ToxSci Advance Access originally published online on October 5, 2006
Toxicological Sciences 2007 95(1):136-146; doi:10.1093/toxsci/kfl127
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Structure-Toxicity Analysis of Type-2 Alkenes: In Vitro Neurotoxicity
* Department of Anesthesiology, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York 10467
Center for Environmental and Human Toxicology, University of Florida, Gainesville, Florida 32611-0885
Department of Chemistry, Iona College, New Rochelle, New York 10804
1To whom correspondence should be addressed at Montefiore Medical Center, Moses Research Tower-7, 111 East 210th Street, Bronx, NY 10467. Fax: (718) 515-4903. E-mail: lopachin{at}aecom.yu.edu.
Received July 12, 2006; accepted September 23, 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Acrylamide (ACR) is a conjugated type-2 alkene that produces synaptic toxicity presumably by sulfhydryl adduction. The
,ß-unsaturated carbonyl of ACR is a soft electrophile and, therefore, adduction of nucleophilic thiol groups could occur through a conjugate (Michael) addition reaction. To address the mechanism of thiol adduct formation and corresponding neurotoxicological importance, we defined structure-toxicity relationships among a series of conjugated type-2 alkenes (1µM–10mM), which included acrolein and methylvinyl ketone. Results show that exposure of rat striatal synaptosomes to these chemicals produced parallel, concentration-dependent neurotoxic effects that were correlated to loss of free sulfhydryl groups. Although differences in relative potency were evident, all conjugated analogs tested were equiefficacious with respect to maximal neurotoxicity achieved. In contrast, nonconjugated alkene or aldehyde congeners did not cause synaptosomal dysfunction or sulfhydryl loss. Acrolein and other ,ß-unsaturated carbonyls are bifunctional (electrophilic reactivity at the C-1 and C-3 positions) and could produce in vitro neurotoxicity by forming protein cross-links rather than thiol monoadducts. Immunoblot analysis detected slower migrating, presumably derivatized, synaptosomal proteins only at very high acrolein concentrations (
25mM). Exposure of synaptosomes to high concentrations of ACR (1M), N-ethylmaleimide (10mM), and methyl vinyl ketone (MVK) (100mM) did not alter the gel migration of synaptosomal proteins. Furthermore, hydralazine (1mM), which blocks the formation of protein cross-links, did not affect in vitro acrolein neurotoxicity. Thus, type-2–conjugated alkenes produced synaptosomal toxicity that was linked to a loss of thiol content. This is consistent with our hypothesis that the mechanism of ACR neurotoxicity involves formation of Michael adducts with protein sulfhydryl groups. Key Words: distal axonopathy; acrolein; acrylamide; adduct formation; neurodegeneration; synapse.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Acrylamide (ACR) is a ubiquitous neurotoxicant that is used extensively in various industrial settings (e.g., water and wastewater management, ore processing, and dye synthesis) and in scientific laboratories for the electrophoretic separation of macromolecules. ACR is also a food contaminant formed during high-temperature preparation of certain potato- or grain-based products. Although the polymer is nontoxic, exposure to monomeric ACR can produce mental status changes, ataxia, and skeletal muscle weakness in humans and experimental animal models (reviewed in LoPachin et al., 2003
). Accumulating evidence suggests that these neurological deficits are mediated by an early disruption of neurotransmission followed by nerve terminal degeneration in the peripheral and central nervous systems (reviewed in LoPachin et al., 2002, 2003). Although the mechanism of synaptic toxicity is unknown, results from recent molecular and proteomic determinations suggest that ACR selectively adducts thiol groups on cysteine residues of several presynaptic proteins (Barber and LoPachin, 2004; LoPachin et al., 2004b, 2006). Thiol adduction has significant neurotoxic implications since it is now recognized that the regulatory functions of certain synaptic proteins (e.g., SNAP-25, v-ATPase, N-ethylmaleimide sensitive factor [NSF]) are determined by the redox states of specific sulfhydryl groups (reviewed in LoPachin and Barber, 2006). Based on this evidence, we have hypothesized that ACR alkylation of essential thiol groups impairs the function of presynaptic proteins that regulate neurotransmission (reviewed in LoPachin et al., 2002, 2003).
ACR is an ,ß-unsaturated carbonyl derivative and is, therefore, classified as a conjugated type-2 alkene, that is, an electrophilic group linked to an alkene carbon, which forms a conjugated structure (Kemp and Vellaccio, 1980). Since the pi electrons in a conjugated system are highly polarizable, the ,ß-unsaturated carbonyl of ACR is categorized as a soft electrophile. Characteristically, soft electrophiles will preferentially form adducts with soft nucleophiles, which in biological systems are primarily sulfhydryl groups on cysteine residues (Coles, 1984–1985; Hinson and Roberts, 1992; LoPachin and DeCaprio, 2005; Pearson and Songstad, 1967). Our proposed mechanism of ACR-induced nerve terminal damage is based on adduction of functionally critical protein thiol groups. However, whether such adduct formation is a principal neuropathogenic step in ACR neurotoxicity has not been determined conclusively and, in fact, previous research suggests that sulfhydryl alkylation is not mechanistically relevant (Hoshimoto and Aldridge, 1970; Lapin et al., 1982; Martenson et al., 1995). To define the thiol adduct chemistry of ACR and its relevance to corresponding neurotoxicological mechanisms, we determined the structure-toxicity relationships for a series of conjugated type-2 alkenes and their nonconjugated analogs. Thus, if thiol adduction is neurotoxic and is mediated by the polarizability (softness) of the conjugated type-2 alkene system, then other conjugated analogs should also produce thiol-based neurotoxicity. Conversely, structural analogs that are nonconjugated and are, therefore, not soft electrophiles will not form thiol adducts and should be devoid of neurotoxic activity. Acrolein is structurally similar to ACR and is prototypical of conjugated type-2 alkenes (Fig. 1). Similarly, N-ethylmaleimide (NEM) is also an ,ß-unsaturated carbonyl derivative and is a well-documented sulfhydryl alkylating agent. We therefore determined the effects of these conjugated alkenes and several additional structural analogs (Fig. 1) on selected neurochemical parameters of striatal synaptosomes. As a quantitative index of sulfhydryl adduct formation, the free thiol content of toxicant-exposed synaptosomes was measured. Cysteine adduct formation was verified by mass spectrometric analysis of synaptosomal protein exposed in vitro to acrolein.
