ToxSci Advance Access originally published online on October 5, 2005
Toxicological Sciences 2006 89(1):224-234; doi:10.1093/toxsci/kfj005
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Acrylamide Inhibits Dopamine Uptake in Rat Striatal Synaptic Vesicles
* Department of Anesthesiology, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York 10467; and Center for Environmental and Human Toxicology, University of Florida, Gainesville, Florida 32611-0885
1 To whom correspondence should be addressed at Montefiore Medical Center, Moses Research Tower-7, 111 E. 210th St., Bronx, NY 10467. Fax: (718) 515-4903. E-mail: lopachin{at}aecom.yu.edu.
Received July 25, 2005; accepted September 24, 2005
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ABSTRACT |
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Evidence suggests that acrylamide (ACR) neurotoxicity is mediated by decreased presynaptic neurotransmitter release. Defective release might involve disruption of neurotransmitter storage, and therefore, we determined the effects of in vivo and in vitro ACR exposure on 3H-dopamine (DA) transport into rat striatal synaptic vesicles. Results showed that vesicular DA uptake was decreased significantly in rats intoxicated at either 50 mg/kg/day x 5 days or 21 mg/kg/day x 21 days. ACR intoxication also was accompanied by a reduction in KCl-evoked synaptosomal DA release, although consistent changes in presynaptic membrane transport were not observed. Silver stain and immunoblot analyses suggested that reduced vesicular uptake was not due to active nerve terminal degeneration or to a reduction in the synaptic vesicle content of isolated striatal synaptosomes. Nor did the in vivo presynaptic effects of ACR involve changes in synaptosomal glutathione concentrations. In vitro exposure of striatal vesicles showed that both ACR and two sulfhydryl reagents, N-ethylmaleimide (NEM) and iodoacetic acid (IAA), produced concentration-dependent decreases in 3H-DA uptake. Although ACR was significantly less potent than either NEM or IAA, all three chemicals caused comparable maximal inhibitions of vesicular uptake. Kinetic analysis of DA uptake showed that in vitro exposure to either ACR or NEM decreased Vmax and increased Km. Determination of radiolabel efflux from 3H-DA-loaded vesicles indicated that in vitro ACR did not affect neurotransmitter retention. These data suggest that ACR impaired neurotransmitter uptake into striatal synaptic vesicles, possibly by interacting with sulfhydryl groups on functionally relevant proteins. The resulting disruption of neurotransmitter storage might mediate defective presynaptic release.
Key Words: acrylamide; toxic axonopathy; nerve terminal; synaptic vesicles; neurotransmitter uptake; adduct formation.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionsREFERENCES |
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Acrylamide (ACR) is a water-soluble, vinyl monomer that is used to manufacture polyacrylamides with different physical and chemical properties. These polymers are used extensively in various chemical industries (e.g., water and wastewater management, ore processing, and dye synthesis) and in laboratories for the electrophoretic separation of macromolecules. ACR monomer is also a contaminant formed during the high-temperature preparation of certain potato- or grain-based foods. Although the polymer is not toxic, monomeric ACR produces ataxia and skeletal muscle weakness in occupationally exposed humans and in experimental animal models (Gold and Schaumburg, 2000; LoPachin et al., 2003
). Substantial evidence now suggests that ACR neurotoxicity is mediated by injury to nerve terminals and cerebellar Purkinje cells (reviewed in LoPachin, 2004; LoPachin et al., 2002a, 2003). Although the mechanisms of these effects are not well understood, damage at both sites is characterized by a buildup of tubulovesicular profiles (Cavanagh and Gysbers, 1983; DeGrandchamp et al., 1990; Tsujihata et al., 1974). In addition, the number of docked synaptic vesicles is reduced at the presynaptic active zones of CNS and PNS synapses (DeGrandchamp et al., 1990; Prineas, 1969; Tsujihata et al., 1974). These ultrastructural changes suggest that ACR interferes with the fusion of vesicles at corresponding cognate membrane sites (Lin and Scheller, 2000). Although the molecular mechanism of this presumed ACR effect is unknown, membrane fusion processes such as neurotransmission are highly sensitive to inhibition by sulfhydryl alkylation (Lonart and Sudhof, 2000). Since ACR is a soft electrophile that forms adducts with nucleophilic sulfhydryl groups on protein cysteine residues (Barber and LoPachin, 2004; Cavins and Friedman, 1968; LoPachin et al., 2004), adduction of functionally important proteins in nerve cells and terminals could produce fusion-incompetent vesicles (e.g., synaptic or Golgi-derived vesicles). Subsequent impairment of membrane fusion could promote neuronal injury and/or presynaptic dysfunction.
