Synthesis and Cellular Effects of an Intracellularly Activated Analogue

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Chem. Res. Toxicol. 2002, 15, 40-47

Synthesis and Cellular Effects of an Intracellularly Activated Analogue of 4-Hydroxynonenal M. Diana Neely,* Venkataraman Amarnath, Carl Weitlauf, and Thomas J. Montine Department of Pathology and the Centers for Molecular Neuroscience and Molecular Toxicology, Vanderbilt University Medical School, MCN U4216, 21st Avenue South, Nashville, Tennessee 37232-2561 Received July 16, 2001

4-Hydroxy-2-nonenal (HNE) has been recognized as reactive product of lipid peroxidation and has been suggested to play a role in the pathogenesis in several common diseases as well as injuries caused by environmental toxicants. Although formed intracellularly in vivo, for practical reasons this molecule is applied extracellularly in order to analyze its biological effects. The focus of this study was to develop an approach that would enable intracellular HNE production in the absence of the many other products and processes that occur in cells experiencing generalized oxidative stress. To this end, we synthesized 1,1,4-tris(acetyloxy)2(E)-nonene (HNE[Ac]3), a triester analogue of HNE that is itself unreactive but could be hydrolyzed intracellularly presumably by lipases and/or esterases into the highly reactive HNE. In vitro lipase rapidly converted HNE(Ac)3 initially to 4-acetyloxy-2-nonenal (HNE[Ac]1) and then to HNE. Neuro 2A cell lysate also caused a rapid hydrolysis of HNE(Ac)3 into HNE(Ac)1 and HNE. Incubation of BSA with HNE(Ac)3 resulted in protein-adduct formation only in the presence of lipase. We demonstrated adduction of HNE to proteins in Neuro 2A cells exposed to HNE(Ac)3 by immunoblotting and immunocytochemistry using antibodies specific for HNEMichael adducts on proteins. We have previously shown that microtubule organization is very sensitive to HNE. Analysis of Neuro 2A cell microtubules showed that this cytoplasmic organelle is similarly sensitive to HNE and HNE(Ac)3.

Introduction There is increasing evidence that oxidative stress, and lipid peroxidation in particular, contributes to disease states as diverse as atherosclerosis, neurodegenerative diseases, and to injuries resulting from environmental toxicants (1-6). Reactive aldehydes generated during the process of lipid peroxidation are believed to play important roles in mediating the pathophysiological effects of oxidative stress (7-16). 4-Hydroxy-2-nonenal (HNE)1 is one of the main aldehydes formed during lipid peroxidation and has received considerable attention due to its bioactivity. This R,β-unsaturated aldehyde is a strong electrophile which readily forms adducts with cellular nucleophiles such as glutathione, cysteine, lysine, and histidine of proteins, and also with nucleic acids (17, 18). Analysis of HNE modified proteins in cells exposed to HNE or experiencing oxidative stress has not revealed a consistent major cellular target, but rather suggests an abundance of targets the identities of which likely vary with cell type and type of oxidative insult. Microsomal, mitochondrial, plasma membrane, and cytoplasmic targets have been identified (17). High concentrations of HNE are cytotoxic, whereas at lower concentrations, this aldehyde can cause disruption of cytoskeletal integrity, * To whom correspondence should be addressed. Phone: (615) 3223699. Fax: (615) 343-9825. E-mail: [email protected]. 1 Abbreviations: BSA, bovine serum albumin; FBS, fetal bovine serum; HNE, 4-hydroxy-2-nonenal; HNE(Ac)1, 4-acetyloxy-2-nonenal; HNE(Ac)3, 1,1,4-tris(acetyloxy)-2(E)-nonene; PBS, phosphate buffered saline.

impairment of mitochondrial respiration, inhibition of DNA-, RNA-, and protein synthesis, stimulation of neutrophil chemotaxis, modulation of platelet aggregation and changes in responses of second messenger pathways (17, 19, 20). To study the effects of HNE, cells or organelles can be subjected to oxidative insults and the formation of HNE correlated with change in a particular activity; alternatively, exogenous HNE can be applied to cells or organelles and changes in cellular functions or enzyme activities measured. Esterbauer and colleagues have compared the inhibition of glucose-6-phosphatase by endogenous HNE formed in microsomes subjected to oxidative insults and by HNE externally applied to microsomes. They measured an ID50 of 1.5 µM HNE in oxidatively stressed microsomes; however, in microsomes exposed to exogenous HNE, the ID50 for glucose-6phosphatase inhibition was 100-200-fold higher (150300 µM) (21). One interpretation of these experiments is that endogenously generated HNE and exogenously applied HNE may have access to different targets. Several studies have reported that HNE is unequally distributed between the lipid and aqueous phase (22, 23) and HNE concentrations in the millimolar range have been measured in biological membranes exposed to oxidative stress (21, 24). This suggests that the majority of endogenously formed HNE remains with intracellular membranes and encounters intracellular targets before it diffuses out of the cell and has access to targets on the cell surface. Targets of exogenously applied HNE are