|
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Chemicals and Materials
Unless otherwise indicated, all reagents were HPLC grade or better, and water was doubly distilled and deionized. Acrylamide (99% purity), NEM, acrolein, MVK, methyl acrylate (MA), allyl choride, allyl alcohol, propanal, Krebs-Henseleit buffer, urea, SDS, iodoacetamide, bovine serum albumin (fraction v), Protease Inhibitor Cocktail, dithiobis[succinimidyl propionate], and Percoll were purchased from the Sigma/Aldrich Chemical Company (St Louis, MO). Tris (2-carboxylethyl) phosphine (TCEP) was purchased from Bio-Rad (Hercules, CA). 3H-dopamine (DA) (specific activity 23.5 Ci/mmol) was obtained from American Radiolabeled Chemicals (St Louis, MO). Sequencing grade trypsin was from Promega (Madison, WI). C18 ZipTip microcolumns were acquired from Millipore (Bedford, MA). A 15-cm x 75-µM id PepMap C18 column was purchased from LC Packings (San Francisco, CA). A 100 x 4.6-mm id Polysulfoethyl A strong cation exchange column was purchased from PolyLC, Inc. (Coumbia, MD). Antibodies against synaptophysin (mouse monoclonal) and synaptobrevin (VAMP; mouse monoclonal) were from Calbiochem (San Diego, CA) and Chemicon International, Inc. (Temecula, CA), respectively. Antibodies for synaptosomal associated protein of 25 kDa (SNAP-25; rabbit polyclonal) were purchased from Affinity BioReagents (Golden, CO). Antibodies for NSF (rabbit polyclonal) were purchased from Upstate Laboratories (Lake Placid, NY). Whatman GF/F filter paper and Whatman GF/B filter disc were purchased from the Brandel Company (Gaithersburg, MD).
Animals
All aspects of this study were in accordance with the National Institutes of Health (NIH) Guide for Care and Use of Laboratory Animals and were approved by the Montefiore Medical Center Animal Care Committee. Adult male rats (Sprague-Dawley, 300–325 g; Taconic Farms, Germantown, NY) were used in this study. Rats were housed individually in polycarbonate boxes, and drinking water and Purina Rodent Laboratory Chow (Purina Mills, Inc., St Louis, MO) were available ad libitum. The animal room was maintained at approximately 22°C and 50% humidity with a 12-h light/dark cycle.
Preparation of Striatal Synaptosomes and Synaptic Vesicles
The striatum was chosen as a source for synaptosomes and synaptic vesicles based on previous research showing that ACR intoxication of rats was associated with early structural and functional damage to nerve terminals in this brain region (Lehning et al., 2003; LoPachin et al., 2006). Rat brain striatal synaptosomes were isolated by the Percoll gradient method of LoPachin et al. (2004b). In brief, bilateral striata (100–120 mg wet weight tissue) were rapidly removed from anesthetized (isoflurane inhalation) rats and minced in cold (4°C) buffer containing sucrose 0.32M, EDTA 1mM, and dithiothreitol 0.25mM (SED gradient buffer; pH 7.4). Tissue was gently homogenized in SED buffer (10 passes in a Teflon-glass homogenizer; 700 rpm), and the resulting homogenate was centrifuged at 1000 x g (10 min, 4°C). The pellet (P1) was washed once, and supernatants (S1 and S2) were combined. Protein content of the pooled supernatant was determined by the Bradford assay using bovine serum albumin as standard. The protein concentration of the supernatant was adjusted with SED to 5 mg/ml and then layered on top of a freshly prepared four-step discontinuous Percoll gradient (3, 10, 15, and 23% Percoll in SED, pH 7.4). Gradients were centrifuged at 32,000 x g for 6 min, and synaptosomes were collected at the last interface (15%/23%). Synaptosomes were washed twice in Kreb's buffer containing NaCl 140mM, KCl 5mM, NaHCO3 5mM, MgCl2 1mM, NaH2PO4 1.2mM, glucose 10mM, and Hepes 10mM (pH 7.4), pelleted, and then resuspended.
Striatal synaptic vesicles were prepared according to LoPachin et al. (2006). In brief, rat striata were homogenized in ice-cold 0.32M sucrose in a glass homogenizer using 10 strokes of a Teflon pestle. All centrifugation steps were preformed at 4°C. The homogenate was centrifuged at 800 x g for 12 min, and the resulting supernatant was centrifuged at 22,000 x g for 10 min. The P2 crude synaptosomal pellet was subjected to osmotic shock (5 min) by homogenization (five strokes with Teflon pestle) in 2 ml of distilled water. Osmolarity was restored by addition (1 ml) of a HEPES (0.25M)-potassium tartrate (1M) buffer (pH 7.5). The suspension was centrifuged at 20,000 x g for 20 min, and the supernatant was centrifuged at 55,000 x g for 60 min. MgSO4 (1mM) buffer was added to the supernatant, which was then centrifuged at 100,000 x g for 50 min. The P5 pellet, which contained the isolated synaptic vesicles (20–30 µg protein), was resuspended in vesicle assay buffer containing (in mM): HEPES (25), potassium tartrate (100), ascorbic acid (1.7) EGTA (0.5), EDTA (0.1), MgSO4 (2), and KCl (10mM).
In Vitro Effects of Structural Analogs on Synaptosomal and Vesicular Function
We determined the in vitro effects of ,ß-unsaturated carbonyl derivatives on (1) KCl-evoked 3H-DA release from striatal synaptosomes, (2) 3H-DA transport into striatal synaptosomes, and (3) 3H-DA transport into striatal synaptic vesicles.
Synaptosomal release.
