NeuroToxicology 25 (2004) 349–363

In Vivo and In Vitro Effects of Acrylamide on

Synaptosomal Neurotransmitter
Uptake and Release
Richard M. LoPachin*, Aron I. Schwarcz, Christopher L. Gaughan,
Shirley Mansukhani, Soma Das
Department of Anesthesiology, Albert Einstein College of Medicine, Montefiore Medical Center,
Moses Research Tower-7, 111 E. 210th Street, Bronx, NY 10467, USA
Received 28 January 2003; accepted 14 July 2003

Abstract

Evidence suggests acrylamide (ACR) neurotoxicity is mediated by impaired presynaptic transmission. To assess the
effects of ACR on nerve terminal function, [3 H]glutamate release and uptake were determined in brain synaptosomes
isolated from intoxicated rats (50 mg/kg per day
8 days, i.p. or 21 mg/kg per day
21 days, p.o.). Regardless of ACR
dose-rate, a significant reduction in synaptosomal Kþ-stimulated, Ca2þ-dependent release was detected, whereas kinetic
analysis of Naþ-dependent uptake did not reveal consistent changes. Immunoblot analysis showed normal protein levels
(e.g. SNAP-25) in dysfunctional synaptosomes isolated from ACR-intoxicated rats. This suggests that defective release
does not involve changes in protein synthesis and/or anterograde delivery of presynaptic constituents. To identify
potential targets, synaptosomes were exposed in vitro to [14 C]-ACR and radiolabeled proteins were separated by
gel electrophoresis and detected by autoradiography. [14 C]-ACR labeling of distinct synaptosomal protein bands
(10.5–154,000 kDa) was blocked by the sulfhydryl alkylating agent, N-ethylmaleimide (NEM; 4 mM) but not by the non-
neurotoxic structural analog propionamide (10 mM). In vitro characterization of synaptosomal [3 H]glutamate uptake
and release showed that ACR, NEM and iodoacetic acid (IAA) produced concentration-dependent decreases in each
parameter that were highly correlated to reductions in free sulfhydryl content. All three chemicals were equiefficacious
with respect to reducing sulfhydryl content and neurotransmitter uptake/release, although the relative potencies differed;
NEM > IAA > ACR. Kinetic analysis of uptake showed that in vitro exposure to ACR, IAA or NEM at their respective
IC50’s caused similar reductions in Vmax. These data suggest that ACR-induced synaptic dysfunction involves adduction of
presynaptic protein thiol groups and subsequent reduction in neurotransmitter release.
# 2003 Elsevier Inc. All rights reserved.

Keywords: Acrylamide; Distal axonopathy; Synaptosomes; Neurotransmitter release; Uptake;

Protein thiol groups; SNARE core complexes

INTRODUCTION ore processing, dye synthesis) and in laboratories for

Acrylamide (ACR) is a water-soluble, vinyl mono-
mer that is used primarily to produce polyacrylamides
with different physical and chemical properties. These
polymers are used extensively in various chemical
industries (e.g. water and wastewater management,
*
Corresponding author. Tel.: þ1-718-920-5054;
fax: þ1-718-515-4903.
E-mail address: lopachin@aecom.yu.edu (R.M. LoPachin).
the electrophoretic separation of macromolecules
(Gold and Schaumburg, 2000; Spencer and Schaum-
burg, 1974a; U.S. Environmental Protection Agency,
1988). ACR monomer is also a contaminant formed by
an as yet unidentified mechanism during the high-
temperature preparation of certain potato- or grain-
based foods (Tareke et al., 2000). Although the poly-
mer is non-neurotoxic, long-term, low-level intoxica-
tion with monomeric ACR produces ataxia and skeletal
muscle weakness in occupationally exposed humans

