4-Oxo-2-nonenal Is Both More Neurotoxic and More Protein Reactive

Chem. Res. Toxicol. 2005, 18, 1219-1231

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4-Oxo-2-nonenal Is Both More Neurotoxic and More Protein Reactive than 4-Hydroxy-2-nonenal De Lin,† Hyoung-gon Lee,‡ Quan Liu,‡ George Perry,‡ Mark A. Smith,‡ and Lawrence M. Sayre*,†,‡ Departments of Chemistry and Pathology, Case Western Reserve University, Cleveland, Ohio 44106 Received March 21, 2005

Electrophilic aldehydes, generated from oxidation of polyunsaturated fatty acyl chains under conditions of oxidative stress, bind to proteins and polynucleotides and can lead to cell death. 4-Hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) have been shown here to be toxic to human neuroblastoma cells in culture at low micromolar concentrations. ONE is 4-5 times more neurotoxic at concentrations near the threshold of lethality. The reactions of these two aldehydes with two model proteins, ribonuclease A and β-lactoglobulin, and their Lys -dimethylamino derivatives, have been followed spectrophotometrically. On the basis of t1/2 measurements for the disappearance of the R,β-unsaturated chromophore, ONE is 6-31 times more reactive with these proteins. The fastest reaction of ONE with proteins involves Schiff base formation at Lys -amino groups, whereas Schiff base formation is not spectroscopically apparent for HNE. Detailed kinetic studies of the initial reactions of HNE and ONE have been carried out with amino acids and amino acid surrogates. Whereas the reactions with imidazole and thiol nucleophiles involve straightforward Michael adduct formation, kinetics analyses reveal the reversibility of both the HNE Michael adduction of amines and the ONE Schiff base adduction of amines. Although ONE is more reactive than HNE toward conjugate addition of imidazole and thiol nucleophiles, it is less reactive than HNE toward Lys/amine Michael adduction. The greater neurotoxicity of ONE could reflect in part the different reactivity characteristics of ONE as compared to HNE.

Introduction The past two decades have witnessed a tremendous research effort focused on the role of lipid oxidation in aging and diseases associated with oxidative stress (15). The condition of oxidative stress results in damage to all biomacromolecules, but the polyunsaturated acyl chains found in membranes and lipoproteins are particularly susceptible to free radical-mediated oxidation, leading to unsaturated lipid hydroperoxides and the breakdown of these to release reactive bifunctional aldehydes such as malondialdehyde, glyoxal, acrolein, and 4-hydroxy-2-nonenal (HNE)1 (6-9). These aldehydes can act locally or can enter the general circulation. Recent studies have shown that peroxidation of linoleoyl chains also produces a seemingly more reactive cousin of HNE, 4-oxo-2-nonenal (ONE) (10, 11). A marker for in vivo generation of ONE has been recently reported (12). Initial interest in HNE centered around its purported involvement in the oxidative modification of LDL and the role of oxLDL in atherosclerosis (13-16). However, there is substantial evidence for increased HNE (and lipid peroxidation in general) also in neurodegenerative diseases, in particular Alzheimer’s disease (17-20) and * To whom correspondence should be addressed. Tel: 216-368-3704. Fax: 216-368-3006. E-mail: [email protected]. † Department of Chemistry. ‡ Department of Pathology. 1 Abbreviations: BCA, bicinchoninic acid; β-LG, β-lactoglobulin; BSA, bovine serum albumin; HNE, 4-hydroxy-2-nonenal; LDH, lactate dehydrogenase; M-β-LG, lysine methylated β-lactoglobulin; M-RNase, lysine methylated ribonuclease A; ONE, 4-oxo-2-nonenal; pEKY, poly(Glu,Lys,Tyr); RNase, ribonuclease A.

Parkinson’s disease (21-23), and in other disease processes associated with an oxidative stress insult (24, 25). HNE readily modifies nucleophilic protein side chains and DNA bases, and its bifunctionality results in protein cross-linking (26, 27). The structures of HNE-protein adducts and cross-links have been investigated through model studies (28-32), immunochemical studies (33-35), and mass spectrometry (36-43). Similarly, the structures of DNA adducts have been elucidated (44-47). The R,βunsaturation in HNE makes it particularly susceptible to Michael addition reactions, especially with thiols, and its primary route of detoxification is via glutathione transferase-mediated conjugation to GSH (48-51), although reduction and oxidation of the C-1 aldehyde group also represent metabolic detoxification. HNE exhibits a wide array of biological activities, including inhibition of the proteasome (52-56), and several studies have defined its cytotoxic effects to represent primarily induction of apoptosis (57, 58). In addition, there is increasing evidence that HNE may act as a signaling molecule (e.g., altering gene expression) (59-64) and that even the GSH adducts of HNE may have biological activity (65). Although the mechanisms responsible for signaling and other biological properties of HNE remain unclear (e.g., whether they represent covalent or noncovalent interactions), HNE has become, overall, the most heavily studied small molecule lipoxidation product (66). With significant interest now being focused on ONE, studies in our laboratories (39, 67) and others (68-71) have characterized its side chain modifying chemistry

10.1021/tx050080q CCC: $30.25 © 2005 American Chemical Society Published on Web 07/06/2005

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Chem. Res. Toxicol., Vol. 18, No. 8, 2005

and led to the conclusion that it is a more reactive protein modification and cross-linking agent than HNE. Also, ONE readily modifies DNA bases, and the structures of these adducts have been studied (72-76). Thus, to whatever extent the biological actions of HNE reflect its chemical reactivity, the more reactive ONE may exert similar biological activities but at even lower concentrations. A recent study showed that both ONE and HNE are toxic to colorectal cancer cells by the same apoptotic mechanism (77). Although not stressed by these authors, ONE was about 2-fold more potent than HNE in some of the indices of apoptosis. In the current study, we exposed two different neuronal cell lines to ONE and HNE in an effort to determine their relative neurotoxicity. At the same time, we report on studies to define differences in the rates and nature of the initial reactions of ONE and HNE with proteins. Our results show that at moderately low concentration (310 µM), ONE is more toxic than HNE, consistent with its higher reactivity. Not only is ONE more reactive than HNE in Michael addition chemistry toward His and Cys, but we now show for the first time that ONE exhibits a uniquely rapid Schiff base formation with Lys and that this reaction represents the first observable reaction with proteins. These findings together support the contention that ONE is an extremely important oxidative stress mediator that could play a role on par with (or perhaps surpassing) that of HNE in human pathophysiology.

