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Department of Clinical and Experimental Pharmacology (P.C.B.), School of Physics and Chemistry (S.M.P.), The University of Adelaide, Adelaide, Australia
Received September 6, 2005; accepted December 20, 2005
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Abstract |
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,
-unsaturated aldehyde formed during lipid peroxidation cascades (Esterbauer et al., 1991
; Uchida, 1999). It also forms during the incomplete combustion of a diverse range of organic matter including timber, plastics, and other construction materials (Vermeire, 1992). Oxidative metabolism of allyl alcohol in hepatocytes also generates acrolein, accounting for the overt periportal hepatotoxicity of this substance (Belinsky et al., 1985). A powerful electrophile, the toxicity of acrolein reflects the pronounced reactivity of its unsaturated bond, which is readily attacked by nucleophilic centers in protein and DNA to form carbonyl-retaining adducts (Michael adducts) (Uchida et al., 1998a; Burcham and Fontaine, 2001). Such reactivity ensures that acrolein disrupts a wide range of cellular and molecular processes in exposed tissues, including alterations in the activity of various transcription factors, enzyme activities, mitochondrial processes, and redox regulatory pathways (Biswal et al., 2002; Ranganna et al., 2002; Luo and Shi, 2004; Yang et al., 2004). Consistent with endogenous acrolein production via lipid peroxidation cascades, acrolein-adducted proteins have been detected in the affected tissues of a number of degenerative conditions known to involve oxidative stress, including Alzheimer's disease, atherosclerosis, dermal photodamage, and spinal cord trauma (Uchida et al., 1998b; Calingasan et al., 1999; Tanaka et al., 2001; Luo et al., 2005).
Given the role of acrolein in a wide range of health conditions, we commenced a search for low-molecular weight, nitrogen-containing nucleophiles that block the toxicity of this substance. An expectation was that such compounds would intercept free acrolein within the intracellular environment, preventing adduction of macromolecules and the progression of cell death. Our efforts identified the vasodilatory antihypertensive hydralazine as a potent inhibitor of acrolein-mediated toxicity in isolated murine hepatocytes (Burcham et al., 2002). These properties were consistent with the carbonyl-trapping actions of hydralazine, because the drug is known to scavenge pyruvate and other endogenous carbonyl compounds in humans (Reece et al., 1985). Therefore, we recently characterized two isomeric hydrazones formed during interactions between acrolein and hydralazine (Kaminskas et al., 2004a).
In addition to scavenging free acrolein, hydralazine also readily forms hydrazones with acrolein-adducted cell proteins, a reaction we term "adduct-trapping" (Burcham et al., 2004). Using electrospray ionization mass spectrometry to characterize products formed upon exposure to an acrolein-modified model peptide, we found that hydralazine readily trapped several species of carbonyl-retaining adducts formed by acrolein at lysine (Burcham et al., 2004). Using rabbit antiserum against drug-labeled adducts, we also detected strong adduct-trapping in a wide range of cell proteins from mouse hepatocytes treated briefly with allyl alcohol and cytoprotective concentrations of hydralazine (e.g., 2 to 50 µM; see Burcham et al., 2004). Moreover, intense protein adduct-trapping occurred in the livers of allyl alcohol-intoxicated mice that received hepatoprotective doses of hydralazine (Kaminskas et al., 2004b).
Taken together, these findings imply two distinct mechanisms may contribute to hydralazine cytoprotection against acrolein-mediated cellular injury (Fig. 1). In the first instance, direct acrolein scavenging is expected to diminish the intracellular availability of acrolein, thereby reducing alkylation of intracellular targets such as proteins, glutathione, and DNA. In the alternative mechanism, hydralazine targets carbonyl-retaining Michael adducted-proteins, forming hydrazones that may prevent participation by modified proteins in nucleophilic additions that generate inter- and intramolecular cross-links (Fig. 1).
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; Gotte and Libonati, 2004). Third, having established conditions for the induction of protein cross-links by acrolein, the effect of hydralazine on protein oligomerization was explored using Western blotting to assess the role of adduct-trapping in any inhibitory outcomes. Finally, the efficacies of two endogenous compounds as inhibitors of acrolein-induced protein cross-linking were explored, namely glutathione and carnosine. Both agents trap a range of ,-unsaturated aldehydes, including acrolein (Burcham et al., 2002; Carini et al., 2003).
