Prostaglandin H Synthase-1-Catalyzed Bioactivation of

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Prostaglandin H Synthase-1-Catalyzed Bioactivation of Neurotransmitters, Their Precursors, and Metabolites: Oxidative DNA Damage and Electron Spin Resonance Spectroscopy Studies Luı´sa L. Gonçalves,†,‡ Annmarie Ramkissoon,† and Peter G. Wells*,†,§ Faculty of Pharmacy, UniVersity of Toronto, 144 College Street, Toronto, Ontario M5S 3M2, and Department of Pharmacology and Toxicology, UniVersity of Toronto, 1 Kings’ College Circle, Toronto, Ontario M5S 1A8, Canada ReceiVed NoVember 10, 2008

The role of prostaglandin H synthase-1 (PHS-1) and a related model enzyme, horseradish peroxidase (HRP), in catalyzing the bioactivation of dopamine (DA) and epinephrine and their precursors and metabolites to potential neurodegenerative free radical intermediates was examined. To determine the potential contribution of PHS-dependent reactive oxygen species (ROS) formation, the neurotransmitter DA or its precursor and metabolites were incubated in Vitro with purified ovine PHS-1 and calf thymus DNA. DA, its L-dihydroxyphenylalanine (L-DOPA), precursor, and its dihydroxyphenylacetic acid (DOPAC) metabolite were excellent PHS-1 substrates, resulting in PHS-1-dependent ROS formation that initiated oxidative DNA damage, selectively quantified as 8-oxo-2′-deoxyguanosine. Most substrates generated isotropic electron spin resonance (ESR) spectra with a resolved hyperfine structure attributable to ortho-semiquinone free radical intermediates upon autoxidation at pH 6, with up to a 18-fold increase via HRP-catalyzed oxidation. Remarkably, HRP-mediated oxidation of DOPAC and dihydroxymandelic acid (DHMA) produced asymmetric ESR spectra characteristic of an immobilized radical, possibly due to free radical intermediates and melanin or melanin-like polymers. These results show that the precursors and metabolites of endogenous neurotransmitters, while inactive in receptor binding assays, may actually play an important role in free radical formation. Additionally, ROS generated by PHS-catalyzed bioactivation produce oxidative DNA damage in the central nervous system, which may initiate neurodegeneration associated with aging. Introduction The brain dopaminergic system is implicated in a variety of physiological and pathological processes. An imbalance between dopaminergic neurotransmission and dopamine (DA)1 receptors is associated with numerous neuropsychiatric (e.g., schizophrenia and depression) and neuropathological disorders, such as Parkinson’s, Alzheimer’s, and Huntington’s diseases (1, 2). Although it is generally accepted that free radicals are involved in the neurodegenerative process, the exact mechanism of neurodegeneration in ViVo is not fully understood. It has been postulated that the oxidation of endogenous substrates, particularly the oxidation of catecholamine neurotransmitters (such as DA, epinephrine, and norepinephrine) may contribute to the degeneration of selective brain regions, * To whom correspondence should be addressed. Tel: 416-978-3221. Fax: 416-267-7797. E-mail: [email protected]. † Faculty of Pharmacy. ‡ Present address: Instituto de Cieˆncias da Sau´de Egas Moniz, Campus Universita´riosQuinta da Granja, Monte de Caparica, 2829-511 Caparica, Portugal. § Department of Pharmacology and Toxicology. 1 Abbreviations: ESR, electron spin resonance; UV/vis, ultraviolet/visible; HPLC-EC, high-performance liquid chromatography with electrochemical detection; CNS, central nervous system; hfcc, hyperfine coupling constant; HRP, horseradish peroxidase; H2O2, hydrogen peroxide; PHS, prostaglandin H synthase; AA, arachidonic acid; ETYA, 5,8,11,14-eicosatetraynoic acid; DTPA, diethylenetriaminepenta-acetic acid; DMPO, 5,5-dimethyl-1-pyrroline-1-oxide; PBN, R-phenyl-N-tert-butylnitrone; ROS, reactive oxygen species; SOD, superoxide dismutase; MS, mass spectrometry; DA, dopamine; L-DOPA, L-dihydroxyphenylalanine; DOPAC, dihydroxyphenylacetic acid; DHMA, dihydroxymandelic acid; HVA, homovanillic acid; MTA, 3-methoxytyramine; 8-oxo-dG, 8-oxo-2′-deoxyguanosine; 2′-dG, 2′-deoxyguanosine; U, units.

as observed for example in the nigro-striatal system of Parkinson’s patients (3-8). Approximately 95% of the released DA is quickly taken up into the neurons (or nerve terminals) and then into the storage vesicles for reuse (2). Although it is not known exactly what percentage of DA molecules inside a neuron is taken up by the storage vesicles, it has been suggested that the fraction of the intracellular DA that is not taken up into the vesicles might be the cause of cytotoxic damage to dopaminergic neurons (2). DA can autooxidize via the loss of one electron, generating semiquinone free radical intermediates that rapidly react to produce reactive oxygen species (ROS) such as superoxide and hydroxyl radicals (6, 9, 10). The ensuing redox cycling between the catechols/catecholamines and their quinones/semiquinones can continuously produce large amounts of ROS. Both DA quinone and semiquinone intermediates and superoxide and hydroxyl radicals are cytotoxic and potentially genotoxic (11-14). Alternatively, several types of oxidizing enzymes (e.g., cytochromes P450) and peroxidases such as myeloperoxidase, lactoperoxidase, horseradish peroxidase (HRP), and prostaglandin H synthase (PHS) can bioactivate DA and other biogenic amines to the same reactive intermediates, which may bind proteins and DNA (15, 16). Additionally, these biogenic amines, when metabolized by peroxidases, are known to form prooxidant radicals, which cooxidize cellular antioxidants, such as ascorbate, NADH, or cysteine (17, 18). The involvement of HRP and PHS in the bioactivation of xenobiotics (19-25) and endogenous substrates (7, 15, 26, 27) is well-documented. PHS is a dual-function enzyme, having a cyclooxygenase and a hydroperoxidase component. It is constitutively expressed in

10.1021/tx800423s CCC: $40.75  2009 American Chemical Society Published on Web 04/17/2009

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the brain, and increased brain expression and activity of PHS have been reported in aging and neurodegenerative diseases, such as Parkinson’s and Alzheimer’s diseases (7, 15). It is generally accepted that the oxidation of a substrate, designated by AH, by the hydroperoxidase component of PHS in the presence of peroxide (AOOH), includes the following initial steps (ref 28 and references cited therein),

whether HRP and PHS-1 catalyze the bioactivation of these endogenous substrates to the same reactive intermediates. PHS-1 and DNA were coincubated with DA, its precursor, and metabolites in Vitro to determine whether the free radical intermediates formed could enhance ROS formation and cause oxidative DNA damage.