Striatal synaptosomes (10 µg protein) were incubated with 3H-DA (0.30µM) for 3 min at 30°C (see LoPachin et al. [2004b] for methodological details). Labeled synaptosomes were trapped on glass fiber filters (Whatman GF/B filter disc) in a 12-well superfusion chamber (Brandell Superfusion 10000 system, Gaitherburg, MD) interfaced with a fraction collector. Synaptosomes were equilibrated (30 min) with oxygenated (95% 02/5% CO2) Krebs-Hepes buffer (0.6 ml/min) and then exposed to control (Krebs-Hepes buffer) solutions or to graded concentrations of chemical for 15 min. Four 2-min fractions (1.2 ml total volume) were then collected as a measure of basal DA efflux. Preliminary studies indicated that toxicant incubation did not affect baseline efflux relative to control (data not shown). Ca2+-dependent, 3H-DA release was stimulated by a 90 s pulse of control (toxicant free) 40mM KCl in modified Krebs buffer. Superfusate fractions from exposed and control synaptosomes were collected throughout the poststimulus period (30 min), and the isotope contents of each fraction and of the corresponding filter-trapped synaptosomes were determined by scintillation counting. 3H-DA release was calculated as percent fractional release, and data are expressed as mean percent of control ± SEM. For each chemical tested, the concentration-response data for release were fitted by nonlinear regression analysis (r2 for all curves > 0.90), and the IC50's (toxicant concentration inhibiting 50% of control response) and respective 95% confidence intervals were calculated by the Cheng-Prusoff equation (Prism, GraphPad Software, San Diego, CA).
Synaptosomal membrane transport.
Striatal synaptosomes (10 µg protein) were incubated with graded concentrations of toxicant (Fig. 1) or Krebs-Hepes buffer for 15 min at 30°C (LoPachin et al., 2004b). Synaptosomes were then washed, filter trapped by rapid filtration through a cell harvester (see above), and superfused (3 min) with Krebs-Hepes buffer containing 3H-DA (0.30µM). To correct for low-affinity Na+-independent transport, uptake was measured in the presence and absence (equimolar choline chloride substitution) of sodium ions. Synaptosomes were then washed, and corresponding radioactivity was measured by scintillation counting. The concentration-response data for transport were fitted by nonlinear regression analysis and the IC50's (95% confidence intervals) were calculated by the Cheng-Prusoff equation (Prism, GraphPad Software).
Vesicular transport.
Synaptic vesicles (3 µg protein) were exposed (15 min) to graded concentrations of toxicant or control assay buffer. Vesicles were then incubated in assay buffer (200 µl) containing Mg2+-ATP (2mM) and 0.30µM 3H-DA for 3 min at 30°C (LoPachin et al., 2006). The uptake reaction was terminated by addition of cold assay buffer (1 ml), and vesicles were collected onto Whatman GF/F glass fiber filters (previously soaked in 0.5% polyethylenimine for 120 min) by rapid filtration through a Brandel cell harvester. Nonspecific uptake was determined by measuring vesicular 3H-DA transport at 4°C in the absence of Mg2+-ATP. Filters were washed, and trapped radioactivity was counted by scintillation spectroscopy. To determine the IC50 for acrolein, the concentration-response data for vesicular transport were fitted by nonlinear regression analysis (r2 for all curves > 0.90). IC50's and respective 95% confidence intervals were calculated by the Cheng-Prusoff equation (Prism, GraphPad Software).
Kinetic Analysis of In Vitro Acrolein Neurotoxicity: Synaptosomal and Vesicular Transport
In separate studies, we determined the effects of in vitro acrolein on the kinetic parameters of DA transport in striatal synaptosomes and synaptic vesicles (for methodological details see LoPachin et al., 2004b, 2006, respectively). Briefly, synaptosomes (10 µg protein) were exposed (15 min x 30°C) to the corresponding IC50 of acrolein (50 µm) or to Krebs-Hepes buffer. Acrolein-exposed and control synaptosomes were washed, filter trapped, and superfused with graded 3H-DA concentrations (50nM–1.7µM). To measure the effects of acrolein on the kinetics of vesicular transport, synaptic vesicles (3 µg protein) were preexposed (15 min x 30°C) to the corresponding IC50 (200µM) and then incubated (3 min) with the graded 3H-DA concentrations (50nM–1.7µM). For these studies, kinetic parameters (Km, Vmax) were determined by nonlinear regression analysis (Prism, GraphPad Software). Respective kinetic data for the control and experimental groups were compared statistically (p < 0.05) by a two-tailed Student t-test (InStat, GraphPad Software).
Measurement of Free Thiols
The concentration-dependent effects of structural analogs (Fig. 1) on free sulfhydryl content in synaptosomes and synaptic vesicles were determined by the method in LoPachin et al. (2004b). Following incubation (15 min) with graded concentrations of toxicant or control buffer solutions, synaptosomes (200 µg protein) or synaptic vesicles (50 µg) were solubilized with 1% SDS (5 min). 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB; 3mM) was added and, following equilibration (5 min, 25°C), absorbance was read at 412 nm using a Jenway 6305 spectrophotometer. A reagent blank without DTNB was used to zero the spectrophotometer. The concentration of 3-carboxylato-4-nitrothiophenolate, the thiol anion released during adduction of sulfhydryl groups by the disulfide reagent DTNB, was calculated by the molar extinction coefficient, 1.36 x 104M–1 cm–1. Sulfhydryl data were calculated as nmol/mg synaptosomal protein, and the toxicant effects were expressed as mean percent of control ± SEM. The free thiol data for each toxicant were fitted by nonlinear regression analysis (r2 for all curves 0.90), and respective IC50's and 95% confidence intervals were calculated by the Cheng-Prusoff equation (Prism 3.0, GraphPad Software). Linear regression analysis was used to assess the relationship between analog-induced changes in synaptosomal free sulfhydryl groups and changes in the measured functional parameters (e.g., vesicular uptake). Corresponding coefficients of determination (r2) were calculated from the Pearson correlation coefficient (InStat 3.0, GraphPad Software).
Western Blot Analysis
Striatal synaptosomes were prepared and exposed (15 min) to graded concentrations of ACR (10mM–1M), acrolein (0.5–100mM), NEM (0.1–10mM), or MVK (0.5–100mM). Synaptosomes were washed, and proteins (20 µg) were resolved by SDS-PAGE and then transferred to nitrocellulose membranes. After transfer, membranes were blocked with 5.0% dried nonfat milk in TBS (Tris-HCl 20mM, NaCl 0.5M, pH 8.3) for 45 min and then rinsed. Membranes were incubated for 2 h at 25°C with the appropriate primary antibody diluted in 5.0% dried milk/TBS: antibodies to NSF (1:2000), SNAP-25 (1:1000), synaptobrevin (1:1000), and synaptophysin (1:1000). Following primary antibody incubation, membranes were washed in TBS and incubated for 1 h at 25°C with an appropriate alkaline phosphatase–conjugated secondary antibody. Membranes were washed with TBS, and bound secondary antibody was visualized with a ProtoBlot II AP system.
