Iron- and Manganese-Catalyzed Autoxidation of ... - ACS Publications

824

Chem. Res. Toxicol. 1998, 11, 824-837

Iron- and Manganese-Catalyzed Autoxidation of Dopamine in the Presence of L-Cysteine: Possible Insights into Iron- and Manganese-Mediated Dopaminergic Neurotoxicity Xue-Ming Shen and Glenn Dryhurst* Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019 Received February 26, 1998

Iron(II/III) and manganese(II) both catalyze the autoxidation of the neurotransmitter dopamine (DA) in the presence of L-cysteine (CySH) in buffered aqueous solution at pH 7.4. Fe2+/Fe3+ and CySH together generate the hydroxyl (HO‚) and cysteinyl thiyl (CyS‚) radicals. DA is oxidized by HO‚ to DA semiquinone radical species that either react with CyS‚ to give 5-S-cysteinyldopamine (5-S-CyS-DA), 2-S-CyS-DA, and 6-S-CyS-DA or disproportionate to DAo-quinone that reacts with CySH to give the same cysteinyl conjugates of DA. The major product of this initial reaction is 5-S-CyS-DA. However, 5-S-CyS-DA can be further oxidized by HO‚ to an o-quinone (2) that undergoes intramolecular cyclization to an o-quinone imine (3). The latter intermediate is the precursor of the dihydrobenzothiazine (DHBT) 7-(2aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1) and several other cyclized products. However, cysteinyl conjugates of DA can also be oxidized by HO‚ in a one-electron abstraction reaction that leads to DA thiyl radicals. Reactions of these radicals with CyS‚ or DA semiquinone radicals lead to some novel DA disulfides and thioethers, respectively. The Mn(II)-catalyzed oxidation of DA generates DA-o-quinone that is scavenged by CySH to give 5-S-CyS-DA (major initial product) with lower yields of other cysteinyldopamines. Subsequent Mn(II)-catalyzed oxidation of 5-S-CyS-DA gives o-quinone 2 and thence o-quinone imine 3 that serve as the precursors of DHBT-1 and several other DHBTs. Organic or oxygen radicals do not play significant roles in the Mn(II)-catalyzed oxidation of DA in the presence of CySH. Recent studies have demonstrated that DHBT-1 can be accumulated by brain mitochondria and evoke irreversible inhibition of NADH-coenzyme Q reductase (complex I). Furthermore, iron, manganese, and alterations in glutathione and CySH metabolism have been implicated in the selective degeneration of nigrostriatal dopaminergic neurons in idiopathic and chemically induced Parkinson’s disease (PD). Because DHBT-1 is formed in both the ironand manganese-catalyzed oxidation of DA in the presence of CySH and a defect in mitochondrial complex I respiration contributes to dopaminergic neuronal cell death in PD, the results of this investigation are discussed in terms of their possible implications to an understanding of the neuropathological processes in idiopathic and chemically induced parkinsonism.

Introduction The major pathology of idiopathic Parkinson’s disease (PD)1 is the degeneration of neuromelanin-pigmented dopaminergic cells in the zona compacta of the substantia nigra (SNc). The mechanisms responsible for this neurodegeneration are unknown although oxidative stress may contribute to the selective loss of nigrostriatal dopamine (DA) neurons (1). Compared to age-matched controls, the total concentration of iron in the parkinsonian SNc is significantly increased (2, 3), levels of the iron-binding protein ferritin are decreased (4), and a * Address correspondence to this author. Tel: (405) 325-4811. Fax: (405) 325-6111. E-mail: [email protected]. 1 Abbreviations: Parkinson’s disease (PD); substantia nigra pars compacta (SNc); dopamine (DA); hydroxyl radical (HO‚); superoxide radical anion (O2-•); γ-glutamyl transpeptidase (γ-GT); glutathione (GSH); glutathione disulfide (GSSG); L-cysteine (CySH); cystine (CySSCy); 5-S-cysteinyldopamine (5-S-CyS-DA); dihydrobenzothiazine (DHBT); 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1); trifluoroacetic acid (TFA); benzothiazine (BT); cysteinylthiyl radical (CyS‚); cystine radical anion (CySSCy-•); 5-Scysteinyl-3,4-dihydroxyphenylalanine (5-S-CyS-DOPA).

neuromelanin-iron complex has been detected (5). Because of the ability of low-molecular-weight iron species or the neuromelanin-iron complex to catalyze formation of the hydroxyl radical (HO‚) from H2O2 and superoxide anion radical (O2-•) (6, 7), it has been suggested that iron is an endotoxin that mediates SNc cell death in PD (7, 8). Indeed, infusions of iron salts into the SNc of rats evoke progressive degeneration of nigrostriatal DA neurons (9, 10). Chronic exposure to manganese also causes the degeneration of nigrostriatal DA neurons and a parkinsonian syndrome (11, 12). One hypothesis concerning the dopaminergic neurotoxicity of manganese is that Mn2+ is first oxidized to Mn3+ that then potentiates the autoxidation of DA in a reaction that generates oxygen radicals and cytotoxic DA-o-quinone (11, 13). However, the manganese-catalyzed autoxidation of DA involves redox cycling of Mn2+ and Mn3+ in a reaction that generates H2O2 and DA-o-quinone (14). Furthermore, Mn2+ can scavenge and detoxify O2-•, and neither Mn2+

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Autoxidation of Dopamine

nor Mn3+ can generate HO‚ from H2O2 and/or O2-• by Fenton-type or Haber-Weiss-type reactions (13, 15). It has also been suggested that an elevated rate of autoxidation of cytoplasmic DA in the parkinsonian SNc contributes to dopaminergic cell death by formation of a cytotoxic quinone and oxygen radicals (16, 17). However, dopaminergic SNc cells are normally pigmented with black neuromelanin deposited in the cytoplasm of these neurons as a result of autoxidation of DA to DA-o-quinone that subsequently polymerizes (16). Thus, in both idiopathic PD and manganese-induced parkinsonism, an increased rate of DA autoxidation, in the absence of any other factor, should result in heavier pigmentation of dying SNc cells. However, degenerating SNc neurons in PD (18-20) and manganism (13) contain less neuromelanin pigment than in the healthy brain. In addition to depletion of nigrostriatal DA, chronic manganese exposure also leads to a marked decrease of glutathione (GSH) levels and an upregulation of γ-glutamyl transpeptidase (γ-GT) (21, 22). These observations are of interest because not only has a massive (40-50%) loss of nigral GSH been consistently reported in PD (3, 23), but also this is the earliest known change in the parkinsonian brain (24), is specific to the SNc, and is not accompanied by a corresponding increase of glutathione disulfide (GSSG) levels. Furthermore, γ-GT is also significantly upregulated in the parkinsonian SNc, whereas activities of other enzymes associated with GSH biosynthesis and metabolism are normal (25). It has been suggested that PD might be a disorder caused by reduced nigral GSH levels that predispose SNc neurons to damage by oxidative stress (26). However, several lines of evidence argue against this suggestion. For example, inhibition of GSH biosynthesis in rat brain does not lead to the degeneration of nigrostriatal DA neurons (27). Indeed, a direct role for reduced GSH levels in the cytoplasm of dopaminergic SNc neurons in the pathogenesis of idiopathic or chemically induced parkinsonism is improbable because neuronal cell bodies normally contain low concentrations of the tripeptide (28, 29). The preceding lines of evidence have led us to propose that a very early event in the pathogenesis of idiopathic PD is the release of GSH from nigral glia, its principal storage compartment (28, 29), and the upregulation of γ-GT with resultant translocation of CySH into pigmented dopaminergic SNc neurons (30-32). The entry of certain environmental, occupational, or xenobiotic chemicals into the SNc of genetically susceptible individuals might trigger such a translocation of CySH (3032). Such a hypothetical γ-GT-mediated translocation of CySH into pigmented SN cells should divert the neuromelanin pathway by scavenging DA-o-quinone to give 5-S-cysteinyldopamine (5-S-CyS-DA) that is easily oxidized to other cysteinyldopamines and dihydrobenzothiazines (DHBTs) (30-32). Such a hypothetical sequence of events might account for the irreversible loss of nigral GSH without increased GSSG levels (3, 23-25), an increased 5-S-CyS-DA/DA concentration ratio (17), and depigmentation of surviving dopaminergic cell bodies (18-20) that all occur in the parkinsonian SNc. Furthermore, a major early product of the in vitro oxidation of DA in the presence of CySH, 7-(2-aminoethyl)-3,4dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1), can be accumulated by intact rat brain mitochondria and irreversibly inhibit complex I (NADH-

