The Toxicologist - Society of Toxicology

ane. Our goal is to determine whether a nitrogen mustard injury heals more
slowly than an equivalent UVB injury. To accomplish this, corneal organ cultures
were exposed to different levels of UVB to determine conditions that would produce
a 24 hr post-UVB phenotype equivalent to the 24 hr post-exposure phenotype
of a 60 min exposure to 100 nmol NM. Corneal organ cultures were irradiated
14 cm from the light source for various times to produce different intensities of
UVB: 5 min resulted in 100 mJ/cm2, 20 min, 400 mJ/cm2; 40 min, 800 mJ/cm2;
60 min, 1200 mJ/cm2; 80 min, 1600 mJ/cm2; and 100 min resulted in a 2000
mJ/cm2 exposure. To compare these organ cultures to those exposed for 60 min to
100 nmol NM, corneas were embedded in O.C.T. medium, frozen, then sectioned
for H&E staining. Micrographs of sections were overlapped to make composites
covering the entire diameter of the cornea. From the composites, the length of denuded
cornea was calculated as a percentage of the entire width. Our results
demonstrated that a 60 min exposure to 100 nmol NM resulted in the epithelium
separating from the stroma across 58.3% of the corneal diameter. A nearly equivalent
phenotype (producing a 61.7% separation between the cell layers) resulted
from the 2000 mJ/cm2 UVB exposure. The time for the recovering epithelium to
completely migrate over the stroma was determined, and was shown to take 4 days
after NM exposure. In contrast, the equivalent damage from UVB exposure recovered
in only 3 days. Supported by RO1 EY009056, U54 AR055073 and P30
ES005022.
2034 SIGNALING MECHANISMS THAT CAN CONTRIBUTE
TO THE NEUROPROTECTION AFFORDED BY
GALANTAMINE IN GUINEA PIGS EXPOSED TO THE
NERVE AGENT SOMAN.
G. Kulkarni 1 , M. Akkerman 1 , Y. Aracava 1 , E. X. Albuquerque 2, 1 and E. R.
Pereira 1, 2 . 1 Department of Epidemiology and Public Health, University of
Maryland School of Medicine, Baltimore, MD and 2 Department of Pharmacology
and Experimental Therapeutics, University of Maryland School of Medicine,
Baltimore, MD.
Treatment of guinea pigs with galantamine, a drug used to treat Alzheimer’s disease,
prevents the neurodegeneration induced by organophosphorus nerve agents, including
soman [PNAS 103:13220, 2006]. This study was designed to identify
mechanisms that may contribute to the effectiveness of galantamine. Male guinea
pigs (30-32 days old) were treated with: (i) saline (0.5 ml/kg, im), (ii) galantamine
(8 mg/kg, im), (iii) soman (28 μg/kg, sc), (iv) galantamine followed 30 min by
soman, or (v) soman followed 15 min by galantamine. All animals, except those
challenged with soman alone, survived. Only 50% of the soman-challenged, untreated
guinea pigs survived; about 75% of them had mild or no signs of intoxication
whereas the remaining 25% presented severe signs of acute toxicity. At various
times after each treatment, animals were euthanized, and their brains removed for
subsequent protein analysis. At 2 h after treatment with galantamine, levels of heat
shock protein 25 (Hsp-25), and ratios of phospho-Akt to total Akt and phospho
(ser-136) Bad to total Bad were higher than those measured in brain extracts from
saline-injected animals. Both Hsp-25 and phospho (ser-136) Bad are known to
promote cell survival. Levels of Hsp-25 were also found to be elevated in brain extracts
obtained at 2 h after the soman challenge of guinea pigs that survived with
only mild or no signs of acute toxicity. This may represent a compensatory mechanism
triggered by the stress induced by soman. Brain levels of Bcl-2 progressively
decreased from 2 to 24 h after the soman challenge. Treatment of the guinea pigs
with galantamine prevented soman-induced loss of Bcl-2. The results presented
herein provide a mechanistic basis for the neuroprotection afforded by galantamine
in animals exposed to soman. (Support: NIH Grant UO1NS059344)
2035 GALANTAMINE COUNTERACTS COGNITIVE
DEFICITS IN GUINEA PIGS CHALLENGED WITH THE
NERVE AGENT SARIN.
J. Mamczarz 1 , Y. Aracava 1 , E. R. Pereira 1, 2 and E. X. Albuquerque 2, 1 .
1 Department of Epidemiology and Public Health, University of Maryland School of
Medicine, Baltimore, MD and 2 Department of Pharmacology and Experimental
Therapeutics, University of Maryland School of Medicine, Baltimore, MD.
Epidemiological studies have reported chronic memory decline among victims and
first responders of the 1995 Tokyo terrorist attack with the nerve agent sarin
(Environ Health Perspect 109:A542, 2001; Environ Health Perspect 109:1169,
2001). This study was designed to test the hypothesis that a single exposure of
guinea pigs to a low dose (0.6xLD50) of sarin induces delayed cognitive impairments
that are counteracted by treatment with galantamine, a drug that protects
436 SOT 2011 ANNUAL MEETING
guinea pigs against the acute toxicity of nerve agents (PNAS 103:13220, 2006).