Experimental Procedures General Methods and Materials. Unless otherwise stated, the solvents and reagents were of commercially available analytical grade. β-Lactoglobulin (β-LG) and ribonuclease A (RNase) were from Sigma. Lysine methylated β-lactoglobulin (M-β-LG) and RNase (M-RNase) were prepared by reductive methylation (HCHO, NaCNBH3) of the parent proteins (78), using a procedure that we have shown removes all trinitrobenzenesulfonic acid detectable amino groups (79). Poly(Glu,Lys,Tyr) (pEKY) sodium salt (6:3:1), MW 23300-47000, was from Sigma. Polyclonal rabbit antiserum to the products of HNE adduction to keyhole limpet hemocyanin, was obtained from Alexis Biochemicals. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was obtained from ICN. HNE and ONE were from our previous studies. All preparative reactions were carried out at ambient temperature (24-26 °C) unless stated otherwise. All column chromatography was carried out using flash grade silica gel. For 1H NMR (200 or 400 MHz) and 13C NMR (50 MHz) spectra, tetramethylsilane or the solvent peak served as an internal standard for reporting chemical shifts, expressed on the δ scale. Attached proton test designations for 13C NMR spectra are given in parentheses. High-resolution mass spectra (HRMS) were obtained at 20 eV on a Kratos MS-25A instrument. Kinetics of Protein Modification by HNE and ONE. To 160 µL of a solution of RNase or M-RNase (1.25 mg/mL) in 62.5 mM, pH 7.4, sodium phosphate buffer at 23 °C was added 40 µL of a solution of HNE or ONE (1.25 mM) in ethanol. To 160 µL of a solution of β-LG or M-β-LG (1.25 mg/mL) in 62.5 mM, pH 7.4, sodium phosphate buffer at 23 °C was added 40 µL of a solution of HNE or ONE (0.5 mM) in ethanol. The reaction of ONE was also monitored spectrophotometrically with pEKY. To 192 µL of a solution of pEKY (1.04 mg/mL) in 50 mM, pH 7.4, sodium phosphate buffer (or 50 mM, pH 8.5, borate buffer) at 23 °C was added 8 µL of solution of ONE (12.5 mM) in ethanol. All reactions were monitored in a 0.1 cm cuvette by repetitive spectral scan over the range of 210-300 nm. Reactions of ONE with all three proteins exhibited an initial red shift of the ONE chromophore. The half-times for these shifts, representing the

Lin et al. Table 1. Reaction of HNE or ONE with RNase or β-LG or Methylated Derivativesa t1/2 for reaction with HNE (h)

t1/2 for reaction with ONE (min)

kONE/ kHNE

RNase

26 ( 3

M-RNase β-LG

95 ( 7 14 ( 3

M-β-LG

33 ( 4

5.8 ( 0.3 (early); 250 ( 14 255 ( 21 2.9 ( 0.1 (early); 43 ( 4 63 ( 4

269 (early); 6.2 22 290 (early); 20 31

a

Reactions were carried out at 23 °C in pH 7.4 sodium phosphate buffer containing 20% ethanol. Final concentrations of protein (1 mg/mL) result in [Lys] ) 1.0 mM and [His] ) 0.29 mM for RNase and [Lys] ) 0.82 mM, [His] ) 0.11 mM, and [free Cys] ) 0.054 mM for β-LG. Final concentrations of aldehydes were 0.25 mM for reactions with RNase and M-RNase and 0.10 mM for reactions with β-LG and M-β-LG. The data listed are the average of three experiments ( SD. Table 2. Rate Constants for the Reactions of HNE or ONE with Imidazole and Thiol Nucleophilesa nucleophile

reaction rate with HNE (M-1 s-1)

reaction rate with ONE (M-1 s-1)

kONE/ kHNE

imidazole NR-Ac-L-His butanethiol NR-Ac-L-Cys

5.45 ( 0.35 × 10-3 2.90 ( 0.27 × 10-3 8.41 ( 0.73 × 10-1 5.93 ( 0.95 × 10-1

17.6 ( 0.54 × 10-3 9.90 ( 0.33 × 10-3 213.0 ( 7.1 84.0 ( 8.9

3.2 3.4 253 142

a Reactions were conducted at 23 °C in 50 mM, pH 7.4, sodium phosphate buffer containing 20% ethanol.

rapid phase of ONE reactivity, are listed in Table 1. The halftimes of the slower bleaching of the HNE or ONE chromophores were calculated by estimating the absorbance at very long reaction times. Kinetics of Reaction of Model Nucleophiles with HNE or ONE. Kinetics were performed with [nucleophile] > 10 × [HNE] or [ONE] so that the calculated kobs values represent pseudo-first-order rate constants. We used 5 mM imidazole nucleophiles and 5-25 mM butylamine to react with 0.05 mM aldehydes, whereas for the more reactive thiol nucleophiles, we used 2 mM thiol with 0.01 mM HNE and 0.2 mM thiol with 0.01 mM ONE. The reactions were executed in 50 mM, pH 7.4, sodium phosphate buffer containing 20% ethanol, where the pH was adjusted after addition of the nucleophile by addition of 1 M NaOH. Some reactions were also carried out in the same buffer with 1% CH3CN. The reactions were monitored at 23 °C by repetitive spectral scan from 210 to 300 nm. Once we determined that only reactions with butylamine require full spectral scans, reactions with other nucleophiles were monitored by observing the decrease over time in absorbance at 224 and 226 nm due to the R,β-unsaturation of HNE and ONE, respectively. The pseudo-first-order plots of the reactions of imidazole and thiol nucleophiles with HNE and ONE were linear to at least 3 half-lives, and the first-order rate constants kobs were determined from the linear regression of ln[(A∞ - A0)/(A∞ - At)] vs time, where A∞ was determined by estimation of the plot of A vs time. In the cases of the slower reactions, we also calculated rate constants by choosing values of A∞ that gave optimal linearity of the first-order plot (rate constants obtained this way were within 5% of those obtained by estimation of A∞). Firstorder kobs values were converted to second-order rate constants by dividing by the concentration of nucleophile used, and the values listed in Table 2 represent the average of at least two rate determinations. Verification of the bimolecular nature of the reactions was obtained for reactions of HNE and ONE with NR-Ac-L-Cys, where the latter concentration was varied from 2 to 5 mM for HNE and from 0.2 to 0.5 mM for ONE. kobs values were determined for four concentrations, and the second-order rate constants listed in Table 2 represent the slopes of these plots that passed through the 0-0 origin.