As a model system for the study of membrane fusion processes, we have determined the effects of ACR on nerve terminal exocytosis. Neurotransmitter release involves Ca2+-stimulated fusion of synaptic vesicles with corresponding presynaptic membrane targets. Membrane fusion leads to transmembrane pore formation and the release of vesicular contents (Stevens, 2003). In previous studies, in vivo and in vitro ACR exposure significantly decreased KCl-evoked, Ca2+-dependent glutamate release from rat brain synaptosomes (LoPachin et al., 2004). These data are consistent with results from earlier electrophysiological studies, which showed that ACR intoxication inhibited neurotransmission at CNS and PNS synapses (Goldstein and Lowndes, 1979, 1981; Tsujihata et al., 1974). Recent proteomic and biochemical determinations suggested that ACR formed cysteine adducts on nerve terminal proteins, some of which (e.g., N-ethylmaleimide sensitive factor, synaptosomal-associated protein of 25 kDa) are critically involved in the presynaptic membrane fusion process (Barber and LoPachin, 2004; LoPachin et al., 2004). Based on this evidence, we have hypothesized that ACR impairs membrane fusion by adduction of proteins that play a role in this process (LoPachin, 2004, LoPachin et al., 2002a, 2003). However, ACR forms cysteine adducts with many nerve terminal proteins (Barber and LoPachin, 2004; LoPachin et al., 2004), and consequently, other pathophysiologically relevant targets might exist. ACR adduction and inhibition of these additional protein targets could contribute to the failure of neurotransmission. In this regard, the loading of transmitter into synaptic vesicles is critically important to presynaptic release. This process relies upon the activity of a vacuolar proton pump (v-ATPase) that establishes an electrochemical proton gradient (
µH+) for carrier-mediated transport of neurotransmitter. v-ATPase activity is highly sensitive to inhibition by sulfhydryl reagents (Fernandez-Chacon and Sudhof, 1999) and is, therefore, a potential target for ACR. Disruption of vesicular storage could explain the reduction in presynaptic neurotransmitter release observed in previous studies of ACR neurotoxicity (LoPachin et al., 2004). Based on this precedence, we have determined the effects of in vivo and in vitro ACR exposure on the uptake of neurotransmitter into synaptic vesicles. In our study, the transport of 3H-dopamine (DA) was measured in synaptic vesicles isolated from striatum of ACR-intoxicated and control rats. The striatum exhibits significant damage in ACR-intoxicated rats (Lehning et al., 2003), and the kinetics of vesicular 3H-DA have been well characterized in this brain region of rodents (Teng et al., 1998).
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MATERIALS AND METHODS |
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Chemicals and materials.
Unless otherwise stated, all reagents were HPLC grade or better, and water was doubly distilled and deionized. Acrylamide (99% purity), N-ethylmaleimide and iodoacetic acid (98% purity), 2,4-dinitrofluorobenzene, and propionamide were all purchased from the Sigma Chemical Company (St. Louis, MO). 3H-dopamine (specific activity 23.5 Ci/mmol) was obtained from American Radiolabeled Chemicals (St. Louis, MO). Antibodies to synaptobrevin (VAMP; mouse monoclonal) were obtained from Chemicon International, Inc (Temecula, CA). For HPLC determination of glutathione, a Phenosphere NH2 amino column was purchased from Phenomenex, Torrance, CA. Whatman GF/F filter paper and Whatman GF/B filter disc were purchased from the Brandel Company (Gaithersburg, MD). The ProtoBlot II AP system was purchased from Promega (Madison, WI).
Animals and ACR intoxication.
All aspects of this study were in accordance with the 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. Unless otherwise stated, randomly assigned groups of animals (six rats/exposure group) were exposed to ACR at daily dose-rates of either 50 mg/kg/day (ip) or 21 mg/kg/day (po). These daily dose-rates and corresponding routes have been well characterized with respect to neuropathological expression (quantitative morphometrics and silver stain analysis), neurological deficits (gait, grip strength, hindlimb extensor thrust, foot splay), and toxicokinetics (Barber et al., 2001; Lehning et al., 1998, 2002a,b, 2003; LoPachin et al., 2002a). In the present study, body weight and gait scores were determined 2–3 times per week as indices of developing neurotoxicity. Gait scoring involved observation of spontaneous open field locomotion, which included evaluations of ataxia, hopping, rearing, and hindfoot placement (LoPachin et al., 2002a). To assess locomotion, rats were placed in a clear plexiglass box (90 x 90 cm) and were observed for 3 min. Following observations, a gait score was assigned from 1 to 4 where: 1 = a normal gait; 2 = a slightly abnormal gait (slight ataxia, hopping gait, and foot splay); 3 = moderately abnormal gait (obvious ataxia and foot splay with limb abduction during ambulation); 4 = severely abnormal gait (inability to support body weight and foot splay). For each ACR dosing regimen, groups of age-matched control rats (n = 6 rats/group) were weighed, and gait scores were determined. Control rats for the higher ACR dose-rate group received daily ip injections of 0.9% saline (3 ml/kg). A trained, blinded observer who was not involved in animal care or ACR exposure performed the testing.
Preparation of striatal synaptic vesicles for in vivo or in vitro ACR experiments.
Striatal synaptic vesicles were prepared according to a modification of Teng et al. (1998). In brief, rat striata (100–120 mg wet wt. tissue) were homogenized in ice-cold 0.32 M 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 (5 strokes with Teflon pestle) in 2 ml of distilled water. Osmolarity was restored by addition (1 ml) of a HEPES (0.25 M)-potassium tartrate (1 M) 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 (1 mM) 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 (10).
