The Toxicologist - Society of Toxicology

currents have shown that SCBIs have no apparent effect on Na+ channels at hyperpolarized
membrane potentials at which channels occupy resting conformations.
However, prolonged depolarizations to potentials that promote non-conducting
slow-inactivated states increase the affinity for SCBIs, resulting in a slow
reduction (“block”) of peak Na+ current amplitude that typically develops over a
period of several minutes. These results imply that SCBIs act by selectively stabilizing
Na+ channels in slow-inactivated conformations. The state-dependent action
of SCBIs is functionally analogous to that of local anesthetic, class I anticonvulsant
and class I antiarrhythmic drugs, which preferentially block open and fast-inactivated
Na+ channels. These drugs bind to the local anesthetic (LA) receptor, which
is formed by residues in the S6 transmembrane segments of at least three of the
four Na+ channel homology domains that form the inner ion pore. Several lines of
evidence suggest that SCBIs and therapeutic Na+ channel blockers may share a
common site of action. In particular, mutagenesis experiments show that residues
in Na+ channel DIV-S6 identified previously as elements of the LA receptor, also
are important determinants of SCBI binding and action. However, the larger molecular
size of SCBIs compared to therapeutic drugs acting at the LA receptor suggests
that SCBI binding determinants exist outside the LA receptor and still have
yettobedetermined.
Supported by NIH grant R01-ES014591.
1354 GABA A RECEPTOR ANTAGONISTS INCREASE FIRING,
BURSTING, AND SYNCHRONY OF SPONTANEOUS
ACTIVITY IN NEURONAL NETWORKS GROWN ON
MICROELECTRODE ARRAYS (MEAS): A STEP
TOWARDS CHEMICAL “FINGERPRINTING”.
A. M. Johnstone 1 , J. Turner 1 , C. Mack 1 , L. Burgoon 2 and T. J. Shafer 1 .
1 Integrated Systems Toxicology, U.S. EPA, Research Triangle Park, NC and 2 Research
Cores Unit, U.S. EPA, Research Triangle Park, NC.
Assessment of effects on spontaneous network activity in neurons grown on MEAs
is a proposed method to screen chemicals for potential neurotoxicity. In addition,
differential effects on network activity (chemical “fingerprints”) could be used to
classify chemical modes of action. To test this, we examined effects of 4 GABA A antagonists
(bicuculline (BIC), lindane, picrotoxin (PTX), RDX) on network activity
in neocortical neurons and compared them to verapamil (VER; Ca 2+ channel antagonist),
fluoxetine (FLU; 5HT 3 reuptake inhibitor) and muscimol (MUS;
GABA A agonist). Concentration-response relationships for effects on network spike
rates were determined using MEAs, followed by analysis of bursting patterns and
firing synchrony using Neuroexplorer and custom written software, respectively.
GABA A receptor antagonists increased network activity (EC 50 (μM): BIC, 0.41;
lindane, 1.9; PTX, 15.2; RDX, 12.3), while the other compounds decreased it
(IC 50 (μM): VER 6.9; FLU, 5.4; MUS, 0.4). GABA A receptor antagonists, but not
other compounds, also altered #bursts/min, burst duration, and %spikes in bursts.
GABA A receptor antagonists increased synchrony of firing (EC 50 (μM): PTX
0.045; RDX, 8; lindane, 0.15). By contrast, MUS (IC 50 = 7.7 nM), VER (IC 50 =
1.0 uM), FLU (IC 50 =1.5 uM) decreased synchrony. While additional chemical
classes need to be evaluated, these data confirm that MEAs are useful to screen
chemicals for neuroactivity and indicate that analysis of chemical effects on spike
rates, burst parameters and synchrony may be useful to develop chemical fingerprints.
This approach will improve the predictive nature of MEA data from uncharacterized
chemicals by providing mode of action information. This abstract does
not reflect Agency policy
1355 EFFECTS OF FIPRONIL ON THE EEG OF LONG-
EVANS RATS.
D. F. Lyke, K. L. McDaniel, V. C. Moser and D. W. Herr.
NB/TAD/NHEERL/ORD, U.S. EPA, Research Triangle Park, NC .
We have reported that the non-stimulus driven EEG is differentially altered by
deltamethrin or permethrin (Lyke and Herr, Toxicologist, 114(S-1):265, 2010). In
the current study, we examined the ability to detect changes in EEG activity produced
by fipronil, a phenylpyrazole pesticide that inhibits GABA receptors. Adult
male Long-Evans rats were dosed with fipronil (po). Range-finding studies (40 or
50 mg/kg, n=5/dose) were conducted using observations of neurological signs, grip
strength, and landing foot splay. The data suggested increased handling reactivity,
mild tremors and gait changes, circling, decreased hindlimb grip strength, and increased
foot splay. Maximal effects were evident about 5 to 8 hours after dosing.