10.1021/tx010115w CCC: $22.00 © 2002 American Chemical Society Published on Web 12/15/2001

An Intracellularly Activated HNE Analogue

likely different and probably involve extracellular domains of proteins and lipids in the plasma membrane to a greater extent. This suggests that the effects of endogenously formed HNE may not always be accurately mimicked in experiments with externally added HNE. To develop a tool that would allow us to compare cellular effects of exogenously applied and endogenously formed HNE, we synthesized 1,1,4-tris(acetyloxy)-2(E)nonene (HNE[Ac]3). This triester analogue of HNE is itself not reactive, but once it diffuses into cells it is hydrolyzed to the reactive HNE and its analogue, 4-acetyloxy-2-nonenal (HNE[Ac]1). This molecule therefore, allowed us to compare the effects of intracellularly formed and exogenously applied HNE on a cytoplasmic organelle, the microtubule network.

Materials and Methods Materials. Chemicals required for the synthesis of 4-hydroxy2(E)-nonenal (HNE) and 1,1,4-tris(acetyloxy)-2(E)-nonene (HNE[Ac]3) were from Aldrich (Milwaukee, WI). Materials used for cell culture were from Life Technologies (Grand Island, NY). Reagents for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were obtained from Bio-Rad (Hercules, CA). All other chemicals were from Sigma (St. Louis, MO), unless otherwise indicated. Synthesis of 4-Hydroxy-2(E)-nonenal (HNE). Unlabeled HNE was synthesized using Gardner’s procedure (25). To synthesize 4-[3H]HNE, we prepared 1,1-dimethoxynon2-en-4one as described (26) and purified it by column chromatography (4:1 hexanes-ethyl acetate). The above ketone (100 mg, 0.5 mmol) was reduced with NaB3H4 (26) (2 mCi/mg, American Radiolabeled Chemicals, Inc., St. Louis, MO), hydrolyzed with 2.5% H2SO4 in tetrahydrofuran, and purified by column chromatography (silica, 2:1 hexanes-ethyl acetate). The specific activity was 15.5 µCi/µM. Synthesis of 1,1,4-Tris(acetyloxy)-2(E)-nonene (HNE[A]3). Although extensive work has been done on protecting aldehydes as acetals, only a handful of reports are available for converting an aldehyde group to 1,1-diacetate (27-30), and they all involve treatment with acetic anhydride in the presence of Lewis acids. When we added HNE to a solution of acetic anhydride and BF3-O(C2H5)2 at room temperature (27), we were unable to isolate a distinct product from the dark mixture. The hydroxyl group at the 4-position likely was oxidized, and the resulting 4-oxononenal underwent further reactions. This was avoided when the initial mixing was done in ether at low temperature to form the 4-acetyloxy-2-nonenal that was converted to 1,1-diacetate on further reaction. Therefore, acetic anhydride (0.36 mL, 3.6 mmol) and BF3OEt2 (30 µL) were stirred in dry ether (5 mL) and cooled to 40 °C in dry ice-2-propanol bath. HNE (160 µL, 1 mmol) was added and the resulting solution was warmed to room temperature. The reaction mixture was heated to reflux in an oil bath kept at 50 °C. After 2 h, we mixed the reaction solution with 10 mL of 1 M phosphate buffer (pH 8.0), separated the layers, and extracted the aqueous layer with ether (2 × 10 mL). The combined ether layers were dried, concentrated, and purified by column chromatography (silica: hexane, 3:1 hexanes-ethyl acetate); yield 120 mg (40%); 1H NMR δ 7.10 (d, 1 H, J ) 6.0 Hz, C1-H), 5.92 (dd, 1 H, J ) 15. 7 and 6.0 Hz, C3-H), 5.68 (dd, 1 H, J ) 15.7 and 6.0 Hz, C2-H), 5.26 (dt, 1 H, J ) 6.2, C4-H), 2.05 (s, 9 H, CH3CdO), 1.6-0.8 (11 H, alkyl chain); 13C NMR δ 170.2 (C4OCdO), 168.6 (C1OC)O), 135.0 (C3), 124.6 (C2), 88.6 (C1), 72.6 (C4), 33.9 (C5), 31.4 (C7), 24.5 (C6), 22.4 (C8), 21.1 and 20.8 (CH3CdO), 13.9 (C9); GC-MS m/z 300 (1, M+), 241 (2, M CH3CO2), 198 (25, 241 - CH3CO), 169 (50, 198 - CHO), 156 and 155 (100, HNE). The conversion of 4-[3H]HNE to 4-[3H]HNE(Ac)3 paralleled the above preparation on a smaller scale. The purification of the crude product was carried out on a small (10 × 2 cm) column of silica (3:1 hexanes-ethyl acetate). We