4-Oxo-2-nonenal and 4-Hydroxy-2-nonenal In contrast to the reactions with imidazole and thiol nucleophiles, the reactions of HNE with butylamine and the initial fast reaction of ONE with butylamine did not proceed to completion, and the data were fitted according to a reversible equilibrium as described (80), which required obtaining kobs as a function of [butylamine]. According to this method, for reactants a and b, the general equation kobs ) {[k2 + k1(a + b)]2 - 4abk12}1/2 reduces to kobs ) k2 + k1b under the conditions b . a that we used. Thus, kobs was determined as above at five concentrations of amine; k1 and k2 were calculated from the slope and intercept of the plots of kobs vs [butylamine]. NMR Tube Scale Reaction of ONE and Butylamine. To a solution of ONE (7.0 mg, 0.045 mmol) in CD3CN/D2O (350 µL/140 µL) in an NMR tube was added butylamine (3.7 mg, 0.05 mmol) in CD3CN (210 µL), to give a final apparent pH of 10.5. The reaction in the tube was monitored immediately (∼1 min). The aldehyde proton of ONE was gone, and the 1H NMR spectrum showed >90% conversion to the Schiff base [downfield signals at δ 8.01 (dtd, 1H, J ) 9.2, 1.2, and 0.4 Hz), 7.01 (dd, J ) 16.4 and 9.2 Hz), 6.54 (dd, J ) 16.4 and 0.4 Hz)]. However, the 13C NMR spectrum could not be obtained due to development of a precipitate. In a second NMR tube reaction, to a solution of butylamine (3.7 mg, 0.05 mmol) in CD3CN (210 µL) were added NaOH (0.1% in D2O) (140 µL) and a solution of ONE (7.0 mg, 0.045 mmol) in CD3CN (350 µL). After 30 min at room temperature, NMR spectra of the Schiff base were recorded as follows. 1H NMR: δ 0.85 (t, 3H, J ) 7.6 Hz), 0.87 (t, 3H, J ) 7.6 Hz), 1.20-1.32 (m, 6H), 1.50-1.59 (m, 4H), 2.64 (t, 2H, J ) 7.6 Hz), 3.48 (td, 2H, J ) 7.6 and 1.2 Hz), 6.54 (dd, 1H, J ) 16.4 and 0.4 Hz), 7.01 (dd, 1H, J ) 16.4 and 8.8 Hz), 8.01 (dtd, 1H, J ) 9.2, 1.2, and 0.4 Hz). 13C NMR: δ 14.19 (-), 14.36 (-), 21.08 (+), 23.23 (+), 24.47 (+), 32.05 (+), 33.31 (+), 41.38 (+), 61.83 (+), 138.80 (-), 140.24 (-), 163.32 (-), 201.48 (+). Preparative Reaction of HNE and ONE with Butylamine. To a solution of 2 mmol of butylamine (146 mg) in a mixture of 160 mL of 62.5 mM, pH 7.4, sodium phosphate buffer and 40 mL of ethanol was added 0.2 mmol of HNE (31.2 mg) or ONE (30.8 mg). The reaction mixture was stirred at room temperature for 20 min and was then treated with 74 mg (2 mmol) of NaBH4. After 1 h, 1.0 g of NH4Cl was added to break up the borate salts. The mixture was then made basic with 10% aqueous NaOH to pH 11.5 and extracted with CH2Cl2. The combined organic extracts were dried (Na2SO4) and evaporated. The resulting residue was applied to a preparative silica gel TLC plate (0.5 mm), which was eluted with 1:2 EtOAc-MeOH (1% NH3). The major amine-containing band (Rf ) 0.30 for HNE and 0.28 for ONE) was extracted with MeOH, and the extract was filtered and evaporated. The product isolated from HNE [3-(butylamino)-1,4-nonanediol] was identical to our earlier report (29). The product isolated from ONE is as follows. (E)-1-(Butylamino)-2-nonen-4-ol. 1H NMR (CDCl3): δ 0.810.92 (m, 6H), 1.20-1.58 (m, 10H), 1.60-1.65 (m, 2H), 2.57 (t, 2H, J ) 7.2 Hz), 3.20 (d, 2H, J ) 5.6 Hz), 4.05 (q, 1H, J ) 6.4 Hz), 5.60 (dd, 1H, J ) 15.6 and 6.4 Hz), 5.71 (dt, 1H, J ) 15.6 and 6.0 Hz). 13C NMR (CDCl3): δ 14.08 (-), 14.10 (-), 20.55 (+), 22.68 (+), 25.20 (+), 31.82 (+), 32.22 (+), 37.27 (+), 49.24 (+), 51.26 (+), 72.70 (-), 129.41 (-), 134.93 (-). HRMS (FAB) calcd for C13H28NO (MH+), 214.2171; found, 214.2169. The reaction of HNE and ONE (final concentration, 0.5 mM) and butylamine (final concentration, 5 mM) was carried out as for the kinetic studies described above. The reaction with ONE was quenched with NaBH4 at the end of the rapid initial “red shift” stage of the reaction (20 min), whereas the reaction with HNE was quenched after 24 h. Following the workup as above and extraction with CH2Cl2, the 1H NMR spectrum of the entire crude product was recorded. For ONE: It showed only the reduced Schiff base (E)-1-(butylamino)-2-nonen-4-ol and the known ONE reduction product (E)-2-nonene-1,4-diol (81). For HNE: It showed the reduced Michael adduct (major), the reduced HNE, and the reduced bis-butylamine Schiff base Michael adduct (minor) (29, 31). As there were no other

Chem. Res. Toxicol., Vol. 18, No. 8, 2005 1221 materials apparent, we took the ratio among these products (integration) to represent the product distribution (total 100%). Preparation of 3-(2-Methoxyethylamino)nonane-1,4diol (MEANAD). To a solution of 2 mmol of 2-methoxyethylamine (152 mg) in a mixture of 80 mL of 62.5 mM, pH 7.4, sodium phosphate buffer and 20 mL of acetonitrile was added 2 mmol of HNE (312 mg). The reaction mixture was stirred at room temperature for 24 h and was then treated with 740 mg (20 mmol) of NaBH4. After 1 h, 4.0 g of NH4Cl was added to break up the borate salts. The mixture was then made basic with 10% aqueous NaOH to pH 11.5 and extracted with CH2Cl2. The combined organic extracts were dried (Na2SO4) and evaporated. The resulting residue was applied to a 0.5 mm silica gel TLC plate, which was eluted with 1:2 hexanes-EtOAc. The major amine-containing band (Rf ) 0.28) was extracted with MeOH, and the extract was filtered and evaporated. 1H NMR (CDCl3): δ 0.86 (t, 3H, J ) 6.8 Hz), 1.20-1.35 (m, 6H), 1.381.50 (m, 2H), 1.52-1.65 (m, 2H), 2.60-2.72 (m, 2H), 2.85-2.94 (m, 1H), 3.32 (s, 3H), 3.44 (t, 2H, J ) 5.6 Hz), 3.67-3.72 (m, 1H), 3.73-3.78 (m, 1H). 13C NMR (CDCl3): δ 14.10 (-), 22.66 (+), 26.14 (+), 29.45 (+), 31.94 (+), 33.59 (+), 46.36 (+), 58.89 (-), 62.01 (+), 62.10 (-), 70.38 (-), 72.31 (+). Preparation of (E)-1-(2-Methoxyethylamino)-2-nonen4-ol (MEANEO). To a solution of 2 mmol of 2-methoxyethylamine (152 mg) in a mixture of 160 mL of 62.5 mM, pH 7.4, sodium phosphate buffer and 40 mL of ethanol was added 0.2 mmol of ONE (30.8 mg). The reaction mixture was stirred at room temperature for 20 min and then treated with 74 mg (2 mmol) of NaBH4. After 1 h, 1.0 g of NH4Cl was added to break up the borate salts. The mixture was then made basic with 10% aqueous NaOH to pH 11.5 and extracted with CH2Cl2. The combined organic extracts were dried (Na2SO4) and evaporated. The resulting residue was applied to a 0.5 mm silica gel TLC plate, which was eluted with 1:2 EtOAc-MeOH (1% NH3). The major amine-containing band (Rf ) 0.31) was extracted with MeOH, and the extract was filtered and evaporated. 1H NMR (CDCl3): δ 0.86 (t, 3H, J ) 6.8 Hz), 1.10-1.55 (m, 8H), 2.102.15 (m, 2H), 2.75 (t, 2H, J ) 3.4 Hz), 3.23 (d, 2H, J ) 3.6 Hz), 3.34 (s, 3H), 3.48 (t, 2H, J ) 3.4 Hz), 4.05 (q, 1H, J ) 4.2 Hz), 5.57-5.78 (m, 2H). 13C NMR (CDCl3): δ 14.10 (-), 22.67 (+), 25.23 (+), 31.83 (+), 37.28 (+), 48.76 (+), 51.13 (+), 58.87 (-), 71.91 (+), 72.48 (-), 128.93 (-), 135.35 (-). HRMS (FAB) calcd for C12H26NO2 (MH+), 216.1964; found, 216.1981. Immunodots for Protein Modification by HNE and ONE. A solution of M-β-LG (2.08 mg/mL, 960 µL) in 50 mM, pH 7.4, sodium phosphate buffer was incubated with a solution of either HNE or ONE in ethanol (50 mM, 40 µL) or with ethanol (40 µL) alone at 37 °C. After 2 h, the incubation was quenched with NaBH4 (final concentration, 25 mM) for 30 min. Then, the solution was dialyzed against 50 mM, pH 7.4, sodium phosphate buffer three times in 8 h intervals. The bicinchoninic acid (BCA) assay using bovine serum albumin (BSA) as standard indicated a lower protein concentration after dialysis. The concentration of the modified proteins was adjusted to 1.0 mg/mL using 50 mM, pH 7.4, sodium phosphate buffer. Then, 2.0 µL protein samples were applied on each dot on Immobilon membrane (Millipore), which was air-dried and rinsed with methanol. Membranes were blocked with 10% milk for 1 h, washed with Tris-buffered saline, and incubated with the HNE Michael adduct antibody (1:100) for 16 h at 4 °C, and after five 5 min washes in Tris-buffered saline-Tween, the HRP-conjugated goat anti-rabbit antibody (1:1000) was applied for 1 h at 37 °C. After they were rinsed again as described above, the blots were developed together for the same length of time using the luminol ECL reagent (Santa Cruz Biotechnology) and imaged by using image acquisition and analysis software (Ultra-Violet Products Ltd.). Immunodots for pEKY Modification by HNE and ONE. A solution of pEKY (2.08 mg/mL, 192 µL) in 50 mM, pH 7.4, sodium phosphate buffer was incubated with a solution of HNE or ONE in ethanol (50 mM, 8 µL) at 37 °C for 16 h or at 23 °C for 15 min, respectively. Also, a solution of pEKY (2.08 mg/mL,