Chem. Res. Toxicol., Vol. 11, No. 7, 1998 825

coenzyme Q reductase) (33). This may be significant because a defect in mitochondrial respiration at the complex I level has been observed in the parkinsonian SNc (34). In our earlier investigations, the influence of CySH on the electrochemically driven oxidation of DA was studied (30-32). In view of the possible involvement of lowmolecular-weight iron species in the mechanisms that underlie SNc neurodegeneration in idiopathic PD (2-8) and at least one form of the disorder having a genetic basis (35), in addition to the dopaminergic neurotoxicity of iron salts (9, 10), it was of interest to understand the influence of Fe2+/Fe3+ on the autoxidation of DA in the presence of CySH. Similarly, because manganese exposure evokes many changes that are also observed in idiopathic PD, it was also of interest to compare the Mn2+-catalyzed autoxidation of DA in the presence of CySH with the Fe2+/Fe3+-catalyzed reaction.

Materials and Methods Chemicals. Dopamine hydrochloride (DA‚HCl) and L-cysteine (CySH) were obtained from Sigma (St. Louis, MO). 2-SCysteinyldopamine (2-S-CyS-DA), 5-S-CyS-DA, 6-S-cysteinyldopamine (6-S-CyS-DA), 2,5-di-S-cysteinyldopamine (2,5-di-SCyS-DA), 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4benzothiazine-3-carboxylic acid (DHBT-1) and its 6-S-cysteinyl conjugate (DHBT-2) and 6,8-di-S-cysteinyl conjugate (DHBT4), and 8-(2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-5) and its 6-S-cysteinyl conjugate (DHBT-6) and 6,7-di-S-cysteinyl conjugate (DHBT-7) were synthesized according to published methods (30, 31). All other chemicals used were of the highest commercial grade available and were used without further purification. Spectroscopy. NMR spectra were recorded on Varian (Palo Alto, CA) XL-300, Inova-400, or VXR-500 spectrometers. Lowand high-resolution fast atom bombardment mass spectrometry (FAB-MS) employed a VG Instruments (Manchester, U.K.) model ZAB-E spectrometer. UV-visible spectra were recorded on a Hewlett-Packard (Palo Alto, CA) 8452A diode array spectrophotometer. High-Performance Liquid Chromatography. HPLC was carried out with a Gilson (Middleton, WI) gradient system equipped with dual model 302 pumps (10-mL pump heads), a Rheodyne (Cotati, CA) 7125 loop injector, and a Waters (Milford, MA) model 440 UV detector set at 254 nm. HPLC methods I-III utilized three mobile phase solvents. Solvent A was prepared by addition of concentrated trifluoroacetic acid (TFA) to deionized water until the pH was 2.15. Solvent B was prepared by adding TFA to a 1:1 (v/v) solution of HPLC grade acetonitrile (MeCN) and deionized water until the measured pH was 2.15. Solvent C was prepared by addition of TFA to deionized water until the pH was 1.50. A preparative reversedphase column (Bakerbond C18, 10 µm, 250 × 21.2 mm; P. J. Cobert Associates, St. Louis, MO) was employed. HPLC method I employed the following binary mobile phase gradient: 0-15 min, 100% solvent A; 15-158 min, linear gradient to 40% solvent B; 158-161 min, linear gradient to 100% solvent B; 161-172 min, 100% solvent B. HPLC method II: 0-20 min, 100% solvent A; 20-140 min, linear gradient to 26% solvent B; 140-144 min, linear gradient to 100% solvent B; 144-156 min, 100% solvent B. HPLC method III: 0-15 min, 100% solvent C; 15-87 min, linear gradient to 26% solvent B; 87-89 min, linear gradient to 100% solvent B; 89-100 min, 100% solvent B. HPLC methods all employed a flow rate of 7.0 mL min-1. Oxidation Reaction Procedures. Reaction solutions were incubated in pH 7.4 phosphate buffer (µ ) 0.2) at 37 °C in a thermostatically controlled water bath. The reaction vessel was open to the atmosphere, and reaction solutions were stirred with a magnetic stirring bar. Concentrations of all additives and