Chem. Res. Toxicol., Vol. 15, No. 1, 2002 41 identified the fractions containing the pure triester by gas chromatography, then combined and evaporated them to obtain 4-[3H]HNE(Ac)3. The specific activity of 4-[3H]HNE(Ac)3 was 12.3 µCi/µM. HNE(Ac)3 Hydrolysis. We analyzed spontaneous and lipaseinduced hydrolysis of HNE(Ac)3. HNE(Ac)3 (28.6 mM) in phosphate buffer (20 mM) was incubated in the absence or presence of 2.9 mg/mL lipase (isolated from Pseudomonas fluorescens, 3414 units/mg, Fluka, NY, no. 62321) at 37 °C and 1 µL aliquots were removed from the reaction mixture every 15 min for analysis by gas-liquid chromatography performed with a Hewlett-Packard 5890 Series II GC-FID equipped with a HPINNOWax capillary column (15 m × 0.53 mm) (HewlettPackard, Wilmington, DE). The carrier gas flow (helium) was 8 mL/min and the temperature was programmed to hold at 80 °C for 2 min and then increase to 200 °C at 20 °C/min and then hold at 200 °C. Acetic acid (CH3COOH), 4-acetyloxy-2-nonenal (HNE[Ac]1), HNE, and HNE(Ac)3 eluted after 3.4, 6.7, 7.9, and 9.1 min, respectively (see Figures 1 and 2). To analyze HNE(Ac)3 hydrolysis by cell lysate, we cultured Neuro 2A cells exactly as described below, then washed them twice with PBS, and harvested and sonicated (2 times for 20 s at 20 W) the cells of two T-25 flasks in 1.2 mL of PBS. Five microliters of a 100 mM HNE(Ac)3 stock solution were added to 400 µL of this cell lysate (1.4-2.0 mg/mL protein) and incubated at 37 °C. We removed 70 µL samples at different times and extracted them with 100 µL of ethyl acetate. The extracts were analyzed by gas-liquid chromatography as described above. In Vitro Reactivity of HNE(Ac)3. We examined the in vitro reactivity of HNE(Ac)3 by analyzing the modification of bovine serum albumin (BSA) (fraction V, Sigma, St. Louis, MO) after incubation with HNE(Ac)3 or HNE in the presence and absence of lipase by immunoblotting and autoradiography. For analysis by immunoblotting BSA (1.6 mg/mL) in 20 mM phosphate buffer was incubated with HNE(Ac)3 or HNE both at 4.5 mM for 2 h at 37 °C in the presence or absence of lipase (3 mg/mL). For analysis by autoradiography BSA was incubated with [3H]HNE(Ac)3 at 4.5 mM or [3H]HNE at 3.6 mM. We stopped the reactions by adding Laemmli sample buffer and subjected the samples to SDS-PAGE (31). Immunoblotting was performed as described below using an antibody specific for HNE-Michael protein adducts (Calbiochem, CA, no. 393205). For autoradiography, we treated the SDS-PAGE gels with an autoradiography enhancer (New England Nuclear, Massachusetts, no. NEF 992) according to the manufacturer’s instructions, dried them and exposed them to film (Kodak X-OMAT AR, Sigma, Missouri) for 3 days at - 80 °C. Cell Culture. Neuro 2A neuroblastoma cells (American Type Culture Collection, Rockville, MD) were grown in growth medium (Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (1:1) (DMEM/F12) with 10% (v/v) FBS and penicillinstreptomycin at 100 units/mL and 100 µg/mL respectively. Two days before the experiment, we subcultured the cells in growth medium and left them to adhere overnight. For immunocytochemical analysis Neuro 2A cells were subcultured at 5 × 104 cells/mL in the wells of 35 mm glass bottom microwell dishes (MatTek Corp., MA) and for analysis of HNE(Ac)3 hydrolysis or protein modification in T-25 culture flasks at 20 × 104 cells/ mL. The next morning, we removed the serum containing growth medium and replaced it with N2-medium (DMEM/F12 medium containing penicillin-streptomycin and N-2 supplement [Life Technologies, Grand Island, NY]). After 24 h culturing in N2-medium, we exposed the cells to HNE or HNE(Ac)3 for 1 h at 37 °C and 5% CO2. Distribution of Radioactivity into Protein and Lipid Fraction of Neuro 2A Cells Exposed to [3H]HNE or [3H]HNE(Ac)3. Cellular protein and lipid were extracted from Neuro 2A cells exposed to 250 µM [3H]HNE or [3H]HNE(Ac)3 for 1 h. After exposure, the cells were pelleted and washed two times with Hank’s balanced salt solution. We dissolved the final cell pellet in 2 mL of chloroform:methanol (2:1, vol:vol) and incubated it at 4 °C for 1 h with occasional vortexing. Then, we