Additional rats were implanted with epidural screw electrodes. After about 1 week
recovery, non-restrained animals were gavaged with corn oil and tested for 2 days to
290 SOT 2011 ANNUAL MEETING
allow acclimation. On day 3, the rats were dosed with 1 ml/kg vehicle (corn oil), 25
mg/kg fipronil, or 50 mg/kg fipronil and tested 6 h later. EEG was recorded as 30
segments of 2 s durations, transformed using a FFT, and the spectra averaged.
Treatment with 25 and 50 mg/kg fipronil decreased the area (17-25% decrease) associated
with Gamma activity when recorded between the visual cortex and frontal
cortex. The peak amplitude of this Gamma activity was decreased (20% decrease)
by 50 mg/kg fipronil. These results differ from the previously reported increases in
Gamma activity following treatment with permethrin (11-25% increase in amplitude-area),
and lack of changes after dosing with deltamethrin. The data show that
fipronil can alter CNS activity as measured by EEG, and the alterations may differ
from some other pesticides. This is an abstract of a proposed presentation and does not
necessarily reflect EPA policy.
1356 TREATMENT OF CAENORHABDITIS ELEGANS WITH
GLYPHOSATE OR MANCOZEB SUGGESTS SELECTIVE
NEURONAL DEGENERATION.
R. Negga, M. Machen, J. Salva and V. A. Fitsanakis. Biology, King College,
Bristol, TN.
Previous studies demonstrate a positive correlation between high levels of pesticide
usage and Parkinson’s disease (PD), a disease that preferentially targets dopaminergic
(DAergic) neurons. In order to better characterize the potential relationship between
two common pesticides and specific neurodegeneration, we chronically (24
hours) or acutely (30 min) exposed two different Caenorhabditis elegans (C. elegans)
strains to varying concentrations (LC 25 , LC 50 or LC 75 ) of TouchDown (TD:
glyphosate) or Manzate (MZ: manganese/zinc ethylene-bis-dithiocarbamate). To
further model environmental exposure, additional populations of worms were exposed
first to TD for 30 min, followed by a 30-min incubation with varying MZ
concentrations. Initially, studies were carried out in NW1229 worms (pan-neuronal::green
fluorescent protein [GFP] construct). Pixel counts from fluorescence
photomicrographs of worms treated with individual pesticides indicated statistically
significant decreases (p < 0.05)in the number of green pixels in the nerve ring
(primarily DAergic), ventral nerve cord (primarily GABAergic or chonlinergic) or
both regions. This was in contrast to data from the dual treatment, where a statistically
significant decrease (p < 0.05) in pixel number was only observed at 20% MZ.
In order to further examine the specificity of neurodegeneration following treatment
with these agrochemicals, we used EG1285 worms (GABAergic
neurons::GFP construct) to verify GABAergic neurodegeneration. Results indicated
a statistically significant decrease in number of green pixels associated with
GABAergic neurons in both chronic treatment (p < 0.01) paradigms, and in the
acute MZ treatment only at 30% (p < 0.001). Visual inspection of photomicrographs
suggested that the decrease in pixel number was due to morphological
changes in ventral nerve cord GABAergic neurons. Taken together, our data suggest
that exposure to TD or MZ promotes neurodegeneration, including that of
GABAergic neurons, in the model organism, C. elegans.
1357 EFFECTS OF ATRAZINE AND ITS METABOLITE
DIAMINOCHLOROTRIAZINE ON
UNDIFFERENTIATED AND DIFFERENTIATING N27
DOPAMINERGIC CELLS.
Z. Lin, C. A. Dodd, I. I. Georgieva and N. M. Filipov. Physiology and
Pharmacology, University of Georgia, Athens, GA.
Multiple in vitro and in vivo studies have shown that excessive exposure to the herbicide
atrazine (ATR) results in dopaminergic neurotoxicity, suggesting that ATR
targets the dopaminergic circuitry preferentially. However, effects of ATR or of its
major mammalian metabolite diaminochlorotriazine (DACT) on dopaminergic
cell differentiation are unknown. Hence, we sought to determine the impact of
ATR and DACT on undifferentiated and differentiating dopaminergic cells.
Immortalized dopaminergic cells (N27) were exposed for 24 or 48 h to a concentration
range of ATR or DACT with and without the presence of a differentiating
agent (dbcAMP) in the exposure medium. Exposure to 300 μM ATR or DACT
caused time-dependent cytotoxicity and decreased ATP production in undifferentiated
N27 cells. On the other hand, 48, but not 24 h, exposure to 300 μM ATR or
DACT increased the ADP: ATP ratio in both undifferentiated and differentiated
N27 cell, indicating that longer exposure to ATR or DACT in these cells may be
apoptotic. Morphological examination indicated that exposure to ATR (≥ 60 μM)
during differentiation decreased soma size as early as 24 h. In contrast, DACT disrupted
N27 cell differentiation by decreasing thin neurite formation at concentrations
as low as 12 μM, but only after 48 h incubation. These data demonstrate that