enzyme + AOOH f E-I + AOH

(1)

Materials and Methods

E-I + AH f E-II + A•

(2)

E-II + AH f enzyme + A•

(3)

where E-I and E-II are compounds I and II, respectively. In this reaction, a broad spectrum of chemicals can serve as electron donors, such as aromatic amines, polycyclic aromatic hydrocarbons, and phenolic compounds (including catechols and catecholamines) (29). Our laboratory has previously shown in Vitro and in ViVo that xenobiotics such as phenytoin and amphetamines are cosubstrates during the reduction of hydroperoxides in eicosanoid biosynthesis, the reducing cosubstrate being oxidized by PHS and/or lipoxygenases to N- and C-centered reactive free radical intermediates, characterized by electron spin resonance (ESR) spectroscopy (22, 23, 25). These intermediates generate ROS that oxidatively damage brain DNA, proteins, and lipids causing dopaminergic nerve terminal degeneration and functional deficits. Additionally, in Vitro studies have shown that DA and serotonin can serve as cofactors for PHS-1 (15). More recently, a preliminary study in our laboratory found that, in addition to DA, its precursor, L-dihydroxyphenylalanine (L-DOPA), and epinephrine were excellent PHS-1 substrates, resulting in PHSdependent ROS formation that initiated the oxidation of 2′-deoxyguanosine (2′-dG) to 8-oxo-2′-deoxyguanosine (8-oxodG) (30). Accordingly, we postulated that the precursors and metabolites of neurotransmitters, while inactive in binding to neurotransmitter receptors, may nevertheless be bioactivated by PHS to ROS-generating free radical intermediates that cause oxidative macromolecular damage, possibly contributing to neurodegenerative diseases associated with aging. This hypothesis is consistent with several observations, including the following: (1) Some neurotransmitter precursors (e.g., L-DOPA), although inactive in receptor binding assays, are bioactivated by PHS-1 (30). (2) Neurotransmitter precursors and metabolites are distributed throughout the brain and are found in higher amounts in the striatum, substantia nigra pars compacta, and hippocampus (31-34). (3) Among different brain regions, there is a positive correlation between increasing levels of PHS and increasing levels of oxidative DNA damage (25). To investigate this hypothesis, we determined the efficacy of neurotransmitters, their precursors, and metabolites to serve as substrates for HRP- and PHS-1-catalyzed bioactivation to free radical intermediates using an in Vitro system, as successfully employed for phenytoin and amphetamines (22, 25). The chemical nature of the putative free radical intermediates was evaluated by spin stabilization- and spin trapping-ESR spectroscopy and by ultraviolet/visible (UV/vis) spectroscopy. An ESR-spin stabilization approach allows the scavenging of a semiquinone by divalent metal ions such as Zn2+ (35, 36), which form chelation complexes with the free radicals to enhance their stability. The use of conventional spin traps such as nitroso-compounds and nitrones to detect these intermediary species do not usually produce detectable signals in the ESR time scale (35-37). UV/vis spectroscopy was used to determine

Materials. All chemicals used in this study, including DA, 3-methoxytyramine (MTA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), dihydroxymandelic acid (DHMA), normetanephrine, 2′-dG, hematin, calf thymus DNA, arachidonic acid (AA), 5,8,11,14-eicosatetraynoic acid (ETYA), 5,5dimethyl-1-pyrroline-1-oxide (DMPO), R-phenyl-N-tert-butylnitrone (PBN), and nuclease P1 were of analytical or HPLC grade, were purchased from Sigma-Aldrich (Oakville, ON, Canada), and were used as received. Redistilled phenol was from Aldrich Chemical Co. (Oakville, ON, Canada). Calf intestine alkaline phosphatase was obtained from Roche (Laval, Quebec, Canada). Hydrogen peroxide (H2O2), obtained as a 30% (wt/wt) solution, was freshly prepared, and the final concentration was determined using ε(230 nm) ) 72.4 M1 cm-1 (38). HRP (type VI lyophilized powder; EC 1.11.1.7; donor-H2O2 oxidoreductase; 250-330 U/mg of solid), superoxide dismutase (SOD) from horseradish (EC 1.15.1.1; 1218 U/mg of solid), and catalase (E.C.1.11.1.6; 3260 U/mg solid) were also from Sigma-Aldrich. These enzymes were used as received, and dilutions from the stock solutions in Na2HPO4 buffer (100 mM, pH 7.4) were performed as needed. Purified ovine PHS-1 (EC.1.14.99.1, 185714 U/mg) and 8-oxo-dG were obtained from Cayman Chemical Co. (Ann Arbor, MI). A stock solution of PHS-1 (10000 U/mL) in phosphate buffer (80 mM, pH 7.9) was prepared, preserved at -80 °C, and subsequently used in the incubations. All of the substrates and the spin traps were freshly prepared, flushed with nitrogen, and stored at -80 °C until the moment that they were used. ESR Spectroscopy. ESR-spin stabilization measurements were carried out at room temperature in solutions contained in a quartz flat cell, using a Bruker BioSpin GmbH X band spectrometer. Instrument parameters typically used to obtain the spectra were as follows: microwave power, 31.7 mW; modulation amplitude, 0.2 G; time constant, 5.120 ms; conversion time, 5.120 ms; sweep time, 5.243 s; receiver gain, 5.0 × 10(5); center field, 3486 G; frequency 9.765 GHz; and number of scans, 100. Spin trapping experiments were also made at room temperature in solutions contained in capillary tubes, using a Bruker ER-200 X band spectrometer equipped with an ER 4123D_188r resonator. Instrument parameters typically used to obtained the spectra were as follows: microwave power, 31 mW; modulation amplitude, 0.99 G; scan time, 5 or 30 min; time constant, 2.56 s; sweep time, 5.243 s; receiver gain, 5.0 × 10(5); center field, 3473 G; and frequency 9.77 GHz. The spectrometer was initially calibrated using the Strong Pitch standard. Hyperfine coupling constants (hfcc) were measured (to (0.1 G) directly from the magnetic field separation and confirmed by computer simulation. ESR spectra were simulated using WINEPR SimFonia software from Bruker. Estimates of the relative radical production were made through the comparison of the normalized double integrals (DI/N) of the ESR-spin stabilization spectra obtained upon autoxidation and HRP-mediated oxidation of the neurotransmitters, their precursors, and metabolites. ESR-Spin Stabilization Experiments. Experimental conditions used in these assays were adapted from the literature (36). To evaluate the ESR signal resulting from the autoxidation of the neurotransmitter substrates, a solution containing the neurotransmitters, their precursors, and metabolites (6 mM) and zinc acetate (0.5 M) in acetate buffer (200 mM, pH 6.0) was prepared, and the spectra were immediately recorded. The enzymatic oxidation of the substrates was initiated with the stepwise addition of H2O2 (5 mM) and HRP (100 U/mL), and the spectra were immediately recorded. Because of the formation of a dark brown precipitate, L-DOPA,

