Cysteinylnorepinephrine Are Irreversible Inhibitors of Mitochondrial C

also evokes time- and concentration-dependent irreversible inhibition of NADH-coenzyme Q1 ... 19) might represent a combination of factors that evoke...
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Chem. Res. Toxicol. 2000, 13, 749-760

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Oxidative Metabolites of 5-S-Cysteinylnorepinephrine Are Irreversible Inhibitors of Mitochondrial Complex I and the r-Ketoglutarate Dehydrogenase and Pyruvate Dehydrogenase Complexes: Possible Implications for Neurodegenerative Brain Disorders Wenkuan Xin, Xue-Ming Shen, Hong Li, and Glenn Dryhurst* Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019 Received October 5, 1999

The major initial product of the oxidation of norepinephrine (NE) in the presence of L-cysteine is 5-S-cysteinylnorepinephrine which is then further easily oxidized to the dihydrobenzothiazine (DHBT) 7-(1-hydroxy-2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid (DHBT-NE-1). When incubated with intact rat brain mitochondria, DHBT-NE-1 evokes rapid inhibition of complex I respiration without affecting complex II respiration. DHBT-NE-1 also evokes time- and concentration-dependent irreversible inhibition of NADH-coenzyme Q1 (CoQ1) reductase, the pyruvate dehydrogenase complex (PDHC), and R-ketoglutarate dehydrogenase (R-KGDH) when incubated with frozen and thawed rat brain mitochondria (mitochondrial membranes). The time dependence of the inhibition of NADH-CoQ1 reductase, PDHC, and R-KGDH by DHBT-NE-1 appears to be related to its oxidation, catalyzed by an unknown component of the inner mitochondrial membrane, to electrophilic intermediates which bind covalently to active site cysteinyl residues of these enzyme complexes. The latter conclusion is based on the ability of glutathione to block inhibition of NADH-CoQ1 reductase, PDHC, and R-KGDH by scavenging electrophilic intermediates, generated by the mitochondrial membranecatalyzed oxidation of DHBT-NE-1, forming glutathionyl conjugates, several of which have been isolated and spectroscopically identified. The possible implications of these results to the degeneration of neuromelanin-pigmented noradrenergic neurons in the locus ceruleus in Parkinson’s disease are discussed.

Introduction Massive decrements of striatal dopamine (DA),1 the result of the degeneration of neuromelanin (NM)pigmented dopaminergic neurons in the substantia nigra * To whom correspondence should be addressed. Telephone: (405) 325-4811. Fax: (405) 325-6111. E-mail: gdryhurst@ chemdept.chem.ou.edu. 1 Abbreviations: Asp, L-aspartate; BSA, bovine serum albumin; BT, benzothiazine; BT-NE-1, 7-(1-hydroxy-2-aminoethyl)-5-hydroxy-1,4benzothiazine-3-carboxylic acid; BT-NE-2, 7-(1-hydroxy-2-aminoethyl)5-hydroxy-1,4-benzothiazine; BT-NE-2R, 7-(1-hydroxy-2-aminoethyl)3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine; CD, circular dichroism; CoQ1, coenzyme Q1; CysGly, cysteinylglycine; CySH, L-cysteine; DA, dopamine; DHBT, dihydrobenzothiazine; DHBT-NE-1, 7-(1-hydroxy2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic acid; DP, dipeptidases; DTT, D,L-dithiothreitol; EDTA, ethylenediaminetetraacetic acid; ESI-MS, electrospray ionization mass spectrometry; FAB-MS, fast atom bombardment mass spectrometry; Glu, L-glutamate; GSH, glutathione; γ-GT, γ-glutamyl transpeptidase; HO•, hydroxyl radical; KCN, potassium cyanide; R-KGDH, R-ketoglutarate dehydrogenase; LC, locus ceruleus; MeCN, acetonitrile; MeOH, methanol; MPP+, 1-methyl-4-phenylpyridinium; MPTP, 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine; NaBH4, sodium borohydride; NAD+, nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; NE, norepinephrine; NM, neuromelanin; NMDA, Nmethyl-D-aspartate; O2-•, superoxide anion radical; PD, Parkinson’s disease; PDHC, pyruvate dehydrogenase complex; 5-S-CyS-DA, 5-Scysteinyldopamine; 2-S-CyS-NE, 2-S-cysteinylnorepinephrine; 5-SCyS-NE, 5-S-cysteinylnorepinephrine; SNc, substantia nigra pars compacta; SOD, superoxide dismutase; TFA, trifluoroacetic acid; TPP, thiamine pyrophosphate; 5, 6-S-glutathionyl-DHBT-NE-1; 6, 8-Sglutathionyl-DHBT-NE-1; 8, (2R)-2-S-glutathionyl-BT-NE-1; 9, (2S)2-S-glutathionyl-BT-NE-1; 10, (2R,3R)-2-S-glutathionyl-DHBT-NE-1; 11, (2S,3S)-2-S-glutathionyl-DHBT-NE-1.

