Amyloid beta  Peptides Do Not Form Peptide-derived Free Radicals Spontaneously, but Can Enhance Metal-catalyzed Oxidation of Hydroxylamines to Nitroxides*

Sergey I. DikalovDagger §, Michael P. Vitek, Kirk R. Maplesparallel , and Ronald P. MasonDagger

From the Dagger  Laboratory of Pharmacology and Chemistry, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, the  Division of Neurology, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, and parallel  Centaur Pharmaceuticals, Inc., Sunnyvale, California 94086

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Amyloid beta  (Abeta ) peptides play an important role in the pathogenesis of Alzheimer's disease. Free radical generation by Abeta peptides was suggested to be a key mechanism of their neurotoxicity. Reports that neurotoxic free radicals derived from Abeta -(1-40) and Abeta -(25-35) peptides react with the spin trap N-tert-butyl-alpha -phenylnitrone (PBN) to form a PBN/·Abeta peptide radical adduct with a specific triplet ESR signal assert that the peptide itself was the source of free radicals. We now report that three Abeta peptides, Abeta -(1-40), Abeta -(25-35), and Abeta -(40-1), do not yield radical adducts with PBN from the Oklahoma Medical Research Foundation (OMRF). In contrast to OMRF PBN, incubation of Sigma PBN in phosphate buffer without Abeta peptides produced a three-line ESR spectrum. It was shown that this nitroxide is di-tert-butylnitroxide and is formed in the Sigma PBN solution as a result of transition metal-catalyzed auto-oxidation of the respective hydroxylamine present as an impurity in the Sigma PBN. Under some conditions, incubation of PBN from Sigma with Abeta -(1-40) or Abeta -(25-35) can stimulate the formation of di-tert-butylnitroxide. It was shown that Abeta peptides enhanced oxidation of cyclic hydroxylamine 1-hydroxy-4-oxo-2,2,6,6-tetramethylpiperidine (TEMPONE-H), which was strongly inhibited by the treatment of phosphate buffer with Chelex-100. It was shown that ferric and cupric ions are effective oxidants of TEMPONE-H. The data obtained allow us to conclude that under some conditions toxic Abeta peptides Abeta -(1-40) and Abeta -(25-35) enhance metal-catalyzed oxidation of hydroxylamine derivatives, but do not spontaneously form peptide-derived free radicals.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

As the leading cause of dementia, Alzheimer's disease (AD)1 is characterized by a loss of memory and neurons. These characteristics have been associated with brain lesions known as neurofibrillary tangles composed of Tau protein and amyloid plaques, which consist of amyloid beta  (Abeta ) peptide. In a small number of families, mutations in the genes for the amyloid peptide precursor (APP, chromosome 21) to Abeta peptide for presenilin-1 (PSEN-1, chromosome 14), presenilin-2 (PSEN-2, chromosome 1) and for an anonymous gene on chromosome 12 have been associated with AD. In contrast, Saunders and colleagues (1) discovered that a significant percentage of patients inheriting one or more of the epsilon-4 alleles of apolipoprotein-E (APOE4, chromosome 19) were at risk of acquiring AD at an earlier age than their counterparts expressing the more common epsilon-3 allele of APOE. The observation that AD patients with mutant genes for APP, PSEN-1, or PSEN-2 have higher levels of Abeta peptide than non-diseased controls (2, 3) argues that Abeta peptide may cause AD. Further supporting this idea is the observation that APOE4 patients with AD display more amyloid plaques than those with APOE3 (4). Based on neuropathological, genetic, and biochemical associations, the presence of amyloid beta  peptide is a key component of AD.

A crucial link between Abeta peptide and AD was provided by Yankner and colleagues (5), who showed that fibrillar aggregates of Abeta peptide were toxic to neurons. This seminal observation inspired a global quest to define the mechanism by which fibrillar Abeta peptide mediated neurotoxicity. One of the mechanisms proposed suggests that neurons exposed to Abeta peptide suffer severe oxidative stress that may lead to their death. Clear evidence of oxidative stress in Alzheimer's disease has been provided by Smith et al. (6), who found that neurons of AD patients contained nitrotyrosine modifications of proteins, which were not detected in age-matched control brains. Increased levels of lipid peroxides (7), reactive aldehydes such as hydroxynonenal (8), and oxidized DNA (9) have also been reported in AD brains, providing additional evidence of an oxidative stress component in the disease.

The exact nature of the radical species generated in Alzheimer's diseased brains is unknown. In one line of investigation, Behl et al. (10) demonstrated that cells exposed to fibrillar Abeta peptide responded by releasing hydrogen peroxide and dying, a process that could be inhibited by the application of catalase to degrade the released peroxide. Subsequent reports have demonstrated cellular release of superoxide (11) and nitric oxide (12) in response to Abeta peptide treatment. The effect of these radicals may be potentiated since Abeta peptides also appear to inhibit the cellular redox mechanisms that normally protect cells from oxidative stress (13).

In a provocative hypothesis, Hensley et al. (14) suggest that the Abeta peptide itself spontaneously generates free radicals that can damage cells. By mixing the spin trap N-tert-butyl-alpha -phenylnitrone (PBN) with neurotoxic forms of the Abeta peptide such as Abeta -(1-40) and Abeta -(25-35), they report the generation of ESR-detectable radical adducts in cell-free solutions (14-17). They suggest that these radicals are generated by methionine oxidation (14, 15) or by the fragmentation of Abeta peptide into smaller oligopeptide radicals (14, 15) that act as "shrapnel" to damage and eventually kill cells. Recently, these results were described as possibly artifactual due to contaminants in some of the preparations (18).

