The Toxicologist - Society of Toxicology

goal of this study is to purify and evaluate the protective efficacy of human and rabbit
serum PON1 against sarin and soman. Large amount of human and rabbit
serum PON1 was purified using multiple chromatographies and characterized by
gel electrophoresis and Western blotting. Purified PON1 was highly pure and exhibited
catalytic activity against multiple organophosphate substrates including
CWNAs. The protective efficacy of catalytically active purified PON1 was evaluated
against CWNAs using a microinstillation inhalation exposure model in guinea
pigs. Pretreatment with 5 - 10 units of purified PON1 showed marginal increase in
the blood level but significantly increased the survival rate and reduced symptoms
of soman and sarin (1.2 LCt50) exposure. The blood levels of PON1 in pre-treated
animals after exposure to nerve agent was higher than that of control animals. The
protective efficacy highly correlated with the increased activity of AChE and BChE
in the blood and tissues of survived animals. In summary, purified human and rabbit
serum PON1 efficiently hydrolyze organophosphates and CWNAs in vitro and
significantly protect against sarin and soman exposure in guinea pigs. These data
support the development of PON1 as an efficient catalytic bioscavenger to protect
soldiers and civilians against exposure to lethal doses of CWNAs.
2025 EVALUATION OF NOVEL NERVE AGENT
ANTICONVULSANTS AND THEIR EFFECTS ON BRAIN
GLUTAMATE CONCENTRATION IN RATS.
J. Guarisco, R. K. Kan, J. K. Chandler, A. J. Wegener, T. M. Shih and J. H.
McDonough. USAMRICD, Aberdeen Proving Ground, MD.
High brain glutamate concentrations are observed after nerve agent poisoning and
are believed to be the primary cause of nerve agent-induced neuropathology. This
study was performed to determine the effects of delayed nerve agent treatment and
to evaluate standard treatments plus novel anticonvulsant adjuncts on brain glutamate
concentration. Rats were surgically prepared with electroencephalographic
(EEG) recording electrodes and a biosensor guide cannulae directed at the basolateral
amygdala. A week later the animals were pretreated with HI-6 (125 mg/kg, IP)
and 30 min later challenged with the nerve agent soman (180 μg/kg, SC). EEG and
a glutamate biosensor were monitored for seizure onset and brain glutamate concentration,
respectively. At 20 min after seizure onset the animals received standard
medical therapies, 0.45 mg/kg atropine sulfate, 25 mg/kg 2-PAM and 2.0 mg/kg
diazepam, IM, either alone or with an adjunctive drug, procyclidine, ketamine or
midazolam. In the absence of an adjunct drug, all animals continued to display
EEG seizure activity following standard medical therapies: 8 of 8 (100%) were still
seizing at 24 hr. In these animals, glutamate concentrations began to increase immediately
following seizure onset, rose to levels 183% above preseizure baselines,
and remained elevated for the remainder of the recording time. Adjunctive treatment
with procyclidine, ketamine, or midazolam in addition to standard medical
therapies stopped seizure activity even after a 20-min delay. Successful seizure control
with the adjunct drugs resulted in a decrease of glutamate concentrations back
to and below baseline levels over the remainder of the recording time. These data
provide evidence that successful seizure control will counteract the neurotoxic increase
of glutamate in the brain of soman-exposed animals.
2026 THE HAIRLESS MOUSE BACK MODEL AND THE
MEVM HAVE SIMILAR MOLECULAR PROFILES.
J. D. Wang 2 , M. Soriano 1 , N. Singer 1 , R. P. Casillas 3 , D. R. Gerecke 1, 2 and Y.
Chang 1 . 1 Pharmacology & Toxicology, Ernest Mario School of Pharmacy, Rutgers
University, EOHSI, Piscataway, NJ, 2 Pharmacology & Toxicology, Ernest Mario
School of Pharmacy, Joint Graduate Program in Toxicology, Rutgers University,
EOHSI, Piscataway, NJ and 3 Battelle Biomedical Research Center, Columbus, OH.
Sulfur mustard (bis-2-chloroethyl sulfide, SM) and Nitrogen mustard (bis-2chloroethyl
ethylamine, NM) are potent vesicant blistering agents. Both chemicals
are highly reactive bifunctional alkylating agents, which crosslink proteins, DNA
and other cellular components, resulting in disruption of tissue structure and loss of
biological activity. We compared sulfur mustard (SM) in the Mouse Ear Vesicant
Model (MEVM) to nitrogen mustard (NM) in the hairless mouse back model to
study chemical irritant-induced damage over time after exposure. Our endpoints
were erythema, edema, histopathology, as well as biochemical and inflammatory
mediators. Both models produced statistically significant dose- and time-dependent
changes in edema and histopathological features that included severe inflammation
and separation of the dermis from the epidermis. Both models showed prolonged
neutrophil presence and increased numbers of macrophages. Matrix metalloproteinase
9 (MMP-9) was significantly increased in both models as determined by
QT-PCR and western blot analysis. Both models had significant hyperplasia at 3
days post exposure and increased expression of the keratinocyte proliferation
marker, keratin 5. Expression of the wound healing protein, laminin γ2 was also
similar in the two models. We conclude that NM exposure in the hairless mouse
back model is a viable alternative to the MEVM exposed to SM. This work is supported
by ES007148, ES005022 and AR055073.