Vesicular 3H-dopamine uptake and spontaneous efflux.
Vesicular dopamine (DA) uptake was determined following in vivo or in vitro ACR exposure by incubating 3 µg of synaptic vesicle protein in assay buffer (200 µl) containing Mg2+-ATP (2 mM) and 3H-DA (see ahead) for 3 min at 30°C (Teng et al., 1998). 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 (Gaithersburg, MD). Nonspecific uptake was determined by measuring vesicular 3H-DA transport at 4°C in the absence of ATP. Filters were washed, and trapped radioactivity was counted by scintillation spectroscopy.
For kinetic analysis of DA uptake in synaptic vesicles isolated from striata of ACR-intoxicated rats or their age-matched controls (Fig. 1), aliquots of vesicles (3 µg protein) were incubated over a range of 3H-DA concentrations (50 nM–1.7 µM, final concentrations), and corresponding radioactivity content was determined. ACR intoxication did not significantly alter the yield of striatal synaptic vesicle protein (mean ± SEM); e.g., control = 55.9 ± 3.6 µg protein versus 50 mg/kg/day ACR = 61.3 ± 5.1 µg protein. To measure the effects of in vitro ACR exposure on the kinetic parameters of uptake (Fig. 8), synaptic vesicles were isolated from control rat brains, preexposed (3 min) to the IC50 (see ahead) of ACR (243 mM) and then incubated (3 min) with the graded 3H-DA concentrations (50 nM–1.7 µM). For comparative purposes, kinetic parameters were determined in control synaptic vesicles exposed in vitro to the IC50 of N-ethylmaleimide (NEM; 6.2 µM). For these studies, kinetic parameters (Km, Vmax) were determined by nonlinear regression analysis (PrismTM, GraphPad Software, San Diego, CA). Respective kinetic data for the control and experimental groups were compared statistically (p < 0.05) by a two-tailed Student's t-test (InStatTM, GraphPad Software, San Diego, CA).
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To determine the IC50 concentrations for ACR, NEM, and an additional sulfhydryl reagent, iodoacetic acid (IAA), synaptic vesicles were isolated from control rat brains. Vesicles were preexposed (3 min) to graded concentrations of each chemical (ACR = 0.015–2.0M; NEM = 100 nM–1 µM; IAA= 10 µM–100 mM) and then incubated with 0.30 µM 3H-DA for an additional 3 min. Resulting curves for each chemical (Fig. 7) were fitted by nonlinear regression analysis (r2 for all curves >0.98) and the IC50's and respective 95% confidence intervals were calculated by the Cheng-Prusoff equation and were compared statistically by Student's t-test (PrismTM, GraphPad Software, San Diego, CA). Synaptic vesicles were also exposed in vitro to propionamide (0.25–1.0 M), a structural analog of ACR that lacks the C2-C3 double bond (CH3CH2CONH2).
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To determine the effects of in vitro ACR exposure on DA efflux, synaptic vesicles were prepared from control rat striata and then incubated in assay buffer containing Mg2+ ATP (2 mM) and 3H-DA (0.3 µM) for 8 min at 30°C (Teng et al., 1998
). Following 3H-DA loading, vesicles were collected onto Whatman GF/B filter disc by rapid filtration through a Brandel Superfusion 1000 System (Gaithersburg, MD) that was interfaced with a fraction collector. Vesicles were washed with assay buffer and then exposed in vitro to ACR (final concentrations 250 mM, 500 mM or 1 M) during a 3 minute superfusion period (superfusion rate = 0.6 ml/min). For kinetic analysis of spontaneous efflux, 1 minute fractions were collected during 20 minutes of superfusion. At the end of the collection period, radioactivity in the superfusate fractions and filter discs was counted by scintillation spectroscopy. The respective efflux t1/2 was calculated for control and each ACR concentration (PrismTM, GraphPad Software, San Diego, CA) and mean data were expressed as percent of control efflux (Fig. 9).
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Amino-cupric silver staining and light microscopic examination.
At specific times during ACR intoxication (50 mg/kg/day = 5 and 11 days; 21 mg/kg/day = 21 and 36 days), neurotoxicant-exposed rats and respective age-matched controls were heparinized (1000 units/rat ip) and then deeply anesthetized with pentobarbital (50 mg/kg; ip). Rats were perfused through the aorta with 0.9% saline buffer containing 2 mM cacodylate, 22 mM dextrose, 22 mM sucrose and 2 mM CaCl2 at pH 7.4. Rats were then perfused with fixative that contained 4% paraformaldehyde, 90 mM sodium cacodylate and 115 mM sucrose (Lehning et al., 2003
). Brains were removed and stored in fixative until embedding. Detailed descriptions of the embedding and silver stain procedures are provided in Lehning et al. (2003). CNS sections were viewed on a Nikon Eclipse E400 light microscope and were photographed with a Nikon Coolpix 995 digital camera. Background levels of silver staining were established in forebrain sections from control rats (Figs. 3 and 4) and specific degenerative events were determined in silver stained sections from CNS of ACR-intoxicated rats (for details see Lehning et al., 2003). The density of degeneration in each silver stained slide was rated according to the following scale: 0 = None, 1 = Rare, 2 = Occasional, 3 = Slight, 4 = Moderate or 5 = Heavy.