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some incubation mixtures were filtrated through a LC13 PVDF membrane (0.2 µm) before acquiring the spectra. ESR-Spin Trapping Experiments. DMPO was dissolved in Na2HPO4 buffer (100 mM, pH 7.4) in the dark to a final concentration of 2.2 M. Stock solutions were kept under argon and stored at -80 °C. The DMPO solutions and the incubation mixtures were also kept in foil-covered tubes, and room light was kept to a minimum. A different spin trap, PBN, was also used to confirm the structural assignments of the free radicals. In this case, PBN was dissolved in deionized H2O to a final concentration 56 mM. Stock solutions were freshly prepared, flushed with argon, and kept in ice in the dark until the moment that they were used. Typical reaction mixtures for spin trapping experiments with DMPO contained the substrate (1 mM), HRP (100 U/mL), diethylenetriaminepenta-acetic acid (DTPA, 100 µM) to chelate residual redox active metals, spin trap (100 mM), and H2O2 (4 mM) in sodium phosphate buffer (100 mM, pH 7.4). All incubations were performed in the dark at room temperature. The reaction was initiated by addition of H2O2 to a mixture containing all other components. Samples were protected from light with aluminum foil and immediately analyzed by ESR spectroscopy. Spectral assignments were confirmed using 75 mM substrate and 30 mM H2O2. PHS-1 (1000 U/mL) was incubated with hematin (1.0 µM) for 1 min at 37 °C in 80 mM potassium phosphate buffer, pH 7.9. After the addition of the substrate (1 mM) and the free radical spin trap DMPO or PBN (1 mM), AA (140 µM) was added to initiate the reaction. These conditions have been used previously to reliably activate the enzyme while limiting its ability to self-inactivate (22, 25). Samples were covered with foil and subsequently analyzed by ESR and by UV/vis spectroscopy as described in the following section. To detect the presence of paramagnetic impurities in the incubations, control experiments were also performed using the same experimental conditions as described above. In this case, we ran several assays where each of the following reagents was omitted from the incubations at the time: neurotransmitter, their precursor or metabolite, and HRP or PHS-1. Additionally, to evaluate the contribution from autoxidation of each substrate, we ran time-course experiments (5 and 30 min) where only the substrate and DMPO were present in the incubation. To block PHS-1-catalyzed bioactivation of L-DOPA, the PHS-1 inhibitor ETYA (40 µM) was incubated with the enzyme at 37 °C for 1 min prior the addition of the substrate and AA. Spectrophotometric Assays. UV/vis spectra were recorded in a SpectraMAX Plus 384 (Molecular Devices Corp.). Reaction mixtures obtained upon HRP and PHS-1-mediated oxidation were analyzed in a 96 well plate under aerobic conditions at 37 °C. Spectra were scanned (λmax ) 220-700 nm) and recorded every 2 min for 30 min against control samples where the substrate and phenol were omitted from the incubations. Experimental conditions used for each of the enzymes were as follows: HRP (100 U/mL); H2O2 (0.3 mM); neurotransmitters, their precursors, and metabolites (0.2 mM) in phosphate buffer (100 mM, pH 7.4); PHS-1 (1000 U/mL); hematin (1 µm); phenol (0.5 mM); AA (140 µM); and neurotransmitters, their precursors, and metabolites (0.2 mM) in phosphate buffer (80 mM, pH 7.9). Ovine PHS-1-Dependent DNA Oxidation by Neurotransmitters, Their Precursors, and Metabolites. Calf thymus DNA was dissolved overnight in 80 mM potassium phosphate buffer, pH 7.9, at 65 °C. PHS-1 (1000 U/mL) was incubated in 80 mM potassium phosphate buffer, pH 7.9, with phenol (0.5 mM) and hematin (1 µM) for 1 min at 37 °C. Neurotransmitters, precursors, or metabolites (500 µM in 80 mM potassium phosphate buffer, pH 7.9) were added to the mixture along with dissolved DNA (2 mg/ mL). AA (140 µM) was added to initiate the reaction or omitted to inhibit PHS-1 activity. The reaction mixture was incubated at 37 °C with gentle agitation for 30 min. The controls for the incubations contained all reagents except the drug (vehicle control) and all reagents except the enzyme PHS-1. The reaction was stopped on ice, and DNA was precipitated with cold ammonium acetate (10 M) and 100% ethanol. Samples were spun at 10000g for 10 min,

GonçalVes et al. and the supernatant was discarded by aspiration. The pellet was washed twice with 70% ethanol and spun at 10000g for 5 min, and the supernatant was discarded by aspiration. The DNA pellet was then dissolved overnight in sodium acetate (20 mM, pH 4.8) at 65 °C. DNA was digested with nuclease P1 (8 U) at 37 °C. After 30 min, Tris-HCl (1 M, pH 8) was added followed by alkaline phosphatase (8 U), and this was incubated at 37 °C for 1 h. Samples were then filtered using Microcon-YM 10 filters (Millipore Canada Ltd.) and were stored at -80 °C until analysis. Detection of 8-Hydroxy-2′-deoxyguanosine and 2′-dG. 8-Hydroxy-2′-deoxyguanosine was quantified using an isocratic Series 200 HPLC system (Perkin-Elmer Instruments LLC, Shelton, CT) with electrochemical detection. It was equipped with a 5 µm Exsil 80A-ODS C-18 column (50 mm × 4.6 mm, Jones Chromatography, Ltd.), an electrochemical detector (Coulochem II), a guard cell (model 5020), an analytical cell (model 5010) (Coulochem, ESA Inc., Chelmsford, MA), and an integrator (Perkin-Elmer NCI 900 Interface). The filtered samples were injected into the HPLC system and eluted using a mobile phase containing ammonium acetate buffer (50 mM, pH 5.2) with 5% methanol at a flow rate of 0.8 mL/min, with a detector oxidation potential of +0.4 V. Chromatographs were analyzed using the TotalChrom chromatography software version 6.2.0 (Perkin-Elmer Instruments LLC). 2′-dG was quantified using HPLC with UV/vis detection at 260 and 280 nm (Perkin-Elmer Instruments LLC) and a mobile phase containing potassium phosphate buffer (50 mM, pH 5.5) with 5% methanol. Statistical Analysis. Multiple comparisons among groups were analyzed by one-way ANOVA with a subsequent Tukey’s test (GraphPad InStat3.05, GraphPad Software, Inc., San Rafael, CA). The level of significance was determined to be at P < 0.05.