0161-813X/$ – see front matter # 2003 Elsevier Inc. All rights reserved.
doi:10.1016/S0161-813X(03)00149-9
350 R.M. LoPachin et al. / NeuroToxicology 25 (2004) 349–363
and in experimental animal models (LeQuesne, 1980,
1985; LoPachin et al., 2002a; Spencer and Schaum-
burg, 1974a). Early morphological studies suggested
that this neurotoxicity was mediated by nerve damage
classified as a central–peripheral distal axonopathy
(LoPachin and Lehning, 1994; Spencer and Schaum-
burg, 1974b; 1976, 1980; Tilson, 1981). Distal axon
swelling and subsequent degeneration were considered
to be the hallmark morphological features of this toxic
axonopathy and, accordingly, research over the past 25
years has focused on putative axonal sites of action
(reviewed in LoPachin and Lehning, 1994; LoPachin
et al., 2002a; Miller and Spencer, 1985; Sabri and
Spencer, 1980). However, other contemporary research
demonstrated that ACR can produce neurological toxi-
city in the absence of axonopathy; i.e. whereas equiva-
lent neurotoxicity can be induced by intoxication over
a wide range of daily dose-rates (e.g. 10–50 mg/kg per
day
 28 days), axon degeneration in PNS and
CNS occurred only during long-term exposure to lower
ACR dose-rates (e.g. 21 mg/kg per day
42 days;
Cavanagh, 1982a; Crofton et al., 1996; Lehning et al.,
1998, 2002a,b, 2003; LoPachin et al., 2002b). The
dissociation between neurological dysfunction and
the presumed underlying morphological lesion suggests
that axonopathy might not be importantly involved
in the pathophysiological process leading to ACR neu-
rotoxicity (see reviews by LoPachin et al., 2000, 2002a,
2003).
Nerve terminals have been suggested as an alter-
native site of ACR action based on early structural and
functional changes as in PNS and CNS (reviewed in
LoPachin et al., 2002a, 2003). Recently completed
morphological studies (Lehning et al., 2002a,b,
2003) using a contemporary amino-cupric silver stain-
ing method to detect degenerating neurons and their
processes (i.e. dendrites, axons, nerve terminals) indi-
cated that intoxication at a higher ACR dose-rate
(50 mg/kg per day
5–11 days) produced selective,
widespread degeneration of nerve terminals in rat brain
and spinal cord regions. Intoxication at a lower dose-
rate (21 mg/kg per day
7–38 days) was associated
with initial nerve terminal degeneration followed later
by preterminal axon degeneration. These findings are
consistent with other evidence suggesting that, regard-
less of dose-rate, nerve terminal degeneration was
an initial consequence of ACR intoxication in both
CNS (Cavanagh, 1982a,b; Ghetti et al., 1973; Prineas,
1969; Spencer and Schaumburg, 1977a,b) and PNS
(Cavanagh, 1982a; DeGrandchamp and Lowndes,
1990; DeGrandchamp et al., 1990; Schaumburg
et al., 1974; Tsujihata et al., 1974). A series of elec-
trophysiological studies by Lowndes and Goldstein
revealed a defect in neurotransmission at primary
afferent terminals (PAT) in spinal cord of ACR-intoxi-
cated cats (DeRojas and Goldstein, 1987; Goldstein,
1985; Goldstein and Lowndes, 1979, 1981; Lowndes
et al., 1978a,b; see also Tsujihata et al., 1974). This
effect developed prior to PAT degeneration, failure of
corresponding somatosensory receptor function (i.e.
muscle spindles) and before the onset of neurological
symptoms. The authors suggested that defective neu-
rotransmission might be related to impaired transmitter
release and might involve changes in presynaptic
uptake, synthesis, or vesicle storage (Goldstein,
1985; Goldstein and Lowndes, 1979, 1981). As mor-
phological correlates of presynaptic dysfunction, sev-
eral studies have shown that peripheral and central
nerve terminals in ACR-intoxicated laboratory animals
exhibited early reductions in docked synaptic vesicles
(DeGrandchamp and Lowndes, 1990; DeGrandchamp
et al., 1990; Prineas, 1969; Tsujihata et al., 1974).
Moreover, lengthening of the postjunctional endplate
in PNS and increases in receptor number and affinity in
CNS also occurred and are indicative of postsynaptic
denervation hypersensitivity (Agrawal et al., 1981a,b;
Bondy et al., 1981; DeGrandchamp and Lowndes,
1990; DeGrandchamp et al., 1990; Uphouse and Rus-
sell, 1981). Based on the early development of these
structural and functional defects, we have hypothesized
that synaptic dysfunction and eventual nerve terminal
degeneration mediate ACR neurotoxicity (LoPachin
et al., 2002a, 2003). Therefore, as an initial investiga-
tion of potential presynaptic mechanisms, we have
determined the in vivo and in vitro effects of ACR
exposure on KCl-evoked, Ca2þ-dependent neurotrans-
mitter release and on high affinity, Naþ-dependent
uptake in rat brain synaptosomes.
MATERIALS AND METHODS
Chemicals
ACR (99% purity), propionamide (97% purity), N-
ethylmaleimide (NEM), iodoacetic acid (IAA, 98%
purity), 5,50 -dithiobis(2-nitrobenzoic acid) (DTNB)
and Percoll (for formation of density gradients) were
all purchased from the Sigma (St. Louis, MO). [14 C]-
ACR (specific activity 5 mCi/mmol) and L-[3 H]gluta-
mate (specific activity 60 Ci/mmol) were obtained from
American Radiolabeled Chemicals (St. Louis, MO).
Antibodies against syntaxin 1 (mouse monoclonal),
growth-associated protein of 43 kDa (GAP-43; mouse
 R.M. LoPachin et al. / NeuroToxicology 25 (2004) 349–363
monoclonal) and synatophysin (mouse monoclonal)
were from Calbiochem (SanDiego, CA). Antibodies
to synaptobrevin (VAMP; mouse monoclonal) and
b-tubulin (mouse monoclonal) were obtained from
Chemicon International Inc. (Temecula, CA). Antibo-
dies for synaptosomal-associated protein of 25 kDa
(SNAP-25; rabbit polyclonal) and rabphilin 3a (Rab-
3a, rabbit polyclonal) were purchased from Affinity
BioReagents (Golden, CO) and Zymed (San Francisco,
CA), respectively.
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, 250–275 g; Taconic Farms, Germantown,
NY) were used in this study. Rats were housed indi-
vidually 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 8C
and 50% humidity with a 12 h light/dark cycle. For
studies using synaptosomes isolated from different
brain regions of intoxicated rats (see below), randomly
assigned groups of animals (four to six rats per expo-
sure group) were exposed to ACR at daily dose-rates of
either 50 mg/kg per day
8 days (i.p.) or 21 mg/kg per
day
21 days (p.o.). These daily dose-rates and cor-
responding routes have been used in mechanistic stu-
dies conducted over the past 30 years and are 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; LoPachin et al.,
2002b; Lehning et al., 1998, 2002a,b, 2003; reviewed
in LoPachin and Lehning, 1994; LoPachin et al., 2000,
2003). In the present study, body weight and gait scores
were determined two to three 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., 2002b). To
assess locomotion, rats were placed in a clear plexiglass
box (90 cm
90 cm) and were observed for 3 min.
Following observations, a gait score was assigned
from 1 to 4 where: 1 ¼ a normal gait; 2 ¼ a slightly
abnormal gait (slight ataxia, hopping gait and foot
splay); 3 ¼ moderately abnormal gait (obvious ataxia
and foot splay with limb abduction during ambulation);
351
4 ¼ severely abnormal gait (inability to support body
weight and foot splay). For each ACR dosing regimen,
groups of age-matched control rats (n ¼ 4 rats per
group) were weighed and gait scores were determined.
Control rats for the higher ACR dose-rate group received
daily i.p. 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 Synaptosomes
Depending upon study conditions, synaptosomes
were isolated from cortical and diencephalon-midbrain
regions of brains from ACR-intoxicated rats and their
age-matched controls or from brains of normal rats
(250–275 g). Previous silver degeneration studies of
ACR-intoxicated rats have shown that these brain
regions exhibit progressive nerve terminal degenera-
