroxylation products. To determine the relative reducing capacity of DOPAL and DOPEGAL we used cyclic voltammetry to measure their reduction potentials. Results indicate that DOPAL, but not DOPEGAL, DA or other DA metabolites, generates hydroxyl radicals. Atomic absorption spectroscopy and heavy metal screening indicate that this result is not due to contamination of DOPAL with iron or other heavy metals. DOPAL reduction potential (161 mV) is lower than that of DOPEGAL (235 mV). DOPAL is present in human substantia nigra. The implications of these findings to CA neuronal death in degenerative brain diseases are discussed. 2001 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neurotoxicity Keywords: 3,4-Dihydroxyphenylacetaldehyde; Free radical; Neuronal death; Parkinson’s disease
1. Introduction Blashko predicted that monamine oxidase (MAO) metabolites of amines would be highly reactive and toxic in tissues in which they are formed [5]. We have chemically synthesized and characterized the MAO metabolites of norepinephrine (NE) and dopamine (DA), 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL) [26] and 3,4dihydroxyphenylacetaldehyde (DOPAL) [27]. Using electron paramagnetic resonance (EPR), we confirmed Blashko’s prediction by showing that DOPEGAL generates a free radical in the presence of H 2 O 2 [7]. This radical gives *Corresponding author. Tel.: 11-314-577-8026; fax: 11-314-2685101. E-mail address:
[email protected] (W.J. Burke).
a different EPR signal than the hydroxyl radical and appears to be derived from DOPEGAL itself [7,12]. In this paper we determine if DOPAL generates a free radical either by mechanisms similar to DOPEGAL or by a different mechanism. We used the highly sensitive salicylic acid (SA) method to detect the formation of free hydroxyl radicals [16,28]. In this method SA is converted by hydroxyl radical to 2,3-dihydroxybenzoic acid (2,3DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA). These products are separated and detected using microcolumn high-performance liquid chromatography with electrochemical detection (mHPLC–EC). We also determine if DA or its other oxidative or methylated metabolites form hydroxyl radicals. To examine the relative reducing capacity of DOPAL and DOPEGAL we compare their redox potentials. Finally, we measure DOPAL levels in
0169-328X / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 01 )00120-6
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human substantia nigra (SN), the region of brain affected in Parkinson’s disease (PD). The implication of these findings to PD pathogenesis are discussed.
optimal detector potential for 2,3-DHBA, 2,5-DHBA and DOPAL was between 10.69 and 10.72 V versus an Ag–AgCl reference electrode. The output range was set at 0.2–2 nA.
2. Materials and methods
2.4. Generation and trapping of hydroxyl radicals
2.1. Materials
The Fenton reaction system was used for generating hydroxyl radicals. In order to ascertain the ability of salicylic acid to trap this radical, the reaction was carried out as follows: to 310 ml of phosphate citrate buffer, pH 7.2, was added 40 ml of 3.6 mM sodium salicylate, 10 ml of 50 mM H 2 O 2 , 40 ml of 2 mM ferrous sulfate. The mixture was incubated at 378C for 15 min. The salicylate was converted by the hydroxyl radical to 2,3-DHBA and 2,5-DHBA, which were identified using standard samples of 2,3- and 2,5-DHBA and detected by HPLC–EC. This reaction was also run in the absence of ferrous sulfate. A similar reaction was run containing 1.5 mM DOPAL, 310 ml of phosphate citrate buffer, pH 7.2 (93 mM disodium phosphate, 7 mM citric acid and 10 mM EDTA), 10 ml 50 mM H 2 O 2 and 40 ml of 3.6 mM sodium salicylate in the presence or absence of ferrous sulfate. An identical reaction without H 2 O 2 was also tested. In addition, we tested DOPAL in the absence of EDTA and EDTA in the absence of DOPAL. We also tested Cu 21 as CuSO 4 in place of Fe 21 . To determine if DA or its other metabolites generate the hydroxyl radical, a 1.5 mM concentration of DA, 3,4-dihydroxyphenylacetic acid (DOPAC), 3,4dihydroxyphenylethanol (DOPET), homovanillic acid (HVA), or DOPEGAL were tested individually in the presence of H 2 O 2 and sodium salicylate.