We have reinvestigated the spin-trapping studies of spontaneous free radical formation by Abeta peptides. In contrast to previous literature (14-17), we now report that neurotoxic Abeta -(1-40) and Abeta -(25-35) in the presence of the spin trap PBN do not form ESR-detectable radical adducts spontaneously. Amyloid/PBN radical adducts (14-17) reported earlier were found to be di-tert-butylnitroxide and tert-butylhydronitroxide, which are formed by oxidation of the corresponding hydroxylamines. We have investigated the possibility that toxic Abeta may potentiate metal-catalyzed oxidation of hydroxylamine derivatives. Transition metals like iron and copper frequently contribute to reactions where molecules are oxidized, but the transition metal oxidation of hydroxylamine derivatives has not been previously studied. A body of emerging work demonstrates that copper and iron are significantly associated with amyloid plaques in the brains of patients with AD, but not with the neuropil lacking plaques nor with control neuropil from healthy brains (19). Since Abeta is retained on metal chelate columns charged with copper (20), and copper dramatically increases the rate of Abeta aggregation into amyloid fibrils (21), then complexes of Abeta and copper may contribute to the pathological oxidative stress associated with AD. We now report the effects of neurotoxic Abeta -(1-40) and Abeta -(25-35) and non-toxic Abeta -(40-1) on the oxidation of hydroxylamine derivatives in the presence and absence of metals. The data obtained allows us to conclude that the previously reported formation of amyloid/PBN radical adducts (14-17) in the Sigma PBN treated with Abeta peptides can be explained as Abeta peptide-stimulated, metal-catalyzed oxidation of the corresponding hydroxylamine impurities in Sigma PBN.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- PBN was obtained from the Oklahoma Medical Research Foundation (OMRF) Spin Trap Source (Oklahoma City, OK). Two different lots (101H3696 and 87H36602) of PBN from Sigma were used. Deferoxamine (Desferal) was purchased from Sigma. Di-tert-butylnitroxide (Aldrich), N-tert-butylhydroxylamine (TCI America, Portland, OR) and analytical grade Chelex-100 (Bio-Rad) were used. Synthetic Abeta -(1-40), Abeta -(25-35) and Abeta -(40-1) peptides were purchased from Bachem (Torrance, CA). These lots of Abeta -(1-40) and Abeta -(25-35), but not the Abeta -(40-1) peptides, have been previously shown to kill neurons in a cell culture system (data not shown). TEMPONE-H was obtained from Alexis Corporation (Läufelfingen, Switzerland) (22). All other reagents were analytical grade.

Preparation of PBN Stock Solution-- Stock solutions of PBN (150 mM) in HPLC grade water were used to prepare final 50 mM PBN solutions.

Preparation of TEMPONE-H Stock Solutions-- TEMPONE-H was dissolved in oxygen-free HPLC grade water (30-min argon bubbled water) with 1 mM Desferal. Desferal was used to decrease the spontaneous oxidation of hydroxylamines catalyzed by traces of transition metal ions. The concentration of TEMPONE-H in the stock solutions was 1 mM. Prior to the experiments, the stock solution was kept under a flow of argon in a cool, air-tight place.

Preparation of tert-Butylhydronitroxide Solution-- tert-Butylhydronitroxide was obtained by auto-oxidation of 1 mM N-tert-butylhydroxylamine in water or by oxidation of 10 µM solution of N-tert-butylhydroxylamine with 10 µM K3[Fe(CN)6] in 0.15 M phosphate buffer at pH 7.4.

ESR Spin-trapping Experiments-- The ESR spectra were recorded using a Bruker ECS-106 spectrometer operating at 9.76 GHz with a modulation frequency of 50 kHz and a TM110 cavity. Spin-trapping experiments were started by solubilizing Abeta peptides into a solution of PBN or TEMPONE-H. All ESR samples were placed in the 17-mm flat cell, which contained Abeta peptides (1 mg/ml), PBN (50 mM), or TEMPONE-H (0.3 mM) in sodium phosphate buffer (0.15 M) at pH 7.4. In order to perform experiments with a lower concentration of transition metals, phosphate buffer was treated with 5 g of Chelex-100/100 ml of solution for 2 h, followed by filtration using Millex-HA 0.45-µm filters (Millipore Corp., Bedford, MA). The ESR instrumental settings for experiments with PBN were as follows: field sweep, 70 G; microwave frequency, 9.76 GHz; microwave power, 40 milliwatts; modulation amplitude, 0.32 G; conversion time, 1310 ms; time constant, 1310 ms; receiver gain, 1 × 105. ESR signals were monitored for 6 h. The ESR instrumental settings for TEMPONE-H experiments were the following: field sweep, 50 G; microwave frequency, 9.76 GHz; microwave power, 20 milliwatts; modulation amplitude, 1 G; conversion time, 656 ms; time constant, 2620 ms; receiver gain, 1 × 105.