pars compacta (SNc), are believed to underlie the classic motor symptoms of Parkinson’s disease (PD) (1). On the basis of pathobiochemical changes in the parkinsonian SNc and in the nigrostriatal system of animals exposed to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) (2), we recently proposed a new mechanism for the neurotoxic process (3). The first step in this mechanism was proposed to be a large but transient impairment of DA neuron energy metabolism. In the case of MPTP, this would be caused by its active metabolite, 1-methyl-4-phenylpyridinium (MPP+) (4), a reversible inhibitor of mitochondrial complex I (5, 6) and R-ketoglutarate dehydrogenase (R-KGDH) (7). The normal decline of neuron energy metabolism with age (8), periodic exposure to environmental toxins that interfere with mitochondrial respiratory enzymes (6, 9-12), particularly in view of both age-based (13) and genetically based impairments of xenobiotic metabolism (14-17), together with systemic mitochondrial enzyme defects (18, 19) might represent a combination of factors that evoke a profound depletion of neuronal energy in the parkinsonian SNc. This energy impairment with resultant depolarization of both the neuronal and mitochondrial membranes was proposed to mediate a massive release of DA (20) together with both cytoplasmic and mitochondrial glutathione (GSH) (21-23). Furthermore, the profound energy impairment and relief of the Mg2+ block of N-methyl-D-aspartate (NMDA) receptors was proposed

10.1021/tx990170t CCC: $19.00 © 2000 American Chemical Society Published on Web 07/08/2000

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to mediate their activation by basal extracellular levels of L-glutamate (Glu) and L-aspartate (Asp) (24) with resultant superoxide (O2-•) generation in excess of the scavenging capacity of superoxide dismutase (SOD) (25). During the DA neuron energy impairment, O2-• was proposed to release low-molecular weight Fe2+ from ironcontaining proteins (26-28) that catalytically decomposes H2O2 in the presence of ascorbate, forming hydroxyl radical (HO•) which oxidizes extracellular GSH (basal and that released from DA neurons). This was proposed to trigger release of GSH from glia as a mechanism for protecting neighboring neurons against damage by extracellular HO• (29). When the neuron energy impairment begins to subside, increasing ATP production would initiate reuptake of DA (30) that is oxidized by and hence would scavenge O2-• (31), thus blocking Fe2+ mobilization, HO• formation, and oxidation of extracellular GSH. However, replenishing intraneuronal GSH, released from DA neurons during the energy impairment, requires its continued release from glia (29, 32) and degradation initially by γ-glutamyl transpeptidase (γ-GT) to Glu and cysteinylglycine (CysGly) which is further hydrolyzed by dipeptidases (DP) to L-glycine (Gly) and L-cysteine (CySH) (33). CysGly and CySH are then translocated into DA neurons to provide CySH, normally employed for intraneuronal GSH biosynthesis (32, 34, 35). Indeed, when perfusions of neurotoxic concentrations of MPP+ into the rat striatum or SNc are discontinued, a massive elevation of extracellular GSH levels occurs, believed to reflect its release from glia (3). The subsequent decline of the extracellular GSH level from its peak concentrations is accompanied by elevation of the extracellular Glu level (36) and a smaller increase in the extracelular CySH level, the latter effect possibly reflecting its rapid translocation into DA neurons (3). During the period of recovering but still reduced DA neuron ATP production, partial relief of the Mg2+ block of NMDA receptors should permit their continued activation by elevated extracellular levels of Glu with resultant O2-• generation that oxidizes returning DA to DA-oquinone which, we propose, reacts with translocated CySH forming, initially, 5-S-cysteinyldopamine (5-S-CySDA) (31). Indeed, 5-S-CyS-DA, normally a minor metabolite of DA, becomes a major metabolite in the parkinsonian SNc as indicated by a large increase in the 5-S-CySDA/DA concentration ratio (37). However, 5-S-CyS-DA is more easily oxidized than DA (31) to dihydrobenzothiazines (DHBTs) and benzothiazines (BTs) (38) that include irreversible inhibitors of mitochondrial complex I (39, 40) and R-KGDH (41). This raises the possibility that DHBTs and BTs, oxidative metabolites of 5-S-CySDA, might be endogenously generated toxicants that contribute to the mitochondrial complex I (42) and R-KGDH (43) deficiencies and dopaminergic SNc cell death in PD. NM-pigmented noradrenergic cells in the locus ceruleus (LC) also degenerate in PD (44). Resultant decrements of norepinephrine (NE) levels in projection areas of LC neurons have been implicated with dementia, depression, and other nonmotor symptoms of PD (4547). However, little is known about pathobiochemical changes that occur in the parkinsonian LC. Nevertheless, the fact that LC and SNc cells both contain easily oxidized catecholamine neurotransmitters and degenerate in PD suggests that similar neuropathological mechanisms may be involved. In other words, intraneuronal O2-•-mediated