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Western blot analysis.
Striatal synaptic vesicles were prepared from brains of rats intoxicated at 50 mg/kg/day x 5 days or 21 mg/kg/day x 21 days. Proteins (7 µg) were resolved by SDS-PAGE and then transferred to nitrocellulose membranes. After transfer, membranes were blocked with 5.0% dried non-fat milk in TBS (Tris-HCl 20 mM, NaCl 0.5 M, 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. 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. Immunoreactive vesicular protein bands were scanned with a densitometer, digitized, and quantified using the NIH Imaging Program. Statistical differences (p < 0.05) between control and experimental group means were determined by one-tailed Student's t-test (InStatTM, GraphPad Software, San Diego, CA). Data are expressed as mean percent of control ± SEM.
Preparation of synaptosomes.
Striatal synaptosomes were isolated from brains of ACR-intoxicated rats or their age-matched controls by the Percoll gradient method of Dunkley et al. as modified by LoPachin et al. (2004). In brief, brains were rapidly removed and minced in cold (4°C) buffer containing sucrose 0.32 M, EDTA 1 mM, and dithiothreitol 0.25 mM (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 1,000 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 140 mM, KCl 5 mM, NaHCO3 5 mM, MgCl2 1 mM, NaH2PO4 1.2 mM, glucose 10 mM, and Hepes 10 mM (pH 7.4), pelleted, and then resuspended.
Measurement of synaptosomal dopamine uptake and release following in vivo ACR exposure.
To determine the effects of ACR on transmitter release, isolated synaptosomes (see above) were incubated with 3H-DA (0.30 µM) for 3 min (see LoPachin et al., 2004, for methodological details). Labeled synaptosomes (10 µg protein) 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 superfused (0.6 ml/min) with oxygenated (95% 02/5% CO2) Krebs-Hepes buffer, and following equilibrium (30 min), four 2-min fractions (1.2 ml total volume) were collected as a measure of basal DA efflux. Ca2+-dependent, 3H-DA release was stimulated by a 90-s pulse of 40 mM KCl in modified Krebs buffer. Superfusate fractions were collected throughout the post-stimulus 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. Mean group data were analyzed statistically (p < 0.05) by a paired Student's t-test.
3H-DA uptake was measured in striatal synaptosomes isolated from ACR-intoxicated rats and their age-matched controls (for methodological details see LoPachin et al., 2004). Briefly, synaptosomes (10 µg) were filter trapped by rapid filtration through a cell harvester (see above) and then superfused (3 min) with Krebs-Hepes buffer containing 3H-DA (50 nM–1.7 µM). 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 washed, and corresponding radioactivity was measured by scintillation counting. Kinetic parameters (Km, Vmax) for 3H-DA uptake by control and ACR-exposed synaptosomes were determined by nonlinear regression analysis (PrismTM, GraphPad Software, San Diego, CA), and respective data were compared statistically (p < 0.05) by a two-tailed Student's t-test (InStatTM, GraphPad Software, San Diego, CA).
Measurement of synaptosomal glutathione content following in vivo ACR exposure.
Synaptosomal glutathione (GSH) was determined by HPLC analysis as described by Fariss and Reed (1987). Briefly, 
-glutamylglutamate was added as an internal standard to synaptosomal samples, which were then deproteinated by addition of perchloric acid. Reduced glutathione was derivatized with iodoacetic acid to prevent artifactual oxidation. Samples were then derivatized with 2,4-dinitrofluorobenzene, separated on an amino column (Phenosphere NH2 5 µm, 4.6 x 150 mm), and quantified by UV detection at 365 nm. Statistical differences (p < 0.05) between control and experimental group means were determined by one-way ANOVA and Dunnett's multiple range test (InStatTM, GraphPad Software, San Diego, CA). Data are expressed as mean percent of control ± SEM.
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RESULTS |
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Body Weight Changes and Neurological Toxicity
ACR intoxication at the 50 mg/kg daily dose-rate produced a progressive loss of body weight and changes in gait (LoPachin et al., 2002b
). Rats in this dose-rate group had a mean (±SEM) starting body weight of 303 ± 6 g, and after 5 days of ACR intoxication, mean weight decreased by 10% to 273 ± 10 g. Body weight continued to decline as ACR intoxication at the higher dose-rate progressed; i.e., by day 11, rats in this exposure group has lost approximately 25% (74 ± 6 g) of original body weight. Gait scores for the higher exposure group increased from 1.4 ± 0.4 on day 5, to 2.7 ± 0.5 on day 8, to 3.5 ± 0.5 on day 11 of intoxication. This score at 11 days represents a statistically significant, severe gait abnormality (see numerical gait scales in Materials and Methods). During the same time period, age-matched, saline-injected control rats exhibited no significant changes in gait (data not shown) and gained approximately 28% in body weight; i.e., starting body weight for the control group was 311 ± 7 g, and after 11 days the body weight increased to 399 ± 11 g. ACR intoxication at the lower daily dose-rate (21 mg/kg) slowed the normal rate of daily weight gain and caused significant increases in gait scores (LoPachin et al., 2002b). Age-matched control rats for this exposure group had a starting mean body weight of 312 ± 4 g, which increased to 483 ± 12 g by day 28 of the exposure paradigm. This represents a 55% increase in body weight at a rate of approximately 5.4 g per day. In contrast, rats intoxicated at the 21 mg/kg dose-rate exhibited a significantly slower rate of weight gain; i.e., rats in this exposure group had a starting weight of 307 ± 4 g, and after 28 days of ACR intoxication body weight rose to 384 ± 9 g. This represents a 25% increase in weight at a daily rate of approximately 2.5 g per day. Age-matched control rats did not exhibit changes in gait during the 28-day experimental period. In contrast, gait scores for ACR-intoxicated rats (21 mg/kg/day) increased steadily from an initial score of 1.3 ± 0.1 to 2.7 ± 0.3 on day 21 and then to approximately 3.5 on both days 28 (3.3 ± 0.7) and 35 (3.6 ± 0.4) of exposure. The later gait scores represented moderate-to-severe levels of neurotoxicity.