DOPAL and DOPEGAL were prepared as we have described [26,27]. 2,3-Dihydroxybenzoic acid and 2,5dihydroxybenzoic acid, sodium salicylate, EDTA and all other chemicals were purchased from Sigma (St. Louis, MO, USA). The EDTA was free of contaminating Fe 21 or transition metals on analysis by Sigma. Triple deionized water (Millipore, Bedford, MA, USA) was used for all preparations. The microbore liquid chromatographic system consisted of a Waters Model 515 HPLC pump, a Unijet injector equipped with 5-ml sample loop and a LC-4C amperometric detector (Bioanalytical System). The detector was set at 0.7 V where 2,3- and 2,5-dihydroxybenzoic acid (2,3DHBA and 3,5-DHBA) and DOPAL produced a maximum current and salicylic acid was not detectable. The salicylate hydroxylation products, 2,3- and 2,5-DHBA, were separated using a Unijet microbore reversed-phase column (C 18 , 15031 mm I.D., 5 mm) with glassy carbon working electrode and Ag–AgCl reference electrode. The output signal from LC–EC was recorded via data acquisition interface with the BAS DA-5 chromatography control and data system (Bioanalytical Systems, West Lafayette, IN, USA).
2.2. Atomic absorption spectroscopy and determination of heavy metals in DOPAL and DOPEGAL Atomic absorption spectroscopy for detection of iron was carried out on 5.0-mg samples of DOPAL and DOPEGAL using a Perkin-Elmer 5100 PC atomic absorption spectrometer with double beam absorption and electrothermal atomizer according to standard procedures [30]. The concentration of standard iron solution was from 50 to 200 ng / ml with sample volumes of 10 ml. The detection limit was up to 0.1 pg. Detection of other heavy metals in 5.0 mg samples of DOPAL and DOPEGAL was performed using a standard procedure [31]. The detection limit was 10 ng.
2.3. Chromatography The mobile phase consisted of 0.095 M disodium phosphate, 0.094 M citric acid, 10 mM EDTA, 1 mM sodium heptanesulfonate and 3.5% acetonitrile in 1000 ml, with a final pH of 3.35. The buffer was filtered through a Rainin nylon-66 0.2-mm filter and degassed prior to use. The flow-rate of mobile phase was 0.04 ml / min. The
2.5. Redox potential measurements The working electrodes were 0.1986-cm 2 glassy carbon disk electrodes (Pine Instruments). Electrodes were polished successively with 1.0- and 0.05-mm alumina on polishing cloths (Buehler). The electrodes were soaked in concentrated nitric acid and thoroughly rinsed with Nanopure water before use. The reference electrodes were standard calomel electrodes (Fisher). The counter electrodes were platinum gauze electrodes. The platinum gauze electrodes were soaked in concentrated nitric acid and thoroughly rinsed with Nanopure water before use. The formal reduction potentials of DOPAL and DOPEGAL were determined through cyclic voltammetry [3]. Cyclic voltammetry was performed using a CH650A potentiostat (CH Instruments, Austin, TX, USA) interfaced to a Pentium computer. The working, reference and counter electrodes were equilibrated in solutions of 0.05 mM redox species and a 0.01 M phosphate buffer (pH 7.4) containing 0.0027 M potassium chloride and 0.137 M sodium chloride. The buffer acts as the electrolyte. Data were collected in three electrode modes. Scan rates were varied from 50 to 1000 mV/ s. The electrodes were allowed
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to equilibrate for at least 2 min between scans. CH Instruments software was used to acquire and analyze the voltammetric data. Peak currents and potentials were determined by the software. The peak currents and potentials were used to calculate the formal reduction potential as previously described [3,21].