Xin et al. Scheme 1

oxidation of NE in the presence of translocated CySH as a noradrenergic neuron energy impairment subsides might lead to endogenous toxicants that contribute to LC cell death. The in vitro oxidation of NE generates NEo-quinone that is scavenged by CySH to give, initially, 5-S-cysteinylnorepinephrine (5-S-CyS-NE) (Scheme 1) (48). However, 5-S-CyS-NE is then further oxidized to o-quinone 1 which rapidly cyclizes to o-quinone imine 2. 5-S-CyS-NE is then oxidized by 2 to give radical 3 forming radical 4. Disproportionation of 3 forms 5-S-CySNE and 1. Disproportionation of radical 4 forms 2 and 7-(1-hydroxy-2-aminoethyl)-3,4-dihydro-5-hydroxy-2H1,4-benzothiazine-3-carboxylic acid (DHBT-NE-1). In view of the possibility that endogenously generated oxidative metabolites of 5-S-CyS-NE might contribute to the pathogenesis of LC cell death in PD, this communication describes the effects of DHBT-NE-1 on mitochondrial respiration and the activities of NADH-coenzyme Q1 (CoQ1) reductase, R-KGDH, and the pyruvate dehydrogenase complex (PDHC).

Materials and Methods Chemicals. NE (hydrochloride salt and free base), CySH (free base), GSH, SOD, catalase, 2-aminoethanediol, nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH), adenosine 5′-diphosphate (ADP, sodium salt), L-(-)-malic acid (disodium salt), R-ketoglutaric acid (sodium salt), succinic acid (disodium salt), pyruvic acid (sodium salt), glycylglycine, ethylenediaminetetraacetic acid (potassium salt, K2EDTA), mannitol, Trizma hydrochloride, coenzyme A, D,L-dithiothreitol (DTT), thiamine pyrophosphate (TPP), fatty acid-free bovine serum albumin (BSA), Triton X-100, and asolectin (phospholipid from soybean) were obtained from Sigma (St. Louis, MO). MPP+ (iodide salt) was obtained from RBI (Natick, MA) and rotenone from Aldrich (Milwaukee, WI). These and all other commercially available chemicals were of the highest purity available and were used without further purification. Coenzyme Q1 (ubiquinone-1) was synthesized as described previously (39). Spectroscopy. NMR spectra were recorded on a Varian (Palo Alto, CA) VXR-500 spectrometer. Low- and high-resolution fast atom bombardment mass spectrometry (FAB-MS) employed a VG Instruments (Manchester, U.K.) ZAB-E spectrometer. Electrospray ionization mass spectrometry (ESI-MS) employed a PE Sciex (Thornhill, ON) API III biomolecular mass analyzer. Circular dichroism (CD) spectra were obtained with an AVIV (Lakewood, NJ) 62DS spectropolarimeter. UV/visible spectra

