The Toxicologist - Society of Toxicology

1751 SULFUR MUSTARD VAPORS INDUCE DNA DAMAGE
AND APOPTOSIS IN MOUSE SKIN.
L. B. Joseph 1 , D. R. Gerecke 1 , A. T. Black 1 , R. P. Casillas 2 , N. D. Heindel 3 , P.
J. Sinko 1 , D. E. Heck 4 , D. L. Laskin 1 and J. D. Laskin 5 . 1 Rutgers University,
Piscataway, NJ, 2 Battelle, Columbus, OH, 3 Lehigh University, Bethlehem, PA,
4 New York Medical College, Valhalla, NY and 5 UMDNJ-Robert Wood Johnson
Medical School, Piscataway, NJ.
Sulfur mustard (SM, bis (2-chloroethyl) sulfide) is a bifunctional alkylating agent
that causes dermal inflammation, edema and blistering. To investigate its mode of
action, we used a vapor cup model which provides an occlusive environment in
which SM vapor is in constant contact with the skin. The backs of SKH-1 hairless
male mice were exposed for 6 min to SM or control; skin was analyzed 1, 3, 7, and
14 days post-treatment. At 1 day, SM caused epidermal thickening, stratum
corneum shedding, and basal cell karyolysis; macrophages and neutrophils accumulated
in the dermis. Cleaved caspase-3, an apoptotic marker, and phosphorylated
histone 2AX (γH2AX), a DNA damage marker, were identified in basal cells.
Whereas PCNA expression was contiguous in basal cells of control skin, it was discontinuous
after SM treatment. Three days after SM exposure, epithelial cell hypertrophy,
edema, parakeratosis, and loss of normal epidermal structures were evident.
Increased epidermal expression of enzymes generating pro-inflammatory
mediators including cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase
(iNOS) was also noted. After 7 days, microblisters were evident at the dermal/epidermal
junction, and expression of keratin-10, an epithelial cell differentiation
marker was upregulated in keratinocytes. Trichrome staining revealed increased collagen
deposition in the dermis. PCNA expression in the epidermis was associated
with hyperplasia, hyperkeratosis, and parakeratosis. By 14 days, cleaved caspase-3,
COX-2 and iNOS expression was decreased, while γH2AX was still evident in the
stratum spinosum and stratum granulosum. These findings are similar to reports in
humans exposed to SM and show that the SM vapor cup model is appropriate for
investigating mechanisms of vesicant-induced DNA damage and apoptosis in the
skin. Supported by CA132624, CA093798, ES004738, ES005022, GM034310
and AR055073.
1752 MECHANISMS OF VESICANT-INDUCED
CYTOTOXICITY IN LUNG EPITHELIAL CELLS.
Y. Wang 1 , D. E. Heck 3 , D. L. Laskin 2 and J. D. Laskin 1 . 1 Environmental &
Occupational Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway,
NJ, 2 Pharmacology & Toxicology, Rutgers University, Piscataway, NJ and
3 Environmental Health Sciences, New York Medical College, Valhalla, NY.
Inhalation of vesicants such as sulfur mustard can cause significant damage to the
respiratory system including inflammation, upper and lower obstructive disease,
and acute respiratory distress syndrome. A major factor contributing to vesicant-induced
lung injury is cytotoxicity and oxidative stress. In the present studies, we
used nitrogen mustard (NM, mechlorethamine), a bifunctional alkylating agent
and model sulfur mustard vesicant, to characterize cytotoxicity and oxidative stress
in A549 cells, a human type II lung epithelial cell line. NM was found to cause a
concentration-dependent inhibition of A549 cell growth (IC50 = 1 μM).
Pretreatment of the cells with 20 μM buthionine [S, R] sulfoximine (BSO) for 6 hr,
which depletes glutathione (GSH), was found to enhance NM-induced growth inhibition
(IC50 = 0.2 μM). Cell cycle analysis revealed that 27.1 ± 0.7 % of A549
cells were in the S phase, 18.0 ±1.0 in G2M and 53.4 ± 0.5% in GoG1. Twentyfour
hr after treatment of the cells with NM (30 μM, 30 min), we observed a Sphase
block, 64.6 ± 1.4% of the cells were in S phase, 25.2 ± 1.5% in G2M and 9.3
± 0.4 in GoG1. Depletion of GSH in cells had no effect on NM-induced cell cycle
arrest. NM (30-300 μM) also enhanced the generation of intracellular hydrogen
peroxide, as determined by flow cytometry in conjunction with the hydroperoxysensitive
probe 2’,7’-dichlorofluorescein. Western blotting showed that while NM
had no effect on expression of the antioxidant enzymes catalase or heme oxygenase-
1; it cross-linked superoxide dismutase, forming a modified 32,000 molecular
weight homodimeric protein. Taken together, these data indicate that NM induced
cytotoxicity in lung epithelial cells is associated with oxidative stress and alterations
in antioxidants, processes that can contribute to vesicant-induced tissue injury.
Supported by NIH grants ES004738, ES005022 and AR055073.
1753 HOUSEKEEPING GENE EXPRESSION IN NORMAL
HUMAN EPIDERMAL KERATINOCYTES IS ALTERED
BY SULFUR MUSTARD EXPOSURE.
S. L. Beach, J. F. Dillman and A. L. Ruff. U.S. Army Medical Research Institute of
Chemical Defense, Aberdeen Proving Ground, MD.