826 Chem. Res. Toxicol., Vol. 11, No. 7, 1998 other details of experimental conditions are given in figure legends or tables. Reactions were stopped by adding TFA until the solution was pH 2.15. In some experiments, reaction solutions were reduced with NaBH4 for 1 min prior to acidification to pH 2.15. Stock solutions of DA‚HCl, CySH, ferrous ammonium sulfate [Fe(NH4)2(SO4)2‚6H2O], FeCl3‚6 H2O, MnCl2‚6H2O, and ethylenediaminetetraacetic acid (potassium salt, K2EDTA‚2 H2O) were prepared fresh in deionized water immediately before use. Spectroscopic evidence bearing on the structures of new products is presented below. All 1H NMR assignments were confirmed by homonuclear decoupling and two-dimensional (2D) correlated spectroscopy experiments. 13C NMR assignments were based on 2D heteronuclear multiple-quantum coherence and heteronuclear multiple-bond correlation experiments. Full chemical names are given for all new compounds. However, for simplicity, proton and carbon assignments based on NMR experiments employ the atom-numbering systems shown in Schemes 3-5. 7-(2-Aminoethyl)-3,4-dihydro-5,8-dioxo-1,4-benzothiazine-3-carboxylic acid (6). Compound 6 was isolated as a blue solid. Dissolved in the HPLC mobile phase (pH 2.15) 6 exhibited UV-visible bands at λmax ) 584, 322 (sh), and 264 nm. FABMS (3-nitrobenzyl alcohol matrix) gave m/z ) 269.0579 (MH+, 15%, C11H13N2O4S1; calcd m/z ) 269.0596). 1H NMR (300 MHz, D2O) gave δ 2.72-2.80 (m, 2H, C(β)-H2), 2.94 (dd, J ) 13.2, 3.6 Hz, 1H, C(2)-H), 3.17 (t, J ) 7.2 Hz, 2H, C(R)-H2), 3.29 (dd, J ) 13.2, 3.6 Hz, 1H, C(2)-H), 4.66 (t, J ) 3.6 Hz, 1H, C(3)-H), 6.52 (s, 1H, C(6)-H). 7-(2-Aminoethyl)-2-[7-(2-aminoethyl)-5-hydroxybenzo1,4-thiazin-2-ylidene]benzo-1,4-thiazin-5-ol (7). Compound 7 was isolated as a dark solid. Dissolved in the HPLC mobile phase (pH 2.15) 7 gave a violet solution that exhibited UVvisible bands at λmax ) 562, 486 (sh), 404 (sh), 362 (sh), 312, 266, and 236 (sh) nm. FAB-MS (3-nitrobenzyl alcohol matrix) gave m/z ) 413.1096 (MH+, 20%, C20H21N4O2S2; calcd m/z ) 413.1106). 1H NMR (500 MHz, Me2SO-d6) gave δ 2.80 (t, J ) 7.5 Hz, 2 × 2H, C(β)-H2 and C(β′)-H2), 3.05 (m, 2 × 2H, C(R)-H2 and C(R′)-H2), 6.72 (d, J ) 2.0 Hz, 2 × 1H, C(6)-H and C(6′)-H), 6.80 (d, J ) 2.0 Hz, 2 × 1H, C(8)-H and C(8′)-H), 7.99 (m, 2 × 3H, 2 x NH3+), 8.92 (s, 2 × 1H, C(3)-H and C(3′)-H). 13C NMR (125 MHz, Me2SO-d6) gave δ 32.81 (C-β and C-β′), 39.46 (C-R, C-R′), 115.36 (C-6, C-6′), 116.33 (C-8, C-8′), 119.69 (C-2, C-2′), 121.93 (C-8a, C-8a′), 125.54 (C-4a, C-4a′), 139.74 (C-7, C-7′), 142.25 (C-3, C-3′), 155.01 (C-5, C-5′). 6-(2-Aminoethyl)-4-hydroxy-1,3-benzothiazole (12). Compound 12 was a very pale yellow solid that, when dissolved in the HPLC mobile phase (pH 2.15), exhibited UV bands at λmax ) 302, 268 (sh), 260 (sh), 252 (sh), and 228 nm. FAB-MS (glycerol matrix) gave m/z ) 195.0583 (MH+, 100%, C9H11N2O1S1; calcd m/z ) 195.0592). 1H NMR (500 MHz, D2O) gave δ 2.87 (t, J ) 7.5 Hz, 2H, C(β)-H2), 3.14 (t, J ) 7.5 Hz, 2H, C(R)-H2), 6.71 (d, J ) 1.5 Hz, 1H, C(5)-H), 7.26 (d, J ) 1.5 Hz, 1H, C(7)H), 9.07 (s, 1H, C(2)-H). 13C NMR (125 MHz, D2O) gave δ 32.49 (C-β), 40.08 (C-R), 112.02 (C-5), 113.39 (C-7), 134.83 (C-7a), 136.20 (C-6), 138.56 (C-3a), 148.79 (C-4), 155.57 (C-2). 5-(3-Dithioalanyl)dopamine (19). Compound 19 was a white solid that in the HPLC mobile phase (pH 2.15) exhibited a single UV band at λmax ) 298 nm. FAB-MS (3-nitrobenzyl alcohol matrix) gave m/z ) 305.0623 (MH+, 100%, C11H17N2O4S2; calcd m/z ) 305.0630). 1H NMR (500 MHz, D2O) gave δ 2.83 (t, J ) 7.5 Hz, 2H, C(β)-H2), 3.15 (dd, J ) 15.0, 8.5 Hz, 1H, C(a)-H), 3.18 (t, J ) 7.5 Hz, 2H, C(R)-H2), 3.34 (dd, J ) 15.0, 4.0 Hz, 1H, C(a)-H), 4.39 (dd, J ) 8.5, 4.0 Hz, 1H, C(b)-H), 6.82 (d, J ) 2.0 Hz, 1H, C(2)-H), 7.04 (d, J ) 2.0 Hz, 1H, C(6)-H). 13C NMR (125 MHz, D O) gave δ 31.58 (C-β), 36.38 (C-a), 40.10 2 (C-R), 51.55 (C-b), 117.38 (C-2), 121.70 (C-5), 124.32 (C-6), 129.13 (C-1), 143.12 (C-4), 144.80 (C-3) 170.74 (C-c). 6-(3-Dithioalanyl)dopamine (20). Compound 20 was isolated as a white solid that in the HPLC mobile phase (pH 2.15) exhibited a UV band at λmax ) 296 nm. FAB-MS (3-nitrobenzyl alcohol matrix) gave m/z ) 305.0610 (MH+, 14%, C11H17N2O4S2;

Shen and Dryhurst calcd m/z ) 305.0630). 1H NMR (500 MHz, D2O) gave δ 3.08 (t, J ) 7.5 Hz, 2H, C(β)-H2), 3.17 (dd, J ) 15.0, 8.0 Hz, 1H, C(a)-H), 3.20 (t, J ) 7.5 Hz, 2H, C(R)-H2), 3.29 (dd, J ) 15.0, 4.0 Hz, 1H, C(a)-H), 4.39 (dd, J ) 8.0, 4.0 Hz, 1H, C(b)-H), 6.85 (s, 1H, C(2)-H), 7.21 (s, 1H, C(5)-H). 13C NMR (125 MHz, D2O) gave δ 30.10 (C-β), 36.04 (C-a), 39.78 (C-R), 51.42 (C-b), 117.85 (C-2), 121.51 (C-5), 124.19 (C-6), 131.39 (C-1), 143.18 (C-4), 145.92 (C-3), 170.57 (C-c). 4-(2-Aminoethyl)-5-[5-(2-aminoethyl)-2,3-dihydroxyphenylthio]benzene-1,2-diol (21). Compound 21 was a white solid that in the HPLC mobile phase (pH 2.15) exhibited UV bands at λmax ) 290 and 250 (sh) nm. FAB-MS (glycerol/TFA matrix) gave m/z ) 337.1223 (MH+, 100%, C16H21N2O4S; calcd m/z ) 337.1222). 1H NMR (500 MHz, D2O) gave δ 2.67 (t, J ) 7.5 Hz, 2H, C(β)-H2), 2.93 (t, J ) 7.5 Hz, 2H, C(β′)-H2), 3.04 (t, J ) 7.5 Hz, 2H, C(R)-H2), 3.12 (t, J ) 7.5 Hz, 2H, C(R′)-H2), 6.23 (d, J ) 2.0 Hz, 1H, C(6)-H), 6.68 (d, J ) 2.0 Hz, 1H, C(2)H), 6.87 (s, 2H, C(5′)-H and C(2′)-H). 13C NMR (125 MHz, D2O) gave δ 30.33 (C-β′), 31.66 (C-β), 39.69 (C-R′), 40.12 (C-R), 114.56 (C-2), 117.76 (C-2′), 120.69 (C-6), 121.63 (C-6′), 121.64 (C-5′), 124.18 (C-5), 129.60 (C-1), 131.25 (C-1′), 140.45 (C-4), 143.62 (C-4′), 144.43 (C-3), 144.89 (C-3′). 4-(2-Aminoethyl)-3-[6-(2-aminoethyl)-3,4-dihydroxyphenylthio]benzene-1,2-diol (22). Compound 22 was a white solid that in the HPLC mobile phase (pH 2.15) exhibited UV bands at λmax ) 294 and 250 (sh) nm. FAB-MS (glycerol/TFA matrix) gave m/z ) 337.1240 (MH+, 42%, C16H21N2O4S; calcd m/z ) 337.1222). 1H NMR (400 MHz, D2O) gave δ 2.85-2.95 (m, 6H), 3.13 (t, J ) 7.2 Hz, 2H), 5.99 (s, 1H, C(2)-H or C(5)-H), 6.66 (s, 1H, C(5)-H or C(2)-H), 6.76 (d, J ) 8.4 Hz, 1H, C(5′)-H or C(6′)-H), 6.89 (d, J ) 8.4 Hz, 1H, C(6′)-H or C(5′)-H). 7-(2-Aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine (BT-2R). Compound BT-2R was isolated as a white solid that in the HPLC mobile phase (pH 2.15) exhibited UV band at λmax ) 292, 286, 256 (sh), 236 (sh), and 218 nm. FAB-MS (3nitrobenzyl alcohol matrix) gave m/z ) 211.0921 (MH+, 71%, C10H15N2OS; calcd m/z ) 211.0905). 1H NMR (300 MHz, D2O) gave δ 2.84 (t, J ) 7.5 Hz, 2H, C(β)-H2), 3.19 (t, J ) 7.5 Hz, 2H, C(R)-H2), 3.26-3.31 (m, 2H, C(2)-H2), 3.70-3.75 (m, 2H, C(3)H2), 6.64 (d, J ) 1.8 Hz, 1H, C(6)-H), 6.70 (d, J ) 1.8 Hz, 1H, C(8)-H).