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192 µL) in 50 mM, pH 8.5, sodium borate buffer was incubated with a solution of ONE in ethanol (50 mM, 8 µL) at 23 °C for 6 min. After each reaction time, the incubation was quenched with NaBH4 (final concentration, 25 mM) for 30 min at room temperature, and then, NH4Cl (to make 0.25 M) was added to decompose the borate salts. All samples were diluted to 1 mg/ mL based on pEKY concentration. Then, 2.0 µL protein samples were used for each immunodot as described above. The specificity of the antibody was ascertained by incubating for 2 h at 37 °C with or without the competing small-molecule antigens MEANEO and MEANAD at a concentration of 0.4 mM. Absorbed and unabsorbed antibody incubations were applied to immnunodots for 16 h at 4 °C, with followup as described above. Cell Culture and Cytotoxicity Assays. Human BE(2)-M17 and SH-SY5Y neuroblastoma cells (82) were kindly provided by Dr. Robert Petersen (Pathology, Case Western Reserve University). Cells were cultured in Opti-MEM media (Life Technologies or Invitrogen) with 5% donor calf serum and 1% penicillin/streptomycin with fungizone (Life Technologies). For cytotoxicity in M17 cells, 2 mL aliquots (1 × 105 cells/mL) of cells were placed in each well in six well plates. After overnight incubation, the media were changed and the cells were treated with different concentrations of ONE or HNE for defined times. Trypan blue was diluted at 0.8 mM in PBS. After all of the pretreatments, cells were pipeted off and mixed 1:1 with trypan blue solution. The cells were counted on a hemocytometer. Viable cells exclude trypan blue, while dead cells stain blue due to trypan blue uptake. For cytotoxicity in SH-SY5Y cells, the cells were plated at a density of 2 × 104 cells/well onto 96 well plates [for lactate dehydrogenase (LDH) assay] or at a density of 3 × 104 cells/well six well plates (for microscopy) and cultured overnight. The cell culture medium was then switched to serumfree Opti-MEM and treated with different concentrations of ONE or HNE for 24 h. The cytotoxicity of ONE and HNE was evaluated by the LDH assay kit (Roche), according to the instructions. Briefly, cell media were collected after each treatment and the collected media were mixed with LDH substrate in a 96 well plate. After incubation for 30 min at room temperature, the optical density was measured at 490 nm using a microplate reader (Molecular Devices). The measured optical density was converted after standardization with low (no treatment; 0% toxicity) and high controls (1% Triton X-100; 100% toxicity) by the following equation: cytotoxicity (%) ) [(experimental value - low control)/(high control - low control)] × 100.

Results Reactions of HNE and ONE with Proteins. The reactions of HNE and ONE with RNase, β-LG, and their lysine permethylated derivatives were followed spectrophotometrically in pH 7.4 sodium phosphate buffer containing 20% (v/v) ethanol. We used ethanol to ensure a homogeneous solution over the course of the several days for which these reactions were monitored. The concentration of aldehydes chosen was adjusted so that it would not be in excess of the concentration of either Lys or His groups theoretically available in the native protein (from the primary sequence) for reaction. There are 14 Lys and four His in RNase and 15 Lys and two His in β-LG. There is also one free Cys (out of five) in β-LG (83) that is modifiable by HNE (36). We verified that the free Cys was present to an extent of 1.1 residues per protein monomer, by the N-ethylmaleimide spectrophotometric assay according to a modification (84) of the standard method (85). Two spectral phenomena were observed. For reaction of ONE with the native proteins, within the first several minutes, there was observed a red shift of the ONE R,βunsaturated chromophore, with an isosbestic point near

Lin et al.

Figure 1. Early stage of UV spectral changes for RNase A (1 mg/mL) exposed to ONE (0.25 mM) in 50 mM, pH 7.4, sodium phosphate buffer containing 20% ethanol at 23 °C.

236 nm (Figure 1 shows the reaction with RNase). This was also seen in the absence of ethanol as cosolvent (data not shown). The red shift was not observed with HNE nor in the reaction of ONE with the Lys methylated proteins. In all of these other cases, a simple bleaching of the R,β-unsaturated chromophore occurred, which was also true for the reaction of native proteins with ONE following the initial rapid red shift. We interpret the red shift to be indicative of Schiff base formation between the ONE aldehyde moiety and the -amino groups of protein Lys residues. The direction of λmax shift is consistent with expectations for substituting O by the less electronegative N. Because of the presence of multiple types of nucleophiles on the proteins with differing reactivity, we made no effort to analyze the reactions according to particular kinetic models. Instead, we simply determined the halflives for the two spectral phenomena as the time needed to reach 50% of the overall changes in absorption (Table 1). For both proteins, it can be seen that the rapid Schiff base reaction with ONE is by far the fastest process, whereas even the slower phases of the ONE reactions (bleaching of the red-shifted chromophore) occur more rapidly than the reactions with HNE. Interestingly, ignoring the initial Schiff base formation stage, the difference in reactivity between ONE and HNE is greater for the reductively methylated proteins. This suggests a greater divergence in reactivity between ONE and HNE for His and Cys vs Lys residues. In addition, despite use of a lower [aldehyde] for the reactions with β-LG and M-βLG, the t1/2 values were shorter, especially with ONE. We ascribe this to the preferential depletion of aldehyde by Michael addition of the one available Cys in this protein. The latter conclusion is supported by the fact that there was a clearly discernible biphasic nature of the time course of bleaching of the R,β-unsaturated chromophores for β-LG. Reactions of HNE and ONE with Individual Side Chain Nucleophiles. In an effort to better understand the relative protein reactivity data, we studied the reactions of the two aldehydes with nucleophiles representing protein side chains. Like HNE, ONE has been found under preparative conditions (buffered aqueous CH3CN, room temperature) to form adducts with the side chains of Cys, His, and Lys, although we could not detect significant Arg adduct formation (67). In studies aimed at determining the relative Michael addition reactivity of side chain nucleophiles with ONE and HNE by