Study of Iron-catalyzed Oxidation of Hydroxylamines-- The presence of hydroxylamine impurities in Sigma PBN was determined by studying iron's effect on nitroxide formation in solutions of PBN with K3[Fe(CN)6] and the iron-chelating agent Desferal. Oxidation of hydroxylamine impurities in Sigma PBN was performed using 5 µM K3[Fe(CN)6]. Inhibition of iron-catalyzed oxidation of hydroxylamine impurities in Sigma PBN was carried out in the presence of 2 mM Desferal.

Study of Ferric-catalyzed Oxidation of Hydroxylamines-- The oxidation of TEMPONE-H by ferric ions was confirmed by the formation of TEMPONE nitroxide during the incubation of TEMPONE-H with ferric ions. The effect of 1 µM Fe(NH4)(SO4)2 and chelating agent Desferal on the TEMPONE nitroxide formation from 0.1 mM TEMPONE-H was studied. Inhibition of iron-catalyzed oxidation of TEMPONE-H was carried out in the presence of 1 mM Desferal, which has its highest affinity for ferric ion.

Study of Cupric-catalyzed Oxidation of Hydroxylamines-- The oxidation of TEMPONE-H by cupric ions was confirmed by the formation of TEMPONE nitroxide during the incubation of TEMPONE-H with cupric ions. In this experiment the effect of 1 µM CuSO4 and chelating agent Desferal on the TEMPONE nitroxide formation from 0.1 mM TEMPONE-H was studied. Inhibition of cupric-catalyzed oxidation of TEMPONE-H was carried out in the presence of 1 mM Desferal.

Computer Simulation-- Computer simulations and spin-trap data base searches were performed using a computer simulation program,2 the details of which have been described elsewhere (23).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reinvestigation of Spontaneous Free Radical Formation by Abeta Peptides-- The ability of Abeta peptides to spontaneously form free radicals was studied by ESR using the spin trap PBN from the OMRF Spin Trap Source. Abeta -(1-40), Abeta -(40-1), and Abeta -(25-35) peptides were tested in parallel with a control that did not contain Abeta peptides (Fig. 1). ESR spectra were collected every 40 min over 6 h, and no ESR signals were observed in any mixtures (Fig. 1, A-D). Therefore, Abeta peptides do not form ESR-detectable radical adducts spontaneously.


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Fig. 1.   ESR spectra of Abeta with high quality OMRF PBN (A-D) or with Sigma PBN (E-H). A, ESR spectrum obtained after 6 h of incubation of 50 mM Sigma PBN in Chelex-100-treated 0.15 M sodium-phosphate buffer (pH 7.4) without Abeta peptides. B, ESR spectrum obtained after 6 h of incubation of 50 mM PBN with 1 mg/ml Abeta -(1-40). C, ESR spectrum obtained after 6 h of incubation of 50 mM PBN with 1 mg/ml Abeta -(40-1). D, ESR spectrum obtained after 6 h of incubation of 50 mM PBN with 1 mg/ml Abeta -(25-35). E, ESR spectrum obtained after 6 h of incubation of 50 mM Sigma PBN in Chelex-100-treated 0.15 M sodium-phosphate buffer (pH 7.4) without Abeta peptides. F, ESR spectrum obtained after 6 h of incubation of 50 mM PBN with 1 mg/ml Abeta -(1-40). G, ESR spectrum obtained after 6 h of incubation of 50 mM PBN with 1 mg/ml Abeta -(40-1). H, ESR spectrum obtained after 6 h of incubation of 50 mM PBN with 1 mg/ml Abeta -(25-35).

In contrast to the OMRF PBN results, ESR spectra from mixtures of Sigma PBN alone, plus Abeta -(1-40), plus Abeta -(40-1), or plus Abeta -(25-35) peptides all yielded a triplet ESR spectrum with the Abeta peptide inhibiting radical formation (Fig. 1, E-H). This triplet spectrum has a nitrogen hyperfine-coupling constant of 17.14 G. The formation of this triplet spectrum with Sigma PBN, but not OMRF PBN, is consistent with the spectrum arising from an impurity in Sigma PBN.

Formation of Di-tert-butylnitroxide and tert-Butylhydronitroxide from Impurities in Sigma PBN-- PBN from Sigma was checked for the presence of impurities that might lead to the formation of ESR signals. After a 6-h incubation of 50 mM aqueous Sigma PBN, a strong ESR spectrum was observed (Fig. 2A) that consisted of ESR spectra from two nitroxides (Fig. 2, B-D). One nitroxide has a triplet ESR spectrum (Fig. 2C) with a nitrogen hyperfine coupling constant of 17.16 G, which is very close to the reported nitrogen hyperfine coupling constant for di-tert-butylnitroxide. The second nitroxide has a four-line ESR spectrum (Fig. 2D) that is consistent with a nitrogen hyperfine coupling constant of 14.61 G and a hydrogen hyperfine coupling constant of 13.93 G, which are very close to the reported hyperfine coupling constants for tert-butylhydronitroxide (24, 25).


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Fig. 2.   Formation of di-tert-butylnitroxide and tert-butylhydronitroxide in an aqueous solution prepared using Sigma PBN. A, ESR spectrum obtained after a 6-h incubation of 50 mM Sigma PBN. B, computer simulation of A obtained by adding spectra of two nitroxides. C, computer simulation of ESR spectrum of nitroxide with a nitrogen hyperfine coupling constant of 17.16 G. D, simulation of ESR spectrum of nitroxide using hyperfine coupling constants of 14.61 G for nitrogen and 13.93 G for hydrogen. E, ESR spectrum of 200 nM di-tert-butylnitroxide. F, ESR spectrum of tert-butylhydronitroxide from 10 µM N-(tert-butyl)hydroxylamine.