Mass Spectrometric Analysis of Adducts in Acrolein-Exposed Synaptosomes
Synaptosomes (200 µg) were incubated with acrolein (1mM) in Krebs buffer (pH 7.0) for 15 min at 37°C. Following incubation, synaptosomal protein was precipitated by adding 9 volumes of acetone acidified to pH 3 with HCl. Samples were stored at –20°C for 30 min to ensure complete protein precipitation followed by centrifugation at 18,000 x g for 20 min at 4°C. The pellet was washed with acetone and resuspended in a buffer containing 0.1% SDS, 200mM Tris-HCl (pH 8.3), 5mM EDTA, and 6M urea. Samples were reduced with TCEP, alkylated with iodoacetamide, and digested with trypsin as described previously (Schrimpf et al., 2005). Resulting peptides were separated into 10 fractions by strong cation exchange (SCX) chromatography (Polysulfoethyl A) on the HPLC using a potassium phosphate gradient (5–400mM). Capillary reverse phase HPLC separation of each SCX fraction was performed on a 15-cm x 75-µm id PepMap C18 column in combination with an Ultimate Capillary HPLC System (LC Packings) operated at a flow rate of 200 nl/min. Inline mass spectrometric analysis of the column eluate was accomplished by a hybrid quadrupole time-of-flight instrument (QSTAR, Applied Biosystems, Foster City, CA) equipped with a nanoelectrospray source. Fragment ion data generated by Information Dependent Acquisition via the QSTAR were searched against the International Protein Index sequence database. Tandem mass spectra were extracted by ABI Analyst version 1.1. Charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK) and X! Tandem (http://www.Thegpm.org). Scaffold (version Scaffold-01-05-06, Proteome Software, Inc., Portland, OR) was used to validate MS/MS–based peptide and protein identification. Peptide identifications were accepted if they could be established at greater than 95% probability as specified by the Peptide Prophet algorithm. Protein identifications were accepted if they could be established at greater than 90% probability and contained at least one identified peptide. Protein probabilities were assigned by the Protein Prophet algorithm. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Acrolein adducts were identified by the addition of 56 amu to an amino acid residue. Adducts were validated by manual interpretation of the MS/MS data.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Effects of Acrolein, ACR, or NEM on Synaptosomal Function and Free Thiol Content
The effects of ACR on synaptosomal function and thiol content have been the focus of previous studies from this laboratory (LoPachin et al., 2004b, 2006
). Therefore, to simplify the presentation of data, the ACR results are provided in Table 1. Figure 2 shows the in vitro effects of acrolein and NEM on three parameters of nerve terminal function presented in blue, that is, synaptosomal neurotransmitter release (Fig. 2A), membrane neurotransmitter uptake (Fig. 2B) and synaptic vesicle transport (Fig. 2C). Parallel measurements of synaptosomal and vesicular free thiol contents are also presented in red.
|
|
Synaptosomal release.
Figure 2A shows that in vitro exposure of striatal synaptosomes to acrolein or NEM produced parallel concentration-dependent decreases in evoked 3H-DA release. The loss of function induced by each toxicant corresponded to graded decreases in synaptosomal free thiol content. In vitro exposure to ACR produced a parallel concentration-dependent decrease in release that was closely correlated to a reduction in synaptosomal thiol content (Table 1; see also LoPachin et al., 2004b). The rank order of the respective concentration curves indicated that NEM and acrolein were significantly more potent than ACR with respect to release inhibition, that is, the respective IC50's are 10µM, 811µM, and 688mM (Table 1).
Synaptosomal transport.
Exposure of striatal synaptosomes to acrolein or NEM resulted in parallel concentration-dependent reductions in membrane 3H-DA transport (Fig. 2B). The inhibition of transport caused by in vitro exposure to these analogs was closely correlated (r2 = 0.91 and 0.96, respectively) to a reduction in synaptosomal free thiol content (Fig. 2B). ACR exposure produced a parallel graded decrease in membrane transport that corresponded (r2 = 0.96) to a graded reduction in sulfhydryl content (Table 1; see also Fig. 4). Acrolein and NEM were of comparable potency (IC50's = 53 vs. 47µM, respectively), and both were significantly more potent than ACR with respect to transport inhibition (IC50 = 438mM; Table 1). Kinetic analysis of the in vitro acrolein effect on synaptosomal transport revealed a statistically significant decrease in Vmax, with no change in Km (Table 2).
|
|
Vesicular transport.
In vitro incubation of synaptic vesicles with acrolein or NEM produced concentration-dependent decreases in 3H-DA transport that were correlated (r2 = 0.90 and 0.94, respectively) to a reduction in vesicular sulfhydryl content (Fig. 2C). ACR incubation caused similar parallel effects on vesicular transport and thiol content (Table 1). The order of potency for inhibition of vesicular transport was NEM (6.2µM)> acrolein (213µM) >> ACR (233mM; Table 1). Kinetic analysis indicated that acrolein significantly decreased Vmax of vesicular transport and increased corresponding Km (Table 2).
These data and results from previous research (LoPachin et al., 2004b, 2006) show that in vitro exposure to the ,ß-unsaturated carbonyl derivatives, acrolein, NEM, or ACR, produced parallel, concentration-dependent inhibitions of synaptosomal function. The inhibitory effects of these structural analogs were closely correlated to reductions in synaptosomal sulfhydryl content. With respect to inhibition of synaptosomal function, NEM was generally more potent than acrolein. Both toxicants, however, were substantially more potent than ACR, that is, compare the respective IC50's for a given parameter (Table 1). Finally, our kinetic analyses revealed that the nerve terminal effects of in vitro acrolein were due to a reduction in Vmax. ACR and NEM also produced similar kinetic changes (LoPachin et al., 2004b, 2006), which together represent noncompetitive inhibition due to irreversible (covalent) protein-chemical interactions (reviewed in LoPachin and DeCaprio, 2005).