4-Oxo-2-nonenal and 4-Hydroxy-2-nonenal

monitoring bleaching of the R,β-unsaturated carbonyl chromophore, Doorn and Petersen found a rate order Cys . His > Lys, with ONE being more reactive than HNE in each case (68, 69). In addition, although no reaction between Arg and HNE was apparent, these workers reported that Arg reacts with ONE at a rate 10-fold slower than with Lys. The Blair group has also reported on ONE-Arg adduct formation (70, 71). Our observation of apparent Schiff base reactivity of ONE above led us to reexamine the kinetics of the reactions of HNE and ONE, with monitoring of the spectrum between 210 and 300 nm in pH 7.4 buffered 20% aqueous ethanol at 23 °C. Kinetic studies were conducted using a large excess (pseudo-first-order kinetics conditions) of the simple side chain surrogates butylamine, imidazole, and butanethiol, as well as with NRAc-L-His and NR-Ac-L-Cys. The reaction of HNE and ONE with imidazole and thiol nucleophiles resulted in a straightforward decrease in intensity of the broad R,βunsaturated chromophore centered at 224 and 226 nm, respectively. The data in Table 2 compare second-order rate constants calculated from the observed pseudo-firstorder rates, except in the case of NR-Ac-L-Cys, where we verified the bimolecular nature of the reaction by measuring kobs as a function of [NR-Ac-L-Cys]. The values given in Table 2 in this latter case represent the linear regression of plots that intersect the 0-0 origin (Supporting Information). In all cases, the NR-acetyl amino acids reacted about half as rapidly as did the simple side chain surrogate nucleophiles. The thiol nucleophiles were found to be more reactive than the imidazole nucleophiles, although whereas the difference was ∼200-fold in the case of HNE, the difference in the case of ONE was ∼10000-fold. This reflects a digression from the usual reactivity-selectivity principle, since the more reactive ONE is apparently more rather than less discriminating in reacting preferentially with the more reactive nucleophile. Hard-soft Lewis acidbase effects may be responsible. Doorn and Petersen observed a similar trend (68, 69), and this observation may explain the data in Table 1 showing that the enhanced reactivity of ONE over HNE is greater for the protein (β-LG) that contains a free Cys. For butylamine, whereas the reaction with HNE resulted in no shift in the HNE chromophore and after a few minutes in its simple bleaching, the reaction with ONE resulted in an initial isosbestic shift of the band with λmax at 226 nm to a new band with λmax at 230 nm (isosbestic point at 232 nm), although with some reduction in intensity (Figure 2A). As was concluded above from the reactions with proteins, we attribute the red shift to the generation of the C-1 Schiff base between ONE and butylamine. The rate of this conversion was most conveniently monitored by the increase in absorbance at 250 nm, where there was maximal amplitude difference between starting aldehyde and Schiff base, although the rate obtained by monitoring the decrease at 226 nm was essentially identical (inset of Figure 2A). Upon increasing the concentration of butylamine from 5 (Figure 2A) to 25 mM (Figure 2B), the red shift occurred more rapidly and became more pronounced (the λmax now comes near 240 nm). This is suggestive of a reversible Schiff base equilibrium, where an increased fraction of ONE is present as the Schiff base at higher [amine]. Additional evidence for reversibility is that when the Schiff base was allowed to form at high concentration of

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Figure 2. Incubation of ONE (0.05 mM) and butylamine at 23 °C in 50 mM, pH 7.4, sodium phosphate buffer containing 20% ethanol, showing isosbestic conversion to the Schiff base. (A) [Butylamine] ) 5 mM. (B) [Butylamine] ) 25 mM. Inset in panel A: ln[(A∞ - A0)/(A∞ - At)] vs time plots for the decrease of ONE absorbance at 226 nm and the increase in Schiff base absorbance at 250 nm.

Figure 3. Reversal of ONE-amine Schiff base formation. ONE (64 mM) and butylamine (71 mM) were allowed to react in CH3CN-H2O (8:2, v/v) with 0.1% NaOH for 30 min. Then, an aliquot (2 µL) was withdrawn and diluted 1:1280 into 50 mM, pH 7.4, sodium phosphate buffer containing 20% ethanol, and the spectral change was monitored over time.

ONE and butylamine and then an aliquot was diluted into fresh buffer, a reversal of the red shift was observed over time (Figure 3). Plots of kobs as a function of [butylamine] for the reaction with HNE and for the fast reaction with ONE are shown in Figure 4. In neither case does the plot pass through the 0-0 origin. In fact, because the plots are linear in the region where [butylamine] . [ONE], the data could be treated in terms of the expectations for a reversible equilibrium (79), where the forward and reverse rate constants k1 and k2 can be determined from the slope and intercept of the plot of kobs vs [amine]. The calculated rate constants are listed in Table 3. We also determined how the Schiff base equilibrium between

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Figure 5. Conversion of ONE to the butylamine C-1 Schiff base (1H NMR spectrum in CD3CN-D2O, 4:1).

Scheme 1

Figure 4. Plots of kobs vs [butylamine] for the reactions with 0.05 mM ONE (A) or HNE (B) at 23 °C in 50 mM, pH 7.4, sodium phosphate buffer containing 20% ethanol. Table 3. Rate Constants for the Reaction of HNE or ONE with Butylaminea pH

k1 (10-3 M-1 s-1)

k2 (10-3 s-1)

K ) k1/k2 (M-1)

HNE (20% EtOH) ONE (0% EtOH) ONE (20% EtOH) ONE (20% EtOH) ONE (20% EtOH) ONE (50% EtOH)

7.4 7.4 6.9 7.4 7.9 7.4

3.37 90.7 82.8 112 197 194

0.00995 5.57 4.48 2.50 1.72 0.430

339 16.3 18.5 44.8 115 451

kONE/kHNE (20% EtOH)

7.4

33

251

a

Reactions were conducted at 23 °C in 50 mM sodium phosphate buffer at different pH values and different ethanol contents.

ONE and butylamine was affected by the nature of the solvent. Thus, in addition to data obtained for buffer containing 20% ethanol, data were obtained also for 0% and for 50% ethanol. The effect of pH on the reaction in buffer-ethanol 8:2 was also investigated (Table 3). Because the reaction of HNE with butylamine exhibited no significant Schiff base formation, the decrease in absorbance is concluded to represent Michael addition, and it is notable that the forward rate constant is similar to that observed for the Michael addition of imidazole nucleophiles to HNE. The equilibrium constant is large, but the finding of kinetic reversibility is consistent with the conclusion that we made several years ago when attempts to isolate the HNE Lys/amine Michael adducts in model reactions afforded recovered HNE and amine (29). In this study, confirmation of C-3 Michael adduct presence prior to attempted isolation was obtained byproduct isolation following NaBH4 quenching. The Schiff base formation between ONE and butylamine represents a less favorable equilibrium, but k1 is markedly improved and k2 is slowed with increasing ethanol content in the reaction solution and with increas-