Mitochondrial Toxicity of Cysteinylnorepinephrine were recorded on a Hewlett-Packard (Palo Alto, CA) 8452A diode array spectrophotometer. Analytical HPLC. Analytical HPLC employed a Gilson (Middleton, WI) gradient system equipped with dual model 302 pumps, a Rheodyne (Cotati, CA) 7125 loop injector (20 µL sample loop), and a Waters (Milford, MA) 440 UV detector (254 nm). Two mobile phase solvents were employed. Solvent A was prepared by adding 10 mL of concentrated ammonium hydroxide (NH4OH) to 4 L of deionized water, the pH being adjusted to 2.2 by addition of concentrated trifluoroacetic acid (TFA). Solvent B was prepared by adding 10 mL of NH4OH to 2 L of HPLC grade methanol (MeOH) and 2 L of deionized water, the pH being adjusted to 2.2 with TFA. A reversed phase column (Columbus C18, 5 µm, 100 mm × 3.2 mm, Phenomenex, Torrance, CA) was employed. The following mobile phase gradient was employed: from 0 to 2 min, 100% solvent A; from 2 to 43 min, linear gradient to 21.5% solvent B; from 43 to 45 min, linear gradient to 100% solvent B; and from 45 to 48 min, 100% solvent B. The flow rate was 0.6 mL/min. A standard calibration curve of HPLC peak height versus DHBT-NE-1 concentration was employed for analysis studies. This analytical HPLC method was used to monitor the oxidation of DHBT-NE-1 and the effects of GSH on this reaction. In a typical experiment, DHBT-NE-1 (1.0 mM) was incubated with rat brain mitochondrial membranes (2 µg of mitochondrial protein/µL) in 20 mM potassium phosphate buffer (pH 8.0) at 30 °C for times of e2 h. After the predetermined incubation time, an aliquot of the reaction mixture (50 µL) was removed and immediately diluted to 500 µL with ice-cold deionized water and analyzed by analytical HPLC. In experiments designed to assess benzothiazine (BT) metabolites, the diluted incubtion mixture was treated with NaBH4 immediately before HPLC analysis. NaBH4 reduces BT-NE-1 to DHBT-NE-1 and BT-NE-2 to BT-NE-2R. Calibration curves prepared with authentic samples of DHBTNE-1 and BT-NE-2R were employed for quantitative HPLC analysis of these compounds. Peak identification was based on coelution and peak fitting with authentic samples. Preparative HPLC. Preparative HPLC employed a Gilson gradient system equipped with dual model 306 pumps, a reversed phase column (Bakerbond C18, 10 µm, 250 mm × 21.2 mm, P. J. Cobert Associates, St. Louis, MO), and a UV detector (254 nm). Six mobile phase solvents were employed. Solvent C was prepared by adding 7.5 mL of NH4OH to 1 L of deionized water and then adjusting the pH to 2.2 with concentrated HCl. Solvent D was 1:1 (v/v) acetonitrile (MeCN) in deionized water adjusted to pH 2.2 with HCl. Solvent E was 1:1 (v/v) MeCN in deionized water. Solvent F was deionized water adjusted to pH 2.15 with TFA. Solvent G was 1:1 MeCN in deionized water adjusted to pH 2.15 with TFA. Solvent H was 1:1 MeOH in deionized water adjusted to pH 2.15 with TFA. Preparative HPLC method I, used to separate and purify cysteinyl conjugates of NE, employed solvents C and D and the following gradient: from 0 to 2 min, 100% solvent C; and from 2 to 30 min, linear gradient to 15% solvent D. Preparative HPLC method II, used to separate and purify DHBT-NE-1 and for initial isolation of BT-NE-2R, employed solvents D and E and the following gradient: from 0 to 2 min, 100% solvent D; and from 2 to 30 min, linear gradient to 30% solvent E. Preparative HPLC method III, used to purify BT-NE-2R, employed solvents F and G and the following gradient: from 0 to 20 min, 100% solvent F; from 20 to 50 min, linear gradient to 3% solvent G; from 50 to 53 min, linear gradient to 100% solvent G; and from 53 to 68 min, 100% solvent G. Preparative HPLC method IV employed solvents F and H and the following gradient: from 0 to 20 min, 100% solvent F; from 20 to 175 min, linear gradient to 20% solvent H; from 175 to 179 min, linear gradient to 100% solvent H; and from 179 to 190 min, 100% solvent H. Method IV was used to separate and purify the 6-(S)-5 and 8-(S)-6 glutathionyl conjugates of DHBT-NE-1, for isolation and purification of metabolites formed by the mitochondrial membranecatalyzed oxidation of DHBT-NE-1 in the presence of GSH, and

Chem. Res. Toxicol., Vol. 13, No. 8, 2000 751 for isolation and purification of glutathionyl conjugates 10 and 11. The flow rate for methods I-IV was 7 mL/min. Synthesis of 5-S-CyS-NE and DHBT-NE-1. NE (24.7 mg, free base) dissolved in aqueous 0.1 M HCl (100 mL) was oxidized by controlled potential electrolysis (1.0 V vs SCE) at pyrolytic graphite electrodes using equipment described previously (48). The electrolysis was terminated after 30 min, and CySH (18 mg) was added to the bright yellow solution of NE-o-quinone, causing the color to rapidly fade to pale yellow. The major products that formed were 5-S-CyS-NE together with smaller yields of 2-S-CyS-NE (48). These conjugates were separated using preparative HPLC method I when 5-S-CyS-NE eluted with a retention time (tR) of 19 min. The solution eluted under this peak was collected and purified by preparative HPLC using 1:1 MeCN in deionized water as the mobile phase. The solution eluted under the peak for 5-S-CyS-NE was collected and then freeze-dried to give a chromatographically pure (analytical HPLC) white solid. The 1H NMR and mass spectra of 5-S-CySNE were identical to those previously reported (48). To synthesize DHBT-NE-1, an aqueous solution of 5-S-CySNE (ca. 5 mM) was adjusted to pH 10-11 with a solution of concentrated KOH and stirred at room temperature for 30 min. The resultant product solution was adjusted to pH 2.2 by addition of HCl and then injected onto the reversed phase column, and components were separated using preparative HPLC method II. The solution eluted under the peak corresponding to DHBT-NE-1 was collected and then desalted by preparative reversed phase HPLC using solvent E as the mobile phase. The 1H NMR and mass spectra of chromatographically pure DHBT-NE-1 were identical to those reported previously (48). Synthesis of 7-(1-Hydroxy-2-aminoethyl)-3,4-dihydro5-hydroxy-2H-1,4-benzothiazine (BT-NE-2R). NE (1 mM) and 2-aminoethanol (1 mM) dissolved in 30 mL of potassium phosphate buffer (pH 7.4; µ ) 0.2) were oxidized by controlled potential electrolysis at pyrolytic graphite electrodes (70 mV vs SCE). After 60 min, the entire pale yellow product solution was introduced onto the preparative reversed phase column, and components were separated using HPLC method II. The tR for BT-NE-2R was 44 min. The solution eluted under this peak was collected at -80 °C (dry ice/acetone bath). Following several repetitive experiments, the combined solutions containing BTNE-2R were purified by preparative HPLC method III and then freeze-dried. BT-NE-2R was isolated as a very pale yellow solid that in the HPLC mobile phase (pH 2.15) exhibited UV bands (λmax) at 294, 288, 254 (sh), and 218 nm. FAB-MS (thioglycerol matrix): m/z 227.0900 (MH+, 3%, C10H15N2O2S; calcd m/z 227.0854). 1H NMR (Me2SO-d6): δ 9.60 [br s, 1H, C(5)-OH], 7.85 (br s, 3H, NH3+), 6.48 [d, J ) 1.5 Hz, 1H, C(8)-H], 6.40 [d, J ) 1.5 Hz, 1H, C(6)-H], 4.51 [dd, J ) 9.5, 3.0 Hz, 1H, C(β)-H], 3.50-3.47 [m, 2H, C(3)-H2], 2.99-2.96 [m, 2H, C(2)-H2], 2.92-2.86 [m, 1H, C(R)-H], 2.77-2.70 [m, 1H, C(R)-H]. Synthesis of 6-S-Glutathionyl-7-(1-hydroxy-2-aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine-3-carboxylic Acid (5) and 8-S-Glutathionyl-7-(1-hydroxy-2aminoethyl)-3,4-dihydro-5-hydroxy-2H-1,4-benzothiazine3-carboxylic Acid (6). DHBT-NE-1 (1 mM) and GSH (5 mM) dissolved in 30 mL of potassium phosphate buffer (pH 7.4; µ ) 0.2) were oxidized by controlled potential electrolysis at pyrolytic graphite electrodes (70 mV vs SCE). After 60 min, the entire pale yellow solution was introduced onto the preparative reversed phase column, and components were separated by HPLC method IV. Two major products eluted at a tR of 112 min (6) and a tR of 141 min (5). The solutions eluted under these peaks were collected and stored separately at -80 °C. Following purification by HPLC method IV, the resulting solutions were freeze-dried. Compound 5 was isolated as a pale yellow solid that in the HPLC mobile phase (pH 2.15) exhibited UV bands (λmax) at 318, 282 (sh), and 248 nm and an ESI-MS with m/z 576 (MH+, 100%). FAB-MS (dithiothreitol/dithioerythritol matrix): m/z 576.1452