2.6. Preparation and quantitation of DOPAL from human substantia nigra by mHPLC–EC Brains were obtained from three men at autopsy. Age at death was 7063.5 years (mean6S.E.M.) and the postmortem interval was 7.160.7 h. None of the brains contained evidence of degenerative disease. One brain had an old right occipital temporal cystic infarct. The substantia nigra were dissected, frozen, and pulverized to a fine powder as we have described [8]. Samples of SN were placed in 1.5-ml polypropylene Eppendorf centrifuge tubes and 200 ml of 0.1 M perchloric acid (PCA) containing 50 ng of internal standard, 3,4-dihydroxybenzylamine, and 0.5% sodium metabisulfite as an antioxidant were added. The tissue was homogenized in the centrifuge tubes using a closely fitting 8 mm O.D. PTFE pestle. Tissue homogenates were maintained in an ice bath for 10 min and centrifuged at 14 000 g in a microcentrifuge for 20 min at 58C. Supernatants were adsorbed to alumina columns washed, extracted and DOPAL separated and quantitated using mHPLC–EC as we have described [8].
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2.7. Statistics Each experimental group, including the control, was replicated three to five times. The HPLC retention time of each peak was determined from seven to ten injections (C.V.,4%). The HPLC–EC chromatograms were representative of the experiments. The cyclic voltammetry measurements were repeated at scan rates of 50–1000 mV/ s.
3. Results No iron or other heavy metal was found in the purified preparations of DOPAL or DOPEGAL using atomic absorption spectroscopy and heavy metal determination. A mixture of salicylate and hydrogen peroxide produced no detectable DHBA. However, when Fe 21 was added to this mixture, both 2,3- and 2,5-DHBA peaks, indicative of hydroxyl radical, were evident at 386 and 490 s, respectively (Fig. 1). The mixture of salicylate, H 2 O 2 and DOPAL produced the same salicylate hydroxylation products either in the absence or presence of EDTA. (Fig. 2A). There were no DHBA peaks when DOPAL was incubated with salicylate with or without Fe 21 in the absence of H 2 O 2 (data not shown). DOPAL with Fe 21 alone or DOPAL with H 2 O 2 without salicylate produced no DHBA peaks (data not shown). Neither EDTA or Cu 21 produced
Fig. 1. HPLC–EC chromatogram of Fenton reactants and salicylate. Reactants, including 40 ml of 2 mM Fe 21 and 10 ml of 50 mM H 2 O 2 were incubated in 310 ml of phosphate citrate buffer, pH 7.2, in the presence of 40 ml of 3.6 mM salicylate at 378C for 15 min (see Methods). The chromatogram shows salicylate hydroxylation products [2,5-dihydroxybenzoic acid (peak 1) and 2,3-dihydroxybenzoic acid (peak 2)] — indicative of hydroxyl radicals.
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Fig. 2. HPLC–EC chromatogram of DOPAL or DOPEGAL with salicylate and H 2 O 2 . DOPAL at 1.5 mM (A) or 1.5 mM DOPEGAL (B) were incubated with 310 ml of phosphate buffer, pH 7.2, containing 10 mM EDTA, 10 ml 40 mM H 2 O 2 and 40 ml 3.6 mM sodium salicylate at 378C for 15 min (see Methods). (A) Salicylate hydroxylation products [2,5-dihydroxybenzoic acid (peak 1) and 2,3-dihydroxybenzoic acid (peak 2)] — indicative of hydroxyl radical and DOPAL (peak 3). (B) DOPEGAL (peak 1) without salicylate hydroxylation products.
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Fig. 3. Structures of chemicals tested for production of hydroxyl radicals using the method described in Fig. 2.
DHBA peaks in this system (data not shown). In addition, the closely related aldehyde, DOPEGAL, did not give evidence of hydroxyl radical formation when incubated with H 2 O 2 and salicylate (Fig. 2B). Following the same procedure, DA, DOPAC, HVA and DOPET (Fig. 3) were examined but no peaks of the dihydroxybenzoic acids were found. The reduction potentials for DOPAL and DOPEGAL were 0.161 and 0.235 mV, respectively. The level of DOPAL in three human SN was 354623 pg / mg wet weight.