3H-Dopamine Uptake in Synaptic Vesicles Isolated from Striata of ACR-Intoxicated Rats and Their Age-Matched Controls
Figure 1 shows the DA transport process in striatal synaptic vesicles prepared from brains of control rats (age-matched) and rats exhibiting slight neurotoxicity (gait score = 1.4 ± 0.4) following 5 days of exposure to the higher ACR dose-rate (50 mg/kg/day). The substrate–velocity data (Fig. 1A) closely fit a Michaelis–Menten model (r2 = 0.98), and for the age-matched controls, kinetic analyses revealed a Vmax (95% confidence interval) = 34 (33–35) fmol/µg/min and a Km (95% confidence interval) = 284 (267–307) nM. These kinetic findings are similar to data from previous studies of 3H-DA uptake in rat striatal synaptic vesicles (Brown et al., 2000; Staal et al., 2000). ACR intoxication at the higher exposure rate (Fig. 1A) significantly lowered Vmax to 12 (11–13) fmol/µg/min and increased the Km (95% confidence interval) = 469 (408–530) nM. Figure 1B shows the substrate–velocity curves for 3H-DA uptake in synaptic vesicles isolated from striata of rats intoxicated at the 21 mg/kg/day (x21 days) dose-rate and their saline-treated, age-matched controls. Rats intoxicated at this lower exposure rate exhibited slight-to-moderate neurotoxicity (gait score = 2.7 ± 0.3). ACR intoxication did not change the Km, although the Vmax (95% confidence interval) was significantly reduced; i.e., control = 35 (34–36) fmol/µg/min versus ACR = 20 (19–21) fmol/µg/min (Fig. 1B).
3H-Dopamine Release and Uptake in Striatal Synaptosomes Isolated from ACR-Intoxicated Rats and Their Age-Matched Controls
We have previously shown that synaptosomal 3H-glutamate release was inhibited in several brain regions of ACR-intoxicated rats (LoPachin et al., 2004). To assess effects of ACR intoxication on presynaptic function in striatum, we measured KCl-evoked synaptosomal 3H-DA release. The data presented in Table 1 show that both ACR dose-rates produced significant decreases in neurotransmitter release. A reduction in transmitter release could involve a nonvesicular effect such as ACR inhibition of presynaptic membrane monoaminergic transport. To address this possibility, high-affinity, Na+-dependent 3H-DA uptake was determined in striatal synaptosomes isolated from intoxicated rats and their age-matched controls (Fig. 2). Intoxication at the higher ACR dose-rate (Fig. 2A) did not cause statistically significant changes in transport kinetic parameters, whereas the lower dose-rate (Fig. 2B) was associated with a small, but statistically significant decrease in Vmax.
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Silver Stain Analysis and Vesicular Synaptobrevin Content
Rather than being a direct chemical effect of ACR, impaired vesicular transport and synaptosomal transmitter release could be nonspecific consequences of nerve terminal degeneration (Lehning et al., 2003
). We therefore used silver stain methods to assess nerve terminal degeneration in striata of ACR-intoxicated rats. In silver-stained sections, synaptic degeneration appeared as variably shaped punctate black debris against a pink background (Figs. 3 and 4). Figure 3 shows a striatal section from an age-matched control rat of the 50 mg/kg/day dose-rate group. At the experimental time when synaptic vesicle function was determined (i.e., day 5; see Fig. 1 above), the relative level (mean ± SEM) of nerve terminal degeneration was assessed to be 1.4 ± 0.3 (Fig. 3). For comparative purposes, at day 11 of the higher dose-rate, rats exhibited severe neurotoxicity (gait score = 3.5 ± 0.5), and silver stain analysis of corresponding striata revealed an argyrophilic density of 4.1 ± 0.5 or moderate nerve terminal degeneration (Fig. 3). Rats intoxicated at the lower dose-rate (21 mg/kg/day) did not exhibit significant argyrophilic changes in striatum up to day 28 (Fig. 4). Figure 4 shows a representative striatal section from a rat intoxicated at the 21 mg/kg/day dose-rate for 21 days. The results indicate that, at the time-point when the corresponding vesicular uptake kinetics were determined (i.e., 21 days; see Fig. 2), synaptic degeneration was rare. However, after 35 days of exposure to the lower ACR dose-rate, the relative density of argyrophilic nerve terminals was increased significantly (3.0 ± 0.4; Fig. 4). Together, these data indicate that the neurochemical changes observed in striatum of ACR-intoxicated rats (Figs. 1 and 2) preceded the onset of nerve terminal degeneration (Figs. 3 and 4).