tion (Lehning et al., 2002b, 2003). For each dose-rate
(50 mg/kg per day
8 days; 21 mg/kg per day
21
days), the selected experimental time-points preceded
the onset of frank nerve terminal degeneration (Lehn-
ing et al., 2002b, 2003). Synaptosomes were prepared
by the Percoll gradient method of Dunkley et al.
(1988). In brief, brains were rapidly removed and
specific tissue regions were dissected and minced in
cold (4 8C) buffer containing sucrose 0.32 M, EDTA
1 mM and dithiothreitol 0.25 mM (SED gradient buf-
fer; pH 7.4). Tissue was gently homogenized in SED
buffer (10 passes in a Teflon-glass homogenizer;
700 rpm) and the resulting homogenate was centri-
fuged at 1000
g (10 min, 4 8C). The pellet (P1) was
washed once and supernatants (S1 and S2) were com-
bined. 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
g for 6 min and synaptosomes were col-
lected at the last interface (15/23%). Characterization
of the Percoll method demonstrated a relative enrich-
ment of synaptosomal markers (syntaxin 1, synapto-
brevin) at the collection interface (data not shown).
Synaptosomes were washed twice in oxygenated
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 in oxygenated Kreb’s buffer.
ACR intoxication of rats did not alter the final synap-
tosomal protein yield; i.e. 21 mg/kg per day
21
352 R.M. LoPachin et al. / NeuroToxicology 25 (2004) 349–363
days ¼ 8:74  0:64 mg protein/ml; 50 mg/kg per
day
8 days ¼ 8:47  0:66 mg protein/ml; combined
age-matched control ¼ 7:96  0:96 mg protein/ml.
Binding of [14C]-ACR to Synaptosomal Proteins
As an initial investigation of putative targets and
chemical mechanisms, [14 C]-ACR–protein binding
was characterized in synaptosomes prepared from
cerebrocortical or midbrain-diencephalon regions of
control rat brains. Synaptosomes (30 mg protein) were
preincubated for 30 min at 37 8C in oxygenated control
Krebs–Hepes buffer or in buffer containing either the
sulfhydryl alkylating agent, N-ethylmaleimide (4 mM)
or the non-neurotoxic ACR analog, propionamide
(10 mM). At the end of the preincubation period,
[14 C]-labeled ACR (7 mCi per tube or 58 nM ACR)
was added to all tubes, which were then incubated at
37 8C for 60 min. Following incubation, samples were
snap-frozen in liquid nitrogen and stored at 180 8C
until analyzed. For each synaptosomal sample, labeled
and unlabeled proteins were resolved by sodium dode-
cyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE; 7.5, 10.0 or 16.5% gels) and then transferred
overnight to nitrocellulose membranes. Autoradio-
graphs of radiolabeled proteins were obtained by
exposure of the dried membranes to X-ray film (Super
RX Fuji film) for 6 weeks (80 8C). Molecular weights
(kDa) of radiolabeled proteins were estimated based on
the migration of standard proteins. Fig. 2 shows a
representative PAGE (7.5% gel) and corresponding
autoradiographic evidence from a total of three sepa-
rate trials.
Determination of Synaptosomal L-[3H]glutamate
Release, Free Sulfhydryl Concentrations and
Protein Content
Effects of In Vivo ACR on Neurotransmitter
Release and Protein Content
Synaptosomes were prepared from brains of
ACR-intoxicated rats or their age-matched controls
(see above ‘‘Preparation of Synaptosomes’’) and were
loaded with L-[3 H]glutamate (10 mCi per tube or
167 nM glutamate) for 10 min at 36 8C. Labeled
synaptosomes (250 mg protein) were trapped on glass
fiber filters (Whatman GF/B filter disc) in a 12-well
superfusion chamber (Brandel Suprafusion 1000 sys-
tem, Gaithersburg, MD) interfaced with a fraction
collector. Synaptosomes were superfused at 0.6 ml/
min with oxygenated (95% O2/5% CO2) Krebs–Hepes
buffer (NaCl 124 mM, MgSO4 2.4 mM, KCl 4 mM,
NaH2PO4 1 mM, CaCl2 2.5 mM, glucose 10 mM,
Hepes 25 mM, pH 7.4) and, following an equilibration
period (60 min), four 2 min fractions were collected as
a measure of basal [3 H]-neurotransmitter efflux. Ca2þ-
dependent, [3 H]glutamate release was stimulated by a
90 s pulse of 40 mM KCl in modified Krebs buffer.
Preliminary studies established the optimal pulse dura-
tion, KCl concentration and Ca2þ-dependency of
evoked neurotransmitter release from synaptosomes
(data not shown). Superfusate fractions were collected
at 2 min intervals (1.2 ml total volume) throughout the
entire experiment; i.e. prestimulus, stimulus and post-
stimulus periods. The isotope contents of each col-
lected fraction and of the corresponding filter-trapped
synaptosomes were determined by scintillation count-
ing. [3 H]glutamate exocytosis was calculated as per-
cent fractional release, which is integrated area under
the release curve (AURC) divided by total synaptoso-
mal counts (filter þ effluent fractions). The effects of
ACR on neurotransmitter release were expressed as
mean percent of control  S:E:M: and mean group
data were analyzed statistically (P < 0:05) by a paired
Student’s t-test.
Our studies of synaptosomes derived from brains or
ACR-intoxicated rats revealed a significant defect in
neurotransmitter release (see ahead). To investigate the
possibility that this effect was due to decreased synth-
esis and/or impaired kinesin-based fast anterograde
axonal transport of presynaptic proteins involved in
the release process (Sickles et al., 2002), the levels of
selected proteins were determined in synaptosomes
isolated from brain regions of ACR-intoxicated rats
(see above ‘‘Animals and ACR Intoxication’’). This
experimental approach was based on previous studies
showing that impairment of rapid axonal transport
(e.g. antisense-reduced kinesin synthesis; kif1Bþ/
knockout) resulted in decreased delivery of proteins
(e.g. synaptophysin, synaptotagmin) to distal axons and
nerve terminals as detected by immunoblot analysis
(e.g. see Amaratung et al., 1995; Zhao et al., 2001).
Synaptosomal proteins 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 8C 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 8C
with an appropriate alkaline phosphatase-conjugated
secondary antibody. Membranes were washed with
TBS and bound secondary antibody was visualized
 R.M. LoPachin et al. / NeuroToxicology 25 (2004) 349–363
with the ProtoBlot II AP system (Promega, Madison,
WI). Immunoreactive synaptosomal 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  S:E:M.
Effects of In Vitro ACR, Propionamide and
Sulfhydryl Reagents on Neurotransmitter Release
and Free Sulfhydryl Concentrations
As a potential model for exploring the mechanism of
nerve terminal dysfunction, the in vitro concentration-
dependent effects of ACR and two sulfhydryl reagents
(N-ethylmaleimide or iodoacetic acid, IAA) on synap-
tosomal Ca2þ-dependent, KCl-evoked [3 H]glutamate
release were determined. Synaptosomes were prepared
from control rats, equilibrated at 36 8C in oxygenated
Krebs–Hepes buffer and then loaded with [3 H]gluta-
mate and placed in a superfusion chamber as described
above. Following equilibration, synaptosomes were
exposed to varying concentrations of N-ethylmalei-
mide, iodoacetic acid or ACR and, at the end of
toxicant exposure (30 min), [3 H]glutamate release
was evoked by a 90 s pulse of 40 mM KCl Krebs–
Hepes buffer. Synaptosomes were also exposed in vitro
to equimolar concentrations of the structural analog
propionamide. Fractional release was calculated as
previously described. In parallel studies, the concen-
tration-dependent effects of ACR, propionamide, NEM
or IAA on free synaptosomal sulfhydryl content were
determined by the method of Ando and Steiner (1973).
Following incubation with ACR, propionamide or the
sulfhydryl chemicals (see above in vitro exposure
protocol), synaptosomes (250 mg protein) were solu-
bilized with 1% SDS (5 min). 5,50 -Dithiobis(2-nitro-
benzoic acid) (3 mM) was added and, following
equilibration (5 min, 25 8C), 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-carboxy-