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(MH+, 2%, C21H30N5O10S2; calcd m/z 576.1434). 1H NMR (D2O): δ 6.71 [s, 1H, C(8)-H], 5.27 [m, 1H, C(β)-H], 4.45 [m, 1H, C(3)H], 4.27 [dd, J ) 8.5, 4.5 Hz, 1H, C(d)-H], 3.75 [d, J ) 18.0 Hz, 1H, C(f)-H], 3.70 [d, J ) 18.0 Hz, 1H, C(f)-H], 3.68 [t, J ) 6.5 Hz, 1H, C(a)-H], 3.18 [m, 1H, C(2)-H], 3.10-3.05 [m, 2H, C(2)H, C(R)-H], 3.02-2.92 [m, 3H, C(e)-H2, C(R)-H], 2.34-2.22 [m, 2H, C(c)-H2], 1.97-1.92 [m, 2H, C(b)-H2]. Compound 6 was a pale yellow solid that in the HPLC mobile phase exhibited UV bands λmax at 318 and 246 nm and an ESIMS with m/z 576 (MH+, 100%). FAB-MS (thioglycerol matrix): m/z 576.1452 (MH+, 1%, C21H30N5O10S2; calcd m/z 576.1434). 1H NMR (D O): δ 6.70 [s, 1H, C(6)-H], 5.39 [dd, J ) 8.0, 4.0 2 Hz, 1H, C(β)-H], 4.40 [dd, J ) 4.5, 3.5 Hz, 1H, C(3)-H], 4.30 [dd, J ) 8.5, 4.5 Hz, 1H, C(d)-H], 3.73 [d, J ) 18.0 Hz, 1H, C(f)H], 3.69 [d, J ) 18.0 Hz, 1H, C(f)-H], 3.65 [t, J ) 6.5 Hz, 1H, C(a)-H], 3.20 [dd, J ) 13.0, 4.5 Hz, 1H, C(2)-H], 3.11-3.01 [m, 3H, C(2)-H, C(e)-H, C(R)-H], 2.99-2.93 [m, 2H, C(e)-H, C(R)H], 2.32 [t, J ) 7.5 Hz, 2H, C(c)-H2], 2.01-1.90 [m, 2H, C(b)H2]. Isolation of Metabolites Formed by the Mitochondrial Membrane-Catalyzed Oxidation of DHBT-NE-1 in the Presence of GSH. DHBT-NE-1 (1 mM), GSH (5 mM), and rat brain mitochondrial membranes (2 µg of mitochondrial protein/ µL) were incubated in 10 mL of 20 mM potassium phosphate buffer (pH 8.0) at 30 °C for 4-12 h. Following centrifugation (10000g for 10 min at 2 °C) and filtration of the resulting supernatant (0.45 µm Millipore HA-type filter), the filtrate was injected onto the reversed phase column and components were separated by HPLC method IV. The solutions eluted under the peaks corresponding to 6 (tR ) 112 min), 8 (tR ) 136 min), 5 (tR ) 141 min), and 9 (tR ) 150 min) were separately collected, immediately frozen, and stored at -80 °C. Following several repetitive experiments, collected solutions containing 5 or 6 were purified (HPLC method IV) and then freeze-dried. Efforts to isolate 8 and 9 as pure solids were unsuccessful because of their decomposition during the final freeze-drying step. Accordingly, vigorously stirred, freshly chromatographed solutions of 8 or 9 were treated with solid NaBH4 until the pH reached 6-7. After 10 min, the resulting solution was adjusted to pH 2.15 with TFA and then pumped directly onto the reversed phase column and components were separated by preparative HPLC method IV. The reduced (DHBT) forms of 8 and 9, i.e., 10 and 11, respectively, eluted at 165 and 163 min, respectively. The solutions containing 10 or 11 were purified (HPLC method IV) and freeze-dried. Rat Brain Mitochondrial Preparations. Brain mitochondria were prepared from male albino Sprague-Dawley rats (Harlan Sprague-Dawley, Madison, WI) using procedures described in detail elsewhere (39). The final mitochondrial pellet was suspended in medium A [300 mM mannitol, 15.1 mM Trizma hydrochloride, 15 mM glycylglycine, and 0.1 mM K2EDTA (pH 7.4)]. For oxygen consumption (mitochondrial respiration) experiments, mitochondria were prepared fresh each day and were stored on ice prior to use. For NADH-CoQ1 reductase, R-KGDH, and PDHC assays, mitochondria were stored at -80 °C until they were needed. The amount of mitochondrial protein was determined by the method of Lowry et al. (49). Oxygen Electrode Studies. Oxygen consumption by intact rat brain mitochondria was assessed with a YSI (Yellow Springs Instrument Co., Yellow Springs, OH) model 5300 biological oxygen monitor equipped with a model 5357 micro oxygen probe assembly (600 µL volume) and thermostated at 30 °C. Mitochondria (200-400 µg of protein) were suspended in 600 µL of air-saturated medium B [300 mM mannitol, 10 mM Trizma hydrochloride, 10 mM KCl, 5 mM potassium phosphate, 0.1 mM K2EDTA, and 1.0 mg/mL BSA (pH 7.2)] without (control) or with known concentrations of DHBT-NE-1 for a predetermined incubation time (2-10 min) prior to measurement of oxygen consumption. Then, 2.0 µL of pyruvate with malate (each at concentrations of 0.75 M to give a final concentration of 2.5 mM) was added, and oxygen consumption was measured for 2 min.