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Materials and Methods |
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). Adult male Swiss mice (5-6 weeks old) were obtained from Animal Services at the Waite Institute of The University of Adelaide and housed under normal conditions until use as hepatocyte donors. All procedures involving animal use were approved by the institutional Animal Ethics Committee and were compliant with principles outlined in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.
Cell Culture Experiments. Hepatocytes were prepared by collagenase digestion of mouse livers as outlined previously (Burcham and Fontaine, 2001). Freshly isolated cells were washed four times in collagenase-free Krebs-Henseleit buffer and then resuspended at 1 x 106 cells per ml in RPMI 1640 media supplemented with 0.2% bovine serum albumin, 0.03% L-glutamine, penicillin (50 U/ml), and streptomycin (50 µg/ml). The cells were then layered on collagen-coated 60-mm polystyrene dishes and placed in a humidified CO2 incubator at 37°C (5% CO2). After 2 h, the dishes were washed twice with phosphate-buffered saline (PBS) to remove nonadherent cells before fresh aliquots of Krebs-Henseleit buffer, pH 7.3, supplemented with glucose (5 g/l) and pyruvate (1 mM) were added to each dish. The use of this amino acid-free media was intended to minimize extracellular side-reactions between free acrolein and nucleophilic buffer constituents. Hydralazine, MESNA, and allyl alcohol were dissolved directly in culture media immediately before use. After the addition of reagents, dishes were returned to the incubator and 100-µl media samples were taken at 30 min (for the assessment of extracellular acrolein) or at 180 min [for the estimation of lactate dehydrogenase (LDH) leakage]. Some dishes collected at 30 min were also used for the immunochemical detection of hydralazine-trapped adducts and protein carbonyls as described below. Cell lysates used in these immunochemical assays were prepared using recently described procedures (Burcham et al., 2004).
Biochemical Procedures. LDH activity was measured using a spectrophotometric procedure based on the enzymatic reduction of NAD+ in the presence of l-lactate (Burcham and Fontaine, 2001). For the estimation of total LDH activity, cells were lysed by adding 250 µl of 5% Triton X-100 to each dish before they were sonicated for 15 s using a Labsonic 1510B Cell Disrupter (Braun Melsungen AG, Melsungen, Germany). Separate dishes were used for glutathione determination via the method of Saville (1958). In brief, cell monolayers that had been exposed to allyl alcohol in the presence and absence of various concentrations of hydralazine were washed three times with ice-cold PBS. Next, 1 ml of ice-cold trichloroacetic acid (6.5%) was added to each plate, and then the contents were transferred to 1.5-ml Eppendorf tubes. After centrifugation at 3000g for 10 min at 4°C, the glutathione content of 0.25-ml aliquots of supernatant was measured against a standard curve constructed using reduced glutathione dissolved in 6.5% trichloroacetic acid (standards spanned a 10 to 200 µM concentration range).
Acrolein Determination. Acrolein concentrations were determined in aliquots of culture media or RNase A reaction mixtures (see below) using a HPLC-based procedure described recently (Kaminskas et al., 2004a). Depending on the acrolein concentration present within the sample, sample aliquots were diluted with from 4 to 249 volumes of mobile phase before HPLC analysis. The HPLC system comprised an ODS Hypersil column (150 x 4.6 mm, 5 µm; Thermo Electron, Waltham, MA) connected to a GBC LC1150 pump (Dandenong, Australia) and fitted with an online ERC 3415 degasser and a Hewlett Packard series 1100 UV detector (Hewlett Packard, Palo Alto, CA) that monitored the absorbance of column eluate at 210 nm. Output from the detector was collected using Delta Junior chromatography analysis software (Version 5009; Digital Solutions, Queensland, Australia). The mobile phase (20% aqueous methanol, v/v) was maintained at a flow rate of 1 ml/min, with acrolein eluting from the column at a retention time of 3.0 min. Aldehyde concentrations were determined by comparing sample peak areas to those obtained by analyzing standard solutions of acrolein prepared in mobile phase over a concentration range of 1 to 16 µM.