lato-4-nitrothiophenolate, the thiol anion released dur-
ing adduction of sulfhydryl groups by the disulfide
reagent DTNB, was calculated by the molar extinction
coefficient, 1:36
104 M1 cm1. Sulfhydryl data
were calculated as nmol/mg synaptosomal protein
and the effects of ACR, propionamide, NEM or IAA
were expressed as mean percent of control  S:E:M.
Both the release and sulfhydryl curves (Fig. 3A) were
fitted by nonlinear regression analysis (r2 for all
curves > 0:98) and respective IC50’s and 95% confi-
353
dence intervals were calculated by the Cheng–Prusoff
equation and were compared statistically by Student’s
t-test (PrismTM 3.0, GraphPad Software, San Diego,
CA). The effects of each chemical on free sulfhydryl
groups and [3 H]glutamate release were compared by
linear regression analysis. Corresponding coefficients
of determination (r2) were calculated from the Pearson
correlation coefficient (InStatTM 3.0, GraphPad Soft-
ware, San Diego, CA).
Determination of Synaptosomal Glutamate
Uptake and Kinetic Analysis
Effects of In Vivo ACR on Neurotransmitter
Uptake
Glutamate uptake was measured in synaptosomes
isolated from ACR-intoxicated rats and their age-
matched controls (see above ‘‘Animals and ACR Intox-
ication’’) according to the method of Robinson et al.
(1991). Synaptosomes were trapped on filters in a
superfusion system, equilibrated at 36 8C and then
superfused with Krebs–Hepes buffer containing
labeled and unlabeled glutamate at final concentrations
ranging from 1 to 500 mM. To correct for Naþ-inde-
pendent, low affinity transport (Robinson et al., 1991),
uptake was measured in the presence and absence
(equimolar choline chloride substitution) of sodium
ions. Following the uptake period (3 min), filter-
trapped synaptosomes were washed (5 min) with ice-
cold buffer and radioactivity was determined by liquid
scintillation counting. Kinetic parameters (Km, Vmax)
for synaptosomal glutamate uptake were determined
by nonlinear regression analysis (PrismTM, GraphPad
Software, San Diego, CA). Respective control and
ACR kinetic parameters were compared statistically
(P < 0:05) by two-tailed Student’s t-test (InStatTM;
GraphPad Software, San Diego, CA).
Effects of In Vitro ACR, Propionamide, NEM
and IAA on Neurotransmitter Uptake
To characterize effects of in vitro toxicant exposure,
synaptosomes were incubated with varying concentra-
tions of ACR, propionamide, NEM or IAA (see above
exposure protocols for neurotransmitter release). Fol-
lowing chemical incubation, Naþ-dependent synapto-
somal uptake was initiated by incubation with Krebs–
Hepes buffer containing [3 H]glutamate (0.417 mM
[3 H]glutamate). Uptake was terminated by rapid super-
fusion of the filter-trapped synaptosomes with normal
ice-cold Krebs–Hepes buffer and radioactivity was
determined by liquid scintillation counting. Coeffi-
cients of determination, IC50’s and 95% confidence
354 R.M. LoPachin et al. / NeuroToxicology 25 (2004) 349–363
intervals were calculated as described above. To deter-
mine how ACR and the other sulfhydryl reagents
affected the kinetic parameters for in vitro glutamate
uptake, synaptosomes were exposed to the correspond-
ing IC50’s for each toxicant (i.e. ACR 187 mM; NEM
35 mM; IAA 9.5 mM) followed by incubation with
varying glutamate concentrations (see above). Kinetic
constants (Km, Vmax) were computed and statistically
compared as described above.
RESULTS
Neurological Evaluation
ACR intoxication at 50 mg/kg daily dose-rate pro-
duced a progressive loss in body weight and abnormal
changes in gait (LoPachin et al., 2002b). Rats in this
dose-rate group had a mean (S.E.M.) starting body
weight of 274  4 g and after 8 days of ACR intoxica-
tion, mean weight decreased by 14% to 236  8 g. Gait
scores for the higher exposure group increased from
1:3  0:4 on day 5 to 2:3  0:1 on day 8 of intoxica-
tion. This score represents statistically significant,
slight-to-moderate changes in gait (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 19% in
body weight; i.e. starting body weight for the control
group was 266  3 g and after 8 days the body weight
increased to 316  4 g. ACR intoxication at the lower
daily dose-rate (21 mg/kg) slowed the normal rate of
daily weight gain and induced significant increases in
gait scores (LoPachin et al., 2002b). Age-matched
control rats had a starting mean body weight of
269  3 g, which increased to 358  4 g by day 21
of the exposure paradigm. This represents a 35%
increase in body weight at a daily rate of approximately
4.5 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 264  4 g and after 21 days of ACR
intoxication body weight rose to 312  5 g. This repre-
sents an 18%increase in weight at a daily rate of
approximately 2.3 g per day. Age-matched control rats
did not exhibit changes in gait during the 21-day experi-
mental period, whereas the gait score for ACR-intoxi-
cated rats (21 mg/kg per day) increased steadily from an
initial score of 1:1  0:1 to 2:8  0:2 on day 21 of
exposure. This gait score represents a slight-to-moderate
level of neurotoxicity.
Table 1
Effects of in vivo ACR intoxication on synaptosomal neurotrans-
mitter release
Parameter 21 mg/kg per 50 mg/kg per
day
21 days day
8 days
AURC 65  5 49  8
Fractional release 81  1 67  9
Synaptosomes were prepared from diencephalon-midbrain region of brains
from ACR-intoxicated rats or their age-matched controls (n ¼ 4–6 rats per
group). Isolated synaptosomes were loaded with [3 H]glutamate, placed in
a superfusion system and Ca2þ-dependent, release was stimulated by a 90 s
pulse of 40 mM KCl-containing Krebs buffer. Superfusate was collected
continuously and radioactivity was determined by liquid scintillation
counting. Data are presented as mean percent of control  S:E:M: and ()
indicates significantly (P < 0:05) different from control. AURC: inte-
grated area under the release curve (control ¼ 9023  436); fractional
release ¼ percent fractional release calculated as AURC/total counts
(control ¼ 21  1%).
In Vivo Effects of ACR on Synaptosomal Protein
Content and Neurotransmitter Release and Uptake
We measured several parameters of Ca2þ-dependent,
[3 H]glutamate release in synaptosomes prepared from
ACR-intoxicated and age-matched control rats. Mean
(S.E.M.) fractional release evoked by superfusion of
control synaptosomes with a KCl (40 mM) pulse was
21  1%, which is consistent with previous release
studies (Nedvetsky et al., 2000). Results presented in
Table 1 indicate that intoxication at either ACR dose-
rate produced significant decreases in percent release
and corresponding integrated area under the release
curve. Fig. 1 shows Naþ-dependent, [3 H]glutamate
uptake in synaptosomes isolated from brain regions
of ACR-intoxicated and age-matched control rats. The
hyperbolic substrate–velocity graphs for control data
(Fig. 1A and B) demonstrates typical substrate satura-
tion kinetics expected of Naþ-dependent, glutamate
transport in synaptosomes (Robinson et al., 1991). The
data could be closely fit to a Michaelis–Menten model
as indicated by an r2 value of 0.98. Intoxication at the
higher ACR dose-rate (Fig. 1A) was not associated
with statistically significant changes in uptake kinetic
parameters. The lower dose-rate (Fig. 1B) produced a
significant decrease in Km but did not affect Vmax.
Table 2 presents the levels of several proteins in
synaptosomes isolated from ACR-intoxicated rats.
Except for b-tubulin (see ahead), the proteins measured
in this study are delivered to nerve terminals by fast
anterograde transport (FAT) (Li et al., 1995, 1996;
Shiff and Morel, 1997; Skene and Willard, 1981)
and they participate in synaptic vesicle cycling/exocy-
tosis (Calakos and Scheller, 1996; Lin and Scheller,
2000). In contrast, b-tubulin is a polypeptide subunit of
 R.M. LoPachin et al. / NeuroToxicology 25 (2004) 349–363 355