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added 4 mL of ice-cold ether to the solution and, after rigorous vortexing, removed the upper (ether) phase and stored it on ice. The proteins in the chloroform/methanol phase were pelleted and re-extracted with 2 mL of ice cold ether. We combined the two ether fractions, evaporated the ether, and redissolved the lipids in hexane. Protein pellets were dissolved in Laemmli sample buffer, and radioactivity associated with lipids and proteins was determined using a Packard 1900CA liquid scintillation counter (Packard, Meriden, CA). Immunoblot Analysis and Autoradiography. After exposure to HNE or HNE(Ac)3 Neuro 2A cells were scraped from the culture flask with a rubber policeman, pelleted, and solubilized in Laemmli sample buffer. The proteins were then separated by SDS-PAGE (31) and transferred to a PVDF membrane (Immobilon-P, Millipore, Bedford, MA), and the membrane was incubated with “blotto” (4% dry milk in trisbuffered saline with 0.05% Tween 20) for at least 2 h at room temperature to block unspecific antibody binding sites. We then immunoreacted the proteins with anti-HNE Michael adducts antibody (Calbiochem, San Diego, CA, no. 393205) diluted 4000fold in “blotto” overnight at 4 °C. After incubation with an HRPconjugated anti-rabbit IgG (Sigma, St. Louis, MO, no. A0545) diluted 1:2000 in “blotto” for 2 h at room temperature, we visualized the signal using a chemiluminescence reagent (NEN, Boston, MA, no. NEL100). For autoradiography the SDS-PAGE gels were treated with an autoradiography enhancer (New England Nuclear, MA, no. NEF 992) according to the manufacturer’s instructions, dried, and then exposed to film (Kodak X-OMAT AR, Sigma, MO) for 3 weeks at - 80 °C. The films from immunoblots and autoradiography were scanned with a GS710 densitometer (Bio-Rad, Hercules, CA) and the signals quantified using Quantity One Quantitation Software (Bio-Rad, Hercules, CA). Immunocytochemistry. After HNE or HNE(Ac)3 exposure Neuro 2A cells were immediately fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 30 min at room temperature and then permeabilized with 1% Triton X-100 in PBS containing 2% FBS for 20 min at room temperature. Microtubules were visualized with an anti-β-tubulin antibody (Boehringer Mannheim, Indianapolis, IN, no. 1111876) at 0.25 µg/ mL in PBS with 2% FBS overnight at 4 °C, followed by a FITCcoupled anti-mouse IgG (Cappel, West Chester, PA, no. 55514) at 3 µg/mL in PBS with 2% FBS for 1-2 h at room temperature. To visualize HNE-modified proteins antibody no. 672 (specific for HNE-protein adducts) (32) diluted 1:500 in PBS with 2% FBS or anti-HNE Michael adducts antibody (Calbiochem, San Diego, CA, no. 393205) diluted 1:200 in PBS with 2% FBS was applied overnight at 4 °C. The secondary antibody, a FITCcoupled affinity-purified goat anti-rabbit antibody (Cappel, West Chester, PA no. 55662), was diluted 1:100 (final concentration of 7.3 µg/mL active antibodies) in PBS with 2% FBS, and the cells were incubated for 1.5 h at room temperature. We carefully removed the glass slides with the cells from the 35 mm dishes with a razor blade and mounted them in ProLong mounting medium (Molecular Probes, Eugene, OR, no. P-7481). Immunofluorescence was analyzed with a Zeiss Axiovert 135 microscope (Carl Zeiss, INC., Thornwood, NY) using a Plan-Apochromat 100×/1.4 objective. For semiquantitative analysis of the microtubule organization, microscope slides were scanned along a fixed y-coordinate using an electronically controlled microscope stage and the microtubule organization of 200-500 cells/slide were categorized as either normal, mildly disrupted, or severely disrupted. The analysis was done blinded to exposure conditions. ANOVA, followed by repeated t-tests with Bonferroni correction for multiple comparisons were used to compare the effects of HNE and HNE(Ac)3. We acquired confocal images of cellular fluorescence using a confocal laser-scanning microscope (Zeiss LSM 410) equipped with a Plan-Apochromat 63×/1.4 objective.

Results Hydrolysis of HNE(Ac)3 by Lipase and Neuro 2A Cell Lysate. To analyze lipase-induced hydrolysis, we

Neely et al.