RNase A Oligomerization Assay. Protein cross-linking reactions were performed in 50 mM sodium phosphate buffer, pH 7.0, in a final volume of 0.2 ml using 0.2-ml polypropylene polymerase chain reaction tubes (the use of maximal reaction volumes helped minimize acrolein loss into headspace at 37°C). The RNase A concentration was 2 mg/ml, conferring a final lysine concentration of 1.5 mM. In reactions exploring the concentration dependence of RNase A cross-linking by acrolein, the latter was added to achieve aldehyde/lysine ratios of 0.5, 1, 2, 4, and 8 (i.e., final concentrations of 0.75, 1.5, 3, 6, and 12 mM). In related experiments, the time course of protein cross-linking and adduction by acrolein was studied using 5-ml glass vials fitted with a rubber septum, enabling aliquot collection without undue loss of acrolein vapors. In brief, vials containing 9 mg of RNase A dissolved in 4.9 ml of 50 mM phosphate buffer, pH 7.0, were prewarmed at 37°C. A 100-µl aliquot of acrolein was then added to give a final concentration of 3 mM (i.e., achieving a 2:1 acrolein/lysine ratio). At various time intervals (0, 15, 30, 45, 60, 90, 120, 150, and 180 min), 100-µl aliquots of reaction mixture were removed and diluted 3:1 with 5x gel electrophoresis sample buffer (Bollag et al., 1996). The samples were then chilled on ice until 20-µl volumes (i.e., 30 µg of protein) were resolved via gel electrophoresis as outlined below.
In experiments in which the role of primary amines in protein adduction and oligomerization was explored, reductively methylated RNase A (Me-RNase A) was substituted for native protein. Me-RNase A was prepared using a half-scale version of a procedure described by others (Xu et al., 1999). In brief, RNase A (17 mg, 1.25 µmol) was dissolved in 490 µl of sodium phosphate buffer (0.1 M, pH 7.2). A 100-fold molar excess of formaldehyde was then added (125 µmol), followed by 7.9 mg of sodium cyanoborohydride (125 µmol). After 18 h at room temperature, unreacted reagents were removed via dialysis for 24 h against two changes of 0.1 M sodium phosphate buffer (pH 7.2, 500 ml) using Pierce Slide-A-Lyser cassettes (3.5-kDa cut-off; Pierce Chemical, Rockford, IL). Confirmation that the number of accessible primary amines was diminished by the formaldehyde/cyanoborohydride treatment was obtained by comparing Me-RNase A with native protein using a spectrophotometric assay for protein amine groups (Spadaro et al., 1979).
Oligomerization Blocking Experiments. To examine the efficacy of hydralazine as an inhibitor of oligomerization, and also any time dependence of susceptibility to the drug, assay substrate was prepared by reacting RNase A (2.1 mg/ml) with 3.2 mM acrolein in 2-ml volumes of buffer in Eppendorf tubes at 37°C. At either 30 or 90 min, 190-µl aliquots were removed and added to 0.2-ml polymerase chain reaction tubes containing either 0, 1-, 3-, or 10-µl volumes of 60 mM hydralazine (diluted to a 10-µl volume with buffer), achieving respective hydralazine concentrations of 0, 0.3, 1, and 3 mM. Reactions were allowed to proceed for a further 120 min at 37°C, after which the reaction mixtures were diluted 3:1 with 5x sample buffer. Aliquots containing 30, 5, or 3 µg of protein were then resolved on separate 14% acrylamide gels for respective analysis of protein oligomerization via Coomassie Blue staining (gel 1), lysine adduction (gel 2), and adduct-trapping (gel 3).
In related experiments, the effects of various reagents (e.g., sodium borohydride, sodium cyanoborohydride, glutathione, and carnosine) on protein oligomerization were examined as described for hydralazine using RNase A that had been premodified by acrolein for 30 min only. After the secondary incubation, reaction mixtures were assessed for oligomers and lysine adducts as described below. In addition, the protein carbonylation status of 2.5-µg aliquots of modified protein was assessed using an immunochemical 2,4-dinitrophenylhydrazine-based procedure (Keller et al., 1993).