Results Influence of CySH on the Uncatalyzed Autoxidation of DA. In the absence of CySH, the autoxidation of DA (1.0 mM) in pH 7.4 phosphate buffer at 37 °C was slow, a very faint precipitate of dark indolic melanin polymer (16) appearing after 3-4 h. However, HPLC analysis of this solution revealed only a very small decrease in the DA peak compared to that measured in the freshly prepared solution. The rate of the autoxidation was accelerated by addition of Fe2+ or Fe3+ (0.1 mM) and was further accelerated in the presence of K2EDTA (0.1 mM) based upon the time required to observe the first appearance of melanin polymer. Again, however, HPLC analysis revealed only very minor decreases of DA concentration at the latter point. The autoxidation of DA (1.0 mM) in the presence of CySH (5.0 mM) at pH 7.4 was also very slow. Thus, following a 4-h incubation, the only detectable product (HPLC method II) was 5-S-CySDA present at ca. 10-20 µM concentrations. At longer times (e.g., 12 h) yields of 5-S-CyS-DA slowly increased and three additional products appeared at much lower concentrations: 2-S-CyS-DA > 6-S-CyS-DA > 2,5-di-SCyS-DA. These four cysteinyl conjugates of DA were the only detectable products during the initial 24 h of the autoxidation reaction. At this time, the reaction solution was colorless but a white precipitate of CySSCy was present. After g30 h HPLC analysis of the solution



Figure 1. HPLC chromatograms (method I) of the product solutions formed following the Fe2+-EDTA (0.1 mM)-catalyzed oxidation of DA (1.0 mM) in the presence of CySH (5.0 mM) in pH 7.4 phosphate buffer (µ ) 0.2) at 37 °C for (A) 0.5 h, (B) 2.5 h, and (C) 2.5 h after reduction with NaBH4. Injection volume: 10 mL.

revealed a number of extremely small chromatographic peaks, one of which had the same retention time as DHBT-1 although identification of this compound, using UV spectrophotometry for example, was impractical owing to its very low concentration. Iron-Catalyzed Autoxidation of DA in the Presence of CySH. Oxidations of DA (1.0 mM) in the presence of CySH (5.0 mM) and K2EDTA (0.1 mM) in stirred air-saturated pH 7.4 phosphate buffer at 37 °C were initiated by addition of ferrous ammonium sulfate

Figure 2. HPLC chromatograms (method I) of the product solutions formed following the Fe2+-EDTA (10 µM)-catalyzed oxidation of DA (100 µM) in the presence of CySH (500 µM) in pH 7.4 phosphate buffer (µ ) 0.2) at 37 °C for (A) 1 h, (B) 2.5 h, and (C) 2.5 h after NaBH4 reduction. Injection volume: 10 mL.

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Shen and Dryhurst

Table 1. Relative Yields of Products Formed upon Incubation of DA (1.0 mM) with CySH (5.0 mM) and Fe2+-EDTA (0.1 mM) in pH 7.4 Phosphate Buffer (µ ) 0.2) at 37 °C chromatographic peak heightsa (arbitrary units)bfor time (h)

5-S-CyS-DA

2-S-CyS-DA

6-S-CyS-DA

2,5-di-S-CyS-DA

DHBT-1

BT-1

BT-2

7

12/6

19/22

20

21

0.5 1 1.5 2 2.5 3 3.5

104.0 133.2 140.5 112.2 58.3 31.0 17.5

11.0 16.5 22.5 18.1 12.3 8.2 5.2

4.5 6.0 7.5 6.9 6.4 5.8 5.0

17.5 33.0 45.0 36.7 33.7 32.3 26.0

0 0 0 18.5 51.5 45.5 36.0

0 0 0 1.0 8.0 9.5 11.0

0 0 0 53.5 89.0 69.0 49.0

0 0 0 7.0 18.2 11.0 6.0

0 1.0 2.0 5.0 17.7 24.8 26.8

0 1.0 4.0 6.0 7.0 8.8 12.2

0 1.0 5.0 7.0 8.2 11.5 16.8

2.0 4.2 12.0 14.8 13.5 13.0 13.0

a

Peak heights measured using HPLC method I. b Average of three replicate measurements.