200 (A) ACR (50mg/kg/d x 8d) Table 2

Relative protein contents of synaptosomes isolated from ACR-
intoxicated rats
nmol/mg/min

150
Protein 21 mg/kg per 50 mg/kg per
day
21 days day
8 days
100
Cerebral cortex
( )Control ( ) ACR b-Tubulins 89  7 96  11
50 Gap-43 121  11 111  7
V m ax 131.0 134.7
SNAP-25 105  9 103  5
Km 45.8 67.4
0
Rab-3a 94  7 109  8
0 100 200 300 400 500 600 Synaptophysin 103  4 101  10
Synaptobrevin 100  6 93  6
Gl ( M)
Midbrain-diencephalon
b-Tubulin 91  11 90  6
300 (B) ACR (21mg/kg/d x 21d) Gap-43 107  2 92  5
SNAP-25 111  11 103  4
250 Rab-3a 100  13 98  3
nmol/mg/min

Synaptophysin 104  9 95  5
200
Synaptobrevin 116  10 101  5
150 Synaptosomes were isolated from brain regions of ACR-intoxicated rats
and their age-matched controls (n ¼ 4–6 rats per group). Synaptosomal
100 proteins were separated by SDS-PAGE and were then transferred to
( )Control ( ) ACR
nitrocellulose membranes and probed with indicated antibody. Immunor-
50 V m ax 219.8 236.3
eactive bands were scanned by a densitiometer, digitized and quantified by
Km 129.6 70.8 * image analysis (NIH Imaging Program). Data are presented as mean
0 percent ofcontrol  S:E:M. Statistical analysis did not indicate statistically
0 100 200 300 400 500 600
significant (P < 0:05) changes in protein content relative to respective
Glu ( M) control. Gap-43: growth-associated protein of 43 kDa; SNAP-25: synap-
tosomal-associated protein of 25 kDa; Rab-3a: Rabphilin 3a.
Fig. 1. Kinetic analysis of high affinity, Naþ-dependent transport
of [3 H]glutamate by synaptosomes isolated from ACR-intoxicated
rat brains. Rats were intoxicated with ACR at two different daily
dose-rates [(A) 50 mg/kg per day, i.p. or (B) 21 mg/kg per day
p.o.] until a slight-to-moderate level of neurotoxicity was attained
(days 8 or 21, respectively). Graphed data in A and B are
presented as mean nmol glutamate/mg synaptosomal protein/
min  S:E:M. Corresponding kinetic analyses (Vmax ¼ nmol/mg;
Km ¼ mM) are provided in the insert table. Asterisk indicates
significantly different from age-matched control (P < 0:05).

cytoskeletal microtubules and is transported in slow

component a (SCa; Sullivan, 1988). Data in Table 2
indicate that, despite changes in neurotransmitter
release, neither ACR dosing-regimen altered synapto-
somal protein content as measured by immunoblot
analysis.
In Vitro Chemical Adduction of Synaptosomal
Proteins
Lanes 2 and 3 of Fig. 2 show the electrophoretic
separation (7.5% gel) of proteins from cerebrocortical
synaptosomes. Lane 3 is derived from synaptosomes
exposed to non-labeled ACR in vitro (10 mM
60 min).
The comparable Coomassie blue staining patterns
(compare lanes 2 and 3) indicate that in vitro ACR
exposure did not affect migration of synaptosomal
Fig. 2. This figure shows autoradiographic patterns of [14 C]-
labeled synaptosomal proteins associated with different experi-
mental conditions. Lane 1: molecular weight standards; lane 2:
distribution of Coomassie blue-stained proteins in 7.5% poly-
acrylamide gel, proteins were isolated from synaptosomes
incubated in control conditions; lane 3: polyacrylamide gel
(7.5%) showing the migration pattern of proteins from synapto-
somes incubated with ACR (10 mM); lanes 4 and 5: autoradio-
graph showing labeled protein bands following incubation of
synaptosomes with [14 C]-ACR (58 nM, 7 mCi); lanes 6 and 7:
autoradiograph showing an absence of labeled protein bands
following incubation of synaptosomes with NEM (4 mM) and
[14 C]-ACR (dark band at the top of the autoradiograph represents
non-migrating [14 C]-ACR); lanes 8 and 9: autoradiograph showing
labeled protein bands following incubation of synaptosomes with
propionamide (10 mM) and [14 C]-ACR.
356 R.M. LoPachin et al. / NeuroToxicology 25 (2004) 349–363

proteins through the gel. Additional studies showed (A) Release

110
that preincubation with NEM or propionamide also did 100

% Control ± SEM
not alter gel migration of synaptosomal proteins (data 90
not shown). The corresponding autoradiographs (lanes 80
70
4 and 5) show that [14 C]-ACR labeled several distinct 60 NEM IAA ACR
SH Content SH Content SH Content
protein bands ranging from an estimated MW of 10.5– 50 (31µM) (2.7mM) (356mM)
Release Release Release
154,000 kDa (Fig. 2). ACR labeling was selective since 40 (13µM) (2.1mM) (480mM)

many protein bands on the gels were not associated 30

20
with autoradiographic counterparts (e.g. lane 3; band at 10
31.7 kDa). Certain protein bands that were heavily 0
stained by Coomassie blue (e.g. lane 3; band at -8 -7 -6 -5 -4 -3 -2 -1 0 1
110,000 kDa) exhibited correspondingly dense [14 C]- Log Concentrations
labeled autoradiograph bands (lane 4). Other heavily
stained proteins (e.g. lane 3; band at 38,000 kDa) were 100 (B) Regression Analysis
matched to lightly labeled autoradiographic bands 90

Fractional Release
80
(lane 4) suggesting limited ACR binding affinity for
r 2 = 0.9594