To confirm the chemical structure of nitroxide radicals observed in the solution of Sigma PBN, we obtained experimental ESR spectra of di-tert-butylnitroxide (Fig. 2E) and tert-butylhydronitroxide (Fig. 2F). Experimental ESR spectra of di-tert-butylnitroxide and tert-butylhydronitroxide were identical to the ESR spectrum of the PBN solution shown above (Fig. 2). The ESR spectra from the Sigma PBN solution (Fig. 2A) can be described as a combination of two specific ESR spectra: those of di-tert-butylnitroxide (Fig. 2E) and tert-butylhydronitroxide (Fig. 2F). Thus, during the incubation of Sigma PBN in water, both di-tert-butylnitroxide and tert-butylhydronitroxide were formed.

In order to analyze the mechanism of nitroxide formation in the PBN solution, the effects of the chelating agent Desferal and of ferric iron addition were studied. Fresh solutions of Sigma PBN in Chelex-100-treated phosphate buffer contained trace amounts of di-tert-butylnitroxide as identified by its ESR spectrum (Fig. 3A). After just 2 h of incubation, the ESR signal was increased dramatically (Fig. 3B). A parallel 2-h incubation in the presence of the chelating agent Desferal significantly inhibited formation of di-tert-butylnitroxide (Fig. 3C). Addition of 5 mM K3[Fe(CN)6] to the fresh PBN solution caused greater nitroxide formation (Fig. 3D) than that of the 2-h incubation (Fig. 3B).


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Fig. 3.   Formation of di-tert-butylnitroxide during transition metal-catalyzed auto-oxidation of di-tert-butylhydroxylamine in a solution of Sigma PBN. A, ESR spectrum obtained after 10 min of incubation of 50 mM Sigma PBN in Chelex-100-treated phosphate buffer. B, ESR spectrum obtained after 2 h of incubation. C, ESR spectrum obtained after 2 h of incubation of 50 mM Sigma PBN with 2 mM Desferal; D, ESR spectrum obtained after addition of 5 µM K3[Fe(CN)6] to A.

Linewidth Dependence on the ESR Instrumental Settings-- Previously, it was reported that Abeta -(1-40) and Abeta -(25-35) peptides formed radical adducts with identical triplet ESR spectra and nitrogen hyperfine coupling constants of 17.1 G (16), which is actually the same as that reported for di-tert-butylnitroxide (Fig. 2E). Based on the much greater linewidth of the Abeta peptide/PBN reaction product (1.6 G), these authors concluded that this product was not di-tert-butylnitroxide (17). However, the linewidth is not a specific parameter of a nitroxide ESR spectrum (26). Linewidth is very dependent on the ESR instrumental settings, mainly modulation amplitude and microwave power. In order to show that authentic di-tert-butylnitroxide and the nitroxide derived from Sigma PBN can have the same ESR linewidth as has been described for a PBN/·Abeta peptide radical adduct, the ESR spectra were obtained using different modulation amplitude settings (Fig. 4). The linewidth of the ESR spectra of di-tert-butylnitroxide was equal to that of the nitroxide from Sigma PBN (Fig. 4, A and D) when ESR spectra were collected under identical instrument settings. Moreover, both spectra displayed the same linewidth dependence on the modulation amplitude setting. We observed peak-to-peak linewidths of 0.51, 0.96, and 1.56 G when using modulation amplitudes of 0.32, 1.00, and 1.59 G, respectively (Fig. 4).


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Fig. 4.   Dependence of ESR spectra of di-tert-butylnitroxide on modulation amplitude. ESR spectra of 50 mM Sigma PBN after 6 h of incubation in Chelex-100-treated phosphate buffer using the following modulation amplitudes: A, 0.32 G; B, 1.00 G; C, 1.59 G. ESR spectra of 200 nM di-tert-butylnitroxide using the following modulation amplitudes: D, 0.32 G; E, 1.00 G; F, 1.59 G.

Previously, it was also reported that non-toxic Abeta -(40-1) peptide formed a radical adduct with a quartet ESR spectrum and equivalent nitrogen and hydrogen hyperfine coupling constants of 14.5 G (16, 17), which are very close to those reported for tert-butylhydronitroxide (Fig. 2F). Based on the much greater linewidth of the PBN/·Abeta peptide reaction product, it was concluded that this product was not tert-butylhydronitroxide (17). In order to show that tert-butylhydronitroxide and the nitroxide from Sigma PBN solution could have the same ESR spectrum linewidth as the radical described as the PBN/·Abeta peptide-(1-40) reaction product, the ESR spectra were obtained using different modulation amplitude settings for the ESR spectrometer (Fig. 5). The linewidth of the ESR spectrum of tert-butylhydronitroxide was the same as for the nitroxide species from the Sigma PBN solution (Fig. 5, A and D). Moreover, the dependence of linewidth on the modulation amplitude was the same. Using modulation amplitudes of 0.32, 1.00, and 1.59 G, the widths of the low-field line were observed to be 0.76, 0.98, and 1.53 G, respectively (Fig. 5).