Prepubertal male guinea pigs received: (i) saline (0.5 ml/kg, im), (ii) sarin (25.5
μg/kg, sc), or (iii) sarin followed 1 h later by galantamine (8 mg/kg, im). Three
months after the injections, the cognitive performance of the guinea pigs was first
tested in the Morris water maze (MWM). Animal received training to a fixed platform
position. Training consisted of four consecutive trials a day over 6 consecutive
days followed by the probe test on day 7. Compared to saline-injected animals,
sarin-challenged guinea pigs showed slower acquisition of the MWM task; only on
the 6th day of training did sarin-challenged animals perform as well as control animals.
There was also no significant difference in the probe test between sarin- and
saline-injected guinea pigs. In the 8-arm radial water maze, the performance of
sarin- and saline-injected animals was comparable. Thus, the cognitive impairment
developed by sarin-challenged animals is related to the procedural component of
learning. Sarin-challenged guinea pigs that were treated with galantamine presented
no cognitive impairment in any test. It is, therefore, concluded that: (i) a single exposure
to a low dose of sarin triggers the development of cognitive deficits, and (ii)
galantamine is an effective antidote to counteract the delayed neurotoxic effects of
sarin. (Support: NIH Grant UO1NS059344)
2036 ANALYSIS OF MAP KINASE SIGNALING IN SULFUR
MUSTARD TOXICITY USING PHARMACOLOGIC
INHIBITORS AND GENE EXPRESSION PROFILING.
C. C. Rothwell, C. M. Crum, P. A. Everley and J. F. Dillman. Cell and Molecular
Biology, USAMRICD, Aberdeen Proving Ground, MD.
Sulfur mustard (SM) is a potent alkylating agent and vesicant. SM exposure activates
numerous signaling cascades such as the mitogen-activated protein kinase
(MAPK) cascades. These include the ERK, p38, and JNK pathways, which are involved
in cell growth, inflammation, and stress signaling. The precise roles of these
pathways in SM toxicity have not been elucidated. We used Western blotting along
with pharmacological inhibitors and microarray analysis to examine the activation
and role of each pathway after SM exposure in primary human keratinocytes.
Western blotting revealed increased phosphorylation of p38 and JNK after SM exposure;
however phosphorylation of ERK was equivocal, suggesting that growth
conditions may impact ERK activation by SM. We used pharmacologic inhibitors
to target each MAPK and then compared gene expression profiles to identify SMinduced
genes regulated by each MAPK. Cells were pretreated with SB203580 (p38
inhibitor), PD98059 (ERK inhibitor), or SP600125 (JNK inhibitor) before SM exposure.
Cells were harvested at 1, 4, and 8h post-exposure, and RNA was extracted
for gene expression profiling. Analysis of variance identified genes significantly
modulated due to pharmacologic inhibition in SM-exposed cells. Pathway mapping
confirmed alterations in SM-induced MAPK signaling due to pharmacologic inhibition.
SM-induced expression of IL-6, IL-8, and TNF-alpha inflammatory cytokines
was decreased by p38 MAPK inhibition, but not by inhibition of other
MAPKs. Based on the number of significant pathways mapped to each MAPK in
the presence and absence of inhibitors, the p38 pathway appeared to be the MAPK
cascade most responsive to SM. Interestingly, pathway mapping of the microarray
data identified potential cross-talk between MAPK pathways and other pathways
involved in SM-induced signaling. Mining of these results will increase our understanding
of the role of MAPK cascades in SM-induced signaling and may identify
additional therapeutic targets for the development of SM medical countermeasures.
2037 THE NEUROPROTECTANT EFFECTS OF DIAZEPAM
ALONE AND COMBINATION WITH HISTONES
DEACETILASES INHIBITORS IN A RAT MODEL OF
STATUS EPILEPTICUS INDUCED BY
ORGANOPHOSPHORUS EXPOSURE.
F. Rossetti 1 , M. Furtado 1 , M. Addis 1 , B. Robertson 1 , M. Moffet 2 , L. Lumley 2
and D. Yourick 1 . 1 Walter Reed Army Institute of Research, Silver Spring, MD and
2 U.S. Army Medical Research Institute of Chemical Defense, Aberdeen Proving
Ground, MD.
The occurrence of status epilepticus (SE) is considered the main cause of brain lesions
and morphological alterations that could result in chronic epilepsy. SE caused
by nerve agents such as soman (organophosphorus family of chemicals) begins rapidly,
continues for hours, and contributes to prolonged physical incapacitation and
neuropathology in both rodent and non-human primate species. Identification of
neuroprotectants with efficacy against nerve agent-induced brain injury is the subject
of the present study. Male Sprague-Dawley (n=69) had SE-induced by LD50 of
soman (110 μg/kg, s.c.) and neuropathological effects analyzed 72 hours after.