ing pH. In the latter case, there was an approximately 2.5-fold increase in K for Schiff base formation for each 0.5 pH unit increase. This increase is unlikely to reflect a pH effect on the true Keq constant but instead the simple fact that a greater fraction of butylamine exists as the free base at higher pH. Because the local dielectric constant in proteins is believed to be somewhat similar to ethanol, the data in Table 3 suggest that Schiff base formation of ONE with protein Lys residues will be more favorable, although reversible, in less polar environments, especially if the Lys side chain exists more as the free base. “Early” HNE and ONE Amine Adduction Chemistry Studied by NMR Spectroscopy. Confirmation of our conclusions from kinetics studies, that under physiomimetic conditions amines interact with HNE primarily through Michael adduction but with ONE primarily through C-1 Schiff base formation, was sought through NMR studies. First, to confirm that the Schiff base occurs at C-1, we recorded the 1H NMR spectrum of ONE in CD3CN and then again following addition of 1.1 equiv of butylamine and D2O to give a final solution CD3CN-D2O 4:1. Analysis of the spectral changes in the downfield ppm region (Figure 5) reveals clean Schiff base formation at C-1, as expected. The extent of Schiff base formation under these unbuffered, mainly organic solvent conditions would not extrapolate to buffered, mainly aqueous solution. However, we also carried out incubations of HNE or ONE (1 mM) with butylamine (10 mM) in 50 mM, pH 7.4, sodium phosphate buffer (20% ethanol), with quenching of the reactions after 20 min using NaBH4. Following workup, isolation of the aminecontaining organic products (preparative TLC) revealed nearly exclusively the reduced Michael adduct in the case of HNE and nearly exclusively the reduced Schiff base in the case of ONE (Scheme 1, R ) Bu). With the reaction products now characterized, the reactions of HNE and ONE with butylamine were repeated under the same kinetics conditions (this time with 5 mM amine and 0.5 mM aldehyde), with analysis


of the entire crude product by 1H NMR following NaBH4 quenching. For the HNE reaction, the 1H NMR spectrum after 24 h of reaction revealed the presence of 67% of the reduced Michael adduct, 28% of the reduced HNE, and 5% of the reduced bis-amine Schiff base Michael adduct (29, 31). This distribution compares favorably with the equilibrium distribution predicted from the value of K listed in Table 3 (339 M-1) using these concentrations: Solving of the quadratic gives 63% Michael adduct and 37% HNE. For the ONE reaction, the 1H NMR spectrum after 20 min of reaction revealed the presence of 16% of the reduced ONE-butylamine Schiff base and 84% of reduced ONE (2-nonene-1,4-diol). This distribution compares favorably with the equilibrium distribution predicted from the value of K listed in Table 3 (44.8 M-1) using these concentrations: Solving of the quadratic gives 18% Schiff base and 82% ONE. Although we did not carry out the companion preparative scale reaction at the highest concentration of butylamine used (25 mM), one can calculate in the same manner for K that ONE distributes 55% as the Schiff base. With the finding under this condition that the λmax of the red-shifted chromophore comes at 240 nm (see Figure 2B), one can calculate that the pure Schiff base should display a λmax of ∼251 nm. Late Stage Reaction of ONE with Amine Nucleophiles. Although the fastest reaction of ONE with amines represents conversion to the minor equilibrium fraction of Schiff base, at longer times, the ONE chromophore (a mixture of free aldehyde and Schiff base) should be depleted if free ONE and/or the Schiff base can undergo Michael addition by amine. Indeed, following the initial rapid isosbestic stage, the reaction between ONE and butylamine became nonisosbestic, with a general loss of the R,β-unsaturated chromophore. However, this ∆A occurred with a half-life (8 h) that is comparable to that observed under the same conditions for ONE in the absence of nucleophile (15 h). The latter reaction represents ONE decomposition that is actually second-order in ONE (Lin and Sayre, unpublished results). The nature and products of the ONE decomposition reaction will be discussed separately. The important point is that the first-order plots of the late stage ONE disappearance are sufficiently linear in the initial phase of the reaction to give the appearance of a pseudo-first-order reaction of ONE with the nucleophile, if one is present. The true reaction of nucleophile, however, must be extracted by correcting for the loss of ONE due to decomposition. If the major reaction occurring with butylamine in competition with ONE decomposition represents Michael addition, then quenching of this reaction with NaBH4 at an appropriate time should reveal, upon workup, some of the reduced Michael adduct or reduced Schiff base Michael adduct. In both cases, two regioisomers are possible, one of each being the same as that generated from HNE, for which we have authentic standards (29, 31). We obtained 1H NMR spectroscopic and TLC information on the reactions of ONE with butylamine under kinetic conditions (pH 7.4) quenched with NaBH4 at the t1/2 (8 h) and after 24 h. In neither case could we identify more than a trace of products representing Michael addition of butylamine. Interestingly, however, when the reaction of ONE with butylamine was conducted at higher pH and quenched with NaBH4 after 20 min, we could observe both the reduced Schiff base and the reduced Michael adduct. At pH 11.5, TLC indicated a nearly equal amount of the two products when the


reaction was run at 0 °C, whereas the reduced Michael adduct predominated when the reaction was run at pH 11.5 at room temperature. Thus, Michael addition of amine to ONE appears to be very sensitive to the reaction conditions. Our finding that the reactions of ONE with slower nucleophiles occur in competition with ONE decomposition led us to reinvestigate the reaction of ONE under the aqueous buffer conditions (50 mM, pH 7.4, sodium phosphate) employed by Doorn and Petersen (68, 69). Data were obtained for the reaction of ONE (0.05 mM) alone and with 5 mM NR-acetyl derivatives of L-Arg, L-Lys, L-Glu, or L-Ile. The plots of ∆A226 vs time for ONE alone and for ONE with NR-Ac-L-Glu and NR-Ac-L-Ile overlapped exactly, indicating not only a lack of reactivity of these side chains but also that ONE decomposition is not catalyzed by NR-acetyl amino acids. In contrast, the decrease in ∆A226 was more rapid for NR-Ac-L-Arg and NR-Ac-L-Lys (we ignored the initial rapid Schiff base formation in the latter case). The kinetic data were treated in terms of a simultaneous pseudo-first-order reaction (ONE + excess nucleophile) and second-order reaction (bimolecular ONE decomposition). This analysis yielded pseudo-first-order rate constants, which at [nucleophile] ) 5 mM could be converted to second-order rate constants of 5.3 × 10-4 and 4.7 × 10-4 M-1 s-1 for NRAc-L-Arg and NR-Ac-L-Lys, respectively (average of two determinations). The former value is a little slower than that reported by Doorn and Petersen (68, 69) for Ac-Argamide but more than an order of magnitude slower than what was reported for Ac-Lys-amide. We are unable to explain this latter apparent discrepancy, but it could reflect differences in procedure, since these workers did not report the actual concentrations used in their studies, nor did they appear to take into account Schiff base formation or ONE decomposition. Under the same aqueous buffer conditions, the reactions of 0.05 mM HNE with 5 mM NR-Ac-L-Lys and NRAc-L-His gave values of 8.3 × 10-4 M-1 s-1 and 19.3 × 10-4 M-1 s-1. These data lead to the surprising conclusion that despite ONE being substantially more reactive than HNE toward imidazole and thiol nucleophiles, it reacts nearly 2-fold slower than HNE toward Michael addition of NR-Ac-L-Lys. This conclusion is consistent with the data in Table 1 showing that whereas Lys permethylation substantially slows down the reaction of proteins with HNE, it has very little or no effect on the rate of reaction of the proteins with ONE. Immunochemical Analysis of HNE and ONE Protein Modification. We sought to obtain confirmatory evidence for the differential nature of protein modification by HNE and ONE through immunochemical studies. The polyclonal “anti-HNE” antibody obtained my immunizing rabbits with HNE-treated keyhole limpet hemocyanin is usually believed to recognize principally HNE Michael adducts on Cys, His, and Lys (86). However, in reality, it should recognize a variety of HNE adducts, and we have unpublished observations that the commercial anti-HNE antibody (Alexis) exhibits some cross-reactivity with borohydride-reduced HNE Michael adducts on proteins. Because borohydride reduction of the major regioisomers of ONE Michael adducts (at C-3, 67) gives the same diol structures as generated by borohydride reduction of HNE Michael adducts, the antibody should additionally recognize ONE-treated protein following borohydride reduction if the modification

