The Toxicologist - Society of Toxicology

and 1.2 LD50 exposure groups displayed robust signs of nerve agent intoxication
and also had cardiac lesions; animals at the 0.6 or 0.8 X LD50 doses showed mild
or no signs of intoxication and were free of cardiac damage. Next, rats were challenged
with 1.2 X LD50 soman, and different groups of animals were euthanized at
progressively longer delays following agent challenge. Plasma obtained at the time
of euthanasia was assayed for norepinephrine, epinephrine and troponin, and the
heart tissue was prepared with Masson’s trichrome to detect cardiac lesions. The results
showed that 15-30 min after nerve agent exposure there were significant increases
in epinephrine and norepinephrine levels, but no changes in cardiac troponin
levels. At 2-4 hr after intoxication, cardiac troponin levels increased and
elevations persisted at least 24 hr after exposure. The differential timing of the catecholamine
and troponin increases supports the hypothesis that the release of catecholamines
triggers the subsequent cardiac damage. This may result from a direct
overstimulation of the sympathetic nervous system by the high levels of acetylcholine.
The elicitation of convulsive activity by the nerve agent may be contributory,
since only animals that developed pronounced convulsions developed cardiac
lesions, whereas animals that displayed no or only minor signs of intoxication all
had normal cardiac tissue.
2016 CENTRALLY ACTING THERAPEUTIC ADJUNCTS FOR
NERVE AGENT INTOXICATION.
I. Koplovitz*, J. H. McDonough and T. Shih. Research Division, U.S. Army
Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, MD.
The brain is a major target for the toxic action of organophosphorus nerve agents
(NA). Two therapeutic approaches were conducted to assess the ability of centrally
active therapies to improve treatment outcomes following lethal NA intoxication.
One approach was to investigate tertiary oxime (MINA) that can readily penetrate
into the brain to determine whether reactivation of central AChE inhibited by NA
will increase survival and terminate seizures. The other approach was to evaluate the
survival benefit of adding scopolamine (SCP) to current treatment regimens. All
studies were conducted in male Hartley guinea pigs exposed s.c. to lethal and
seizure-inducing doses of various NA. In reactivation studies, the tertiary oxime was
administered 15 min after 1 x LD50 of sarin, soman, cyclosarin, or VR; peripheral
tissue, blood and regional brain oxime dose-dependent AChE activity was determined
45 min later. Oxime doses from these studies were used as treatments 1 min
after NA with or without atropine (ATS) and/or 2-PAM to assess the influence on
24-hr survival. Various oxime doses were evaluated for antizeizure activity when administered
at the time of and up to 40 min after seizure onset. SCP studies were
conducted to determine its survival benefit as an adjunct to ATS plus quaternary
oxime (2-PAM or MMB-4) treatment administered 1 min after 2 x LD50 of various
NAs with or without pyridostigmine (PB) pretreatment. Human relevant doses
of SCP, ATS, 2-PAM, and PB were evaluated. Tertiary oxime resulted in dose-dependent
increases in AChE activity in the brain, improved survival and terminated
seizures. Seizures could be terminated even when the tertiary oxime was administered
40 min after seizure onset. SCP increased survival at 24 hr, which appeared to
be dependent on recovery/restoration of AChE activity either through oxime reactivation
or decarbamlyation of PB-protected AChE. Both centrally acting oxime
and SCP appear to be viable therapeutic approaches as adjunct treatments to mitigate
the toxic effects of NA and improve treatment outcomes.
2017 RICIN TOXICITY IN BALB/C 3T3 CELLS: DOSE
DEPENDENT PROTEIN EXPRESSION DETERMINED
BY MASS SPECTROMETRY BASED PROTEOMICS.
V. H. Bevilacqua 1 , S. Deshpande 2 , J. S. Madren-Whalley 3 , R. Jabbour 4 , K.
Jones 5 , L. M. Reilly 6 and J. S. Rice 7 . 1 Point Detection Branch, Edgewood Chemical
Biological Center, Aberdeen Proving Ground, MD, 2 STC, Edgewood, MD,
3 Biotechnology Branch, Edgewood Chemical Biological Center, Aberdeen Proving
Ground, MD, 4 SAIC, Gunpowder, MD, 5 NuSep, Bogart, GA, 6 Physical Science
Department, Bethany College, Bethany, WV and 7 Elona Biotechnologies, Inc.,
Greenwood, IN. Sponsor: J. Sekowski.
Ricin has remained a significant potential biological threat agent due in part to its
wide availability and ease of extraction from the castor bean. It is a glycoprotein
made up of A- and B-chains with the B-chain facilitating transport of the lectin
into the cell. Once inside, it kills the cell by inhibiting protein synthesis. Ricin is
considered extremely toxic to man by multiple routes of exposure. Proteomics
based on mass spectrometry (MS) data can yield biomarker and relative protein expression
information for interrogation of physiological changes in toxin-dosed cells.
We have analyzed trypsinized cellular protein extracts from BALB/c 3T3 murine fibroblasts
dosed with three concentrations of ricin (IC 20 , IC 50 , IC 80 ) and a vehicle
control by liquid chromatography tandem MS. Nine hundred proteins were characterized
employing bioinformatics methods including in-house (ABOID,
Edgewood Chemical Biological Center) and external (ProteoIQ, NuSep) software.