It is possible that the delivery of synaptic vesicles and other nerve terminal components are reduced by ACR inhibition of fast axonal transport (Sickles et al., 1996). Such an effect could explain the observed changes in transmitter release and vesicular uptake kinetics (Figs. 1 and 2). To investigate this possibility, the content of synaptobrevin (a vesicular marker) in striatal P5 fractions was measured by semiquantitative immunoblot analysis. Figure 5 shows representative immunoblots for vesicular synaptobrevin from ACR-intoxicated rats or their age-matched controls. Corresponding densitometric measurements indicate, on day 5 of the 50 mg/kg/day dose-rate, the synaptobrevin content (mean percent of control ± SEM) was significantly (p < 0.05) increased by 31% ± 8%. On day 21 of the lower dose-rate (21 mg/kg/day), the mean synaptobrevin content was decreased slightly (–9% ± 4%), although not significantly. These findings suggest that striatal syanptosomes contain normal vesicular contents. This is inconsistent with the possibility that ACR-induced syanptic toxicity is mediated by decreased fast anterograde delivery of these proteins.
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GSH Levels in Synaptosomes Isolated from ACR-Intoxicated Rats
ACR exposure can lower cellular GSH levels (e.g., see Shivakumar and Ravindranath, 1992
), and therefore, the resulting oxidative shift in redox potential could promote sulfhydryl oxidation and loss of vesicular transporter function (see Discussion). To test this possibility, we measured the GSH content of synaptosomes isolated from ACR-intoxicated rats. Figure 6A shows that, in rats intoxicated at the higher dose-rate (50 mg/kg/day), no changes in synaptosomal GSH content were detected through day 8 of exposure. At day 11, a small but statistically significant decrease was noted (Fig. 6A). When rats were exposed to ACR at the lower intoxication rate (21 mg/kg/day), a statistically significant increase in synaptosomal GSH levels was detected at day 14. However, by day 21 of ACR exposure, the GSH content returned to control values (Fig. 6B).
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In Vitro Effects of ACR, Propionamide, N-Ethylmaleimide, and Iodoacetic Acid on 3H-Dopamine Uptake in Synaptic Vesicles
Figure 7 shows that in vitro exposure of striatal vesicles to ACR produced graded, concentration-dependent decreases in 3H-DA uptake. We also determined the in vitro effects of two sulfhydryl reagents, N-ethylmaleimide (NEM) and iodoacetic acid (IAA), on striatal vesicular uptake. Both chemicals produced concentration-dependent reductions in vesicular 3H-DA uptake (Fig. 7). NEM and IAA were significantly more potent than ACR with respect to inhibiting uptake; i.e., the respective IC50s were 6.2 µM, 3.7 mM, and 243 mM. In corroborative studies, in vitro incubation of synaptic vesicles with the structural analog, propionamide, caused a statistically significant increase in 3H-DA uptake at the lower concentration (250 mM), whereas the higher concentrations (500–1000 mM) did not affect vesicular transport (Table 2). Kinetic analysis of in vitro uptake inhibition showed that both ACR and NEM exposure produced a significant decrease in Vmax and an increase in Km (Fig. 8). These in vitro data indicate that ACR can directly affect vesicular transport in a concentration-dependent fashion. The ACR- and NEM-induced changes in kinetic parameters are consistent with irreversible, uncompetitive inhibition of the transport process. The correspondence of effect among ACR, IAA, and NEM suggests a common mechanism involving sulfhydryl adduction.