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Figure 6. Anti-HNE antibody recognition of NaBH4-reduced HNE- and ONE-modified M-β-LG. M-β-LG was exposed to 2 mM ONE or HNE (or buffer alone, control) for 2 h, treated with NaBH4, dialyzed, and then applied to the PVDF membrane. Modifications were detected by the anti-HNE antibody from Alexis (top panel). The bottom panel shows quantitation of the oxidized luminal ECL reagent in the dots by image acquisition for three such experiments.

involves a significant Michael addition. Whether the antibody also recognizes reduced Schiff bases is unknown. We used the reductively methylated preparation M-βLG as a protein source of strictly His and Cys nucleophiles. The data in Figure 6 show that treatment of M-βLG with 2 mM HNE or ONE for 2 h followed by borohydride reduction results in immunorecognition, but the much greater immunreactivity for ONE is consistent with the greater Michael reactivity of ONE than HNE toward His and Cys. We next used poly(Glu,Lys,Tyr) (6:3:1) (pEKY) as a protein source of only Lys nucleophiles. Spectrophotometric monitoring of the reaction of pEKY with ONE at 23 °C demonstrated that the red shift indicative of rapid Schiff base formation (see above) was essentially complete in 15 min at pH 7.4 or 6 min at pH 8.5 before any significant decrease in the absorption indicative of conjugate addition to the R,β-unsaturation had occurred (data not shown). Quenching with NaBH4 at these time points should then afford a preparation of ONE-modified Lys that involves strictly the reduced Schiff base, although the equilibrium extent of Schiff base formation was greater at the higher pH, consistent with the model kinetics studies (Table 3). The data in Figure 7 compare the immunorecognition by the anti-HNE antibody toward these two ONE-pEKY preparations to that seen for pEKY treated with 2 mM HNE for 24 h at 37 °C with or without subsequent NaBH4 reduction. It can be seen that the antibody recognizes reduced HNE-Lys Michael adducts (B) nearly as well as unreduced HNE-Lys Michael adducts (C) but that it also recognizes the reduced ONE-Lys Schiff base adduct (D and E) (which is the same as the reduced HNE-Lys Schiff base, but the latter is insignificantly formed on protein, as discussed above). One possibility for the cross-reactivity is that the principal antigenic determinant of antibody recognition is the C5H11CH(OH)- fragment common to

Lin et al.

Figure 7. Anti-HNE antibody recognition of HNE- and ONEmodified pEKY. pEKY (2 mg/mL) was exposed to 2 mM HNE in pH 7.4 phosphate buffer for 24 h at 37 °C with (B) or without (C) subsequent treatment with NaBH4 or to 2 mM ONE at 23 °C for 15 min in pH 7.4 phosphate buffer (D) or for 6 min in pH 8.5 borate buffer (E) with subsequent NaBH4 quenching. After workup with NH4Cl, these preparations and the control protein (A) were applied to the PVDF membrane. Modifications were detected by the anti-HNE antibody (top panel). The bottom panel shows quantitation of the oxidized luminal ECL reagent in the dots by image acquisition for three experiments.

Scheme 2

all of these structures (Scheme 2), although there are clearly multiple immunogenic proteins in the polyclonal antiserum that could have different or overlapping recognition features. We next compared immunoreactivity to reduced HNEand ONE-treated pEKY, using the anti-HNE antibody with and without competitive absorption by the reduced Michael adduct or reduced Schiff base of 2-methoxyethylamine (MEANAD and MEANEO, see Scheme 1; the NMR spectra of MEANAD indicated that it was a single diastereomer). As shown in Figure 8, whereas antibody binding to both reduced HNE- and ONE-treated pEKY preparations is nearly completely inhibited by the reduced Schiff base MEANEO, reduced Michael adduct MEANAD inhibits antibody binding only to the reduced HNE-treated pEKY. The finding that MEANAD does not inhibit antibody binding to the ONE-treated pEKY preparation is consistent with this preparation containing nearly entirely the reduced ONE Schiff base, in turn suggesting that ONE modification of protein Lys residues is dominated by formation of Schiff base rather than Michael adducts. Neurotoxicity. The relative cytotoxicity of HNE and ONE was evaluated in two human neuroblastoma cell



Figure 8. Absorption of anti-HNE antibody recognition of HNE- and ONE-modified pEKY. pEKY (2 mg/mL) was exposed to 2 mM HNE in pH 7.4 phosphate buffer for 24 h at 37 °C or 2 mM ONE in pH 8.5 borate buffer for 6 min at 23 °C. The preparations were then treated with NaBH4 and then NH4Cl. The reduced HNE, HNE(R), and ONE, ONE(R), preparations and the control protein were then applied to the PVDF membrane. Modifications were detected (left panel) by the anti-HNE antibody (A) or after preabsorption with MEANAD (B) or MEANEO (C). The right panel shows quantitation of the oxidized luminal ECL reagent in the dots by image acquisition for three experiments.

Figure 9. Effect of 4 h treatment with HNE and ONE (concentrations of 0-500 µM) on the viability of human BE(2)M17 cells as determined using the trypan blue assay.

Figure 10. Viability of human BE(2)-M17 cells (trypan blue assay) following exposure to 50 µM HNE and ONE for 1, 2, or 4 h.

lines. We first evaluated acute toxicity over a 4 h period in M17 cells. In can be seen (Figure 9) that there is minimal acute toxicity of either aldehyde at less than 3 µM, whereas at a concentration of 0.5 mM, less than 10% of the cells remained viable in either case. On the other hand, at midconcentration ranges, there was significant discrimination showing a greater toxicity of ONE, especially at the 10 µM concentration. In addition, in a separate experiment, at a somewhat higher concentration (50 µM), where ONE is essentially completely lethal, the time course of cell death (Figure 10) shows that most of the cytotoxicity of ONE is already expressed after 1 h, whereas for the more weakly toxic HNE, there is a progressive loss of cell viability over the 4 h period. We

Figure 11. Effect of overnight treatment with HNE and ONE (1-10 µM) on the viability of SH-SY5Y cells, as assessed by the LDH assay.

also used SH-SY5Y cells to evaluate the longer term toxicity (24 h) of HNE and ONE, using the LDH assay (Figure 11). Again, whereas at the low concentration of 1 µM and the higher concentration of 10 µM there was either very little or nearly complete cell killing for both aldehydes, at the intermediate concentration of 3 µM, there was a 4-5-fold greater toxicity of ONE over HNE. The 24 h cytotoxicity of ONE and HNE at different concentrations (1-10 µM) was also observed by microscopy (Figure 12). At 3 µM, HNE-treated cells did not show any abnormality in morphology, consistent with the LDH assay data. However, the same concentration of ONEtreated cells exhibited the typical morphologies of cell death (i.e., rounding up and fragmentation) and the number of those cells was increased in a concentrationdependent fashion. At 10 µM concentration, both HNE and ONE treatment caused a similar level of cell death, consistent with the LDH assay data.

