The Toxicologist - Society of Toxicology

2011 IDENTIFICATION OF A NOVEL VX METABOLITE
FROM MINIPIG LIVER MICROSOMES VIA LC-Q-TOF
MASS SPECTROMETRY.
J. M. McGuire 1 , M. E. Parrish 2 , E. M. Jakubowski 1 and S. A. Thomson 1 . 1 U.S.
Army Edgewood CB Center, Aberdeen Proving Ground, MD and 2 SAIC,
Gunpowder, MD.
VX(O-Ethyl-S-[2-(diisopropylamino)ethyl] methylphosphonothioate is an extremely
toxic organophosphate chemical warfare nerve agent (CWNA) that rapidly
inhibits acetylcholinesterase. While the use of CWNAs is forbidden by an international
convention, nerve agents have been stockpiled in numerous countries and remain
a threat to both civilian and military personnel. The biochemical reactions at
the tissue and organism levels induced by CWNAs are very complex. If
metabolomic work can identify key metabolites present in a given condition, this
information can more adequately describe mechanisms of toxicity and potential
biochemical points for developing effective CWNA countermeasures. To that end,
we have been examining the metablolites formed following incubation of
Göttingen Minipig liver microsomes with VX. Although previous studies have been
done with various liver tissue homogenates from several animal species, we have focused
on isolating individual functional networks, such as the cytochrome P450
(CYP) superfamily of oxidative hemoprotein enzymes. Activating the CYP enzymes
with NADPH cofactor produced a novel metabolite of VX,(O-Ethyl-S-[2-(isopropylamino)ethyl]
methylphosphonothioate, which was identified using liquid
chromatography coupled with quadrupole/time-of-flight mass spectrometry.
Although this mono-isopropyl derviative of VX has been previously identified in
VX-ozone and UV irradiation reaction mixtures, to our knowledge this is the first
time this compound has been identified in a biochemical reaction.
2012 COMPARISON OF THE EFFECT UPON AND
RECOVERY OF ACETYLCHOLINESTERASE (ACHE)
ACTIVITY IN VARIOUS TISSUES AND BLOOD AFTER
SUB-LETHAL INHALATION AND SUBCUTANEOUS
CYCLOSARIN (GF) EXPOSURES.
C. E. Whalley 1 , L. A. Lumley 2 , J. O’Donnell 2 , D. Miller 1 , W. Muse 1 and T.
Shih 2 . 1 ECBC, Aberdeen Proving Ground, MD and 2 USAMRICD, Aberdeen
Proving Ground, MD .
AChE activity data was collected following either subcutaneous (SC) or whole body
inhalation (IH) exposures using two different sub-lethal GF doses (0.1 or 0.4 x
LD50/LCt50) in male Hartley guinea pigs from various tissues and blood at sequential
time points. Following the higher IH GF exposure, blood AChE was 84%
depressed and at 48hr was 60% depressed; 7 days later blood AChE was still 49%
depressed. A similar profile occurred with the lower IH exposure. With both SC exposure
concentrations AChE was also depressed but the AChE recovery process
began much earlier (24-48hr). AChE activity tissue results: Following IH exposure,
eye, lung, heart, kidney and liver activity was significantly depressed (time intervals
varied) but brain (prefrontal cortex/striatum) and diaphragm activity was essentially
unaffected; following SC exposure, brain, eye, lung, heart, diaphragm, and
kidney activity was significantly depressed (effective GF exposure concentration
and time intervals varied) while liver activity was essentially unaltered. In conclusion,
sub-lethal IH and SC GF exposures depressed blood AChE activity substantially
with slow recovery. Blood AChE recovery was not totally complete 48-72hr
after either SC or IH exposures. With tissue AChE activity, both exposure routes
and both GF concentrations caused significant reductions in AChE activity in eye,
lung and heart. Both IH exposure concentrations significantly inhibited activity in
kidney and liver but not in brain or diaphragm. In contrast, only the higher SC GF
exposure significantly inhibited AChE activity in these four tissues. Recovery time
for tissue AChE activity varied somewhat, 24-168hr (IH exposure) and from ~4hr
to >48hr (SC exposure). Data will be utilized to improve physiologically based
pharmacokinetic-pharmacodynamic models. Information generated by this study is
extremely relevant to force health protection concerns that also include individual
protection, decontamination and environmental surveillance.
2013 A DEEP DERMAL SKIN INJURY FOLLOWING
EXPOSURE OF HAIRLESS GUINEA PIGS TO SULFUR
MUSTARD VAPOR.
S. Dachir, M. Cohen, L. Tverya, R. Sahar, H. Gutman, R. Gez, R. Brandeis,
V. Horwitz and T. Kadar. Pharmacology, Israel Institute for Biological Research, Ness
Ziona, Israel. Sponsor: J. Graham.