Mechanism of Formation of Hydralazine Adducts in Protein. To assess the reactivity of 1-APH with the model protein, RNase (2 mg/ml final concentration) was treated with 0.1, 0.3, or 1 mM concentrations of 1-APH in 50 mM sodium phosphate buffer, pH 7.0,. The final reaction volume was 90 µl. 1-APH solutions were prepared freshly in methanol and added to tubes to obtain a final methanol concentration of 16.6%. Control tubes received equivalent concentrations of neat methanol. Tubes were then placed in an incubator at 37°C for 30 min, after which RNase was processed for the presence of hydralazine adducts via Western blotting as outlined below. To prepare acrolein-adducted RNase A that was free of unbound acrolein for use as a positive control in these experiments, 1.5 mg of RNase was dissolved in 500 µl of sodium phosphate buffer (50 mM, pH 7.0) and then treated with an equimolar concentration of acrolein (relative to the lysine concentration of 2.25 mM) for 30 min at 37°C. Unincorporated acrolein was then removed via dialysis for 18 h against 50 mM sodium phosphate buffer, pH 7.0, three 1-liter changes using Pierce Slide-A-Lyser cassettes (3.5-kDa cut-off). The resulting modified protein was then treated with 0.1, 0.3, or 1 mM concentrations of hydralazine under the same conditions outlined above for the reaction with 1-APH. The presence of trapped adducts was then assessed via Western blotting.
Gel Electrophoresis and Western Blotting. After heat denaturation for 6 to 8 min at 90°C, protein samples were loaded onto a 14% acrylamide minigel (4% stacking gel) before electrophoresis at 200 V for 45 min. Samples comprising cell lysates (i.e., used to detect trapped adducts and protein carbonyls) were resolved overnight at 120 V on a large gel (12% acrylamide, 4% stacking gel) using a Protean II apparatus (Bio-Rad, Hercules, CA). After transfer to nitrocellulose (100 V, 30 min), membranes were blocked with 7.5% nonfat milk in PBS for 30 min and then treated for 60 min with 1/1000 dilutions of rabbit antiserum [raised against either acroleinmodified hemocyanin (Burcham et al., 2003), hydralazine-stabilized acrolein-modified hemocyanin (Burcham et al., 2004) or commercial anti-dinitrophenyl serum (Keller et al., 1993)]. After washing and exposure to horseradish peroxidase-coupled goat anti-rabbit IgG serum (1/10,000 dilution, 30 min; Pierce Immunopure), membranes were developed using Pierce Super Signal West Pico chemiluminescence reagent and BioMax Light film (Eastman Kodak, Rochester, NY). The resulting images were analyzed via densitometry using Kodak Digital Science software.
Statistical Analysis. Relevant experimental data (LDH leakage, acrolein concentrations, glutathione contents) were analyzed by oneway analysis of variance followed by Tukey's multiple comparison test using Prism software (ver. 4.01; GraphPad Software Inc., San Diego, CA).
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), adduct trapping was detected in a 
130-kDa mass protein in control cells exposed to 100 µM hydralazine only, indicative of hydralazine reactions with endogenously carbonylated proteins (Fig. 2E, lane 2). At low drug concentrations in cells coexposed to allyl alcohol and hydralazine, adduct-trapping reactions predominantly involved highmass proteins, although smaller proteins (
30 to 50 kDa) were targeted at higher drug concentrations (Fig. 2E). Despite the strong adduct-trapping produced by hydralazine, the drug did not minimize the extensive protein carbonylation occurring in cells during a 30-min coexposure to allyl alcohol (Fig. 2F). Protein carbonyl groups were detected by derivatizing cell proteins with 2,4-dinitrophenylhydrazine followed by denaturing polyacrylamide electrophoresis and Western blotting using anti-dinitrophenyl serum (Keller et al., 1993). Protective concentrations of hydralazine also failed to prevent the marked depletion of hepatocyte glutathione occurring during a 30-min exposure to 100 µM allyl alcohol (Fig. 2G). The fact that protective concentrations of hydralazine had no effect on extracellular acrolein concentrations, glutathione depletion, or protein carbonylation suggests that acrolein scavenging and minimization of the adduction of intracellular targets is not central to the cytoprotective actions of the drug. This conclusion focused attention on possible ways the adduct-trapping reactivity of hydralazine might contribute to its cytoprotective efficacy. The next phase of the project thus explored the cross-linking properties of acrolein as well as the ability of hydralazine ability to disrupt these reactions.