Figure 1. Synthesis and hydrolysis of HNE(Ac)3. Chemical structures of HNE, and HNE(Ac)3 and HNE(Ac)1, the triester and monoester analogues of HNE are shown. HNE(Ac)3 was synthesized by incubation of HNE with acetic anhydride in the presence of an acid catalyst. Unlike its parent compound, this triester analogue lacks an aldehyde group and therefore does not share the electrophilicity of HNE. The initial product of the lipase-catalyzed hydrolysis of HNE(Ac)3 is the monoester HNE(Ac)1, which is then further cleaved into HNE.

incubated HNE(Ac)3 in phosphate buffer in the absence or presence of lipase and followed hydrolysis by gas chromatography. We did not observe any hydrolysis of HNE(Ac)3 in the absence of lipase during 2 h incubation (Figure 2A). In the presence of lipase, HNE(Ac)3 was initially hydrolyzed to 4-acetyloxy-2-nonenal (HNE[Ac]1), the monoester of HNE, which was then further hydrolyzed to HNE (Figure 2B, see also Figure 1). After 3 min of incubation, 11% of HNE(Ac)3 was converted into HNE(Ac)1, after 15 min 100% of HNE(Ac)3 was hydrolyzed into HNE(Ac)1 (57%) and HNE (43%). After 60 min incubation, the reaction mixture contained HNE(Ac)1 (28%) and HNE (72%). We observed only little additional conversion of HNE(Ac)1 into HNE after the initial hour of incubation (not shown). The results shown are representative of several experiments performed. These observations show that HNE(Ac)3 does not undergo spontaneous hydrolysis within the time frame relevant for this study and that lipase hydrolyzes HNE(Ac)3 initially into HNE(Ac)1 and then into HNE (Figure 1). The hydrolysis of HNE(Ac)3 by Neuro 2A cell lysates was analyzed in the same way. We added HNE(Ac)3 to Neuro 2A cell lysates in molar ratios [moles of HNE(Ac)3 per mg of cell protein] comparable to the ones used in the experiments described below (equivalent to 100 µM) and followed its hydrolysis by gas chromatography (Figure 3). Hydrolysis of HNE(Ac)3 was rapid, such that 5.6% of HNE(Ac)3 was converted to HNE(Ac)1 upon adding HNE(Ac)3 to the cell lysate. After 30 min, 88% of HNE(Ac)3 was hydrolyzed into HNE(Ac)1 (86%) and HNE (2%). After 60 min, the hydrolysis of HNE(Ac)3 was complete with the conversion of 94.4% of HNE(Ac)3 into HNE(Ac)1 (90%) and HNE (4.4%). Within the next 60 min, a small fraction of HNE(Ac)1 (2.8%) was hydrolyzed to HNE, resulting in a total yield of 7.2% HNE. The sum of the peak areas on the gas chromatography chromatograms [HNE(Ac)3, HNE(Ac)1, and HNE] stayed constant, implicating that the amount of HNE(Ac)1 or HNE lost through metabolism or adduction to cellular components was too low to be detected with this method. Therefore, Neuro 2A cells convert HNE(Ac)3 largely into HNE(Ac)1, which differs from HNE only by having an acetyloxyinstead of a hydroxyl-group on the C4 position (see Figure 1), and therefore shares the electrophilic character of

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Figure 3. HNE(Ac)3 hydrolysis by Neuro 2A cell lysates. HNE(Ac)3 was added to Neuro 2A cell lysate [600-800 nmol of HNE(Ac)3/mg of cell protein] and incubated for 2 h. Aliquots were removed at different times, extracted, and analyzed by gas chromatography. The amount of HNE(Ac)3, HNE(Ac)1, and HNE are plotted as a percentage of the total peak area (area under peaks of all three compounds). The total peak area did not change significantly over the time course of the experiment. Values are means ( SEM; n ) 8 (some of the SEM-bars are hidden by the symbols).

Figure 2. Lipase-catalyzed hydrolysis of HNE(Ac)3. HNE(Ac)3 in 20 mM phosphate buffer was incubated at 37 °C in the absence (A) or presence (B) of lipase and hydrolysis followed by gas chromatography. No hydrolysis was observed during 2 h of incubation in the absence of lipase (A). In the presence of lipase HNE(Ac)3 was quickly converted first to HNE(Ac)1 and then to HNE.

HNE. When the ratio of HNE(Ac)3/mg of protein was increased 2 or 4-fold, the hydrolysis of HNE(Ac)3 was incomplete, such that within a 2 h time period only 60 or 27% of HNE(Ac)3 was hydrolyzed, respectively. Cell lysates which had been boiled for 7 min before the assay did not hydrolyze HNE(Ac)3 (data not shown). In Vitro Modification of Protein by HNE(Ac)3 Depends on the Presence of Lipase. In vitro reactivity of HNE(Ac)3 was tested and compared to HNE. To mimic exposure conditions used in cell culture experiments described below, HNE(Ac)3 and HNE were incubated with BSA in cell culture medium (N2 medium) for 1-2 h at 37 °C in the presence or absence of lipase. Our immunoblot analysis using an antibody specific for HNE-protein adduct demonstrates that, while incubation of BSA (1.6 mg/mL) with HNE (4.5 mM) resulted in adduction of this aldehyde to BSA regardless of the presence of lipase, no BSA adducts were observed in samples incubated with HNE(Ac)3 (4.5 mM) in the absence of lipase. When lipase was present, HNE-BSA adducts were detectable (Figure 4A). This shows that HNE(Ac)3 does not adduct to protein, is not spontaneously hydrolyzed in solution to HNE, and that the formation of HNE-BSA adducts depends on the hydroly-