Structure-Toxicity Relationships
For a comprehensive analysis of structure-toxicity relationships, we selected 3H-DA transport as a representative synaptosomal parameter and determined the corresponding effects of several conjugated and nonconjugated structural analogs (Figs. 3A and 4A). These functional studies were correlated with measurements of synaptosomal sulfhydryl content (Figs. 3B and 4B). Figure 3 shows that when synaptosomes were exposed to a relatively broad concentration range (1µM–10mM) of nonconjugated analogs, that is, alkenes (allyl chloride and allyl alcohol) or aldehydes (propanal), no changes in either neurotransmitter uptake (Fig. 3A) or free thiol content (Fig. 3B) were observed. With respect to allyl chloride and allyl alcohol, the lack of in vitro effects is in contrast to the documented ability of these alkenes to produce toxicity in exposed laboratory animals (e.g., see Jaeschke et al., 1987; Nagano et al., 1991). Since the induction of in vivo toxicity by allyl chloride or allyl alcohol requires metabolic biotransformation (He et al., 1995; Jaeschke et al., 1987), our finding that these nonconjugated analogs did not affect in vitro synaptosomal activity is consistent with the limited capacity of neurons for xenobiotic metabolism (Minn et al., 1991). Conjugated ,ß-unsaturated analogs, however, produced parallel, concentration-dependent decreases in synaptosomal transport (Fig. 4A). The decreases in neurotransmitter transport induced by each structural congener were highly correlated to corresponding reductions in sulfhydryl content (Fig. 4B), that is, linear regression analysis of synaptosomal uptake versus thiol content revealed the following r2 values: acrolein = 0.91, NEM = 0.96, ACR = 0.96, MA = 0.93, and MVK = 0.96. Acrolein and NEM were similar in relative potency, whereas ACR and MA were least potent, that is, compare the respective IC50's in Figures 4A and 4B. MVK was slightly less potent when compared to acrolein (Figs. 4A and 4B). Although differences in potency are evident, all conjugated analogs exhibited comparable neurotoxic efficacy, that is, each was capable of producing maximal inhibitions of the measured neurochemical parameters and correspondingly depleted thiol contents (e.g., Fig. 2). These structure-toxicity data indicate that the synaptosomal toxicity of the ,ß-unsaturated derivatives (ACR, acrolein, NEM, MVK, MA) is related to their common type-2–conjugated alkene structure, that is, nonconjugated alkenes or aldehydes did not cause toxicity. These data also indicate that loss of synaptosomal free thiols is importantly involved in the inhibition of neurotransmitter uptake induced by these analogs.
|
Cross-Link Formation by Acrolein and Structural Congeners
Acrolein and other
,ß-unsaturated carbonyls are bifunctional electrophiles and, therefore, initial adduct formation at the ß-carbon atom might be followed by attack from a neighboring nucleophile at the reactive carbonyl carbon atom resulting in the formation of protein cross-links (Esterbauer et al., 1991; Kurtz and Lloyd, 2003). To identify cross-links that might form during our study of synaptosomal function, the presence of higher molecular weight derivatized proteins was detected by electrophoretic separation and immunoblot analysis. This approach has been used in previous studies of the neurofilament cross-links formed during 2,5-hexanedione intoxication (e.g., see LoPachin et al., 2004a). Results show that exposure of synaptosomes to lower concentrations of acrolein (0.5–10mM) did not affect the migration of four nerve terminal proteins (Figs. 5 and 6): SNAP-25 (Fig. 5A), synaptophysin (Fig. 5B), VAMP (Fig. 6A, synaptobrevin), or NSF (Fig. 6B). However, altered migration patterns were evident for all four proteins in synaptosomes exposed to a higher acrolein concentration (100mM; Figs. 5 and 6). Slower migrating, presumably higher molecular weight, derivatives of native SNAP-25 and synaptophysin were evident at acrolein concentrations 25mM. The migration of VAMP and NSF was completely truncated in synaptosomes exposed to these higher acrolein concentrations (Fig. 6). In contrast to acrolein, high concentrations of NEM (10mM), ACR (1M), or MVK (100mM) did not affect protein migration (Fig. 7). Hydralazine is a "protein adduct-trapping" drug that has been shown to offer some protection against the production of in vitro hepatocyte toxicity by acrolein (Kaminskas et al., 2004). Therefore, in the present studies, we also determined the ability of hydralazine to prevent protein cross-links and acrolein synaptosomal toxicity. Hydralazine (1mM) alone did not alter synaptosomal protein migration (Figs. 5 and 6) or thiol content (data not shown), whereas it did reduce control 3H-DA transport by 40% (data not shown). Our research indicates that hydralazine did not abolish in vitro acrolein neurotoxicity, that is, hydralazine (1mM) did not prevent the formation of higher molecular weight protein derivatives (Figs. 5 and 6) and it potentiated, rather than reduced, the inhibitory effect of acrolein on synaptosomal membrane transport (Fig. 8).
|
|
|
|
Mass Spectrometric Detection of Adducts in Acrolein-Exposed Synaptosomes
Two-dimensional chromatographic separation of synaptosomal peptides resulted in positive identification of 207 proteins. The majority of acrolein adducts were on cysteine residues of proteins such as Na/K-ATPase alpha 3; malate dehydrogenase 2 (Fig. 9), 14-3-3 protein, glyceraldehyde-3-phosphate dehydrogenase, citrate synthase, fructose bisphosphate aldolase, guanine nucleotide–binding protein ß-subunit, succinyl coA ligase, and tubulin ß4. Acrolein adducts of lysine or histidine were also identified on several proteins. These MS data, in conjunction with our previous analysis of ACR adduct formation (Barber and LoPachin, 2004), demonstrate that conjugated type-2 alkenes can form thiol adducts and that this accounts for the loss of free sulfhydryl groups detected in the respective DTNB assays (e.g., see Fig. 4B).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Results of the present structure-toxicity analyses indicate that exposure of striatal synaptosomes to ACR or its conjugated analogs produced parallel concentration-dependent decreases in several neurochemical parameters. For each active congener, the neurotoxic effect was highly correlated to graded decreases in synaptosomal free thiol content. In contrast, nonconjugated analogs did not cause changes in either synaptosomal function or sulfhydryl groups. This lack of neurotoxic effect is consistent with previous studies of propionamide (CH3CH2CONH2), a nonconjugated ACR analog that did not affect synaptosomal function or thiol content (LoPachin et al., 2004b, 2006
). Together, these data indicate that the ,ß-unsaturated carbonyl structure of ACR and other type-2 alkenes is responsible for the corresponding in vitro neurotoxicity. The pi electrons of this conjugated structure are mobile and, as a consequence, the type-2 alkene is a soft (polarizable) electrophile that will form adducts with soft (polarizable) nucleophiles via the Michael addition reaction (Friedman, 1973; Kemp and Vellaccio, 1980). In biological systems, sulfhydryl groups are the preferential soft nucleophilic targets of ACR and other conjugated alkenes (Calleman, 1996; Coles, 1984–85; Esterbauer et al., 1991; Friedman, 1973; Hinson and Roberts, 1992; LoPachin and DeCaprio, 2005; Witz, 1989). This is substantiated by our present findings that ACR, acrolein, and other conjugated type-2 analogs reduced the free sulfhydryl content of exposed synaptosomes (see also LoPachin et al., 2004b). In addition, mass spectrometric analyses have shown that ACR (Barber and LoPachin, 2004; Bordini et al., 2000; Hall et al., 1993) and acrolein (present study) can alkylate protein thiol groups. In nonconjugated alkene (e.g., allyl alcohol) or aldehyde systems (i.e., propanal), the pi electrons are not part of a resonance system and are, instead, restricted to the alkene or carbonyl functions, respectively. The carbonyl carbon of the aldehyde is a relatively hard electrophile, whereas alkenes are not electrophilic and neither group is, therefore, likely to function as a Michael acceptor for thiol adduction. Predictably, the nonconjugated compounds used in this study did not alter synaptosomal free thiol content or 3H-DA transport (Fig. 3).