Results In Vitro Bioactivation of Neurotransmitters, Their Precursors, and Metabolites to a Free Radical Intermediate by HRP and PHS-1. In the absence of HRP, the incubation of the two neurotransmitters DA and epinephrine and their precursors and metabolites L-DOPA, DOPAC, and DHMA gave rise to an isotropic ESR spectrum with resolved hyperfine structure, reflecting autoxidation (Figure 1B). Despite considerable overlapping of the spectral lines, computer-simulated spectra helped to identify these radical species as noncyclized Zn2+-complexed primary ortho-semiquinones (36, 37, 39-41). Computer simulation of the spectra (Figure 1C) gave the ESR parameters shown in Table 1. These results are in good agreement with published data for zinc-complexed primary semiquinones for epinephrine (36), DA (37), and related molecules (36, 37, 41). The isotropic g value found for all radicals was 2.0039, suggesting their identical structure. HVA and normetanephrine did not generate radical intermediates due to autoxidation. In the presence of HRP, the addition of H2O2 to the incubation mixture containing the above-mentioned substrates and Zn2+ in acetate buffer produced marked changes in the ESR spectral patterns as compared to those from autoxidation (Figure 1A). These changes included enhanced signal intensity as compared to those obtained upon autoxidation, along with an increased line width and/or unresolved hyperfine splittings. These latter observations may reflect the formation of more complex structures, such as dimers and/or melanin-like polymers (37, 42-44). The most visible change was observed for DOPAC and DHMA, where asymmetric ESR spectra characteristic of an immobilized radical were observed. Measurements of the relative radical production for Zn2+-complexed semiquinones obtained by autoxidation (Figure 2) revealed that all neurotransmitters, precursors, and metabolites, except HVA and normetanephrine, exhibited limited autoxidation to free radical inter-



Figure 1. ESR spectra of Zn2+-complexed o-semiquinones generated during HRP-catalyzed oxidation (A) by autoxidation (B) and by computer simulation (C) of (1) DOPAC, (2) DHMA, (3) DA, (4) L-DOPA, (5) epinephrine, (6) HVA, and (7) normetanephrine. Control without substrate (D). Spectra were acquired according to conditions described in Materials and Methods.

Table 1. ESR Data for Zn2+-Complexed o-Semiquinones Obtained from the Autooxidation of Neurotransmitters, Their Precursors, and Metabolitesa hfccb (G) neurotransmitter L-DOPA

DA DOPAC HVA epinephrine DHMA normetanephrine

derived radical +

-

A, RdCH2CH(N H3)COO A, RdCH2CH2N+H3 A, RdCH2COOH B, RdCH2COOH A, RdCH(OH)CH2N+H2CH3 A, RdCH(OH)COOH B, RdCH(OH)CH2N+H3

A3H

A5H

A6H

aβH

aγH

0.37 c c

3.81 3.88 3.86

0.61 0.67 0.62

3.24 (1H)2.66(1H) 3.36 (2H) 3.26 (2H)

c c

2.0039 2.0039 2.0039

0.37 0.30

3.61 3.71

0.64 0.57

3.10 (1H) 1.99 (1H)

0.20 (2H)

2.0039 2.0039

g

a Radicals were generated from 6 mM solutions of substrate in acetate buffer (200 mM, pH 6) containing Zn2+ (0.5 M). b The ring protons are numbered as 3, 5, and 6 (the side chain substituent is at position 4), and protons in the side chain are designated as β (attached to the carbon atom adjacent to the ring) and γ. c Not detected.

mediates (ortho-semiquinones). This limited radical production via autoxidation was in contrast to substantial HRP-catalyzed oxidation, with a maximal 18-fold increase for HRP-catalyzed radical production from DOPAC, as shown by the normalized double integral ESR signal. HRP-catalyzed free radical formation from DA, its precursor L-DOPA, and its metabolite DOPAC was 3-8 times greater than that from epinephrine and its DHMA metabolite. To explore the formation of superoxide and/or hydroxyl free radical intermediates, we also ran spin trapping experiments using HRP, PHS-1, DMPO, and PBN. Unlike PBN, DMPO yields distinct and more persistent spin adducts with the primary O2•- and HO• radicals (DMPO-OOH and DMPO-OH, respectively). DMPO-OH may also arise from the initial trapping of O2•-, giving DMPO-OOH, which is reported to readily decompose to DMPO-OH at physiological pH (45, 46). Spectral confirmation for HRP-catalyzed formation of free radical spin adducts was obtained for DA and its precursor L-DOPA (Figure

3). Elimination of any component of the spin trapping reaction mixture resulted in the complete loss or diminution of the spectra. The ESR signal revealed the presence of two different radical spin adducts (RSA): a triplet (RSA1) and a large doublet of triplets (RSA2). The RSA2 was observed for DA, L-DOPA, and MTA when incubated with active HRP (Table 2). Additionally, in the spectra of DA, L-DOPA, epinephrine, and normetanephrine incubated with HRP, a quartet was also observed at aN ) aβH ) 14.88 G (RSA3) characteristic of DMPO-OH• spin adducts (Table 2). Considering that artifacts with DMPO have been reported under a variety of experimental conditions (47-50), we assigned the ESR signal corresponding to RSA1 to an aminoxyl radical from DMPO, which is an artifact due to an oxidized derivative of DMPO and/or due to oxidized DMPO impurities, such as hydroxylamine. Similarly, we also consider the large doublet of triplets signal produced by RSA2, noteworthy for further investigation.

846


Figure 2. Relative radical production for Zn2+-complexed semiquinones obtained by autoxidation and by HRP-mediated oxidation of DA, its precursor, and metabolites and of epinephrine and its metabolites. The relative radical production was estimated using the normalized double integral (DI/N) obtained from the spin stabilization ESR spectra.