Figure 4. In vitro protein modification by HNE(Ac)3 is lipase dependent. HNE adduction to BSA incubated with HNE (lanes 1 and 2) or HNE(Ac)3 (lanes 3 and 4) in the presence (+) or absence (-) of lipase was analyzed. (A) BSA was incubated with HNE or HNE(Ac)3 and HNE adduction to BSA analyzed by immunoblotting using an antibody specific for HNE-protein adducts. (B) BSA was incubated with [3H]HNE or [3H]HNE(Ac)3 and protein modification analyzed by 3H-autoradiography.

sis of HNE(Ac)3 by lipase. To detect protein adducts not recognized by the anti-HNE antibody, we incubated BSA with [3H]HNE(Ac)3 and [3H]HNE and analyzed BSA adduction by autoradiography. While HNE-adduction to BSA was clearly detectable in the absence or presence of lipase, binding of radioactivity to BSA incubated with [3H]HNE(Ac)3 was dependent on lipase (Figure 4B). Distribution of HNE(Ac)3 and Its Products into the Cellular Lipid and Protein Pools. We next compared the distribution of radioactivity from [3H]HNE and [3H]HNE(Ac)3 into cellular protein and lipid fractions

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Figure 5. Comparison of HNE modified proteins in Neuro 2A cells exposed to HNE and HNE(Ac)3. (A) Neuro 2A cells were exposed to vehicle only (lane 1), 25 µM HNE (lane 2), 250 µM HNE(Ac)3 (lane 3), or 500 µM HNE(Ac)3 (lane 4) for 1 h. Protein modification of the cell lysates was analyzed by immunoblotting using an anti-HNE-protein adduct antibody as described in the materials and methods section. Molecular weight markers (kDa) are shown on the left. Arrowheads indicate two proteins that were modified by HNE, but not detectably modified by HNE(Ac)3. A protein modified preferentially in cells exposed to HNE(Ac)3 is indicated by an arrow. No protein modification was observed in cells exposed to vehicle only (lane 1). Three and a half times as much cell lysate was applied from cells exposed to HNE(Ac)3 (lanes 3 and 4) than HNE treated cells (lane 2). (B) Neuro 2A cells were exposed to [3H]HNE (50 µM, lane 1; 100 µM lane 2) or [3H]HNE(Ac)3 (250 µM, lane 3; 500 µM, lane 4) for 1 h and protein modifications analyzed by autoradiography. Molecular weight markers (kDa) are shown on the left. Equal amounts of protein were applied to all lanes.

[3H]HNE,

of Neuro 2A cells. After 1 h exposure to 250 µM 2.4 ( 0.5% of the total radioactivity applied to the cultures was associated with cells. Of this cell-associated radioactivity 93.5 ( 0.8% (2.2% of total label) remained with the protein fraction, while 6.5 ( 0.8% (0.2% of total label) was associated with the lipid fraction. In cells incubated with 250 µM [3H]HNE(Ac)3 for 1 h, 1.7 ( 0.3% of the added radioactivity was associated with the cells of which 79.9 ( 2.1% (1.4% of total label) was partitioned into the protein fraction and 20.1 ( 2.1% (0.3% of total label) into the lipid fraction. Thus, the total amount of cell-associated label was not significantly different after [3H]HNE or [3H]HNE(Ac)3 treatment. However, a statistically significantly larger fraction of the radioactive label was associated with the proteins in cells incubated with [3H]HNE than in cells exposed to [3H]HNE(Ac)3 (p e 0.001). HNE(Ac)3 Forms HNE-Protein Adducts in Neuro 2A Cells. Exposure of Neuro 2A cells to HNE(Ac)3 resulted in protein modifications detectable with antibodies specific for HNE-protein adducts (Figure 5A). This confirms intracellular conversion of HNE(Ac)3 to HNE. Although the patterns of HNE-adducted proteins in cells exposed to HNE and HNE(Ac)3 were similar, they were not identical. Two proteins, one with MW > 212 kDa (Figure 5A, single arrowhead) and another with a 43.8 kDa < MW < 86 kDa (Figure 5A, double arrowhead) were adducted by HNE, but not detectably modified by HNE(Ac)3. Another protein with MW < 43.8 kDa (Figure 5A,

Figure 6. Subcellular localization of HNE-protein adducts in Neuro 2A cells exposed to HNE and HNE(Ac)3. Neuro 2a cells were exposed to 25 µM HNE (A) or 250 µM HNE(Ac)3 (B) for 1 h. Immunofluorescence was performed using an antibody specific for HNE-protein adducts as described in the Materials and Methods and analyzed by confocal laser scanning microscopy. No staining was observed in cells incubated with vehicle only. Arrows point to cellular nuclei.