4. Discussion Our results indicate that the hydroxyl radical is generated in the presence of DOPAL and H 2 O 2 . We were unable to test this result using electron paramagnetic resonance (EPR) spin trapping technique due to the relative insolubility of DOPAL in aqueous solution and because the EPR method is not sensitive enough to detect the signal of such low concentrations of hydroxyl radical (our unpublished observations). The hydroxyl radical generated in the presence of DOPAL was not attributable to a contaminant for several reasons. The DOPAL was 99.5% pure by HPLC [12]. Atomic absorption spectrometry (with a detection limit of 0.1 pg) and heavy metal determination (with a detection limit of 10 ng) of DOPAL and DOPEGAL showed no evidence of Fe or other heavy metals in the preparation which could generate radicals in the presence of H 2 O 2 . EDTA was added to bind metals including transition metals without blocking the Fenton reaction triggered by Fe 21 [33]. The EDTA contained no Fe 21 or transition metals and did not produce hydroxyl radicals in
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the presence of H 2 O 2 . In addition, transition metals do not produce hydroxyl radicals in the presence of H 2 O 2 [2]. In particular, Cu 21 , a transition metal, did not produce hydroxyl radicals either directly or through a chain reaction. Salicylate, with or without the other test compounds, in the presence of H 2 O 2 did not form the hydroxyl radical. Finally, DOPEGAL, which was purified by the same procedure as DOPAL [27] and was reacted with H 2 O 2 in the same EDTA buffer solution as DOPAL, did not produce the hydroxyl radical. The data presented here indicate that DOPAL is a cofactor in the generation of hydroxyl radical in the presence of H 2 O 2 . DOPAL in the absence of H 2 O 2 , even in the presence of Fe 21 , did not produce the hydroxyl radical. However, H 2 O 2 in the presence of either Fe 21 or DOPAL produced the hydroxyl radical. This data suggests that DOPAL acts like Fe 21 in the formation of hydroxyl radical from H 2 O 2 , i.e. DOPAL acts like a reducing agent and is oxidized in the process. The low DOPAL reduction potential, measured by cyclic voltammetry, supports this view. We postulate that DOPAL generates a free hydroxyl radical through formation of a quinone, a process previously described for another DA derivative, 6-hydroxydopamine [14]. The precise chemical mechanisms by which the free hydroxyl radical is formed, and whether it involves redox recycling and continuous free hydroxyl generation or is a one turn event, is not yet known. This chemical mechanism is a topic for further research. DOPAL, like 6-hydroxydopamine, is toxic to DA neurons in vivo [12]. However, unlike 6-hydroxydopamine, we found DOPAL present in normal human brain, a finding which confirmed and extended our earlier report [8]. In contrast to DOPAL, DOPEGAL, a structurally related CA-derived aldehyde, did not form hydroxyl radical in the presence of H 2 O 2 . Our previous reports [7,12] suggest that DOPEGAL itself in the presence of H 2 O 2 produces a free radical which, like the hydroxyl radical, forms an adduct with the spin trapping agent 5,5-dimethyl-1-pyroline-Noxide (DMPO). However, neither DOPAL nor any precursor or oxidative, reduced or methylated metabolite of DOPEGAL, form an adduct with DMPO [11]. Furthermore, 3,4-dimethoxyphenylglycolaldehyde, which lacks the ring hydroxyls found in DOPEGAL, still generates an EPR signal [11]. These data suggest that the free radical generated by DOPEGAL requires the b-hydroxyl group which is absent in DOPAL. These observations are supported by our earlier finding that DOPEGAL, in the presence of H 2 O 2 , gives an EPR signal which differs from that of hydroxyl radical [7]. Our present redox potential results support the view that DOPAL and DOPEGAL, both MAO metabolites of CA, have different mechanisms for generating free radicals and that they both differ from other CA metabolites in their capacity to generate free radicals. For instance, DOPAL has a lower redox potential than DOPEGAL, which may contribute to its capacity to form the hydroxyl radical. On the contrary, neither DA nor