or ferric chloride such that the concentration of Fe2+ or Fe3+, respectively, initially added to the reaction solution was 0.1 mM. However, the rates and products of the subsequent reaction and spectral changes that occurred were the same whether Fe2+ or Fe3+ was added. Immediately upon addition of Fe2+ or Fe3+ a very pale bluered color (λmax ) 527 ( 1 nm) developed in the solution. Over the subsequent 80-100 min, the solution became increasingly red and the visible absorption band shifted to λmax ) 504 ( 2 nm. At longer times, this band slowly decreased in intensity and the solution became a yellowred color with no clearly evident visible absorption bands. After about 4 h, a small amount of a yellow-brown precipitate formed that was readily soluble in dilute KOH solution. When the reaction was carried out in the absence of CySH, the black indolic melanin precipitate that formed was insoluble in KOH solution. For preparative purposes higher concentrations of DA (4 mM), CySH (16 mM), Fe2+ or Fe3+ (0.4 mM), and K2EDTA (0.4 mM) were incubated at pH 7.4 (37 °C). However, under these conditions, a white precipitate of CySSCy appeared in the reaction. The same precipitate was also observed when CySH (16 mM) was incubated with K2EDTA (0.4 mM) and Fe2+ or Fe3+ (0.4 mM) in the absence of DA under otherwise identical conditions. Termination of the Fe2+/Fe3+-EDTA-catalyzed autoxidation of DA in the presence of CySH after 30 min, by adjusting the pH to 2.15 with TFA, caused the bright red solution to immediately become colorless. Following filtration, HPLC analysis (method I) of the solution revealed that the major product was 5-S-CyS-DA (Figure 1A). Lower yields of other cysteinyl conjugates of DA were formed in the order 2,5-di-S-CyS-DA > 2-S-CySDA > 6-S-CyS-DA. Acidification of the yellow-red reaction solution formed after 2.5 h caused it to become yellow. Following filtration, HPLC analysis (method I) of the solution revealed a significant decrease in the yield of 5-S-CyS-DA (Figure 1B) compared to that observed after 30 min (Figure 1A) and the appearance of many additional products. These included DHBT-1, 7, 20, and 21. Using HPLC method III, it was possible to separate and isolate 19 (major) and 22 (minor), and 12 (major) and 6 (minor) (data not shown). The solution eluted under the last peak in Figure 1B had the characteristic yellow color of benzothiazines (BTs) and their decomposition products in acidic solution (31, 36). Previous studies demonstrated that the isolation of BTs is difficult owing to their instability, particularly in acidic solution such as the HPLC mobile phased employed in the present investigation. However, in neutral solution BTs can be readily reduced by NaBH4 to their corresponding DHBTs (36, 37). Figure 1C, therefore, is a chromatogram of an aliquot of the same

product solution analyzed in Figure 1B that had been reduced with NaBH4 for 60 s prior to acidification with TFA and filtration. Thus, following NaBH4 treatment, the BTs peak was significantly decreased, the peak for 7 disappeared, a major new peak appeared corresponding to BT-2R (i.e., the DHBT expected upon reduction of BT2), and the peak height for DHBT-1 increased indicating that BT-1 was reduced (36). The relative changes in product yields, based on chromatographic peak heights, over a 3.5-h time period during which DA (1.0 mM) was oxidized in the presence of CySH (5.0 mM) and Fe2+/Fe3+-EDTA (0.1 mM) are shown in Table 1. Thus, with increasing reaction time the yields of 5-S-CyS-DA, 2-S-CyS-DA, 6-S-CyS-DA, 2,5di-S-CyS-DA, DHBT-1, BT-2 (measured as BT-2R following borohydride reduction), 7, and 21 initially increased, reached maximal values, and then declined. In contrast, yields of BT-1 (measured by the increased DHBT-1 peak after borohydride reduction), 12 + 6, 19 + 22, and 20 steadily increased over the 3.5-h reaction period. The unlabeled peaks present in the chromatograms shown in Figure 1B,C were due to products that either were formed in such low yields and/or were too unstable to permit their isolation and identification. Oxidation of lower concentrations of DA (100 µM) in the presence of CySH (500 µM) and Fe2+/Fe3+-EDTA (10 µM) also gave 5-S-CyS-DA as the major initial product with lower yields of 2,5-di-S-CyS-DA > 2-S-CyS-DA > 6-S-CyS-DA (Figure 2A). Other major products observed after longer reaction times were 7, 12 (Figure 2B), and particularly BT-2 (measured as BT-2R following NaBH4 reduction; Figure 2C) with smaller yields of 19-22. Only minor yields of DHBT-1 and BT-1 were formed. Several other products were formed but were not identified. In the absence of K2EDTA the autoxidation of DA in the presence of CySH and Fe2+/Fe3+ was appreciably slower. To illustrate, incubation of DA (100 µM), CySH (500 µM), and Fe2+ or Fe3+ (10 µM) required 5-6 h to give a product profile and yields equivalent to that observed within 1 h in the presence of 10 µM K2EDTA (i.e., a chromatogram virtually the same as that shown in Figure 2A). Approximately 8-9 h was necessary to reach the same stage in the reaction achieved in 2.5 h in the presence of K2EDTA (i.e., equivalent to that shown in Figure 2B,C). The rates of autoxidation reactions in which K2EDTA was replaced by 10 µM concentrations of citrate were virtually the same as in the absence of K2EDTA. Furthermore, citrate had no significant effect on either the identities or relative yields of products. Manganese(II)-Catalyzed Autoxidation of DA in the Presence of CySH. The initial phase of the Mn2+catalyzed autoxidation of DA in the presence of CySH



Figure 3. HPLC chromatograms (method II) of the production solutions formed following the Mn2+ (0.1 mM)-catalyzed oxidation of DA (1.0 mM) in the presence of CySH (1.0 mM) in pH 7.4 phosphate buffer (µ ) 0.2) at 37 °C for (A) 3 h, (B) 4.25 h, and (C) 4.25 h after NaBH4 reduction. Injection volume: 10 mL.

was somewhat slower and appreciably less complex in terms of products than the Fe2+/Fe3+-catalyzed reaction. To illustrate, oxidation of DA (1.0 mM) in the presence of CySH (1.0 mM) and Mn2+ (0.1 mM) resulted in the significant accumulation of only cysteinyl conjugates of the neurotransmitter during the initial 3 h of the reaction (Figure 3A). In order of decreasing yields, these were 5-S-CyS-DA . 2,5-di-S-CyS-DA > 2-S-CyS-DA > 6-SCyS-DA. During this initial phase of the reaction, the solution was colorless and no precipitates were formed. However, at longer times reaction solutions rapidly

Figure 4. HPLC chromatograms (method II) of the product solutions formed following the Mn2+ (0.1 mM)-catalyzed oxidation of DA (1.0 mM) in pH 7.4 phosphate buffer (µ ) 0.2) at 37 °C for 4 h in the presence of (A) 1.0 mM CySH, (B) 3.0 mM CySH, and (C) 5.0 mM CySH. Injection volume: 10 mL.

developed an increasingly intense yellow color. To illustrate, Figure 4A is a chromatogram (method II) of the solution formed 4 h after initiating the reaction and reveals the presence of several DHBTs in order of decreasing yield DHBT-1 . DHBT-2 > DHBT-6 >


Shen and Dryhurst

Table 2. Relative Yields of Products Formed upon Incubation of DA (1.0 mM) with CySH (5.0 mM) and Mn2+ (0.1 mM) in pH 7.4 Phosphate Buffer (µ ) 0.2) at 37 °C for 3.5-5.5 h chromatographic peak heighta(arbitrary units)bfor time (h)

5-S-CyS-DA

2-S-CyS-DA

6-S-CyS-DA

2,5-di-S-CyS-DA

DHBT-1

DHBT-2

DHBT-4

DHBT-5

DHBT-6

DHBT-7

3.5 4 5 5.5

81.2 87.0 33.5 0

14.0 14.5 8.0 0.5

6.0 6.5 8.0 7.0

49.5 53.0 13.0 1.5

1.0 2.0 83.0 88.5

22.5 37.5 61.5 39.0

10.0 17.0 23.0 19.0

0.5 1.0 18.0 32.0

57.2 97.5 185.5 140.5

99.0 184.0 245.0 241.0

a

Peak heights measured using HPLC method II. b Average of three replicate measurements.