Xin et al. State 3 (complex I) respiration was then initiated by addition of 2.0 µL of 0.27 M ADP (final concentration of 0.9 mM). NADH-CoQ1 Reductase Assays. Rat brain mitochondria were exposed to six freeze-thaw cycles immediately before use to ensure maximal NADH-CoQ1 reductase activity. The activity of NADH-CoQ1 reductase was measured by monitoring the decrease in NADH concentration with time at 30 °C using a Beckman DU640 spectrophotometer as described previously (39, 42). Rat brain mitochondrial membranes were g95% rotenone sensitive in NADH-CoQ1 reductase assays. Method I. This method was employed to follow the time course of NADH-CoQ1 reductase inhibition by the compounds of interest. Frozen and thawed rat brain mitochondria (200400 µg of protein) were incubated with (sample) or without (control) DHBT-NE-1 in 250 µL of 20 mM potassium phosphate buffer (pH 8.0) at 30 °C for times ranging from 0 to 120 min. Then 25 µL of the resulting solution (containing 20-40 µg of mitochondrial protein) was transferred to the assay solution that consisted of 850 µL of 20 mM potassium phosphate buffer (pH 8.0), 50 µL of 30 mM KCN, 50 µL of asolectin (15 mg/mL), and 50 µL of 1 mM CoQ1 (in 10% ethanol/water, v/v) contained in a 0.5 cm optical path length quartz UV cell. With the exception of CoQ1, all other reagents were dissolved in 20 mM potassium phosphate buffer (pH 8.0). The solution was incubated at 30 °C for 5 min, and then 25 µL of 10 mM NADH was added to initiate the reaction. The activity of NADH-CoQ1 reductase (nanomoles of NADH per minute per milligram of mitochondrial protein) was based on the decrease in the concentration of NADH measured at 340 nm using a molar absorptivity of 6.22 mM-1 cm-1. Method II. This method was employed to investigate the irreversible inhibition of NADH-CoQ1 reductase. Frozen and thawed rat brain mitochondria (300-400 µg of protein) were incubated at 30 °C for 60 min in a total volume of 200 µL of 20 mM potassium phosphate buffer (pH 8.0) in the absence (control) or presence of DHBT-NE-1. In some experiments, GSH, catalase, or SOD was included in this incubation mixture. After 60 min, 400 µL of 20 mM phosphate buffer (pH 8.0) was added and then the mixture was centrifuged (150000g for 10 min at 5 °C). The supernatant was discarded and the pellet gently resuspended in 100 µL of 20 mM phosphate buffer (pH 8.0). A 25 µL aliquot of this solution (containing 50-100 µg of mitochondrial protein) was removed and assayed for NADH-CoQ1 reductase activity as described in Method I. PDHC and R-KGDH Assay. Procedures for PDHC and R-KGDH assays were based on those described by Tabatabaie et al. (50) and Lai and Cooper (51), respectively. Briefly, to 500 µL of a solution containing 6 mM NAD+, 2 mM MgCl2, and 0.4 mM TPP in 50 mM potassium phosphate buffer (pH 7.4) were added 100 µL of CoA (6 mM), 50 µL of pyruvic acid (20 mM, for the PDHC assay) or R-ketoglutarate (20 mM, for the R-KGDH assay), 50 µL of 2% Triton X-100, and 60 µL of DTT (5 mM), all dissolved in 50 mM phosphate buffer (pH 7.4), and 200 µL of 50 mM phosphate buffer (pH 7.4). The resultant solution, in an optical quartz UV cuvette, was thoroughly mixed, and after thermal equilibrium had been attained (5 min at 30 °C), 40 µL of frozen and thawed rat brain mitochondria in medium A (containing 80 µg of mitochondrial protein) was added to initiate the reaction. The activity of PDHC or R-KGDH was based on the rate of increase of NADH production (nanomoles of NADH formed per minute per milligram of mitochondrial protein) measured at 340 nm between 30 and 240 s after initiation of the reaction. The effect of DHBT-NE-1 on the activity of PDHC or R-KGDH was investigated by incubating this putative metabolite in 50 mM potassium phosphate buffer (pH 7.4) with frozen and thawed rat brain mitochondria (2 µg/µL of protein) for times of e120 min. At predetermined times, a 40 µL aliquot of this solution was transferred into the normal assay solution to determine PDHC activity.