these proteins. When synaptosomes were preincubated
with the sulfhydryl alkylating agent, NEM, followed
by [14 C]-ACR exposure, the corresponding autoradio-
graphs (lanes 6 and 7) lacked protein bands (compare
lanes 5 and 6) and a broad dark stain was present at the
top of the respective lanes. Corroborative gel electro-
phoresis studies with [14 C]-ACR alone (no protein)
revealed similar positioning of a dark stain on corre-
sponding autoradiographs indicating that unbound
radiolabel did not migrate during electrophoresis (data
not shown). This, and the observation that NEM
adduction does not affect the gel migration pattern
(see above), suggest that preincubation with NEM-
blocked binding of [14 C]-ACR to synaptosomal pro-
teins. Accordingly, the dark band at the top of lanes 6
and 7 represents unbound, non-migrating [14 C]-ACR.
In contrast, preincubation with propionamide, a non-
neurotoxic structural analog of ACR, did not affect
ACR–protein binding; i.e. compare lanes 5 and 8
(Fig. 2). Similar results were obtained when the same
[14 C]-ACR incubation conditions were followed by
separation of synaptosomal proteins on 10 or 16.5%
gels (n ¼ 3 per gel; data not shown).
In Vitro Effects of ACR, Propionamide, NEM or
IAA on Synaptosomal Neurotransmitter Release
and Free Sulfhydryl Groups
In this series of studies, we determined the acute in
vitro effects of ACR, propionamide, NEM and IAA
on both synaptosomal [3 H]glutamate release and free
sulfhydryl content. Results (Fig. 3A) show that ACR,
NEM and IAA each produced concentration-depen-
dent decreases in synaptosomal neurotransmitter
release and, over the same concentration ranges, all
chemicals produced highly correlated reductions in
% Control
70
60
50
40
30
20
10
0
0 10 20 30 40 50 60 70 80 90 100
% Control SH Content
Fig. 3. Concentration-dependent effects of NEM, IAA and ACR
on synaptosomal KCl-evoked, Ca2þ-dependent [3 H]glutamate
release and free sulfhydryl group content are presented in (A)
Data are presented as mean percent control  S:E:M:; control free
sulfhydryl content: 88  14 pmol/mg synaptosomal protein; con-
trol fractional release: 21  1%. Calculated IC50’s are provided in
parentheses. Regression analysis of pooled neurotransmitter
release and sulfhydryl content data is presented in (B). Coefficient
of determination (r2) is provided and dashed lines denote the 95%
confidence interval.
sulfhydryl contents. In contrast, propionamide did
not affect synaptosomal [3 H]glutamate release or sulf-
hydryl content (data not shown). Specifically, ACR
exposure produced a concentration-dependent decrease
in neurotransmitter release that was nearly identical to
the effects on sulfhydryl content over a wide concen-
tration range (Fig. 3A; 50 mM–2 M). Linear regression
analysis revealed that the reduction of synaptosomal
neurotransmitter release induced by in vitro ACR
exposure was closely correlated to the decrease in free
sulfhydryl groups (r 2 ¼ 0:9705). In addition, the
respective IC50’s for release and sulfhydryl content,
480 mM versus 356 mM, did not differ significantly
(Fig. 3A, P ¼ 0:9331). NEM was the most potent of
the three chemicals tested and decreased both release
and sulfhydryl content over a concentration range of
100 nM–500 mM (Fig. 3A). The respective IC50’s for
 R.M. LoPachin et al. / NeuroToxicology 25 (2004) 349–363 357

the NEM-induced effects on release and sulfhydryl 110 (A) Uptake

content were not statistically different; i.e. 13 and 100

% Control ± SEM
31 mM (P ¼ 0:7162). The concentration-dependent 90
80
inhibition of [3 H]glutamate release induced by NEM
70
was closely correlated to the reduction of sulfhydryl 60 NEM IAA ACR
content (r 2 ¼ 0:9425). IAA was of intermediate 50 SH Content
(31µM)
SH Content
(2.7mM)
SH Content
(356mM)
potency and reduced both release and sulfhydryl con- 40 Uptake
(35µM)
Uptake
(9.5mM)
Uptake
(187mM)
tent over a dose-range of 10 mM to 100 mM (Fig. 3). 30
20
The respective IC50’s, 2.7 and 2.1 mM, did not differ
10
statistically (P ¼ 0:6117), and the effects of IAA on 0
release and sulfhydryl content were highly correlated -8 -7 -6 -5 -4 -3 -2 -1 0 1
(r 2 ¼ 0:9625). Linear regression analysis of the pooled Log Concentrations
release and sulfhydryl content data for each chemical
tested indicated a high degree of correlation among the (B) Regression Analysis
100
respective data sets (Fig. 3B; r 2 ¼ 0:9594). 90