The addition of SOD (1 µM) to the incubations did not measurably affect RSA2 or RSA3 spin adducts but produced an increase in the signal amplitude of RSA1 for DA and L-DOPA. In contrast, the addition of catalase (62 µM) inhibited RSA2 and RSA3 spin adducts (results not shown), suggesting that both radical species are formed through a peroxidative mechanism and are H2O2-dependent. These results may also suggest that HO• may be formed by a Fenton-like reaction. Similar results were obtained when PHS-1 was used instead of HRP (Figure 3). PHS-1-catalyzed oxidation of DA, L-DOPA, epinephrine, and normetanephrine produced spectra with the same hyperfine splitting pattern: a triplet that corresponded to RSA1 of HRP reactions and a large doublet of triplets that corresponded to RSA2 of HRP reactions summarized in Table 3. Additionally, in the spectra of DA, L-DOPA, epinephrine, and normetanephrine incubated with PHS, a quartet was also observed at aN ) aβH ) 14.88 G (RSA3) characteristic of DMPO-OH• spin adducts (Table 3). In contrast, the ESR spectra for the control experiments showed that neither RSA1 nor RSA2 are formed in phosphate buffer containing only DMPO (Figure 3F) or without substrate (Figure 3G). Preincubation of PHS-1 with the PHS/lipoxygenase inhibitor ETYA (40 µM) abolished the ESR signal corresponding to RSA2 and RSA3 (Figure 3E). The 40 µM concentration of ETYA is above the Ki value for PHS inhibition in isolated cells and purified enzyme preparations (51). To evaluate the presence of radical species generated by autoxidation of the substrates, we also ran time-dependent control incubations (5 and 30 min) in which only the substrate, DMPO, and phosphate buffer were present, without a bioactivating enzyme. The spectra presented for these controls (without either HRP or PHS-1) (Figure 3H,I) did not show the presence of any radical other than a triplet corresponding to RSA1 in the first 5 min (results not shown). These results suggest that the radical species RSA1 is mainly produced via HRP- or PHS1-catalyzed oxidation. Weak ESR signals corresponding to RSA2 were initially visible only after 30 min (Figure 3). To investigate the genesis of the postulated C-centered free radical corresponding to RSA2, we repeated the assays with PHS-1, epinephrine, and L-DOPA using PBN. The ESR signal obtained for epinephrine (Figure 4B) is composed of two superimposed, overlapping spectra, with hfcc values that are in good agreement

GonçalVes et al.

with published data for C-centered spin adducts (52). The ESR spectra of L-DOPA also indicated the presence of C-centered radicals with hfcc values similar to those observed for epinephrine. There was no detectable radical adduct of PBN in the control incubations (without substrate) for these compounds (Figure 4A). On the basis of the results of these studies, we believe that the RSA2 detected in reactions with DMPO and HRP or PHS-1 are C-centered free radicals that are derived from the oxidation of the substrates DA, L-DOPA, MTA, epinephrine, and normetanephrine. Spectrophotometric Assays. The spectrophotometric profile of the incubation mixtures resulting from HRP and PHS-1catalyzed oxidation of DA and epinephrine precursors and metabolites was analyzed by UV/vis spectroscopy between 220 and 700 nm, under physiological pH, in a time scale of 30 min. The UV/vis spectra obtained for the 2 min time point are shown in Figures 5 (HRP) and 6 (PHS-1). Upon catalysis, both enzymes produced colored incubation mixtures, from light yellow to brown, depending on the substrate and the enzyme. Although conversion of the substrates to the putative intermediates (orthosemiquinones/quinones and aminochromes) occurred at higher rates for HRP, time-dependent incubations did not show noticeable differences in the nature of the chromophores detected for each enzyme in a time scale of 30 min (results not shown). Neutral aqueous solutions of catechols and catecholamines are known to have absorption spectra in the UV region with two maxima at ca. 230 and 280 nm (44). In fact, upon HRP- and PHS-1-catalyzed oxidation of substrates, the absorbance was shifted to longer wavelengths, suggesting the formation of new products/chromophores. Time-dependent incubation of L-DOPA and DA with HRP revealed the early (within 2 min) existence of a chromophore with λmax similar to that given in the literature for their aminochromes (a cyclized quinone) at 300 and 480 nm (Table 4 and Figure 5) (42, 53). Nevertheless, the products of DOPAC and DHMA at λmax ) 400 nm, respectively, are assumed to be ortho-quinones (42). DHMA produced an intense absorption band at λmax ) 340 nm (Figure 5), which is near to λmax ) 335 nm observed for human melanin pigment (54). HVA and normetanephrine did not produce detectable amounts of either intermediate. As indicated above, with PHS-1, important differences in the rates of oxidation were observed among the substrates tested (compare, for example, Figures 5 and 6). PHS-1catalyzed oxidation of L-DOPA produced a band at λmax ) 400 nm assumed to be due to its ortho-quinone, with no detectable amounts of aminochrome. In contrast, for DA, new peaks were formed for both the quinone at λmax ) 400 nm and the aminochrome at λmax ) 480 nm.2 Similarly, the product of DOPAC, an ortho-quinone, was observed at λmax ) 400 nm. No absorption bands were detected for HVA and normetanephrine. Ovine PHS-1-Dependent DNA Oxidation by Neurotransmitters, Their Precursors, and Metabolites. DA, L-DOPA, and DOPAC (500 µM) all were bioactivated by ovine PHS-1 in Vitro to a free radical reactive intermediate that enhanced ROS formation, resulting in the oxidation of DNA to produce 8-oxo-dG (Figure 7). While there was evidence of DA autoxidation, as illustrated in the reaction without AA, reactions containing active PHS-1 showed a 72% increase in DNA oxidation as compared to reactions containing inhibited enzyme (p < 0.05). Similarly, L-DOPA with active PHS-1 caused a 70% 2 The absorption band of the parent catecholamine presumably hindered the band at ca. λmax ) 400 nm.



Figure 3. X-band ESR spectra of radical species trapped with DMPO: (A) DA, (B) L-DOPA, (C) DOPAC, (D) epinephrine, (E) incubation with L-DOPA and ETYA, (F) control with DMPO, (G) control without substrate, (H) control without HRP, and (I) control without PHS-1. Radical species are identified as ([) RSA1, (*) RSA2, and (X) RSA3. Spectra were acquired according to conditions described in Materials and Methods.