Skin injuries following exposure to sulfur mustard (HD) are characterized by various
symptoms including inflammatory response, vesications, necrosis and ulcerations
of the exposed skin that are dose and exposure duration dependent. Although
HD was first introduced during WW1, no effective treatment is yet available, and
very often HD-induced injuries result in long-term complications. In order to develop
a treatment that will improve the healing process and prevent skin damage
and long-term effects, an experimental model of a deep dermal skin injury following
exposure to HD was established and characterized in hairless guinea pigs
(HGP). Clinical evaluation was conducted using reflectance colorimetry, trans-epidermal
water loss and wound area measurements. Analysis of prostaglandin E2
(PGE2) content and metalloproteinase-2 & 9 (MMP) activity, as well as
histopathological and immunohistochemical assessment of the extent of the injury
were conducted up to four weeks post-exposure. Typical symptoms of HD skin injury
developed including erythema and edema, impairment of skin barrier and severe
ulcers. PGE2 content and MMP activity were elevated during the wound development
and during the healing process. Histological evaluation revealed severe
damage to the epithelium and deep dermis and vesications. At four weeks after exposure
healing was not completed: significantly impaired stratum corneum, inflammatory
cells, hemorrhages, absence of hair follicles and epithelial hyperplasia were
observed. These results confirm the use of the deep dermal skin injury in the HGP
as a suitable model that can further be utilized for the investigation of the pathological
processes of acute as well as long term injuries and for testing treatments efficacy.
This work was supported by the U.S. Army Medical Research and Material
Command under Award#: W81XWH-08-2-0128.
2014 REACTIVATION OF ORGANOPHOSPHATE INHIBITED
ACETYLCHOLINESTERASE (ACHE) BY NOVEL
PYRIDINIUM OXIMES IN THE CENTRAL NERVOUS
SYSTEM OF RATS.
E. C. Meek 1 , H. W. Chambers 1 , J. Gearhart 2 , R. B. Pringle 1 and J. E.
Chambers 1 . 1 Center for Environmental Health Sciences, Mississippi State University,
Mississippi State, MS and 2 AFRL, Wright-Patterson AFB, OH.
Organophosphate (OP), e.g. nerve agent, exposure resulting in the inhibition of
AChE with subsequent overstimulation of the nervous system, may result in neurological
deficits and death. Traditional therapies for OP exposure include atropine
and an oxime (2-PAM) to reactivate the OP inhibited AChE. A weakness of the
currently approved oximes is their limited ability to cross the blood-brain barrier
(BBB). A series of novel pyridinium oximes has been synthesized to incorporate
moieties increasing BBB penetration. Oximes were evaluated in vitro for their abilities
to reactivate AChE inhibited by nerve agent surrogates, a sarin surrogate, phthalimidyl
isopropyl methylphosphonate (PIMP), or a VX surrogate, nitrophenyl
ethyl methylphosphonate (NEMP), which leave AChE phosphorylated with the
same moieties as sarin or VX, respectively. Rat brain homogenate was incubated
with PIMP or NEMP yielding about 80% AChE inhibition, followed by an oxime
0.1mM, and AChE activity measured. In vitro reactivation varied among oximes
but was similar for surrogates, ranging from 21-82% for PIMP and 29-76% for
NEMP, with 2-PAM yielding about 90%. Oxime lipophilicities (n-octanol/water
partition coefficients) ranged from 0.009 to 2.244 and were greater than 2-PAM
(0.006). A subset of oximes that demonstrated in vitro reactivation greater than
40% was selected for testing in vivo. Rats were administered 0.325mg/kg ip of a
sarin surrogate, nitrophenyl isopropyl methylphosphonate (NIMP) or 0.4mg/kg of
NEMP yielding 70-80% brain AChE inhibition, followed by im administration of
2-PAM or novel oxime (0.1mmole/kg) at the time of peak brain AChE inhibition.
Ten of the 16 novel oximes tested yielded 10-35% brain AChE reactivation, indicating
their ability to cross the BBB and reactivate OP inhibited brain AChE,
demonstrating their therapeutic potential for exposure to nerve agents. Supported
by Defense Threat Reduction Agency: 1.E0056_08_AHB_C
2015 CATECHOLAMINE OVERLOAD AND CARDIAC
LESION FORMATION IN RATS FOLLOWING
EXPOSURE TO THE NERVE AGENT SOMAN.
J. H. McDonough, K. E. Van Shura, M. E. Lyman, B. A. Capacio, J. C.
O’Donnell and K. A. Whitten. U.S. Army Medical Research Institute of Chemical
Defense, Aberdeen Proving Ground, MD.
Nerve agents are potent inhibitors of the enzyme acetylcholinesterase. The resulting
cholinergic over-stimulation causes respiratory distress, convulsions, seizures, and
eventually death. Several studies have shown that nerve agent exposure can result in
cardiac damage. We investigated if this cardiac damage may be due to nerve agentinduced
excessive release of catecholamines. We first determined the incidence of
cardiac lesions 24 hr following administration of 0.6, 0.8, 1.0, or 1.2 X LD50 of
the nerve agent soman or saline to different groups of rats. Only animals in the 1.0
SOT 2011 ANNUAL MEETING 431