Sulfur mustard (SM) is a potent alkylating agent that can cause severe pulmonary,
ocular, and cutaneous injury. Many of the molecular mechanisms involved in the
response to SM exposure have yet to be fully elucidated, and knowledge of these
376 SOT 2011 ANNUAL MEETING
mechanisms is necessary for the development of therapeutics for this injury. For
many molecular assays, it is important to normalize data across samples against
housekeeping gene expression. Housekeeping genes are those genes that play an important
role in general cell function and are constitutively expressed. The long-held
belief that their constitutive expression is not affected by experimental conditions
has led to their widespread use for data normalization. Studies are emerging, however,
which suggest that experimental conditions can affect the expression levels of
housekeeping genes. A previous study by our laboratory showed that the mRNA
levels of many housekeeping genes changed significantly in rat lung following SM
exposure. Following this observation, we sought to determine if housekeeping gene
expression at the mRNA and protein level is affected by SM using the in vitro cutaneous
model normal human epidermal keratinocytes (NHEK). Cells were exposed
to SM and samples were collected over a 24-hour time period. Housekeeping gene
expression was then evaluated in SM-exposed samples and unexposed timematched
controls by microarray and Western blot analysis. Our results suggest that
the expression of many housekeeping genes is affected by SM exposure, whereas the
expression of others is not. If carefully selected, some housekeeping genes may be
appropriate for data normalization in SM-exposed samples. Disclaimer: The opinions
or assertions contained herein are the private views of the author(s) and are not
to be construed as official or as reflecting the views of the Army or the Department
of Defense. This research was supported by the Defense Threat Reduction Agency
– Joint Science and Technology Office, Medical S&T Division.
1754 DEVELOPMENT OF A SULFUR MUSTARD NON-
HUMAN PRIMATE INHALATION EXPOSURE MODEL.
W. M. Weber, J. D. McDonald, L. F. Blair, M. R. Lehman, D. A. Kracko, C.
Cox, M. Duncan, J. Baldwin, J. Seagrave, T. March and G. R. Grotendorst.
Lovelace Respiratory Research Institute, Albuquerque, NM.
Sulfur mustard (SM) is a chemical threat agent for which its effects have no current
treatment. In a wartime environment, the inhalation exposure scenario is the most
likely route of administration to a soldier. Several laboratories are working to develop
therapeutics to alleviate SM induced damage. FDA guidance defines the requirement
of both small and large animal efficacy to gain approval of a new therapeutic.
Small animal inhalation models in rats have previously been reported.
Recently, we have developed a non-human primate (NHP) inhalation model for
sulfur mustard induced lung injury using cynomolgus macaques. Methods have
been developed for the generation and characterization of SM atmospheres through
an intubated exposure delivery system to maximize the amount of injury in the
lower respiratory tract. Male NHP’s were exposed to 100 or 150 mg/m3 of SM for
10 minutes (1000 or 1500 mg*min/m3). Animals were monitored and euthanized
at predetermined time points between 3 and 180 days post exposure (DPE).
The pathology induced by the SM inhalation was similar to that in our F344 rat
model of SM inhalation injury. Epithelial necrosis and sloughing is apparent along
the length of the airway. By 28 DPE there is some regeneration of the airway epithelium
but an absence of differentiated airway epithelial cells. Submucosal fibrotic
nodules are present and there is an appearance of Collagen V (Col V) leading
to the development of auto-antibodies to Col V which are detected in the NHP
serum at 28 DPE. This strongly indicates an immunologically based pathological
mechanism is being established. The occurrence of the rare pulmonary disorder in
human victims of SM exposure and our observations in the NHP model suggest
that immunomodulatory therapies will be required for successful treatment of SM
inhalation injury. This work was supported by grant number U54 NS058185-04
(S1) from the NIN/National Institute of Neurological Disorders and Stroke.
1755 A TOXICOLOGICAL, BIOCHEMICAL, AND
PHYSIOLOGICAL ASSESSMENT OF LUNG AND
SYSTEMIC INJURY IN RATS EXPOSED TO INHALED
SULFUR MUSTARD ACROSS DOSE AND TIME.
D. S. Olivera 1 , K. A. Whitten 2 , R. K. Kan 3 , J. L. Collins 1 , J. B. Simons 1 , A. M.
Rodriguez 1 , C. M. Bowens 1 , A. M. Witriol 1 and A. M. Sciuto 1 . 1 Analytical
Toxicology, USAMRICD, Gunpowder, MD, 2 Research Support, USAMRICD,
Gunpowder, MD and 3 Research, USAMRICD, Gunpowder, MD.
Sulfur mustard (HD) is a blistering agent (vesicant) that causes severe chemical
burns to the skin, eyes, and airways. HD was used as a chemical warfare agent
(CWA) in the Iran/Iraq conflict, and more than half of surviving HD-exposed casualties
suffer from permanent lung injuries. HD remains a serious threat with no
effective antidote. Despite research in the 90 years since HD was developed as a
CWA, the mechanisms and timing of the development of these pathologies are
poorly defined. To model the mouth-breathing human, rats were intubated and
ventilated for 10 min with nebulized HD or vehicle to achieve total doses of 0, 0.5,
1.75, 2.25, and 3 mg/kg. Pulmonary function was analyzed by whole-body
plethysmography. Rats were euthanized at various time-points ≤ 6 months post-ex-