Discussion The central nervous system is particularly vulnerable to oxidative stress on account of the high rate of oxygen utilization and high content of unsaturated lipids. Thus, there has been much focus on possible roles of lipoxidation-derived aldehydes in contributing to neuronal dysfunction in neurodegenerative diseases associated with oxidative stress. Among such aldehydes, HNE has become the most studied cytotoxic product of lipid peroxidation (66) and appears to be a major signaling molecule

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Figure 12. Cytotoxicity in SH-SY5Y cells exposed to HNE or ONE. Cells were plated at a density of 3 × 104 cells/well and cultured overnight. The cell culture medium was then switched to serum-free Opti-MEM and treated with different concentrations of HNE or ONE for 24 h.

in the pathogenesis of neurodegenerative disease (e.g., it may mediate Aβ toxicity). Whereas modest concentrations of HNE can induce apoptosis (57, 58), induce differentiation, impair proteasomal function (52-56), and alter signal transduction, including activation of adenylate cyclase, JNK, PKC, and caspase 3, lower concentrations of HNE appear to promote cell proliferation (65, 66). As there has been no evidence to suggest that HNE can exert biological activity through a noncovalent process, one must presume that the various deleterious biological activities ascribed to HNE reflect its ability to bind covalently to protein targets, such as in signaling cascades. It will be important to verify the nature of HNE covalent binding that results in modulating the enzymatic activity of, e.g., various protein kinases (86). Recently, the two-electron oxidized cousin of HNE, ONE, has come under scrutiny with the discovery that it is a product of lipid oxidation in its own right (10, 11) and has been detected in human plasma (12). ONE is more reactive than HNE in modifying and cross-linking proteins (67-69) and thus might be expected to exert similar (or perhaps more pronounced) biological activities as does HNE. In a recent study, ONE and HNE were found to execute a similar death response in the human colorectal carcinoma cell line RKO at roughly comparable doses over the same time frame (77). The apoptotic response was shown to involve activation of caspases, proteolysis of the downstream caspase targets PARP, DFF45, and DFF35, and nucleosomal DNA fragmentation. HNE-induced apoptosis in this cell line had previously been shown to involve cytochrome c release from mitochondria and activation of caspase-9 and caspase-3. Although some of the indices of apoptosis in RKO cells suggested that ONE was about 2-fold more toxic than HNE (77), these workers did not call attention to this relatively small difference as compared to expectations of a much greater divergence based on findings that ONE displays 10-100-fold greater Michael adduct reactivity in simple chemical reactions (68, 69). It was suggested that metabolism of ONE by detoxification enzyme systems in the RKO cells (and presumably in vivo) would have a “leveling effect” on toxicity.

In the current study using two human neuroblastoma cell lines, we show that ONE is about 4-5 times more toxic than HNE in a concentration range near the threshold of lethality. We have not investigated the mechanisms of toxicity, but on the basis of studies of other workers described above, we presume that the toxicity that manifests over a 24 h exposure to low concentrations of the aldehydes reflects principally apoptosis. The acute toxicity seen at 4 h with somewhat higher concentrations may additionally involve a necrotic action of the aldehydes. The expected complexity in the reactions of HNE and ONE with protein side chains led us to reinvestigate the kinetics of reactions with individual side chain nucleophiles and to attempt to correlate these data with observations made on reactions with model proteins. The most profound new finding is that whereas the initial chemistry of HNE is dominated by Michael adduction chemistry, ONE very rapidly forms observable Schiff bases on proteins that are reversible but which would be maintained at equilibrium at concentrations of ONE (we used 50 µM in our kinetics studies) that are not unreasonable to be found in tissues exposed to oxidative stress. ONE is believed to be produced at similar levels as HNE (10), and some workers have suggested that HNE can reach millimolar levels in certain tissues. In addition to our demonstrating the distinctive tendency of ONE (vs HNE) to form Schiff bases, we have here also kinetically defined the reversibility of HNE amine Michael adducts. In this regard, it is of interest to point out the possible effect that such reversible modifications can have in regulating protein interactions or in responding to other dynamic posttranslational modifications of proteins. For example, there is evidence to suggest that HNE Michael adduction to Lys residues in the KSP repeats of the tails of the medium and especially heavy subunit of neurofilaments is a dynamic process in normal axons that occurs throughout life to an extent that is governed by the extent of serine phosphorylation in these KSP repeats (87). Ignoring the rapid equilibrium Schiff base chemistry of ONE, ONE still appears to react with protein nucleophiles through conjugate addition at a rate 6-31 times


faster than does HNE (Table 1). However, the most surprising finding of the current study is that the greater conjugate addition reactivity of ONE resides almost entirely in the greater rate of reaction with His and Cys, since Michael addition of Lys/amine to ONE is actually slower than to HNE. It is clear that conjugate addition by Lys residues is not a dominant component of the reaction with HNE or ONE for even the Lys-rich proteins β-LG and RNase. For HNE, reaction with Lys residues does at least contribute somewhat to the overall reaction profile, because reductive methylation of Lys results in a 2-4-fold slowing of the reaction (Table 1). On the other hand, the rate of reaction with ONE is only slightly slowed (β-LG) or not at all (RNase) by Lys reductive methylation. Further studies will be needed to understand the remarkable distinction between HNE and ONE in terms of their reactivity toward conjugate addition of amine vs imidazole and thiol nucleophiles. In conclusion, the biological activity of electrophilic aldehyde products of lipid peroxidation can arise from a combination of noncovalent and covalent chemical processes. Although there has been substantial focus on biological endpoints that arise from irreversible covalent modification of proteins, we would like to point out that reversible covalent modifications should not be excluded as potential culprits of deleterious biological effects, even though these modifications are unlikely to survive the mass spectroscopic analyses that have become so widespread today. The fraction of protein side chain nucleophiles that must be modified to rationalize any particular biological effect may be quite low, in which case reversible modifications that are defined by sizable equilibrium constants cannot not be discounted. Although there need be no necessary correlation between reactivity indices and toxicity, because of the many biochemical steps between protein modification and ultimate cell death, it is tempting to consider that the greater neurotoxicity of ONE than HNE reported here, although modest, could reflect in part the greater Cys and His reactivity of ONE or the different nature of protein Lys modification by these two similar aldehydes.

Acknowledgment. This work was supported by Grants HL 53315 (to L.M.S.) and AG 14249 (to L.M.S. and G.P.) from the National Institutes of Health. We thank Professor John Stuehr for guidance with the kinetics manipulations. Supporting Information Available: Plots of kobs vs [NRAc-L-Cys] for the reactions with HNE and ONE; 1H and 13C NMR spectra for (E)-1-(butylamino)-2-nonen-4-ol, 3-(2-methoxyethylamino)nonane-1,4-diol, and (E)-1-(2-methoxyethylamino)2-nonen-4-ol; and 13C NMR spectrum for Schiff base formation between ONE and butylamine. This material is available free of charge via the Internet at http://pubs.acs.org.

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4-Oxo-2-nonenal Is Both More Neurotoxic and More Protein Reactive

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