In contrast to thiol selectivity, some previous research has emphasized the formation of lysine adducts (e.g., FDP-lysine, bis-Michael adducts) by acrolein and other ,ß-unsaturated carbonyls (Kaminskas et al., 2005; Uchida et al., 1999). The initial step of these reactions involves nucleophilic addition of the lysine amino group at the double bond (C-ß). However, acrolein is a soft electrophile, whereas the 
-amino group of the lysine residue is a relatively hard nucleophile and, although a Michael addition between a soft electrophile and a hard nucleophile can occur, it is not a favored reaction (Coles, 1984–85; Hinson and Roberts, 1992). This is consistent with our current mass spectrometric findings that, although acrolein formed lysine adducts, they were not prevalent. Because ,ß-unsaturated carbonyls are bifunctional (i.e., electrophilic reactivity at the ß-carbon and carbonyl carbon atoms), acrolein and other chemicals in this class could disrupt synaptosomal activity by cross-linking proteins rather than by forming thiol monoadducts (Esterbauer et al., 1991, Kurtz and Lloyd, 2003). However, our immunoblot analyses (Figs. 5–7
) and studies with pharmacological cross-link blockers (Fig. 8) do not support a mechanistic role for protein cross-links in the production of alkene synaptosomal toxicity.
Results of this study show that the conjugated type-2 toxicants differed significantly in relative potency. This difference is likely ascribed to variations in the degree of relative "softness" among the conjugated carbonyl compounds used. Specifically, the "hard and soft, acids and bases" (HSAB; Pearson and Songstad, 1967) theory suggests that soft-soft interactions can be described by frontier orbital interactions. Thus, with respect to thiol adduct formation, electrons from the highest occupied molecular orbital (HOMO) of the sulfhydryl group (donor) will interact with the lowest unoccupied molecular orbital (LUMO) of, for example, the ß-alkene carbon of ACR (acceptor). The ease (rate of reaction) of adduct formation is determined by the extent to which the respective frontier orbitals correspond energetically, that is, HOMO energy of the thiol group versus LUMO energy of the ,ß-unsaturated carbonyl. Based on the HSAB theory, the observed rank order of in vitro potencies among the type-2 alkenes (see Fig. 4) should be related to the respective LUMO energies or degree of relative softness. Indeed, in preliminary studies Gavin, Geohagen and LoPachin (unpublished data), we have used a commercially available program for quantum chemical calculations (Spartan; Wavefunction, Inc., Irvine, CA) to calculate the respective LUMO energies of the alkenes and their nonconjugated analogs. Our initial results (Table 3) indicate that the LUMO energies for this chemical series are directly related to corresponding abilities to deplete synaptosomal thiols and impair function. Thus, defining LUMO-toxicity relationships could provide a mechanistic explanation, at the atomic level, for the relative in vitro differences in type-2 alkene neurotoxic potencies. Whereas the conjugated toxicants differed in potencies, their respective abilities to produce maximal inhibitions (efficacy) of synaptosomal or vesicular function were equivalent (e.g., see Figs. 2 and 4). This means that regardless of the electrophile (e.g., ACR or acrolein), subsequent formation of thiol adducts results in equivalent pathophysiological consequences, that is, inhibition of synaptosomal 3H-DA transport (see LoPachin and DeCaprio, 2005 for additional discussion).
|
Although the degree of polarizability (softness) might be the chemical basis for the low electrophilic reactivity of ACR, the relatively high (mM) concentrations needed in this study to produce in vitro neurotoxicity are in contrast to the predicted micromolar tissue concentrations achieved during in vivo intoxication (e.g., see Martenson et al., 1995
). However, in accordance with the mass action kinetics of chemicals with lower reactivity, higher ACR (or MA) concentrations (mM) are required during acute (15 min) in vitro exposures to drive adduct formation and initiate synaptosomal toxicity. That the adducts formed in vitro have neurotoxicological relevance is indicated by our mass spectrometric analyses which showed that the thiol adduct content of ACR-exposed synaptosomes was highly correlated (r2 = 0.99) to the induction of neurotoxicity (Barber and LoPachin, 2004; LoPachin et al., 2006). The higher concentrations of ACR and MA used in vitro are, therefore, in accordance with their relatively low electrophilic reactivities. The lower adduct potency of ACR is also consistent with the cumulative neurotoxicity produced by in vivo intoxication, that is, the progression of neurological deficits in rats requires daily exposure to relatively high-dose rates (e.g., 10–50 mg/kg) for extended durations (e.g., 90–11 days; LoPachin et al., 2004b). Proteomic analyses have indicated that the formation of protein thiol adducts in nerve terminals is also cumulative and highly correlated to the onset and development of neurotoxicity in ACR-intoxicated rats (Barber and LoPachin, 2004; LoPachin et al., 2004b). These data indicate that, although adduct potency is relatively low, cumulative adduction of sulfhydryl groups is an important neuropathogenic component of ACR neurotoxicity. Thus, the characteristic in vitro and in vivo toxicodynamics of ACR are clearly related to lower electrophilic reactivity (see more detailed discussion in LoPachin and DeCaprio, 2005).