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Fig. 5.   Dependence of ESR spectra of tert-butylhydronitroxide on modulation amplitude. ESR spectra of 50 mM Sigma PBN after 6 h of incubation in phosphate buffer using the following modulation amplitudes: A, 0.32 G; B, 1.00 G; C, 1.59 G. ESR spectra of tert-butylhydronitroxide using the following modulation amplitudes: D, 0.32 G; E, 1.00 G; F, 1.59 G.

Abeta Peptide Stimulation of Oxidation of Di-tert-butylhydroxylamine Impurity in Sigma PBN-- The ability of Abeta peptides to stimulate oxidation of di-tert-butylhydroxylamine was studied by ESR using PBN from Sigma. For these experiments, solutions of Abeta -(1-40), Abeta -(25-35), and Abeta -(40-1) with 50 mM PBN and 1 mM Desferal were tested in parallel with a control that did not contain Abeta peptides (Fig. 6). Incubation of PBN (Sigma) with Abeta peptides for 2 h in phosphate buffer with Desferal gave rise to the ESR spectra shown in Fig. 1. Neurotoxic Abeta -(1-40) and Abeta -(25-35) produced triplet ESR spectra (Fig. 6, A and B) in agreement with Butterfield and co-workers (14-17). Computer simulation of these ESR spectra (Fig. 6) revealed an ESR spectrum of one nitroxide compound which displays a triplet ESR spectrum with a nitrogen hyperfine coupling constant of 17.16 G, which is identical to the nitrogen hyperfine coupling constant for di-tert-butylnitroxide. Non-toxic Abeta -(40-1) produced a much weaker nitroxide triplet and the four-line tert-butylhydronitroxide (Fig. 6C). ESR spectra of a control sample without Abeta peptides did not reveal any nitroxide formation (Fig. 6D). Under these conditions the formation of this triplet spectrum with Sigma PBN was dependent on the presence of toxic Abeta -(1-40) and Abeta -(25-35).


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Fig. 6.   Formation of di-tert-butylnitroxide during the incubation of Abeta peptides with Sigma PBN. ESR spectra obtained after a 2-hr incubation of 50 mM PBN in 0.15 M sodium phosphate buffer (pH 7.4) plus 1 mM Desferal: A, with 1 mg/ml Abeta -(1-40); B, with 1 mg/ml Abeta -(25-35); C, with 1 mg/ml Abeta -(40-1); D, without Abeta . ESR receiver gain was 5 × 105.

It was previously shown that di-tert-butylnitroxide can be formed in a solution of PBN by the metal-catalyzed auto-oxidation of di-tert-butylhydroxylamine. Therefore, in the presence of 1 mM Desferal in phosphate buffer, Abeta -(1-40) and Abeta -(25-35) stimulate the oxidation of the di-tert-butylhydroxylamine impurity to form di-tert-butylnitroxide.

Abeta -stimulated Oxidation of TEMPONE-H-- In order to study the mechanism of Abeta -stimulated hydroxylamine oxidation, we used TEMPONE-H (22). The time course of ESR spectra and the effect of removing transition metals from the phosphate buffer by treatment with chelating resin Chelex-100 were studied.

TEMPONE-H was incubated with Abeta peptides in phosphate buffer with 1 mM Desferal (Fig. 7). It was found that after a 1-h incubation of TEMPONE-H (0.3 mM) with Abeta -(25-35) and Abeta -(1-40), the ESR amplitude of TEMPONE nitroxide increased up to 4 times in comparison with the control (Fig. 7, A, B, and D). Incubation of TEMPONE-H (0.3 mM) with Abeta -(40-1) increased the ESR amplitude in the range of 50-100% compared with the control (Fig. 7 C, D). Therefore, neurotoxic Abeta -(1-40) and Abeta -(25-35) significantly stimulated oxidation of TEMPONE-H to TEMPONE nitroxide (Fig. 7, A and B). Non-toxic Abeta -(40-1) caused relatively minor changes in the formation of TEMPONE nitroxide (Fig. 7C) compared with a control sample without Abeta peptides (Fig. 7D).


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Fig. 7.   Effect of Abeta peptides on the TEMPONE formation during transition metal-catalyzed oxidation of TEMPONE-H. A-D, 0.15 M sodium phosphate buffer plus 1 mM Desferal (DF). E-H, Chelex-100-treated 0.15 M sodium phosphate buffer plus 1 mM DF. ESR spectra obtained after a 1-h incubation of 0.3 mM TEMPONE-H with Abeta (1 mg/ml). ESR receiver gain was 1 × 105.

In order to clarify the role of transition metals in Abeta -mediated stimulation of hydroxylamine oxidation, phosphate buffer was pretreated for 1 h with chelating resin Chelex-100 to remove trace-metal contaminants. ESR spectra obtained after the incubation of TEMPONE-H (0.3 mM) with Abeta peptides in Chelex-100-treated phosphate buffer are shown in Fig. 7, E-H. In Chelex-100-treated phosphate buffer, Abeta peptides did not stimulate oxidation of TEMPONE-H, implying that Abeta peptides were not the primary source of trace transition metals.