Table 2. Hyperfine Splitting Constants Obtained by EPR-spin Trapping Experiments for HRP-Catalyzed Oxidation of Neurotransmitters, Their Precursors and Metabolitesa substrate

radical species

hyperfine splitting constants (G)

L-DOPA

RSA1 RSA2 RSA3 RSA1 RSA2 RSA3 RSA1 RSA2 RSA3 RSA1 RSA2 RSA3 RSA1 RSA2 RSA3

aN ) 14.73 aN ) 15.74, aβH ) 23.03 aN ) aβH ) 14.88 a ) 14.73 aN ) 15.88, aβH ) 23.61 aN ) aβH ) 14.88 aN ) 14.73 b b aN ) 14.73 aN ) 15.59, aβH) 23.46 b aN ) 14.73 b b

DA DOPAC MTA HVA

a Radicals were generated from 1 mM solutions of substrate in phosphate buffer, pH 7.4, by HRP-mediated oxidation (100 U/mL). The in Vitro system contained H2O2 (4 mM), substrate, and DMPO (100 mM). b Not detected.

Table 3. Hyperfine Splitting Constants Obtained by EPR-spin Trapping Experiments for PHS-Catalyzed Oxidation of Neurotransmitters, Their Precursors, and Metabolitesa substrate L-DOPA

DA DOPAC epinephrine normetanephrine

radical species

hyperfine splitting constants (G)

RSA1 RSA2 RSA3 RSA1 RSA2 RSA3 RSA1 RSA2 RSA3 RSA1 RSA2 RSA3 RSA1 RSA2 RSA3

aN ) 14.73 aN ) 15.88, aβH ) 23.18 aN ) aβH ) 14.88 aN ) 14.73 aN ) 15.59, aβH ) 23.32 aN ) aβH ) 14.88 aN ) 14.73 b b aN ) 14.73 aN ) 15.74, aβH ) 23.03 aN ) aβH ) 14.88 aN ) 14.73 aN ) 15.45, aβH ) 23.03 aN ) aβH ) 14.88

a Radicals were generated from 1 mM solutions of substrate in phosphate buffer, pH 7.9, by PHS-1-catalyzed oxidation (1000 U/mL). The in Vitro system contained PHS-1 and hematin (1.0 µM). After preincubation for 1 min at 37 °C, substrate, DMPO (1mM), and AA (140 µM) were added to initiate the reaction. b Not detected.

increase in DNA oxidation as compared to reactions with inhibited enzyme (p < 0.05). DOPAC also showed a 38-62% increase in DNA oxidation as compared to reactions containing no PHS-1 (p < 0.05) and no AA (p < 0.05), respectively. Under these reaction conditions at 500 µM, HVA and MTA did not lead to a PHS-1-catalyzed increase in DNA oxidation.

Discussion We investigated the hypothesis that the precursors and metabolites of endogenous neurotransmitters, in addition to the neurotransmitters themselves, may serve as substrates for PHS1-catalyzed bioactivation to potential neurodegenerative free radical intermediates. The neurodegenerative potential of neurotransmitter precursors and metabolites is particularly important because such endogenous molecules are generally considered biologically inactive based upon neurotransmitter receptor binding assays. The free radical intermediates formed via PHScatalyzed bioactivation of such endogenous substrates can initiate the formation of ROS that oxidize cellular macromolecules like DNA, protein, and lipids, which have been implicated

Figure 4. X-band ESR spectra of radical species derived from epinephrine trapped with PBN: (A) control without substrate, (B) experimental spectrum obtained upon incubation of epinephrine with PHS-1, and (C) computer simulation of the X band ESR spectrum obtained in B.

in cellular degeneration in the brain (25). Our results accordingly may provide new insights into neurodegenerative effects associated with aging and mechanisms underlying neurodegenerative diseases like Alzheimer’s and Parkinson’s diseases. In particular, we examined the role of mammalian PHS-1 and a related model enzyme, HRP, in catalyzing the bioactivation of

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GonçalVes et al. Table 4. UV/Vis Spectral Characterization of Products Obtained upon HRP- and PHS-1-Mediated Oxidation of Neurotransmitters, Their Precursors, and Metabolitesa product(s) λmax substrate L-DOPA

DA DOPAC DHMA

oxidizing conditions HRP PHS-1 HRP PHS-1 HRP PHS-1 HRP PHS-1

HVA

Figure 5. UV/vis spectra of the products obtained by HRP-mediated oxidation of neurotransmitters, their precursors, and metabolites. HRPmediated oxidation was performed at 37 °C using the following experimental conditions: HRP (100 U/mL), H2O2 (0.3 mM), and neurotransmitters, their precursors, and metabolites (0.2 mM) in phosphate buffer (100 mM, pH 7.4). Spectra presented were scanned from 220 to 700 nm and recorded at 2 min.

Figure 6. UV/vis spectra of the products obtained by PHS-1-mediated oxidation of neurotransmitters, their precursors, and metabolites. PHS1-mediated oxidation was performed at 37 °C using the following experimental conditions: PHS-1 (1000 U/mL), hematin (1 µm), phenol (0.5 mM), AA (140 µM), and neurotransmitters, their precursors, and metabolites (0.2 mM) in phosphate buffer (80 mM, pH 7.9). Spectra presented were scanned from 220 to 700 nm and recorded at 2 min.

DA and epinephrine, as well as their precursors and metabolites to potentially neurodegenerative free radical intermediates. The in Vitro studies herein using ESR, UV/vis spectroscopy, and high-performance liquid chromatography with electrochemical detection (HPLC-EC) provide direct evidence that neurotransmitters, precursors, and metabolites can be bioactivated by HRP and PHS-1 to form not only semiquinone radicals but also carbon-centered free radicals and ROS, such as hydroxyl and/ or superoxide free radicals. This PHS-dependent bioactivation resulted in ROS-mediated oxidative DNA damage measured by 8-oxo-dG formation, which has been implicated in neurodegenerative processes (25). Previous studies with epinephrine have demonstrated some of these reactive outcomes during autoxidation or HRP-catalyzed bioactivation (26, 36, 55-57). Our work confirms those studies and presents novel ESR, UV/ vis spectroscopy, and HPLC-EC data characterizing reactive intermediates from PHS-1-catalyzed bioactivation of the LDOPA precursor and DOPAC metabolite of the neurotransmitter DA, as well as DA itself, resulting in oxidative DNA damage. Spin stabilization ESR experiments with Zn2+ proved useful for trapping semiquinone radical species formed by one-electron oxidation of the substrates. With the exception of HVA and

normetanephrine

HRP PHS-1 HRP PHS-1

UV/vis (nm) 300 400 300 400 b 400 400 400 340 c 340 c c c c

480 (aminochrome) (o-semiquinone) 480 (aminochrome) (o-semiquinone) 480 (o-semiquinone) (o-semiquinone) (o-semiquinone)

c c c c

a

The wavelengths of maximal absorption obtained at 2 min of incubation time are given for HRP-mediated oxidation at pH 7.4 (100 mM phosphate buffer) and for PHS-1-mediated oxidation at pH 7.9 (80 mM phosphate buffer). Other experimental conditions are described in the Materials and Methods. b Not determined. c No signal.