arrow) was preferentially modified in cells treated with HNE(Ac)3. In addition to these qualitative differences, we also observed a quantitative difference in protein modification. To achieve protein modification detectable by immunoblot analysis, exposure of cells to higher concentrations of HNE(Ac)3 than HNE were necessary. Thus, a 20-fold higher concentration of HNE(Ac)3 (500 µM) resulted in less detectable protein modification than what was observed after HNE (25 µM) treatment of the cells (Figure 5A, compare lane 2 and 4). This difference in signal intensity could be due to the specificity of the antiHNE-protein adduct antibody used in the immunoblot analysis, which may not recognize a protein-adduct formed by HNE(Ac)1. We therefore performed autoradiography of Neuro 2A cells exposed to tritiated HNE or HNE(Ac)3 (Figure 5B). Total radioactivity associated with the proteins resolved by SDS-PAGE was 3-4-fold less in extracts of cells incubated with [3H]HNE(Ac)3 than in cells incubated with [3H]HNE, a difference much less dramatic than the one observed on immunoblots. As observed on immunoblots, the patterns of proteins modified in Neuro 2A cells exposed to [3H]HNE and

An Intracellularly Activated HNE Analogue

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Figure 7. HNE and HNE(Ac)3 disrupt the microtubule network. Neuro 2A cells were exposed to HNE or HNE(Ac)3 for 1 h and their microtubule organization analyzed by immunocytochemistry using an anti-β-tubulin antibody. In control cells the microtubule network is dense with microtubules radiating out to the periphery of the cells (A). HNE at 25 µM causes a severe loss of the microtubule network (B). HNE(Ac)3 applied at 25 µM induces a more moderate change, with many cells showing loss of mainly the peripheral microtubules (C). 50 µM HNE(Ac)3 leads to a near complete loss of the microtubule network in the majority of the cells (D). Bar 20 ) µm. (E) Neuro 2A cells were exposed to HNE or HNE(Ac)3 and the degree of microtubule disruption quantified as described in the materials and methods section. Values are means ( SEM; n ) 5 (control); n ) 4 (25 µM HNE); n ) 4 (25 µM HNE[Ac]3); n ) 3 (50 µM HNE[Ac]3.

[3H]HNE(Ac)3 are similar, but not identical. The most obvious difference again was a protein of MW > 212 kDa that was modified by HNE, but not detectably by HNE(Ac)3. Whether or not that band represents the same high molecular weight protein observed to be preferentially modified by HNE by immunoblotting (Figure 5A) is not known at this time. We analyzed the subcellular distribution of HNEprotein adducts by immunocytochemistry using an antibody specific for HNE-Michael protein adducts and observed a striking difference in the staining pattern in cells treated with HNE and HNE(Ac)3. Exposure to HNE for 1 h resulted in HNE-protein adducts in the plasma membrane as well as cytoplasm, but the nucleus was spared (Figure 6A, arrow). In contrast, cells incubated with HNE(Ac)3 showed clear nuclear immunoreactivity in addition to cytoplasmic and plasma membrane immunofluorescence (Figure 6B, arrow). As observed in the immunoblot analysis study described above, higher concentrations of HNE(Ac)3 than HNE were required to obtain a signal detectable with the anti-HNE-protein adduct antibody. Effects of HNE(Ac)3 on Cellular Microtubule Organization. HNE causes disruption of microtubules in Neuro 2A cells at low concentrations and after short exposure times (33). We exposed Neuro 2A cells to HNE(Ac)3 or HNE for 1 h and characterized the microtubule network as normal (Figure 7A), mildly disrupted (Figure 7C), or severely damaged (Figure 7, panels B and D). A majority of control cells (83.9 ( 8.5%) displayed a normal dense network of microtubules radiating out to the periphery of the cell (Figure 7, panels A and E); the remaining 14.7 ( 8.9% showed moderate changes in the microtubule distribution. After 1 h exposure to 25 µM HNE(Ac)3, 36.6 ( 11.7% of Neuro 2A cells showed severe and 63.2 ( 11.5% moderate microtubule disruption with

loss mainly from the cellular periphery (Figure 7, panels C and E) (0.2% of the cells had normal microtubule network). Prolonging the time of exposure to 25 µM HNE(Ac)3 did not increase the percentage of cells with severe disruption of the microtubule network (data not shown). At 50 µM HNE(Ac)3 89.6 ( 0.6% of the cells had lost all or almost all of their microtubules (Figure 7D, E), with the remaining 10.4 ( 0.6% cells displaying a moderate disruption of the microtubule network. This is comparable to the effect of 25 µM HNE which results in 87.7 ( 2.9% showing severe and 12.3 ( 2.9% moderate disturbance of the microtubules (Figure 7, panels B and E). Thus, HNE(Ac)3 causes disruption of microtubules with approximately 2-fold less efficacy than HNE.