Protein Cross-Linking by Acrolein. Coomassie Blue staining of gels obtained after electrophoresis of acroleinmodified ribonuclease A (RNase A) allowed facile detection of oligomeric species formed via cross-linking reactions (Fig. 3A). Exposure for 3 h at 37°C to 0.75, 1.5, 3, 6, and 12 mM acrolein resulted in concentration-dependent accumulation of RNase A dimers to respective values of 11, 32, 41, 52, and 51% of starting material (Fig. 3A, lanes 2 to 6). Masses corresponding to RNase A trimers represented 7.7, 21, and 22% of starting material at 3, 6, and 12 mM concentrations of acrolein (lanes 4 to 6). Protein cross-linking was accompanied by extensive protein modification, as detected using rabbit antiserum selective for acrolein-adducted lysine residues (Fig. 3B). Immunorecognition of oligomeric RNase A species was particularly intense (Fig. 3B, lanes 4 to 6), suggesting that acrolein-adducted lysine residues are key participants in the formation of aggregated proteins in this system. Moreover, subjecting the model protein to reductive methylation before acrolein exposure abolished both the accumulation of oligomeric RNase A (Fig. 3A, lanes 8 and 9) and the immunodetection of acrolein-adducted protein (Fig. 3B, lanes 8 to 9). Reductive methylation reduced the primary amine content of the RNase A by over 90% relative to native protein, as determined using a picrylsulfonic acid-based spectrophotometric assay (Spadaro et al., 1979). The finding that methylated RNase A resisted cross-linking indicates a role for carbonyl-retaining adducts at the 
-amine group of lysine in protein oligomerization. This was reinforced by the finding that delayed (30 min) addition of the strong, carbonyl-reducing reagent sodium borohydride diminished oligomer levels after a subsequent 120 min reaction (compare lanes 5 to 7 with lane 4). In contrast, a milder reducing agent that is inactive at carbonyl groups (sodium cyanoborohydride) was less effective in this regard (Fig. 3E, lanes 8 to 10).
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). Densitometry of Fig. 3C revealed that dimeric RNase A comprised 8.4% of starting material at 45 min, increasing to 29% by 180 min (Fig. 3C, lanes 4 and 9). The generation of oligomeric species was again accompanied by extensive lysine adduction (Fig. 3D). Consistent with a role for lysine adducts in the cross-linking chemistry, adducts were detected in monomeric RNase A at early time points that preceded the appearance of oligomeric RNase species (e.g., at 15 min after commencing acrolein exposure as per lane 2 in Fig. 3D).
Inhibition of Acrolein-Induced Protein Oligomerization by Hydralazine. Added concurrently with acrolein, hydralazine (0.1 to 3 mM) produced strong concentration-dependent inhibition of acrolein-induced RNase A oligomerization during a 3-h reaction at 37°C (Fig. 4). Densitometry of Coomassie Blue-stained proteins (Fig. 4A) revealed hydralazine concentrations of 0.3, 1, and 3 mM decreased dimer abundance by 25, 56, and 100%, respectively (Fig. 4A). The effects of hydralazine on levels of RNase A trimers were particularly striking, because levels of this product were reduced by 39, 100, and 100% at these drug concentrations (Fig. 4A). Hydralazine also produced concentration-dependent declines in the immunoreactivity of acrolein-modified lysine residues within each form of RNase A (Fig. 4B), and these effects were accompanied by parallel formation of hydralazine-trapped protein adducts (Fig. 4C). Acrolein modification strongly increased the carbonyl content of monomeric, dimeric, and trimeric forms of RNase A (Fig. 4D), with hydralazine suppressing this effect only at the 3 mM concentration (lane 7). Consistent with its efficient acrolein-scavenging properties, 0.1, 0.3, 1, and 3 mM hydralazine decreased free acrolein concentrations by 18, 31, 65, and 100%, respectively, 30 min after commencing concurrent exposure to acrolein and hydralazine (Fig. 4E). This indicates both acrolein scavenging and adduct trapping probably contributed to the inhibitory effects of hydralazine on protein oligomerization during coexposure of RNase A to acrolein and the drug.