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Table 1. Time-Dependent Inhibition of NADH-CoQ1 Reductase by DHBT-NE-1 When Incubated with Frozen and Thawed Rat Brain Mitochondriaa incubation time (min)

control

0 20 40 60 80

100 ( 1.9d 92.7 ( 1.4 89.5 ( 1.5 83.4 ( 1.6 73.9 ( 1.9

NADH-CoQ1 reductase activity expressed as % of control activity at time zerob,c 0.1 mM DHBT-NE-1 0.5 mM DHBT-NE-1 2.0 mM DHBT-NE-1 5.0 mM DHBT-NE-1 10 mM DHBT-NE-1 99.9 ( 4.1 88.0 ( 3.3 83.7 ( 1.8 74.5 ( 3.0 64.9 ( 2.3*

96.2 ( 5.9 91.8 ( 4.2 80.2 ( 4.0 67.5 ( 5.5* 54.5 ( 5.8**

100.0 ( 8.6 77.5 ( 3.9 65.0 ( 5.9* 50.8 ( 3.6*** 47.9 ( 5.6***

93.8 ( 4.2 79.5 ( 4.2 66.5 ( 4.3* 53.1 ( 5.0*** 45.9 ( 1.7***

87.1 ( 3.3 77.3 ( 4.9 65.0 ( 5.9* 50.8 ( 3.6*** 37.2 ( 2.3***

a Frozen and thawed rat brain mitochondria (ca. 1.2 µg/µL of mitochondrial protein) were incubated in 20 mM potassium phosphate buffer (pH 8.0) in the absence (control) or presence of DHBT-NE-1 at the specified concentration. Aliquots were assayed by method I at the indicated times for NADH-CoQ1 reductase activity. b Data are means ( SEM (n g 4). c *p < 0.05. **p < 0.001. ***p < 0.0001 (statistical analysis compared the effects of DHBT-NE-1 on the activity of NADH-CoQ1 reductase to control activity measured at the same time). d The control activity at time zero of NADH-CoQ reductase was 221.8 ( 4.3 nmol of NADH min-1 (mg of mitochondrial protein)-1. 1