Scheme 1

DHBT-7 ≈ DHBT-5, a large BTs peak, and a small peak corresponding to 12. After 4.25 h, the 5-S-CyS-DA peak decreased significantly, the DHBT-1 peak increased, and a very large increase in the BTs peak occurred (Figure 3B). Borohydride reduction of the product solution formed after 4.25 h revealed that the major component eluted under the BTs peak was BT-2 as evidenced by the appearance of the BT-2R peak (Figure 3C). The increased peak height for DHBT-1 following NaBH4 reduction indicated that BT-1 was also formed (Figure 3C). Under the HPLC conditions (method II) employed to analyze product mixtures formed in the Mn2+-catalyzed oxidation of DA in the presence of CySH, dimer 7 eluted under the BTs peak. HPLC method II was employed to analyze these reaction mixtures because using method I was not possible to separate and identify many DHBTs. With increasing concentrations of CySH (1-5 mM), the Mn2+ (0.1 mM)-catalyzed oxidation of DA (1.0 mM) for periods up to 3.5 h gave only cysteinyl conjugates of DA. However, after 4 h of reaction, increasing CySH concentrations resulted in decreased yields of yellow BTs (and 7) and increased yields of DHBT-2, DHBT-6, and particularly DHBT-7 (Figure 4A-C). The changes in the relative yields of products when DA (1.0 mM) was oxidized in the presence of Mn2+ (0.1 mM) and CySH (5.0 mM) for 3.5-5.5 h are shown in Table 2. Thus, with increasing reaction time the yields of 5-S-CyS-DA, 2-SCyS-DA, 6-S-CyS-DA, 2,5-di-S-CyS-DA, DHBT-2, DHBT4, and DHBT-6 initially increased, reached maximal values, and then declined. By contrast, yields of DHBT1, DHBT-5, and most particularly DHBT-7 increased with reaction times up to 5.5 h. At longer reaction times, yields of all identified products declined and the BTs peak

increased very dramatically although the identities of all the numerous compounds coeluted under this peak remain to be found. HPLC analysis (method I) of product solutions formed as a result of the Mn2+-catalyzed oxidation of DA in the presence of CySH failed to show significant peaks corresponding to 19-22.

Discussion The autoxidation of CySH in buffered aqueous solution in the physiological pH range is catalyzed by redox metal ions such as Fe3+ and Cu2+ and generates superoxide radical anion (O2-•), H2O2, hydroxyl radical (HO‚), cysteinylthiyl radical (CyS‚), and cystine radical anion (CySSCy-•) in addition to cysteinesulfinic and -sulfonic acids (38, 39). The key reactions associated with the ironcatalyzed autoxidation of CySH relevant to the present investigation are summarized in Scheme 1 (38). The predominant radical species generated are HO‚ and CyS‚ . Thus it is likely that HO‚ is the species responsible for oxidation of DA although, in fact, the neurotransmitter can also be oxidized by O2-• (43). Initiation of the oxidation of DA in the presence of CySH by addition of Fe2+ or Fe3+ results in the same catalytic effects and products. When the reaction is initiated with Fe3+, direct reduction by CySH generates Fe2+ (and CyS‚) (44). Thus, after this initial phase of the reaction the same Fe3+/Fe2+ equilibrium must be achieved (44). Iron-EDTA complexes exert a more powerful catalytic effect than iron-phosphate or iron-citrate complexes presumably because of the lower redox poten-


Chem. Res. Toxicol., Vol. 11, No. 7, 1998 831 Scheme 2

tial of the Fe3+-EDTA/Fe2+-EDTA system (44, 45) that favors both reduction of Fe3+ to Fe2+ and hence CyS‚ formation and oxidation of Fe2+ to Fe3+ by H2O2 with resultant HO‚ generation. The one-electron oxidation of DA by HO‚ would be expected to initially generate the DA semiquinone radical, represented by 1a-e in Scheme 2, that upon reaction with CyS‚ gives 5-S-CyS-DA, 2-S-CyS-DA, and 6-S-CySDA. However, the bimolecular disproportionations of radicals such as 1 into the parent catechol and o-quinone have second-order rate constants of approximately 108 M-1 s-1 (46). Thus, cysteinyl conjugates of DA might also be formed by nucleophilic addition of CySH to DA-oquinone (Scheme 2). The initial blue tinge that develops upon initiation of the iron-catalyzed autoxidation of DA in the presence of CySH corresponds to the Fe3+-DA complex (47). The subsequent red color primarily reflects formation of the Fe3+-5-S-CyS-DA complex (λmax ) 500 ( 2 nm) (48). Further oxidation of 5-S-CyS-DA (and 2-SCyS-DA) then leads to 2,5-di-S-CyS-DA by similar reaction pathways. A previous study of the iron-EDTAcatalyzed autoxidation of DA in the presence of CySH reported formation of only 5-S-CyS-DA and traces of 2-SCyS-DA (49). However, in accord with the results of this investigation (Table 1), yields of 5-S-CyS-DA initially increased and subsequently declined suggesting further oxidation of the latter conjugate. In part, such decreased yields of 5-S-CyS-DA reflect formation of 2,5-di-S-CySDA. However, oxidation of 5-S-CyS-DA also leads to the formation of DHBT-1 and other cyclized products. Previous investigations have demonstrated that the oxidation of 5-S-CyS-DA generates o-quinone 2 that undergoes a

very rapid intramolecular cyclization to o-quinone imine 3 (Scheme 3) (30, 31). This reactive intermediate undergoes several parallel reactions. For example, 3 can oxidize 5-S-CyS-DA to o-quinone 2 forming radical 4 that disproportionates to DHBT-1 and 3. The structure of the blue p-quinone 6 suggests that it results from further oxidation of DHBT-1. Indeed, incubations of DHBT-1 (0.5 mM) or 5-S-CyS-DA (0.5 mM) with Fe2+ or Fe3+ (50 µM), and K2EDTA (50 µM) form low yields of 6. Accordingly, it seems probable that HO‚ addition to DHBT-1 generates the 5,8-dihydroxydihydrobenzothiazine 5 that is oxidized to 6 (Scheme 3). Borohydride reduction of the product solutions formed from the Fe2+/Fe3+-catalyzed oxidation of DA in the presence of CySH results in a decrease of the yellow BTs peak observed by HPLC (compare Figure 1B,C), growth of the DHBT-1 peak, and appearance of a major peak corresponding to BT-2R. These observations are consistent with the rearrangement of o-quinone imine 3 to BT-1 (reduced by NaBH4 to DHBT-1), a minor route, and rearrangement/decarboxylation to BT-2 (reduced by NaBH4 to BT-2R), a major route. Similar reactions have been reported for an o-quinone imine intermediate generated by oxidation of 5-S-cysteinyl-3,4-dihydroxyphenylalanine (5-S-CyS-DOPA) (37). The latter reaction also forms a 1,3-benzothiazole structurally similar to 12 (50) and, following acidification of the product solution, a dimer similar to 7 (37). By analogy with these earlier studies, it can be concluded that BT-2 is oxidized to 1,4benzothiazine 8 that upon nucleophilic attack by water (or HO-) at C(2) forms the hemithioketal 9 (Scheme 3). Rearrangement of 9 affords aldehyde 10 that is oxida-