normetanephrine, all substrates tested generated ortho-semiquinone free radical intermediates upon autoxidation. This was consistent with the known oxidation of catecholamines involving molecular oxygen under physiological conditions (55-57). HRP-catalyzed free radical formation from DA, its precursor L-DOPA, and its metabolite DOPAC was 3-8 times greater than that from epinephrine and DHMA. DA was a slightly better substrate for HRP than L-DOPA, but more importantly, DOPAC was a better substrate than DA for HRP-catalyzed oxidation to free radical intermediates. Kinetics studies using the same ESR approach and enzymatic system also found that DA was a better substrate for HRP than L-DOPA (35), at both pH 8 and pH 5. In spin stabilization ESR experiments, the unresolved hyperfine structure observed in the spectrum of DA and epinephrine may be a consequence of the overlapping of ESR signals due to the formation of secondary semiquinone radicals. In the case of DA and epinephrine, this observation may reflect higher rates of cyclization of their ortho-semiquinones, which are the intermediate molecular products in the pathway to aminochromes, and/or perhaps a higher stability of their aminochromes, from which the secondary radicals are derived. UV/vis spectral characterization of the metabolites generated by HRP-catalyzed oxidation of DA and L-DOPA, using similar experimental conditions, revealed the formation of a chromophore with λmax at 300 and 480 nm (Table 4), characteristic of their aminochromes (42, 53). Previous studies (26, 35, 36) established that oxidation of epinephrine and norepinephrine by HRP-H2O2 generates ortho-semiquinones as a primary one-electron oxidation product, which by disproportionation may give rise to orthoquinones. With deprotonation of the amino group in the side chain, ortho-quinones undergo 1,4-intramolecular cyclization to form leukoadrenochrome. Interestingly, HRP-catalyzed bioactivation of DOPAC and DHMA produced asymmetric ESR spectra, characteristic of an immobilized radical. Similar ESR spectra have been obtained from enzymatic oxidation of the DOPA derivative 5-S-cysteinyldopa to synthetic pheomelanins (58). Synthetic melanins are characterized by an ESR spectrum with a single broad line with g ) 2.004 and a line width of 4-10 G. The putative DOPAC- and DHMA-derived polymers have g values of 2.004 and 2.003, respectively, and a dark brown precipitate was



Figure 7. Ovine PHS-1-dependent DNA oxidation by neurotransmitters, their precursors, and metabolites. Groups include DA, L-DOPA, DOPAC, HVA, and MTA. Each reaction contained 1000 U/mL PHS-1, 1.0 µM hematin, and 0.5 mM phenol. After preincubation for 1 min at 37 °C, 500 µM neurotransmitter and precursor or metabolite, 2 mg/mL DNA, and 140 µM AA were incubated for 30 min at 37 °C. DNA was precipitated and digested, and oxidative DNA damage was quantified by the formation of 8-oxo-2′-deoxyguanosine (8-oxo-dG)/µg 2′-dG. The vehicle control incubation contained all components except the substrate. The No AA group represents inhibition of PHS-1 activity.

observed frequently during the course of the ESR-spin stabilization experiments. Particularly in the case of DOPAC and DHMA, filtration of the incubations mixtures through a PVDF membrane was necessary prior to spectral acquisition. Although the mechanism of melanin formation is not thought to involve the direct reaction of free radicals per se, the formation of melanin is a well-documented characteristic in the process of catecholamine oxidation (37, 42-44, 58). As described in the literature (42), neuromelanin deposited in the substantia nigra and locus ceruleus is generated from polymerization of oxidized products of DA and norepinephrine. For DOPAC and DHMA, the 1,4-intramolecular cyclization of their ortho-quinones is hindered, so their bioactivation pathway is devoid of leukoadrenochrome and aminochrome formation. The direct attack of nucleophiles and/or other radical species to the aromatic ring of primary ortho-semiquinone free radicals may lead to the formation of oligomeric or polymeric structures that may account for the observed ESR signal. This hypothesis would be consistent with the further detection of UV/vis absorption bands characteristic of ortho-semiquinone free radicals (Table 4) under similar in Vitro conditions. More importantly, the intense absorption peak observed at λmax ) 340 nm for DHMA was assigned to melanin or a melanin-like polymer, since human melanin has a characteristic λmax ) 335 nm (54). These results may provide the first direct evidence for the formation of free radical intermediates and melanin or melanin-like polymers in the peroxidase-dependent bioactivation of neurotransmitter metabolites, such as DOPAC and DHMA, which might contribute to the neurodegenerative process. As mentioned previously, catecholamine bioactivation by peroxidases is also associated with the generation of ROS. ESR-spin trapping experiments investigated the formation of free radical intermediates such as HO•, O2•-, and other putative

radical species, such as C- and N-centered free radicals (25). Although the generation of superoxide radicals during autoxidation of the semiquinones is generally accepted (8, 10), the superoxide radical spin adduct formed with DMPO is relatively unstable in aqueous solution. The half-life of its ESR signal is approximately 80 s at pH 6 and 35 s at pH 8 (59). The HO• radical, and usually C-centered radicals, forms stable spin adducts with DMPO in aqueous solutions, with the resulting signal often lasting hours or even days, depending on the temperature. In our studies, the production of hydroxyl free radicals via PHS-1- and HRP-catalyzed oxidation of DA, L-DOPA, and epinephrine was confirmed by spin trapping-ESR spectroscopy (Tables 2 and 3). The quartet, RSA3, having an intensity ratio of 1:2:2:1 and aN ) aβH ) 14.88 G is a wellknown characteristic of DMPO/OH• spin adducts (18). The addition of SOD did not affect the RSA3 intensity, suggesting that HO• was not generated by a Haber-Weiss reaction. To the contrary, the inhibition of RSA3 after addition of catalase suggests that HO• could be generated by Fenton chemistry. However, we used lower concentrations of SOD, below its catalytic range (33 µM). Because catalase is a more effective inhibitor than SOD (26), lower concentrations of SOD may have not produced a detectable change in the ESR signal. Moreover, Fenton reactions usually require the presence of catalytic concentrations of metal ions, such as Fe2+. The use of DTPA should render the Fenton reaction a minor contributor to the production of HO•. The PHS inhibitor ETYA abolished the signal of the DMPO-OH spin adduct (Figure 3), negating the possibility of exclusive production of HO• by Fenton chemistry, and alternatively suggesting that the generation of HO• is peroxidase-mediated. One cannot discard completely the possibility that HO• may also be formed by homolytic cleavage of