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In Vitro Effects of ACR on Vesicular 3H-Dopamine Efflux
Rather than affecting vesicular uptake, ACR might alter 3H-DA efflux or the ability to retain neurotransmitter (e.g., see Teng et al., 1998
). In the preceding studies, such an effect would lower vesicular 3H-DA content and thereby lead to a misinterpretation of altered uptake. Therefore, we determined the effects of in vitro ACR exposure on efflux from striatal synaptic vesicles. This possibility could not be examined during in vivo intoxication, since the observed vesicular uptake defect would confound interpretation of the efflux data. The data presented in Figure 9 fit a monophasic decay model with a control t1/2 of 4.5 min (Fig. 9). Incubation of synaptic vesicles with graded concentrations of ACR (250–1000 mM) did not alter the exponential rate of 3H-DA loss (Fig. 9). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionsREFERENCES |
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We originally studied neurotransmitter release as a model system to test our hypothesis that membrane fusion was a primary site of action for ACR (reviewed in LoPachin et al., 2003
). Corresponding results supported this hypothesis and indicated that ACR exposure decreased synaptosomal neurotransmitter release, presumably through direct chemical adduction of SNARE components (e.g., SNAP-25) and other presynaptic proteins (e.g., NSF) that are involved in membrane fusion (Barber and LoPachin, 2004; LoPachin et al., 2004). To assess selectivity of the release defect, in the present study we determined the effects of ACR exposure on a presynaptic process that is not based on membrane fusion; i.e., vesicular neurotransmitter uptake. Results show that both in vivo and in vitro ACR exposures inhibit vesicular transport (Figs. 1 and 2). Although the functional relationship between defective vesicular uptake and impaired release is unknown (see ahead), several lines of evidence suggest that the presynaptic actions of ACR are specific and neurotoxicologically relevant. Thus, we have demonstrated that significant changes in vesicular transport and transmitter release develop prior to the onset of nerve terminal degeneration (Figs. 3 and 4) and gross neurological dysfunction (see also Barber and LoPachin, 2004; LoPachin et al., 2004). The early temporal appearance of synaptic toxicity suggests an involvement in the neuropathogenic process. Moreover, evidence from this (Fig. 5) and previous studies (LoPachin et al., 2004) suggest that ACR-induced synaptic toxicity is not a consequence of decreased anterograde delivery of vesicles or related proteins to the nerve terminal (reviewed in LoPachin, 2002). Our research has also demonstrated that ACR intoxication at either dose-rate did not cause significant changes in synaptosomal GSH content (Fig. 6). This finding, in conjunction with previous measurements of cytoplasmic pyridine nucleotide ratios (NAD+/NADH, NADP+/NADPH) in different brain regions of intoxicated rats (Johnson and Murphy, 1977), suggests that the mechanism of presynaptic dysfunction does not involve primary oxidative stress. These data suggest that impaired vesicular transport and defective transmitter release are early, specific neurotoxic effects that might play a causal role in ACR neurotoxicity.
Whereas the molecular mechanism of synaptic toxicity is unknown, the well-described adduct chemistry of this neurotoxicant can provide possible mechanistic insight. ACR is an 
,ß-unsaturated carbonyl chemical with electrophilic reactivity at the C3 carbon atom. As a soft electrophile, ACR will form covalent adducts with nucleophilic centers via a Michael condensation reaction across the C2–C3 double bond (reviewed in Calleman, 1996; LoPachin and DeCaprio, 2005, Friedman, 1973). Our findings that in vitro propionamide exposure did not affect vesicular uptake (this study) or synaptosomal 3H-glutamate release (LoPachin et al., 2004), strongly support Michael adduction as a relevant step in the molecular mechanism of ACR neurotoxicity; i.e., propionamide lacks the C2–C3 vinyl group that is critical for Michael condensation reactions. The similar kinetic changes in vesicular DA uptake (increased Km, decreased Vmax) following in vitro ACR or NEM exposure are consistent with irreversible covalent bond formation with sulfhydryl groups. That sulfhydryl adduct formation is linked to nerve terminal dysfunction is implicated by previous observations that the synaptosomal levels of cysteine adducts were correlated to the development of in vivo and in vitro neurotoxicity (Fig. 10; see also Barber and LoPachin, 2004). These data suggest that the formation of sulfhydryl adducts is a necessary step in the molecular mechanism of ACR neurotoxicity. Although the corresponding protein targets are unknown, the respective activities are likely to be importantly involved in the transport process and modulated by the redox state of specific resident cysteines (reviewed in Stamler et al., 2001). For example, the uptake of DA and other catecholamines in striatal synaptic vesicles is dependent upon an inwardly directed proton gradient, which is established by a vacuolar proton pump (v-ATPase). The vesicular monoamine transporter-2 (VMAT-2) uses this electrochemical gradient to drive neurotransmitter uptake (reviewed in Fernandez-Chacon and Sudhof, 1999). VMAT-2 contains cysteines (Lesch et al., 1993), although it is not known whether these residues play a role in transport function. In contrast, v-ATPase activity is highly sensitive to inhibition by NEM, and adduction of a single cysteine residue (Cys 254) located in the A subunit has been shown to be responsible for NEM sensitivity (Feng and Forgac, 1992). Adduct-inhibition of the v-ATPase would lead to dissipation of the electrochemical proton gradient and subsequent inhibition of neurotransmitter uptake. Thus, v-ATPase is a cysteine-directed protein that is critically important to the vesicular uptake mechanism and is, therefore, a rational ACR target. However, it is important to note that ACR forms sulfhydryl adducts with numerous synaptosomal proteins (LoPachin et al., 2004). Therefore, the suggestion that v-ATPase is an important neuropathogenic target implies that the majority of adduction is nonspecific; i.e., ACR adducts many cysteine residues that do not play a regulatory role in protein function (see Barber and LoPachin, 2004; LoPachin et al., 2004).