Shen and Dryhurst Scheme 3

tively decarboxylated via 11 to 12. The low pH of the HPLC mobile phase employed to separate the products formed by oxidation of DA in the presence of CySH, by analogy with previous studies (50), promotes the acidcatalyzed oxidative coupling of BT-2 to violet dimer 7. Formation of DA thioethers 21 and 22 and disulfides 19 and 20 suggests formation of a DA-thiyl radical intermediate similar to that formed by homolytic cleav-

age of the S-CH2 bond of 5-S-DOPA by UV irradiation (51). In the Fe2+/Fe3+-EDTA-catalyzed oxidation of DA in the presence of CySH, a one-electron abstraction from the sulfur residues of 5-S-CyS-DA, 6-S-CyS-DA, and 2-SCyS-DA by HO‚ generates cation radicals 13, 14, and 15, respectively (Scheme 4). Subsequent cleavage of the S-CH2 bonds with formation of the aziridine 23 and hence serine (24) leads to the isomeric DA-thiyl radicals


Chem. Res. Toxicol., Vol. 11, No. 7, 1998 833 Scheme 4

16-18. Coupling of radical 17 or 18 with 1 then leads to the DA thioether 22. Similarly, of between thiyl radical 17 or 16 with 1 accounts for formation of thioether 21. Couplings between radicals 16 and 17 with CyS‚ lead to disulfides 19 and 20, respectively. It is likely that additional DA thioethers and disulfides are formed but apparently in such low yields that they were very difficult to isolate and identify. The Mn2+-catalyzed autoxidation of DA in the presence of CySH does not result in detectable amounts of thioethers 21 and 22, disulfides 19 and 20, or indeed CySSCy. These observations argue against the participation of DA or cysteinyl radicals in this reaction. Using ESR spectroscopy and kinetics studies, Lloyd (14) has provided convincing evidence for the Mn2+-catalyzed oxidation of

DA following the pathway shown in eqs 1-4

D(OH)2 + Mn2+ h DO2-MnII + 2H+

(1)

DO2-MnII + O2 f DO2-MnIII+ + O2-•

(2)

DO2-MnIII+ + O2-• f ‚DO2-MnIII2+ + O22- (3) ‚DO2-MnIII2+ f DA-o-quinone + Mn2+

(4)

D(OH)2 + O2 f DA-o-quinone + H2O2

(5)

overall


Shen and Dryhurst Scheme 5

where D(OH)2 ) DA, ‚DO2 ) DA semiquinone radical, and DO2-Mn ) DA-manganese complex. Thus, catalysis of the oxidation of DA is related to the ability of Mn2+ and Mn3+ to redox cycle. This mechanism implies that the concentration of free DA-semiquinone radicals is lower in the presence of manganese than even in the uncatalyzed autoxidation reaction (14). Furthermore, while O2-• is formed in the Mn2+-catalyzed autoxidation of DA (eq 2) it is subsequently consumed (eq 3) (14). Indeed, Mn2+ can scavenge and detoxify O2-•, and neither Mn2+ nor Mn3+ can mediate HO‚ formation from H2O2/ O2-• by Fenton or Haber-Weiss types of reactions (13, 52). Overall, therefore, Mn2+ catalyzes autoxidation of DA directly to DA-o-quinone (eq 5) that is scavenged by CySH to give primarily 5-S-CyS-DA along with smaller yields of other cysteinyldopamines in the initial stages of the reaction. In the presence of low concentrations of CySH relative to DA, the major subsequent reaction is the oxidation of 5-S-CyS-DA to o-quinone 2 that cyclizes to o-quinone imine 3, the precursor of DHBT-1, BT-1, BT2, and 12 (Scheme 3, Figure 3A,B). Because 5-S-CyS-

DA retains a DA residue, it is probable that Mn2+ catalyzes its oxidation by a mechanism similar to that shown in eqs 1-5. With increasing concentrations of CySH, relative yields of DHBT-1 decline dramatically (Figure 4) as do the compounds that contribute to HPLC peak BTs (BT-1, BT-2, and other BTs). These effects derive from two causes. A relatively minor reaction pathway is nucleophilic addition of CySH to o-quinone imine 3 to give DHBT-2 that can be further oxidized to 25 and attacked by CySH to give DHBT-4 (Scheme 5) (31). A more important reaction, however, is nucleophilic addition of CySH to o-quinone 2 to give 2,5-di-S-CyS-DA, the most easily oxidized cysteinyl conjugate of DA (31). This oxidation generates o-quinone 26 that, in part, reacts with CySH to give 2,5,6-tris-S-CyS-DA. However, the predominant reaction of 26 is intramolecular cyclization to o-quinone imine 27 that is reduced by 2,5-di-SCyS-DA via a radical intermediate (not shown) to DHBT-6 or attacked by CySH to give DHBT-7 (Scheme 5). o-Quinone 26 can also cyclize to o-quinone imine 25, a minor pathway (31), a precursor of DHBT-2 and DHBT-