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Hydralazine Traps "Early" Acrolein-Protein Adducts. To clarify the role of adduct-trapping in the effects of hydralazine on acrolein-mediated protein oligomerization, the time dependence of susceptibility to the drug was examined (Fig. 5). By delaying hydralazine addition until 30 min, reactivity with adducts present on monomeric RNase A could be explored. Figure 5A indicates that 0.3 to 3 mM hydralazine elicited a concentration-dependent inhibitory effect when added 30 min after the commencement of RNase A modification by acrolein (compare lanes 4 to 6 with lane 3). For example, 1 mM hydralazine (lane 5) diminished dimer abundance by 67% relative to acrolein-modified RNase alone (lane 3). However, if drug addition was delayed until 90 min after starting acrolein exposure, hydralazine had negligible effect upon levels of oligomeric RNase A present after a secondary 120-min incubation (Fig. 5A, lanes 7 to 9). This suggests that hydralazine targets Michael adducts formed during the early stages of protein modification by acrolein, but that cross-links formed during subsequent reaction of these species with neighboring nucleophiles resist attack by the drug.
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Mechanism of Formation of Hydralazine Adducts in Protein. The main isolable product of reactions between free acrolein and hydralazine, 1-APH (Kaminskas et al., 2004a), possesses an ,-unsaturated bond that might be attacked by protein nucleophiles to form hydralazine-labeled adducts (Scheme 1). To assess this possibility, the yield of adducts generated during reactions of RNase A with 1-APH was compared with those obtained via the adduct-trapping route (i.e., where hydralazine reacts directly with acrolein-adducted protein). To assess the latter under conditions that precluded 1-APH formation, acrolein-adducted RNase A was subjected to extended dialysis to remove unreacted acrolein (i.e., before the addition of hydralazine). The resulting "unbound acrolein-free" modified RNase A was then reacted with a range of hydralazine concentrations (0.1, 0.3, and 1 mM) for 30 min at 37°C. Results obtained during immunochemical analysis of the resulting protein, as well as RNase A that was treated with a range of concentrations of 1-APH, are shown in Fig. 6. The data confirm that the reaction of hydralazine with acrolein-adducted protein (Fig. 6, lanes 4 to 6) was by far the favored route for the introduction of hydralazine moieties into the model protein. In contrast, comparable concentrations of 1-APH exhibited no reactivity with the protein (Fig. 6, lanes 7 to 9).
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), but an alternative strategy involves using nucleophilic drugs to intercept toxic carbonyls (Shapiro, 1998). Our group has recently explored the latter option, giving attention to drugs that neutralize acrolein, the most reactive carbonyl compound formed during oxidative stress. We identified the antihypertensive hydralazine, among the strongest nucleophiles in clinical use, as a powerful inhibitor of acrolein toxicity (Lalich and Paik, 1975; Burcham et al., 2002). Although its protective efficacy seemed to be related to its acrolein-scavenging properties in cell-free systems (Kaminskas et al., 2004b), we found that hydralazine also reacts extensively with adducts formed during protein modification by acrolein (Burcham et al., 2004; Kaminskas et al., 2004b). The present work breaks new ground by establishing that the latter adduct-trapping reactivity is more closely related to hydralazine cytoprotection than the direct acrolein-scavenging reaction (Fig. 1). This finding was not a foregone conclusion, because our previous studies in hepatocytes were optimized to detect adduct-trapping events (Burcham et al., 2004). Thus, the cells were preloaded with acrolein adducts, and then, immediately before the irreversible loss of membrane integrity, they were washed free of acrolein precursor before subsequent incubation with fresh media containing a range of drug concentrations. This ensured hydralazine was added to cells under conditions where levels of adducted proteins were high relative to concentrations of free, unadducted acrolein (Burcham et al., 2004). Although extensive adduct-trapping occurred under such conditions, it is unknown whether adduct-trapping reactions also occur where ongoing acrolein generation would ensure direct acroleinscavenging reactions would compete for hydralazine alongside trapping reactions with adducted proteins. Indeed, if the former was highly efficient within the cellular environment, it is conceivable that this reaction might entirely prevent protein modification by acrolein, thereby precluding protein adduct-trapping by hydralazine. The current findings resolve this issue by confirming that intense adduct trapping occurred in cells exposed simultaneously to allyl alcohol and hydralazine, indicating the reaction with adducted proteins is a significant intracellular fate of hydralazine.