434 SOT 2011 ANNUAL MEETING
2027 USE OF GAMMA-H2AX AS A BIOMARKER TO
EVALUATE THE ANTI-GENOTOXIC ACTIVITY OF
COMPOUNDS IN CULTURED HUMAN SKIN CELLS
FOLLOWING CEES EXPOSURE.
A. L. Miller, N. K. Waraich, C. L. Gross, E. W. Nealley, O. E. Clark, K. L.
Rodgers and W. J. Smith. Research, USAMRICD, Aberdeen Proving Ground, MD.
CEES (2-chloroethyl ethyl sulfide) is monofunctional analog of sulfur mustard (2-
2’-dichlorodiethyl sulfide, SM) a chemical warfare agent. CEES was used in this
study to form a base line of DNA damage. The skin serves as a principal target site
for in vivo toxicity of SM exposure resulting in the formation of blisters and inflammation.
Previous studies show the genotoxic effects of SM using normal
human epidermal keratinocytes (NHEK, Lonza Corp., MD) as an in vitro model
by observing the presence of γ-H2AX and micronuclei formation. γ-H2AX is a
phosphorylated derivative of the H2AX histone and is tightly bound to double
stranded DNA break sites. Cells were exposed to 0, 50, 200, 500, 1000 or 1500
μM concentration of CEES for 2 hrs. Various compounds (anti-oxidants, anti-inflammatories,
plant extracts), which were described in the literature as providing
protection against the genotoxic manifestations of chemical toxicants, were added 2
hrs after CEES exposure and analyzed by flow cytometry after 24 hrs. CEES exposure
resulted in a dose dependent formation of γ-H2AX. Preliminary data show
that some of these compounds (N-acetylcysteine and Sulforaphane) have potential
to minimize the DNA damage caused by CEES. The changes of this biomarker will
be a useful technique for the study of SM toxicity and for the development of potential
therapeutic approaches. This research was supported by the Defense Threat
Reduction Agency – Joint Science and Technology Office, Medical S&T Division.
2028 ROLE OF ENDOGENOUS CANNABINOIDS IN
VESICANT-INDUCED SKIN INJURY.
I. M. Wohlman 1 , D. E. Heck 2 , N. D. Heindel 3 , M. Huang 4 , D. R. Gerecke 1 , P.
J. Sinko 5 , C. J. Lacey 3 , D. L. Laskin 1 and J. D. Laskin 6 . 1 Pharmacology &
Toxicology, Rutgers University, Piscataway, NJ, 2 Environmental Health Sciences, New
York Medical College, Valhalla, NY, 3 Chemistry, Lehigh University, Bethlehem, PA,
4 Chemical Biology, Rutgers University, Piscataway, NJ, 5 Pharmaceutics, Rutgers
University, Piscataway, NJ and 6 Environmental & Occupational Medicine, UMDNJ-
Robert Wood Johnson Medical School, Piscataway, NJ.
As a potent skin vesicant, sulfur mustard rapidly induces inflammation and blistering.
Recent studies indicate that endogenous cannabinoids including the fatty acid
amides palmitoylethanolamide and anandamide mediate dermal inflammatory reactions,
a process that is down-regulated by enzymes that degrade these compounds,
in particular, fatty acid amide hydrolase (FAAH). In the present studies we
determined if FAAH inhibitors were effective in reducing inflammation using a
mouse ear vesicant model (MEVM) and the sulfur mustard analog 2-chloroethyl
ethyl sulfide. Carbamates with a fatty (aliphatic) side chain are sufficiently similar
in structure to fatty acid amides [R-CO-NH-R’ where R’ is a lipophilic construct]
that they inhibit FAAH. Two types of carbamates were evaluated as inhibitors in the
MEVM, Type 1 or vanilloid-CH 2 NHCOO-R and Type 2 or vanilloid-CH 2 -O-
CO-NHR. When R = phenyl in the Type 1 class, the IC50 for FAAH inhibition
was 2.5 μM, this derivative was found to inhibit inflammation by more than 75%
in the MEVM. Conversely, when R = methoxyethyl there was no activity against
FAAH and minimal effects on inflammation in the MEVM. When R = geranyl in
the Type 2 class, the IC50 was 140 μM, inflammation was suppressed 34%; when
R = phenoxyformyl, the IC50 was 120 μM and the inflammation suppression was
62%; when R = perillyl, the IC50 was 17 μM and inflammation suppression 53%.
These results indicate that FAAH inhibitors are active as suppressors of vesicant-induced
inflammation in the MEVM and may be useful as sulfur mustard countermeasures.
Moreover, endocannabinoids appear to be important mediators of vesicant-induced
inflammatory reactions in the skin. Supported by AR055073,
ES004738 and ES005022.