It is not known how thiol adduct formation mediates synaptosomal dysfunction. One possibility is that ,ß-unsaturated carbonyl derivatives deplete glutathione and other nonprotein reducing equivalents and thereby promote secondary oxidative stress (e.g., Dixit et al., 1984; Luo and Shi, 2005; Pocernich et al., 2001; Shivakumar and Ravindranath, 1992). Whereas this could be a contributing factor, other evidence indicates that the toxicity of ACR and type-2 congeners is more complex and that the formation of protein adducts is critically involved (e.g., Barber and LoPachin, 2004; Beiswanger et al., 1993; Biswal et al., 2003; Kaminskas et al., 2004; Ku and Billings, 1986; LoPachin et al., 2006; Park et al., 2002; Patel and Block, 1993; reviewed in Kehrer and Biswal, 2000; Reed, 1990). That chemical alkylation of protein thiol groups is likely to have significant functional consequences is suggested by increasing evidence that the redox states of specific cysteine sulfhydryl groups regulate the activities of many rate-limiting nerve terminal proteins (e.g., Cys 264 of NSF). Therefore, adduction of these essential thiols is likely to inhibit the activities of proteins that regulate important synaptic processes (reviewed in LoPachin and DeCaprio, 2005; LoPachin and Barber, 2006).
Our previous research has indicated that in vivo or in vitro exposure to ACR inhibits nerve terminal function and that this effect is mediated by formation of protein thiol adducts as revealed by mass spectrometric analyses and measurements of free sulfhydryl content (Barber and LoPachin, 2004; LoPachin et al., 2004b, 2006). These data support our original hypothesis that ACR alkylation of essential thiol groups impairs the function of proteins that regulate the synaptic vesicle cycle and other presynaptic processes (LoPachin et al., 2002, 2003). The current results also support our hypothesis and provide additional mechanistic information that has led to a better understanding of ACR adduct chemistry and resulting neurotoxicity. Specifically, we have shown that a series of structural analogs can produce synaptosomal dysfunction and thiol loss that parallel the effects of in vitro ACR exposure. These data emphasize the pathophysiological relevance of sulfhydryl adduction and show clearly that such adduct formation is a product of the conjugated type-2 system of ACR and other ,ß-unsaturated carbonyl derivatives. In a broader sense, our in vitro findings suggest that conjugated alkenes, as a chemical class, could produce neurotoxicity via a common molecular mechanism: thiol adduction and subsequent nerve terminal dysfunction. However, it should be noted that the in vivo neurotoxicity of acrolein and other conjugated ,ß-unsaturated carbonyl and acrylic acid derivatives (e.g., MVK, MA, acrylonitrile) has not been adequately documented. Such research is clearly important and the results could have significant economic and regulatory implications based on the broad industrial applications of type-2 alkenes (Beauchamp et al., 1985; Witz, 1989). With respect to risk management, our results could provide a rational basis for predicting the human neurotoxic potential of these and structurally related chemicals. Finally, a growing body of evidence suggests that presynaptic dysfunction in the hippocampus and certain other brain regions are responsible for the early memory loss that characterizes Alzheimer's disease (AD; e.g., see Coleman and Yao, 2003). Results from other research suggest that endogenous generation of neurotoxic type-2 alkenes (i.e., acrolein, 4-hydroxy-2-nonenal) secondary to lipid peroxidation might be a common pathogenic mechanism of AD (Uchida, 1999). If the effects of in vitro acrolein on synaptosomal neurotransmitter release, uptake, and vesicular storage mirror its in vivo neurotoxic actions, our data implicate an AD mechanism involving acrolein-induced synaptic toxicity. Such a mechanism would be susceptible to aggravation by environmental or occupational exposure to ACR or other conjugated alkene chemicals.
|
ACKNOWLEDGMENTS |
|---|
Research presented in the manuscript was supported by an NIH grant from the National Institute of Environmental Health Sciences (RO1 ES03830-20). The authors would like to express their sincere thanks to Dr Joseph Ross (Ross Toxicology) and Dr Lisa Opanashuk (University of Rochester) for their helpful comments and criticisms during the preparation of the manuscript.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Barber DS and LoPachin RM. (2004) Proteomic analysis of acrylamide-protein adduct formation in rat brain synaptosomes. Toxicol. Appl. Pharmacol. 201:120–136.[CrossRef][ISI][Medline]
Beauchamp RO, Andjelkovich DA, Kligerman AD, Morgan KT, Heck Hd'A. (1985) A critical review of the literature on acrolein toxicity. In Golberg L (Ed.). Critical Reviews in Toxicology. Vol. 14 (4) pp. 309–380.[Medline]
Beiswanger CM, Mandella RD, Graessle TR, Reuhl KR, Lowndes HE. (1993) Synergistic neurotoxic effects of styrene oxide and acrylamide: Glutathione-independent necrosis of cerebellar granule cells. Toxicol. Appl. Pharmacol. 118:233–244.[CrossRef][ISI][Medline]
Biswal S, Maxwell T, Rangasamy T, Kehrer JP. (2003) Modulation of benzo[a]pyrene-induced p53 activity by acrolein. Carcinogenesis 24:1401–1406.
Bordini E, Hamdan M, Righette PG. (2000) Probing acrylamide alkylation sites in cysteine-free proteins by matrix-assisted laser desorption/ionisation time-of-flight. Rapid Commun. Mass Spectrom. 14:840–848.[CrossRef][ISI][Medline]
Calleman CJ. (1996) The metabolism and pharmacokinetics of acrylamide: Implications for mechanisms of toxicity and human risk estimation. Drug Metab. Rev. 28:527–590.[ISI][Medline]
Coleman PD and Yao PJ. (2003) Synaptic slaughter in Alzheimer's disease. Neurobiol. Aging 24:1023–1027.[CrossRef][ISI][Medline]
Coles B. (1984–1985) Effects of modifying structure on electrophilic reactions with biological nucleophiles. Drug Metab. Rev. 15:1307–1334.
Dixit R, Das M, Seth PK, Mukhtar H. (1984) Interaction of acrylamide with glutathione in rat erythrocytes. Toxicol. Lett. 23:291–298.[CrossRef][ISI][Medline]
Esterbauer H, Schaur RJ, Zollner H. (1991) Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic. Biol. Med. 11:81–128.[CrossRef][ISI][Medline]
Friedman M. (1973) Nucleophilic additions. The Chemistry and Biochemistry of the Sulfhydryl Group in Amino Acids, Peptides and proteins(Pergamon Press, New York) pp. 88–134 Chapter 4.