The finding that acrolein levels in the media of allyl alcohol-treated cells were unaffected by cytoprotective hydralazine concentrations reinforces the conclusion that reactions with unadducted aldehyde are not a major cellular fate of hydralazine. This conclusion concurs with our recent finding of only low levels of the major hydrazone formed during reactions between free acrolein and hydralazine, 1-APH, in the culture media of hepatocytes after a 30-min incubation with allyl alcohol, and the same drug concentrations used in the present study (Kaminskas et al., 2004a). For example, the concentration of 1-APH in cells after a 30-min exposure to 100 µM hydralazine and 100 µM allyl alcohol was 0.38 ± 0.1 nmol/ml—an order of magnitude lower than the level of free acrolein measured under the same conditions in our present study (3.6 ± 0.8 nmol/ml; see Fig. 2C). Thus, even at this high drug concentration, direct acrolein scavenging cannot account for the cytoprotection afforded by hydralazine.
Although the present work confirmed that adduct-trapping occurred under conditions of concurrent exposure to hydralazine and allyl alcohol, the experimental design that was used raised the possibility of alternative mechanisms for the incorporation of drug moieties into protein. One possibility was that the hydralazine-containing adducts might have been formed via the intermediacy of 1-APH, the product of scavenging reactions between hydralazine and free acrolein (Scheme 1). For example, an attack on the ,-unsaturated bond of 1-APH would yield ternary protein-aldehyde-drug complexes that structurally resemble species formed by hydralazine when attacking Michael adducts in acrolein-modified proteins. The present findings discount this mechanism, because exposure of RNase A to high concentrations of 1-APH (0.1 to 1 mM) for 30 min resulted in no detectable adducts (Fig. 6). The 30-min reaction duration was used because this was the time after which extensive adduct trapping was detected in hepatocytes (Fig. 2). Moreover, repeating the experiment using 3 to 30 mM concentrations of 1-APH failed to generate detectable hydralazine-containing adducts (data not shown). Therefore, the intense adduct trapping occurring in hepatocytes exposed simultaneously to allyl alcohol and hydralazine in Fig. 2 almost certainly reflects trapping of Michael-adducted proteins by free hydralazine rather than protein adduction by 1-APH.
Exactly how adduct-trapping reactivity confers cytoprotective efficacy on hydralazine is unclear, although the possibility that it involves interference with cross-link formation is strengthened by the present work. The protein cross-linking reactivity of acrolein is poorly characterized, and the chemistry underlying such reactions is unknown. Because the present work established a role for lysine in protein crosslinking by acrolein, future work should clarify whether these residues account for both nucleophilic centers participating in the formation of a given cross-link (i.e., Lys-ACR-Lys) or whether other nucleophiles known to be modified by acrolein also participate (e.g., Cys-ACR-Lys, His-ACR-Lys, etc.). In analogous work, clarification of the chemistry of protein cross-linking by the lipid peroxidation product 4-hydroxynonenal facilitated development of antibody-based assays to detect cross-linking in oxidized low-density lipoproteins (Xu et al., 2000). An immunochemical strategy was also used to detect cross-linking of cytoskeletal proteins in neuroglial cells after brief (3 h) exposures to 10 to 1000 µM concentrations of 4-hydroxynonenal (Montine et al., 1996). Because the cross-linking reactivity of acrolein toward RNase A in the present study resembled that described for 4-hydroxynonenal under comparable conditions (Liu et al., 2003), it is likely that acrolein will facilitate protein cross-linking in the cellular environment in a similar manner to 4-hydroxynonenal (Montine et al., 1996). Ongoing work in our laboratory is aimed at establishing the identity of the major protein targets for acrolein adduction in exposed tissues, using a proteomics-based approach comparable with that which recently enabled identification of targets for 4-hydroxynonenal in rodent liver (Carbone et al., 2004). Because adducted proteins are precursors to cross-link formation, identifying proteins most prone to adduction by acrolein will allow development of immunochemical approaches to detect aggregated proteins in acrolein-exposed tissues.