In Vitro Effects of ACR, Propionamide, NEM or
IAA on Synaptosomal Neurotransmitter Uptake
and Free Sulfhydryl Groups
Our analysis indicates that ACR, NEM and IAA
each produced concentration-dependent decreases in
synaptosomal glutamate uptake, whereas propiona-
mide was without effect (data not shown). Fig. 4A
shows that in vitro exposure of synaptosomes to ACR
produced progressive decreases in both Naþ-depen-
dent, [3 H]glutamate uptake and free sulfhydryl content
over the same concentration range. The respective
IC50’s, 356 and 187 mM, did not differ statistically
(P ¼ 0:0634). Moreover, the concentration-dependent
reduction in free sulfhydryl groups produced by ACR
was closely correlated to the decrease in uptake
(r 2 ¼ 0:9705). NEM produced a graded reduction of
both synaptosomal [3 H]glutamate uptake and sulfhy-
dryl groups over the same concentration range
(Fig. 4A). The respective IC50’s were 35 and 31 mM
and were not statistically different (P ¼ 0:7162). Like
ACR, the concentration-dependent reduction in sulf-
hydryl content induced by NEM was closely correlated
to the progressive inhibition of [3 H]glutamate uptake
(r 2 ¼ 0:9661). The ability of IAA to produce concen-
tration-dependent inhibition of uptake was also similar
to the corresponding relative potency for reduction of
sulfhydryl content (Fig. 4A). The respective IC50’s, 9.5
and 2.7 mM, did not differ statistically (P ¼ 0:0652)
and the effects of IAA on sulfhydryl content and uptake
were highly correlated (r2 ¼ 0:9276). When the sulf-
hydryl and uptake data for ACR, NEM and IAA were
pooled and analyzed by linear regression, the r2 value
was 0.9266 (Fig. 4B). To determine how in vitro
exposure to ACR, NEM and IAA affected the kinetic
properties of uptake, synaptosomes were exposed to
% Control Uptake
80
70 r2= 0.9266
60
50
40
30
20
10
0
0 10 20 30 40 50 60 70 80 90 100
% Control SH Content
Fig. 4. Concentration-dependent effects of NEM, IAA and ACR
on synaptosomal high affinity, Naþ-dependent [3 H]glutamate
uptake and free sulfhydryl group content are presented in (A).
Data are presented as mean percent control  S:E:M:; control free
sulfhydryl content ¼ 88  14 pmol/mg synaptosomal protein;
control uptake ¼ 57:4  6:1 pmol glutamate/mg protein/min.
Calculated IC50’s are provided in parentheses. Regression analysis
of pooled neurotransmitter release and sulfhydryl content data is
presented in (B). Coefficient of determination (r2) is provided and
dashed lines denote the 95% confidence interval.
the respective IC50’s for each chemical and transport
was measured as a function of [3 H]glutamate concen-
tration. Results shown in Fig. 5 indicate that each
chemical produced a significant comparable decrease
in Vmax, whereas, with the exception of NEM, these
chemicals did not affect Km.
Data presented in Figs. 3A and 4A indicate that in
vitro exposure of synaptosomes to ACR, NEM or IAA
caused graded, concentration-dependent decreases in
synaptosomal neurotransmitter release, uptake and free
sulfhydryl groups. For each parameter measured, the
rank order of respective IC50’s for these chemicals (e.g.
Fig. 3A) indicates relative differences in potency; i.e.
NEM > IAA > ACR. However, it is important to note
that NEM, IAA and ACR were equivalent with respect
to maximal efficacy; i.e. all chemicals were capable of
358 R.M. LoPachin et al. / NeuroToxicology 25 (2004) 349–363
300
250
nmol/mg/min
200
150
100
V m ax K m
Control 259.7 55.33
50 ACR 122.8 * 27.83
NEM 124.7* 16.47 *
IAA 154.4* 45.81
0 Although these proteins were synthesized in the nerve
0 100 200 300
Glu (µM)
Fig. 5. Kinetic analysis of high affinity, Naþ-dependent transport
of [3 H]glutamate by synaptosomes exposed in vitro to the res-
pective IC50 concentrations of ACR, NEM or IAA. Graphed data
are presented as mean nmol glutamate/mg synaptosomal protein/
min  S:E:M. Corresponding kinetic analyses (Vmax ¼ nmol/mg;
Km ¼ mM) are provided in the insert table. Asterisk indicates
significantly different from age-matched control (P < 0:05).
reducing sulfhydryl group content below detectable
limits and completely blocking neurotransmitter
release and uptake (e.g. Fig. 3A).
DISCUSSION
In Vivo Effects of ACR on Synaptosomal
Function and Protein Content
Data from electrophysiological, morphological and
neurochemical studies implicate defective neurotrans-
mission at central and peripheral synapses of ACR-
intoxicated animals (reviewed in LoPachin et al.,
2002a, 2003). Goldstein and Lowndes (1979, 1981)
have suggested that this defect might be mediated by
changes in presynaptic transmitter uptake, synthesis,
vesicle storage or release. As an initial investigation we
focused on neurotransmitter uptake and release as
possible ACR targets. Results show that Kþ-evoked,
Ca2þ-dependent glutamate release was significantly
decreased in synaptosomes isolated from ACR-intoxi-
cated rats, whereas the kinetic parameters of high
affinity, Naþ-dependent glutamate uptake were not
consistently affected (see ahead). These findings there-
fore offer a possible explanation for ACR-induced
defective neurotransmission (decreased presynaptic
release) and suggest an in vitro model system (evoked
synaptosomal transmitter release) for mechanistic stu-
dies (see ahead). It is possible that the observed
reduction in transmitter release was secondary to
decreased perikaryal synthesis and/or kinesin-based
fast anterograde transport of materials to distal axon
regions (Cavanagh, 1964, 1979; Sickles et al., 1996;
Stone et al., 2000). Subsequent depletion of nerve
terminal components critically involved in the synaptic
vesicle cycle (e.g. SNAP-25, synaptobrevin) could be
responsible for synaptic dysfunction. Based on this
possibility, we measured the levels of several proteins
in release incompetent synaptosomes isolated from
different brain regions of ACR-intoxicated rats.
400 500 cell body and the majority were delivered to nerve
terminals by FAT, we found no changes in correspond-
ing content (Table 2). These findings are consistent
with previous research from this and other laboratories,
which showed that induction of ACR neurotoxicity was
not associated with significant changes in peripheral
nerve axonal Naþ/Kþ-ATPase protein content/activity,
subaxonal ion regulation or other distal axon para-
meters that are dependent on FAT for supply of critical
components (reviewed in LoPachin, 2002; LoPachin
et al., 2000, 2002a,b, 2003). If impaired synthesis and/
or fast anterograde transport were pathophysiologi-
cally important, secondary alterations in these para-
meters should have occurred. The absence of observed
changes suggests that ACR does not disrupt perikaryal
or axonal processes and might, instead, cause presy-
naptic dysfunction by acting directly at nerve terminal
sites (see ahead).
Identification of Putative Nerve Terminal
Protein Targets
To identify possible nerve terminal targets and che-
mical interactions, we characterized [14 C]-ACR label-
ing of synaptosomal proteins. Results show that certain
protein bands were variably labeled by in vitro expo-
sure to [14 C]-ACR. Preincubation of synaptosomes
with the sulfhydryl-alkylating agent NEM completely
prevented radiolabeling of proteins, which suggests
that ACR–protein interactions were mediated by thiol
adduction. Although the neurotoxicological relevance
has been questioned (Hashimoto and Aldridge, 1970;
Martenson et al., 1995a,b), adduction of protein thiol
groups is, nonetheless, consistent with the chemical
structure of ACR. ACR is an a,b-unsaturated carbonyl
chemical with electrophilic reactivity at the carbonyl
and b-alkene carbon atoms. As such, ACR will interact
with nucleophilic centers via a carbonyl condensation
reaction known as the Michael addition (Calleman,
1996; Friedman, 1973; Kemp and Vellaccio, 1980). As
an electrophile, ACR could interact with biological
nucleophilic centers formed by amines, imidazoles and
 R.M. LoPachin et al. / NeuroToxicology 25 (2004) 349–363
thiol groups. However, the nucleophilic reactivity of
sulfur is significantly greater than that of either carbon,
oxygen or nitrogen (Friedman, 1973; Kemp and
Vellaccio, 1980) and, consequently, the preferential in
vivo target nucleophile for ACR is thiol groups on, for
example, protein cysteine residues (see review by Calle-
man, 1996). The relative affinity of ACR for sulfhydryl
groups has been demonstrated in previous in vivo and in
vitro studies of protein adduct formation (Bergmark
et al., 1991, 1993; Calleman, 1996; Cavins and Fried-
man, 1968; Dixit et al., 1986; Sega et al., 1989) and by
ACR-inhibition of zinc iodide–osmium staining of pro-
tein thiol groups at the rat neuromuscular junction
(Kemplay and Cavanagh, 1984a,b; Kemplay et al.,
1988). In our studies, preferential thiol adduction was
demonstrated by NEM blockade of [14 C]-ACR protein
labeling and by ACR reduction of synaptosomal free
sulfhydryl groups. In addition, we found that propiona-