Hall SC, Smith DM, Masiarz RR, Soo VW, Tran HM, Epstein LB, Burlinggame AL. (1993) Mass spectrometric and Edman sequencing of lipocortin I isolated by two-dimensional SDS/PAGE of human melanoma lysates. Proc. Natl. Acad. Sci. U.S.A. 90:1927–1931.
Hashimoto K and Aldridge WN. (1970) Biochemical studies on acrylamide, a neurotoxic agent. Biochem. Pharmacol. 19:2591–2604.[CrossRef][ISI][Medline]
He Y, Nagano M, Yamamoto H, Miyamoto E, Futatsuka M. (1995) Modifications of neurofilament proteins by possible metabolites of allyl chloride in vitro. Drug Chem. Toxicol. 18:315–331.[ISI][Medline]
Hinson JA and Roberts DW. (1992) Role of covalent and noncovalent interactions in cell toxicity: Effects on proteins. Annu. Rev. Pharmacol. Toxicol. 32:471–510.
Jaeschke H, Kleinwaechter C, Wendel A. (1987) The role of acrolein in allyl alcohol-induced lipid peroxidation and liver cell damage in mice. Biochem. Pharmacol. 36:51–57.[CrossRef][ISI][Medline]
Kaminskas LM, Pyke SM, Burcham PC. (2004) Strong protein adduct trapping accompanies abolition of acrolein-mediated hepatotoxicity by hydralazine in mice. J. Pharmacol. Exp. Toxicol. 310:1003–1010.
Kaminskas LM, Pyke SM, Burcham PC. (2005) Differences in lysine adduction by acrolein and methyl vinyl ketone: Implications for cytotoxicity in cultured hepatocytes. Chem. Res. Toxicol. 18:1627–1633.[CrossRef][ISI][Medline]
Kehrer JP and Biswal SS. (2000) The molecular effects of acrolein. Toxicol. Sci. 57:6–15.
Kemp DS and Vellaccio F. (1980) Conjugated alkenes. Organic Chemistry(Worth Publishers, New York) pp. 650–684 Chapter 19.
Ku RH and Billings RE. (1986) The role mitochondrial glutathione and cellular protein sulfhydryls in formaldehyde toxicity in glutathione-depleted rat hepatocytes. Arch. Biochem. Biophys. 247:183–189.[CrossRef][ISI][Medline]
Kurtz AJ and Lloyd RS. (2003) 1,N2-deoxyguanosine adducts of acrolein, crotonaldehyde and trans-4-hydryoxynonenal cross-link to peptides via Schiff base linkage. J. Biol. Chem. 278:5970–5976.
Lapin EP, Weissbarth S, Maker HS, Lehrer GM. (1982) The sensitivities of creatine and adenylate kinases to the neurotoxins acrylamide and methyl n-butyl ketone. Environ. Res. 28:21–31.[Medline]
Lehning EJ, Balaban CD, Ross JF, LoPachin RM. (2003) Acrylamide neuropathy. III. Spatiotemporal characteristics of nerve cell damage in rat forebrain. Neurotoxicology 24:125–136.[CrossRef][ISI][Medline]
LoPachin RM, Balaban CD, Ross JF. (2003) Acrylamide axonopathy revisited. Toxicol. Appl. Pharmacol. 188:135–153.[CrossRef][ISI][Medline]
LoPachin RM and Barber DS. (2006) Synaptic cysteine sulfhydryl groups as targets of electrophilic neurotoxicants. Toxicol. Sci. (in press).
LoPachin RM, Barber DS, He D, Das S. (2006) Acrylamide inhibits dopamine uptake in rat striatal synaptic vesicles. Toxicol. Sci. 89:224–234.
LoPachin RM and DeCaprio AP. (2005) Protein adduct formation as a molecular mechanism in neurotoxicity. Toxicol. Sci. 86:214–225.
LoPachin RM, He D, Reid ML, Opanashuk LA. (2004a) 2,5-Hexanedione-induced changes in the monomeric neurofilament protein content of rat spinal cord fractions. Toxicol. Appl. Pharmacol. 198:61–73.[CrossRef][ISI][Medline]
LoPachin RM, Ross JF, Lehning EJ. (2002) Nerve terminals as the primary site of acrylamide action: A hypothesis. Neurotoxicology 23:43–60.[CrossRef][ISI][Medline]
LoPachin RM, Schwarcz AI, Gaughan CL, Mansukhani S, Das S. (2004b) In vivo and in vitro effects of acrylamide on synaptosomal neurotransmitter uptake and release. Neurotoxicology 25:349–363.[CrossRef][ISI][Medline]
Luo J and Shi R. (2005) Acrolein induces oxidative stress in brain mitochondria. Neurochem. Int. 46:243–252.[CrossRef][ISI][Medline]
Martenson CH, Sheetz MP, Graham DG. (1995) In vitro acrylamide exposure alters growth cone morphology. Toxicol. Appl. Pharmacol. 131:119–129.[CrossRef][ISI][Medline]
Minn A, Ghersi-Egea J-F, Perrin R, Leininger B, Siest G. (1991) Drug metabolizing enzymes in the brain and cerebral microvessels. Brain Res. Rev. 16:65–82.[CrossRef][Medline]
Nagano M, Hitoshi T, Futatsuka M. (1991) Neurotoxicity of allyl chloride in rats. Dose-effect relationship following long-term exposure. Jpn. J. Ind. Health 33:73–81.
Park J, Kamendulis LM, Friedman MA, Klaunig JE. (2002) Acrylamide-induced cellular transformation. Toxicol. Sci. 65:177–183.
Patel JM and Block ER. (1993) Acrolein-induced injury to cultured pulmonary artery endothelial cells. Toxicol. Appl. Pharmacol. 122:46–53.[CrossRef][ISI][Medline]
Pearson RG and Songstad J. (1967) Application of the principle of hard and soft acids and bases to organic chemistry. J. Am. Chem. Soc. 89:1827–1836.[CrossRef]
Pocernich CB, Cardin AL, Racine CL, Lauderback CM, Butterfield DA. (2001) Glutathione elevation and its protective role in acrolein-induced protein damage in synaptosomal membranes: Relevance to brain lipid perioxidation in neurodegenerative disease. Neurochem. Int. 39:141–149.[CrossRef][ISI][Medline]