The finding that physiological concentrations of glutathione inhibited acrolein-mediated protein oligomerization raises the possibility that this tripeptide acts as an endogenous adduct-trapping reagent. Although glutathione is a highly efficient scavenger of free acrolein, the delayed administration of glutathione to the assay system in this study (i.e., 30 min after commencement of acrolein modification) ensured that adduction had already occurred on the model protein. This rules out the possibility that scavenging of free acrolein explains the low yield of protein oligomers seen in the presence of glutathione. The chemistry underlying the adduct-trapping efficacy of glutathione is unclear, but it is noteworthy that Uchida and associates (Furuhata et al., 2002) found that glutathione adds rapidly to the cyclic unsaturated bond of FDP-lysine, the product of sequential addition of two molecules of acrolein to a given lysine side chain. This implies FDP-lysine is a reactive intermediate able to participate in reactions with nucleophiles in addition to glutathione (e.g., the thiolate anion of cysteine residues within proteins might react with FDP-lysine to generate interior intramolecular cross-links). However, another explanation for the cross-link blocking efficacy of glutathione could be that the terminal amine of the tripeptide participates in Schiff reactions with carbonyl adducts generated by acrolein. Work is underway in our laboratory to characterize the relative contributions of N-versus S-glutathiolation pathways in reactions of glutathione with acrolein-adducted proteins.
As a mechanism of drug action, the "cross-link blocking" action of hydralazine documented in this study resembles the "cross-link breaker" compounds that are under exploration for efficacy against the role of toxic carbonyls in diabetic complications (Zieman and Kass, 2004). The chemical diversity of AGE production is striking, but a major consequence of these reactions is the cross-linking of collagen molecules in connective tissues and especially vascular tissue, a key factor in the arterial inelasticity accompanying diabetes (Thorpe and Baynes, 2003). The prototype compounds in the crosslink breaker class, N-phenacylthiazolium bromide and 4,5-dimethyl-3-(2-oxo-2-phenylethyl)-thiazolium chloride (ALT-711), improve a number of disease endpoints in animal models of diabetes as well as aged nondiabetic animals, producing reductions in AGE levels in renal and vascular tissue, declines in myocardial and aortic stiffness, and diminished collagen deposition in a range of tissues (Cooper et al., 2000; Vaitkevicius et al., 2001). At present, the extent to which "cross-link cleavage" contributes to the clinical improvements in these animal models is subject to debate, because these compounds improve a number of biological endpoints in target tissues (e.g., reduction in inflammatory markers, profibrotic cytokines, etc.) but may not cleave cross-linked proteins in diabetic tissues (Yang et al., 2003). Nevertheless, because carbonyl-retaining protein adducts contribute to collagen cross-linking during AGE reactions, the possibility that adduct-trapping compounds might produce beneficial outcomes in these conditions is worthy of attention.
In conclusion, this work sheds light on the mechanisms underlying protection by hydralazine against acrolein-mediated toxicity and confirms that the drug is able to readily "trap" reactive Michael adducts in proteins. More work is needed to establish how such adduct trapping might suppress the onset of cell death, but the present work indicates that prevention of protein cross-linking is one outcome of this mechanism of drug action.
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ABBREVIATIONS: MESNA, 2-mercaptoethanesulfonic acid; RNase A, ribonuclease A; 1-APH, 1-acrylaldehyde phthalazin-1-ylhydrazone; PBS, phosphate-buffered saline; LDH, lactate dehydrogenase; HPLC, high-performance liquid chromatography; Me-RNase A, reductively methylated ribonuclease A; ACR, acrolein; ACR-RNase, acrolein-adducted ribonuclease A; AGE, advanced glycation end products; FDP-lysine, -(3-formyl-3,4-dehydropiperidino)lysine.
Address correspondence to: Assoc. Prof. Philip C. Burcham, Pharmacology Unit, School of Medicine and Pharmacology, The University of Western Australia, Nedlands, Western Australia, 6009. E-mail: pburcham{at}meddent.uwa.edu.au
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