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In this study we have shown that vesicular DA transport is impaired in nerve terminals isolated from ACR-intoxicated rats (Fig. 1). In vitro studies showed that ACR produced similar effects on the kinetic parameters of DA uptake (Fig. 8) and have provided corroborative evidence that ACR acts by forming thiol-based Michael adducts (Table 2). Although the comparable vesicular toxicity suggests that the in vitro system is a relevant model for studying molecular mechanisms, the relatively high in vitro ACR concentrations used in this and other studies (0.001–1.0 M; Barber and LoPachin, 2004
; LoPachin et al., 2004) are difficult to reconcile with the in vivo toxicokinetics; i.e., peak ACR plasma concentration of 422 µM after 11 days of intoxication at 50 mg/kg/day (Barber et al., 2001). The use of high in vitro ACR concentrations is, nonetheless, consistent with the known adduct chemistry of ACR. ACR is a weak electrophile (Cavins and Freidman, 1968; Friedman, 1973), and based on the mass action kinetics of chemicals with low reactivity, high ACR concentrations are necessary to generate toxic intrasynaptosomal levels of adduct during acute in vitro experiments. The data presented in Figure 10 provide direct support for this concept; i.e., high in vitro ACR concentrations (0.001–1.0 M) produce graded increases in the synaptosomal ACR adduct of cysteine, carboxyethylcysteine (CEC; Barber and LoPachin, 2004), that are inversely related to the inhibition of both synaptosomal glutamate release (LoPachin et al., 2004) and vesicular uptake (this study). The high degree of correlation among these data (mean r2 = 0.99) suggests that in vitro cysteine adduct formation mediates ACR-induced synaptosomal toxicity. To place these data in perspective, the in vitro toxicodynamics of ACR are not unique, since other axonopathic neurotoxicants that act through protein adduct formation such 2,5-hexanedione also exhibit low electrophilicity and require relatively high in vitro exposure conditions for induction of neurotoxicity (DeCaprio et al., 1982; Graham et al., 1982; reviewed in LoPachin and DeCaprio, 2005).
Goldstein and Lowndes (1981) suggested that defective neurotransmission in ACR-intoxicated laboratory animals might be mediated by changes in transmitter synthesis, storage, uptake, or release (reviewed in LoPachin, 2004; LoPachin et al., 2002a, 2003). The present findings indicate that in striatum of ACR-intoxicated rats both neurotransmitter storage and release are disrupted. Although the effects of ACR exposure on striatal neurotransmission have not been determined experimentally, previous research showed that corresponding DA receptor binding was increased in this brain region (Bondy et al., 1981; Uphouse and Russell, 1981). This likely represents a compensatory upregulation of postsynaptic dopaminergic receptors in response to decreased presynaptic transmitter release. Disruption of nigrostriatal communication could play a role in ACR-induced gait abnormalities, given the importance of the basal ganglia in complex motor behavior. However, striatal dysfunction is unlikely to be solely responsible for the expression of ACR neurotoxicity. Indeed, impaired neurotransmission has been identified at several peripheral and central synapses including the neuromuscular junction and the spinal primary afferent terminal of ACR-intoxicated laboratory animals (Goldstein and Lowndes, 1979, 1981; Tsujihata et al., 1974). We have also recently shown that evoked 3H-glutamate release was decreased significantly in synaptosomes isolated from several brain regions of ACR-intoxicated rats (LoPachin et al., 2004). These combined data suggest that presynaptic dysfunction is a global consequence of ACR intoxication. The correspondingly widespread, but later developing, nerve terminal degeneration that characterizes ACR neuropathy (Lehning et al., 2002a,b, 2003) is likely due to the disruption of membrane fusion processes that mediate plasmalemmal turnover (reviewed in LoPachin et al., 2003).
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Conclusions |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConclusionsREFERENCES |
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Results suggest that both vesicular neurotransmitter uptake and release are defective in striatum of ACR-intoxicated rats. Inhibition of vesicular transport is likely to induce defective presynaptic neurotransmitter storage, which would secondarily affect release. However, our evidence suggests that ACR also has direct effects on the release mechanism (Barber and LoPachin, 2004
; LoPachin et al., 2004). Therefore, ACR inhibits membrane fusion (transmitter release) and nonfusion (vesicular transport) processes in nerve terminals. Presumably, these inhibitory actions involve ACR adduction of multiple cysteine-regulated proteins (e.g., NSF, v-ATPase, VMAT, SNAP-25) which, together, promote the failure of neurotransmission. In a larger context, many of the predicted ACR targets are also effectors for neuronal nitric oxide (NO) signaling (reviewed in LoPachin, 2004; Stamler et al., 2001). NO forms reversible nitrosothiol adducts with cysteine groups and can thereby reduce the functionality of critical proteins involved in nerve terminal processes (reviewed in Stamler et al., 2001). We therefore propose that ACR adducts NO-regulated cysteine residues on proteins that are critically involved in presynaptic function. In contrast to enzymatic denitrosylation that presumably terminates the NO effect, the covalent formation of ACR–cysteine adducts cannot be reversed, and as a consequence, presynaptic neurotoxicity develops as the NO-inhibitory actions of ACR persist. |
ACKNOWLEDGMENTS |
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Research presented in this manuscript was supported by an NIH grant from the National Institute of Environmental Health Sciences (RO1 ES03830–19). The authors would like to express their sincere thanks to Dr. Annette Fleckenstein (University of Utah) for her methodological assistance and for her helpful comments during the preparation of this manuscript.
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