Approximately 5% of the observed proteins were common to all dose levels, indi-
432 SOT 2011 ANNUAL MEETING
cating that ricin did not affect relative expression of these proteins, while more than
10% of observed proteins were unique to each specific dose level. These results indicate
a dose-dependent response of relative protein expression to ricin exposure by
3T3 cells. Ongoing pathway analysis may reveal the survival mechanism for cells
dosed at low levels, which in turn will provide insight into the related 3T3 physiological
changes required to overcome ricin dosing. Funding: The Edgewood
Chemical Biological Center In-House Laboratory Independent Research Program.
2018 ASSESSMENT OF PRO-2-PRALIDOXIME (PRO-2-PAM)
THERAPY FOR EXPOSURE TO ORGANOPHOSPHATE
AGENTS IN GUINEA PIGS.
J. C. DeMar 1 , S. M. Somerville 1 , R. H. Ratcliffe 1 , T. C. Ku 1 , N. M. Nur 1 , B.
M. Ursic 1 , S. M. Schulz 2 , K. H. Smith 2 , E. D. Clarkson 2 and R. K. Gordon 1 .
1 Regulated Laboratories, Walter Reed Army Institute of Research, Silver Spring, MD
and 2 Medical Toxicology Branch, U.S. Army Medical Research Institute of Chemical
Defense, Aberdeen Proving Ground, MD.
Novel therapeutics to overcome neuro-toxic effects of organophosphate (OP)
agents are needed due to past use in combat and terrorism. Standard exposure therapy
includes pralidoxime chloride (2-PAM), which restores OP-inhibited PNS
acetylcholinesterase (AChE) activity. Since 2-PAM is charged, it cannot cross into
the brain to restore CNS AChE. In guinea pigs, we evaluated the ability of pro-2-
PAM, a non-polar pro-drug of 2-PAM, to protect the brain after OP-agent exposure.
First, animals were pre-treated with pyridostigmine bromide and then subcutaneously
given 1 × LD 50 of the OP-agent diisopropylfluorophosphate (DFP) and
treated with pro-2-PAM and PNS acting atropine methyl bromide. Seizure (by
EEG radiotelemetry) was alleviated, brain AChE activity (by enzyme assay) was increased,
and hippocampal cell death (by histopathology at 24 h post-exposure) was
prevented. In contrast, 2-PAM did not afford this protection. Pro-2-PAM reduced
DFP-induced brain damage with treatment delays up to 60 min and at recoveries to
7 days. Next, we evaluated pro-2-PAM therapy to the chemical warfare OP-agents
soman (GD), VR, and VX. In pyridostigmine pre-treated animals cutaneously exposed
to GD, VR, or VX and treated with pro-2-PAM and CNS active atropine
sulfate, there was reduced hippocampal damage to 10, 3, and 3 × LD 50 , respectively,
at 24 h. Brain AChE activity also increased compared to exposed non-treated
controls. 2-PAM, however, did not show similar CNS-protection. In conclusion,
pro-2-PAM is a promising therapeutic for treating the detrimental effects of OPagent
poisoning in the brain.
Support: CounterACT NINDS U01 NS058166-01 and DTRA
1.E0033_07_WR_C. Opinions or assertions contained herein are private views of
the authors, and are not to be construed as official or as reflecting views of the NIH,
US Army, or DoD.
2019 SULFUR MUSTARD-INDUCED RESPONSES OF AIRWAY
AND SKIN IN VITRO.
J. Seagrave, W. Weber and G. Grotendorst. Lovelace Respiratory Research Institute,
Albuquerque, NM.
Sulfur mustard (SM) is a vesicant that has been used in warfare and is a potential
threat to civilians either from acts of terrorism or through accidental exposure to
abandoned military caches of the chemical. Exposure of the skin produces blisters
and slow-healing wounds resulting in scarring, while exposure of the lung causes
short-term sloughing of the epithelium and longer term fibrotic responses.
Inflammatory responses are also involved. Currently, few useful therapeutic agents
are available to ameliorate the adverse reactions to this agent. In vitro models of
human tissues could provide faster and more cost-effective screening prior to definitive
testing in an animal model. As preliminary model development, we exposed
human full thickness models of the skin and airway epithelium (EpiDerm and
EpiAirway cultures; MatTek Corp.) to SM vapor or to air. Half of the cultures were
then transferred to the appropriate medium and the others were transferred to
medium containing approximately 10 million freshly isolated human peripheral
blood leukocytes. The cultures were returned to the incubator. After 24 h, the
medium was harvested for analysis of matrix metalloproteinase (MMP) activity and
cytokines. The cell layers were cut into two parts: one was fixed for histopathology
and the other was frozen in lysis buffer. Epithelial sloughing (airway) and microblister
formation (dermal) was observed in the SM-exposed cultures. The results
showed upregulation of several key cytokines, with some modulation of the responses
in cultures with leukocytes during the post-exposure incubations. In many
cases, the responses closely mirrored responses in the lungs of rats exposed to SM
vapors. MMP activity was increased in the medium as well as the cell lysates of the
dermal model. These results provide preliminary information for further development
of a relevant model for testing potential therapeutics.