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its other oxidative, reduced or methylated metabolites generate hydroxyl radicals. Rather, DA and its other metabolites scavenge hydroxyl radicals [14,28] and have relatively high redox potentials compared to that of DOPAL [19]. Interestingly, Blashko [5] predicted that the aldehyde metabolite of amines, unlike all other metabolites, would be highly reactive and toxic. Our results support Blashko’s longstanding hypothesis [5] and provide a molecular basis for a unique role for these CA-derived aldehydes as cell death messengers [6,7,12]. Our earlier reports described in vitro and in vivo selective toxicity of the aldehyde metabolites of CA without toxicity of CA themselves or their other oxidative or methylated metabolites [6,12]. In this regard, we have shown that the free radical sensitive permeability transition (PT) pore [22,32] plays a role in death of CA neurons [6,7,12]. These findings have important implications for the pathogenesis of CA neuron death in PD and in Alzheimer’s disease (AD) where DA and NE neurons are lost in the SN and locus ceruleus (LC), respectively. In these sets of CA neurons, DOPAL and DOPEGAL along with H 2 O 2 are formed on the outer mitochondrial membrane adjacent to the PT pore [14,16,22] by MAO action on DA and NE, respectively. Activation of mitochondrial PT is implicated in the apoptotic type of neuron death [29] which occurs in PD and AD [1,25]. In support of a free radical mediated cell death mechanism for CA neurons in these degenerative diseases, DOPAL and DOPEGAL have several unique characteristics which make them candidates for neuronal death messengers. They are located on the outer mitochondrial membrane in proximity to the free radical sensitive PT pore. They are the only CA metabolites which generate free radicals. They are found in CA neurons, which die in degenerative diseases [1,8,9]. Finally, they both activate mitochondrial PT [7,12]. Both DOPAL and DOPEGAL are major metabolites in CA neurons in the normal human brain [8,9]. There is growing evidence that these aldehydes may play a role in neuropathology of CA neurons in degenerative disease. We showed that DOPEGAL and its synthesizing enzymes accumulate in cell bodies of dying NE LC neurons in AD [9]. There are several mechanisms which could lead to accumulation of these aldehydes in CA neuronal cell bodies in degenerative diseases. First, we provided evidence that the accumulation of DOPEGAL in NE neuronal cell bodies is due to defective axonal transport of its biosynthetic enzymes. Second, L-dihydroxyphenylalanine ( L-DOPA), the drug used to treat PD, is a precursor of both DOPAL and DOPEGAL and could increase the synthesis of these toxic aldehyde metabolites in SN and LC neurons which are affected in PD [19]. In support of this view, L-DOPA is toxic to DA neurons both in vivo and in vitro [15]. Third, there may be a defect in catabolism of these aldehydes in degenerative diseases. A recent animal model for PD using chronic rotenone injections produces degene-
ration of SN neurons which mimics changes found in PD SN [4]. The authors attribute the selective neuropathological changes in DA neurons to free radical damage [4]. Rotenone, a mitochondrial complex I inhibitor, blocks production of NAD. NAD is a cofactor for aldehyde dehydrogenase (ALDH), an enzyme found in mitochondria [23], which oxidizes DOPAL to its non toxic metabolite, DOPAC [12]. In fact, recent studies show that exposure of PC 12 cells to rotenone produces a several-fold increase in DOPAL levels [24]. Interestingly, such a complex I deficiency has been reported in PD SN [29]. Related to this complex I deficit, oxidative modification of a-synuclein, a pathological protein marker of PD, has been described. The authors suggest accumulation of a-synuclein in PD may be due to this oxidative damage [18]. The fact that DOPAL generates free hydroxyl radicals, is toxic in vivo [10] and in vitro [10,24] and increases with complex I deficit suggests that DOPAL could play a role in oxidative processes leading to degeneration of DA neurons in PD [4,18]. Further studies are needed to determine if DOPAL levels increase in SN in PD to levels which are toxic in vivo [10,13].
Acknowledgements This study was supported by grants from the Veterans Affairs Research Program, Missouri Alzheimer’s Disease Program and the Souer’s Stroke Institute. The authors thank Dr. Dana M. Spence, Department of Chemistry, Saint Louis University, for analysis of DOPAL and DOPEGAL by atomic absorption spectroscopy and test for heavy metals.
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