Fig. 8 shows the time dependence of TEMPONE formation during the incubation of TEMPONE-H with Abeta peptides. Formation of TEMPONE nitroxide in the samples with Abeta -(1-40) and Abeta -(25-35) was significantly faster than with Abeta -(40-1) and in the control (Fig. 8A). Removing transition metals from the solution by pretreatment of phosphate buffer with Chelex-100 strongly inhibited Abeta stimulation of TEMPONE formation (Fig. 8B). Therefore, in phosphate buffer with 1 mM Desferal, Abeta -(1-40) and Abeta -(25-35) enhanced TEMPONE-H oxidation, which was inhibited by Chelex-100 treatment.


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Fig. 8.   Time course of TEMPONE formation during incubation of TEMPONE-H with Abeta peptides (1 mg/ml). A, formation of TEMPONE in 0.15 M sodium phosphate buffer plus 1 mM Desferal. B, formation of TEMPONE in Chelex-100-treated 0.15 M sodium phosphate buffer plus 1 mM Desferal.

Effect of Ferric Ions on TEMPONE-H Oxidation-- The potential role of ferric ion in Abeta -stimulated hydroxylamine oxidation was studied (Fig. 9). ESR spectra of the fresh solution of TEMPONE-H revealed only a relatively small amount of TEMPONE (Fig. 9A). Addition of 1 µM Fe3+NH4(SO4)2 to TEMPONE-H led to a 10-fold increase in the ESR amplitude of the TEMPONE spectra (Fig. 9B). The chelating agent Desferal (1 mM) completely inhibited oxidation of TEMPONE-H in solutions containing 1 µM Fe3+NH4(SO4)2 (Fig. 9C). Therefore, ferric ion can oxidize hydroxylamine TEMPONE-H to form nitroxide TEMPONE and, presumably, ferrous ion (Reaction 1), which is air-oxidized (Reaction 2) (27). Hydrogen peroxide did not directly oxidize the hydroxylamine TEMPONE-H in solution containing Desferal (data not shown).
<UP>Fe<SUP>3+</SUP></UP>+<UP>TEMPONE-H → Fe<SUP>2+</SUP></UP>+<UP>TEMPONE</UP>
<UP>Fe<SUP>2+</SUP></UP>+<UP>O<SUB>2</SUB> → Fe<SUP>3+</SUP></UP>+<UP>O&cjs1138;<SUB>2</SUB></UP>
<UP><SC>Reactions</SC> 1 and 2</UP>


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Fig. 9.   Effect of ferric ion on TEMPONE-H oxidation. A, control. B, in the presence of 1 µM Fe3+NH4(SO4)2; C, in the presence of 1 µM Desferal plus 1 µM Fe3+NH4(SO4)2. ESR spectra were recorded after a 20-min incubation. ESR receiver gain was 1 × 105.

Effect of Cupric Ions on TEMPONE-H Oxidation-- The possible role of cupric ions in Abeta -stimulated hydroxylamine oxidation was studied (Fig. 10). Addition of 1 µM Cu2+SO4 to TEMPONE-H led to a drastic increase in the ESR amplitude of the TEMPONE spectrum (Fig. 10, A, and D), which was much more pronounced than the effect of 1 µM Fe3+NH4(SO4)2 (Fig. 10B). The chelating agent Desferal (1 mM) completely inhibited oxidation of TEMPONE-H by 1 µM Cu2+SO4 (Fig. 10C). Therefore, cupric ion can oxidize hydroxylamine TEMPONE-H to form nitroxide TEMPONE and cuprous ion (Reaction 3), which is oxidized by oxygen (Reaction 4) (28).
<UP>Cu<SUP>2+</SUP></UP>+<UP>TEMPONE-H → Cu<SUP>+</SUP></UP>+<UP>TEMPONE</UP>
<UP>Cu<SUP>+</SUP></UP>+<UP>O<SUB>2</SUB> → Cu<SUP>2+</SUP></UP>+<UP>O&cjs1138;<SUB>2</SUB></UP>
<UP><SC>Reactions</SC> 3 and 4</UP>


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Fig. 10.   Effect of cupric ion on TEMPONE-H oxidation. A, in the presence of 1 µM Cu2+SO4. B, in the presence of 1 µM Fe3+NH4(SO4)2. C, in the presence of 1 mM Desferal plus 1 µM Cu2+SO4. D, control. ESR spectra were recorded after a 20-min incubation. ESR receiver gain was 1 × 104.

Auto-oxidation of Hydroxylamines-- In order to clarify the mechanism of auto-oxidation of hydroxylamine derivatives, formation of TEMPONE was determined in the presence of superoxide dismutase, catalase, or Desferal (Fig. 11). TEMPONE-H was incubated in phosphate buffer for 30 min. ESR spectra of the control sample of TEMPONE-H revealed a small amount of TEMPONE (Fig. 11A). The addition of superoxide dismutase (100 units/ml) to TEMPONE-H led to a 2-fold decrease in the ESR amplitude of the TEMPONE spectra (Fig. 11B). The effect of superoxide dismutase supports the role of Reaction 5 in the auto-oxidation of hydroxylamines (22).
<UP>O&cjs1138;<SUB>2</SUB></UP>+<UP>TEMPONE-H → H<SUB>2</SUB>O<SUB>2</SUB></UP>+<UP>TEMPONE</UP>
<UP><SC>Reaction</SC> 5</UP>
Addition of catalase (10 µg) to TEMPONE-H did not significantly change the content of TEMPONE in the sample (Fig. 11C). Addition of the chelating agent Desferal (100 µM) to TEMPONE-H greatly inhibited the TEMPONE formation (Fig. 11D). Therefore, transition metals (Mn+1) can oxidize hydroxylamine TEMPONE-H to form TEMPONE (Reaction 6). Reduced transition metals (Mn+) are oxidized by oxygen (Reaction 7). Decomposition of hydrogen peroxide by catalase did not affect the oxidation of hydroxylamine TEMPONE-H, excluding a role for a direct oxidation of TEMPONE-H by hydrogen peroxide.
<UP>M</UP><SUP>n<UP>+1</UP></SUP>+<UP>TEMPONE-H → M</UP><SUP>n<UP>+</UP></SUP>+<UP>TEMPONE</UP>
<UP>M</UP><SUP>n<UP>+</UP></SUP>+<UP>O<SUB>2</SUB> → M</UP><SUP>n<UP>+1</UP></SUP>+<UP>O&cjs1138;<SUB>2</SUB></UP>
<UP><SC>Reactions</SC> 6 and 7</UP>