mide did not alter synaptosomal function nor free
sulfhydryl content. Propionamide is a non-neurotoxic
structural analog that lacks the unsubstituted alkene
function of ACR and has little electrophilic reactivity.
Together, these findings indicate that protein thiol
groups are the principal target nucleophile for ACR.
In Vitro Effects of ACR on Synaptosomal
Neurotransmitter Release and Possible
Neurotoxicological Relevance
Sulfhydryl content and neurotransmitter release
were determined following in vitro exposure of brain
synaptosomes to ACR or two sulfhydryl alkylating
agents, NEM and IAA. Results show that all three
compounds produced parallel, concentration-depen-
dent decreases in neurotransmitter release that were
highly correlated to respective reductions in synapto-
somal-free sulfhydryl content (Fig. 3A and B). The
finding that both in vivo and in vitro exposure to ACR
caused inhibition of neurotransmitter release suggests
that the in vivo effect was due to the direct actions of
ACR at nerve terminal sites (see ahead). Furthermore,
this correspondence indicates that in vitro inhibition
represents a specific neurotoxicological effect with
possible mechanistic relevance. Specificity is substan-
tiated further by the observation that release was
inhibited in a graded, concentration-dependent fashion
(Fig. 3A). The sequential and parallel nature of the
concentration–response curves for NEM, IAA and
ACR implies that these compounds interact with a
common sulfhydryl-regulated component of the pre-
synaptic release mechanism. The rank order of these
curves indicates that ACR has relatively low potency
359
for this interaction. However, the ability to produce a
maximum effect (e.g. inhibition of transmitter release)
equivalent to those of NEM or IAA denotes the full
efficacy of ACR (Fig. 3A). The low potency/high
efficacy of ACR is a critical character and indicates
that, although higher in vitro concentrations are needed
to promote protein adduction, the pathophysiological
outcome (i.e. inhibition of release) of the ACR adducts
is comparable in magnitude to that induced by NEM–
or IAA–protein adducts. This has significant implica-
tions for in vivo toxicodynamics (see ahead).
Whereas these in vitro characteristics implicate a
specific neurotoxic action, it is nonetheless difficult to
reconcile the relatively high ACR concentrations
needed to produce such in vitro effects (i.e. the ACR
IC50 for release is 480 mM) with the in vivo exposure
conditions (i.e. the peak ACR plasma concentration
after 11 days at 50 mg/kg is 422 mM; Barber et al.,
2001). This disparity could be related to the relatively
low potency of ACR for sulfhydryl adduction and the
distinctive toxicodynamics of adduct formation during
in vivo and in vitro exposure. Specifically, regardless of
exposure conditions, graded inhibition of transmitter
release should occur as the fractional presynaptic
concentration of adducted, functionally impaired pro-
tein increases (Goldstein and Lowndes, 1979, 1981;
Hinson and Roberts, 1992). However, during acute in
vitro exposure of synaptosomes, high ACR concentra-
tions will be necessary to generate the requisite toxic
levels of adducted protein based on the lower sulfhy-
dryl potency of this toxicant and the law of chemical
mass action. In contrast, in vivo intoxication is inher-
ently more complex. Although ACR has low potency
for the formation of sulfhydryl adducts, the process is
an irreversible covalent reaction (see above). Therefore,
in vivo, detoxification or removal of this adduct will
depend upon the corresponding half-life of the protein.
For proteins with long half-lives, nerve terminal adduct
levels would increase during in vivo exposure by a
cumulative process. Accordingly, many key regulatory
proteins associated with neurotransmitter release have
relatively long half-lives (Calakos and Scheller, 1996)
and are highly sensitive to inhibition by sulfhydryl
alkylation (see ahead). These proteins would therefore
be susceptible to additive, exposure-dependent adduc-
tion by ACR and subsequent functional inhibition. Low
sulfhydryl potency and cumulative protein adduction are
consistent with the in vivo neuropathokinetics of ACR;
i.e. relatively high mg/kg daily dose-rates and extended
exposure are required for cumulative induction of neu-
rological deficits in laboratory animals (see Goldstein
and Lowndes, 1979, 1981; Kuperman, 1958; LoPachin
360 R.M. LoPachin et al. / NeuroToxicology 25 (2004) 349–363
et al., 2002b). Resolving the in vivo–in vitro disparity is
important for validating our in vitro model system and
for understanding the mechanism of ACR neurotoxicity.
Accordingly, we are currently using mass spectroscopy
techniques to assess synaptosomal protein adduct
formation during in vivo and in vitro ACR exposure.
The above scenario is analogous to that proposed for
2,5-hexanedione (HD); i.e. HD produces cumulative
neurotoxicity in laboratory animals, which is presumed
to be mediated by cumulative adduction of long-lived
cytoskeletal proteins, despite demonstrated low in vitro
potency for this molecular effect (reviewed in LoPachin
and Lehning, 1997).
In Vitro Effects of ACR on Synaptosomal
Neurotransmitter Uptake and Possible
Neurotoxicological Relevance
Our results have shown that in vitro exposure of
synaptosomes to ACR, NEM or IAA inhibited Naþ-
dependent L-glutamate uptake and this effect was
correlated to graded reductions in free sulfhydryl
groups. In brain, glutamate uptake is mediated by
one neuronal (EAAC1) and two glial (GLT1, GLAST)
subtypes (Kanai and Hediger, 1992; Storck et al., 1992).
The Percoll gradient procedure used in the present study
produces a relatively homogeneous synaptosomal pre-
paration, although some glial contamination is expected
(Dunkley et al., 1988). Regardless, the EAAC1 subtype
contains two conserved cysteine residues (Bjoras et al.,
1996) that appear to function as sulfhydryl redox-sen-
sing sites and thereby regulate transmitter uptake (Trotti
et al., 1997a,b). Kinetic analysis revealed that the
respective IC50’s for ACR, NEM and IAA significantly
decreased the uptake Vmax, which is consistent with
noncompetitive inhibition by irreversible adduction of
cysteine residues (Garret and Grisham, 1999). However,
the neurotoxicological relevance of inhibited in vitro
uptake is unclear, since we did not observe correspond-
ing in vivo changes (see above). This reservation could
be related to methodological problems such as a low in
vivo signal-to-noise ratio (e.g. a low percentage of
affected nerve terminals in a given brain region), which
make detection of subtle but mechanistically important
effects difficult.
Mechanistic Implications
The present results suggest that ACR impairs synap-
tosomal neurotransmitter release, possibly through
adduction of thiol groups on nerve terminal proteins.
These data support the proposal that disruption of
neurotransmission identified in previous electrophy-
siological studies of intoxicated laboratory animals
(e.g. Goldstein and Lowndes, 1979, 1981; Tsujihata
et al., 1974) was mediated by a direct effect of ACR at
presynaptic sites. Although specific targets have not
been identified, many proteins involved in neurotrans-
mitter release are thiol-rich and are therefore putative
candidates for ACR adduction. Indeed, neurotransmit-
ter release has been shown to be highly sensitive to
inhibition by thiol alkylating chemicals such as NEM
(Lonart and Sudhof, 2000; Nedvetsky et al., 2000; Pan
et al., 1995, 1996; Sequeira et al., 1997). The corre-
sponding molecular mechanism likely involves alkyla-
tion of SNAP-25 (synaptosomal-associated protein of
25 kDa), N-ethylmaleimide-sensitive factor (NSF)
and/or other presynaptic cysteine-containing proteins
that regulate the exocytotic process (Meffert et al.,
1996; Sollner et al., 1993; Tolar and Pallanck, 1998;
Washbourne et al., 2001). Based on this evidence, we
have proposed that ACR adducts sulfhydryl groups on
key regulatory proteins (e.g. NSF) and thereby impairs
neurotransmitter release (LoPachin et al., 2002a,
2003). Our results also suggest that in vitro ACR
exposure inhibited synaptosomal neurotransmitter
uptake. If this effect is active in vivo, it might play
a role in promoting synaptic dysfunction. The present
studies provide a foundation for ongoing research
designed to identify molecular targets and decipher
mechanisms of ACR-induced synaptic dysfunction.
Finally, in addition to the obvious neurotoxicological
implications, the current findings are consistent with
the rapidly developing concept that neurotransmission
is regulated by redox modulation of cysteine sulfhydryl
groups on specific proteins (e.g. NSF; Broillet, 1999;
Gilbert, 1982; Stamler, 1994).
ACKNOWLEDGEMENTS
Research presented in this manuscript was supported
by a NIH grant from the National Institute of Environ-
mental Health Sciences (RO1 ES03830-17).
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