Discussion The goal of this study was to develop a tool that would allow the comparison of cellular effects caused by endogenously produced and externally applied HNE. We designed an HNE analogue that itself is unreactive by converting the aldehyde into a diacetate. Once inside the cell, enzyme-catalyzed hydrolysis of the ester groups (34) was expected to release the aldehyde group. At this point, we have no knowledge of the cellular site(s) and enzyme(s) responsible for the hydrolysis of HNE(Ac)3. Comparison of the sites of HNE delivery resulting from HNE(Ac)3 metabolism and HNE production after intracellular generation of reactive oxygen species would be complex, since the site of HNE production by the latter pathway likely depends on the type of oxidative insult as well as cell type. Indeed, the pattern of HNE-adducted proteins is different in synaptosomes exposed to FeSO4 or Aβ (35). Analyzing the dynamics of HNE(Ac)3 hydrolysis, we observed that Neuro 2A cell lysates hydrolyze 94.5% of HNE(Ac)3 into HNE(Ac)1 (87.3%) and HNE (7.2%) within

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a 2 h period relevant for this study. To avoid intracellular formation of HNE(Ac)1, we attempted to synthesize 1,1bis(acetyloxy)-2(E)-nonene [HNE(Ac)2], a diester of HNE which retains the hydroxyl group on the C4 position of the aldehyde and would therefore be hydrolyzed solely into HNE. However, our attempts to synthesize HNE(Ac)2 were not successful. HNE(Ac)1 differs from HNE solely by having an acetyloxy- instead of a hydroxyl-group on the C4 position. HNE(Ac)1 and HNE are both R,βunsaturated aldehydes and therefore are electrophilic molecules with comparable reactivity toward nucleophilic side chains. While performing immunoblot and immunocytochemical analysis using an antibody specific for HNE-protein adducts, we observed that the signal resulting from protein modifications of Neuro 2A cells exposed to HNE(Ac)3 was substantially less intense than the one observed in cells exposed to HNE. This is likely a result of the specificity of the antibodies used and not due to a difference in reactivity of HNE(Ac)1 and HNE. The antiHNE-protein adduct antibodies used require a 4-hydroxyl group on the C4 position of the aldehyde (32, 3638). These antibodies, therefore, only recognize HNEadducted proteins, and adducts formed by HNE(Ac)1 are not likely to be recognized efficiently on immunoblots or by immunocytochemistry. This explanation is substantiated by our observations that (1) the difference in proteinassociated radioactivity is less than 2-fold in cells exposed to [3H]HNE(Ac)3 (1.4%) and cells exposed to [3H]HNE (2.2%) and (2) HNE(Ac)3 is only two times less efficient than HNE in disrupting cellular microtubules. The proteins modified in Neuro 2A cells exposed to HNE or HNE(Ac)3 were similar, but not identical. At least two proteins were preferentially modified by HNE, and one was preferentially modified by HNE(Ac)3. As discussed above, the proteins revealed on the immunoblots represent only HNE-adducted proteins, while the proteins on the autoradiographs reveal HNE- and HNE(Ac)1-adducted proteins. The nature of these proteins and the relevance of their modification to biological effects are not known at present. A similar observation was made by Keller and colleagues who found that the pattern of HNE-modified proteins was different in each of three synaptosome preparations exposed to externally applied HNE, FeSO4, or Aβ (35). In addition, using these antibodies specific for HNE-adducted proteins for immunocytochemistry, we observed more nuclear staining in Neuro 2A cells exposed to HNE(Ac)3 than in cells treated with HNE. These observations support the hypothesis that the compartmentalization of proteins in relation to the site of HNE generation may be important with respect to the protein species that are modified. Other investigators have demonstrated that exogenously added HNE reacts first with plasma membrane proteins (39, 40), whereas HNE formed endogenously in cells or organelles exposed to oxidative stress is present in high concentrations in intracellular membranes (21, 24). In conclusion, in this study we developed a tool that allows us to form intracellularly HNE. We, therefore, have for the first time the possibility to observe the effects of intracellularly formed HNE in the absence of the plethora of other products and processes that occur in cells undergoing oxidative stress. This molecule allows us to test the appropriateness of extracellular HNE exposure to study effects of this aldehyde on functions associated with different cellular compartments, includ-

Neely et al.

ing plasma membrane, cytoplasm, and nucleus. Using an antibody specific for HNE-adducted proteins, we have presented evidence that the degree of protein modification in at least one subcellular compartment, the nucleus, is different in cells exposed to exogenously added HNE and cells producing endogenous HNE. In addition, the pattern of HNE-adducted proteins although similar, was not identical in Neuro 2A cells exposed to HNE and HNE(Ac)3. This new tool will allow us to focus on the relevant targets and cellular functions affected by HNE, one of the major and most reactive products formed during lipid peroxidation (17).

Acknowledgment. This work was supported by NIH Grants AG00774, ES10196, and AG16835 to Dr. Thomas J. Montine.

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