Figure 1. Effect of (A) 1 mM and (B) 2 mM DHBT-NE-1 on malate- and pyruvate-supported state 3 (complex I) oxygen consumption (respiration) after preincubation for the times indicated with intact rat brain mitochondria (ca. 1.2 µg of protein/µL) in medium B (pH 7.2). Oxygen consumption was assessed with a Clark-type oxygen electrode assembly. Data are means ( SEM (n g 6). One asterisk indicates p < 0.05; two asterisks indicate p < 0.001. Statistics. All results were obtained at least in triplicate and are presented as means ( SEM. A Student’s t test was employed to determine statistical significance with a p of 60 min with mitochondrial membranes before the inhibition of NADHCoQ1 reductase reached statistical significance. However, even the highest concentration of DHBT-NE-1 (10 mM) that was employed required approximately incubation for 40 min with mitochondrial membranes before the inhibition of NADH-CoQ1 reductase reached statistical significance. Irreversible Inhibition of NADH-CoQ1 Reductase by DHBT-NE-1. When mitochondrial membranes were washed following incubation (60 min) with DHBT-NE-1 (0.1-10 mM), NADH-CoQ1 reductase activity remained significantly reduced compared to control activities (Figure 2). These results indicate that DHBT-NE-1 evokes a time-dependent and irreversible inhibition of NADHCoQ1 reductase. When SOD (1.2 units/µL) and/or catalase (4.7 or 13 units/µL) were included in the initial incubation of mitochondrial membranes (2 µg of protein/µL) with DHBT-NE-1 for 60 min, NADH-CoQ1 reductase remained inhibited after the membranes were washed (data not shown). However, when no lower than equimolar GSH was included in the initial incubation mixture, the

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Table 2. Rat Brain Mitochondrial Membrane-Catalyzed Oxidation of DHBT-NE-1 (1 mM) and Irreversible Inhibition of NADH-CoQ1 Reductasea,b DHBT-NE-1 oxidizedc

time (min) 0 60 120

autoxidation (nmol) 0 21 ( 3 43 ( 7

mitochondrial membrane-catalyzed oxidation (nmol)

BT-NE-1d formed (nmol)

11 ( 2

NDf

NDf

1(1 0(0

110 ( 6 41 ( 2

185 ( 1 200 ( 0

BT-NE-2e formed (nmol)

NADH-CoQ1 reductase activity expressed as % of control activity at time zero control experimental 100 ( 2 (216 ( 5)g 84 ( 3 74 ( 4

93 ( 6 62 ( 5** 35 ( 4***

a DHBT-NE-1 (1 mM, 200 nmol) in 20 mM potassium phosphate buffer (pH 8.0) at 30 °C was incubated with frozen and thawed rat brain mitochondria (2 µL of protein/µL). At the indicated times, aliquots were removed for analytical HPLC analysis for DHBT-NE-1, BT-NE-1, and BT-NE-1 and assayed for irreversible inhibition of NADH-CoQ1 reductase activity as described in Materials and Methods. b Data are means ( SEM (n ) 6). c The amount of DHBT-NE-1 remaining in the incubation solution was determined by analytical HPLC analysis of an aliquot of the incubation solution (50 µL) following 10-fold dilution. d The amount of BT-NE-1 that formed was measured in a 10-fold-diluted aliquot of the incubation solution following NaBH4 reduction by the increase of the magnitude of the DHBT-NE-1 peak in analytical HPLC. e The amount of BT-NE-2 was measured in a 10-fold-diluted aliquot of the incubation solution following NaBH4 reduction by the height of the resultant BT-NE-2R peak in analytical HPLC. f Not determined. g Control activity of NADH-CoQ1 reductase at time zero (nanomoles of NADH per minute per milligram of mitochondrial protein). **p < 0.001. ***p < 0.0001.

Figure 3. Effects of GSH on the irreversible inhibition of NADH-CoQ1 reductase by 10 mM DHBT-NE-1. Frozen and thawed rat brain mitochondria (ca. 2 µg of protein/µL) were incubated for 60 min in 20 mM potassium phosphate buffer (pH 8.0) together with GSH at the indicated concentrations. After the mitochondrial membranes had been washed, NADH-CoQ1 reductase activities were measured via method II (Materials and Methods). Data are means ( SEM (n g 6). One asterisk indicates p < 0.05, and two asterisks indicate p < 0.001.

irreversible inhibition of NADH-CoQ1 reductase normally evoked by DHBT-NE-1 was completely blocked (Figure 3). Effects of DHBT-NE-1 on PDHC and R-KGDH Activities. When incubated with rat brain mitochondrial membranes in 50 mM phosphate buffer (pH 7.4), DHBTNE-1 evoked a time-dependent inhibition of both PDHC (Figure 4A) and R-KGDH (Figure 4B). Neither SOD nor catalase was able to block or attenuate the inhibition of PDHC or R-KGDH by DHBT-NE-1. However, no lower than equimolar GSH completely blocked the time-dependent inhibition of PDHC and R-KGDH evoked by DHBT-NE-1 (data not shown). Mitochondrial Membrane-Catalyzed Oxidation of DHBT-NE-1. In the presence of rat brain mitochondrial membranes, the oxidation of DHBT-NE-1 was appreciably more rapid than its autoxidation (Table 2). The rate of oxidation of DHBT-NE-1 increased with increasing concentrations of mitochondrial protein (data not shown), indicating that mitochondrial membranes catalyze the oxidation reaction. Analytical HPLC chromatograms of product solutions formed at various times during incubation of DHBT-NE-1 (1 mM) with rat brain mitochondrial membranes at pH 8 are presented in Figure 5A-C. Thus, the HPLC peak for DHBT-NE-1 completely disappeared in