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Fig. 11.   Formation of TEMPONE nitroxide during the auto-oxidation of TEMPONE-H. ESR spectra obtained after a 30-min incubation of 0.1 mM TEMPONE-H in 0.15 M sodium phosphate buffer (pH 7.4). A, control. B, TEMPONE-H (0.1 mM) + SOD (100 units/ml). C, TEMPONE-H (0.1 mM) + catalase (10 µg/ml). D, TEMPONE-H (0.1 mM) + Desferal (0.1 mM). ESR receiver gain was 2 × 105.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Despite many studies on Abeta peptide-mediated neurotoxicity, its exact mechanism of action is still undefined. One intriguing hypothesis suggests that Abeta peptides in solution spontaneously form free radicals which damage and kill cells (14, 15). We did not observe any radical adducts when the Abeta peptides Abeta (1-40), Abeta -(40-1), and Abeta -(25-35) were mixed with high quality PBN (OMRF Spin Trap Source).

Previously, it was reported that ESR signals formed during the incubation of Abeta peptides with PBN were due to PBN/·Abeta peptide radical adducts (14-17). However, according to the current literature, there are no reported radical adducts of PBN that have similar ESR parameters (aN = 17.1 G). Using Sigma PBN, both the triplet and quartet spectra of di-tert-butyl- and tert-butylhydronitroxides, respectively, were detected. These nitroxides had previously been misinterpreted as novel radical adducts formed from Abeta radicals (14-17).

Recently, it was suggested that PBN/·Abeta peptide radical adducts decomposed to an alkoxyl nitroxide (15, 17). This alkoxyl nitroxide would have the same structure as the alkoxyl adduct of 2-methyl-2-nitrosopropane. This assignment is also an error, because the nitrogen hyperfine coupling constant of alkoxyl adducts of 2-methyl-2-nitrosopropane is about 27 G (29, 30), which is quite different from the 17.1 G for the radical observed. Moreover, according to the data base (http://epr.niehs.nih.gov/), the hyperfine coupling constant of 17.1 G is very specific to di-tert-butylnitroxide, which is directly supported by the ESR experiments with synthetic di-tert-butylnitroxide presented here (Fig. 2E).

We show in this work that these ESR signals do not appear during the incubation of Abeta peptides with high quality OMRF PBN. Moreover, these ESR signals did appear during the auto-oxidation of impurities only in the Sigma PBN. These data strongly suggest that the ESR spectra reported as PBN/·Abeta peptide radical adducts actually result from the formation of di-tert-butylnitroxide and tert-butylhydronitroxide radicals. These nitroxide radicals are formed during metal-catalyzed auto-oxidation of di-tert-butylhydroxylamine and N-tert-butylhydroxylamine (Scheme 1), which are impurities in PBN from Sigma.


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Scheme 1.  

The spin trap PBN is usually synthesized by condensation of benzaldehyde with N-tert-butylhydroxylamine (31). Therefore, the trace amount of N-tert-butylhydroxylamine in preparations of PBN is probably due to incomplete purification.

The appearance of di-tert-butylhydroxylamine in PBN can be explained by the presence of di-tert-butylhydroxylamine in the N-tert-butylhydroxylamine commonly used for the synthesis of PBN. Scheme 2 illustrates the mechanism of di-tert-butylhydroxylamine formation from N-tert-butylhydroxylamine. N-tert-butylhydroxylamine is a good reducing agent, which, after two one-electron oxidations, produces 2-methyl-2-nitrosopropane. 2-Methyl-2-nitrosopropane is an unstable compound, which is readily decomposed by heat or light to the tert-butyl radical and nitric oxide (32, 33). The tert-butyl radical will react with 2-methyl-2-nitrosopropane (which is actually a well known spin trap) to form di-tert-butylnitroxide (32, 33). Di-tert-butylnitroxide can easily be reduced to di-tert-butylhydroxylamine even by excess N-tert-butylhydroxylamine. The presence of di-tert-butylhydroxylamine in the sample of N-tert-butylhydroxylamine was supported by the fact that the ESR spectra of N-tert-butylhydroxylamine oxidized by K3[Fe(CN)6] contained small traces of the triplet signal of di-tert-butylnitroxide (Fig. 3F). Therefore, trace amounts of di-tert-butylhydroxylamine are present in many preparations of PBN. Due to the intrinsic sensitivity of ESR, oxidation of trace levels of these impurities can lead to readily detected ESR signals. The assignment of the structure of these nitroxides to specific compounds excludes the possibility that these ESR spectra are derived from amyloid beta  peptides.