850


H2O2, as HO• has been observed in buffered (pH 7.2) reaction mixtures containing DMPO and H2O2 after UV irradiation (60, 61). PHS-1-catalyzed bioactivation of DA, its precursor L-DOPA, epinephrine, and normetanephrine generated another ESR signal, possibly due to a C-centered or a phenoxyl DMPO-spin adduct. Inhibition of the ESR signal by the PHS inhibitor ETYA indicated that the bioactivation of L-DOPA to a free radical intermediate was catalyzed by PHS-1. ETYA is a dual inhibitor of PHSs and lipoxygenases (62, 63) and has been shown in Vitro at the same concentration to inhibit the PHS-catalyzed bioactivation of xenobiotics and neurotransmitters (21, 22, 30). The absence of the signal in the control spectrum (Figure 3) when PHS-1 was omitted from the medium also indicated that bioactivation was due to the active enzyme rather than other components of the system. Interestingly, the hyperfine structure and the hfcc values were very similar to those of a DMPO-spin adduct derived from DA previously reported (54). Computer simulation of the ESR signal obtained in assays with PBN confirmed the existence of C-centered free radicals for L-DOPA (data not shown). A C-centered free radical with similar hfcc values (aN ) 15.21 G and aβH ) 23.48 G) was also detected by others (4) in an ESR-spin trapping study using RCHT cells with DMPO, but this radical spin adduct was not identified. Our results suggest that this radical species is probably due either to the formation of C-centered free radicals on the side chain of the molecule and/or to a radical species delocalized over the aromatic ring. This hypothesis is consistent with published studies reporting the generation of putative N- and C-centered free radicals in the bioactivation of 3,4-methylenedioxyamphetamine by PHS-1 (25), where the oxidation occurred initially at the primary amino group in the side chain of the molecule. Furthermore, hfcc values found for this DMPO-radical species are in the range of values described in the literature for DMPO C-centered free radicals (49) and for DMPO-CH(OH)CH3 types of adducts (63, 64). Our attempts to determine superhyperfine coupling constants by decreasing the modulation amplitude resulted in a decrease of the signal intensity, which prevented the determination of a more accurate structure for these radical species. Our results suggest that different competing pathways could account for the generation of superoxide and hydroxyl free radicals and C-centered radicals during the peroxidasemediated bioactivation of catecholamines. One pathway involves the generation of semiquinone free radical intermediates that rapidly react to produce superoxide radicals and other damaging ROS. Redox cycling between catechols/catecholamines and their quinones/semiquinones can continuously produce large amounts of ROS, the chemical basis of which is provided elsewhere (6, 9, 10). Although this might be a minor competing pathway in the overall process, peroxidase-catalyzed bioactivation of endogenous substrates, the oxidation of the amino group, and subsequent formation of a C-centered free radical may also play an important role in the formation of damaging ROS. The generation of a C-centered free radical and its reaction with O2 and the subsequent oxidation by a peroxidase or by a Fentonlike mechanism may generate ROS capable of oxidizing macromolecules. DNA oxidation is a sensitive measure of oxidative stress that can occur in neurodegenerative conditions, and this molecular lesion may cause genomic instability and/or aberrant cell signaling leading to cellular dysfunction and cell death (25). In our DNA oxidation studies, in the absence of PHS-1, DA showed higher overall DNA oxidation as compared to L-DOPA and DOPAC due to autoxidation. However, in the presence of

GonçalVes et al.

enzyme, DNA oxidation was further enhanced over that due to autoxidation, and PHS-1 activity was equally effective in increasing DNA oxidation for DA and L-DOPA (i.e., both had increases of ∼70%). The increase in PHS-1-dependent DNA oxidation for the catechol DOPAC was lower (an increase of ∼40%) as compared to DA and L-DOPA, which may be due to its lack of ability to cyclize to form an aminochrome. MTA and HVA did not lead to PHS-1-dependent DNA oxidation under these reaction conditions, possibly because these chemicals are not catechols. While spin trapping-ESR spectroscopy revealed that MTA produced a free radical, it may not be sufficiently reactive or abundant at these concentrations to initiate ROS formation and DNA oxidation. Higher concentrations of the substrates may be necessary to detect oxidized DNA or to trap free radicals directly by ESR. The DA concentration in nerve terminals is about 50 mM, but this is stored in vesicles (2, 65). However, total catechol levels of precursors and metabolites present in the cytosol can be in the millimolar range (66); hence, the neurodegenerative potential of the precursors and metabolites is distinct from the actual neurotransmitters. In summary, we have shown that DA, its precursor L-DOPA, and its metabolite DOPAC, as well as epinephrine and its metabolite DHMA, were excellent HRP and PHS-1 substrates, resulting in HRP- and PHS-1-catalyzed bioactivation to various free radical intermediates that initiate ROS formation and DNA oxidation. This oxidative damage to DNA and other cellular macromolecules, in addition to ROS-mediated signal transduction, may contribute to the neurodegenerative process (25, 67). In addition, we provide spectroscopic evidence for the potential bioactivation of neurotransmitter precursors and metabolites by PHS-1 to ortho-semiquinones and aminochromes. The differences in rates of cyclization of the ortho-semiquinones and/or the absence of an amino group in the side chain of the substrate molecules observed in this and other studies (15, 16) imply that precursors and metabolites may have the opportunity to react with external nucleophiles and/or radical species rather than undergo internal cyclization, which might contribute to the neurodegenerative process as well. Perhaps most importantly, our results show that the precursors and metabolites of endogenous neurotransmitters, which are generally considered inactive based upon receptor binding assays, may actually play an important role in free radical-initiated neurodegeneration associated with aging. Acknowledgment. This research was supported by a grant from the Canadian Institutes of Health Research (CIHR). L.L.G. was supported by a Postdoctoral Fellowship from the Portuguese Foundation for Science and Technology (FCT). A.R. was supported by a Doctoral Fellowship from the CIHR/Rx&D Health Research Foundation. We thank Prof. Miguel Teixeira (ITQB Metalloproteins and Bioenergetics Laboratory, Portugal) for his kind access to the EPR facility and Dr. Smilja Todorovic for assistance in running some ESR spectra.

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