4. DHBT-5 is formed by oxidation of 2-S-CyS-DA (31), both of which are minor products. The results of this and an earlier study (49) demonstrate that the Fe3+/Fe2+ redox system potentiates oxidation of DA in the presence of CySH. The catalytic effect is most pronounced when the iron species are complexed with EDTA because of the influence of this chelating agent on the Fe3+/Fe2+ system redox potential (44, 45). CySH appears to play a key role by reducing Fe3+ to Fe2+ with concomitant formation of CyS‚. Both CyS‚ and Fe2+ then participate in the reactions responsible for formation of O2-•, H2O2, and, particularly, HO‚ (Scheme 1) (44). It is probably the latter radical that is responsible for oxidation of DA. Other investigators have demonstrated that incubations of Fe3+/Fe2+ and H2O2 with DA generate the dopaminergic neurotoxin 6-hydroxydopamine (6OHDA) or its quinone oxidation product (53, 54). However, in the present investigation no evidence for formation of 6-OHDA or its cysteinyl conjugates was obtained, although it is not possible to entirely discount the possibility that such compounds are among the many minor products formed. The major initial product of the Fe3+/Fe2+-catalyzed oxidation of DA in the presence of CySH is 5-S-CyS-DA by the pathways presented in Scheme 2. Subsequent oxidation of 5-S-CyS-DA generates the key cyclic o-quinone imine intermediate 3 that serves as the precursor of DHBT-1, BT-1, BT-2, 6, 7, and 12 (Scheme 3). Compounds BT-2, 6, 7 and 12 have not previously been identified as products of DA oxidation in the presence of CySH. Unlike the electrochemically driven (30, 31, 36) and Mn2+-catalyzed reactions, the Fe3+/Fe2+-catalyzed oxidation of DA in the presence of CySH gives only a single major DHBT, i.e., DHBT-1. In part, this results from the oxidation of CySH by Fe3+ (and HO‚) to give CySSCy. Thus, less CySH is available in the Fe3+/Fe2+-catalyzed reaction to attack o-quinone 2 or o-quinone imine 3 to give more complex DHBTs. Furthermore, under the highly oxidizing conditions generated in the Fe2+/Fe3+-catalyzed reaction, HO‚ also consumes cysteinyldopamines to form disulfides 19 and 20 and thioethers 21 and 22 (Scheme 4). The latter compounds have not previously been reported and appear to be products unique to the Fe2+/Fe3+-catalyzed reaction. The results of this investigation of the Fe2+/Fe3+catalyzed oxidation of DA in the presence of CySH may have relevance to the pathogenesis of PD in view of the fact that the parkinsonian SNc contains increased levels of iron (2, 3, 5) and decreased levels of ferritin (4) compared to controls. How or why iron accumulates to excess in the parkinsonian SNc is unknown (55) and may occur only late in the disease (56). Nevertheless, it is rather widely accepted that elevated nigral iron would catalyze the decomposition of O2-• and H2O2, normal byproducts of the synthesis, catabolism, and autoxidation of DA, by Fenton or Haber-Weiss chemistry leading to increased fluxes of HO‚ with resultant lipid peroxidation and other forms of oxidative damage. Indeed, increased lipid peroxidation has been measured in the SNc of PD patients (57), and low-molecular-weight iron induces membranous lipid peroxidation (reviewed in ref 10). However, this and other investigations (43) indicate that DA is a good oxygen radical scavenger. Thus, if elevated nigral iron serves simply to potentiate HO‚ formation in dopaminergic SNc cells, then DA would be expected to be a primary target resulting in an elevated rate of its oxidation to neuromelanin. Thus, dying dopaminergic


neurons in the PD brain should be more heavily melanized than such cells in the healthy SNc. However, several studies report that normally more heavily pigmented cells degenerate preferentially in PD and that dying SNc cells contain less neuromelanin pigment than their counterparts in the nondiseased brain (18-20). To rationalize these observations, we have hypothesized that, perhaps in response to the entry of certain environmental, occupational, or xenobiotic chemicals into the SNc, an early event in the pathogenesis of PD is an elevated release of GSH from nigral glia and upregulation of γ-GT (30-32). These responses would lead to elevated extracellular levels of CySH that is translocated into pigmented dopaminergic cell bodies that normally contain little GSH or CySH (28, 29). Translocation of CySH into these cells would be expected to divert the neuromelanin pathway by scavenging DA-o-quinone to give, initially, 5-S-CyS-DA. Increased cytoplasmic levels of iron or the iron-neuromelanin complex would, on the basis of the present investigation, potentiate this reaction. Some support for this sequence of events in idiopathic PD is provided by the increased 5-S-CyS-DA/ DA concentration ratio (17), upregulation of γ-GT (25), irreversible loss of GSH (23-25), and progressive depigmentation of dopaminergic cells (18-20) that all occur in the parkinsonian SNc. However, 5-S-CyS-DA, similar to 5-S-CyS-DOPA (48), is also an efficient scavenger of HO‚ in a reaction that in part leads to DHBT-1 (Scheme 3). Thus, elevated intraneuronal iron together with cytoplasmic DA and increased CySH provides conditions favorable for HO‚ formation. However, the present study suggests that rather than directly inflicting damage to membrane lipids and other macromolecules, HO‚ might be scavenged by DA to give 5-S-CyS-DA that, in turn, also reacts with the radical to give DHBT-1. These reactions may be relevant in view of the recent report that oxygen radical-mediated damage in the parkinsonian SNc probably occurs only very late in PD (58). Furthermore, DHBT-1 is a mitochondrial NADH-CoQ reductase toxicant (33), and a defect in mitochondrial respiration at the complex I level probably contributes to dopaminergic SNc cell death in PD (34). Interestingly, the irreversible inhibition of complex I by DHBT-1 is not related to oxygen radical-mediated damage (33) but is dependent on its oxidation, catalyzed by an unknown component of the inner mitochondrial membrane (59). Thus, efforts to detect this putative endotoxin in brain tissue from PD patients will probably be difficult. However, in the event that iron, CySH, and DA play roles in the pathogenesis of PD, then this investigation suggests that other marker molecules of reactions that lead to DHBT-1 should be formed (Schemes 3 and 4). The Mn2+-catalyzed oxidation of DA generates DA-oquinone and H2O2 (eq 5) without significant formation of free organic or oxygen radical species (14). The present investigation supports this conclusion because in the presence of CySH the Mn2+-catalyzed autoxidation of DA does not generate significant yields of products expected for an oxygen radical (HO‚)-mediated reaction such as CySSCy, 19-22. The Mn2+-catalyzed autoxidation of DA in the presence of CySH generates DHBT-1 as a major product when CySH concentrations are relatively low (Figure 3A,B). With high CySH concentrations relative to DA, o-quinone 2 is preferentially scavenged to give 2,5-di-S-CyS-DA and thence DHBT-6 and DHBT-7 rather than intramolecular


cyclization to o-quinone imine 3 and thence DHBT-1 (Scheme 5). Chronic exposure of experimental animals to manganese leads to the degeneration of nigrostriatal DA neurons (11, 12), as do infusions of iron salts into the SNc (9, 10). Although little further information is available concerning iron-induced parkinsonism in laboratory animals, Mn2+ exposure leads to a large decrease of nigrostriatal levels of GSH and upregulation of γ-GT (21, 22). Using microdialysis experiments, we have recently observed that perfusions of Fe2+ or Mn2+ into the rat SNc evoke a significant but transient elevation of extracellular GSH and more prolonged increases of extracellular CySH levels (ref 32 and unpublished results). Although additional work remains to be done, these observations are compatible with iron- or manganese-stimulated release of GSH, probably primarily from glia (28, 29), and upregulation of γ-GT leading to elevation of extracellular CySH levels. Subsequent translocation of CySH and lowmolecular-weight iron or manganese into dopaminergic SNc cells would, based on the results of this investigation, provide intraneuronal conditions favorable for formation of 5-S-CyS-DA and the mitochondrial complex I toxin DHBT-1. Whether such reactions do in fact occur in vivo or, indeed, if iron- or manganese-induced parkinsonism in experimental animals results in DHBT-1-mediated mitochondrial complex I inhibition that contributes to the degeneration of nigrostriatal dopaminergic neurons remains to be established. However, recent epidemiological studies provide a strong link between occupational exposure to manganese, iron, and other redox-active metals such as copper and a significant increase in the incidence of PD in humans (60-63). Such observations imply that these metals are able to enter the brain. The particular susceptibility of the nigrostriatal dopaminergic system to iron, manganese, or copper exposure might be related to the ineffective blood-brain barrier in the SNc (64), their ability to stimulate elevation of extracellular CySH, and subsequent translocation of both the metal ions and CySH into SNc cells where they react to give DHBT-1.

Acknowledgment. This work was supported by Grant No. NS-29886 from the National Institutes of Health. Additional support was provided by the Vice President for Research and Research Council at the University of Oklahoma.

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