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Scheme 2.  

In this work we investigated the effect of neurotoxic Abeta -(1-40) and Abeta -(25-35) and non-toxic Abeta -(40-1) on the oxidation of hydroxylamine derivatives. We previously showed that, in the presence of Desferal, incubation of Abeta -(1-40) and Abeta -(25-35) with PBN (Sigma) led to the formation of di-tert-butylnitroxide, which was produced after oxidation of di-tert-butylhydroxylamine. Stimulation of oxidation of hydroxylamine derivatives by Abeta -(1-40) or Abeta -(25-35) was also demonstrated using TEMPONE-H. Oxidation of TEMPONE-H to TEMPONE nitroxide in the presence of Abeta -(1-40) or Abeta -(25-35) was significantly more intensive than in the control without Abeta . The treatment of phosphate buffer with chelating resin Chelex-100 inhibits the Abeta effect on TEMPONE-H oxidation. These data support a catalytic role of phosphate buffer-derived transition metal contaminants in the Abeta -stimulated oxidation of hydroxylamine derivatives.

It is known that ferricyanide is able to oxidize hydroxylamine derivatives (34). Cupric ion is another candidate for the oxidation of hydroxylamines. In this work we checked the possible role of both ferric and cupric ions in oxidation of hydroxylamine derivatives. It was found that both ferric and cupric ions were effective in oxidation of TEMPONE-H. Moreover, oxidation of TEMPONE-H by cupric ions was 40-fold more effective than ferric ions. Therefore, both ferric and cupric ions could be involved in Abeta -stimulated metal-catalyzed oxidation of hydroxylamine derivatives.

The data obtained allow us to conclude that, under some conditions, the toxic Abeta peptides Abeta -(1-40) and Abeta -(25-35) enhance transition metal-catalyzed oxidation of hydroxylamine derivatives. Therefore, the previously reported formation of radical adducts with Abeta peptides can be explained as an Abeta -enhanced, transition metal-catalyzed oxidation of hydroxylamine derivatives found as impurities in commercial PBN. In addition, our data (Fig. 7) demonstrate that Abeta peptides can compete with Desferal for transition metals such as copper, which implies that Abeta peptides may bind redox-active transition metals in vivo where trace metal concentrations are extremely low. Any biological significance of the catalytic role of the toxic Abeta peptides in the transition metal-mediated oxidation of hydroxylamine is yet to be determined. Although these hydroxylamines do not occur naturally, other easily oxidized substances exist in vivo such as ascorbate and GSH.

Moreover, it is known that copper and iron ions catalyze protein damage and may be partly responsible for the alterations of protein damage in vivo (35). Metal-catalyzed protein damage is associated with oxidative modification of amino acids, for example, formation of protein carbonyl groups (35). Therefore, the previously reported inactivation of glutamine synthetase and creatine kinase incubated with Abeta -(25-35) (14) could be explained on the basis of metal-catalyzed protein damage.

Previously, it was shown that iron facilitates Abeta toxicity to cultured cells (36). Moreover, it was found that iron metabolism is altered in Alzheimer's disease (37), which, in combination with accumulation of Abeta peptides, could result in peroxidative stress in the brains of Alzheimer's disease patients. It was suggested that lowering the level of available iron could provide a therapeutic approach to Alzheimer's disease (38). Furthermore, it was shown that Abeta -(25-35) enhances iron- and copper-catalyzed generation of reactive oxygen species (39). Therefore, our data concerning toxic Abeta -stimulated metal-catalyzed oxidation of hydroxylamine derivatives could give insight into the synergetic toxicity of transition metals and Abeta peptides.

The data obtained lead to the conclusion that Abeta peptides do not form ESR-detectable radical adducts spontaneously. Our spin-trapping results and interpretation differ from the previous investigations (14-17). Our data do not support the conclusion that Abeta peptides spontaneously form free radicals, but demonstrate that hydroxylamine impurities in the spin trap preparations are responsible for the observed ESR spectra. Therefore, the reported spontaneous generation of PBN radical adducts by Abeta peptides has been reinterpreted. Although the spontaneous free radical model of Abeta neurotoxicity has been criticized by Sayre et al. (40), the previous spin-trapping results and interpretations have not been challenged until now.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Laboratory of Pharmacology and Chemistry, NIEHS/National Institutes of Health, 111 Alexander Dr., P. O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-2687; Fax: 919-541-1043; E-mail: dikalov{at}niehs.nih.gov. Permanent address: Institute of Chemical Kinetics and Combustion, Novosibirsk 630090, Russia.

2 This program is available to the public via the World Wide Web (http://epr.niehs.nih.gov/).

    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; Abeta , amyloid beta ; ESR, electron spin resonance; OMRF, Oklahoma Medical Research Foundation; PBN, N-tert-butyl-alpha -phenylnitrone; APP, amyloid peptide precursor; PSEN, presenilin; APOE, apolipoprotein E; HPLC, high pressure liquid chromatography; TEMPONE-H, cyclic hydroxylamine 1-hydroxy-4-oxo-2,2,6,6-tetramethylpiperidine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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